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Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis
Author(s) -
Wittrup Larsen Marianne,
Zielinska Dorota F.,
Martinelle Mats,
Hidalgo Aurelio,
Jensen Lars Juhl,
Bornscheuer Uwe T.,
Hult Karl
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200900743
Subject(s) - hydrolysis , candida antarctica , lipase , chemistry , acylation , nucleophile , active site , mutant , hydrolase , butanol , substrate (aquarium) , catalysis , organic chemistry , stereochemistry , biochemistry , enzyme , biology , ethanol , ecology , gene
A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 m M butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild‐type lipase. Mutants with a blocked tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.