Patent Publication Number: US-11396648-B2

Title: Stable protease variants

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of priority under 35 U.S.C. § 371 of International Application No. PCT/EP2017/084452, filed on Dec. 22, 2017, which claims the benefit of the filing date of European Application No. 16206367.1, filed on Dec. 22, 2016. The content of these earlier filed applications is hereby incorporated by reference herein in its entirety. 
     REFERENCE TO SEQUENCE LISTING 
     The Sequence Listing submitted herewith as a text file named “13318 0043U1 Sequence Listing,” created on Jun. 19, 2019, and having a size of 18,646 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). 
     FIELD OF THE INVENTION 
     The present invention relates to the field of proteases. 
     BACKGROUND 
     Proteases are today used in large array of industrial applications, including animal feed, detergents, fruit and beverage processing, leather processing, production of protein hydrolysates, hard surface cleaning or biofilm cleaning, treatment of necrotic or burned tissue to promote wound healing and/or food preparation including baking dough preparation. 
     In many of these applications, improved stability of the enzyme is a significant advantage. Improved thermostability helps to increase the processability of the respective protease, because the latter oftentimes undergoes thermal treatment during the manufacturing process. 
     This applies, inter alia, for the use of proteases in animal feed where they help to improve the digestibility and nutrient exploitation of the feed. 
     During feed processing, the feed is often subjected to heat, e.g., by application of steam, to reduce or eliminate pathogens, increase storage life of the feed and optimized utilization of the ingredients leading to improved feed conversion. The conditioning time can vary from a few seconds up to several minutes depending on the type and formulation of the feed. The temperature during conditioning typically ranges from 70° C. to 100° C. After conditioning, the feed is sometimes extruded through a pelleting die, which for a short time raises the temperature of the feed incrementally due to heat dissipation caused by friction. 
     Yet in other applications, protease enzymes are exposed to heat as well. This includes the use in detergents (e.g. exposure to hot water during laundry washing), fruit and beverage processing (heat exposure during the squeezing process or due to pasteurization or sterilization), leather processing, production of protein hydrolysates, hard surface cleaning or biofilm cleaning, treatment of necrotic or burned tissue to promote wound healing, processing aid in tissue engineering (sterilization, and denaturation of prion proteins) and/or food preparation including baking dough preparation. 
     Because proteases are proteins, they are susceptible to denaturation by heat and pressure. Denaturing essentially alters the structure of the enzyme, resulting in decreased activity levels and decreased efficacy of the enzyme. 
     There are different ways to improve protease stability or protect proteases from thermal impact. In animal feed applications, one option is Post-pellet liquid application, which is relatively complex and expensive because it requires the purchase and installation of specialized equipment, space in which to store the liquid enzyme and careful calculation of the amount of enzyme to apply. 
     Another option is the application of a protective coating before pelleting of the protease with other ingredients (e.g., in feed or detergents). This approach may reduce the efficacy of the enzyme because the coating may not fully dissolve, e.g., in the washing medium, or in the digestive tract of the animal. It is furthermore difficult to achieve a coating design that can withstand the high heat and moisture content of the pelleting process, but subsequently dissolve in the lower temperature and higher moisture conditions, e.g., in the animal&#39;s gut or the washing machine. 
     Another option is to use intrinsically thermostable proteases. These proteases are derived from thermophilic and hyper-thermophilic organisms and have unique structure and function properties of high thermostability. However, these proteases may suffer from other limitations, like suboptimal activity, specificity, bioavailability, pH-range or processability. 
     It is hence one object of the present invention to provide stable protease variants which do not suffer from the above discussed limitations. 
     SUMMARY OF THE INVENTION 
     These and further objects are met with methods and means according to the independent claims of the present invention. The dependent claims are related to specific embodiments. 
     EMBODIMENTS OF THE INVENTION 
     Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts or structural features of the devices or compositions described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include singular and/or plural referents unless the context clearly dictates otherwise. Further, in the claims, the word “comprising” does not exclude other elements or steps. 
     It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values. 
     It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done. 
     According to one embodiment of the invention, a protease variant is provided which is at least 90% identical to the full length amino acid sequence of a Kumamolisin AS backbone as set forth in any of SEQ ID NOs 1-3, or a fragment, fraction or shuffled variant thereof maintaining proteolytic activity. The protease variant demonstrates altered or improved stability compared to
         (i) the Kumamolisin AS wildtype as set forth in SEQ ID NO 4, or   (ii) the Kumamolisin AS backbone as set forth in any of SEQ ID NOs 1-3.       

     The term “shuffled variant” relates to a combination of such fragment or fraction with one or more fragments from other homologous enzymes, as long as such combination maintains proteolytic activity. 
     The term “homologous enzyme” describes enzymes belonging to the same structural fold as Kumamolisin and at least 40% sequence identity. This category encompasses Sedolisins as discussed below herein. 
     Some mutants of Kumamolisin AS have been described. The discovery of N291D mutant strain of Kumamolisin AS has been discussed to providing a useful treatment against celiac disease. There are many proposals that suggest creating a genetically modified organism that could produce N291D Kumamolisin AS protein in human&#39;s gastrointestinal tracts. See US application US 20140178355 A1. 
     Preferably, the Kumamolisin AS variant according to the invention has 93% identity, more preferably 95% identity, more preferably 98% identity, most preferably 99% identity. 
     The term “Kumamolisin” refers to acid proteases from the Sedolisin family of peptidases, also called S53 (MEROPS Accession MER000995, see also Wlodawer et al, 2003), comprising acid-acting endopeptidases and a tripeptidyl-peptidase. Sedolisins are endopeptidases with acidic pH optima that differ from the majority of endopetidases in being resistant to inhibition by pepstatin (Terashita et al., 1981; Oda et al., 1998). 
     The activation of sedolisins involves autocatalytic cleavage at pH below pH 6.5, better below pH 3.5 (see also patent application EP16176044 and Okubo et al., 2016), which releases one or more peptides to deliver the maturated and active form. Said autocatalytic cleavage is inhibited under alkaline, neutral and lightly acidic conditions. 
     Sedolisins comprise a catalytic triad with Glu, Asp and Ser, which in Kumamolisin AS according to SEQ ID NO 1 reside in positions Glu267, Asp271 and Ser278. The Ser residue is the nucleophile equivalent to Ser in the catalytic triad Asp, His, Ser triad of subtilisin proteases (MEROPS family S8), and the Glu of the triad is a functional substitution for the His general base in subtilisin though not in structural equivalent positions. 
     The protein folds of sedolisins are clearly related to that of subtilisin, and both groups are sometimes called serine proteases. However, sedolisins have additional loops. The amino acid sequences are not closely similar to subtilisins, and this, taken together with the quite different active site residues and the resulting lower pH for maximal activity, justifies the separate families. 
     In one embodiment, a protease variant is provided which comprises an amino acid sequence derived from a Kumamolisin AS as set forth in SEQ ID NO. 1, or a fragment, fraction or shuffled variant thereof maintaining proteolytic activity, which protease variant has one or more amino acid substitutions at one or more residue positions in SEQ ID NO. 1 selected from the group consisting of D447, A449, A517, N510, V502, E453, E360, A514, A460, A392, A386, T301, D199, Q518, G266, P553, E269, R412, S435, G320, T326, T461, Q244, D293, A487, V274, A372, K283, T308, A418, I391, A423, A331, S327, 1219, M333, A329, N515, A378, S434, E421, A433, S230, Q393, D399, Y490, G281, Y287, R516, A475, S354, S315P, W325, L442, A470, S324, Q361, A190, T196, Q202, E228, A229, A242, D251, S262, N291, L297, H305, D306, V314, A328, 1330, L338, A342, A351, D358, G388, D402, V455, E459, A478, K483, Q497, T507, L540, Q542, A548, P551, R166 and/or D265. 
     Note that, while the numbering set forth above refers to SEQ ID NO 1 or 4 (which are almost identical, with 4 being the wildtype and 1 being the actual backbone used for mutagenesis, the difference between the two being the N terminal AA residue), the claimed protease can be a fragment, fraction or shuffled variant thereof maintaining proteolytic activity. In such case, the resulting amino acid sequence is shorter than that of SEQ ID NO 1 or 4, while the numbering of the mutant residues still refers to the full length SEQ ID NO 1 or 4, and has to be translated respectively to the numbering of the shorter form. 
     In one embodiment, the protease variant demonstrates altered or improved stability compared to
         (i) the Kumamolisin AS wildtype as set forth in SEQ ID NO 4, or   (ii) the Kumamolisin AS backbone as set forth in any of SEQ ID NOs 1-3,       

     In one embodiment, the protease variant has at least one amino acid substitution selected from the group consisting of D447S, A449Y, A517T, N510H, E360L, E360V, E360C, V502C, E453W, A514T, A514Y, A514D, A514S, A460W, A386I, A392V, A392L, A392I, A392M, T301S, D199E, Q518G, P553K, E269M, E269T, E269C, E269H, E269Q, G266A, D293Y, G320A, R412Q, E421R, A487Q, T461V, T461C, A331F, A331Y, A329Q, A329H, A329T, S435I, S435R, 5435T, S435V, V274I, A372S, K283L, Q244C, Q244G, T308C, A418W, I391W, A423V, T326R, T326W, T326L, T326K, I219L, S327F, S327L, S327W, M333I, N515G, A378G, S434G, A433G, 5230D, Q393S, D399S, Y490W, A190D, T196S, Q202D, E228Q, A229W, A242S, D251S, S262C, G281R, Y287K, N291T, N291S, D293F, L297T, T301C, T301M, H305F, H305W, D306S, V314M, V314L, 5315P, G320Q, G320S, S324L, S324R, W325K, A328W, A328D, A328R, A328Y, I330L, M333Y, M333L, L338R, A342R, A351S, S354E, S354Q, D358G, Q361C, Q361L, A386L, A386V, A386M, G388C, D402E, R412M, R412E, R412D, L442W, L442W, D447C, D447A, A449L, A449M, A449E, A449N, E453Y, E453F, V455I, V455L, E459W, A460R, A470V, A475V, A478L, K483A, Q497Y, Q497M, Q497D, Q497R, V502T, T507L, R516L, R516E, R516I, A517S, L540V, Q542H, Q542D, Q542S, A548S, P551N, P551R, P553L, R1661, D265T, compared to the Kumamolisin as as set forth in SEQ ID NO 1 or 4. 
     These individual amino acid substitutions are shown in Table 1. Note that, while the numbering set forth above refers to SEQ ID NO 1 or 4, the claimed protease can be a fragment, fraction or shuffled variant thereof maintaining proteolytic activity. In such case, the resulting amino acid sequence is shorter, or longer, than that of SEQ ID NO 1 or 4, while the numbering of the mutant residues still refers to the full length SEQ ID NO 1 or 4. 
     In one embodiment of the invention, the protease variant has at least one amino acid substitution compared to the Kumamolisin AS as set forth in SEQ ID NO 1 or 4, which substitution is selected from the group consisting of:
         A517T or A517S   A514S, A514T or A514D   N510H   V502C   A449Y, A449N or less preferred A449E   D447S or D447C   A392I, A392L, A392V or A392M   E360L, E360V or E360C   E269H, E269T, E269M, E269C or E269Q   Q518G   G320Q, G320A or less preferred G320S   A386I, A386L, A386V or A386M   G266A   A372S   E453Y, E453W or less preferred E453F   A460W   A329Q, A329H or A329T   D293Y   R412E, R412D, R412Q or R412M   T301S   D199E   A331F or A331Y   S435T, S435R or S435I   V274I   D399S   S230D   S434G   M333I or M333L   N515G   A418W   I391W   E421R   A487Q   A378G   A423V   T326K, T326L, T326R or T326W   A433G   D399S   Y490W   R516E or R516I   P553K   V314L   S327W, S327L or S327FA475V   A342R   S354E or S354Q   S315P       

