Patent Document

FIELD OF THE INVENTION 
       [0001]    The present invention relates to a polypeptide with lipolytic enzyme activity and to a method of preparing it. 
       BACKGROUND OF THE INVENTION 
       [0002]    WO8802775 describes  Candida antarctica  lipase B (CALB). Uppenberg, Hansen, Patkar, Jones, Structure 2, 293-308 (1994) describe the amino acid sequence and three-dimensional (3D) structure of CALB. The 3D structure can be found in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCBS PDB) (http://www.rcsb.org/), its identifier being 1TCA. 
         [0003]    CALB variants are described in Zhang et al. Prot. Eng. 2003, 16, 599-605; Lutz. 2004, Tetrahedron: Asymmetry, 15, 2743-2748; Qian and Lutz, JACS, 2005, 127, 13466-13467; and in WO 2004/024954. 
         [0004]    WO9324619 describes a lipase from  Hyphozyma  sp. Amino acid sequences for other lipases can be found in UniProt [the Universal Protein Resource] with accession numbers Q4pep1, Q7RYD2, Q2UE03, Q4WG73, Q6BVP4 and Q4HUY1. 
       SUMMARY OF THE INVENTION 
       [0005]    The inventors performed molecular dynamics (MD) simulation on the 1TCA structure. The analysis reveals two hitherto unknown lids with a marked mobility, Lid 1 consisting of residues from 135 or 136 to 155 or 160, and Lid 2 consisting of residues 267-295. The simulation indicated a more closed like form in water solution and a more fully open form in organic solvent solution. The analysis revealed important areas in the 3D structure for affecting the activity and functionality of the lipase, and the inventors used this to design lipolytic enzyme variants with increased specific activity, particularly towards bulky substrates (e.g. esters of a branched acid or long-chain fatty acid and/or a secondary alcohol) and/or increased activity at high pH (higher pH optimum) and/or increased enantioselectivity. 
         [0006]    Further, the inventors have selected amino acid residues and designed lipolytic enzyme variants based on an alignment of CALB with some homologous lipase sequences. 
         [0007]    Accordingly, the invention provides a method of preparing a polypeptide, comprising 
         [0008]    a) selecting a parent polypeptide which has lipolytic enzyme activity and has an amino acid sequence with at least 30% identity to CALB (SEQ ID NO: 1), 
         [0009]    b) selecting one or more amino acid residues in the sequence corresponding to any of residues 1, 13, 25, 38-51, 53-55, 58, 69-79, 91, 92, 96, 97, 99, 103, 104-110, 113, 132-168, 173, 187-193, 197-205, 215, 223-231, 242, 244, 256, 259, 261-298, 303, 305, 308-313, or 315 of CALB (SEQ ID NO: 1), 
         [0010]    c) altering the selected amino acid sequence wherein the alteration comprises substitution or deletion of the selected residue(s) or insertion of at least one residue adjacent to the selected residue(s), 
         [0011]    d) preparing an altered polypeptide having the altered amino acid sequence, 
         [0012]    e) determining the lipolytic enzyme activity or enantioselectivity towards carboxylic ester bonds of the altered polypeptide, and 
         [0013]    f) selecting an altered polypeptide which has higher lipolytic enzyme activity or a higher enantioselectivity than the parent polypeptide. 
