肠杆菌植酸酶基因的克隆及重组表达  

Cloning and Recombinant Expression of Phytase Gene from Enterobacter asburiae

作　　者：程琪 赵东莹 李杨 包永明 CHENG Qi;ZHAO Dong-ying;LI Yang;BAO Yong-ming(School of Ocean Science and Technology,Dalian University of Technology,Panjin Liaoning 124221,China;School of Bioengineering,Dalian University of Technology,Dalian Liaoning 116024,China)

机构地区：[1]大连理工大学海洋科学与技术学院,辽宁盘锦124221 [2]大连理工大学生物工程学院,辽宁大连116024

出　　处：《沈阳农业大学学报》2022年第4期394-404,共11页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金青年基金项目(52000020)。

摘　　要：植酸酶水解植酸产生可溶性磷酸,广泛应用于饲料、有机及生物肥料;植酸酶性质和植酸酶发酵制备是植酸酶制剂生产的基础。利用实验室前期筛选的降解有机磷Enterobacter asburiae ZSHY1菌株,克隆其植酸酶基因phyE,构建质粒pET28a(+)表达载体,在Escherichiacoli BL21(DE3)中进行异源表达;利用Ni-NTA和Q-Sepharose Fast Flow对重组酶PhyZSHY1-nsp进行纯化,表征其酶学性质及反应动力学参数。结果表明:植酸酶基因phyE全长1359bp,编码452个氨基酸,前20个氨基酸为信号肽。重组酶的酶促反应最适温度和pH值分别为60℃和4.5,在酸性条件下,可以保持60%以上酶活,是中温酸性植酸酶。Mg^(2+)、Cu^(2+)、Zn^(2+)、Al^(3+)等金属离子及SDS对酶促反应有抑制作用,并随着浓度的增加抑制作用增强;植酸钠为底物时,K_(m)为0.859mmol·L^(-1),k_(cat)为16.036s^(-1),k_(cat)/K_(m)为18.66L·s^(-1)·mmol^(-1),说明重组植酸酶PhyZSHY1-nsp具有较好的温度和酸碱稳定性,可被制成酶制剂应用于动物饲料和生物肥料中。Phytase hydrolyzes phytic acid to produce soluble phosphoric acid,which is widely used in feed,organic and biological fertilizer.Enterobacter asburiae ZSH Y_(1) strain with ability degrading phytate was isolated in early research,and its phytase gene phyE was cloned and ligated with plasmid pET28a(+)to construct expression vector.Heterologous expression was performed in Escherichia coli BL21(DE3).Ni-NTA and Q-Sepharose Fast Flow were used to purify recombinant PhyZSHY1-nsp,enzyme properties and kinetics parameters were characterized.The results showed that the phytase gene phyE was 1359 bp and encoded 452 amino acids,the first 20 amino acids were signal peptides.The optimal temperature and pH of the recombinant enzyme were 60℃ and 4.5,respectively.Under weak acid conditions,the recombinant enzyme could keep more than 60%of the activity,which was intermediate temperature acid phytase.Metal ions such as Mg^(2+),Cu^(2+),Zn^(2+),Al^(3+) and SDS could inhibit the enzymatic reaction,and the inhibition increased with the increase of concentration.The K_(m) was 0.859mmol·L^(-1),k_(cat) was 16.036s^(-1) and k_(cat)/K_(m) was 18.66L·s^(-1)·mmol^(-1) when sodium phytate was used as the substrate.So PhyZSHY1-nsp had notable thermostability and pH stability,which can be used as commercial enzyme and applied in animal feed and biological fertilizer.

关 键 词：植酸酶 肠杆菌 重组表达 分离纯化 酶学性质 

分 类 号：Q786[生物学—分子生物学] Q814