多糖奈瑟球菌淀粉蔗糖酶突变株F191M的构建及酶学性质表征  

Construction and Enzymatic Properties of the Mutant F191M of Amylosucrase from Neisseria polysacchare

作　　者：张磊[1,2] 刘春雷 闵伟红[1,2] 孙琳琳[1,2] 方丽[1,2] 

机构地区：[1]吉林农业大学食品科学与工程学院,长春130118 [2]小麦和玉米深加工国家工程实验室,长春130118

出　　处：《中国食品学报》2015年第10期19-25,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：吉林省科技厅资助项目(20110251)

摘　　要：目的:通过定点突变提高淀粉蔗糖酶的活力,研究酶学性质表征,为淀粉蔗糖酶的规模化制备和应用提供试验依据。方法:利用基因工程技术,将来源于多糖奈瑟球菌(Neisseria polysacchare)的AS基因在大肠杆菌BL21上异源表达。对F191位点进行定点突变,筛选获得F191M,再对F191M和野生型淀粉蔗糖酶诱导表达和非变性镍柱纯化后进行酶学性质表征。结果:成功构建突变株F191M,该突变株比酶活为202 U/mg,较野生型提高1.44倍。通过对比F191M与野生型酶学性质发现,F191M最适p H值由野生型的7.0提高为8.0,两者最适温度均为35℃,该温度下,F191M半衰期由野生型的20h降低到19h,F191M对金属离子及有机试剂具有更好的抗性。酶动力学研究显示,F191M的Vmax较野生型提高了1.45倍。结论:突变株F191M在大肠杆菌中得到有效表达,活力较野生型提高了1.44倍。【Objective】 To improve the enzyme activityof amylosucrase（AS） by site-directed mutagenesisand provide experiment basis for large-scale preparation and application of AS by the study of enzymology properties characterization.【Methods】 The gene of AS from Neisseria polysaccharea were cloned and heterologously expressed in E.coli BL21 by using the genetic engineering techniques and F191 M was obtained by site-directed mutagenesis. The enzymology properties of AS from F191 M and wild type were detected after the inducible expression and purification. 【Results】 F191 M was built successfully, and its specific activity was 202 U/mg, increasing by 1.44 times compared with wild type. By comparing the enzymology properties of F191 M and wild type, the optimal p H of F191 M was 8.0 higher than the wild type 7.0.The optimal temperature was the same as the wild type（35 ℃）. The half-life of F191 M and the wild type were 19 h and20 h at 35 ℃, respectively. F191 M had better resistance on metalions and organic solvents than wild type. The Kinetics study showed that the Vmaxof F191 M increased by 1.45 times. 【Conclusions】 The mutant strain F191 M was expressed in E.coli BL21 effectively, and the activity of AS increased by 1.44 times compared with wild type.

关 键 词：多糖奈瑟球菌 淀粉蔗糖酶 定点突变 酶学性质表征 

分 类 号：TS201.25[轻工技术与工程—食品科学]