克雷伯杆菌甘油脱氢酶的分离纯化及性质  

Purification and Characterization of Glycerol Dehydrogenase from Klebsiella pneumoniae

作　　者：陈宏文[1] 吴雅红[2] 吴振华[2] 方柏山[2] 胡宗定[1] 

机构地区：[1]天津大学化工学院 [2]华侨大学生物工程与技术系福建泉州362011

出　　处：《无锡轻工大学学报（食品与生物技术）》2005年第1期1-5,37,共6页Journal of Wuxi University of Light Industry

基　　金：国家自然科学基金项目(20276026);福建省自然科学重点基金项目(D0120002);福建省科学技术重点基金项目(20031020)资助课题.

摘　　要：在有氧条件下,利用Q Sepharose Fast Flow离子交换层析和Blue Sepharose CL 6B亲和层析提纯克雷伯杆菌胞内甘油脱氢酶.酶的纯化倍数和回收率分别为 32.61 倍和 5.83%.通过SDS PAGE电泳测得该酶亚基的相对分子质量约为 34 000.该酶最适表观反应温度和最适反应pH值分别为60 ℃和11.在30 ℃以下及pH值10~12时,该酶具有良好的稳定性.在45 ℃和pH值11条件下,该酶以甘油和NAD+为底物的米氏常数Km分别为 0.75 mmol/L和 0.12 mmol/L.甘油脱氢酶对甘油的生理反应活性最大,对其它醇类如 1,2 丙二醇、乙二醇也有氧化能力.NH4+和Na+对酶有显著激活作用.巯基保护剂可明显地提高酶的活力.Glycerol dehydrogenase from Klebsiella pneumoniae was purified to homogeneity by Q Sepharose Fast Flow ion-exchange chromatography and Blue Sepharose CL-6B affinity chromatography under aerobic conditions. A 32.61 fold purification was obtained with the recovery of 5.83% activity. The subunit molecular weight of enzyme was 34 000. The optimum temperature and pH of the enzyme activity were 60 ℃ and pH 11. Below 30 ℃ and at range of pH 10~12, glycerol dehydrogenase was stable. At 45 ℃ and pH 11, the K_(m) for glycerol and NAD^(+) were 0.75 mmol/L and 0.12 mmol/L, respectively. The enzyme oxidized other alcohols such as 1,2-propanediol and ethylene glycol, besides the physiological substrate glycerol. The enzyme was significantly activated by NH_(4)^+、Na^(+)、K^(+) and Mg^(2+). Reducing agents enhanced glycerol dehydrogenase

关 键 词：甘油脱氢酶 克雷伯杆菌 纯化 

分 类 号：Q936[生物学—微生物学]