摘要
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.
作者
郝瑶
李恒
龚劲松
蒋敏
许正宏
史劲松
HAO Yao;LI Heng;GONG Jin-song;JIANG Min;XU Zheng-hong;SHI Jin-song(Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education,School of Pharmaceutical Sci ence,Jiangnan University,Wuxi,214122,China;School of Biotechnology,Jiangnan University,Wuxi,214122,Chi ha;National Engineering Laboratory for Cereal Fermentation Technology,Wuxi,214122,China)
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2018年第3期227-230,324,共5页
Journal of Nanjing University of Traditional Chinese Medicine
基金
江苏省产学研前瞻性联合研究项目(BY2016022-19)
江苏省重大科技成果转化项目(BA2015006)
四川省重点研发计划项目(2017JZ0043)
国家海洋公益性行业科研专项经费项目(201305007)
关键词
褐藻胶裂解酶
外源表达
诱导条件优化
alginate lyase
exogenous expression
induction condition optimization