     Some of these substitutions cause a high ΔIT50 when introduced individually into the Kumamolisin AS as set forth in SEQ ID NO 1 or 4, and are therefore preferred, while others have a high occurrence in the combinatorial and distinct clones of Tables 2a, 2b and 4 and some combinations, which have a combination of individual substitutions with a high overall ΔIT50. 
     Some can interchangeably be used to stabilize the enzyme and some combinations results in other traits that are relevant for the production or performance in feed, like fermentation titers, the hydrolysis of anti-nutritive factors as protease inhibitors (soy bean Bowman-Birk and Kunitz-type trypsin and/or chymotrypsin inhibitors), pH profile, pH and pepsin stability, or stability against and performance under higher ionic strength. 
     Note that, while the numbering set forth above refers to SEQ ID NO 1 or 4, the claimed protease can be a fragment, fraction or shuffled variant thereof maintaining proteolytic activity. In such case, the resulting amino acid sequence is shorter than that of SEQ ID NO 1 or 4, while the numbering of the mutant residues still refers to the full length SEQ ID NO 1 or 4. 
     In one embodiment of the invention, the protease variant has at least two amino acid substitutions compared to the Kumamolisin AS backbone as set forth in SEQ ID NO 1 or 4. Preferably the protease variant has at least three, more preferably at least four, more preferably at least five and most preferably at least six amino acid substitutions selected from said group. Preferably, these amino acid substitutions are combinations of the individual substitutions discussed above. 
     In one embodiment of the invention, the protease variant has at least two amino acid substitutions compared to the Kumamolisin AS backbone as set forth in SEQ ID NO 1 or 4, the at least 2 amino acid substitutions being at two or more residue positions in SEQ ID NO 1 or 4 selected from the group consisting of 447 and 449, 453, 502, 510, 517, 360, 460, 199, 266, 301, 386 and 514. Preferably the protease variant has at least three, more preferably at least four, more preferably at least five and most preferably at least six amino acid substitutions selected from said group. 
     In one preferred embodiment, the protease variant has at least one, preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, most preferably at least six amino acid substitutions selected from the group consisting of D447S, A449Y, A517T, N510H, E360L, E360V, E360C, V502C, E453W, A514T, A514Y, A460W, A386I, D199E, G266A, T301S. 
     Tables 2a, 2b and 4 show sets of such so-called “distinct clones” or “combinatorial clones” which have combinations of the individual mutations set forth above. 
     As used herein, the term “combinatorial clone or variant” means a clone or variant screened from a recombination library. Such a recombination library contains a population carrying different amounts and mutations selected from the group of table 1. 
     As used herein, the term “distinct clone or variant” means A clone constructed containing a defined set of mutations selected from the group of table 1 in a rational approach. 
     Preferably, said improved stability which the protease variant according to the invention has is improved thermostability (IT50). The thermostability of an enzyme is usually determined by measuring the inactivation temperature (IT 50). The “inactivation temperature” is defined as the temperature at which the residual activity of the enzyme after incubation for a certain duration and subsequent cooling to room temperature is 50% of the residual activity of the same enzyme incubated for the same duration under the same conditions at room temperature. 
     According to one embodiment, the protease variant has a set of substitutions at selected residues in the Kumamolisin AS backbone as set forth in SEQ ID NO 1 or 4, which set is at least one of the following 
     a) 360, 447, 449 and 510 
     b) 447, 449 and 514, and/or 
     c) 447, 449, 453, and 517. 
     These three sets of simultaneously substituted residues occur in three sets of specific distinct or combinatorial clones which are particularly preferred (consensus mutations). See Table 2a/ FIG. 3 , Table 2b/ FIG. 4  and Table 4/ FIG. 5 . For these reasons, these sets of simultaneously substituted residues seem to be particularly synergistic when it comes to improvement of stability. 
     According to one embodiment said improved stability is improved thermostability (IT50) of either the activated enzyme or the zymogen. In one embodiment of, the protease variant has an IT50 of between ≥75 and ≤105° C. 
     In some embodiments, for the activated enzyme an IT50 of between ≥70 and ≤90° C. is provided, while a for the zymogen an IT50 of between ≥80 and ≤105° C. is provided. 
     The Kumamolisin AS wildtype enzyme has an IT50 of 79.6° C.+/−0.4° C. (n=46) as the zymogen, i.e., the inactive zymogen, and an IT50 of 59° C.+/−1° C. (n=10) as the activated enzyme. In the course of this specification, the different variants are either characterized by their IT50, or by ΔIT50 (i.e., the difference compared to the wildtype IT50). 
     According to another embodiment of the invention, a nucleic acid molecule encoding a protease variant according the above description is provided. Furthermore, a plasmid or vector system comprising said nucleic acid molecule is provided, as well as a host cell being transformed with said plasmid or vector and/or comprising said nucleic acid molecule is provided. 
     Further, a method for producing a protease or protease variant is provided, said method encompassing: 
     a) cultivating said host cell, and 
     b) isolating the protease or protease variant from said host cell, or harvesting the protease or protease variant from the medium. 
     According to another embodiment of the invention, a composition comprising a protease variant according to the above description is provided, which composition has a pH of ≥5. 
     Such composition is generally discussed—yet not with the specific protease variants disclosed herein—in EP application No 16176044.2-1375 and later applications claiming the priority thereof, the content of which is incorporated by reference herein. 
     According to another embodiment of the invention, a feed additive, feed ingredient, feed supplement, and/or feedstuff comprising a protease variant or a composition according to the above description is provided. 
     Further, the use of a protease variant according to the above description for the manufacture of a feedstuff is provided. 
     Such feed additive, feed ingredient, feed supplement, and/or feedstuff is preferably meant for monogastric poultry, pig, fish and aquaculture, where it helps to increase protein digestion and absorbance from the feedstuff, plus degrade proteinogenic compounds which are detrimental for animal health or digestion. 
     Furthermore, the use of a protease according to the above description is provided for at least one purpose or agent selected from the group consisting of:
         detergent   fruit and beverage processing   leather processing   production of protein hydrolysates   hard surface cleaning or biofilm cleaning   treatment of necrotic or burned tissue to promote wound healing,   processing aid in tissue engineering and/or   food preparation including baking dough preparation.       

     Likewise, an additive, ingredient or agent for one purpose or agent selected from the group consisting of:
         detergent   fruit and beverage processing   leather processing   production of protein hydrolysates   hard surface cleaning or biofilm cleaning   treatment of necrotic or burned tissue to promote wound healing   processing aid in tissue engineering and/or   food preparation including baking dough preparation.
 
is provided which additive, ingredient or agent comprises a composition according to the above description.
       

     Furthermore, a process of generating a protease variant according to the above description is provided, which process comprises: 
     i) mutagenizing a DNA, cDNA or mRNA encoding a Kumamolisin AS amino acid sequence as set forth in any of SEQ ID NOs 1-4 
     ii) expressing one or more mutants of Kumamolisin AS thus obtained, and 
     iii) testing the one or more mutants of Kumamolisin AS for at least stability, preferably thermostability. 
     Preferably, in said method, the encoding nucleic acid sequence and/or the amino acid sequence of one or variants of Kumamolisin AS is determined. For this purpose, routine methods from the prior art can be used. 
     EXPERIMENTS AND FIGURES 
     While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. Any reference signs should not be construed as limiting the scope. 
     All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5′-&gt;3′. 
     1. Amino Acid Sequences of the Kumamolisin AS Backbone 
     SEQ ID NO 1 shows the proenzyme (propeptide plus enzyme, also called zymogen herein) sequence of the Kumamolisin AS backbone used herein. It is important to understand that, while the wildtype sequence of Kumamolisin AS has an N-terminal M residue, the Kumamolisin AS backbone used herein lacks said M, because the latter was replaced by a signal sequence that was later cleaved off. Such signal sequence is for example, the sacB signal peptide MNIKKFAKQATVLTFTTA LLAGGATQAFA (SEQ ID NO:5). 
     In SEQ ID NO:1, the propeptide hence comprises AAs 2-189 (former N-terminal M which is lacking is yet considered as AA NO 1 in the numbering of SEQ ID NO), and the enzyme comprises AAs 190-553: 
     
       
         
           
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
            
           
         
       
     
     The propeptide is shaded in grey. The catalytic triad SED (=Ser/Glu/Asp) consists of E267, D271 and S467, shown in italics. The positions where the inventors have found mutations that result in altered/improved properties are underlined. 
     2. Amino Acid Sequences of the Kumamolisin AS Backbone Plus Leader Sequence and HisTag 
     In SEQ ID NO:2, the sacB leader sequence comprises AAs 1-29 (wavy underline) and replaces the original N-terminal M of the propeptide. The propeptide (shaded in grey) comprises AA 30-217, the activated enzyme comprises AA 218-581 and the His-tag comprises AAs 582-587 (double underline). 
                                                           
3. Amino Acid Sequences of the Activated Kumamolisin AS Backbone Devoid of Propeptide
 
     In SEQ ID NO:3, the activated Kumamolisin AS backbone enzyme is shown with AAs 1-364: 
                            AAPTAYTPLD VAQAYQFPEG LDGQGQCIAI IELGGGYDEA SLAQYFASLG VPAPQVVSVS    60                   VDGASNQPTG DPSGPDGEVE LDIEVAGALA PGAKFAVYFA PNTDAGFLDA ITTAIHDPTL   120               KPSVVSISWG GPEDSWTSAA IAAMNRAFLD AAALGVTVLA AAGDSGSTDG EQDGLYHVDF   180               PAASPYVLAC GGTRLVASGG RIAQETVWND GPDGGATGGG VSRIFPLPAW QEHANVPPSA   240               NPGASSGRGV PDLAGNADPA TGYEVVIDGE ATVIGGTSAV APLFAALVAR INQKLGKAVG   300               YLNPTLYQLP ADVFHDITEG NNDIANRAQI YQAGPGWDPC TGLGSPIGVRL LQALLPSAS   360               QPQP   364            
4. Amino Acid Sequence of the Kumamolisin AS Wildtype
 
     SEQ ID NO:4 shows the proenzyme (propeptide plus enzyme) sequence of the Kumamolisin AS wildtype, as obtained from  Alicyclobacillus sendaiensis  (GenBank: AB085855.1). SEQ ID NO:4 differs from SEQ ID NO:1, which shows the sequence of the Kumamolisin AS backbone used herein in that the latter lacks the N-terminal M still present in the Wildtype SEQ ID NO:4. This is because the N-terminal M was replaced, in SEQ ID NO:1, by the sacB signal sequence, which was later cleaved off. In SEQ ID NO:4, the propeptide comprises AAs 1-189, and the enzyme comprises AAs 190-553: 
     
       
         
           
               
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
            
           
         
       
     
     Again, the propeptide is shaded in grey. The catalytic triad SED (=Ser/Glu/Asp) consists of E267, D271 and S467, shown in italics. 
    
    
     
       SHORT DESCRIPTION OF THE FIGURES 
         FIG. 1  shows the distribution of mutations in variants optimized for thermal stability of the zymogen and the activated enzyme. 
         FIG. 2  shows the effects of the ionic strength on stability and performance for the WT and top variants #1 to #7 from table 4. 
         FIG. 3-5  show the occurrence of substitutions at AA position in different sets of distinct clones and combinatorial clones. 
     
    
    
     EXAMPLE 1: PROTEASE ACTIVITY ASSAY 
     Protease activity assays were carried out in microtiter plates 
     a) AAPF Assay 96 Well Formate 
     
         
         Assay buffer: 200 mM Sodium Acetate, 1 mM CaCl 2 , 0.01% Triton X-100 at pH 3
       depending on the experiment   
     
         Substrate stock solution: 100 mM in water free DMSO 
         Substrate working solution: Substrate Stock solution diluted 1:50 in assay buffer, 
       
    
     Execution: Load 50 μL of the diluted sample into the wells of a Nunc 96 clear flat bottom plate. Dilution is made in water containing 0.01% Triton-X100 corresponding to the volumetric activity of the sample. Start reaction by adding 50 μL of substrate working solution. Measure kinetics at 37° C. by monitoring the increase in adsorption at 410 nm as a measure for enzymatic activity. The activity was calculated by building a calibration curve with a reference enzyme preparation of the backbone with known proteolytic activity measured by a reference method. 
     For assaying the protease activity at different pH values the following buffers were used, each 200 mM: glycine/HCL between pH 2.0-3.0, trisodium citrate/citric acid between 3.0 and 6.0 and Tris/maleic acid between 6.0 and 7.5. 
     b) IT 50    
     IT 50  defines the temperature where 50% of the activity is inactivated under the conditions described above. Although not equivalent to, it is a measure for the thermal stability in the application, e.g. pelleting conditions or conditions in a detergent application, either dish washing or the cleaning of a fabric or hard surface and other technical applications. 
     The screening of enzyme variants under predictive conditions is essential. For proteases like those described herein, screening for thermally more stable variants by methods as also described herein can be affected by the self-hydrolysis of the protease. As already described in patent application EP16176044 Example 9, screening for variants with higher thermal stability under conditions where the protease is active results in a large number of false positives, as the result of a mixed effect of thermal inactivation and self-hydrolysis. The same applications teaches to circumvent this problem in the absence of small molecule reversible enzyme inhibitors, as is the case for the class of acid protease described herein, by executing the test for thermal stability of the enzyme and enzyme variants in the form of the inactive enzyme zymogen in the way described below. 
     Assay buffers: 50 mM sodium phosphate, 0.25 mM CaCl 2 ) pH6.5 
     
         
         
           
             800 mM glycine/HCl pH2.8 
           
         
       
