来源于铜绿假单胞杆菌(Pseudomonas aeruginosa)所产蛋白酶PT121克隆表达及其在小肽合成上的应用

Cloning and expression of protease PT121 from Pseudomonas aeruginosa and application in peptide synthesis

摘要:

摘要:【目的】基于前期筛选到的P. aeruginosa PT121所产有机溶剂耐受性蛋白酶，本研究对该蛋白酶进行克隆表达，研究了重组蛋白的酶学性质及小肽合成上的应用。【方法】参考文献中报道的相似蛋白酶pseudolysin设计引物从菌株PT121基因组中克隆到耐有机溶剂蛋白酶PT121基因lasB。构建诱导表达重组质粒pET22b-lasB，于大肠杆菌中进行表达。考察蛋白酶PT121酶学性质及小肽合成上的应用。【结果】序列分析表明lasB基因编码信号肽、前肽及成熟肽3个部分，成熟肽部分含有301个氨基酸，分子量约33 kDa，属于金属蛋白酶M4家族。通过破碎条件优化，一步法制备得到较为纯净的重组蛋白酶PT121，其比活力达7700 U/mg，该酶呈现了较高的热稳定性，pH稳定性及溶剂耐受性，与野生菌P．aeruginosa PT121所产蛋白酶性质一致。在50% DMSO体系中高效催化合成了多种小肽，其中阿斯巴甜前体(Cbz-Asp-Phe-NH2)产率高达91%。【结论】蛋白酶PT121基因在大肠杆菌中克隆表达为进一步研究相关催化机理及分子改造奠定了基础。

关键词:关键词:蛋白酶PT121，基因lasB，异源表达，有机溶剂耐受性

Abstract:

Abstract:［Objective］ We studied the cloning and expression of lasB encoding solvent-resistant protease from Pseudomonas aeruginosa PT121．The recombinant protease was then characterized and applied in peptide synthesis．［Methods］The PCR primers were designed to acquire the open read frame (ORF) of lasB according to similar protease gene (pseudolysin) reported in the literature．Inducible expression plasmid pET22b-lasB was constructed and expressed in E.coli BL21 (DE3)．The recombinant protease was then characterized and applied in peptide synthesis．［Results］ The protease PT121 was defined as metalloproteinase M4 family according to sequence blast．Gene sequence analysis shows that lasB encodes signal peptide，pro-peptide and mature peptide．Mature protein contains 301 residues with molecular weight of 33 kDa．One-step preparation of the recombinant proteases PT121 was optimized by breaking cell wall．The specific activity of protease PT121 reached up to 7700U/mg，and it was stable similar with wild type PT121 from P．aeruginosa PT121 in temperature，pH and organic solvent．The synthesis rate of various dipeptides in 50% DMSO was effective，especially productivity of aspartame precursor reached up to 91%．［Conclusions］ Successful hetero-expression of protease PT121 lays the foundation of studying mechanism of catalysis and molecular evolution.

Key words:Keywords: protease PT121，lasB gene，hetero-expression，organic solvent stability