假单胞杆菌D-海因酶的纯化及酶学性质  

PURIFICATION AND SOME PROPERTIES OF D-HYDANTOINASE PRODUCED BY PSEUDOMONAS 2262

作　　者：石亚伟[1] 李汉卿[1] 袁静明[1] 齐延红[2] 李晋川[2] 

机构地区：[1]山西大学生物工程中心,太原030006 [2]山西省生物研究所,太原030006

出　　处：《微生物学报》2001年第5期605-610,共6页Acta Microbiologica Sinica

基　　金：山西省重点行业科技发展项目 ( 9832 2 5)

摘　　要：D 海因酶是工业上生产D 型氨基酸的关键酶 ,用热变性 ,硫酸铵沉淀及SepharoseQfastflow ,Phenyl Sepharosefastflow ,Superose 1 2等柱层析步骤从Pseudomonas 2 2 62菌体中分离纯化了该酶 ,纯化倍数约为 60 ,活力回收约为 1 6%。该酶为同源二聚体 ,分子量约为 1 0 9kD ,亚基分子量约为 53 7kD ,反应最适pH为 8 0 ,最适温度为 70℃ ,在pH6.0～ 1 0 0和温度 60℃以下稳定 ,该酶对巯基试剂敏感 ,大多数二价金属离子如镁、锰离子等能促使酶活提高 ,但高浓度锌离子能抑制酶活 ,以二氢尿嘧啶为底物的米氏常数Km =2 .5× 1 0 - 2 mol L。该酶的N末端1A D\|hydantoinase produced by Pseudomonas 2262 was purified to electrophoretic homogeneity by the steps of thermal treatment, (NH\-4)\-2SO\-4 fractionation and column chromatography with Q\|Sepharose fast flow, phenyl\|Sepharose fast flow and Superose 12. Purification of about 60 fold was achieved with an overall yield of 16%. The relative molecular mass of the native enzyme is 109 kD and that of subunit is 53.7 kD by the analysis of Native and SDS\|PAGE as well as gel filtration respectively. Some properties of the enzyme such as the sensitivity to thiol reagent and the effects of metal ions, for instance inhibited by Zn\+\{2+\} and activited by Mn\+\{2+\}, Mg\+\{2+\} are identical to dihydropyrimidinase. The optimum temperature and pH for enzymatic catalysis are 70 ℃ and 8.0 respectively. The enzyme activity is stable under 60 ℃ and in the pH range of 6～10. The N\|terminal sequence for 10 amino acid residues is MDKLIKNGTI.

关 键 词：假单胞杆菌2262 D-海因酶 纯化 性质 

分 类 号：Q55[生物学—生物化学]