重组褐藻胶裂解酶表达体系的构建及诱导条件优化

郝瑶; 李恒; 龚劲松; 蒋敏; 许正宏; 史劲松

重组褐藻胶裂解酶表达体系的构建及诱导条件优化

郝瑶¹,
李恒^1,,
龚劲松¹,
蒋敏¹,
许正宏^2,3,
史劲松^1,

1.
糖化学与生物技术教育部重点实验室，江南大学药学院，江苏无锡 214122
2.
江南大学生物工程学院，江苏无锡 214122
3.
粮食发酵工艺与技术国家工程实验室，江苏无锡 214122

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出版历程
- 刊出日期: 2018-05-10

Expression System Construction of Recombinant Alginate Lyase and Optimization of Induction Conditions

1.
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, China
2.
School of Biotechnology, Jiangnan University, Wuxi, 214122, China
3.
National Engineering Laboratory for Cereal Fermentation Technology, Wuxi, 214122, China

摘要

摘要: 对褐藻胶裂解酶基因aly-cob进行外源表达，并优化诱导条件以实现褐藻胶裂解酶的高效表达。方法以产褐藻胶裂解酶菌株Cobetia sp.WG-007为出发菌株，对克隆得到的褐藻胶裂解酶基因进行稀有密码子改造，构建重组质粒pET-28a(+)-aly-cob，在宿主Escherichia coli BL21 pLysS中进行诱导表达，对发酵培养基、异丙基-β-D-硫代半乳糖苷（IPTG）、诱导温度、诱导时机、诱导浓度及诱导时间进行系统优化以提高酶活。结果构建了重组菌E.coli BL21 pLysS/pET-28a(+)-aly-cob，最适培养基为SB培养基，最佳诱导条件为OD600为1.0时加入终浓度为0.1mmol/L的IPTG，在22℃下诱导24h后酶活可达2403.93U/mL。结论成功对优化后的褐藻胶裂解酶基因aly-cob进行外源表达，经诱导表达条件优化后酶活为野生菌酶活的15倍，更具有工业化应用的潜力。
- 褐藻胶裂解酶 /
- 外源表达 /
- 诱导条件优化
Abstract: OBJECTIVE To express the optimized alginate lyase gene aly-cob exogenously and optimize the expression conditions to achieve the high expression of alginate lyase. METHODS The alginate lyase gene from Cobetia sp. WG-007 was used to carry out rare codon modification， the recombinant plasmid pET-28a(+)-aly-cob was constructed and induced by isopropyl-β-D-thiogalactoside (IPTG) in Escherichia coli BL21 pLysS. Then medium components and the induction temperature， induction time， final concentration and adding time of IPTG was optimized to improve enzyme activity. RESULTS The recombinant E. coli BL21 pLysS/pET-28a(+)-aly-cob was constructed， the alginate lyase was induced by IPTG， and the optimum induction conditions were determined as follows: SB medium was the optimum medium， when the OD600 value of bacterial reached 1.0， IPTG was added with its final concentration was 0.1mmol/L， and after inducing for 24h at 22℃， the enzyme activity reached to 2403.93U/mL. CONCLUSION The optimized alginate lyase gene aly-cob is successfully expressed in E. coli BL21 pLysS and the expression conditions are optimized. The optimized enzyme activity is 15 times of wild-type enzyme activity， which is more favorable for industrial application.
- alginate lyase /
- exogenous expression /
- induction condition optimization