嗜水气单胞菌J-1株弹性蛋白酶的表达、纯化及特性分析

Expression, purification and molecular characterization of elastase from Aeromonas hydrophila strain J-1

摘要:

摘要：【目的】表达、纯化嗜水气单胞菌J-1株弹性蛋白酶，并对弹性蛋白酶的性质进行分析。【方法】以pET-32a为表达载体将弹性蛋白酶基因ahyB转化至大肠杆菌BL21菌株中进行诱导表达，表达重组酶用His TaqNi2+亲和层析柱纯化并用6 mol/L盐酸胍进行复性；利用硫酸铵分级沉淀、阴离子交换层析和分子筛层析对嗜水气单胞菌培养上清液中的弹性蛋白酶进行纯化。将【结果】从嗜水气单胞菌培养上清液中获得的弹性蛋白酶原酶的最适pH 为8.5，而表达重组酶为 10.0；对热的稳定性，原酶高于表达酶。两种形式酶的性

Abstract:

Abstract: [Objective]The enzyme elastase is an important virulent factor of the opportunistic pathogen Aeromonas hydrophila. To know better about the molecular characterization of this enzyme, we purified the elatase and investigated its property and activity. [Methods] We cloned a structural gene ahyB encoding for the extracellular elastase of A. hydrophila J-1 and expressed it in E. coli BL21 by using pET-32a as vector. We purified the recombinant enzyme using His Bind Resin Purification Kit and recovered the elastolytic activity of the purified protein by incubation in a buffer containing 6 mol/L guanidine HCl and subsequent removal of denaturant by dilution. In addition, we also purified the native enzyme from the culture supernatant of A. hydrophila J-1 by 30 to 60% ammonium sulfate fractionation, anion exchange chromatography and sephaceryl chromatography. We compared the properties of the two elastase preparations. [Results] Native enzyme showed a pH optimum at 8.5, but recombinant enzyme at 10.0. Compared with the native enzyme, the recombinant enzyme was more stable for heat. Both the elastase preparations showed some identical properties concerning inhibitors, which were both mainly inhibited by EDTA, the cation chelator, and OPA, a Zn2+/Fe2+protease inhibitor. However, the recombinant elastase had higher tolerance than native enzyme against the inhibitors. And the metal cation Zn2+ and Fe2+ strongly inhibited the enzyme activity. [Conclusion] The recombinant product showed the similar enzymatic characteristics as the native enzyme from A. hydrophila J-1 and the two elastases belonged to the zinc-and-iron-dependant metalloendopeptidase. This is the first report on the recombinant expression and subsequent molecular chracterization analysis of A. hydrophila elastase.