沙蚕消化道细菌D2胞外蛋白酶的分离纯化及其性质

目的分离纯化沙蚕消化道菌D2胞外蛋白酶,并对其性质进行分析。方法收集48 h发酵液上清,经80%饱和硫酸铵沉淀、DEAE-Sepharose阴离子交换层析、Superdex G-75凝胶过滤层析逐步纯化D2蛋白酶,分析其酶学性质,并用ESI-MS/MS测定其酶解后的部分氨基酸序列。结果经纯化,得到电泳纯的D2蛋白酶,纯化倍数为4.9倍,活性回收率为33%,相对分子质量约42 000,纯度大于99%;最适作用温度为60℃,最适pH为9,在pH6~11及0~60℃具有较好的稳定性,水解酪蛋白的Km值为2.44 mg/ml;Cu2+、Ca2+、Mg2+、K+等金属离子对酶活力有明显促进作用,EDTA和PMSF对酶活力有强烈抑制作用;该酶对尿素、SDS、DTT等变性剂有较好的耐受性,其两个内部肽段经质谱测序,序列分别为AHGFLPLTK和APSATG-GSALYPLEFVVGK。结论该酶为一高温碱性丝氨酸蛋白酶,具有较高的工业开发价值和较大的应用潜力。

Separation, Purification and Property of Extracellular Protease Produced by Strain D2 from Digestive Tract of Perinereis aibuhitensis Grube

Objective To separate and purify extracellular protease from the culture supernatant of strain D2 isolated from the digestive tract of Perinereis aibuhitensis grube and study its property. Methods Collect the culture supernatant of strain D2 48 h after fermentation and purify extracelluar protease by 80% saturated ammonium sulfate precipitation,DEAE-Sepharose anion exchange chromatography and Superdex G-75 gel filtration chromatography.Study the enzymological property of purified protease and analyze the partial amino acid sequence of lysed protease by electrospray ionization tandom mass spectrum(ESI-MS/MS). Results Electrophoretically pure protease was obtained after purification.The purification factor,activity recovery,relative molecular mass and purity of protease were 4.9,33%,42 000 and more than 99% respectively.The optimal temperature and pH value for digestion with the purified protease were 60℃ and 9 respectively.The protease showed good stability at a pH value range of 6～11 and a temperature range of 0～60℃.The Km value of lysed protease was 2.44 mg/ml.The activity of protease was significantly promoted by metal ions such as copper,calcium,magnesium and potassium ions,but significantly inhibited by EDTA and PMSF.The protease showed good tolerance to denaturants such as urea,SDS and DTT.The amino acid sequences of two internal peptides of the protease were AHGFLPLTK and APSATGGSALYPLEFVVGK respectively. Conclusion A novel protease was successfully separated and purified from the culture supernatant of strain D2,which provided a reliable basis for further study on its potential industrial application.