根霉α-半乳糖苷酶的三相分离及其酶学性质  

Three-phase partitioning and characterization of α-galactosidase from Rhizopus sp.A01

作　　者：王剑锋[1] 王璋[2] 李江[1] 饶军[1] 

机构地区：[1]东华理工大学生物系,江西抚州344000 [2]中国食品发酵工业研究院,北京100027

出　　处：《菌物学报》2012年第2期251-257,共7页Mycosystema

摘　　要：根霉Rhizopus sp.A01发酵豆渣产α-半乳糖苷酶,粗酶液依次经过三相分离、Sephadex G-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了6.7倍,总酶活回收率达到46%;凝胶过滤和SDS-PAGE显示该酶为相对分子质量为87.6kDa的单体蛋白。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为5.0,最适温度为55℃,表观Km、kcat/Km分别为2.56mmol/L、47,400L/mol·s;能微弱水解蜜二糖和棉子糖,水解蜜二糖的速率是水解棉子糖速率的3.4倍;水解活性受多种金属离子的明显抑制,其中Ca2+、Cu2+、Fe3+、Hg+、Mn2+等几乎完全抑制酶活性。该酶活性在pH4.5-9.0保持稳定,在55℃时保温120min,酶活残留48%。Using three-phase partitioning flowed by Sephadex G-100 chromatography,a form of α-galactosidase from Rhizopus sp.A01 grown on bean dregs broth was purified to homogeneity with a 6.7-fold increase in specific activity and 46% recovery.The enzyme was showed a monomer with apparent molecular mass of 87.6kDa by SDS-polyacrylamide gel electrophoresis and gel filtration.The α-galactosidase had high activity against p-nitrophenyl-α-d-galactopyranoside（pNPGal） but had slight activity for melibiose and raffinose,and the optimal activity was observed at pH 5.0 and 55℃.The kinetic parameters Km and kcat/Km were 2.56mmol/L and 4.74×104L/mol？s with pNPGal,and the rate of hydrolysise for melibiose was 3.4 times as much as that for raffinose.The enzyme activity was inhibited by all tested metal ions at 5.0mmol/L,and almost completely by Ca2＋,Cu2＋,Fe3＋,Hg＋ and Mn2＋ among them.The α-galactosidase was highly stable over pH range of 4.5-9.0 at 25℃,and its activity retained approximately 48% of the original activity after incubation for 120min at 55℃.

关 键 词：棉子糖 蜜二糖 对硝基苯-α-D-吡喃半乳糖苷 三相分离 

分 类 号：Q814[生物学—生物工程]