GL-7-ACA酰化酶的分离纯化及性质研究  

STUDIES ON PURIFICATION AND PROPERTIES OF GL-7-ACA ACYLASE FROM CU334

作　　者：周宏伟 魏中荻 杨蕴刘[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理研究所,上海200032

出　　处：《微生物学报》1997年第3期196-202,共7页Acta Microbiologica Sinica

摘　　要：CU334是高表达GL-7-ACA酰化酶工程菌,其菌悬液用超声波处理后,经硫酸铵分级沉淀、DEAE-Sephadex A-50离子交换柱层析、DEAE—纤维素DE-52柱层析、Sephadex G-200凝胶过滤及羟基磷灰石吸附柱层析等步骤,得到了凝胶电泳均一的GL-7-ACA酰化酶蛋白,纯化了22倍,得率4.0％,比活力为13.8U/mg。用浓度梯度PAGE测得GL-7-ACA酰化酶的分子量为134kD,用SDS-PAGE测得两个亚基分子量分别为15.5kD和58.4kD。用PI法测得等电点为3.5。GL-7-ACA酰化酶反应最适pH为7.0。反应最适温度为37℃,GL-7-ACA酰化酶对底物GL-7-ACA的K_m值为0.50mmol/L,V_(max)为13.10U·mg^(-1)。Ca^(2+)、EDTA和巯基乙醇对该酶有激活作用,Cu^(2+)、Fe^(2+)和Mg^(2+)等有一定程度的抑制作用。产物7-ACA、戊二酸均为GL-7-ACA酰化酶的反竞争性抑制剂,其K_1值分别为16.58mmol·L^(-1)和9.88mmol·L^(-1)。7β (4-Carboxybutanamido) cephalosporanic acid acylase (GL-7-ACA acylase) was purified from cell-free extracts of a recombinant, CU334 by a procedure invoiving ammonium suifate fractionation and successive column chromatography on DEAE-Sephadex A-50, DEAE-cellulose DE-52, Sephadex G-200 and Hydroxylapatide with 22-fold purification, 4.0% recovery and 13.8 U /mg specific activity. The purified enzyme was homogeneous on polyacrylamide gel disc clectrophoresis. The Mr estimated by concentration gradient - PAGE was 134000 and subunlt Mr determined by SDS-PAGE were 15400 and 58500. The isoelectric point was estimated to be 3.5 by PAG-IEF. The optimum pH was 7.0 for GL-7-ACA acylase. The optimum temperature was about 37℃ . The Michaelis-Menten constant (Km) and Vmax for GL-7-ACA were 0.46mmol /L and 0.51μmol · min-1· mg-1, respectively. The enzyme was found to act on substrates within narrow range. Ca2+, EDTA and β - Mercaptoethanol enhanced the enzyme activity, Mg2+. Mn2+, and Ba2+ inhibited the enzyme activity. The Ki for the reaction products. 7-ACA and glutaric acid were 16-58 mmol /L and 9.88mmol /L, respectively, as determined by Lineweaver-Burk plot and Dixon plot.

关 键 词：酰化酶 GL-7-ACA酰化酶 纯化 性质 

分 类 号：Q555.303[生物学—生物化学]