期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 97, 期 8, 页码 3355-3362出版社
SPRINGER
DOI: 10.1007/s00253-012-4516-z
关键词
Enantioselectivity-activity trade-off; Directed evolution; Molecular dynamics simulation; Site-directed saturation mutagenesis; Kinetic resolution
资金
- Natural Science Foundation of China [21176215]
- Outstanding Young Scholar of Zhejiang Province [R4110092]
- Program for Zhejiang Leading Team of ST Innovation [2011R50007]
Despite directed evolution being a practical and efficient method of improving the properties of enzymes, a trade-off between the targeted property and other essential properties often exists which hinders the efficiency of directed evolution. In our previous work, mutant CVH of an esterase from Rhodobacter sphaeroides with high enantioselectivity was obtained by directed evolution, unfortunately its activity cannot catch another mutant YH. To compensate the trade-off of mutant CVH, site-directed saturation mutagenesis was conducted on four residues, three (Asn62, Met121, and Leu145) were hot spots determined from directed evolution, and one (Tyr27) was introduced to make up the large distance between a mutation (Asn62) and the substrate. A new mutant (HMVY) with high enantioselectivity and comparable activity to YH was obtained. According to the kinetic analysis and molecular dynamics simulations, it was understood that the high enantioselectivity and poor activity of mutant CVH was caused by different decrement of efficiency constants to two isomers, (R)-, (S)-methyl mandelate, and the high enantioselectivity and activity of mutant HWVY was caused by improved activity towards the preferred substrate ((S)-methyl mandelate), which provided the interpretation of the trade-off compensation. This work could provide a way to compensate the trade-off of enantioselectivity and activity in the process of enzyme evolution.
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