双齿围沙蚕蛋白酶的纯化及其性质  

Purification and properties of the protease from Perinereis aibuhitensis Grube

作　　者：汪靖超[1] 赵峰[1] 李荣贵[1] 王斌[2] 

机构地区：[1]青岛大学医学院生物系,山东省青岛市266071 [2]青岛大学医学院微生物学教研室,山东省青岛市266021

出　　处：《世界华人消化杂志》2007年第8期800-806,共7页World Chinese Journal of Digestology

基　　金：国家重点基础研究前期专项基金资助项目;No.2004CCA02400

摘　　要：目的:纯化双齿围沙蚕(Pednereis aibuhitensis Grube)蛋白酶,并对其性质进行研究．方法:将沙蚕匀浆液先后进行Superdex-75凝胶柱层析、MonoQTM阴离子交换柱层析及SP- Sepharose 4B阳离子交换层析进行纯化．将经过非还原性SDS-聚丙烯酰胺凝胶电泳的蛋白酶电转移至含明胶的SDS-聚丙烯酰胺凝胶中,通过观察明胶的分解来确定电泳条带的蛋白酶活性．对纯化后蛋白酶的最适pH值,热稳定性,最适温度,蛋白酶抑制剂的影响,有机溶剂的稳定性,金属离子对酶活性的影响等性质进行了测定．结果:纯化后得到的一种蛋白酶,还原性SDS- PAGE显示纯化的蛋白酶为两条带,分子量分别为Mr34 900和Mr33 900,而非还原性SDS- PAGE出现了分子量分别为Mr 65 600、Mr 57 900、Mr 43 500的三条蛋白带,明胶蛋白酶活性图谱检测显示酶活性主要在约Mr 66 000处．该蛋白酶具较好的热稳定性,在pH 8-10和20-35℃的情况下有较高活性．PMSF对该酶有较强抑制作用,而有机溶剂对该酶没有明显的影响．结论:该蛋白酶由两个通过二硫键连接在一起的亚基构成,两亚基的分子量分别为Mr 33 900和Mr 34 900;本文所采用的新的明胶蛋白酶活性图谱检测法是一种非常灵敏和有效的蛋白酶活性检测方法．AIM： To purify a new protease from the clamworm Perinereis aibuhitensis Grube, and study its properties. METHODS： After homogenization, Superdex-75 chromatography, MonoQTM chromatography, and SP-Sepharose 4B chromatography were used ordinally to purify the protease from the clamworm Perinereis aibuhitensis Grube. A new gelatin zymography method was used to study the proteolytic activity of the protease： protease separated by non-reductive SDS-PAGE was electrophoretically transferred to a gel containing gelatin, and the proteolytic activity of the protease was determined by the clear zone of the electrophoretic protein band. The effect of pH value, temperature, protease inhibitor, organic solvents and metal ions on the proteolytic activity of the protease was studied.RESULTS： After purification, one kind of protease was obtained, which showed two protein bands with molecular weights of 34 900 and 33 900 separately by reductive SDS-PAGE, while three bands with molecular weights of 65 600, 57 900, and 43 500 separately by non-reductive SDS-PAGE. Gelatin zymography showed that the proteolytic activity mainly appeared around the band of Mr 66 000. The protease was stable and displayed high proteolyric activity within the range of pH 8 to 10 and at 20 - 35℃ It was strongly inhibited by phenylmethyl sulfonyl fluoride （PMSF）, while stable in the presence of organic solvents. CONCLUSION： The protease is composed of two subunits, whose molecular weights are 33 900 and 34 900, respectively. Gelatin zymography is sensitive and effective in studying the proteolyric activity of proteases.

关 键 词：纯化 蛋白酶 纯化双齿围沙蚕 性质 

分 类 号：Q814[生物学—生物工程]