In vitro evolution of lipase B from Candida antarctica using surface display in Hansenula polymorpha

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In vitro evolution of lipase B from Candida antarctica using surface display in Hansenula polymorpha
S Y Kim; Jung Hoon Sohn; Y R Pyun; I S Yang; K H Kim; Eui Sung Choi
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 17, no. 8, pp. 1308-1315
Publication Year
Lipase B from Candida antarctica (CalB) displayed on the 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/Leu219 Gln 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.
Ca1Bdirected evolutionhansenula polymorphalipasesurface display
Korea Soc-Assoc-Inst
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Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
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