嗜酸乳杆菌细胞壁蛋白酶的分离纯化及酶学性质的研究

为优化CEP的分离纯化方法,采用聚乙二醇(PEG)-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。Co2+、Mg2+、Ca2+对CEP有激活作用;Ba2+、Mn2+、K+、Na+对CEP的活力影响不大;Zn2+、Ni2+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。

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

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 Co2+,Mg2+,Ca2+;Ba2+,Mn2+,K+,Na+ has little effect on the activity of CEP;Zn2+,Ni2+ 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.