SH-110菌海藻糖合成酶基因克隆、表达及酶学性质研究

为找到理想的工业化生产海藻糖合成酶基因工程菌，以长白山温泉SH-110基因组DNA为模板，PCR扩增编码Tres基因片段，克隆至pET-30a(+)原核表达载体，转化E.coli BL21(DE3)，实验中采用IPTG和乳糖同时进行对比诱导，对表达产物进行SDS-PAGE和酶学性质研究。成功克隆了2912 bp的Tres基因，实验中发现加入IPTG和乳糖后，融合蛋白在胞内都得到了表达，而且添加乳糖诱导的蛋白表达量偏高。表达重组酶Tres相对分子量约为110 kDa，最适作用温度60℃，最适pH7.0。金属离子Ca2+、K+、Zn2+对重组酶有明显激活作用，Ba2+、Cu2+、Mn2+有明显抑制酶活作用。重组酶的动力学常数Km为8.823 mmol/L和Vmax为235.29 mmol/L。已成功在大肠杆菌表达重组Tres，为进一步研究利用基因工程菌生产大量海藻糖合成酶，进行大规模生产海藻糖提供理论依据。

Cloning and Expression of the Tres Gene from SH-110 Strain and Characterization of the Enzyme

In order to find the ideal industrialized production of trehalose synthase gene engineering bacterium, the Tres gene was amplified by PCR with Changbai Mountain hot spring SH-110strain＇ s genomie DNA. The amplified Tres gene was cloned into prokaryotic expression vector pET-30a（＋） and expressed in BL21（DE3） under induction of IPTG and lactose. The expressed product was identified with SDS-PAGE and studied on characterization of the enzyme. The Tres gene at a length of 2912 bp was successfully cloned, in the experiments that joined IPTG and lactose, fusion protein in intracellular had been expressed, and added lactose protein expression induced by high volume. The relative molecular mass of expressed recombinant Tres was about 110 kDa. This enzyme exhibited the highest activity at the conditions of pH 7.0 and 60℃. Furthermore, metal ions such as Ca2＋, K＋, Zn2. could activate the enzyme activity, while metal ions such as Ba2＋, Cu2＋, Mn2＋ decreased the enzyme activity. The kinetic parameters Km and Vmax were 8.823 mmol/L and 235.29 mmol/L. Recombinant Tres was expressed in E.coli, which laid the further study of recombinant gene-engineered strain produce large amounts of trehalose synthase provided theoretical basis for mass production.