         [0014]    The invention also provides a polypeptide which: 
         [0015]    a) has lipolytic enzyme activity, and 
         [0016]    b) has an amino acid sequence which has at least 80% identity (particularly at least 90% or at least 95% identity) to CALB (SEQ ID NO: 1) and has a difference from CALB (SEQ ID NO: 1) which comprises an amino acid substitution, deletion or insertion at a position corresponding to any of residues 1, 13, 25, 38-51, 53-55, 58, 69-79, 91, 92, 96, 97, 99, 103, 104-110, 113, 132-168, 173, 187-193, 197-205, 215, 223-231, 242, 244, 256, 259, 261-298, 303, 305, 308-313, or 315. 
         [0017]    Finally, the invention provides use of the above variant polypeptide in a lipase-catalyzed process. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]      FIG. 1  shows an alignment of amino acid sequences SEQ ID NOS. 1-7. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Parent Polypeptide 
       [0019]    The parent polypeptide has lipolytic enzyme activity and has an amino acid sequence with at least 30% identity (particularly at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%) to  Candida antarctica  lipase B (CALB, SEQ ID NO: 1) which is described in WO8802775, and whose sequence is given in Uppenberg, J., Hansen, M. T., Patkar, S., Jones, T. A., Structure v2 pp. 293-308, 1994. The parent polypeptide may be any of the following lipases. An alignment is shown in  FIG. 1 . 
         [0020]    SEQ ID NO: 1:  Candida antarctica  lipase B (CALB), 1TCA 
         [0021]    SEQ ID NO: 2:  Hyphozyma  sp., WO9324619 
         [0022]    SEQ ID NO: 3:  Ustilago maydis,  UniProt Q4pep1 
         [0023]    SEQ ID NO: 4:  Gibberella zeae  ( Fusarium graminearum ), UniProt Q4HUY1 
         [0024]    SEQ ID NO: 5:  Debaryomyces hansenii,  UniProt Q6BVP4 
         [0025]    SEQ ID NO: 6:  Aspergillus fumigatus,  UniProt Q4WG73 
         [0026]    SEQ ID NO: 7:  Aspergillus oryzae,  UniProt Q2UE03 
         [0027]    SEQ ID NO: 8:  Neurospora crassa  lipase, UniProt Q7RYD2 
         [0028]    The alignment was done using the needle program from the EMBOSS package (http://www.emboss.org) version 2.8.0 with the following parameters: Gap opening penalty: 10.00, Gap extension penalty: 0.50, Substitution matrix: EBLOSUM62. The software is described in  EMBOSS: The European Molecular Biology Open Software Suite  (2000), Rice, P. Longden, I. and Bleasby, A., Trends in Genetics 16, (6) pp 276-277. The program needle implements the global alignment algorithm described in Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453, and Kruskal, J. B. (1983). 
         [0029]    Other parent polypeptides may aligned to the sequences in  FIG. 1  by the same method or by the methods described in D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley. 
       Three-Dimensional (3D) Structure and Lids 
       [0030]    In the 3D structure 1TCA, the inventors identified two lids with high mobility at amino acid residues from 135 or 136 to 155 or 160 (Lid 1) and residues 267-295 (Lid 2) of SEQ ID NO: 1. The MD simulation indicated that the following regions are of particular interest because of a particularly high mobility: residues 141-149 in Lid 1 and the following regions in Lid 2: 267-269, 272, 275-276, 279-280, 282-283, 286-290. 
