Abstract:
The inventors have developed a method of altering the amino acid sequence of a fungal alpha-amylase to obtain variants, and they have used the method to construct such variants. The variants may be useful for anti-staling in baked products. Accordingly, the invention provides a method of constructing fungal alpha-amylase variants based on a comparison of three-dimensional (3D) structures of the fungal alpha-amylase and a maltogenic alpha-amylase. One or both models includes a substrate. The invention also provides novel fungal alpha-amylase variants.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. application Ser. No. 10/536,101 filed on Feb. 22, 2005 (now abandoned), which is a 35 U.S.C. 371 national application of PCT/DK2004/000558 filed Aug. 23, 2004, which claims priority or the benefit under 35 U.S.C. 119 of Danish Application No. PA 2003 01201 filed Aug. 22, 2003 and U.S. Provisional Application No. 60/497,455 filed Aug. 22, 2003, the contents of which are fully incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to the construction of variants of fungal alpha-amylases. 
     BACKGROUND OF THE INVENTION 
     WO 0134784 discloses variants of a fungal alpha-amylase. Pdb files 2AAA, 6taa and 7taa (available at rcsb.org) show the amino acid sequences and three-dimensional structures of fungal alpha-amylases. WO 9943794 discloses the amino acid sequence and three-dimensional structure of a maltogenic alpha-amylase from  Bacillus stearothermophilus , known as Novamyl®. 
     SUMMARY OF THE INVENTION 
     The inventors have developed a method of altering the amino acid sequence of a fungal alpha-amylase to obtain variants with improved anti-staling effect and a higher degree of exo-amylase activity (increased ratio of exo-amylase to endo-amylase), and they have used the method to construct such variants. The variants may be useful for anti-staling in baked products. 
     Accordingly, the invention provides a method of constructing fungal alpha-amylase variants based on a comparison of three-dimensional (3D) structures of the fungal alpha-amylase and a maltogenic alpha-amylase. One or both models includes a substrate. The invention also provides novel fungal alpha-amylase variants and use of the variants in the preparation of dough and baked products. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  shows an alignment of fungal amylases SEQ ID NO: 2, 3 and 4. 
         FIG. 2  shows an alignment of the 3D structures 1QHO for the maltogenic alpha-amylase Novamyl (SEQ ID NO: 1) at top and 6taa for a fungal alpha-amylase (SEQ ID NO: 2) below. Details are described in Example 1. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Fungal alpha-amylase 
     The method of the invention uses an amino acid sequence of a fungal alpha-amylase and a three-dimensional model for the fungal alpha-amylase. The model may include a substrate. 
     The fungal alpha-amylase may be one of the following having the indicated amino acid sequence and a three-dimensional structure found under the indicated identifier in the Protein Data Bank (available at rcsb.org): acid alpha-amylase from  Aspergillus niger  (2AAA, SEQ ID NO: 3), alpha-amylase (Taka amylase) from  Aspergillus oryzae  (6taa or 7taa, SEQ ID NO: 2) or alpha-amylase from  Thermomyces lanuginosus  (SEQ ID NO: 4, WO 9601323). Alternatively, the fungal alpha-amylase may be a variant having at least 70% amino acid identity with SEQ ID NO: 2, e.g. a variant described in WO 0134784. 
     3D structures for other fungal alpha-amylases may be constructed as described in Example 1 of WO 9623874. To develop variants of a fungal alpha-amylase without a known 3D structure, the sequence may be aligned with a fungal alpha-amylase having a known 3D structure. The sequence alignment may be done by conventional methods, e.g. by use the software GAP from UWGCG Version 8.  FIG. 1  shows an alignment of SEQ ID NO: 4 (without a known 3D structure) with SEQ ID NO: 2 and 3 (with known structures). 
     Maltogenic alpha-amylase 
