Characterization of β-glucosidase from Streptomyces sp. GXT6 and its molecular modification of glucose tolerance

Abstract:

[Objective] A gene encoding family 3 β-glucosidase from Streptomyces sp. GXT6 was cloned and expressed. The enzymatic properties of recombinant protein were studied in detail. And the related amino acid residues were modified to improve glucose-tolerance. [Methods] Based on the analysis of the genome sequence of Streptomyces sp. GXT6, one of the genes annotated as glycosyl hydrolase family 3 was cloned into expression vector pSE380. The recombinant plasmid pSE-bgl3 was constructed and transformed into Escherichia coli XL1-blue for induction of expression. The recombinant protein was purified with Ni-NTA. The features of the recombinant protein BGL3-GXT6 were characterized. The recombinant enzyme was modified through site-directed saturation mutation. [Results] A new gene encoding β-glucosidase of glycosyl hydrolase family 3 was cloned from Streptomyces sp. GXT6. The properties of BGL3-GXT6 were identified. Its optimal pH and temperature were 6.0 and 40 μmol/(min·mg), and K_i value was (1.8880±0.1307) mmol/L. BGL3-GXT6 was able to hydrolyze daidzin, genistin, polydatin and icariin. Furthermore, the possible amino acids related to glucose tolerance of BGL3-GXT6 (81-Trp and 233-Trp) were substituted. Then, twenty-five mutants with activity were obtained and further characterized. As a result, it was found K_m and K_i values of mutants of W233 were significantly changed compared with the wild-type BGL3-GXT6. And the glucose tolerance of these mutants was improved to some extent, with the highest increase of 209 times. [Conclusion] The β-glucosidase BGL3-GXT6 in this study has the ability to hydrolyze rubusoside, daidzin, genistin, polydatin and icariin. These characteristics indicate that BGL3-GXT6 has important applications in theoretical research and in industry.