嗜热地衣芽胞杆菌Y6角蛋白酶的异源表达与表征  

Cloning and Heterologous Expression of the Keratinase from Thermophilic Bacillus licheniformis Y6

作　　者：刘建新[1] 姚大伟[1] 刘艳海 杨德吉[1] 

机构地区：[1]南京农业大学动物医学院,南京210095

出　　处：《农业生物技术学报》2018年第2期302-310,共9页Journal of Agricultural Biotechnology

基　　金：农业部"948"研究项目(No.2015-Z40)

摘　　要：嗜热角蛋白酶可在较高的温度下降解角蛋白,具有良好的热稳定性。为了克隆嗜热地衣芽胞杆菌(Bacillus licheniformis)Y6角蛋白酶基因,研究重组角蛋白酶酶学特性,本实验根据角蛋白酶基因设计引物,克隆了一个含完整开放性阅读框的1 140 bp基因片段(Gen Bank No.MF327392),将该片段插入到表达载体p ET-32a,转化至大肠杆菌(Escherichia coli)BL21(DE3),成功诱导表达出重组角蛋白酶,经Ni-亲和柱纯化后对酶学特性进行测定。结果显示Y6菌株的角蛋白酶前体有1个29个氨基酸残基的信号肽,成熟蛋白的理论相对分子量为27.27 k D,等电点为6.57,属于疏水性稳定蛋白质。以酪蛋白为底物,测得重组角蛋白酶最适反应温度为70℃,最适反应pH为8.5,V_(max)为0.149 mmol/(L·min),K_m为0.029 mmol/L,K_(cat)为20.1 S^(-1)。乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)、十二烷基硫酸钠(sodium dodecyl sulfate,SDS)、苯甲基磺酰氟(phenylmethanesulfonyl fluoride,PMSF)、Mn^(2+)、Fe^(3+)对其酶活性有一定的抑制作用,但异丙醇、甘油、二甲基亚砜(dimethyl sulfoxide,DMSO)等有机溶剂对其活性没有影响。该嗜热角蛋白酶具有较大的应用潜力,未来可应用于某些工业生产中。Bacillus licheniformis could secrete a kind of keratinase which can hydrolyze keratin such as feather,hair,nail and so on.In previous work,the results indicated that a thermophilic Bacillus licheniformis Y6 was able to live at elevated temperature,it also could degrade feather rapidly,and lived on feather as the only source of carbon and nitrogen.Based on these properties,this paper finally extracted a kind of thermostable keratinase with excellent characteristics was extracted.Hence,it possess the potential to manage poultry waste and can be exploited in some specific industries.aiming at cloning a keratinase gene from Bacillus licheniformis Y6,this paper designed a pair of primers,and finally obtained the gene（GenBank No.MF327392）which included a complete Open Reading Frame with the size of 1 140 bp.Aligned in NCBI,this keratianse remained to be conserved during evolution,and shared the same catalytic triad Ser-His-Asp with other serine proteases.Analyzed by some online software,this entire gene encoded 379 amino acids with pre-,pro-and mature protein regions,and its mature protein,with the molecular weight 27.27 kD and the theory isoelectric point 6.57,was classified as stable protein.After cloning the gene into vector pET-32a,The recombinant vector pET-32a ker Y6 was transformed into Escherichia coli BL21（DE3）induced with 0.5 mmol/L isopropyl β-D-thiogalactoside（IPTG）,then the precipitation and culture supernatant were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）analysis which indicated that the recombinant keratinase was successfully induced with a band at 58 kD corresponding to expectation,but could not be detected in the broth.In order to detect whether it developed into inclusion body,precipitation and supernatant of bacteria lysate were subjected to SDS-PAGE,results showed no band in the supernatant lane.Subsequently,the recombinant keratinase was purified by Ni-chelating affinity chromatography.Followed by characterization,the recombinant keratinase d

关 键 词：地衣芽胞杆菌 角蛋白酶 克隆 表达 

分 类 号：S188[农业科学—农业基础科学] Q815[生物学—生物工程]