     The method also uses an amino acid sequence of a maltogenic alpha-amylase (EC 3.2.1.133) and a three-dimensional model of the maltogenic alpha-amylase. The model may include a substrate. The maltogenic alpha-amylase may have the amino acid sequence have the amino acid sequence shown in SEQ ID NO: 1 (in the following referred to as Novamyl). A 3D model for Novamyl with a substrate is described in U.S. Pat. No. 6,162,628 and is found in the Protein Data Bank with the identifier 1QHO. Alternatively, the maltogenic alpha-amylase may be a Novamyl variant described in U.S. Pat. No. 6,162,628. A 3D structure of such a variant may be developed from the Novamyl structure by known methods, e.g. as described in T. L. Blundell et al., Nature, vol. 326, p. 347 ff (26 Mar. 1987); J. Greer, Proteins: Structure, Function and Genetics, 7:317-334 (1990); or Example 1 of WO 9623874. 
     Superimposition of 3D Models 
     The two 3D models may be superimposed by aligning the amino acid residues of each catalytic triad by methods known in the art. This may be based on the deviations of heavy atoms (i.e. non-hydrogen atoms) in the active sites, e.g. by minimizing the sum of squares of deviations. Alternatively, the superimposition may be based on the deviations of the three pairs of C-alpha atoms, e.g. by minimizing the sum of squares of the three deviations or by aligning so as to keep each deviation below 0.8 Å, e.g. below 0.6 Å, below 0.4 Å, below 0.3 Å or below 0.2 Å. 
     The structural alignment may be done by use of known software. In the structurally aligned models, pairs of residues from different sequences are considered to be aligned when they are located close to each other. The following software may be used: 
     DALI software, available at ebi.ac.uk/dali. 
     CE software available at cl.sdsc.edu. 
     STAMP software available at compbio.dundee.ac.uk/Software/Stamp/stamp.html. 
     Protein 3Dhome at lecb.ncifcrf.gov/˜tsai. 
     Yale Gernstein Lab—spare parts at bioinfo.mbb.yale.edu/align. 
     Structural alignment server at molmovdb.org/align. 
     Substrate 
     A 3D structure of the enzyme(s) having a substrate or substrate analog in the active site binding cleft. A “substrate” could be a substrate bound in an inactive or active enzyme, or a substrate inhibitor like acarbose bound in the active site, or a modelled substrate in the active site, a docked substrate in the active site, or a substrate superimposed into the enzyme of interest and taken from a homologous 3D structure having such substrate or substrate analog bound in the active site. 
     Selection of Amino Acid Residues 
     In the superimposed 3D models, amino acid residues in the fungal alpha-amylase sequence are selected by two criteria: Firstly, fungal alpha-amylase residues &lt;11 Å from a substrate (i.e. residues having a C-alpha atom located &lt;11 Å from an atom of a substrate) are selected. Secondly, fungal alpha-amylase residues &gt;0.8 Å from any maltogenic alpha-amylase residue (i.e. fungal alpha-amylase residues having a C-alpha atom &gt;0.8 Å from the C-alpha atom of any maltogenic alpha-amylase residue) are selected. 
     Alteration of Fungal Alpha-Amylase Amino Acid Sequence 
     One or more of the following alterations are made to the fungal alpha-amylase sequence: 
     Deletion or Substitution 
     A fungal alpha-amylase residue &lt;11 Å from a substrate and &gt;0.8 Å from any maltogenic alpha-amylase residue may be deleted or may be substituted with a different residue. 
     The substitution may be made with the same amino acid residue as found at a corresponding position in the maltogenic alpha-amylase sequence or with a residue of the same type. The type indicates a positively charged, negatively charged, hydrophilic or hydrophobic residue, understood as follows (Tyr may be hydrophilic or hydrophobic): 
     Hydrophobic amino acids: Ala, Val, Leu, Ile, Pro, Phe, Trp, Gly, Met, Tyr 
     Hydrophilic amino acids: Thr, Ser, Gln, Asn, Tyr, Cys 
     Positively charged amino acids: Lys, Arg, His 
     Negatively charged amino acids: Glu, Asp 
     The fungal alpha-amylase residue may be substituted with a larger or smaller residue depending on whether a larger or smaller residue is found at a corresponding position in the maltogenic alpha-amylase sequence. In this connection, the residues are ranked as follows from smallest to largest: (an equal sign indicates residues with sizes that are practically indistin-guishable):
 
