Evaluation and directed evolution for thermostability improvement of a GH 13 thermostable aα-glucosidase from Thermus thermophilus TC11

Abstract

Background: Thermal stable aα-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, aα-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable aα-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated. Results: The recombinant aα-glucosidase (TtAG) from Thermus thermophilus TC11 was expressed in Escherichia coli BL21 (DE3) and purified. The purified enzyme had a molecular mass of 184 kDa and consisted of 59-kDa subunits; it showed hydrolytic activity for pNP-aα-d-glucopyranoside (pNPG), sucrose, trehalose, panose, and isomaltooligosaccharides and very low activity for maltose. The highest specific activity of 288.96 U/mg was observed for pNPG at 90 °C and pH 5.0; Pb2+ provided a 20 % activity increase. TtAG was stable at 70 °C for more than 7 h and had a half-life of 195 min at 80 °C and 130 min at 90 °C. Transglycosylation activity was also observed with sucrose and trehalose as substrates. TtAG showed differences on substrate specificity, transglycosylation, multimerization, effects of metal ions and optimal pH from other reported Thermus aα-glucosidases. One single-substitution TtAG mutant Q10Y with improved thermostability was also obtained from random mutagenesis library. The site-saturation mutagenesis and structural modelling analysis indicated that Q10Y substitution stabilized TtAG structure via additional hydrogen bonding and hydrophobic interactions. Conclusion: Our findings indicate that TtAG is a highly thermostable and more acidic aα-glucosidase distinct from other reported Thermus aα-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of aα-glucosidases, which may guide molecular engineering of aα-glucosidase and other thermostable enzymes for industrial application.