CALB Variants

Abstract

The invention relates to amino acid sequence variants of a lipase with improved activity for catalyzing synthesis reactions and methods of preparing the variants. The methods include predicting amino acid sites for change based on computational models of the protein structure in non-aqueous conditions, and expressing the protein in a prokaryotic host for subsequent purification and use. The enzyme sequence variants described have a three to nine-fold improvement in synthesis activity over the parent protein sequence.

1 - 41 . (canceled) 
     
     
         42 . A nucleic acid encoding a  Candida antarctica  lipase B (CALB) variant, wherein the CALB variant comprises an amino acid sequence that differs from SEQ ID NO: 2 by two to ten amino acid modifications, wherein the two amino acid modifications are at positions 223 and 227 of SEQ ID NO: 2. 
     
     
         43 . The nucleic acid of  claim 42 , wherein the CALB variant has about a two fold to about fifteen fold improved synthesis activity as compared to a wild type (WT) CALB comprising an amino acid sequence of SEQ ID NO: 2. 
     
     
         44 . The nucleic acid of  claim 43 , wherein the synthesis activity comprises synthesis of an ester or an amide. 
     
     
         45 . The nucleic acid of  claim 42 , wherein the CALB variant further comprises one or more amino acid modifications at positions 141, 146, 188, 189, or 235 of SEQ ID NO: 2. 
     
     
         46 . The nucleic acid of  claim 42 , wherein the CALB variant comprises three or more, four or more, five or more, six or more, or seven or more amino acid modifications. 
     
     
         47 . The nucleic acid of  claim 42 , wherein the amino acid modifications are amino acid substitutions. 
     
     
         48 . The nucleic acid of  claim 47 , wherein the amino acid substitutions are A141T, A146T, E188D, I189V, D223G, S227T, or V235A. 
     
     
         49 . The nucleic acid of  claim 48 , wherein the CALB variant comprises an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22. 
     
     
         50 . The nucleic acid of  claim 42 , wherein nucleic acid comprises SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:
 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21.   
     
     
         51 . A vector comprising the nucleic acid of  claim 42 . 
     
     
         52 . An expression vector comprising the nucleic acid of  claim 42 . 
     
     
         53 . The expression vector of  claim 52 , wherein the expression vector comprises a secretion signal. 
     
     
         54 . A method for expressing a CALB variant, wherein the method comprises transfecting the vector of  claim 52  into a host cell, and culturing the host cell under conditions that allow expression of the CALB variant. 
     
     
         55 . A method for preparing a CALB variant, wherein the method comprises transfecting the vector of  claim 53  into a host cell, culturing the host cell in a culture medium and under conditions that allow expression of the CALB variant, and obtaining supernatant from the culture medium. 
     
     
         56 . The method of  claim 55 , wherein the method further comprises concentrating the supernatant comprising the CALB variant. 
     
     
         57 . The method of  claim 55 , wherein the host cell is  Bacillus subtilis.    
     
     
         58 . An expression vector for expressing CALB in  Bacillus , wherein the expression vector comprises a nucleic acid encoding CALB, one or more origins of replication for replication in  Bacillus , a promoter, a secretion signal, and optionally a selectable marker. 
     
     
         59 . The expression vector of  claim 58 , wherein the expression vector comprises a constitutive promoter. 
     
     
         60 . An expression system comprising the expression vector of  claim 58 , a host strain of  Bacillus , and a culture medium. 
     
     
         61 . A nucleic acid encoding a fusion protein comprising a CALB variant and a heterologous peptide, wherein the CALB variant comprises an amino acid sequence that differs from SEQ ID NO: 2 by two to ten amino acid modifications, wherein the two amino acid modifications are at positions 223 and 227 of SEQ ID NO: 2. 
     
     
         62 . A method of making CALB comprising transfecting the expression vector of  claim 58  into a  Bacillus , and cultivating the  Bacillus  in a culture medium under conditions that allow expression and secretion of CALB in the culture medium. 
     
     
         63 . The method of  claim 62 , wherein the culture medium comprises a non-carbohydrate micronutrient source, a buffering agent for maintaining pH of the medium at a range of about 5 to about 9, a non-hydrolyzable nonionic surfactant, and a nitrogen source. 
     
     
         64 . The method of  claim 63 , wherein the method comprises cultivating the  Bacillus  at a temperature of about 30° C. to about 42° C., about 35° C. to about 40° C., or about 37° C. 
     
     
         65 . The method of  claim 62 , wherein the expression vector comprises a selectable marker, and the method further comprises adding an additive for selection to the culture medium. 
     
     
         66 . The method of  claim 62 , wherein the  Bacillus is B. subtilis, B. cereus, B. brevis, B. licheniformis, B. stearothermophilus, B. pumilis, B. amyloliquefaciens, B. clusii , or  B. megaterium.    
     
     
         67 . The method of  claim 62 , wherein the method further comprises obtaining the supernatant from the culture medium and concentrating the supernatant comprising CALB. 
     
     
         68 . The method of  claim 67 , wherein the supernatant is concentrated by ultrafiltration.