       Selection of Amino Acid Residue 
       [0031]    An amino acid residue may be selected having a non-hydrogen atom within 8 Å of a non-hydrogen atom of a residue in Lid 1 or Lid 2 in a 3D structure. This criterion selects the following residues in the structure 1TCA: 38-51, 53-55, 58, 69-79, 104-110, 113, 132-168, 173, 187-193, 197-205, 223-231, 259, 261-298, 305, 308-313, 315 of SEQ ID NO: 1. 
         [0032]    The residue may particularly be selected within 6 A of the lids, leading to the following residues in 1TCA: 40-42, 46-51, 54, 58, 70-77, 79, 104-107, 109, 133-165, 167, 173, 187-192, 197-203, 223-225, 228-229, 261-297, 308-312. 
         [0033]    An amino acid residue may also be selected by aligning homologous lipolytic enzyme sequences and selecting a residue at a position with variability, i.e. a position where different sequences have different residues. Thus, the following residues in CALB (SEQ ID NO: 1) can be selected by a comparison with  Hyphozyma lipase  (SEQ ID NO: 2) based on the alignment shown in  FIG. 1 : 1, 3, 5, 10, 12-15, 25, 30, 31, 32, 57, 62, 66, 76, 78, 80, 83, 88, 89, 91, 92, 96, 97, 114, 121, 123, 143, 147-149, 159, 163, 164, 168, 169, 174, 188, 194, 195, 197, 199, 205, 210, 214, 215, 221, 223, 229, 238, 242, 244, 249, 251, 254, 256, 261, 265, 268, 269, 272-274, 277-280, 282-284, 287, 303-306, 309, 314, 315, 317. 
         [0034]    The following residues are of special interest: 1, 13, 25, 38, 42, 74, 140, 143, 147, 164, 168, 190, 199, 215, 223, 242, 244, 256, 265, 277, 280, 281, 283, 284, 285, 292, 303, 315 of CALB (SEQ ID NO: 1). 
         [0035]    Corresponding residues in other lipases may be identified from a sequence alignment. An alignment of several sequences is shown in  FIG. 1 . Other sequences may be aligned by known methods, such as AlignX (a component of vector nti suite 9.0.0) using standard settings. 
       Altered Amino Acid Sequence 
       [0036]    The altered amino acid sequence is derived from the parent sequence by making an amino acid alteration at one or more selected positions, and optionally also at other positions. Each amino acid alteration consists of substitution or deletion of the selected residue or insertion of at least one residue adjacent to the selected residue at the N- or C-terminal side. 
       Particular Substitutions 
       [0037]    The following alterations in SEQ ID NO: 1 may optionally be combined:
       K13Q, A25G, P38V,L,S, T42N, N74Q, V78I, Y91S, A92S, N96S, L99V, W104H, D134L,M,N, T138L, L140E, P143S,L, D145S, A146T, L147N,F, A148P, V149P, S150A, W155Q,N, Q157N, T158S, L163F, T164V, R168D, V190,IA, S197L,G, L199P, V215I, D223G, T229Y, R242A, T244P, T256K, L261A, D265P, P268A, E269Q, L277I, P280V, A281S, A283K, A284N, 1285E,D, G288D, N292C,Q, P303K, K308D, ot V315I.   Multiple substitutions:
           1258D G288D   S197G L199P   T164V L163F   V190X Q157X   A281X W155X   D223X A281X   D223X I285X   A281X I285X   A281X W155X A148X   D145X K308X K138X   D223X A281X1285X   
           Insertions: L147FN, G137ASV, V190GAH, L1QL, L1QGPL   Deletion: N97*       
 