    
     Thermal inactivation execution: Samples were diluted corresponding to the volumetric activity in potassium phosphate buffer. The pH of the final solution was checked to be above pH 6.3. The samples were transferred in replicates, 20 μL per well, into a 384 well PCR plate according to the direction of the temperature gradient of the PCR machine. The plates were sealed with an adhesive or hot melting cover foil and incubated on a thermal gradient cycler with a temperature gradient of +/−12° C. around the expected IT50 value for 10 minutes. The samples were cooled to 8° C. before measuring the residual activity of the samples with AAPF-pNA as followed. Samples, 15 μL each from the temperature incubation plate were transferred into a 384 well greiner clear flat bottom PS-microplate and 9 μL of glycine buffer was added to activate the protease during an incubation of 1 hour at 37° C. After the activation of the protease the assay was started by adding 24 μL of an AAPF-pNA solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by following the kinetics at 37° C. The normalized experimental data for residual activity at the inactivation temperatures were fitted to a four parameter logistics function to evaluate the IT50. 
     c) IT50 without Propeptide—Activated Enzyme Protein: 
     Enzyme activation prior to thermal inactivation execution. Samples were diluted corresponding to the volumetric activity in glycine buffer pH 2.8 as described in 2b) and pH was checked to be equal or lower than pH 4.0. Samples were activated by an incubation for 1 hour at 37° C. After the incubation pH was set to above 7.0 by diluting the samples 1:3 in 50 mM sodium phosphate buffer pH 8.0. Thermal inactivation of activated enzyme protein execution. Aliquots of the activated enzyme protein were transferred in replicates, 20 μL per well, into a 384 well PCR plate according to the direction of the temperature gradient of the PCR machine. The plates were sealed with an adhesive or hot melting cover foil and incubated on a thermal gradient cycler with a temperature gradient of +/−12° C. around the expected IT50 value for 10 minutes. The samples were cooled to 8° C. before measuring the residual activity of the samples with AAPF-pNA as followed. Samples, 15 μL each from the temperature incubation plate were transferred into a 384 well greiner clear flat bottom PS-microplate and 9 μL of glycine/HCl buffer was added to adjust the pH to 3.0. The assay was started by adding 24 μL of an AAPF-pNA solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by following the kinetics at 37° C. The normalized experimental data for residual activity at the inactivation temperatures were fitted to a four parameter logistics function to evaluate the IT50. 
     d) pH-Profile—Activated Enzyme Protein 
     Undiluted bacterial supernatant containing enzyme protein was titrated with 1 M HCl to pH 4 and enzyme was activated at 37° C. for 60 min. 20 μl of sample were added to 200 μL Britton Robinson buffer with pH 1.8-7.0 (adjusted to conductivity of 15 mS/cm with NaCl). 20 μL were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet. 
     e) pH/Pepsin-Resistance 
     Undiluted bacterial supernatant containing enzyme protein was titrated with 1 M HCl to pH 2.5. 90 μl were then transferred to a Nunc 96-well clear flat bottom microtiter plate. 10 μl of a 250 μg/mL Pepsin stock solution in pH 2.5 buffer (final concentration in assay 25 μg/mL) or pH 2.5 buffer were added to each well and then incubated at 37° C. for 30 min. Finally, 5 μl of a 100 μM Pepstatin A solution (final concentration 5 μM) was added to each well to stop the pepsin reaction. 25 μl of the sample were transferred in 175 μl glycine/HCl buffer pH 3.0 in a new Nunc 96-well clear flat bottom microtiter plate. 20 μl were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet. 
     f) Conductivity Dependency 
     20 μl undiluted bacterial supernatant was diluted in 180 μL glycine/HCl buffer pH 3.0 adjusted with NaCl to conductivity of 2, 4, 6, 10, 20, 30, 40, 50 mS/cm in a Nunc 96-well clear flat bottom microtiter plate. The samples were incubated at 37° C. for 20 min and then 20 μL sample were then transferred into a 384-well Greiner flat bottom PS-microplate plus 20 μl substrate solution (2 mM AAPF-pNA in water with 0.01% Triton-X100) and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet. 
     g) BBI/KTI Hydrolysis—Functional Trypsin Assay 
     Bowman-Birk and Kunitz-type inhibitors (BBI/KTI) are strong inhibitors of serine proteases which are widely spread in seed of legumes and cereal grains. The assay principle is that a proteolytic degradation of the BBI/KTI by protease activity recovers the natural trypsin activity on Benzyl-Arginine-pNA (Bz-R-pNA) substrate without inhibitors. 90 μL of bacterial supernatant containing enzyme protein was diluted in glycine/HCl buffer to pH 3.0 and then incubated at 37° C. for 30 min. 20 μl of the sample was then mixed with 20 μl inhibitor solution (KTI: 8 μg/mL; BBI: 16 μg/mL; KTI/BBI: 4/8 μg/mL diluted in glycine buffer pH 3.0) and further incubated at 37° C. for 60 min. 15 μl of the sample were transferred into a 384-well Greiner flat bottom PS-microplate and then 15 μl trypsin solution in pH 8.0 (final trypsin concentration 1 μg/mL; final pH 7.0 or pH 7.5) was added to each well and the plate was incubated at 37° C. for 10 min. Finally, 30 μL substrate solution (2 mM Bz-R-pNA in water with 0.01% Triton-X100) was added to each well and activity was measured by monitoring the kinetics at 410 nm and 37° C. as described in example 1a). Each kinetic experiment was run in quadruplet. 
     EXAMPLE 2: GENERATION OF GENETIC DIVERSITY 
     Initial genetic diversity was introduced by randomizing each position of the active enzyme core sequence of SEQ ID NO 1. Mutant enzyme single site saturation libraries were introduced in the gene carried on an  E. coli/Bacillus  shuttle vector using mutagenesis methods as described in Green &amp; Sambrook (eds), Molecular Cloning, 4 th  edition, CSHL and suitable mutagenic PCR methods as disclosed in Cadwell and Joyce (PCR Methods Appl. 3 [194], 136-140. Protease enzyme variants were characterized after heterologous expression in  Bacillus subtilis  and phenotypically optimized variants selected by the screening procedure outlined in Example 3. 
     In general, methods to mutagenize a protein, like an enzyme, to obtain a library of mutated proteins members of which may have altered characteristics, are well established. Methods to mutagenize a protein encompass site directed mutagenesis and others, as described e.g. in Hsieh &amp; Vaisvila (2013), content of which incorporated herein by reference for enablement purposes. 
     Such methods are sometimes called “directed evolution”, namely when the established library is then screened for particular features. Packer &amp; Liu (2015) provide an overview of the respective methodology, content of which incorporated herein by reference for enablement purposes. 
     EXAMPLE 3: PHENOTYPICALLY SCREENING FOR ENZYME VARIANTS WITH INCREASED THERMAL STABILITY 
     The generated genetic diversity either in the initial stage in form of single site saturation libraries or in the subsequent stage in the form of recombination libraries or distinct clones was screened for variants with an optimized phenotype, i.e. increased thermal stability using the method as described in example 1b) with adaptations required to run them in a fully automated robotic workstation at high throughput. These were mainly adaptation in incubation times, volumes, substrate and the main adaptation was to select optimized variants not by the thermal inactivation profile on a temperature gradient but by the residual activity after incubation at a single temperature, the temperature which was set to discriminate optimized variants from the average of the genetic diversity. Protease variants were derived which differed in one or more amino acid positions from SEQ ID NO 2, including two positions, three positions, n positions. Appropriate iterative rounds of the procedures described herein were performed to satisfy the demands of the application 
     EXAMPLE 4 
     The following individual mutations which increase the IT50 compared to the used backbone were identified. The IT50 was analyzed as described above and compared to the IT50 of the used backbone (=wildtype with missing N-terminal methionine) characterizing the variant by the corresponding ΔIT50. The backbone has an IT50 of 79.6° C.+/−0.4° C. (n=46) as zymogen and an IT50 of 59° C.+/−1° C. (n=10) as activated enzyme. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Kumamolisin AS single amino acid substitutions relative to SEQ ID NO 1, 
               
               
                 and their ΔIT50 compared to the backbone for the zymogen and the 
               
               
                 activated enzyme 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 ΔIT50 
                 ΔIT50 activated 
               
               
                   
                 Position 
                 Mutation 
                 Zymogen 
                 Enzyme 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 A190 
                 D 
                 1.5 
                 0.8 
               
               
                   
                 T196 
                 S 
                 0.7 
                 0.3 
               
               
                   
                 D199 
                 E 
                 0.5 
                 1.0 
               
               
                   
                 Q202 
                 D 
                 0.4 
                 −0.3 
               
               
                   
                 I219 
                 L 
                 1.1 
                 0.8 
               
               
                   
                 E228 
                 Q 
                 0.7 
                 0.1 
               
               
                   
                 A229 
                 W 
                 0.2 
                 n.d. 
               
               
                   
                 S230 
                 D 
                 2.8 
                 −0.8 
               
               
                   
                 A242 
                 S 
                 0.3 
                 −0.4 
               
               
                   
                 Q244 
                 C 
                 0.5 
                 −3.6 
               
               
                   
                 Q244 
                 G 
                 0.7 
                 1.5 
               
               
                   
                 D251 
                 S 
                 0.8 
                 −0.3 
               
               
                   
                 S262 
                 C 
                 0.9 
                 −0.3 
               
               
                   
                 G266 
                 A 
                 1.7 
                 0.0 
               
               
                   
                 E269 
                 M 
                 2.4 
                 −0.1 
               
               
                   
                 E269 
                 T 
                 2.6 
                 −0.1 
               
               
                   
                 E269 
                 C 
                 2.1 
                 −1.1 
               
               
                   
                 E269 
                 H 
                 4.0 
                 −0.5 
               
               
                   
                 E269 
                 Q 
                 2.0 
                 −1.4 
               
               
                   
                 V274 
                 I 
                 1.8 
                 1.3 
               
               
                   
                 G281 
                 R 
                 2.0 
                 5.4 
               
               
                   
                 K283 
                 L 
                 0.6 
                 −0.2 
               
               
                   
                 Y287 
                 K 
                 0.2 
                 5.2 
               
               
                   
                 N291 
                 T 
                 0.7 
                 0.5 
               
               
                   
                 N291 
                 S 
                 −0.2 
                 1.0 
               
               
                   
                 D293 
                 Y 
                 0.8 
                 1.0 
               
               
                   
                 D293 
                 F 
                 1.1 
                 1.3 
               
               
                   
                 L297 
                 T 
                 1.2 
                 0.2 
               
               
                   
                 T301 
                 S 
                 0.6 
                 7.6 
               
               
                   
                 T301 
                 C 
                 0.8 
                 1.0 
               
               
                   
                 T301 
                 M 
                 0.7 
                 0.5 
               
               
                   
                 H305 
                 F 
                 0.4 
                 −0.4 
               
               
                   
                 H305 
                 W 
                 0.1 
                 −2.7 
               
               
                   
                 D306 
                 S 
                 0.3 
                 −0.5 
               
               
                   
                 T308 
                 C 
                 0.5 
                 −0.8 
               
               
                   
                 V314 
                 M 
                 0.6 
                 0.3 
               
               
                   
                 V314 
                 L 
                 2.5 
                 0.7 
               
               
                   
                 S315P 
                 P 
                 0.8 
                 3.0 
               
               
                   
                 G320 
                 A 
                 3.0 
                 −0.2 
               
               
                   
                 G320 
                 Q 
                 3.6 
                 1.5 
               
               
                   
                 G320 
                 S 
                 1.0 
                 0.6 
               
               
                   
                 S324 
                 L 
                 0.1 
                 1.3 
               
               
                   
                 S324 
                 R 
                 0.7 
                 2.0 
               
               
                   
                 W325 
                 K 
                 −0.3 
                 2.7 
               
               
                   
                 T326 
                 R 
                 1.7 
                 1.2 
               
               
                   
                 T326 
                 W 
                 0.9 
                 0.2 
               
               
                   
                 T326 
                 L 
                 1.7 
                 1.6 
               
               
                   
                 T326 
                 K 
                 1.9 
                 1.2 
               
               
                   
                 S327 
                 F 
                 1.2 
                 0.6 
               
               
                   
                 S327 
                 L 
                 1.5 
                 1.1 
               
               
                   
                 S327 
                 W 
                 2.0 
                 1.0 
               
               
                   
                 A328 
                 W 
                 0.6 
                 0.5 
               
               
                   
                 A328 
                 D 
                 1.3 
                 1.1 
               
               
                   
                 A328 
                 R 
                 1.1 
                 0.1 
               
               
                   
                 A328 
                 Y 
                 1.5 
                 0.8 
               
               
                   
                 A329 
                 Q 
                 2.8 
                 0.2 
               
               
                   
                 A329 
                 H 
                 2.1 
                 0.3 
               
               
                   
                 A329 
                 T 
                 1.0 
                 0.9 
               
               
                   
                 I330 
                 L 
                 1.1 
                 0.8 
               
               
                   
                 A331 
                 F 
                 2.0 
                 0.6 
               
               
                   
                 A331 
                 Y 
                 1.3 
                 0.6 
               
               
                   
                 M333 
                 I 
                 2.5 
                 −0.7 
               
               
                   
                 M333 
                 Y 
                 0.3 
                 1.0 
               
               
                   
                 M333 
                 L 
                 2.4 
                 −1.0 
               
               
                   
                 L338 
                 R 
                 −0.5 
                 1.5 
               
               
                   
                 A342 
                 R 
                 −0.6 
                 3.9 
               
               
                   
                 A351 
                 S 
                 1.3 
                 −0.9 
               
               
                   
                 S354 
                 E 
                 1.6 
                 3.3 
               
               
                   
                 S354 
                 Q 
                 2.0 
                 0.3 
               
               
                   
                 D358 
                 G 
                 −2.0 
                 0.7 
               
               
                   
                 E360 
                 L 
                 1.4 
                 3.1 
               
               
                   
                 E360 
                 V 
                 2.4 
                 2.9 
               
               
                   
                 E360 
                 C 
                 2.3 
                 2.3 
               
               
                   
                 Q361 
                 C 
                 0.9 
                 1.5 
               
               
                   
                 Q361 
                 L 
                 0.2 
                 0.1 
               
               
                   
