Rational introduction of disulfide bond to enhance optimal temperature of Lipomyces starkeyi alpha-dextranase expressed in Pichia pastoris.

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 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.