产大分子levan的果糖基蔗糖转移酶的原核表达及酶学性质  

Prokaryotic expression and enzymatic properties of macromolecular levan producing levansucrase

作　　者：宋廷富 姜腾飞[1] 李茜[1] 常斐斐 黄国印 付佳琪[1] 乔代蓉[1] 徐辉[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2016年第2期195-200,共6页Chinese Journal of Applied and Environmental Biology

基　　金："十二五"科技支撑计划项目(2014BAD02B02);国家自然科学基金项目(31272659);四川省科技厅项目(213GZX0161-3;2014GZX0005;2013HH0007;2013GZ0058;15010302;2012GE0008;ARRLKF14-04)资助~~

摘　　要：Levan型果聚糖是由微生物通过果糖组成的一个均聚物,寻找可以产均一度高的大分子果聚糖的果糖基蔗糖转移酶对其工业应用具有十分重要的意义.本研究克隆了来自运动发酵单胞菌(Zymomonas mobilis,SCTCC102250)的果糖基蔗糖转移酶基因,并在大肠杆菌BL21(DE3)中成功实现表达.通过His-tag柱层析对表达的果糖基蔗糖转移酶进行纯化,将纯化的酶与底物蔗糖反应,经乙醇沉淀得到多糖样品,利用凝胶色谱法确定了其分子量(Mr)的大小为2×106.多糖酸水解后的样品Rt与果糖标准品的Rt保持一致,表明该聚合物为levan果聚糖.最后,通过研究果糖基蔗糖转移酶的酶学性质,确定其最适温度为40℃,最适pH为6.0,Km值为3.95 mmol/L,Vmax为1 31.58μmol m L-1 min-1.本研究通过酶法合成得到levan果聚糖,为今后制备大分子levan果聚糖提供了理论依据.Levan-type fructan is a homopolymer made up of microorganisms by fructose. Levan-type fructan of different degrees of polymerization is widely used in food, medicine, pharmaceuticals and cosmetics. Enzymatic synthesis of levantype fructan is currently the most widely used method for industrial applications. Therefore it is of great importance to search for levansucrase that can produce fructose of uniform macromoleculres. This research cloned levansucrase from Zymomonas mobilis（SCTCC102250） and successfully expressed in E. coli BL21（DE3）. A single stripe was achieved through t he His-tag column chromatography and purification of expressed protein by SDS-page protein electrophoresis. Polysaccharide samples were obtained by ethanol precipitation after the reaction between purified enzyme and sucrose. Gel permeation chromatography method was used to determine the size of its molecular weight. The molecular weight（Mr） of polysaccharide sample was 2 × 106, indicating it was macromolecular polysaccharide. The Rt value of the sample polysaccharides after acid hydrolysis was the same with the standard Rt of fructose, showing that the polymer was levan fructan. Study of the enzyme properties of the fructose based sugar transferase showed an optimal temperature of 40°C, the optimum pH of 6.0, Km of 3.95 mmol/L, and Vmax of 131.58 μmol m L-1 min-1. This research obtained levan by enzymatic synthesis, and provided a theoretical basis for the preparation of macromolecular levan.

关 键 词：levan果聚糖 果糖基蔗糖转移酶 原核表达 蛋白纯化 酶学性质 

分 类 号：Q55[生物学—生物化学]