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KMID : 0545120070170081308
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 8 p.1308 ~ p.1315
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In Vitro Evolution of Lipase B from Candida antarctica Using Surface Display in Hansenula polymorpha
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Kim So-Young
Sohn Jung-Hoon
Pyun Yu-Ryang
Yang In-Seok
Kim Kyung-Hyun
Choi Eui-Sung
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Abstract
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cell surface of H. polymorpha has been functionally improved for catalytic activity by molecular evolution. CalB was displayed on the cell surface by fusing to a cell-wall anchor motif (CwpF). A library of CalB mutants was constructed by in vivo recombination in H. polymorpha. Several mutants with increased whole-cell CalB activity were acquired from screening seven thousand transformants. The two independent mutants CalB10 and CalB14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB10 showed 6 times greater activity and CalB14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of Leu278Pro in CalB10 and Leu278Pro/Leu219Gln in CalB14. The substituted Pro278 in both mutants was located near the proline site of the α10 helix. This mutation was assumed to induce a conformational change in the α10 helix and increased the kcat value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ (Leu219Gln) was secreted into the culture supernatant at an amount of approximately 3 times more without an increase in the CalB transcript level, compared with the wild-type.
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KEYWORD
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CalB, directed evolution, Hansenula polymorpha, lipase, surface display
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