乳酸乳球菌细胞壁蛋白酶的分离纯化  

Isolation and Purification of Cell-envelope Proteinase from Lactococcus lactis subsp. lactis

作　　者：郭宇星[1] 潘道东[1] 

机构地区：[1]南京师范大学乳品生物技术研究所,江苏南京210097

出　　处：《食品科学》2008年第10期388-391,共4页Food Science

基　　金：国家自然科学基金项目(30571311)

摘　　要：本研究确定了分离纯化乳酸乳球菌细胞壁蛋白酶(CEP)的最佳技术路线。用裂解液(50mmol/LTris-HCl,2mmol/LEDTA-Na2,100mmol/LNaCl,0.5%Tritonx-100,1mg/ml溶菌酶,pH8.5)悬浮菌体(20ml/g),37℃下保温3h,离心后取上清液即为粗酶液。粗酶液通过45%硫酸铵沉淀,DEAE-SephadexA-25和Sephacryl-S-300HR两步层析,可以得到纯化的细胞壁蛋白酶。蛋白酶提纯倍数达到74.048%,最后回收率为14.865%,PAGE电泳检测为一条带,SDS-PAGE检测蛋白酶为单体结构,分子量大约为53kD。用纯化后CEP酶解乳清蛋白,酶解液ACE抑制率为45%。In this study, the isolation and purification process of cell-envelope proteinase （CEP） from Lactococcus lactis subsp. lactis is determined as follows： suspending the washed Lactococcus lactis subsp, lactis cells by 50 mmol/L Tris-HCl （pH7.8） in lysis buffer solution （50 mmol/L Tris-HC1, 2 mmol/L EDTA, 100 mmol/L NaCl, 0.5% Tritonx-100 and 1 mg/ml lysozyme, pH 8.5 ） at the ratio of 1：20 （g/ml） and then incubating the suspension for 3 h at 37℃, centrifugating the suspension and then collecting the supernatant （crude cell-envelope proteinase solution）, fractionally precipitating the cell wall proteinase by adding 25%, 45% and 65% ammonium sulfate into the supernatant, and purifying the crude proteinase by DEAE-Sephadex A- 25 and Sephacryl-S-300 HR column chromatography successively. The enzyme is purified by about 74 folds from the crude cell-envelope proteinase solution and the the recovery of activity is about 14.87%. The purified enzyme showes a single protein band on native PAGE pattern, and it has a monomer structure and the molecular mass of about 53 kD by SAS-PAGE. The angiotensi converting enzyme （ACE） inhibitory rate of whey protein hydrolyzate by the purified enzyme is 45%.

关 键 词：ACE抑制活性 乳酸乳球菌 细胞壁蛋白酶 

分 类 号：TS201.3[轻工技术与工程—食品科学]