         [0053]    Based on an alignment such as that shown in  FIG. 1 , one sequence may be used as a template for alterations in another sequence. Thus, Lid 1 or Lid 2 of one sequence may be substituted with the corresponding lid region of another sequence. The following variants are designed by altering Lid 1 of CALB using the indicated polypeptide as template:
       Q7RYD2 ( Neurospora crassa ) as termplate: Y135F K136H V139M G142Y P143G D145C L147G A148N V149F S150GKVAKAGAPC A151P W155L   Q4HUY1 ( Fusarium graminearum ) as template: V139I G142N P143I L144G D145G L147T A148G V149L S150IN A151T S153A W155V     Hyphozyma  sp. lipase as template: L140E P143L L147F A148G V149L.   Q4PEP1 ( Ustilago maydis ) as template: V139I L140E P143L D145S A146T L147F A148G V149L S150A A151S P152Q.       
 
         [0058]    Each of the above variants may optionally be combined with N292C and/or D223G and/or A281S and/or 1285E. 
         [0059]    The following substitutions may be made in SEQ ID NO: 2 ( Hyphozyma  sp. lipase): V192I, Q159N, D136L,M,N, P41V,L, S50A, N45S, W106H. 
       Nomenclature for Amino Acid Alterations 
       [0060]    In this specification, an amino acid substitution is described by use of one-letter codes, e.g. W155Q. X is used to indicate a substitution with any different residue (e.g. V190X). Multiple substitutions are concatenated, e.g. S197G L199P to indicate a variant with two substitutions. Alternatives are indicated by commas, e.g. W155Q,N to indicate a substitution of W155 with Q or N. An asterisk indicates a deletion. An insertion is indicated as substitution of one residue with two or more residues (e.g. L147FN) 
       Lipolytic Enzyme Activity 
       [0061]    The parent and the variant polypeptides have lipolytic enzyme activity (particularly lipase activity), i.e. they are able to hydrolyze carboxylic ester bonds to release carboxylate (EC 3.1.1), particularly ester bonds in triglycerides (triacylglycerol lipase activity, EC 3.1.1.3). 
         [0062]    The enzyme activity may be expressed as specific activity, i.e. hydrolytic activity per mg of enzyme protein. The amount of enzyme protein can be determined e.g. from absorption at 280 nm or by active-site titration (AST), as described by Rotticci et al. Biochim. Biophys. Acta 2000, 1483, 132-140. 
       Enantioselectivity 
       [0063]    Enantioselectivity is often an important parameter in CaLB catalyzed reactions, both in the hydrolysis and in the synthesis direction. The substrate can be a racemic mixture of two enantiomers, or it can be a prochiral meso form. In both cases a single enantiomer product is often desired. Enantiomeric excess (ee) is measured by quantifying the amount of both product enantiomers, and then calculating ee=(yield of desired enantiomer−yield of other enantiomer)/(sum of both yields). The quantification is often by chiral gas chromatography (GC) or high-performance liquid chromatography (HPLC). 
       Use of Lipolytic Enzyme Variant 
       [0064]    The lipolytic enzyme variant may be used for biocatalysis in a lipase-catalyzed reaction, both in ester hydrolysis and synthesis reactions, e.g. in synthesis of some polymers. The lipase-catalyzed reaction may be: 
         [0065]    a) hydrolysis with a carboxylic acid ester and water as reactants, and a free carboxylic acid and an alcohol as products, 
         [0066]    b) ester synthesis with a free carboxylic acid and an alcohol as reactants, and a carboxylic acid ester as product, 
         [0067]    c) alcoholysis with a carboxylic acid ester and an alcohol as reactants, or 
         [0068]    d) acidolysis with a carboxylic acid ester and a free fatty acid as reactants. 
         [0069]    Like CALB, the variant of the invention may particularly be used in applications where the enzyme&#39;s chemo-, regio-, and/or stereoselectivity, stability and reaction rate or the ability to accept a relatively broad range of substrates is important. The reaction products are typically used in the chemical, fine chemical, pharmaceutical, or agrochemical industry, or as food ingredients. The variant may be immobilized, e.g. by adsorption on an adsorbent resin such as polypropylene. 