G&lt;A=S=C&lt;V=T&lt;P&lt;L=I=N=D=M&lt;E=Q&lt;K&lt;H&lt;R=F&lt;Y&lt;W
 
Insertion
 
     One or more amino acid residues may be inserted at a position in the fungal alpha-amylase sequence corresponding to one or more residues in the maltogenic alpha-amylase sequence which are &lt;11 Å from a substrate and which are &gt;0.8 Å from any fungal alpha-amylase residue. The insertion may be made with the same residue as in the maltogenic alpha-amylase sequence or with another amino acid residue of the same type. The type indicates a positively charged, negatively charged, hydrophilic or hydrophobic residue, as above. 
     Where the maltogenic alpha-amylase sequence contains a consecutive stretch (a peptide loop) of residues which are &gt;0.8 Å from any fungal alpha-amylase residue and of which some are &lt;11 Å from a substrate, the insertion at the corresponding position in the fungal alpha-amylase sequence may consist of an equal number of residues, or the insertion may have one or two fewer or more residues. Thus, in the case of a stretch of 5 such residues in the maltogenic alpha-amylase sequence, the insertion may be made with 3-7 residues, e.g. 3, 4, 5, 6 or 7 residues. Each inserted residue may be the same as one of the maltogenic alpha-amylase residues or of the same type. 
     Optional Further Alterations of the Fungal Alpha-Amylase Sequence 
     Optionally, one or more other residues in the fungal alpha-amylase sequence may be substituted. The substitution may be made as described in WO 0134784 and may improve the thermostability of the variant. 
     A fungal alpha-amylase residue &lt;11 Å of a substrate and &lt;0.8 Å of a maltogenic alpha-amylase residue may be substituted with a residue identical to or of the same type as the corresponding maltogenic alpha-amylase residue, or with a larger or smaller residue depending on whether the corresponding maltogenic alpha-amylase residue is larger or smaller. 
     Degree of Exo-Activity 
     The degree of exo amylase activity is given as a relative activity compared to the endo amylase activity. The endo activity can be measured by a number of well known assays e.g. starch iodine, Phadebas (Amersham now GE Healthcare), or AZCL-amylose (Megazyme). The exo activity is preferably a measure of the small malto-oligomers released from starch at initial phases of hydrolysis. It is preferably measured by total carbohydrate after removal of the remaining starch, by the exo activity assay described below or similar method, but could be measured by other means e.g. the sum of oligomers by HPAEC-PAD (Dionex) or sum of oligomers after size exclusion chromatography. 
     Endo-Amylase Activity Assay: 
     1 mL resuspended Phadebas substrate (0.25 tablets/mL 50 mM sodium acetate, 1 mM CaCl 2 , adjusted to pH 5.7) is incubated with 25 micro-L enzyme for 15 min at 40° C. with agitation. The reaction is stopped by addition of 0.5 mL 1 M NaOH and the mixture is centrifuged in a table centrifuge at 14,000 RPM. The absorbance of the supernatant at 620 nm is measured. The activity is determined by comparing to a standard with declared activity (BAN 480 L, 480 KNU/g) 
     Exo-Amylase Activity Assay: 
     900 μL 3.3% solubilized waxy maize starch (3.3% starch is boiled in 50 mM sodium acetate, 1 mM CaCl 2 , pH 5.7 for 5 min and cooled to 40° C.) is incubated with 100 micro-L enzyme at 40° C. with stirring. After appropriate reaction time the remaining starch is precipitated by addition of 450 micro-L 4° C. 96% ethanol. The precipitate is immediately removed by centrifugation at 3000 G for 20 min. The total carbohydrate in the supernatant is determined by mixing 200 micro-L supernatant with 50 micro-L 2% tryptophan and 900 micro-L 64% sulfuric acid. The mixture is heated for 15 min at 95° C. and the absorbance at 630 nm is measured after cooling to room temperature. The activity is determined by comparing with the absorbance of glucose standards in the same assay. One unit is defined as the amount of enzyme that at initial rates liberates 1 mg oligomeric products (products that are not precipitated by ethanol) per min. 
     Fungal Alpha-Amylase Variants 
     A fungal alpha-amylase variant may be a polypeptide which: 
     a) has an amino acid sequence having at least 70% identity to SEQ ID NO: 2, 3 or 4; and 
     b) comprises an amino acid alteration which is deletion, substitution or insertion as described below, and 
     c) has the ability to hydrolyze starch. 
     The identity may be at least 80%, at least 90% or at least 95%. Amino acid identity may be determined as described in U.S. Pat. No. 6,162,628. 
     Production of Fungal Alpha-Amylase Variants 
