摘要
为进一步拓展褐藻胶裂解酶在枯草芽孢杆菌体系中的高效表达,该研究以来源于海洋嗜盐白蚁菌WX(Isoptericola halotolerans WX)的一段褐藻胶裂解酶基因aly-ih为目的片段,成功构建Bacillus subtilis-pMA5-aly-ih工程菌,并利用启动子改造与培养基优化的方法提高酶催化活性。采用同源重组方法,将内源性启动子HpaⅡ替换为mpr,Aly-IH酶活力较初始酶活力提高至4倍;进一步通过启动子的串联多拷贝提高酶活力,当拷贝数为3时,重组酶酶活力较初始酶活力提高至5.1倍。在此基础上,优化发酵培养基配方,在16 g/L甘油、16 g/L大豆蛋白胨、25 mmol/L Mn^(2+)的培养条件下,Aly-IH酶活力提高到320 U/mL,是初始酶活力的10.7倍。该酶可高效降解褐藻胶得到平均聚合度为2.3的寡糖产物。研究结果表明,经枯草芽孢杆菌表达并改造优化的褐藻胶裂解酶Aly-IH具有良好的催化性能和应用潜力。
For further expanding the high-efficiency expression of alginate lyase in Bacillus subtilis,a segment of the alginate lyase gene named aly-ih from Isoptericola halotolerans WX was employed to construct an engineered strain of B.subtilis-pMA5-aly-ih.The catalytic activity of the enzyme was successively improved by the strategies of promoter modification and medium optimization.Herein,the enzymatic activity was increased by 4-fold compared with the initial enzyme through replacing the endogenous promoter HpaⅡby mpr with homologous recombination method.The activity of the recombinase was further increased by 5.1-fold when the copy number of promoter mpr reached 3.The optimized fermentation medium was as following:16 g/L glycerol,16 g/L soy peptone,25 mmol/L Mn 2+.With the above conditions,the enzymatic activity of Aly-IH reached 320 U/mL which was 10.7 times as much as the initial enzyme.Aly-IH could efficiently degrade alginate to oligosaccharides with an average degree of polymerization of 2.3.Conclusively,the modified engineered Aly-IH in B.subtilis exerted prominent catalytic performance and potential application.
作者
吴雯
朱风帅
俞嘉乐
李恒
龚劲松
许正宏
史劲松
WU Wen;ZHU Fengshuai;YU Jiale;LI Heng;GONG Jinsong;XU Zhenghong;SHI Jinsong(Key Laboratory of the Ministry of Education of Sugar Chemistry and Biotechnology,College of Life Sciences and Health Engineering,Jiangnan University,Wuxi 214122,China;National Engineering Research Center for Food Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;College of Bioengineering,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2023年第2期47-53,共7页
Food and Fermentation Industries
基金
国家重点研发计划专项子课题(2021YFC2102000)
宁夏回族自治区重点研发计划(2020BFH02011)。