                 A372 
                 S 
                 2.4 
                 −0.7 
               
               
                   
                 A378 
                 G 
                 1.5 
                 1.5 
               
               
                   
                 A386 
                 I 
                 3.6 
                 0.5 
               
               
                   
                 A386 
                 L 
                 2.7 
                 1.3 
               
               
                   
                 A386 
                 V 
                 2.1 
                 1.2 
               
               
                   
                 A386 
                 M 
                 1.7 
                 0.0 
               
               
                   
                 G388 
                 C 
                 0.6 
                 −3.5 
               
               
                   
                 I391 
                 W 
                 1.7 
                 0.6 
               
               
                   
                 A392 
                 V 
                 2.8 
                 0.7 
               
               
                   
                 A392 
                 L 
                 3.0 
                 0.9 
               
               
                   
                 A392 
                 I 
                 3.7 
                 2.4 
               
               
                   
                 A392 
                 M 
                 2.3 
                 2.0 
               
               
                   
                 Q393 
                 S 
                 0.9 
                 0.2 
               
               
                   
                 D399 
                 S 
                 2.3 
                 2.1 
               
               
                   
                 D402 
                 E 
                 0.6 
                 1.7 
               
               
                   
                 R412 
                 Q 
                 0.5 
                 2.4 
               
               
                   
                 R412 
                 M 
                 1.5 
                 2.9 
               
               
                   
                 R412 
                 E 
                 1.8 
                 4.4 
               
               
                   
                 R412 
                 D 
                 0.4 
                 3.5 
               
               
                   
                 A418 
                 W 
                 2.8 
                 0.2 
               
               
                   
                 E421 
                 R 
                 1.0 
                 0.5 
               
               
                   
                 A423 
                 V 
                 1.1 
                 0.8 
               
               
                   
                 A433 
                 G 
                 1.4 
                 1.9 
               
               
                   
                 S434 
                 G 
                 1.9 
                 0.7 
               
               
                   
                 S435 
                 I 
                 1.7 
                 1.6 
               
               
                   
                 S435 
                 R 
                 1.8 
                 0.5 
               
               
                   
                 S435 
                 T 
                 2.5 
                 4.7 
               
               
                   
                 S435 
                 V 
                 1.6 
                 2.1 
               
               
                   
                 L442 
                 W 
                 1.4 
                 0.3 
               
               
                   
                 L442 
                 W 
                 −0.7 
                 2.4 
               
               
                   
                 D447 
                 S 
                 4.0 
                 3.2 
               
               
                   
                 D447 
                 C 
                 3.0 
                 1.4 
               
               
                   
                 D447 
                 A 
                 1.6 
                 1.3 
               
               
                   
                 A449 
                 Y 
                 1.7 
                 0.7 
               
               
                   
                 A449 
                 L 
                 0.8 
                 0.3 
               
               
                   
                 A449 
                 M 
                 1.9 
                 −0.9 
               
               
                   
                 A449 
                 E 
                 1.6 
                 0.4 
               
               
                   
                 A449 
                 N 
                 1.6 
                 3.3 
               
               
                   
                 E453 
                 W 
                 2.4 
                 0.0 
               
               
                   
                 E453 
                 Y 
                 2.6 
                 0.7 
               
               
                   
                 E453 
                 F 
                 1.1 
                 −0.5 
               
               
                   
                 V455 
                 I 
                 1.2 
                 0.3 
               
               
                   
                 V455 
                 L 
                 1.8 
                 0.7 
               
               
                   
                 E459 
                 W 
                 0.9 
                 −0.3 
               
               
                   
                 A460 
                 W 
                 2.6 
                 0.5 
               
               
                   
                 A460 
                 R 
                 2.0 
                 −0.6 
               
               
                   
                 T461 
                 V 
                 1.2 
                 0.0 
               
               
                   
                 T461 
                 C 
                 1.2 
                 0.6 
               
               
                   
                 A470 
                 V 
                 0.6 
                 2.3 
               
               
                   
                 A475 
                 V 
                 −0.3 
                 3.7 
               
               
                   
                 A478 
                 L 
                 1.2 
                 0.2 
               
               
                   
                 K483 
                 A 
                 1.5 
                 0.7 
               
               
                   
                 A487 
                 Q 
                 0.0 
                 1.6 
               
               
                   
                 Y490 
                 W 
                 1.5 
                 0.3 
               
               
                   
                 Q497 
                 Y 
                 1.8 
                 1.2 
               
               
                   
                 Q497 
                 M 
                 0.8 
                 0.8 
               
               
                   
                 Q497 
                 D 
                 0.3 
                 1.0 
               
               
                   
                 Q497 
                 R 
                 0.6 
                 0.2 
               
               
                   
                 V502 
                 C 
                 2.3 
                 1.9 
               
               
                   
                 V502 
                 T 
                 1.5 
                 1.6 
               
               
                   
                 T507 
                 L 
                 0.2 
                 1.0 
               
               
                   
                 N510 
                 H 
                 2.4 
                 7.9 
               
               
                   
                 A514 
                 T 
                 2.2 
                 1.3 
               
               
                   
                 A514 
                 Y 
                 1.3 
                 −1.2 
               
               
                   
                 A514 
                 D 
                 1.5 
                 1.2 
               
               
                   
                 A514 
                 S 
                 2.4 
                 0.5 
               
               
                   
                 N515 
                 G 
                 2.0 
                 −0.2 
               
               
                   
                 R516 
                 L 
                 0.5 
                 1.2 
               
               
                   
                 R516 
                 E 
                 1.1 
                 3.5 
               
               
                   
                 R516 
                 I 
                 1.2 
                 4.3 
               
               
                   
                 A517 
                 T 
                 1.3 
                 3.9 
               
               
                   
                 A517 
                 S 
                 0.3 
                 7.7 
               
               
                   
                 Q518 
                 G 
                 1.6 
                 4.1 
               
               
                   
                 L540 
                 V 
                 0.7 
                 0.5 
               
               
                   
                 Q542 
                 H 
                 0.9 
                 −0.2 
               
               
                   
                 Q542 
                 D 
                 1.1 
                 0.4 
               
               
                   
                 Q542 
                 S 
                 0.4 
                 0.5 
               
               
                   
                 A548 
                 S 
                 0.2 
                 n.d. 
               
               
                   
                 P551 
                 N 
                 0.9 
                 −0.4 
               
               
                   
                 P551 
                 R 
                 0.6 
                 0.3 
               
               
                   
                 P553 
                 K 
                 0.5 
                 0.3 
               
               
                   
                 P553 
                 L 
                 0.8 
                 0.2 
               
               
                   
                 R166 
                 I 
                 1.0 
                 0.7 
               
               
                   
                 D265 
                 T 
                 1.7 
                 n.d. 
               
               
                   
                   
               
            
           
         
       
     
     Quite a few distinct clones and combinatorial clones as shown in Table 3 have substitutions in these positions, leading to synergistic effects in thermal stabilization, when two or more residues thereof are mutated simultaneously. 
     EXAMPLE 5 
     Distinct variants were generated by introducing selected distinct mutations into the Kumamolisin AS wild-type sequence via site-directed mutagenesis. Suitable mutagenic PCR methods known in the art and standard cloning techniques as described in Green &amp; Sambrook (eds), Molecular Cloning, 4 th  edition, CSHL were used. Protease enzyme variants were characterized after heterologous expression in  Bacillus subtilis  and phenotypically analysis using the methods described above. 
     Combinatorial libraries, combining mutations identified in the examples provided above and outlined in Table 1 were generated by well-known PCR methods as described in Yolov and Shabarova (1990) and standard cloning techniques as described in Green &amp; Sambrook (eds), Molecular Cloning, 4th edition, CSHL were used. Combinatorial libraries were screened for optimized variants as described in example 3. 
     EXAMPLE 6 
     Distinct clones and combinatorial clones comprising two or more mutations from Table 1 were identified, the IT50 analyzed as described above and compared to the IT50 of the used backbone (=wildtype with missing N-terminal methionine) characterizing the variant by the corresponding ΔIT50. As the IT50 of the backbone was determined in the same experiment as the variant the measured IT50 of the backbone can be slightly different from the average value. Results are shown in the following Table 2a ( FIG. 3  shows results in graphic form): 
     
       
         
           
               
             
               
                 TABLE 2a 
               
               
                   
               
             
            
               
                 Distinct clones comprising selected combinations of mutations from table 1, 
               
               
                 and their ΔIT50 compared to the wildtype 
               
            
           
           
               
               
            
               
                 # 
                 Mutations in distinct clones and selected combinatorial clones 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
               
               
                 2 
                   
                   
                   
                 T301S 
                   
                 E360V 
                 A386I 
                   
               
               
                 3 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392V 
               
               
                 4 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392I 
               
               
                 5 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                 A392I 
               
               
                 6 
                   
                   
                   
                 T301S 
                 G320A 
                 E360L 
                   
                   
               
               
                 7 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A3861 
                 A3921 
               
               
                 8 
                   
                   
                   
                 T301S 
                   
                 E360V 
                   
                 A392I 
               
               
                 9 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 10 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 11 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392I 
               
               
                 12 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                   
               
               
                 13 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392I 
               
               
                 14 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 15 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 16 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                   
               
               
                 17 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392V 
               
               
                 18 
                   
                   
                   
                 T301S 
                   
                 E360V 
                 A386I 
                   
               
               
                 19 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 20 
                   
                   
                   
                 T301S 
                   
                 E360V 
                   
                 A392I 
               
               
                 21 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 22 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 23 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392I 
               
               
                 24 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 25 
                   
                   
                 E269T 
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 26 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392V 
               
               
                 27 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
               
               
                 28 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392I 
               
               
                 29 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                   
               
               
                 30 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392I 
               
               
                 31 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A3861 
                 A392V 
               
               
                 32 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392I 
               
               
                 33 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392I 
               
               
                 34 
                   
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 35 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392V 
               
               
                 36 
                   
                   
                   
                 T301S 
                   
                 E360V 
                 A386I 
                   
               
               
                 37 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 38 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A3861 
                 A392V 
               
               
                 39 
                   
                   
                   
                 T301S 
                   
                 E360V 
                   
                 A392V 
               
               
                 40 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A3861 
                 A392V 
               
               
                   
               
            
           
           
               
            
               
                 ctd&#39;: Distinct clones comprising selected combinations of mutations from table 
               
               
                 1, and their ΔIT50 compared to the wildtype 
               
            
           
           
               
               
            
               
                 # 
                 Mutations in distinct clones and selected combinatorial clones 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 2 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 3 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 4 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 5 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 6 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 7 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 8 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 9 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                   
                 N510H 
               
               
                 10 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 11 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 12 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 13 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 14 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 15 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 16 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 17 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 18 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 19 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 20 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 21 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 22 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 23 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 24 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 25 
                   
                 D447S 
                 A449Y 
                   
                   
                 V502C 
                 N510H 
               
               
                 26 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 27 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 28 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 29 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 30 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 31 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 32 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 33 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 34 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 35 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 36 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 37 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 38 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 39 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 40 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                   
               
            
           
           
               
            
               
                 ctd&#39;: Distinct clones comprising selected combinations of mutations from table 
               
               
                 1, and their ΔIT50 compared to the wildtype 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Mutations in distinct clones and selected  
                 IT50  
                 ΔIT50 
                 IT50 active 
                 ΔIT50 active 
               
               
                 # 
                 combinatorial clones 
                 Zymogen 
                 Zymogen 
                 enzyme 
                 enzyme 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                   
                 A517T 
                   
                   
                 95.5 
                 17.0 
                 90.1 
                 30.6 
               
               
                 2 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 90.1 
                 30.6 
               
               
                 3 
                   
                 A517T 
                   
                   
                 99.5 
                 21.0 
                 89.2 
                 29.7 
               
               
                 4 
                   
                 A517T 
                   
                   
                 97.3 
                 18.8 
                 89.1 
                 29.6 
               
               
                 5 
                   
                 A517T 
                   
                   
                 99.4 
                 20.9 
                 88.8 
                 29.3 
               
               
                 6 
                   
                 A517T 
                   
                   
                 96.4 
                 17.9 
                 88.6 
                 29.1 
               
               
                 7 
                   
                 A517T 
                   
                   
                 96.4 
                 17.9 
                 88.5 
                 29.0 
               
               
                 8 
                   
                 A517T 
                   
                   
                 99.1 
                 20.6 
                 88.5 
                 29.0 
               
               
                 9 
                   
                 A517T 
                 Q518G 
                   
                 97.8 
                 19.3 
                 88.5 
                 29.0 
               
               
                 10 
                   
                 A517T 
                   
                   
                 98.4 
                 19.9 
                 88.4 
                 28.9 
               
               
                 11 
                   
                 A517T 
                   
                   
                 97.7 
                 19.2 
                 88.4 
                 28.9 
               
               
                 12 
                   
                 A517T 
                   
                   
                 98.6 
                 20.1 
                 88.3 
                 28.8 
               
               
                 13 
                   
                 A517T 
                   
                   
                 99.5 
                 21.0 
                 88.2 
                 28.7 
               
               
                 14 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 88.2 
                 28.7 
               