         [0070]    The ester in the lipase-catalyzed reaction may have a bulky acid group or a bulky or secondary alcohol part, such as pNP 2-Me-butyrate, 6,8-difluro-4-methylumbelliferyl octanoate (DiFMU octanoate) or an iso-propyl fatty acid ester (e.g. C 16 -C 18  fatty acid which may be saturated or unsaturated). 
         [0071]    The variant may be used as described for CALB in A. J. J. Straathof, S. Panke, A. Schmid.  Curr. Opin. Biotechnol.  2002, 13, 548-556; E. M. Anderson, K. M. Larsson, O. Kirk.  Biocat. Biotrans.  1998, 16, 181-204; R. A. Gross, A. Kumar, B. Kaira.  Chem. Rev.  2001, 101, 2097-2124). 
       Examples 
     Example 1 
     Selection of Amino Acid Residues by Molecular Dynamics 
       [0072]    From Molecular Dynamics simulations 2 regions were found to be of high importance for the activity of  Candida antarctica  lipase B, as follows. 
         [0073]    CHARMm was used to prepare the 1TCA structure for the simulations. Hydrogen atoms were added to both protein and waters using the command HBUILD. The system was embedded in explicit water molecules and confined to a cubic box of side equal to 90 Angstroms. There were in total 24630 water molecules including those already present in the 1TCA structure. A simulation at constant temperature, 300K, and constant pressure, 1.01325 atmospheres, was performed for a total of 20 nanoseconds using NAMD. Berendsen&#39;s coupling method was used to keep the temperature and the pressure at the desired values. The results of the simulation were then analyzed using CHARMm (References for CHARMM: MacKerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D., Field, M. J., Fischer, S., Gao, J., Guo, H., Ha, S., Joseph-McCarthy, D., Kuchnir, L., Kuczera, K., Lau, F. T. K., Mattos, C., Michnick, S., Ngo, T., Nguyen, D. T., Prodhom, B., Reiher, W. E., Roux, B., Schlenkrich, M., Smith, J. C., Stote, R., Straub, J., Watanabe, M., Wiorkiewicz-Kuczera, J., Yin, D., Karplus, M.  J. Phys. Chem. B  1998, 102, 3586; MacKerell, A. D., Jr., Brooks, B., Brooks, C. L., III, Nilsson, L., Roux, B., Won, Y., Karplus, M. In  The Encyclopedia of Computational Chemistry;  Schleyer, P. v. R. et al., Eds.; John Wiley &amp; Sons: Chichester, 1998; Vol. 1, p 271; Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan, S., Karplus, M.  J. Comput. Chem.  1983, 4, 187). 
         [0074]    The analysis revealed hitherto unknown lids with high mobility. Several regions were found to move when the enzyme is in solution. It was concluded that the enzyme functionality and specificity are dependent on this mobility and the specific structure present in the media of choice for the hydrolysis or the synthesis reaction. The simulation indicated a more closed like form in water solution and a more fully open form in organic solvent solution, i.e. more like the crystal structure in some surfactant containing water solution. 
         [0075]    Using calculation of the isotropic Root Mean Square Displacements for the C-alpha atoms of the residues in CALB along the above mentioned simulation, regions with increased mobility were identified. The mobile lid regions were found to be residues 136-160 for Lid1 and residues 267-295 for Lid2. It was concluded that the residues in the neighborhood of these novel lids interact with the lid mobility and are thus very important for the activity of the enzyme. 
       Example 2 
     Hydrolysis Reactions 
       [0076]    Hydrolytic activity of the variants was evaluated on pNP-butyrate, racemic pNP 2-methylbutyrate, and 6,8-difluro-4-methylumbelliferyl octanoate (DiFMU octanoate). Racemic pNP 2-Me-butyrate was synthesized according to  J. Biol. Chem.  1971, 246, 6019-6023. DiFMU octanoate, purchased from Molecular Probes, has previously been reported by Lutz et al. ( J. Am. Chem. Soc.  2005, 127, 13466-13467) in CALB assays. Whereas pNP 2-Me-butyrate selects variants with improved acceptance of substrates with a bulky acid group, DiFMU octanoate selects variants with improved acceptance of a bulky alcohol part. Reactions were performed in 50 mM aqueous phosphate buffer, pH 7.0 with 0.1% Triton X-100. Reaction kinetic was followed for approx. 15 min in microtiter plates, measuring at 405 nm (pNP) or 350/485 nm (ex/em for DiFMU). Activities were normalized based on enzyme A 280 . 
         [0000]    
       
