LIPASE VARIANTS AND POLYNUCLEOTIDES ENCODING SAME

12236-WO-PCT

LIPASE VARIANTS AND POLYNUCLEOTIDES ENCODING SAME

Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

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BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to lipases variants with improved activity in an amide-bond reaction, polynucleotides encoding the variants, methods of producing the variants, and methods of using

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the variants. More particularly, the present invention relates to modified enzymes with at least 60% homology with Candida antarctica Lipase B (CALB) which have improved activity in an amide-bond reaction as compared to the corresponding unmodified enzyme.

Description of the Related Art

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Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are serine hydrolases that catalyses the hydrolysis of fatty acid esters (triglycerides). They have broad substrate specificity and accommodate a wide range of structurally diverse esters, alcohols and carboxylic acids as substrates. Lipases do not catalyze or are very poor catalysts for the hydrolysis of amides which in part is explained by the lower reactivity of amides compared to esters (Bruice, P.Y. (1998) Organic Chemistry, 678).

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However, a few reports of amide hydrolysis by lipases have been published: Duarte DE., Castillo

E., Bárzana E., & López-Munguía A. (2000) Biotechnol Lett., 22, 1811-1814 describes the hydrolysis of capsaicin, a natural water-insoluble amide, by lipase B from Candida antarctica. Henke E. & Bornscheuer U. T. (2003) Anal Chem., 75, 255-260 describes a fluorophoric assay for the high-throughput determination of amide hydrolysis, which was used to evaluate 22 unmodified 25

lipases and esterases, and to screen 15000 mutants created by error-prone PCR and other random mutagenesis methods. This directed evolution experiment revealed no positive result, that is, new amidase activity.

     In another directed evolution experiment Fujii R., Nakagawa Y., Hiratake J., Sogabe A., & Sakata K. (2005) Protein Eng Des Sel., 18, 93-101 screened 20000 mutants, which were generated

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randomly by error-prone PCR, of Pseudomonas aeruginosa lipase for improved amide hydrolysis and found 3 mutations, namely F207S, A213D and F265L to affected the amidase/esterase activity ratios. The continuation of this work was described by Nakagawa Y., Hasegawa A., Hiratake J. & Sakata K. (2007) Protein Eng Des Sel., 20, 339-346, where they mutated Pseudomonas aeruginosa lipase for enhanced amidase activity and showed that the triple mutant F207S +A213D 35

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12236-WO-PCT

+M252F, but not substitutions of residues M16 or H83, gave an increase in amidase activity. This study also tried to give more insight why lipases do not hydrolyze amides despite some similarity to serine proteases, which are able to hydrolyze amides. Both contain a catalytic triade, which consists of Ser-His-Asp/Glu, and an oxyanion hole. However, there are also consider...