               
                 15 
                   
                 A517T 
                   
                   
                 98.3 
                 19.8 
                 88.1 
                 28.6 
               
               
                 16 
                   
                 A517T 
                   
                   
                 95.8 
                 17.3 
                 88.0 
                 28.5 
               
               
                 17 
                   
                 A517T 
                   
                   
                 97.2 
                 18.7 
                 88.0 
                 28.5 
               
               
                 18 
                   
                 A517T 
                   
                   
                 97.6 
                 19.1 
                 87.8 
                 28.3 
               
               
                 19 
                   
                 A517T 
                   
                   
                 98.5 
                 20.0 
                 87.8 
                 28.3 
               
               
                 20 
                   
                 A517T 
                 Q518G 
                   
                 97.0 
                 18.5 
                 87.8 
                 28.3 
               
               
                 21 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 87.8 
                 28.3 
               
               
                 22 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 87.8 
                 28.3 
               
               
                 23 
                   
                 A517T 
                   
                   
                 97.1 
                 18.6 
                 87.8 
                 28.3 
               
               
                 24 
                   
                 A517T 
                   
                   
                 99.0 
                 20.5 
                 87.8 
                 28.3 
               
               
                 25 
                   
                 A517T 
                   
                   
                 94.0 
                 16.0 
                 87.7 
                 27.0 
               
               
                 26 
                   
                 A517T 
                   
                   
                 97.4 
                 18.9 
                 87.7 
                 28.2 
               
               
                 27 
                   
                 A517T 
                 Q518G 
                   
                 98.0 
                 19.5 
                 87.7 
                 28.2 
               
               
                 28 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 87.6 
                 28.1 
               
               
                 29 
                   
                 A517T 
                   
                   
                 96.5 
                 18.0 
                 87.6 
                 28.1 
               
               
                 30 
                   
                 A517T 
                   
                   
                 99.0 
                 20.2 
                 87.5 
                 28.0 
               
               
                 31 
                   
                 A517T 
                   
                   
                 98.1 
                 19.6 
                 87.5 
                 28.0 
               
               
                 32 
                   
                 A517T 
                 Q518G 
                   
                 97.9 
                 19.4 
                 87.4 
                 27.9 
               
               
                 33 
                   
                 A517T 
                 Q518G 
                   
                 97.1 
                 18.6 
                 87.4 
                 27.9 
               
               
                 34 
                   
                 A517T 
                   
                   
                 95.6 
                 17.1 
                 87.4 
                 27.9 
               
               
                 35 
                   
                 A517T 
                   
                   
                 98.2 
                 19.7 
                 87.4 
                 27.9 
               
               
                 36 
                   
                 A517T 
                   
                   
                 98.5 
                 20.0 
                 87.4 
                 27.9 
               
               
                 37 
                   
                 A517T 
                 Q518G 
                   
                 97.9 
                 19.4 
                 87.4 
                 27.9 
               
               
                 38 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 87.3 
                 27.8 
               
               
                 39 
                   
                 A517T 
                   
                   
                 96.2 
                 17.7 
                 87.2 
                 27.7 
               
               
                 40 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 87.1 
                 27.6 
               
               
                   
               
            
           
           
               
            
               
                 ctd&#39;: Distinct clones comprising selected combinations of mutations from table 
               
               
                 1, and their ΔIT50 compared to the wildtype 
               
            
           
           
               
               
            
               
                 # 
                 Mutations in distinct clones and selected combinatorial clones 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 41 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                   
               
               
                 42 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392I 
               
               
                 43 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                 A392I 
               
               
                 44 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
               
               
                 45 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 46 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                 A392I 
               
               
                 47 
                 D199E 
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 48 
                   
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 49 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392I 
               
               
                 50 
                   
                   
                   
                 T301S 
                   
                 E360V 
                 A386I 
                   
               
               
                 51 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 52 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 53 
                 D199E 
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 54 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                 A392I 
               
               
                 55 
                 D199E 
                 G266A 
                 E269H 
                   
                   
                 E360V 
                   
                 A392L 
               
               
                 56 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 57 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 58 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                 A392I 
               
               
                 59 
                 D199E 
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 60 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                 A392I 
               
               
                 61 
                 D199E 
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 62 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 63 
                 D199E 
                 G266A 
                   
                 T301S 
                   
                 E360L 
                   
                   
               
               
                 64 
                 D199E 
                 G266A 
                 E269T 
                   
                 G320A 
                 E360V 
                   
                 A392L 
               
               
                 65 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 66 
                   
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
               
               
                 67 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
               
               
                 68 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 69 
                 D199E 
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
               
                 70 
                 D199E 
                   
                   
                   
                   
                 E360L 
                   
                   
               
               
                 71 
                 D199E 
                 G266A 
                 E269H 
                 T301S 
                   
                 E360L 
                   
                   
               
               
                 72 
                 D199E 
                   
                   
                   
                   
                 E360L 
                   
                   
               
               
                 73 
                 D199E 
                 G266A 
                   
                   
                   
                 E360V 
                   
                   
               
               
                 74 
                 D199E 
                 G266A 
                 E269H 
                   
                   
                 E360V 
                   
                 A392L 
               
               
                 75 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 76 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392I 
               
               
                 77 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                   
               
               
                 78 
                 D199E 
                   
                   
                   
                   
                 E360L 
                   
                   
               
               
                 79 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 41 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 42 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                   
                 N510H 
               
               
                 43 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 44 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                   
                 N510H 
               
               
                 45 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 46 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 47 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 48 
                 R412E 
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                   
                   
               
               
                 49 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 50 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 51 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 52 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 53 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 54 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 55 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 56 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 57 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 58 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 59 
                   
                 D447S 
                 A449Y 
                   
                   
                 V502C 
                 N510H 
               
               
                 60 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 61 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 62 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 63 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 64 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 65 
                   
                 D447S 
                 A449Y 
                   
                   
                 V502C 
                 N510H 
               
               
                 66 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                   
                   
               
               
                 67 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 68 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 69 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 70 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 71 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 72 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 73 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 74 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                 V502C 
                 N510H 
               
               
                 75 
                   
                 D447S 
                 A449Y 
                   
                 A460W 
                 V502C 
                 N510H 
               
               
                 76 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
                   
                 N510H 
               
               
                 77 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 78 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
                 V502C 
                 N510H 
               
               
                 79 
                   
                 D447S 
                 A449Y 
                   
                   
                 V502C 
                 N510H 
               
               
                   
               
            
           
           
               
            
               
                 ctd&#39;: Distinct clones comprising selected combinations of mutations from table 
               
               
                 1, and their ΔIT50 compared to the wildtype 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Mutations in distinct clones and selected 
                 IT50 
                 ΔIT50 
                 IT50 active 
                 ΔIT50 active 
               
               
                 # 
                 combinatorial clones 
                 Zymogen 
                 Zymogen 
                 enzyme 
                 enzyme 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 41 
                   
                 A517T 
                 Q518G 
                   
                 98.4 
                 19.9 
                 87.0 
                 27.5 
               
               
                 42 
                   
                 A517T 
                   
                   
                 92.7 
                 14.2 
                 86.9 
                 27.4 
               
               
                 43 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 86.9 
                 27.4 
               
               
                 44 
                   
                 A517T 
                   
                   
                 96.9 
                 18.4 
                 86.8 
                 27.3 
               
               
                 45 
                   
                 A517T 
                   
                   
                 93.5 
                 15.1 
                 86.7 
                 26.0 
               
               
                 46 
                   
                 A517T 
                 Q518G 
                   
                 97.4 
                 18.9 
                 86.7 
                 27.2 
               
               
                 47 
                   
                 A517T 
                 Q518G 
                   
                 101.5 
                 23.0 
                 86.6 
                 27.1 
               
               
                 48 
                   
                 A517T 
                 Q518G 
                   
                 100.3 
                 21.8 
                 86.6 
                 27.1 
               
               
                 49 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 86.6 
                 27.1 
               
               
                 50 
                   
                 A517T 
                 Q518G 
                   
                 94.7 
                 16.2 
                 86.5 
                 27.0 
               
               
                 51 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 86.5 
                 27.0 
               
               
                 52 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 86.4 
                 26.9 
               
               
                 53 
                   
                 A517T 
                 Q518G 
                 P553K 
                 102.2 
                 23.7 
                 86.4 
                 26.9 
               
               
                 54 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 86.4 
                 26.9 
               
               
                 55 
                   
                 A517T 
                 Q518G 
                 P553K 
                 101.7 
                 23.2 
                 86.3 
                 26.8 
               
               
                 56 
                   
                 A517T 
                   
                   
                 93.1 
                 14.6 
                 86.2 
                 27.0 
               
               
                 57 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 86.2 
                 26.7 
               
               
                 58 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 86.2 
                 26.7 
               
               
                 59 
                   
                 A517T 
                   
                   
                 92.2 
                 14.1 
                 86.1 
                 25.4 
               
               
                 60 
                   
                 A517T 
                 Q518G 
                   
                 97.2 
                 18.7 
                 86.0 
                 26.5 
               
               
                 61 
                   
                 A517T 
                   
                   
                 93.2 
                 14.9 
                 85.9 
                 26.2 
               
               
                 62 
                   
                 A517T 
                   
                   
                 92.4 
                 13.9 
                 85.9 
                 26.6 
               
               
                 63 
                   
                 A517T 
                   
                   
                 95.7 
                 17.2 
                 85.8 
                 26.3 
               
               
                 64 
                   
                 A517T 
                 Q518G 
                 P553K 
                 100.1 
                 21.6 
                 85.8 
                 26.8 
               
               
                 65 
                   
                 A517T 
                   
                   
                 94.0 
                 15.9 
                 85.8 
                 25.1 
               
               
                 66 
                 R5161 
                 A517T 
                 Q518G 
                   
                 100.1 
                 21.6 
                 85.7 
                 26.2 
               
               
                 67 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 85.7 
                 26.2 
               
               
                 68 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 85.7 
                 26.2 
               
               
                 69 
                   
                 A517T 
                   
                   
                 94.9 
                 16.6 
                 85.4 
                 25.7 
               
               
                 70 
                   
                 A517T 
                   
                   
                 −10.0 
                 −10.0 
                 85.4 
                 25.9 
               
               
                 71 
                   
                 A517T 
                   
                   
                 95.8 
                 17.3 
                 85.4 
                 25.9 
               
               
                 72 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 85.4 
                 25.9 
               
               
                 73 
                   
                 A517T 
                 Q518G 
                   
                 &gt;95 
                 &gt;17 
                 85.4 
                 25.9 
               
               
                 74 
                   
                 A517T 
                 Q518G 
                   
                 100.4 
                 21.9 
                 85.3 
                 25.8 
               
               
                 75 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 85.3 
                 25.8 
               
               
                 76 
                   
                 A517T 
                 Q518G 
                   
                 95.1 
                 16.6 
                 85.2 
                 25.7 
               
               
                 77 
                   
                 A517T 
                   
                   
                 94.9 
                 16.4 
                 85.1 
                 25.5 
               
               
                 78 
                   
                 A517T 
                   
                   
                 &gt;95 
                 &gt;17 
                 85.1 
                 25.6 
               
               
                 79 
                   
                 A517T 
                   
                   
                 93.0 
                 15.0 
                 85.0 
                 25.7 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Some preferred substitutions and their key characteristics 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 G320 
                 A 
                 3.0 
                 −0.2 
                 2 
                 186 
               
               
                   
                   
                 Q 
                 3.6 
                 1.5 
                   
                 46 
               
               
                   
                   
                 S 
                 1.0 
                 0.6 
                   
                 35 
               
               
                   
                 T326 
                 R 
                 1.7 
                 1.2 
                   
                 11 
               
               
                   
                   
                 W 
                 0.9 
                 0.2 
               
               
                   
                   
                 L 
                 1.7 
                 1.6 
                 1 
                 6 
               
               
                   
                   
                 K 
                 1.9 
                 1.2 
                 1 
                 1 
               
               
                   
                 T461 
                 V 
                 1.2 
                 0.0 
                 1 
                 26 
               
               
                   
                   
                 C 
                 1.2 
                 0.6 
                   
                 48 
               
               
                   
                 Q244 
                 C 
                 0.5 
                 −3.6 
                   
                 46 
               
               
                   
                   
                 G 
                 0.7 
                 1.5 
                 1 
                 1 
               
               
                   
                 D293 
                 Y 
                 0.8 
                 1.0 
                 1 
                 24 
               
               
                   
                   
                 F 
                 1.1 
                 1.3 
               
               
                   
                 A487 
                 Q 
                 0.0 
                 1.6 
                 1 
                 24 
               
               
                   
                 V274 
                 I 
                 1.8 
                 1.3 
                   
                 104 
               
               
                   
                 A372 
                 S 
                 2.4 
                 −0.7 
                   
                 82 
               
               
                   
                 K283 
                 L 
                 0.6 
                 −0.2 
                   
                 68 
               
               
                   
                 T308 
                 C 
                 0.5 
                 −0.8 
                   
                 30 
               
               
                   
                 A418 
                 W 
                 2.8 
                 0.2 
                   
                 12 
               
               
                   
                   
                 H 
                 1.1 
                 1.3 
                   
                 16 
               
               
                   
                 I391 
                 W 
                 1.7 
                 0.6 
                   
                 21 
               
               
                   
                 A423 
                 V 
                 1.1 
                 0.8 
                   
                 18 
               
               
                   
