乳酸克鲁维酵母β-半乳糖苷酶的分离纯化及性质研究  

Purification and Characterization of β-galactosidase from Kluyveromyces lactis

作　　者：沈为群[1] 郭杰炎[1] 李永福 陈惠萍 

机构地区：[1]复旦大学微生物学与微生物工程系,上海 200433 [2]新型发酵厂,上海 200437

出　　处：《生物工程学报》1993年第4期348-354,共7页Chinese Journal of Biotechnology

摘　　要：乳酸克鲁维酵母经高压破壁后的粗提液，其β－半乳糖苷酶比活力为５．５６ｕ／ｍｇ．经硫酸铵沉淀，丙酮沉淀，ＰＡＰＭＡ－Ａｅｐｈａｒｏｓｅ ４Ｂ柱层析后，乳糖酶比活力达３７０ｕ／ｍｇ，纯化了６６．２倍，ＳＤＳ－ＰＡＧＥ鉴定为一条带，分子量８５０００Ｄａ。酶作用的最适ｐＨ在６．４－６．８之间，最适温度４０℃，５０℃保温１５ｍｉｎ酶活丧失９０％，以邻硝基苯－β－半乳糖苷为底物的米氏常数为２．Crude extract containing β-galactosidase( E.C. 3.2.1.23 )specific activity of 5.56u/mg was obtained from K.lactis through high pressure homogenizer. β-galactosidase was purified 66.2 fold by (NH_4)_2SO_4 fractionation, acetone precipitation, affinity chromatography, hydroxylapatite chromatography and DEAE-Sephacel chromatography. The purified enzyme gives a specific activity of 370u/mg(using O-nitrophenyl-β-D-galactopyranoside as the substrate). When the purified enzyme was subjected to SDS-PAGE, one band with an apparent molecular weight of 85000Da was observed. The enzyme showed a pH optimum within pH6.4—6.8, and temperature optimum of 40℃. It lost 90% activity when incubated at 50℃ for 15min. The Km for ONPG is 2.78mmol/L. β-galactose, the natural product of this enzyme, has some inhibitive effect while ribose strongly inhibites the enzyme. Fe^(2+), Cu^(2+), Zn^(2+), Ag^+, PCMB and NBS also strongly inactivate the enzyme. The purified enzyme gives maximum activity at the presence of Mg^(2+), Mn^(2+) and 2-mercaptothanol.

关 键 词：乳酸 克鲁维酵母 提纯 半乳糖苷酶 

分 类 号：Q81[生物学—生物工程]