链霉菌GXT6 β-葡萄糖苷酶的酶学性质及葡萄糖耐受性分子改造

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

摘要:

[目的]从经过全基因组测序的链霉菌GXT6中克隆、表达一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因，研究重组酶的酶学性质并进行相关葡萄糖耐受性的氨基酸残基的分子改造，提高其对葡萄糖的耐受性。[方法]根据链霉菌GXT6的全基因测序结果，对其中一个注释为糖基水解酶的基因设计引物，PCR扩增目的基因，以pSE380为表达载体构建重组质粒，转化至大肠杆菌中诱导表达；采用镍亲和层析技术纯化重组蛋白质，对目的蛋白质进行酶学性质研究；采用定点饱和突变的方法对重组酶进行相关氨基酸残基的分子改造。[结果]从链霉菌GXT6中克隆到一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因，并在大肠杆菌中表达。酶学性质研究结果表明该β-葡萄糖苷酶的最适温度为40，最适pH为6.0，K_m值为（0.4712±0.0180）mmol/L，V_max值为（128.000±1.741）μmol/（min·mg），葡萄糖抑制常数K_i值为（1.8880±0.1307）mmol/L。该BGL3-GXT6能够水解黄豆苷、染料木苷、甜茶苷、虎杖苷、淫羊藿苷。还对BGL3-GXT6中与葡萄糖耐受性可能相关的氨基酸残基位点81-Trp和233-Trp进行了定点饱和突变，获得了25个具有酶活的突变酶并对其进行酶学性质研究。其中W233位点饱和突变后获得的突变酶的K_m和葡萄糖抑制常数K_i值与重组酶BGL3-GXT6相比均发生明显变化，葡萄糖耐受性有不同程度的提高，最高的提高了209倍。[结论]本研究获得的BGL3-GXT6对天然底物甜茶苷、黄豆苷、染料木苷、虎杖苷和淫羊藿苷具有水解功能，这些特性表明该β-葡萄糖苷酶在理论研究及在工业中有一定的应用价值。

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.