耐热赖氨酸氨肽酶的原核表达及特性表征

Prokaryotic Expression and Characterization of a Thermo-stable Lysine Aminopeptidase

DOI：10.3969/j.issn.1673-1689.2019.12.016

中文关键词: 赖氨酸氨肽酶 热稳定性 原核表达 酶学特性

英文关键词: lysine aminopeptidase,thermo-stable,gene engineering,enzymatic characteristics

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中文摘要:

铜绿假单胞菌NJ-814（Pseudomonas aeruginosaNJ-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1 461 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。

英文摘要:

Aminopeptidase produced byPseudomonas aeruginosaNJ-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 Ni2+-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.

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