一种枯草芽孢杆菌亮氨酸氨肽酶的提取及部分酶学性质研究

Study on extraction process and partial characterization of the leucine aminopeptidase of Bacillus subtilis

采用(NH4)2SO4盐析法、有机溶剂沉淀法以及超滤浓缩等操作提取亮氨酸氨肽酶,初步建立了一种易于规模化提取枯草芽孢杆菌亮氨酸氨肽酶的工艺。发酵液采用质量分数为15%的(NH4)2SO4澄清,再经30kDa的超滤膜浓缩2.5倍时,纯化倍数为6.1,酶的回收率为87.7%。超滤后的保留液再经质量分数为25%的(NH4)2SO4盐析,可沉淀出保留液中90%的酶。按此工艺超滤浓缩后的液态酶稳定性较好,85d仍能保留80%以上的酶活。Co2+对此亮氨酸氨肽酶有较强的激活作用,Zn2+、Fe2+、Mn2+和Ca2+等几种金属离子对酶有不同程度的抑制作用。

Some measures such as salting-out method with ammonium sulphate,organic solvent percipiitation and concentration with ultrafiltration were taken to extract the leucine aminopeptidase from Bacillus subtilis, finally a extraction process abled to enlarge was established.Preliminary extraction procedures ：the fermentation broth was clarified by using 15% ammonium sulphate, followed with 30kDa-ultrafiltration membrane treatment, so that the enzyme was concentrated by 2.5 fold, with 6.1- fold purification and recovery 87.7%, respectively, 90% of the enzyme in the retention fluid was precipitated by 25% ammonium sulphate.The minopeptidase obtained kept 80% activity after 85d and remained stable. The influence of some metal ions was discussed. Co2＋ had obvious activation, meanwhile,Zn2 ＋ , Fe2 ＋ , Mn2 ＋ and Ca2 ＋ inhibited aminopeptidase activity.