重组褐藻胶裂解酶表达体系的构建及诱导条件优化

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

目的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 BL21pLysS中进行诱导表达,对发酵培养基、异丙基-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 BL21pLysS/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0.1mmol/L的IPTG,在22℃下诱导24h后酶活可达2 403.93U/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。

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 fromCobetiasp.WG-007 was used to carry out rare codon modification,the recombinant plasmid pET-28 a（＋）-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-28 a（＋）-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 hat 22 ℃,the enzyme activity reached to 2 403.93 U/mL.CONCLUSION The optimized alginate lyase gene aly-cobis 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.