     A polypeptide having the resulting amino acid sequence may be produced by conventional methods, generally involving producing DNA with a sequence encoding the polypeptide together with control sequences, transforming a suitable host organism with the DNA, cultivating the transformed organism at suitable conditions for expressing and optionally secreting the polypeptide, and optionally recovering the expressed polypeptide. 
     DNA encoding any of the above fungal alpha-amylase variants may be prepared, e.g. by point-specific mutation of DNA encoding the parent fungal alpha-amylase. This may be followed by transformation of a suitable host organism with the DNA, and cultivation of the transformed host organism under suitable conditions to express the encoded polypeptide (fungal alpha-amylase variant). This may be done by known methods. 
     Optional Screening of Fungal Alpha-Amylase Variants 
     Optionally, one or more expressed polypeptides may be tested for useful properties. This may include testing for the ability to hydrolyze starch or a starch derivative by a conventional method, e.g. a plate assay, use of Phadebas tablets or DSC on amylopectin. Also, the polypeptide may be tested for thermostability, and a more thermostable one may be preferred. Finally, the polypeptide may be tested by adding it to a dough, baking it and testing the firmness of the baked product during storage; a polypeptide with anti-staling effect may be selected as described in WO 9104669 or U.S. Pat. No. 6,162,628. 
     Optional Gene Recombination 
     Optionally, DNA encoding a plurality of the above fungal alpha-amylase variants may be prepared and recombined, followed by transformation of a suitable host organism with the recombined DNA, and cultivation of the transformed host organism under suitable conditions to express the encoded polypeptides (fungal alpha-amylase variants). The gene recombination may be done by known methods. 
     Dough and Baked Product 
     The variants are useful in the preparation of dough and baked products from dough. Particularly, the variant may be added in an amount which is effective to retard the staling of the baked product. 
     The dough may be leavened e.g. by adding chemical leavening agents or yeast, usually  Saccharomyces cerevisiae  (baker&#39;s yeast). The dough generally comprises flour, particularly wheat flour. Examples of baked products are bread and rolls. 
     The dough may comprise an additional enzyme, e.g. a second amylase, a protease or peptidase, a transglutaminase, a lipolytic enzyme, a cellulase, a xylanase or an oxidoreductase, e.g. a carbohydrate oxidase with activity on glucose and/or maltose. The lipolytic enzyme may have triacyl glycerol lipase activity, phospholipase activity and/or galactolipase activity, e.g. as described in WO 9953769, WO 9826057 or WO 0032758. 
     EXAMPLES 
     Example 1 
     Construction of Variants of Fungal Alpha-Amylase from  A. oryzae    
     Two 3D structures with substrates were used: 6taa for a fungal alpha-amylase (SEQ ID NO: 2) and 1QHO for a maltogenic alpha-amylase (Novamyl, SEQ ID NO: 1), wherein the substrates are indicated as ABC for 6taa and as ABD for 1QHO. The two structures were superimposed using the heavy atoms of the three C-alpha atoms at the catalytic triad: D206, E230 and D297 for 6taa, and D228, E256 and D329 for Novamyl. The superimposed structures were analyzed, and the result is shown in  FIG. 2  with the Novamyl sequence at the top and the fungal alpha-amylase sequence below. 
     The following fungal alpha-amylase residues were found to have a C-alpha atom &lt;11 Å from an atom of either substrate: 13, 14, 15, 18, 31, 32, 33, 34, 35, 36, 61, 62, 63,64, 66, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 94, 117,118, 119, 120, 121, 122, 123, 124,125, 126, 127, 151, 152, 153, 154, 155, 156, 157, 158, 160,161, 162, 164, 165,166, 167,168, 169, 170, 171, 172, 173, 174, 175, 204,205, 206, 207, 208, 209, 210, 211,216,228, 229,230, 231, 232, 233, 234, 235, 236, 237, 238, 239,242, 250,251, 252, 253, 254, 255, 256,257, 258, 259, 260, 275, 292, 294,295, 296, 297, 298, 299, 304, 328, 338, 239, 340, 341, 342, 343, 344. They are indicated by the first underlining in  FIG. 2 . 
     The following fungal alpha-amylase residues were found to be included in either of the above subsets (&lt;11 Å from a substrate or in a loop) and to have a C-alpha atom &gt;0.8 Å from the C-alpha atom of any Novamyl residue: 15, 32, 33, 34, 35, 36, 63, 64, 73, 74, 75, 76, 77, 119, 120, 125, 126, 151, 152, 155, 156, 167, 168, 169, 170, 171, 172, 211, 233, 234, 235, 236, 237, 238, 239. They are indicated by the second underlining in  FIG. 2 . 
     Variants were constructed by substituting a selected residue in SEQ ID NO: 2 (fungal amylase) as indicated below: 
     
