分散泛菌胞内脱乙酰酶在大肠杆菌中重组表达条件优化及酶学特性

Optimization of expression conditions and enzymatic properties of intracellular deacetylase from Pantoea dispersa in Escherichia coli

中文关键词:  分散泛菌  N-乙酰氨基葡萄糖脱乙酰酶  重组表达  酶学特性

英文关键词:Pantoea dispersa  N-acetylglucosamine deacetylase  recombinant expression  enzymatic properties

中文摘要:

      目的 对分散泛菌的胞内脱乙酰酶(Pantoea dispersa N-acetylglucosamine deacetylase, Pd-nagA)进行表达条件优化及酶学性质分析。方法 本研究使用聚合酶链式反应(polymerase chain reaction, PCR)技术对分散泛菌全基因组中Pd-nagA基因序列进行全长扩增, 将含有Pd-nagA基因的质粒pET-28a(+)转化进入大肠杆菌BL21(DE3)中诱导表达, 同时采用单因素实验, 优化诱导温度、诱导前菌体生物量、诱导时间以及乳糖类似物异丙基硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG)的浓度, 采用Ni2+-次氮基三乙酸(nitrilotriacetic acid, NTA)进行重组酶的纯化, 再对其酶学特性进行测定。结果 以pET-28a(+)质粒作为表达载体, 大肠杆菌BL21(DE3)成功表达了可溶形式的Pd-nagA蛋白, 分子量约为40 kDa。当诱导温度为28℃, 诱导前菌液OD600值为1.2, IPTG浓度为0.6 mmol/L, 诱导时间为20 h时, Pd-nagA蛋白得到了大量表达。通过薄层层析法分析表明Pd-nagA重组蛋白能够在体外转化N-乙酰氨基葡萄糖生成氨基葡萄糖, Pd-nagA重组蛋白能够在高盐溶液中保持一半的相对酶活性, 该酶4℃储存10 d的相对活性仍然保持在90%以上, 具有良好的储存稳定性, 催化N-乙酰氨基葡萄糖生成氨基葡萄糖反应条件简单快捷。结论 本研究成功异源表达了具有催化活性的Pd-nagA酶, 并对表达条件进行优化及酶学特性探究, 可为工业上单酶催化制备氨基葡萄糖提供一定的理论参考。

英文摘要:

      Objective To optimize the expression conditions and analyze the enzymatic properties of the intracellular Pantoea dispersa N-acetylglucosamine deacetylase (Pd-nagA). Methods In this study, polymerase chain reaction (PCR) was used to amplify the full-length sequence of Pd-nagA gene in the whole genome of Panthera dispersa, and the plasmid pET-28a(+) containing Pd-nagA gene was transformed into Escherichia coli BL21(DE3) to induce expression. At the same time, single factor experiments were used. The induction temperature, biomass before induction, induction time and the concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) were optimized. The recombinant enzyme was purified by Ni2+-nitrilotriacetic acid (NTA), and its enzymatic characteristics were determined. Results The Pd-nagA protein was efficiently synthesized in soluble form in Escherichia coli BL21(DE3) utilizing the pET-28a(+) plasmid as the expression vector, and the recombinant protein had a molecular weight of roughly 40 kDa. The Pd-nagA protein was produced in substantial quantities when the induction temperature was 28°C, the OD600 value of the bacterial fluid before induction was 1.2, the IPTG concentration was 0.6 mmol/L, and the induction period was 20 h. Thin-layer chromatography analysis confirmed the capacity of the Pd-nagA recombinant protein to convert N-acetylglucosamine to glucosamine in vitro. Pd-nagA recombinant protein could maintain half of the relative activity in high salt solution, the enzyme could be stored for 10 days at 4℃ and the relative activity was still more than 90%, with good storage stability, and preparation of glucosamine from N-acetylglucosamine under simple and fast reaction conditions. Conclusion In this study, a catalytically active Pd-nagA enzyme is successfully heterologously expressed, and the optimization of the expression conditions and enzyme properties is investigated, which can provide some theoretical references for the catalytic preparation of glucosamine by a single enzyme in the industry.