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

Background: Thermal stable alpha-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, alpha-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable alpha-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated. Results: The recombinant alpha-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-alpha-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 degrees C and pH 5.0; Pb2+ provided a 20 % activity increase. TtAG was stable at 70 degrees C for more than 7 h and had a half-life of 195 min at 80 degrees C and 130 min at 90 degrees 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 alpha-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 alpha-glucosidase distinct from other reported Thermus alpha-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of alpha-glucosidases, which may guide molecular engineering of alpha-glucosidase and other thermostable enzymes for industrial application.