       
         
               
               
               
               
             
           
               
                   
               
               
                 Selected 
                 Corresponding 
                   
                   
               
               
                 residue in 
                 residue in 
                   
                 Particular 
               
               
                 SEQ ID NO: 2 
                 SEQ ID NO: 1 
                 Criteria for 
                 substitution in 
               
               
                 (fungal amylase) 
                 (Novamyl) 
                 substitution 
                 SEQ ID NO: 2 
               
               
                   
               
             
             
               
                 Q35 
                 K44 
                 larger and/or 
                 Q35K/R 
               
               
                   
                   
                 positive 
                   
               
               
                 Y75 
                 T84 
                 smaller 
                 Y75A/F 
               
               
                 Y155 
                 W177 
                 larger and/or 
                 Y155W 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 L166 
                 F188 
                 larger and/or 
                 L166F 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 G167 
                 T189 
                 larger and/or 
                 G167T 
               
               
                   
                   
                 hydrophilic 
                   
               
               
                 N169 
                 P191 
                 smaller and/or 
                 N169P 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 T170 
                 A192 
                 smaller and/or 
                 T170A 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 L232 
                 Y258 
                 larger 
                 L232Y 
               
               
                 D233 
                 G259 
                 smaller and/or 
                 D233G 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 G234 
                 D260 
                 larger and/or 
                 G234D 
               
               
                   
                   
                 negative 
                   
               
               
                 Y252 
                 F284 
                 smaller and/or 
                 Y252F 
               
               
                   
                   
                 hydrophobic 
                   
               
               
                 Y256 
                 T288 
                 smaller and/or 
                 Y256T 
               
               
                   
                   
                 hydrophilic 
               
               
                   
               
             
          
         
       
     
     Variants were constructed by altering a subsequence with insertion of an additional residue in SEQ ID NO: 2 (fungal amylase) to match the number of residues in SEQ ID NO: 1, as indicated below: 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Alteration In SEQ ID NO: 2 (fungal amylase) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 166LGDNTV171 to FTDPAGF (Novamyl loop (long)) 
               
