Thermotoga petrophila普鲁兰酶基因的异源表达与酶学性质分析  

Heterologous expression and enzymatic characterization of the pullulanase from Thermotoga petrophila

作　　者：邢岩 邱爽 聂慧慧 孙利鹏 邱立友 王明道 XING Yan;QIU Shuang;NIE Huihui;SUN Lipeng;QIU Liyou;WANG Mingdao(Key Laboratory of Enzyme Engineering of Agricultural Microbiology of Miniswy of Agricuhure,College of Sciences,Henan Agricuhural University,Zhengzhou 450002,China;Henan Yangshao Bio-products Co.,Ltd.,Mianchi 472400,China)

机构地区：[1]河南农业大学生命科学学院农业部农业微生物酶工程重点实验室,河南郑州450002 [2]河南仰韶生化工程有限公司,河南渑池472400

出　　处：《河南农业大学学报》2018年第3期404-411,423,共9页Journal of Henan Agricultural University

基　　金：科技部"十二五"农村领域科技计划项目(2013AA102101-2);河南省重点科技攻关计划项目(142102110086)

摘　　要：通过生物信息学筛选出来源于超嗜热菌Thermotoga petrophila(DSM13995)的普鲁兰酶基因,以从德国菌种保藏中心购买的基因组作为出发材料,克隆出普鲁兰酶基因TP-pul A;通过二步PCR方法构建含有该嗜热普鲁兰酶基因的载体pET21a-TP-pul A;将重组质粒转入大肠杆菌Rosetta(DE3)中测定其酶学性质。结果表明,TP-pul A为Ⅰ型普鲁兰酶,最适pH值为6.4,最适温度是85℃,半衰期为26.28 h,Na^+、K^+、Ca^(2+)、Mg^(2+)对其酶活性有明显的促进作用,Mn^(2+)、Co^(2+)、AL^(3+)、Fe^(3+)、SDS及EDTA对酶活性有不同程度的抑制作用,Km、Vmax、Kcat及Kcat/Km值分别为0.72 g·L-1、0.004 9 mmol·L^(-1)·s^(-1)、154.63 s^(-1)、214.76 L·g^(-1)·s^(-1)。本研究采用的生物信息学方法比传统的筛菌方法简单方便耗时短,且获得的嗜热普鲁兰酶的热稳定性好、催化效率高、对普鲁兰糖有较强的底物亲和能力,具有良好的应用前景。The pullulanase gene was screened from the hyperthermophilic bacterium Thermotoga petrophila（ DSM13995） by bioinformatics and the gene TP-pul A was cloned using the genome purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures as the starting material. The carrier pET21 a containing the pullulanase gene was constructed by the two-step PCR method. The recombinant plasmid was transferred into the Escherichia coli Rosetta（ DE3） and the enzymatic properties were determined. The results showed that TP-pul A was a type Ⅰ pullulanase. The optimum pH was 6. 4 and the optimum temperature was 85 ℃. The half-lifetime was 26. 28 h and Na~＋,K~＋,Ca^（2＋）,Mg^（2＋） had a significant enhancement on the enzyme activity. Mn^（2＋）,Co^（2＋）,AL^（3＋）,Fe^（3＋）,SDS and EDTA inhibited the activity of enzymes in different degrees and the value of Km,Vmax,Kcatand Kcat/Kmwas 0. 72 g·L^（-1）,0. 004 9 mmol·L^（-1）·s^（-1）,154. 63 s^（-1）,214. 76 L·g^（-1）·s^（-1）,respectively.The bioinformatics analytical method in this study was simple,convenient,and time-saving,compared with the traditional method of screening. The high thermalstability,catalysis efficiency and substrate affinity of this pullulanase had a good application foreground.

关 键 词：超嗜热菌 普鲁兰酶 THERMOTOGA petrophila 异源表达 酶学性质分析 

分 类 号：Q556.2[生物学—生物化学]