枯草芽孢杆菌高效分泌表达盒的构建和应用  

Construction and application of high-efficiency secretoryexpression box for Bacillus subtilis

作　　者：韩瑞 初文琴 刘振 HAN Rui;CHU Wenqin;LIU Zhen(State Key Laboratory of Marine Food Processing and Safety Control,College of Food Science and Engineering,Ocean University of China,Qingdao 266404,China;Qingdao Key Laboratory of Food Biotechnology,Qingdao 266404,China;Key Laboratory of Biological Processing of Aquatic Products,China National Light Industry,Qingdao 266404,China)

机构地区：[1]中国海洋大学食品科学与工程学院,海洋食品加工与安全控制国家重点实验室,山东青岛266404 [2]青岛市食品生物技术重点实验室,山东青岛266404 [3]中国轻工业水产品生物加工重点实验室,山东青岛266404

出　　处：《食品与发酵工业》2024年第11期9-17,共9页Food and Fermentation Industries

基　　金：国家自然科学基金项目(32172178)。

摘　　要：该工作旨在研究枯草芽孢杆菌表达系统中不同元件之间的适配性,从而构建高效蛋白分泌载体,并进一步应用于提高壳聚糖酶Csn21c的胞外表达量。首先,构建包含不同启动子(P43、P_(shuttle09))、核糖体结合位点(RBS1、RBS5)和信号肽(Snpr B、Spho D、Symz C)的12个质粒,并以分别以3种蛋白(琼胶酶Ag WH50C、壳聚糖酶Csn21c和脂肪酶Lip15)为指示蛋白比较不同元件组合的蛋白分泌水平,发现载体p P435Y*K(P43-RBS5-Symz C)和p P095P*K(P_(shuttle09)-RBS5-Spho D)优于其他载体。相较原载体(p P431N*K),载体p P435Y*K能使琼胶酶Ag WH50C酶活性提高184.25%,脂肪酶Lip15酶活性提高52.8%,壳聚糖酶Csn21c酶活性提高164.62%。最后,通过比较5种不同培养基,确定了TB培养基为壳聚糖酶Csn21c的最适发酵培养基,使用TB培养基壳聚糖酶Csn21c的最高酶活力达到21.14 U/m L,较LB培养基的酶活力提高了128.31%。The purpose of this work was to investigate the compatibility between different elements in the expression system of Bacillus subtilis,constructing an efficient protein secretion vector,which could be further applied to improve the extracellular expression level of chitosanase Csn21c.Firstly,12 plasmids containing different promoters(P43,P shuttle09),ribosomal binding sites(RBS1,RBS5),and signal peptides(SnprB,SphoD,SymzC)were constructed,and the protein secretion levels of different element combinations were compared with three proteins(agarase AgWH50C,chitosanase Csn21c,and lipase Lip15)as indicator proteins,and the vectors pP435YK(P43-RBS5-SymzC)and pP095PK(P shuttle09-RBS5-SphoD)was superior to other vectors.By using vector pP435YK,the activity of agar AgWH50C enzyme increased by 184.25%,the activity of lipase Lip15 enzyme increased by 52.8%,and the activity of chitosanase Csn21c enzyme increased by 164.62%compared with that of the original vector(pP431NK).Finally,by comparing five different media,it was determined that the TB medium was the most suitable fermentation medium for chitosanase Csn21c,and the highest enzyme activity of chitosanase Csn21c in the TB medium reached 21.14 U/mL,which was 128.31%higher than that of LB medium.

关 键 词：枯草芽孢杆菌 分泌 启动子 核糖体结合位点 信号肽 壳聚糖酶 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]