发酵乳杆菌细胞壁蛋白酶的分离纯化及酶学性质研究  

Purification and Enzymatic Properties of Cell Envelope Protease from Lactobacillus fermentum

作　　者：朱姁[1] 潘道东[1,2] 

机构地区：[1]南京师范大学国家乳品加工技术研发分中心,江苏南京210097 [2]宁波大学生命科学与生物工程学院,浙江宁波315211

出　　处：《食品科学》2011年第17期262-268,共7页Food Science

基　　金：国家自然科学基金项目(30972130);江苏省科技支撑计划项目(BE2009366);浙江省重大科技专项(2010C12015)

摘　　要：研究分离纯化发酵乳杆菌细胞壁蛋白酶(cell envelope proteinase,CEP)的方法及其酶学性质。用50mmol/LTris-HCl缓冲液(pH7.8)悬浮菌体,进行超声波破碎,细胞质量浓度为0.06g/mL,破碎功率330W,工作220次(工作时间3s,间隔时间5s),离心取上清液即为粗酶液。60%硫酸铵沉淀,Sephacryl S-300 HR凝胶层析,Native-PAGE割胶回收纯化发酵乳杆菌的CEP。用纯化的CEP酶解脱脂乳,酶解液ACE(angiotensin I-converting enzyme)抑制率为50%。SDS-PAGE检测CEP分子质量为32.5kD,最适酶反应温度为41℃,最适酶反应pH值为8.0。Mg2+、Co2+、Ca2+对CEP活性有激活作用;Zn2+、Ni2+、PMSF、EDTA对CEP活性有抑制作用,说明CEP为丝氨酸蛋白酶且酶的活性中心结构的维持与金属离子有关。The purification process of cell envelope protease（CEP） from Lactobacillus fermentum was explored in this paper.Cells were suspended in 50 mmol/L Tris-HCl（pH 7.8） and subjected to ultrasonication（cell concentration of 0.06 g/mL,ultrasonic power of 330 W,ultrasonic treatment time of 3 s with intermission of 5 s,and repeated ultrasonic treatment number of 220）.The supernatant was collected,precipitated with 60% saturated（NH4）2SO4 solution and separated by Sephacryl S-300 HR chromatography.The active protease was recovered from Native-PAGE gel.The ACE inhibitory rate of purified CEP was 50%.The molecular mass of purified CEP estimated by SDS-PAGE was approximately 32.5 kD.Maximum activity was reached at pH 8.0 and 41℃.The activity of CEP could be activated by Mg2＋,Co2＋ and Ca2＋ and inhibited by Zn2＋,Ni2＋ and PMSF,suggesting that CEP belongs to serine protease.On the other hand,its activity could also be inhibited by EDTA,suggesting that CEP is a metallopeptidase.

关 键 词：发酵乳杆菌 细胞壁蛋白酶 纯化 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]