                 A331 
                 F 
                 2.0 
                 0.6 
                   
                 7 
               
               
                   
                   
                 Y 
                 1.3 
                 0.6 
                   
                 9 
               
               
                   
                 S327 
                 F 
                 1.2 
                 0.6 
               
               
                   
                   
                 L 
                 1.5 
                 1.1 
                   
                 16 
               
               
                   
                   
                 W 
                 2.0 
                 1.0 
               
               
                   
                 I219 
                 L 
                 1.1 
                 0.8 
                   
                 16 
               
               
                   
                 M333 
                 I 
                 2.5 
                 −0.7 
                   
                 16 
               
               
                   
                 A329 
                 Q 
                 2.8 
                 0.2 
                   
                 5 
               
               
                   
                   
                 H 
                 2.1 
                 0.3 
                   
                 3 
               
               
                   
                   
                 T 
                 1.0 
                 0.9 
                   
                 7 
               
               
                   
                 N515 
                 G 
                 2.0 
                 −0.2 
                   
                 13 
               
               
                   
                 A378 
                 G 
                 1.5 
                 1.5 
                   
                 12 
               
               
                   
                 S434 
                 G 
                 1.9 
                 0.7 
                   
                 12 
               
               
                   
                 E421 
                 R 
                 1.0 
                 0.5 
                 1 
                 11 
               
               
                   
                 A433 
                 G 
                 1.4 
                 1.9 
                   
                 11 
               
               
                   
                 S230 
                 D 
                 2.8 
                 −0.8 
                   
                 9 
               
               
                   
                 Q393 
                 S 
                 0.9 
                 0.2 
                   
                 3 
               
               
                   
                 D399 
                 S 
                 2.3 
                 2.1 
                   
                 4 
               
               
                   
                 Y490 
                 W 
                 1.5 
                 0.3 
                   
                 2 
               
               
                   
                 G281 
                 R 
                 2.0 
                 5.4 
               
               
                   
                 Y287 
                 K 
                 0.2 
                 5.2 
               
               
                   
                 R516 
                 I 
                 1.2 
                 4.3 
               
               
                   
                   
                 E 
                 1.1 
                 3.5 
               
               
                   
                   
                 L 
                 0.5 
                 1.2 
               
               
                   
                 A475 
                 V 
                 −0.3 
                 3.7 
               
               
                   
                 S354 
                 E 
                 1.6 
                 3.3 
               
               
                   
                 S315P 
                 P 
                 0.8 
                 3.0 
               
               
                   
                 W325 
                 K 
                 −0.3 
                 2.7 
               
               
                   
                 L442 
                 W 
                 −0.7 
                 2.4 
               
               
                   
                   
                 W 
                 1.4 
                 0.3 
               
               
                   
                 A470 
                 V 
                 0.6 
                 2.3 
               
               
                   
                 S324 
                 R 
                 0.7 
                 2.0 
               
               
                   
                 S324 
                 L 
                 0.1 
                 1.3 
               
               
                   
                 Q361 
                 C 
                 0.9 
                 1.5 
               
               
                   
                 Q361 
                 L 
                 0.2 
                 0.1 
               
               
                   
                 A190 
                 D 
                 1.5 
                 0.8 
               
               
                   
                 T196 
                 S 
                 0.7 
                 0.3 
               
               
                   
                 Q202 
                 D 
                 0.4 
                 −0.3 
               
               
                   
                 E228 
                 Q 
                 0.7 
                 0.1 
               
               
                   
                 A229 
                 W 
                 0.2 
                 n.d. 
               
               
                   
                 A242 
                 S 
                 0.3 
                 −0.4 
               
               
                   
                 D251 
                 S 
                 0.8 
                 −0.4 
               
               
                   
                 S262 
                 C 
                 0.9 
                 −0.3 
               
               
                   
                 N291 
                 T 
                 0.7 
                 0.5 
               
               
                   
                 N291 
                 S 
                 −0.2 
                 1.0 
               
               
                   
                 L297 
                 T 
                 1.2 
                 0.2 
               
               
                   
                 H305 
                 F 
                 0.4 
                 −0.4 
               
               
                   
                 H305 
                 W 
                 0.1 
                 −2.7 
               
               
                   
                 D306 
                 S 
                 0.3 
                 −0.5 
               
               
                   
                 V314 
                 M 
                 0.6 
                 0.3 
               
               
                   
                 V314 
                 L 
                 2.5 
                 0.7 
               
               
                   
                 A328 
                 W 
                 0.6 
                 0.5 
               
               
                   
                 A328 
                 D 
                 1.3 
                 1.1 
               
               
                   
                 A328 
                 R 
                 1.1 
                 0.1 
               
               
                   
                 A328 
                 Y 
                 1.5 
                 0.8 
               
               
                   
                 I330 
                 L 
                 1.1 
                 0.8 
               
               
                   
                 M333 
                 Y 
                 0.3 
                 1.0 
               
               
                   
                 M333 
                 L 
                 2.4 
                 −1.0 
               
               
                   
                 L338 
                 R 
                 −0.5 
                 1.5 
               
               
                   
                 A342 
                 R 
                 −0.6 
                 3.9 
               
               
                   
                 A351 
                 S 
                 1.3 
                 −0.9 
               
               
                   
                 S354 
                 Q 
                 2.0 
                 0.3 
               
               
                   
                 D358 
                 G 
                 −2.0 
                 0.7 
               
               
                   
                 G388 
                 C 
                 0.6 
                 −3.5 
               
               
                   
                 D402 
                 E 
                 0.6 
                 1.7 
               
               
                   
                 V455 
                 I 
                 1.2 
                 0.3 
               
               
                   
                 V455 
                 L 
                 1.8 
                 0.7 
               
               
                   
                 E459 
                 W 
                 0.9 
                 −0.3 
               
               
                   
                 A478 
                 L 
                 1.2 
                 0.2 
               
               
                   
                 K483 
                 A 
                 1.5 
                 0.7 
               
               
                   
                 Q497 
                 Y 
                 1.8 
                 1.2 
               
               
                   
                 Q497 
                 M 
                 0.8 
                 0.8 
               
               
                   
                 Q497 
                 D 
                 0.3 
                 1.0 
               
               
                   
                 Q497 
                 R 
                 0.6 
                 0.2 
               
               
                   
                 V502 
                 T 
                 1.5 
                 1.6 
               
               
                   
                 T507 
                 L 
                 0.2 
                 1.0 
               
               
                   
                 L540 
                 V 
                 0.7 
                 0.5 
               
               
                   
                 Q542 
                 H 
                 0.9 
                 −0.2 
               
               
                   
                 Q542 
                 D 
                 1.1 
                 0.4 
               
               
                   
                 Q542 
                 S 
                 0.4 
                 0.5 
               
               
                   
                 A548 
                 S 
                 0.2 
                 n.d. 
               
               
                   
                 P551 
                 N 
                 0.9 
                 −0.4 
               
               
                   
                 P551 
                 R 
                 0.6 
                 0.3 
               
               
                   
                 P553 
                 L 
                 0.8 
                 0.2 
               
               
                   
                 R166 
                 I 
                 1.0 
                 0.7 
               
               
                   
                 D265 
                 T 
                 1.7 
                 n.d. 
               
               
                   
                   
               
            
           
         
       
     
     It is further to be understood that the mutations can have positive or negative effects on other enzyme parameters, as the producibility in fermentative microbial production systems or the stability against pH-conditions or endogenous proteases of the animal, like pepsin. Testing the stability of feed enzymes at low pH and in the presence of pepsin is a standard for feed enzymes and was performed in this study as outlined in example 1e. The stability against higher ionic strength is not a standard test for feed enzymes though high ion concentrations can interfere with the enzyme stability and with the enzyme performance under such conditions and can be found for example in the gut. The secretion of acid in the gut and the feed ingredients translate to an increased ionic strength. 
       FIG. 2  shows that the wildtype suffers from combined effects of stability and performance reduction in the presence of higher ionic strength.  FIG. 2  also shows the effect of ionic strength on the top variants also shown in table 4, variants #1 to #7. 
     The performance and stability in high ionic strength was tested as described in example 1d. The pH profile was a control parameter and tested as described in example 1f. The digestion of proteinaceous antinutritive factors like the Trypsin/chymotrypsin inhibitors BBI and KTI (Bowman-Birk inhibitors and Kunitz-type inhibitors) is a potential beneficial performance characteristic of a protease which was tested as described in example 1 g. 
     From the 651 individual combinatorial and distinct variants tested in detail, Table 4 describes the variants consolidating a multitude of performance and stability parameters ( FIG. 5  shows results in graphic form). 
     All variants shown in table 4 are better or equally well produced in a microbial production system than the wildtype and have no relevant changes in their pH activity profile tested as described in example 1d. Table 4 ranks these variants based on the thermal stability of the activated enzyme, the pH/pepsin stability and the stability against and the performance under higher ionic strength. 
     It was further found that the best variants can hydrolyze BBI and KTI (Bowman-Birk inhibitors and Kunitz-type inhibitors) as tested in a functional trypsin inhibition assay, which differentiates these variants from the parent enzyme, beside the high thermal stability engineered into these variants. 
     
       
         
           
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Some distinct and combinatorial clones with particularly good performance 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 # 
                 mutant code 
                 Mutations in distinct clones and selected combinatoral clones 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                  1 
                 GIN_3B2_08.42a 
                   
                   
                   
                 T301S 
                   
                 E360V 
                   
                 A392I 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  2 
                 Gin_48a5xa 
                 D199E 
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  3 
                 Gin_85b5xa 
                 D199E 
                 G266A 
                 E269H 
                   
                   
                 E360V 
                   
                 A392L 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  4 
                 Gin_24b5xa 
                 D199E 
                 G266A 
                 E269T 
                   
                 G320A 
                 E360V 
                   
                 A392L 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  5 
                 GIN_3B2_08.34a 
                   
                   
                   
                   
                   
                 E360V 
                 A386I 
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  6 
                 GIN_3B2_08.58a 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  7 
                 Gin_48a4xa 
                 D199E 
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  8 
                 GIN_48a1xbxe 
                   
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                  9 
                 GIN_3B2_08.85c 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 10 
                 GIN_3B2_08.47a 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 11 
                 GIN_3B3_05.84c 
                   
                   
                   
                 T301S 
                   
                 E360L 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 12 
                 GIN_3B2_10.87a 
                   
                   
                   
                 T301S 
                   
                 E360V 
                   
                 A392I 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 13 
                 GIN_3B3_03.04c 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392I 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 14 
                 GIN_3B3_01.11a 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 15 
                 GIN_3B3_02.55a 
                   
                   
                   
                   
                   
                 E360V 
                   
                 A392I 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 16 
                 GIN_3B2_09.09c 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 17 
                 CIN_3B3_05.09c 
                   
                   
                   
                   
                   
                 E360L 
                 A386I 
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 18 
                 GIN_3B2_09.31a 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 19 
                 GIN_3B3_01.52a 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 20 
                 GIN_48a1xbxd 
                   
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
                 R412E 
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 21 
                 GIN_3B3_01.21b 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                 A392I 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 22 
                 Gin_28a5x2xf 
                 D199E 
                 G266A 
                   
                 T301S 
                   
                 E360L 
                   
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 23 
                 Gin_28a5x3xa 
                 D199E 
                 G266A 
                 E269H 
                 T301S 
                   
                 E360L 
                   
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 24 
                 Gin_85b4xa 
                 D199E 
                 G266A 
                 E269H 
                   
                   
                 E360V 
                   
                 A392L 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 25 
                 Gin_28a5x1xf 
                 D199E 
                   
                   
                 T301S 
                   
                 E360L 
                   
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 26 
                 Gin_28a4xa5x 
                 D199E 
                   
                   
                   
                   
                 E360V 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 27 
                 GIN_48a1xbxg 
                   
                 G266A 
                   
                   
                   
                 E360V 
                   
                 A392V 
                 R412D 
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 28 
                 Gin_28a4xa6x 
                 D199E 
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 29 
                 Gin_28a5x1xg 
                 D199E 
                   
                   
                 T301S 
                   
                 E360L 
                   
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 30 
                 GIN_3B3_05.20b 
                   
                   
                   
                   
                   
                 E360L 
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 31 
                 GIN_3B2_08.81c 
                   
                   
                   
                 T301S 
                   
                 E360L 
                 A386I 
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                 32 
                 Gin_28a5x2xd 
                 D199E 
                 G266A 
                 E269T 
                 T301S 
                   
                 E360L 
                   
                   
                   
                 D447S 
                 A449Y 
                 E453W 
                   
               
               
                 33 
                 Gin_48a3xa 
                 D199E 
                 G266A 
                   
                   
                   
                   
                   
                 A392V 
                   
                 D447S 
                 A449Y 
                 E453W 
                 A460W 
               
               
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 pH/ 
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 pepsin 
                 Stability/ 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 IT50 
                 IT50 
                 Stability 
                 perfor- 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 (° C.) 
                 (° C.) 
                 % 
                 mance 
                 Inhibitor  
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 Mutations in distinct clones and selected 
                 Zymo- 
                 acti- 
                 residual 
                 at 25 mS  
                 hydrolysis 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 # 
                 combinatoral clones 
                 gen 
                 vated 
                 activity 
                 cm-1 
                 BBI 
                 KTI 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                  1 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 99.10 
                 88.48 
                  93 
                 62 
                 48% 
                 41% 
               
               
                   
                  2 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                 P553K 
                 102.15 
                 86.37 
                  96 
                 87 
                 58% 
                 46% 
               
               
                   
                  3 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                 P553K 
                 101.73 
                 86.34 
                  92 
                 62 
                 60% 
                 47% 
               
               
                   
                  4 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                 P553K 
                 100.13 
                 85.82 
                  95 
                 77 
                 68% 
                 65% 
               
               
                   
                  5 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 98.48 
                 87.82 
                  99 
                 68 
                 50% 
                 48% 
               
               
                   
                  6 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 98.00 
                 87.65 
                  91 
                 48 
                 45% 
                 40% 
               
               
                   
                  7 
                 V502C 
                 N510H 
                 A514Y 
                 R516I 
                 A517T 
                 Q518G 
                   
                 101.45 
                 86.59 
                  92 
                 81 
                 58% 
                 52% 
               
               
                   
                  8 
                   
                   
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 100.10 
                 85.73 
                  88 
                 98 
                 75% 
                 56% 
               
               
                   
                  9 
                   
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 97.78 
                 88.46 
                  93 
                 33 
                 41% 
                 40% 
               
               
                   
                 10 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 98.33 
                 88.10 
                  92 
                 38 
                 pos. 
                 pos. 
               
