基于信号肽的β-葡萄糖苷酶的分泌表达  

Secretory expression ofβ-glucosidase based on signal peptide

作　　者：李晓楠 王云[2] 王喆 刘小莉[1] 吴寒[1] 周剑忠[1] 夏秀东 LI Xiao-nan;WANG Yun;WANG Zhe;LIU Xiao-li;WU Han;ZHOU Jian-zhong;XIA Xiu-dong(Institute of Agricultural Product Processing,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏省农业科学院农产品加工研究所,江苏南京210014 [2]江苏大学食品与生物工程学院,江苏镇江212013

出　　处：《江苏农业学报》2022年第1期223-231,共9页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省科技计划项目(BE2019355)。

摘　　要：为提高大肠杆菌异源表达β-葡萄糖苷酶的底物可及性,本研究以大肠杆菌BL21(DE3)为宿主细胞,使用信号肽OprI、OsmY、PelB、OmpA将纳豆芽孢杆菌的β-葡萄糖苷酶基因bglH锚定到大肠杆菌BL21(DE3)外膜和周质上。然后使用启动子T7、Trc、LacUV5诱导表达Ⅱ型分泌途径的SecB伴侣蛋白,进而实现全细胞催化糖苷型底物水解。本研究通过表征不同细胞定位的β-葡萄糖苷酶活性,发现信号肽PelB分泌效果明显高于其他信号肽,LacUV5启动子与SecB伴侣蛋白组合表达bglH的重组菌株全细胞催化酶活性最高。此外,添加甘氨酸至质量分数为1.0%时,全细胞催化β-葡萄糖苷酶活性最高。并且通过优化诱导条件确定了β-葡萄糖苷酶的最优发酵条件:初始细胞密度(OD_(600))为1.0,发酵温度为25℃,发酵时间为24 h,发酵pH为6.5,最终全细胞催化β-葡萄糖苷酶活性可达2.55 U/ml。To enhance the substrate accessibility of heterologous expression ofβ-glucosidase in Escherichia coli,E.coli BL21(DE3)was used as the host cell in this study.β-glucosidase related gene(bglH)of Bacillus natto was anchored to the outer membrane and periplasm of E.coli BL21(DE3)by using different signal peptides(OprI,OsmY,PelB,OmpA).Different promoters(T7,Trc,LacUV5)were used to induce the expression of SecB chaperonin in typeⅡsecretory pathway to realize the hydrolysis of glycoside-type substrate from the aspect of whole-cell catalysis.Through representation of the activities ofβ-glucosidase located in different cells,it was found that the secretion effect of signal peptide PelB was obviously higher than other signal peptides,and in the recombinant strain which combined LacUV5 promoter and SecB chaperonin to express bglH,the whole-cell catalytic enzyme activity was the highest.In addition,activity ofβ-glucosidase was the highest under the condition of whole-cell catalysis when the mass fraction of glycine in the solution was 1.0%.The optimum conditions forβ-glucosidase fermentation were determined through optimizing the induction conditions,which were as follows:the initial cell density(OD_(600))was 1.0,the fermentation temperature was 25℃,the fermentation time was 24 h and the fermentation pH was 6.5.Under these conditions,activity ofβ-glucosidase from the aspect of whole-cell catalysis can reach 2.55 U/ml.

关 键 词：Β-葡萄糖苷酶 分泌表达 信号肽 伴侣蛋白SecB 

分 类 号：Q78[生物学—分子生物学]