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Rational Introduction of Disulfide Bond to Enhance Optimal Temperature of Lipomyces starkeyi α-Dextranase Expressed in Pichia pastoris
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
J. Microbiol. Biotechnol. 2009; 19(12): 1506-1513
Published December 28, 2009 https://doi.org/10.4014/jmb.0902.0096
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
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J. Microbiol. Biotechnol. 2009; 19(12): 1506-1513
Published online December 28, 2009 https://doi.org/10.4014/jmb.0902.0096
Copyright © The Korean Society for Microbiology and Biotechnology.
Rational Introduction of Disulfide Bond to Enhance Optimal Temperature of Lipomyces starkeyi α-Dextranase Expressed in Pichia pastoris
Lin Chen , Chao Yu , Xiangshan Zhou * and Yuanxing Zhang
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
Abstract
α-Dextranase, which can hydrolyze dextran, is largely
used in the sugar industry. However, a thermostable α-
dextranase is needed to alleviate the viscosity of syrups
and clean blocked machines. Thus, to improve the optimal
temperature of Lipomyces starkeyi α-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
α-dextranase was constructed using homology modeling.
Four amino acids residues were then selected for site-directed
mutagenesis to cysteine. When compared with the wildtype
dextranase, the mutant DexM2 (D279C/S289C) showed
a more than 13oC 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 Vmax.
Keywords: Lipomyces starkeyi α-dextranase, site-directed mutagenesis, Pichia pastoris, thermostability, disulfide bond
