Rational redesign of Candida antarctica lipase B

• Main Author: Magnusson, Anders
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: KTH, Skolan för bioteknologi (BIO) 2005
• Subjects: Biochemistry; Candida antarctica lipase B; rational redesign; secondary alcohols; substrate-assisted catalysis; S-selective; entropy; aldolase; stereospecificity pocket; oxyanion hole; Biokemi; Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology); Molecular Biology; Microbiology; Biochemistry or Biopharmacy); Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi); molekylärbiologi; mikrobiologi; biokemi eller biofarmaci); Antarc*; Antarctica
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-186

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 ...