Suppression of water as a nucleophile in Candida antarctica lipase B catalysis

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 m...

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Bibliographic Details
Published in:ChemBioChem
Main Authors: Larsen, Marianne Wittrup, Zielinska, Dorota F, Martinelle, Mats, Hidalgo, Aurelio, Jensen, Lars Juhl, Bornscheuer, Uwe T, Hult, Karl
Format: Article in Journal/Newspaper
Language:English
Published: 2010
Subjects:
Amino Acid Substitution
Binding Sites
Biocatalysis
Candida
Catalytic Domain
Computer Simulation
Hydrolysis
Lipase
Mutagenesis
Site-Directed
Recombinant Proteins
Water
Antarc*
Antarctica
Online Access:https://curis.ku.dk/portal/da/publications/suppression-of-water-as-a-nucleophile-in-candida-antarctica-lipase-b-catalysis(2cb9974f-af4f-41ce-8593-0bc374f0b01b).html
https://doi.org/10.1002/cbic.200900743
Description
Summary: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 mM 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.

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