                 
         
             
             
         
       
     
         [0077]    Results are shown below as activity for the various substrates in % of CALB wild-type. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 pNP 
                 pNP 2-Me- 
                 DiFMU 
               
               
                   
                 Variant 
                 butyrate 
                 butyrate 
                 octanoate 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 N74Q 
                 77 
                 113 
                 110 
               
               
                   
                 P143S 
                 50 
                 113 
                 42 
               
               
                   
                 A281S 
                 215 
                 232 
                 208 
               
               
                   
                 P38S 
                 35 
                 107 
                 44 
               
               
                   
                 N292Q 
                 73 
                 158 
                 105 
               
               
                   
                 L1QGPL 
                 63 
                 144 
                 85 
               
               
                   
                 L1QL 
                 65 
                 193 
                 49 
               
               
                   
                 I285E 
                 233 
                 332 
                 236 
               
               
                   
                 L147F 
                 98 
                 232 
                 90 
               
               
                   
                 L147N 
                 79 
                 178 
                 79 
               
               
                   
                 N292C 
                 80 
                 282 
                 90 
               
               
                   
                 L140E 
                 51 
                 151 
                 79 
               
               
                   
                 P143L 
                 79 
                 192 
                 112 
               
               
                   
                 A146T 
                 55 
                 126 
                 42 
               
               
                   
                 P280V 
                 48 
                 100 
                 36 
               
               
                   
                 A283K 
                 104 
                 115 
                 94 
               
               
                   
                 A284N 
                 65 
                 125 
                 19 
               
               
                   
                 T103G, A148P 
                 70 
                 167 
                 0 
               
               
                   
                 W104H, A148P 
                 11 
                 146 
                 0 
               
               
                   
                 N74Q, A281S 
                 88 
                 156 
                 0 
               
               
                   
                 V190A 
                 64 
                 143 
               
               
                   
                 L199P 
                 74 
                 162 
                 75 
               
               
                   
                 T256K 
                 105 
                 120 
                 79 
               
               
                   
                 T42N 
                 35 
                 216 
                 47 
               
               
                   
                 R242A 
                 24 
                 119 
                 39 
               
               
                   
                 V215I 
                 105 
                 133 
                 43 
               
               
                   
                 T164V 
                 75 
                 130 
                 80 
               
               
                   
                 L163F, T164V 
                 81 
                 160 
                 92 
               
               
                   
                 D265P 
                 28 
                 117 
                 44 
               
               
                   
                 P303K 
                 35 
                 108 
                 50 
               
               
                   
                 R168D 
                 62 
                 122 
                 53 
               
               
                   
                 A25G 
                 66 
                 111 
                 26 
               
               
                   
                 V315I 
                 65 
                 102 
                 19 
               
               
                   
                 T244P 
                 56 
                 146 
                 20 
               
               
                   
                 K13Q 
                 56 
                 122 
                 39 
               
               
                   
                 L277I 
                 53 
                 137 
                 51 
               
               
                   
                 Y91S, A92S, N96S, 
                 39 
                 135 
                 76 
               
               
                   
                 N97*, L99V 
               
               
                   
                 D223G 
                 830 
                 3621 
                 820 
               
               
                   
                 Parent (CALB) 
                 100 
                 100 
                 100 
               
               
                   
                   
               
             
          
         
       
     
         [0078]    The results demonstrate that the specific activity towards a bulky substrate (ester with a branched fatty acid) can be increased up to 37-fold by substituting a single selected amino acid residue. 
       Example 3 
     Variants with Lid Replacement 
       [0079]    Variants based on CaLB wild-type (SEQ ID NO: 1) were designed by replacing lid 1 with the corresponding residues of the  Fusarium  lipase (SEQ ID NO: 4), the  Debaryomyces  lipase (SEQ ID NO: 5) or the  Neurospora  lipase (SEQ ID NO: 8). Further variants were designed by combining this with a single substitution of a selected residue (A281S). Results are expressed as activity in % of CALB activity on the same substrate. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 pNP 
                   
               
               
                   
                 pNP 
                 2-Me- 
                 DiFMU 
               
               
                 Variant 
                 butyrate 
                 butyrate 
                 octanoate 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 V139I, G142N, P143I, L144G, D145G, 
                 187 
                 661 
                 836 
               
