摘要
目的 对分散泛菌的胞内脱乙酰酶(PantoeadispersaN-acetylglucosaminedeacetylase,Pd-nagA)进行表达条件优化及酶学性质分析。方法 本研究使用聚合酶链式反应(polymerasechainreaction,PCR)技术对分散泛菌全基因组中Pd-nagA基因序列进行全长扩增,将含有Pd-nagA基因的质粒pET-28a(+)转化进入大肠杆菌BL21(DE3)中诱导表达,同时采用单因素实验,优化诱导温度、诱导前菌体生物量、诱导时间以及乳糖类似物异丙基硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)的浓度,采用Ni2+-次氮基三乙酸(nitrilotriacetic acid, NTA)进行重组酶的纯化,再对其酶学特性进行测定。结果 以pET-28a(+)质粒作为表达载体,大肠杆菌BL21(DE3)成功表达了可溶形式的Pd-nagA蛋白,分子量约为40kDa。当诱导温度为28℃,诱导前菌液OD600值为1.2, IPTG浓度为0.6 mmol/L,诱导时间为20 h时, Pd-nagA蛋白得到了大量表达。通过薄层层析法分析表明Pd-nagA重组蛋白能够在体外转化N-乙酰氨基葡萄糖生成氨基葡萄糖,Pd-nagA重组蛋白能够在高盐溶液中保持一半的相对酶活性,该酶4℃储存10d的相对活性仍然保持在90%以上,具有良好的储存稳定性,催化N-乙酰氨基葡萄糖生成氨基葡萄糖反应条件简单快捷。结论 本研究成功异源表达了具有催化活性的Pd-nagA酶,并对表达条件进行优化及酶学特性探究,可为工业上单酶催化制备氨基葡萄糖提供一定的理论参考。
Objective To optimize the expression conditions and analyze the enzymatic properties of the intracellular Pantoea dispersa N-acetylglucosamine deacetylase(Pd-nagA).Methods In this study,polymerase chain reaction(PCR)was used to amplify the full-length sequence of Pd-nagA gene in the whole genome of Panthera dispersa,and the plasmid pET-28a(+)containing Pd-nagA gene was transformed into Escherichia coli BL21(DE3)to induce expression.At the same time,single factor experiments were used.The induction temperature,biomass before induction,induction time and the concentration of isopropylβ-D-1-thiogalactopyranoside(IPTG)were optimized.The recombinant enzyme was purified by Ni2+-nitrilotriacetic acid(NTA),and its enzymatic characteristics were determined.Results The Pd-nagA protein was efficiently synthesized in soluble form in Escherichia coli BL21(DE3)utilizing the pET-28a(+)plasmid as the expression vector,and the recombinant protein had a molecular weight of roughly 40 kDa.The Pd-nagA protein was produced in substantial quantities when the induction temperature was 28°C,the OD600 value of the bacterial fluid before induction was 1.2,the IPTG concentration was 0.6 mmol/L,and the induction period was 20 h.Thin-layer chromatography analysis confirmed the capacity of the Pd-nagA recombinant protein to convert N-acetylglucosamine to glucosamine in vitro.Pd-nagA recombinant protein could maintain half of the relative activity in high salt solution,the enzyme could be stored for 10 days at 4℃and the relative activity was still more than 90%,with good storage stability,and preparation of glucosamine from N-acetylglucosamine under simple and fast reaction conditions.Conclusion In this study,a catalytically active Pd-nagA enzyme is successfully heterologously expressed,and the optimization of the expression conditions and enzyme properties is investigated,which can provide some theoretical references for the catalytic preparation of glucosamine by a single enzyme in the industry.
作者
杨文韬
庞立
田红
陈晓
易有金
夏菠
YANG Wen-Tao;PANG Li;TIAN Hong;CHEN Xiao;YI You-Jin;XIA Bo(College of Food Science and Technology,Hunan Agricultural University,Changsha 410000,China;College of Horticulture,Hunan Agricultural University,Changsha 410000,China)
出处
《食品安全质量检测学报》
CAS
北大核心
2023年第22期190-199,共10页
Journal of Food Safety and Quality
基金
湖南省自然科学基金项目(2022JJ30299、2022JJ30290)
湖南省教育厅项目(20C0947)
郴州可持续发展议程创新示范区建设专项(2019sfq30)。
关键词
分散泛菌
N-乙酰氨基葡萄糖脱乙酰酶
重组表达
酶学特性
Pantoea dispersa
N-acetylglucosamine deacetylase
recombinant expression
enzymatic properties
