嗜酸乳杆菌细胞壁蛋白酶的分离纯化及酶学性质的研究  

Purification and Enzymatic Characterization of Cell-envelope Proteinase from Lactobacillus Acidophilus

作　　者：潘道东[1,2] 李敏[2] 

机构地区：[1]宁波大学海洋学院食品科学与工程系,浙江宁波315211 [2]南京师范大学国家乳品加工技术研发分中心,江苏南京210097

出　　处：《中国食品学报》2012年第4期48-56,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然基金项目(30972130);宁波市创新基金(2010C92024);浙江省自然基金项目(Z3110211)

摘　　要：为优化CEP的分离纯化方法，采用聚乙二醇（PEG）一20000浓缩粗酶液，用超滤离心管过滤酶液，40％-60％硫酸铵盐析分级沉淀，Sephacryl-S-300HR纯化，并通过Native-PAGE切胶回收得到纯酶。所得CEP的分子质量为28_3ku，比酶活力62．066U／mg，最适温度32℃，最适pH6．5，耐热性比较强，耐酸性明显好于耐碱性。Co^2＋、Mg^2＋、Ca^2＋对CEP有激活作用；Ba^2＋、Mn^2＋,K^＋、Na^＋对CEP的活力影响不大；Zn^2＋、Ni^2＋则表现出较强的抑制CEP活性作用：EDTA对CEP活性有抑制作用，表明CEP是一种金属离子激活酶；PMSF对CEP有显著抑制作用，表明CEP是一种丝氨酸蛋白酶。用嗜酸乳杆菌CEP水解乳清蛋白，其酶解液对ACE的抑制率为56.31％，表明CEP水解乳清蛋白可以产生ACE抑制肽。Cell-envelope Proteinase （CEP） purification methods were optimized, using PEG-20000 concentre the crude enzyme solution, and filter the enzyme solution using ultrafiltration tube. Collection the depositon in the ammonium sulfate fractionation between 40% to 60%, and purification on Sephacryl-S-300 HR chromatogram, obtained pure enzyme by recovery from Native-PAGE gel. The molecular weight and activity of CEP were 28.3 ku and 62.066 U/mg, respectively. Optimum temperature and pH of CEP are 32℃and 6.5, respectively. Heat resistance of CEP is strong, acid resistance is better than alkali resistance. The CEP can be activated by Co^2＋, Mg^2＋, Ca^2＋; Ba^2＋, Mn^2＋, K^＋, Na^＋ has little effect on the activity of CEP; Zn^2＋, Ni^2＋ showed a strong CEP activity inhibition; EDTA inhibited the CEP activity, indicating that CEP is a metal ion activation peptidase; PMSF showed significant inhibition of CEP, indicating that the CEP is a serine protease. Whey protein hydrolyzed with CEP of Lactobacillus acidophilus showed ACE inhibition, ACE inhibition rate was 56.31%, indicating that CEP of Lactobacillus acidophilus can hydrolyze whey protein and produce ACE inhibitory peptides.

关 键 词：嗜酸乳杆菌 细胞壁蛋白酶(CEP) 纯化 酶学性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]