葡萄糖胺-6-磷酸合成酶的异源表达及其酶学性质表征

Heterologous expression and enzymatic characterization of glucosamine-6-phosphate synthase

葡萄糖胺-6-磷酸合成酶(GFA)是己糖胺生物合成中的一个关键酶,为建立一条高效的GFA生产工艺,其研究对象GFA来源于致病菌白色假丝酵母(Candida albicans ATCC 64548),通过基因克隆,然后在大肠杆菌中进行异源表达。结果显示,GFA的最佳异源表达条件为:诱导剂浓度为0.1 mmol×L^(-1),诱导温度为28℃,诱导时间为15 h,诱导时机为菌体浓度OD600达到1.0。其最佳的反应条件为:反应温度为37℃,反应pH为7.2,反应时间为20 min。通过双倒数作图法得到该酶的KM值为0.47 mg×mL^(-1);当反应体系中加入抑制剂(bacilysin)时,GFA转氨酶活性会被显著抑制,酶活降低约54.8%。结果表明,研究所采取的方法能够高效地异源表达葡萄糖胺-6-磷酸合成酶,提高其应用潜力,这对于加快新型抗真菌药物的研发具有重要意义。

Glucosamine-6-phosphate synthase(GFA)is a key enzyme in the biosynthesis of hexosamine.In order to establish an efficient GFA production process,the heterologous expression of GFA from pathogenic bacterium Candida albicans ATCC 64548 was expressed in E.coli BL21(DE3)Codon plus.The results show that the optimal expression conditions of GFA were:the concentration of IPTG 0.1 mmol×L^(-1);induction temperature 28℃;induction time 15 h;induction timing(OD600)reaching 1.0.The optimum reaction conditions were determined as:reaction temperature 37℃;reaction pH 7.2,and reaction time 20 min.The KM value of GFA was determined as 0.47 mg×mL^(-1) by Lineweaver-Burk.When the inhibitor(bacilysin)was added,the transaminase activity of GFA was significantly inhibited and reduced by 54.8%.With the method developed in this study,glucosamine-6-phosphate synthase could be expressed heterologously,which would greatly increase its application and help accelerate the development of new antifungal drugs.