               
                 L147T, A148G, V149L, S150IN, 
               
               
                 A151T, S153A, W155V 
               
               
                 Y135F, V139R, L140M, A141V, 
                 62 
                 306 
                 14 
               
               
                 G142P, P143V, D145C, A146P, 
               
               
                 L147S, A148F, V149P, S150KLSC, 
               
               
                 A151P, W155L 
               
               
                 Y135F, K136H, V139M, G142Y, 
                 341 
                 1223 
                 14 
               
               
                 P143G, D145C, L147G, A148N, 
               
               
                 V149F, S150GKVAKAGAPC, A151P, 
               
               
                 W155L 
               
               
                 V139I, G142N, P143I, L144G, D145G, 
                 1052 
                 1612 
                 631 
               
               
                 L147T, A148G, V149L, S150IN, 
               
               
                 A151T, S153A, W155V, A281S 
               
               
                 Y135F, K136H, V139M, G142Y, 
                 378 
                 2397 
                 76 
               
               
                 P143G, D145C, L147G, A148N, 
               
               
                 V149F, S150GKVAKAGAPC, A151P, 
               
               
                 W155L, A281S 
               
               
                   
               
             
          
         
       
     
         [0080]    The results demonstrate that the specific activity towards a bulky substrate can be significantly increased by replacing the lid of one lipase with the lid of another lipase, and that this can be further increased by combining with a single substitution of a selected residue. 
       Example 4 
     Enantioselectivity 
       [0081]    Hydrolysis reactions were performed in 2 mL scale using 2 mM pNP 2-Me-butyrate as substrate in sodium phosphate buffer, 0.5 M pH 7.0 with 1% Triton X-100. The reactions were stopped by addition of 2 M HCl (0.1 mL), and then extracted into Et 2 O (2 mL). After analysis by chiral GC (Varian CP-Chiralsil-DEX CB 10 m colum, temperature program 80 to 180° C. at 2° C./min), E (enantiomeric ratio) was calculated as E=ln[ee p (1−ee s )/(ee p +ee s )]/ln[ee p (1+ee s )/(ee p +ee s )], with ee s  and ee p  being ee (enantiomeric excess) for substrate and product, respectively. Reactions were performed in triplets for each enzyme (stopped at different conversions) and E reported as an average. 
         [0082]    CALB was tested and compared with variant Y135F, K136H, V139M, G142Y, P143G, D145C, L147G, A148N, V149F, S150GKVAKAGAPC, A151P, W155L. The results were E=2.4 for the variant and E=1.05 for the parent lipase (CALB), showing that CALB is almost entirely non-selective, but the variant has an increased enantioselectivity. 
       Example 5 
     Hydrolysis of Long-Chain Fatty Acid Ester 
       [0083]    Michaelis-Menten constants were determined for a CALB variant with pNP laurate as a long-chain substrate. Experiments were performed in 0.5 M sodium phosphate buffer, pH 7.0, containing 1% Triton X-100 (to avoid turbid solutions at high substrate concentrations). 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                   
                 k cat  /K M   
               
               
                   
                 k cat  (s −1 ) 
                 K M  (micro-M) 
                 (s −1  M −1 ) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Parent (caLB) 
                 3.1 
                 535 
                 0.58 * 10 4   
               
               
                 V139I, G142N, P143I, L144G, 
                 23 
                 170 
                   14 * 10 4   
               
               
                 D145G, L147T, A148G, V149L, 
               
               
                 S150IN, A151T, S153A, W155V 
               
               
                   
               
             
          
         
       
     
