Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis |
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| Authors: | Marianne Wittrup Larsen Dr. Dorota F. Zielinska Mats Martinelle Dr. Aurelio Hidalgo Dr. Lars Juhl Jensen Prof. Uwe T. Bornscheuer Prof. Karl Hult Prof. |
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| Affiliation: | 1. Department of Biochemistry, School of Biotechnology, Royal Institute of Technology, AlbaNova University Center, 106 91 Stockholm (Sweden), Fax: (+46)?8‐5537‐8468;2. Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix‐Hausdorff‐Strasse 4, 17487 Greifswald (Germany);3. Centro de Biología Molecular “Severo Ochoa” (UAM‐CSIC), Nicolas Cabrera 1, 28049 Madrid (Spain);4. Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N (Denmark) |
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| Abstract: | 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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| Keywords: | Candida antarctica lipase B hydrolysis library screening mutagenesis rational design transacylation |
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