共表达分子伴侣蛋白提高Ⅲ型普鲁兰水解酶在短小芽孢杆菌中的分泌表达及发酵优化
Enhanced Extracellular Type Ⅲ Pullulan Hydrolase Production by Co-expressing Molecular Chaperone in Brevibacillus choshinensis and Fermentation Optimization
曾静 1郭建军 1王通 1袁林1
作者信息
- 1. 江西省科学院微生物研究所,江西南昌 330096
- 折叠
摘要
本研究旨在通过共表达分子伴侣蛋白以及优化发酵条件来提高Ⅲ型普鲁兰水解酶(TK-PUL)在短小芽孢杆菌中的分泌表达水平.通过构建共表达TK-PUL和分子伴侣蛋白的多种重组短小芽孢杆菌,并以胞外酶活为指标对其进行筛选,确定最有利于TK-PUL分泌表达的分子伴侣蛋白及对应的重组短小芽孢杆菌.在此基础上采用单因素实验和响应面法优化重组短小芽孢杆菌的发酵条件.结果表明,共表达分子伴侣蛋白PrsABa的重组短小芽孢杆菌的胞外酶活达到 98.79 U/mL,提高了 0.31倍.该重组短小芽孢杆菌的最佳培养基包括 19.65 g/L葡萄糖、21.46 g/L 酵母提取物、12.01 g/L MgCl2·6H2O、9.02 g/L 脯氨酸、0.01 g/L FeSO4·7H2O、0.01 g/L MnSO4·4H2O、0.001 g/L ZnSO4·7H2O.在发酵温度为 35℃、起始发酵pH为 7.0的条件下,该重组短小芽孢杆菌于最佳培养基中培养 66h时,其胞外酶活达到 192.68 U/mL,提高了 1.56倍.本研究通过共表达分子伴侣蛋白和发酵优化实现了TK-PUL在短小芽孢杆菌的高效分泌表达,为TK-PUL的应用提供了基础.
Abstract
This study aimed to increase the expression and secretion of type Ⅲ pullulan hydrolase(TK-PUL)by Brevibacillus choshinensis through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions.By constructing multiple recombinant B.choshinensis co-expressing TK-PUL and molecular chaperone proteins,and screening them using extracellular enzyme activity as an indicator,the most favorable molecular chaperone protein and corresponding recombinant B.choshinensis for TK-PUL secretion and expression were determined.On this basis,single factor experiments and response surface methodology were used to optimize the fermentation conditions of recombinant B.choshinensis.Results showed that,the extracellular enzyme activity of the recombinant B.choshinensis strain co-expressing the molecular chaperone protein PrsABa reached 98.79 U/mL,representing a 0.31-fold increase.The optimal medium for this recombinant B.choshinensis was composed of 19.65 g/L of glucose,21.46 g/L of yeast extract,12.01 g/L of MgCl2·6H2O,9.02 g/L of proline,0.01 g/L of FeSO4·7H2O,0.01 g/L of MnSO4·4H2O,and 0.001 g/L of ZnSO4·7H2O.Culturing the optimized recombinant B.choshinensis in the above-mentioned optimized medium for 66 hours at 35°C and an initial pH of 7.0 increased the extracellular TK-PUL activity to 192.68 U/mL,which represented a 1.56-fold increase.Efficient secretion of TK-PUL in B.choshinensis was achieved through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions.This study provides a foundation for exploring the industrial-scale application of TK-PUL.
关键词
Ⅲ型普鲁兰水解酶/短小芽孢杆菌/胞外分子伴侣蛋白/发酵优化/分泌表达Key words
type Ⅲ pullulan hydrolase/Brevibacillus choshinensis/extracytoplasmic molecular chaperone/fermentation optimization/secretory expression引用本文复制引用
基金项目
国家自然科学基金地区科学基金(32160579)
江西省主要学科学术和技术带头人培养计划(20212BCJ23033)
江西省主要学科学术和技术带头人培养计划(S2021GDQN2403)
江西省科学院基础研究项目(2022YJC2006)
出版年
2024
国家科技期刊平台
NSTL
万方数据