         [0084]    The results show that the variant is 23 times more active than the parent lipase on the long-chain substrate (measured as k cal /K M ). 
       Example 6 
     Hydrolysis of Iso-Propyl Ester 
       [0085]    The variant used in the previous example was also tested in hydrolysis of iso-propyl palmitate. The results showed that the hydrolysis was 26% higher for the variant than for CALB. The hydrolysis was performed as follows: 
         [0086]    As substrate, isopropylpalmitate was added to a concentration of 3 mg/ml in 50 mM NaAcetate pH 5.0 (=buffer), heated to 60° C. for 5 minutes and homogenized by Ultra Turrax for 45 seconds and used immediately after preparation. Purified enzyme preparations were diluted to a concentration corresponding to OD280=0.00016 in desalted water and 10 ppm Triton X-100. In PCR-plates 20 micro-L buffer, 60 micro-L substrate and 20 micro-L enzyme solution were mixed at 800 RPM for 20 seconds and transferred to a PCR thermocycler for 30 minutes reaction at 30 C followed by 5 minutes at 90° C. to inactivate enzymes and addition of 20 micro-L 10% solution of TritonX100 (in desalted water). The amount for fatty acids produced was determined using the NEFA C kit from Wako and results were calculated as an average of 6 determinations and subtraction of enzyme blank. 
       Example 7 
     Activity at High pH 
       [0087]    Lipase activity of two CALB variants was measured at various pH at 30° C. with tributyrin as substrate and gum arabic as emulsifier. The results are expressed as relative activity, taking activity at pH 7.0 as 100. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 pH 5.0 
                 pH 6.0 
                 pH 7.0 
                 pH 8.0 
                 pH 9.0 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Y135F, K136H, V139M, 
                 53 
                 97 
                 100 
                 90 
                 151 
               
               
                 G142Y, P143G, D145C, 
               
               
                 L147G, A148N, V149F, 
               
               
                 S150GKVAKAGAPC, 
               
               
                 A151P, W155L 
               
               
                 V139I, G142N, P143I, 
                 41 
                 76 
                 100 
                 99 
                 148 
               
               
                 L144G, D145G, L147T, 
               
               
                 A148G, V149L, S150IN, 
               
               
                 A151T, S153A, W155V 
               
               
                 Parent lipase (CALB) 
                 47 
                 62 
                 100 
                 60 
                 49 
               
               
                   
               
             
          
         
       
     
         [0088]    The variants are seen to have increased activity at alkaline pH (pH 7-9) and a higher pH optimum. 
       Example 8 
     Synthesis Reactions 
       [0089]    The variants were immobilized on Accurel porous polypropylene by physical adsorption to a loading of 20 mg/g (based on A280). Reactions were performed in Eppendorf tubes with 1 mmol of each reagent, approx. 0.8 mL hexane, and 5 mg immobilized enzyme @ 40° C., 1200 rpm. Samples were withdrawn for analysis by NMR and chiral GC. 
         [0090]    Results from a synthesis reaction with 2-ethyl-1-hexanol and vinyl acetate as reactants are shown below as conversion % (ee %): 
         [0000]    
       
         
               
             
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
             
          
           
               
                   
               
               
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                   
               
             
          
           
               
                 Variant 
                 15 min 
                 30 min 
                 1 h 
                 2 h 
                 3 h 
               
               
                   
               
               
                 Parent (CaLB) 
                  11 (32) 
                 24 (27) 
                 43 (22) 
                 61 (17) 
                 63 (17) 
               
               
                 Y135F K136H V139M 
                   6 (46) 
                 13 (46) 
                 24 (45) 
                 41 (40) 
                 52 (38) 
               
               
                 G142Y P143G D145C 
               
               
                 L147G A148N V149F 
               
               
                 S150GKVAKAGAPC 
               
               
                 A151P W155L 
               
               
                 V139I G142N P143I 
                 0.1 (51) 
                  6 (49) 
                 12 (50) 
                 22 (49) 
                 33 (48) 
               
               
                 L144G D145G L147T 
               
               
                 A148G V149L S150IN 
               
               
                 A151T S153A W155V 
               
               
                   
               
             
          
         
       
     
         [0091]    The enantiomeric ratio was calculated by the formula given above. The results were E=1.9 for the parent lipase (CALB), and E=3.0 and E=3.2 for the two variants. Thus, the results show improved enantioselectivity for the two variants. 
         [0092]    Another experiment was made in the same manner, but with vinyl benzoate and 1-hexanol as reactants. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0093]    After 72 hours, a conversion of 17% was found for the variant 1285E, whereas the parent CALB gave 9%.

Technology Category: 8