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Creation of an Enantioselective Hydrolase by Engineered Substrate-Assisted Catalysis
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
2001 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 123, no 18, 4354-4355 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2001. Vol. 123, no 18, 4354-4355 p.
Keyword [en]
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-5089DOI: 10.1021/ja015604xISI: 000168495800038OAI: diva2:7846
QC 20100907Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2010-09-07Bibliographically approved
In thesis
1. Rational redesign of Candida antarctica lipase B
Open this publication in new window or tab >>Rational redesign of Candida antarctica lipase B
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity.

Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze.

Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis.

The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C.

Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.

Biochemistry, Candida antarctica lipase B, rational redesign, secondary alcohols, substrate-assisted catalysis, S-selective, entropy, aldolase, stereospecificity pocket, oxyanion hole., Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:kth:diva-186 (URN)91-7178-012-2 (ISBN)
Public defence
2005-05-13, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2012-03-21Bibliographically approved

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