               
                   
                 11 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 97.18 
                 87.96 
                  90 
                 32 
                 pos. 
                 pos. 
               
               
                   
                 12 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 96.99 
                 87.81 
                  81 
                 29 
                 pos. 
                 pos. 
               
               
                   
                 13 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 97.06 
                 87.79 
                  68 
                 24 
                 pos. 
                 pos. 
               
               
                   
                 14 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 99.01 
                 87.75 
                 102 
                 49 
                 pos. 
                 40% 
               
               
                   
                 15 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 97.88 
                 87.42 
                  92 
                 32 
                 pos. 
                 pos. 
               
               
                   
                 16 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 98.23 
                 87.38 
                  80 
                 18 
                 pos. 
                 pos. 
               
               
                   
                 17 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 97.85 
                 87.35 
                  68 
                 32 
                 pos. 
                 pos. 
               
               
                   
                 18 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 98.41 
                 86.97 
                  79 
                 29 
                 pos. 
                 pos. 
               
               
                   
                 19 
                   
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 96.85 
                 86.75 
                  61 
                 39 
                 pos. 
                 pos. 
               
               
                   
                 20 
                   
                   
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 100.30 
                 86.59 
                  90 
                 89 
                 58% 
                 41% 
               
               
                   
                 21 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 98.50 
                 86.59 
                  94 
                 34 
                 pos. 
                 pos. 
               
               
                   
                 22 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 95.73 
                 85.83 
                  74 
                 51 
                 pos. 
                 pos. 
               
               
                   
                 23 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 95.81 
                 85.38 
                  75 
                 34 
                 pos. 
                 pos. 
               
               
                   
                 24 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 100.40 
                 85.34 
                  89 
                 57 
                 pos. 
                 pos. 
               
               
                   
                 25 
                 V502C 
                 N510H 
                   
                   
                 A517T 
                   
                   
                 94.77 
                 84.81 
                  95 
                 35 
                 55% 
                 40% 
               
               
                   
                 26 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                 P553K 
                 98.90 
                 84.73 
                  95 
                 52 
                 55% 
                 pos. 
               
               
                   
                 27 
                   
                   
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 96.46 
                 84.30 
                  94 
                 70 
                 50% 
                 pos. 
               
               
                   
                 28 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                 P553K 
                 99.08 
                 84.08 
                 112 
                 33 
                 43% 
                 pos. 
               
               
                   
                 29 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 &gt;97 
                 83.84 
                 n.d. 
                 32 
                 pos. 
                 pos. 
               
               
                   
                 30 
                   
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 &gt;97 
                 83.75 
                  80 
                 30 
                 54% 
                 48% 
               
               
                   
                 31 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                   
                   
                 96.61 
                 83.73 
                  73 
                 28 
                 46% 
                 46% 
               
               
                   
                 32 
                 V502C 
                 N510H 
                   
                   
                 A517T 
                   
                   
                 94.68 
                 83.66 
                  81 
                 44 
                 pos. 
                 pos. 
               
               
                   
                 33 
                 V502C 
                 N510H 
                 A514Y 
                   
                 A517T 
                 Q518G 
                   
                 97.99 
                 82.38 
                 101 
                 42 
                 50% 
                 pos. 
               
               
                   
               
            
           
         
       
     
     The following Table 5 shows the frequency of occurrence of given mutations preferred combinatorial and distinct variants. The frequency of occurrence is a measure for the role and importance of a given mutation. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Frequency of occurrence of given mutations in preferred combinatorial and 
               
               
                 distinct variants. Frequency of occurrence is a measure for the role and importance of a 
               
               
                 given mutation. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Frequency of occurence 
                   
                 Frequency of occurence in preferred 
                   
                   
               
               
                   
                 in preferred 
                   
                 combinatorial and 
                   
                 Frequency of occurence in preferred 
               
               
                   
                 combinatorial and distinct variants 
                   
                 distinct variants zymogen 
                   
                 combinatorial and distinct variants 
               
               
                 Mutation 
                 (activated enzyme (IT50 &gt; 80° C.)) 
                 Mutation 
                 (IT50 &gt; 85° C.) 
                 Mutation 
                 zymogen (IT50 &gt; 85° C.) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 D447S 
                 127 
                 D447S 
                 365 
                 A331F 
                 7 
               
               
                 A449Y 
                 127 
                 A449Y 
                 353 
                 A331Y 
                 9 
               
               
                 A517T 
                 127 
                 A517T 
                 256 
                 A329Q/H/T 
                 15 
               
               
                 N510H 
                 125 
                 N510H 
                 175 
                 S435R/I 
                 11 
               
               
                 E360L/V 
                 125 
                 E360V/L 
                 262 
                 V274I 
                 104 
               
               
                 V502C 
                 120 
                 V502C 
                 185 
                 A372S 
                 82 
               
               
                 E453W 
                 92 
                 E453W 
                 320 
                 K283L 
                 66 
               
               
                 A514Y/T 
                 84 
                 A514Y/T 
                 265 
                 Q244C 
                 46 
               
               
                 A460W 
                 72 
                 A460W 
                 308 
                 T380C 
                 30 
               
               
                 A386I 
                 56 
                 A386I 
                 178 
                 A418W 
                 28 
               
               
                 A392V/I 
                 50 
                 A392V/I 
                 392 
                 I391W 
                 21 
               
               
                 T301S 
                 44 
                 T301S 
                 79 
                 A423V 
                 18 
               
               
                 D199E 
                 43 
                 D199E 
                 30 
                 T326L 
                 16 
               
               
                 Q518G 
                 36 
                 Q518G 
                 250 
                 I219L 
                 16 
               
               
                 P553K 
                 20 
                 P553K 
                 6 
                 S327L 
                 16 
               
               
                 E269T/H 
                 12 
                 E269T/H 
                 133 
                 M333I 
                 16 
               
               
                 G266A 
                 19 
                 G266A 
                 133 
                 N515G 
                 13 
               
               
                 D293Y 
                 1 
                 D293Y 
                 24 
                 A378G 
                 12 
               
               
                 G320A 
                 1 
                 G320A 
                 265 
                 S434G 
                 12 
               
               
                 R412Q 
                 1 
                 R412Q 
                 74 
                 A433G 
                 10 
               
               
                 E421R 
                 1 
                 E421R 
                 11 
                 S230D 
                 9 
               
               
                 A487Q 
                 1 
                 A487Q 
                 24 
                 Q393S 
                 3 
               
               
                 T461V 
                 1 
                 T461V 
                 26 
                 D399S 
                 3 
               
               
                   
                   
                 T461C 
                 48 
                 Y490W 
                 3 
               
               
                   
               
            
           
         
       
     
     The following Table 6 shows the impact of single mutations on ΔIT50 of the zymogen or the activated form. Again, the amount of impact of a single mutation on ΔIT50 is a measure for the role and importance of a said mutation. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Impact of single mutations on ΔIT50 of the zymogen (left) or the activated form (right). 
               
               
                 The amount of impact of a single mutation on ΔIT50 is a measure for the role and  
               
               
                 importance of a said mutation. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 ΔIT50 
                   
                 ΔIT50 
               
               
                   
                 ΔIT50 
                   
                 ΔIT50 
                   
                 activated 
                   
                 activated 
               
               
                 Mutation 
                 Zymogen 
                 Mutation 
                 Zymogen 
                 Mutation 
                 Enzyme 
                 Mutation 
                 Enzyme 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 D447S 
                 4.8 
                 L297T 
                 1.2 
                 A517S 
                 7.7 
                 D358G 
                 0.7 
               
               
                 E269H 
                 4.0 
                 S327F 
                 1.2 
                 N510H 
                 7.6 
                 A331Y 
                 0.6 
               
               
                 A392I 
                 3.7 
                 V455I 
                 1.2 
                 T301S 
                 7.6 
                 S327F 
                 0.6 
               
               
                 G320Q 
                 3.6 
                 T461V 
                 1.2 
                 G281R 
                 5.4 
                 T461C 
                 0.6 
               
               
                 A386I 
                 3.6 
                 T461C 
                 1.2 
                 Y287K 
                 5.2 
                 G320S 
                 0.6 
               
               
                 G320A 
                 3.0 
                 A478L 
                 1.2 
                 S435T 
                 4.7 
                 A386I 
                 0.5 
               
               
                 A392L 
                 3.0 
                 R516I 
                 1.2 
                 R412E 
                 4.4 
                 A460W 
                 0.5 
               
               
                 D447C 
                 3.0 
                 I219L 
                 1.1 
                 R516I 
                 4.3 
                 A514S 
                 0.5 
               
               
                 S230D 
                 2.8 
                 D293F 
                 1.1 
                 Q518G 
                 4.1 
                 S435R 
                 0.5 
               
               
                 A329Q 
                 2.8 
                 A328R 
                 1.1 
                 A517T 
                 3.9 
                 A190D 
                 0.5 
               
               
                 A392V 
                 2.8 
                 I330L 
                 1.1 
                 A475V 
                 3.7 
                 E421R 
                 0.5 
               
               
                 A418W 
                 2.8 
                 A423V 
                 1.1 
                 R516E 
                 3.5 
                 N291T 
                 0.5 
               
               
                 A386L 
                 2.7 
                 E453F 
                 1.1 
                 R412D 
                 3.5 
                 T301M 
                 0.5 
               
               
                 E269T 
                 2.6 
                 R516E 
                 1.1 
                 A342R 
                 3.4 
                 L540V 
                 0.5 
               
               
                 E453Y 
                 2.6 
                 Q542D 
                 1.1 
                 D447S 
                 3.3 
                 A328W 
                 0.5 
               
               
                 A460W 
                 2.6 
                 G320S 
                 1.0 
                 S354E 
                 3.3 
                 Q542S 
                 0.5 
               
               
                 V314L 
                 2.5 
                 A329T 
                 1.0 
                 A449N 
                 3.3 
                 A449E 
                 0.4 
               
               
                 M333I 
                 2.5 
                 E421R 
                 1.0 
                 E360L 
                 3.1 
                 Q542D 
                 0.4 
               
               
                 S435T 
                 2.5 
                 R166I 
                 1.0 
                 S315PP 
                 3.0 
                 A329H 
                 0.3 
               
               
                 E269M 
                 2.4 
                 V410I 
                 1.0 
                 E360V 
                 2.9 
                 S354Q 
                 0.3 
               
               
                 M333L 
                 2.4 
                 S262C 
                 0.9 
                 R412M 
                 2.9 
                 Y490W 
                 0.3 
               
               
                 E360V 
                 2.4 
                 T326W 
                 0.9 
                 W325K 
                 2.7 
                 L442W 
                 0.3 
               
               
                 A372S 
                 2.4 
                 Q361C 
                 0.9 
                 A392I 
                 2.4 
                 V455I 
                 0.3 
               
               
                 E453W 
                 2.4 
                 Q393S 
                 0.9 
                 R412Q 
                 2.4 
                 A449L 
                 0.3 
               
               
                 N510H 
                 2.4 
                 E459W 
                 0.9 
                 L442W 
                 2.4 
                 T196S 
                 0.3 
               
               
                 A514S 
                 2.4 
                 Q542H 
                 0.9 
                 E360C 
                 2.3 
                 V314M 
                 0.3 
               
               
                 E360C 
                 2.3 
                 P551N 
                 0.9 
                 A470V 
                 2.3 
                 P551R 
                 0.3 
               
               
                 A392M 
                 2.3 
                 D251S 
                 0.8 
                 D399S 
                 2.1 
                 P553K 
                 0.3 
               
               
                 D399S 
                 2.3 
                 D293Y 
                 0.8 
                 S435V 
                 2.1 
                 A329Q 
                 0.2 
               
               
                 V502C 
                 2.3 
                 T301C 
                 0.8 
                 A392M 
                 2.0 
                 A418W 
                 0.2 
               
