黑曲霉葡萄糖氧化酶基因在毕赤酵母中的表达及性质研究

Expression and Enzymatic Properties of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris

试验旨在提高葡萄糖氧化酶(glucose oxidase,E.C.1.1.3,GOD)的产量并进一步研究重组葡萄糖氧化酶的酶学性质及其对畜禽常见致病菌的体外抑菌效果。试验选取黑曲霉来源的GOD基因序列GOX4,经密码子优化后电击转入毕赤酵母,构建Pichia pastoris X33/pGAPZαA-GOX4重组菌,而后通过高浓度博来霉素筛选到一代高产葡萄糖氧化酶的重组菌。在此基础上,通过共表达糖酵解途径中的关键限速基因丙酮酸激酶基因BTS成功构建二代高产菌,并研究重组葡萄糖氧化酶的酶学性质,采用牛津杯法进行抑菌试验。结果表明:①第二代高产菌的GOD产量相比第一代提高了60%;②重组葡萄糖氧化酶的最适温度为50℃,最适pH为7.0;③稀释10倍和25倍的重组葡萄糖氧化酶发酵上清液对大肠杆菌、沙门氏菌和铜绿假单胞菌均有一定程度的抑制效果,但效果有所差异。综上所述,共表达丙酮酸激酶基因BTS提高了重组葡萄糖氧化酶的表达,重组葡萄糖氧化酶的酶学性质及体外抑菌试验表明其在畜牧行业具有良好的应用潜力。

This experiment was conducted to increase the production of glucose oxidase(E.C.1.1.3,GOD)and to futher investigated the enzymatic properties of the recombinant protein and its in vitro antibacteri⁃al effect on common pathogenic bacteria in livestock and poultry.The GOD gene sequence derived from Aspergillus niger was selected and electrostatically transferred into Pichia pastoris after codon optimiza⁃tion to construct X33/pGAPZαA-GOX4,which were screened by high concentration of bleomycin to ac⁃quire the first generation of high-yield glucose oxidase recombinant bacteria.On this basis,the secondgeneration high-yielding bacteria were further constructed by co-expressing the pyruvate kinase gene,a key rate-limiting gene in the glycolytic pathway.The enzymatic properties of recombinant glucose oxi⁃dase were further investigated,and the antibacterial test was carried out by the Oxford cup method.The results showed that:①the GOD production of the second generation of high-yielding bacteria increased by 60%compared with the first generation;②The optimal temperature of recombinant glucose oxidase was 50℃and the optimal pH was 7.0;③Recombinant glucose oxidase fermentation supernatant diluted 10-fold and 25-fold showed some degree of anti⁃bacterial effect on E.coli,Salmonella and Pseudo⁃monas aeruginosa,although the effects varied.In conclusion,co-expression of the pyruvate kinase gene BTS increases the expression of recombinant glucose oxidase,and the enzymatic properties of recombinant glucose oxidase and in vitro bacteriostatic tests show its good potential for application in the animal husbandry.