Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Rational Introduction of Disulfide Bond to Enhance Optimal Temperature of Lipomyces starkeyi $\alpha$-Dextranase Expressed in Pichia pastoris

  • Chen, Lin (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Yu, Chao (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Zhou, Xiangshan (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology) ;
  • Zhang, Yuanxing (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
  • Published : 2009.12.31
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Abstract

$\alpha$-Dextranase, which can hydrolyze dextran, is largely used in the sugar industry. However, a thermostable $\alpha$-dextranase is needed to alleviate the viscosity of syrups and clean blocked machines. Thus, to improve the optimal temperature of Lipomyces starkeyi $\alpha$-dextranase expressed by Pichia pastoris, the rational introduction of a de novo designed disulfide bond was investigated. Based on the known structure of Penicillium minioluteum dextranase, L. starkeyi $\alpha$-dextranase was constructed using homology modeling. Four amino acids residues were then selected for site-directed mutagenesis to cysteine. When compared with the wild-type dextranase, the mutant DexM2 (D279C/S289C) showed a more than $13^{\circ}C$ improvement on its optimal temperature. DexM2 and DexM12 (T245C/N248C, D279C/S289C) also showed a better thermal stability than the wild-type dextranase. After the introduction of two disulfide bonds, the specific activity of DexM12 was evaluated and found to be two times higher than that of the wild-type. Moreover, DexM12 also showed the highest $V_{max}$.

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References

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