摘要
铜绿假单胞菌NJ-814(Pseudomonas aeruginosa NJ-814)所产氨肽酶具有良好的热稳定性。克隆出该氨肽酶编码基因(lap),测序得知该基因长度为1461 bp,编码486个氨基酸。将lap与表达载体pET-42a(+)连接构建重组基因pET-42a-lap,重组基因转化进入原核表达宿主E.coil BL21(DE3)构建重组菌BLAP,BLAP在16℃低温诱导下成功表达出重组氨肽酶。通过镍柱(Ni-NTA)亲和层析对重组酶进行分离纯化,得到电泳纯的重组酶酶液,纯化倍数和回收率分别为4.7和83.5%。考察该重组氨肽酶的酶学特性,发现该酶的最适pH为8.3,在pH 6.5~9.5之间具有较好的稳定性;最适反应温度为80℃,经过70℃热处理1 h仍能保留60%以上的酶活性;底物特异性研究表明该重组酶能够水解Arg-pNA、Leu-pNA和Lys-pNA,对Lys-pNA的水解能力分别是其他2种的6.6倍和2.4倍,通过酶动力学分析证实了该酶对赖氨酸底物具有最强的亲和力,与野生菌所产的酶一致属于一种赖氨酸氨肽酶。
Aminopeptidase produced by Pseudomonas aeruginosa NJ-814 shows excellent thermo-stable feature.The gene encoding this peptidase was cloned and symbolized as lap in this paper.Sequencing results revealed lap is consists of 1461 nucleotides coding 486 amino acids.Recombinant gene pET-42a-lap was constructed by connecting lap with pET-42a(+)expression vector.Recombinant strain BLAP was generated by transferring pET-42a-lap into E.coil BL21(DE3),BLAP successfully express aminopeptidase after cultivated in proper condition.The recombinant aminopeptidase was purified 4.7-fold to homogeneity with a recovery of 83.5%from cell free extract using Ni^2+-NAT affinity column chromatography.The properties of the recombinant aminopeptidase were investigated and the results showed that the optimal reaction pH and temperature were pH 9.0 and 80℃respectively,and it was extraordinary stable within pH 7.0~9.5 or below 70℃.According to substrate specificity analysis and enzymatic reaction kinetics,this recombinant enzyme belongs to lysine aminopeptidase,which is same as P.aeruginosa NJ-814 aminopeptidase reported before.
作者
黄浩
周楠迪
田亚平
HUANG Hao;ZHOU Nandi;TIAN Yaping(School of Biotechnology,Jiangnan Universtty,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第12期110-115,共6页
Journal of Food Science and Biotechnology
基金
江苏省产学研前瞻性联合研究项目(BY2014023-22)。
关键词
赖氨酸氨肽酶
热稳定性
原核表达
酶学特性
lysine aminopeptidase
thermo-stable
gene engineering
enzymatic characteristics