               
                   
                 168-171 (DNTV) substituted with DPAGF (Novamyl loop) 
               
               
                   
                 168-171 (DNTV) substituted with DPAGL (Novamyl loop with 
               
               
                   
                 adjustments in last part) 
               
               
                   
                 168-171 (DNTV) substituted with DPAGC (Novamyl loop with 
               
               
                   
                 adjustments in last part) 
               
               
                   
               
             
          
         
       
     
     Further, amino acid alterations were combined as follows: 
     
       
         
               
               
               
             
           
               
                   
               
               
                   
                 Alteration with insertion 
                 Substitutions 
               
               
                   
                 in SEQ ID NO: 2 
                 in SEQ ID NO: 2 
               
               
                   
               
             
             
               
                   
                   
                 D233G + G234D 
               
               
                   
                   
                 Q35K + Y75F + D168Y 
               
               
                   
                   
                 Q35R + Y75F 
               
               
                   
                   
                 Q35R + Y75F + D168Y 
               
               
                   
                 168-171 (DNTV) 
                 Y75A 
               
               
                   
                 substituted with DPAGF 
                   
               
               
                   
                 168-171 (DNTV) 
                 Q35K + Y75A 
               
               
                   
                 substituted with DPAGF 
                   
               
               
                   
                 168-171 (DNTV) 
                 Q35K + Y75A + D233G + G234D 
               
               
                   
                 substituted with DPAGF 
                   
               
               
                   
                 168-171 (DNTV) 
                 Y75A + G234D 
               
               
                   
                 substituted with DPAGF 
                   
               
               
                   
                 168-171 (DNTV) 
                 Y75A + D233G + G234D 
               
               
                   
                 substituted with DPAGF 
                   
               
               
                   
                 166-171 (LGDNTV) 
                 Y75A 
               
               
                   
                 substituted with FTDPAGF 
                   
               
               
                   
                 166-171 (LGDNTV) 
                 Q35K + Y75A 
               
               
                   
                 substituted with FTDPAGF 
                   
               
               
                   
                 166-171 (LGDNTV) 
                 Q35K + Y75A + D233G + G234D 
               
               
                   
                 substituted with FTDPAGF 
               
               
                   
               
             
          
         
       
     
     Example 2 
     Construction of Variants of Acid Amylase from  A. niger    
     The three-dimensional structure 2AAA for the acid alpha-amylase from  Aspergillus niger  (SEQ ID NO: 3) was compared with the structure of Novamyl 1QHO, and variants were constructed by altering the sequence SEQ ID NO: 3 as follows: 
     Q35K 
     Q35R 
     P70K 
     L151F 
     L151D 
     N233G+G234D 
     D75G 
     D75A 
     166-171 (EGDTIV) substituted with FTDPAGF (Novamyl loop (long)) 
     Example 3 
     Construction of Variants of Fungal Amylase from  T. lanuginosus    
     A three-dimensional model of SEQ ID NO: 4 (fungal amylase from  T. lanuginosus ) was constructed from a model of SEQ ID NO: 2 (fungal amylase from  A. oryzae ) using the alignment shown in  FIG. 1 . Residues were selected, and variants were constructed with amino acid alterations to substitute or delete selected residues as follows: 
     G35K 
     G35R 
     A76del+D77del 
     D74del+A78del 
     D74A 
     D74G 
     D77A 
     D77G 
     Y157W 
     L168F+A169T+T171P+P172A+T173G 
     Example 4 
     Anti-Staling Effect of Variants (Straight-Dough Method) 
     Baking tests were made with the following variants of SEQ ID NO: 2 (fungal amylase from  A. oryzae ): 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Alteration in SEQ ID NO: 2 (fungal amylase) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 168-171 (DNTV) substituted with DPAGF 
               
               
                   
                 Y75A 
               
               
                   
