嗜热菌Bacillus fordii3-2海因酶的纯化及性质  

Purification and characterization of hydantoinase from thermophilus Bacillus fordii 3-2

作　　者：孙倩倩[1] 潘瑶[1] 姚忠[1] 何冰芳[1] 

机构地区：[1]南京工业大学制药与生命科学学院,南京210009

出　　处：《生物加工过程》2007年第4期32-36,共5页Chinese Journal of Bioprocess Engineering

基　　金：国家973资助项目(2003CB716004);国家自然科学基金资助项目(20336010)

摘　　要：对自行筛选的海因酶法L-苯丙氨酸生产菌株Bacillus fordii3-2中的海因酶进行了分离纯化及相关性质研究。该海因酶协同L-氨甲酰水解酶是海因酶法生产L-氨基酸的关键酶。且fordii3-2菌悬液经压力破碎离心后取上清液为粗酶液，粗酶液通过硫酸铵分级沉淀、Phenyl FF（high sub）疏水层析以及Source 15Q离子交换层析，经SDS—PAGE分析达到电泳纯。亚基相对分子质量为55×10^3，海因酶的纯化回收率为20．5％，纯化倍数为149．23。该海因酶在pH8．0～10．0的范围内具有较高的活性，在45—70℃具有很高的酶活力，最适反应pH和温度分别为10．0℃和65℃。纯酶易氧化失活，DTT对该酶有一定的保护作用。二价金属离子，Mn^2＋、Co^2＋及Fe^2＋对酶活性有显著的促进作用，而Ca^2＋、Cu^2＋等对酶活性有抑制作用。相关研究可为该菌株及海因酶的进一步开发与应用提供理论指导。The purification and properties of hydantoinase from L-phenylalanine producer Bacillus fordii 3- 2 of hydantoinase process were studied. Hydantoinase and L-carbamoylase are both key biocatalysts in the production of the optically pure L-amino acids. Hydantoinase from Bacillus fordii 3-2 was purified to homogeneity by ammonium sulfate fractionation, Phenyl FF （high sub） hydrophobic interaction chromatogra-phy and Source 15Q ion exchange chromatography with a yield of 20. 5% and 149. 23-fold purification. The high activities of hydantoinase were showed in the range of pH 8.0 - 10.0 and at the temperature of 45 -70℃. The optimal reaction temperature and pH were 65℃ and 10.0, respectively. The supplement of DTT was helpful to the stabilization of pure hydantoinase. Hydantoinase activity was significantly enhanced by Mn^2＋ , Co^2＋ and Fe^2＋ ,and inhibited by Ca^2＋ and Cu^2＋. This study will provide theory basis for the development of the hydantoinase of B.fordii 3-2.

关 键 词：海因酶 Bacillu sfordii 3-2 纯化 酶学性质 

分 类 号：Q55[生物学—生物化学]