               
                 A514T 
                 2.2 
                 S315P 
                 0.8 
                 V502C 
                 1.9 
                 L297T 
                 0.2 
               
               
                 E269C 
                 2.1 
                 A449L 
                 0.8 
                 A433G 
                 1.9 
                 A478L 
                 0.2 
               
               
                 A329H 
                 2.1 
                 Q497M 
                 0.8 
                 S324R 
                 1.9 
                 T326W 
                 0.2 
               
               
                 A331F 
                 2.1 
                 P553L 
                 0.8 
                 D402E 
                 1.7 
                 Q393S 
                 0.2 
               
               
                 A386V 
                 2.1 
                 T196S 
                 0.7 
                 T326L 
                 1.6 
                 P553L 
                 0.2 
               
               
                 E269Q 
                 2.0 
                 E228Q 
                 0.7 
                 S435I 
                 1.6 
                 Q497R 
                 0.2 
               
               
                 G281R 
                 2.0 
                 Q244G 
                 0.7 
                 V502T 
                 1.6 
                 A331F 
                 0.1 
               
               
                 S327W 
                 2.0 
                 N291T 
                 0.7 
                 A487Q 
                 1.6 
                 A328R 
                 0.1 
               
               
                 S354Q 
                 2.0 
                 T301M 
                 0.7 
                 G320Q 
                 1.5 
                 E228Q 
                 0.1 
               
               
                 A460R 
                 2.0 
                 L540V 
                 0.7 
                 A378G 
                 1.5 
                 Q361L 
                 0.1 
               
               
                 N515G 
                 2.0 
                 K283L 
                 0.6 
                 Q361C 
                 1.5 
                 E453W 
                 0 
               
               
                 T326K 
                 1.9 
                 T301S 
                 0.6 
                 Q244G 
                 1.5 
                 G266A 
                 0 
               
               
                 S434G 
                 1.9 
                 V314M 
                 0.6 
                 L338R 
                 1.5 
                 A386M 
                 0 
               
               
                 A449M 
                 1.9 
                 S324R 
                 0.6 
                 D447C 
                 1.4 
                 I391W 
                 0 
               
               
                 V274I 
                 1.8 
                 A328W 
                 0.6 
                 A386L 
                 1.3 
                 T461V 
                 0 
               
               
                 R412E 
                 1.8 
                 G388C 
                 0.6 
                 A514T 
                 1.3 
                 E269T 
                 −0.1 
               
               
                 S435R 
                 1.8 
                 D402E 
                 0.6 
                 V274I 
                 1.3 
                 E269M 
                 −0.1 
               
               
                 V455L 
                 1.8 
                 A470V 
                 0.6 
                 D447A 
                 1.3 
                 Q542H 
                 −0.1 
               
               
                 Q497Y 
                 1.8 
                 Q497R 
                 0.6 
                 D293F 
                 1.3 
                 G320A 
                 −0.2 
               
               
                 G266A 
                 1.7 
                 P551R 
                 0.6 
                 S324L 
                 1.3 
                 N515G 
                 −0.2 
               
               
                 T326R 
                 1.7 
                 D199E 
                 0.5 
                 A386V 
                 1.2 
                 K283L 
                 −0.2 
               
               
                 T326L 
                 1.7 
                 Q244C 
                 0.5 
                 T326K 
                 1.2 
                 S262C 
                 −0.3 
               
               
                 A386M 
                 1.7 
                 T308C 
                 0.5 
                 Q497Y 
                 1.2 
                 E459W 
                 −0.3 
               
               
                 I391W 
                 1.7 
                 R412Q 
                 0.5 
                 T326R 
                 1.2 
                 D251S 
                 −0.3 
               
               
                 S435I 
                 1.7 
                 R516L 
                 0.5 
                 A514D 
                 1.2 
                 Q202D 
                 −0.3 
               
               
                 A449Y 
                 1.7 
                 P553K 
                 0.5 
                 R516L 
                 1.2 
                 P551N 
                 −0.4 
               
               
                 D265T 
                 1.7 
                 Q202D 
                 0.4 
                 S327L 
                 1.1 
                 H305F 
                 −0.4 
               
               
                 S354E 
                 1.6 
                 H305F 
                 0.4 
                 A328D 
                 1.1 
                 A242S 
                 −0.4 
               
               
                 S435V 
                 1.6 
                 R412D 
                 0.4 
                 S327W 
                 1.0 
                 E269H 
                 −0.5 
               
               
                 D447A 
                 1.6 
                 Q542S 
                 0.4 
                 D293Y 
                 1.0 
                 E453F 
                 −0.5 
               
               
                 A449E 
                 1.6 
                 A242S 
                 0.3 
                 T301C 
                 1.0 
                 D306S 
                 −0.5 
               
               
                 A449N 
                 1.6 
                 D306S 
                 0.3 
                 D199E 
                 1.0 
                 A460R 
                 −0.6 
               
               
                 Q518G 
                 1.6 
                 M333Y 
                 0.3 
                 M333Y 
                 1.0 
                 M333I 
                 −0.7 
               
               
                 A190D 
                 1.5 
                 Q497D 
                 0.3 
                 Q497D 
                 1.0 
                 A372S 
                 −0.7 
               
               
                 S327L 
                 1.5 
                 A517S 
                 0.3 
                 T507L 
                 1.0 
                 S230D 
                 −0.8 
               
               
                 A328Y 
                 1.5 
                 A229W 
                 0.2 
                 N291S 
                 1.0 
                 T308C 
                 −0.8 
               
               
                 A378G 
                 1.5 
                 Y287K 
                 0.2 
                 A392L 
                 0.9 
                 A449M 
                 −0.9 
               
               
                 R412M 
                 1.5 
                 Q361L 
                 0.2 
                 A329T 
                 0.9 
                 A351S 
                 −0.9 
               
               
                 K483A 
                 1.5 
                 T507L 
                 0.2 
                 V455L 
                 0.8 
                 M333L 
                 −1.0 
               
               
                 Y490W 
                 1.5 
                 A548S 
                 0.2 
                 A328Y 
                 0.8 
                 E269C 
                 −1.1 
               
               
                 A514D 
                 1.5 
                 S324L 
                 0.1 
                 I330L 
                 0.8 
                 E269Q 
                 −1.4 
               
               
                 E360L 
                 1.4 
                 A487Q 
                 0 
                 A423V 
                 0.8 
                 H305W 
                 −2.7 
               
               
                 A433G 
                 1.4 
                 N291S 
                 −0.2 
                 Q497M 
                 0.8 
                 G388C 
                 −3.5 
               
               
                 L442W 
                 1.4 
                 W325K 
                 −0.3 
                 A392V 
                 0.7 
                 Q244C 
                 −3.6 
               
               
                 A328D 
                 1.3 
                 A475V 
                 −0.3 
                 E453Y 
                 0.7 
                 D265T 
                 n.d. 
               
               
                 A331Y 
                 1.3 
                 L338R 
                 −0.5 
                 V314L 
                 0.7 
                 R166I 
                 n.d. 
               
               
                 A351S 
                 1.3 
                 A342R 
                 −0.6 
                 S434G 
                 0.7 
                 V410I 
                 n.d. 
               
               
                 A514Y 
                 1.3 
                 L442W 
                 −0.7 
                 A449Y 
                 0.7 
                 A229W 
                 n.d. 
               
               
                 A517T 
                 1.3 
                 D358G 
                 −2 
                 K483A 
                 0.7 
                 A548S 
                 n.d. 
               
               
                   
               
            
           
         
       
     
     It is further to be understood that some mutations of Table 1 and Table 6 can interchangeably be used to engineer thermostability in Kumamolisin As. Table 7 shows a set of variants based on variant #1 of Table 7. In the course of engineering the mutations at position 502 and 510 seemed to change the activity at extrem acidic pH, below pH 2. 
     Excluding mutations at 502 and 510 reduced the thermostability significantly below the targeted temperature stability for the activated enzyme, as for example in Table 7, clone #2 which has a 7.8° C. reduction in thermal stability compared to clone #1. A set of distinct variants were constructed by a rational approach taking advantage of the mutations identified and shown in Tables 1 and 6 to compensate for the effect of 502 and 510. With the exception of D399S substitutions can gradually or fully compensate the effect of mutations at 502 and 510. 
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
             
            
               
                 A set of variants based on variant #1 
               
            
           
           
               
               
            
               
                 # 
                 Mutations in distinct clones and selected combinatorial clones 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 G266A 
                   
                   
                 E360V 
                 A392V  
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 2 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 3 
                 G266A 
                   
                   
                 E360V 
                 A392V  
                   
                 R412E 
                   
                   
                 D447S 
                 A449Y 
               
               
                 4 
                 G266A 
                   
                   
                 E360V  
                 A392V  
                   
                 R412D 
                   
                   
                 D447S 
                 A449Y 
               
               
                 5 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                 R412Q  
                   
                   
                 D447S 
                 A449Y 
               
               
                 6 
                 G266A 
                   
                   
                 E360V 
                 A392V  
                   
                   
                   
                 S435I 
                 D447S 
                 A449Y 
               
               
                 7 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                 A433G  
                   
                 D447S  
                 A449Y  
               
               
                 8 
                 G266A 
                   
                 T326L 
                 E360V 
                 A392V  
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 9 
                 G266A 
                   
                   
                 E360V 
                 A392V  
                   
                 R412M 
                   
                   
                 D447S 
                 A449Y  
               
               
                 10 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                   
                 S435T 
                 D447S 
                 A449Y  
               
               
                 11 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 12 
                 G266A 
                   
                 T326K 
                 E360V 
                 A392V 
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 13 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                   
                   
                 D447S  
                 A449Y 
               
               
                 14 
                 Q244G  
                 G266A 
                   
                 E360V 
                 A392V 
                   
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                 15 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                   
                   
                   
                 S435V  
                 D447S 
                 A449Y  
               
               
                 16 
                 G266A 
                   
                   
                 E360V 
                 A392V 
                 D399S 
                   
                   
                   
                 D447S 
                 A449Y 
               
               
                   
               
            
           
           
               
            
               
                 A set of variants based on variant #1 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 IT50 [° C.] 
                 IT50 [° C.] 
               
            
           
           
               
               
               
               
            
               
                 # 
                 Mutations in distinct clones and selected combinatorial clones 
                 Zymogen 
                 activated 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 E453W 
                 A460W 
                   
                 V502C 
                 N510H 
                 A514Y 
                 A517T 
                 Q518G 
                 101.5 
                 86.6 
               
               
                 2 
                 E453W 
                 A460W  
                   
                   
                   
                 A514Y 
                 A517T  
                 Q518G 
                 &gt;95 
                 78.8 
               
               
                 3 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 100.3 
                 86.6 
               
               
                 4 
                 E453W 
                 A460W  
                   
                   
                   
                 A514Y 
                 A517T  
                 Q518G 
                 96.5 
                 84.3 
               
               
                 5 
                 E453W 
                 A460W  
                   
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 97.7 
                 81.8 
               
               
                 6 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 97.4 
                 81.4 
               
               
                 7 
                 E453W  
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 98.1 
                 81.3 
               
               
                 8 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 98.7 
                 81.1 
               
               
                 9 
                 E453W  
                 A460W 
                   
                   
                   
                 A514Y  
                 A517T 
                 Q518G 
                 98.3 
                 81.0 
               
               
                 10 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T  
                 Q518G 
                 99.2 
                 81.0 
               
               
                 11 
                 E453W 
                 A460W  
                 A487Q  
                   
                   
                 A514Y 
                 A517T 
                 Q518G 
                 96.3 
                 81.0 
               
               
                 12 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y  
                 A517T  
                 Q518G 
                 97.2 
                 80.5 
               
               
                 13 
                 E453W  
                 A460W  
                   
                   
                   
                 A514Y 
                 A517T  
                 Q518G 
                 96.2 
                 80.4 
               
               
                 14 
                 E453W 
                 A460W 
                   
                   
                   
                 A514Y 
                 A517T  
                 Q518G 
                 96.3 
                 80.2 
               
               
                 15 
                 E453W  
                 A460W  
                   
                   
                   
                 A514Y  
                 A517T 
                 Q518G 
                 98.6 
                 80.1 
               
               
                 16 
                 E453W 
                 A460W  
                   
                   
                   
                 A514Y  
                 A517T 
                 Q518G 
                 87.90 
                 71.92 
               
               
                   
               
            
           
         
       
     
     REFERENCES 
     
         
         Wlodawer Al, Li M, Gustchina A, Oyama H, Dunn B M, Oda K., Acta Biochim Pol. 2003; 50(1):81-102 
         Terashita, T., Oda, K., Kono, M. &amp; Murao, S., Agric Biol Chem (1981) 45, 1937-1943 
         Oda, K., Takahashi, S., Ito, M. &amp; Dunn, B. M., Adv Exp Med Biol (1998) 436, 349-353 
         Packer &amp; Liu, Methods for the directed evolution of proteins. Nature Reviews Genetics 16, 379-394 (2015) 
         Hsieh &amp; Vaisvila, Protein engineering: single or multiple site-directed mutagenesis. Methods Mol Biol. 2013; 978:173-86 
         Cadwell and Joyce, Mutagenic PCR. PCR Methods Appl. 3, 1994, 136-140 
         Okubo et al, 2006 June; 273(11):2563-76.