枯草芽孢杆菌优化表达蛋白质谷氨酰胺酶的研究  

Study on optimal expression of protein glutaminase in Bacillus subtilis

作　　者：王新秀 尹航 邢天悦 刘紫薇 吴思 张会[1] 李杰[1] 双宝[1] WANG Xinxiu;YIN Hang;XING Tianyue;LIU Ziwei;WU Si;ZHANG Hui;LI Jie;SHUANG Bao(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2024年第4期13-21,31,共10页Journal of Northeast Agricultural University

基　　金：中国博士后科学基金项目(2019M651149)。

摘　　要：蛋白质谷氨酰胺酶(Protein-glutaminase,PG)作用于大分子蛋白质或其小侧链的谷氨酰胺,使其脱酰胺变成谷氨酸可提高蛋白质溶解性和乳化性,在食品行业应用广泛。PG来源于解朊金黄杆菌(Chryseobacterium proteolyticum),但原始解脘金黄杆菌菌株表达PG产量较低。为提高蛋白质谷氨酰胺酶表达水平,将来源于解朊金黄杆菌的蛋白质谷氨酰胺酶基因优化密码子,将密码子换成枯草芽孢杆菌(Bacillus subtilis)偏爱密码子,再将密码子优化前后的PG基因分别连接至表达载体pBSA43,最后转化到枯草芽孢杆菌WB600中进行高效表达。结果显示,密码子优化前重组菌株的PG酶活力为1.83 U·mL^(-1),优化后重组菌株的PG酶活力为2.91 U·mL^(-1)。为进一步提高PG表达水平,经响应面分析优化后,确定最佳发酵条件:发酵时间为62.3 h,发酵温度为32.5℃,培养基初始pH为7.24。在该条件下,密码子优化后重组菌株PG酶活力为5.76 U·mL^(-1)。来源于解朊金黄杆菌的蛋白质谷氨酰胺酶基因经密码子优化后,表达水平明显提高,为蛋白质谷氨酰胺酶在枯草芽孢杆菌中的高效表达提供参考。Protein-glutaminase(PG)acts on glutamine in large proteins or their small side chains to deamidify it to glutamate to improve protein solubility and emulsification.Therefore,this enzyme has a wide range of applications in the food industry.PG is derived from Chryseobacterium proteolyticum,but the production of PG expressed by the original Chryseobacterium proteolyticum is very low.In order to improve the expression level of protein glutaminase,the protein glutaminase gene derived from Bacillus subtilis was codon optimized in this study,and the codon was replaced with a Bacillus subtilis preferred codon,after which the PG genes before and after codon optimization were ligated into the expression vector pBSA43,and finally transformed into Bacillus subtilis WB600 for high efficiency expression.The results showed that the PG enzyme activity of the recombinant strain before codon optimization was 1.83 U·mL^(-1),and that of the recombinant strain after codon optimization was 2.91 U·mL^(-1).In order to further improve the expression level of PG, the optimal fermentation conditions weredetermined after optimization by response surface analysis: fermentation time of 62.3 h, fermentationtemperature of 32.5 ℃, and an initial pH of 7.24 for the medium. Under these conditions, the PG enzymeactivity of the recombinant strain reached 5.76 U·mL^(-1) after codon optimization. The protein glutaminasegene derived from Bacillus subtilis was codon optimized to significantly increase the expression level,which provided a reference for the efficient expression of protein glutaminase in Bacillus subtilis.

关 键 词：密码子优化 枯草芽孢杆菌 蛋白质谷氨酰胺酶 异源表达 响应面分析 

分 类 号：Q786[生物学—分子生物学]