Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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In Vitro Evolution of Lipase B from Candida antarctica Using Surface Display in Hansenula polymorpha

  • Kim, So-Young (Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Sohn, Jung-Hoon (Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB)) ;
  • Pyun, Yu-Ryang (Department of Biotechnology, Yonsei University) ;
  • Yang, In-Seok (Graduate School of Biotechnology, Korea University) ;
  • Kim, Kyung-Hyun (Graduate School of Biotechnology, Korea University) ;
  • Choi, Eui-Sung (Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
  • Published : 2007.08.30
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Abstract

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 CalB 10 and CalB 14 showed an approximately 5 times greater whole-cell activity than the wild-type. When these mutants were made as a soluble form, CalB 10 showed 6 times greater activity and CalB 14 showed an 11 times greater activity compared with the wild-type. Sequence analyses of mutant CALB genes revealed amino acid substitutions of $Leu^{278}Pro$ in CalB10 and $Leu^{278}Pro/Leu^{219}Gln$ in CalB14. The substituted $Pro^{278}$ in both mutants was located near the proline site of the ${\alpha}$10 helix. This mutation was assumed to induce a conformational change in the ${\alpha}$10 helix and increased the $k_{cat}$ value of mutant CalB approximately 6 times. Site-directed mutagenized CalB, LQ ($Leu^{219}Gln$) 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.

Keywords

References

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