玫瑰链霉菌海藻糖合成酶基因克隆、表达及功能鉴定

Cloning,Expression and Characterization of a Novel Gene Encoding Trehalose Synthase from Streptosporangium roseum

目的:克隆玫瑰链霉菌海藻糖合成酶基因(Srt)使其在大肠杆菌XL10-Gold中高效表达,并对重组酶的酶学特性进行研究。方法:利用PCR技术从玫瑰链霉菌中克隆到一段长1 704bp的海藻糖合成酶基因(Srt),构建重组表达质粒pSE380-Srt-treS,将其转化大肠杆菌XL10-Gold中诱导表达,对重组纯酶进行SDS-PAGE分析及酶学特性测定。结果:SDS-PAGE显示在65kDa处有明显单一蛋白条带。该酶可催化麦芽糖和海藻糖之间的可逆反应,海藻糖得率达82%,且含有很低的副产物葡萄糖(5%左右)。最适反应温度和pH分别为30℃、7.5,Cu2+、Zn2+和Tris能明显抑制酶活力。该酶还可催化蔗糖生成一种无龋齿,适合糖尿病患者食用的糖类-海藻酮糖。结论:成功克隆表达了一个海藻糖合成酶基因,该酶转化率高,副产物较少,为工业酶法生产海藻糖奠定基础。

Objective： A novel gene was cloned from Streptosporangium roseum CGMCC 4. 1208 and overexpressed in Escherichia coli XL10 - Gold, and the enzymatic characteristics of recombinant TreS were studied. Method： The gene which had a length of 1 704 bp and enco- ded 567 amion acids was amplified by PCR from Streptosporangium roseum. The recombinant plasmid pSE380 - Srt - treS was constructed and transformed into E. coli XL10 -Gold. The recombinant TreS was purified and characterized. Result： The molecular weight of the re- combinant protein was approximately 65 kDa by SDS - PAGE analysis. The recombinant TreS could catalyze the interconversion of maltose and trehalose, the production of trehalose reached 82 %, accompanied by little byproduct glucose （about 5 % ）. The optimum temperature and pH was at 30 ~C and 7. 5, respectively. Cu2 ~ , Zn2 ~ , and Tris strongly inhibited the enzyme activity. Most importantly, the enzyme could catalyze sucrose to trehalulose, which was an anticarious sugar suitable for diabetics. Conclusion： The trehalose synthase gene was cloned and expressed successfully, it could lay the foundation for industrial production of trehalose by enzymatic method due to its high conversion efficiency and few byproducts.