高活性Co^2＋-替代海因酶中Co^2＋的定量及酶学活性鉴定  

Characterization of a Co^(2+)-substituted D-Hydantoinase with Higher Enzymatic Activity

作　　者：张学尧[1] 陈云霞[2] 史鹏[1] 梁爱华[1] 袁静明[1] 

机构地区：[1]教育部化学生物学与分子工程重点实验室,生物技术研究所,山西大学,太原030006 [2]山西省长治医学院,长治046001

出　　处：《中国生物化学与分子生物学报》2008年第5期469-474,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：山西省自然科学基金资助项目(No.20031042)~~

摘　　要：利用产D-海因酶(HDT)的重组pE-hdt/E.coli菌株,在LB培养基中添加40μmol/L的Co2+,37℃培养10h,表达产物经Q-Sepharose阴离子交换剂和Phenyl-Sepharose疏水层析,获得电泳纯Co2+-D-海因酶(Co2+-HDT).该酶对底物DL-海因的比活性较HDT高约6倍,达21.8U/mg.可见光光谱分析表明,在498nm和568nm处呈现Co2+-海因酶络合物的特征性吸收峰.用ICP-AES测定纯酶金属离子含量,HDT每摩尔亚基含0.93摩尔Zn2+和0.04摩尔Co2+,而Co2+-HDT中每摩尔亚基中含0.17摩尔Zn2+和0.89摩尔Co2+.这一结果表明,HDT中的Zn2+已被Co2+所替代.此外,在动力学常数,pH和温度稳定性,金属螯合剂EDTA的影响等方面,HDT和Co2+-HDT也略有差异.A Co^2＋-substituted D-hydantoinase （Co^2＋ -HDT） was produced by a hydantoinase-expressing recombinant pE-hdt/E, coli under the culture with the presence of 40 μmol/L CoCl2 at 37℃ for 10 hours. The Co^2＋ -HDT was then extracted from the cells and rapidly prepared by a two-step chromatography, Q-Sepharose and Phenyl-Sepharose, then eluted with the buffer containing 40 μmol/L CoCl2 . Two peaks at 498 nm and 568 nm in the visible spectra were unique for the complex of Co^2＋ - HDT. ICP-AES analysis showed that the Co^2＋ - HDT contained 0.17 Zn^2＋ and 0.89 Co^2＋ per molar subunit,while the wildtype HDT were 0.93 Zn^2＋ and 0.04 Co^2＋ , implying that Zn^2＋ in HDT had been substituted with Co^2＋ . The specific activity of the Co^2＋ - HDT reaches 21.8 U/mg, which is six-fold higher than that of HDT. In addition, other notable differences were observed between Co^2＋ -HDT and HDT, such as optimal pH, thermal stability, responses to metal chelators like EDTA, and certain kinetic parameters.

关 键 词：D-海因酶 Co^2+-海因酶 金属离子含量 酶活性鉴定 

分 类 号：Q814[生物学—生物工程] Q819