                 Q35R 
               
               
                   
                 Q35R + Y75F 
               
               
                   
                 168-171 (DNTV) substituted with DPAGC 
               
               
                   
                 L232Y 
               
               
                   
                 168-171 (DNTV) substituted with DPAGF + Y75A 
               
               
                   
                 D233G + G234D 
               
               
                   
                 168-171 (DNTV) substituted with DPAGF + Q35K + Y75A 
               
               
                   
               
             
          
         
       
     
     Doughs were made according to the straight dough method. Bread was baked in lidded pans, and the bread was stored at ambient temperature. Firmness and elasticity were evaluated after 1, 4 and 6 days. Each variant was added at a dosage of 1 mg per kg flour. Controls were made without enzyme, with the parent fungal amylase of SEQ ID NO: 2 and with Novamyl (maltogenic alpha-amylase of SEQ ID NO: 1). 
     The results showed that the fungal alpha-amylase variants and Novamyl improved the elasticity after storage compared to the control without enzyme, whereas the fungal alpha-amylase gave a slightly lower elasticity. All the enzymes tested (variants, fungal amylase and Novamyl) improved the firmness after storage. In conclusion, the amino acid alterations succeeded in changing the functional properties of the fungal amylase to make it more Novamyl-like. 
     Example 5 
     Anti-Staling Effect of Variants (Sponge-and-Dough Method) 
     Baking tests were made with the following variants of SEQ ID NO: 2 (fungal amylase from  A. oryzae ): 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Alteration in SEQ ID NO: 2 (fungal amylase) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 168-171 (DNTV) substituted with DPAGF 
               
               
                   
                 Y75A 
               
               
                   
               
             
          
         
       
     
     Doughs were made by the sponge &amp; dough method, and the variants were tested as in the preceding example. Controls were made without enzyme, with the parent fungal amylase of SEQ ID NO: 2 and with Novamyl (maltogenic alpha-amylase of SEQ ID NO: 1). 
     The variants show comparable softness and improved elasticity relative to the parent amylase, when dosed at optimal dosage in this trial. 
     A sensory evaluation by a small panel agrees with NMR data on mobility of free water and shows that the variants improve the moistness of bread crumb to the same level or slightly better than the parent amylase. 
     In conclusion, the variants showed improved effect (a more Novamyl-like effect) compared to the parent amylase. 
     Example 6 
     Exo/Endo Ratio of  A. oryzae  Amylase variants 
     The following variants of SEQ ID NO: 2 (fungal amylase from  A. oryzae ) were tested: 
     
       
         
               
             
               
               
             
           
               
                   
               
               
                 Alteration in SEQ ID NO: 2 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 168-171 (DNTV) substituted with DPAGF 
               
               
                   
                 Y75A 
               
               
                   
                 168-171 (DNTV) substituted with DPAGC 
               
               
                   
                 Q35R 
               
               
                   
                 Q35R + Y75F 
               
               
                   
               
             
          
         
       
     
     The exo- and endo-amylase activities were determined for each variant by the assays described in the specification, and the parent amylase was tested for comparison. The results showed that each variant had a higher degree of exo-amylase activity (higher exo/endo-amylase ratio) that the parent fungal amylase. 
     Example 7 
     Exo/Endo Ratio of  A. niger  Amylase Variants 
     The following variants of SEQ ID NO: 3 (acid amylase from  A. niger ) were tested: 
     
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 Alteration in SEQ ID NO: 3 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 D75G 
               
               
                   
                   
                 Q35K 
               
               
                   
                   
                 L151F 
               
               
                   
                   
                 L151D 
               
               
                   
                   
                 N233G + G234D 
               
               
                   
                   
               
             
          
         
       
     
     The exo- and endo-amylase activities were determined for each variant by the assays described in the specification, and the parent amylase was tested for comparison. The results showed that each variant had a higher degree of exo-amylase activity (higher exo/endo-amylase ratio) that the parent fungal amylase.