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Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris

Applied Microbiology and Biotechnology volume 67, pages 215–224 (2005)Cite this article

Abstract

Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZαC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6–38.4°C), glucose concentration (0.3–3.7%), yeast extract (0.16–1.84%), and pH (5.3–8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1°C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9±39.7 U/ml, and the actual value was 253.3±18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

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Acknowledgment

We acknowledge financial support by a grant NSC 93-2313-B-001-006 from the National Science Council, Taiwan, to J.F. Shaw.

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Affiliations

  1. Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 202, Taiwan

    S.W. Chang & J. F. Shaw

  2. Institute of Botany, Academia Sinica, Nankang, Taipei, 11529, Taiwan

    S.W. Chang, C. J. Shieh, G. C. Lee & J. F. Shaw

  3. Department of Bioindustry Technology, Dayeh University, Changhua, 51505, Taiwan

    C. J. Shieh

  4. Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, 403, Taiwan

    J. F. Shaw

Authors
  1. S.W. Chang
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  2. C. J. Shieh
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  3. G. C. Lee
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  4. J. F. Shaw
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Correspondence to J. F. Shaw.

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Chang, S., Shieh, C., Lee, G. et al. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris. Appl Microbiol Biotechnol 67, 215–224 (2005). https://doi.org/10.1007/s00253-004-1815-z

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  • DOI: https://doi.org/10.1007/s00253-004-1815-z

Keywords

  • Lipase
  • Response Surface Methodology
  • Lipolytic Activity
  • Lipase Production
  • Candida Rugosa