Improved thermostability of maltooligosyl trehalose hydrolase by computer-aided rational design

Abstract

Trehalose is a widely used and safe natural disaccharide. Maltooligosyl trehalose hydrolase(MTHase) is one of the key enzymes for trehalose preparation by double enzyme method using starch or dextrin as substrate. In industrial production, the thermalstability of MTHase is of great significance. We first heterogeneously expressed MTHase from Arthrobacter in E.coli strains BL21 (DE3). Based on the overall stability of the protein after virtual saturation mutation predicted by FoldX and the evolutionary information from PSSM, 15 mutations were selected and combined. Finally, the combinatorial mutant G589P/A57P was obtained. At 60 ℃, the t _1/2 of G589P/A57P and wild type are 37 min and 19 min, respectively, which is 1.9 times higher than that of wild type. The enzyme kinetic parameters of G589P/A57P were analyzed. The K _M and k _cat are 4.82 mM and 1136 s^{− 1}, respectively, and the results were close to the wild type, indicating that the mutation did not reduce the catalytic efficiency of the enzyme. The molecular dynamics simulation results show that the rigidity and thermal stability of G589P/A57P protein increase in the range of residues 50–100 and 400–500, which may be due to the proline effect caused by the introduction of proline.

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