【摘要】 目的 基于响应面法对四氢嘧啶羟化酶（ectoine hydroxylase, EctD）催化反应体系进行优化，以四氢嘧啶（Ectoine, Ect）为底物，以温度、酸碱度和Ect、EctD浓度为影响因素，利用Box-Benhnken实验法研究各因素及其交互作用对EctD体外催化反应效率的影响，以期获得EctD体外催化合成5-羟基四氢嘧啶（5-Hydroxyectoine, 5-HE）的最优条件。方法 分别以Ect浓度、温度和酸碱度为自变量，在1 mM Fe²⁺、10 mM α-酮戊二酸（α-ketoglutarate）和10 mM TES缓冲液及EctD液组成的反应体系中反应25 min后，利用高效液相色谱仪（HPLC）检测5-HE的合成量，得到各单因素对酶催化反应的影响情况，并利用SPSS 22.0软件对数据进行统计分析；以单因素实验结果为基础，选择X1（温度）、X2（酸碱度）、X3（Ect浓度）、X4（EctD浓度）为影响因素，以5-HE合成量为响应值，设计4因素3水平响应面Box-Benhnken实验29组，每组实验重复3次，其数据以平均值±标准差表示，并用Design-Expert 12.0软件进行统计分析。结果 单因素实验结果显示，EctD在体外催化5-HE的合成量随着酸碱度和温度的增加呈现先升高后降低趋势，当酸碱度为7、温度为35℃时5-HE合成量达到最高值；而Ect浓度在1.25～1.5 mM时，5-HE的合成量无明显变化，说明此时底物Ect与酶催化中心的结合达到饱和。通过Box-Benhnken实验得到的回归方程及结果的方差分析表明，该模型的F为9.41,P<0.0001,失拟项P为0.0634,说明回归方程可信。回归方程一次项系数绝对值的大小依次为X4（EctD浓度）>X3（Ect浓度）>X1（温度）>X2（酸碱度）,说明X4（EctD浓度）是影响EctD催化合成5-HE的显著因素，而X2（酸碱度）对其影响较小。与此同时，X1（温度）与X4（EctD浓度）的交互作用对酶催化反应也具有显著影响。结论 EctD体外催化合成5-HE的最优反应条件：温度为35℃、pH为7、Ect浓度为1 mM、EctD浓度为0.6 U/mL、Fe²⁺浓度为1 mM、α-ketoglutarate浓度为10 mM。温度是影响酶催化的主要外界因素。更多还原

【Abstract】 Objective To optimize the catalytic reaction system of ectoine hydroxylase（EctD）based on response surface method.Taking Ectoine（Ect）as substrate, temperature, pH and concentrations of Ect and EctD as influencing factors, Box-Benhnken experiment was used to investigate the influence of various factors and their interactions on the catalytic reaction efficiency of EctD enzyme in vitro, so as to obtain the optimal conditions for the synthesis of 5-Hydroxyectoine（5-HE）catalyzed by EctD enzyme in vitro.Methods With Ect concentration, temperature and pH as independent variables, the reaction system including 1 mM Fe²⁺,10 mM α-ketoglutarate, 10 mM TES buffer and EctD enzyme solution was reacted for 25 min.The synthesis amount of 5-HE was detected by high performance liquid chromatography（HPLC）,and the influence of each single factor on the enzyme-catalyzed reaction was obtained.The software SPSS 22.0 was used for statistical analysis of the data.Based on the results of single factor experiments, X1（temperature）,X2（pH）,X3（Ect concentration）and X4（EctD concentration）were selected as influencing factors, and the synthesis amount of 5-HE was taken as response value.Twenty-nine groups of Box-Benhnken experiments with four factors and three levels of response surface were designed, and each group of experiments was repeated three times.The data were expressed as mean±standard deviation and were statistically analyzed by the software Design-Expert 12.0.Results The results of single factor experiment showed that the synthesis amount of 5-HE catalyzed by EctD in vitro increased first and then decreased with the increase of pH and temperature.The synthesis amount of 5-HE reached the highest value when pH was 7 and temperature was 35℃.The synthesis amount of 5-HE did not change significantly when the concentration of Ect was 1.25～1.5 mM,indicating that the combination of Ect and enzyme catalytic center was saturated.The regression equation obtained by Box-Benhnken experiment and the variance analysis of the results showed that the F of the model was 9.41,P<0.0001,and the disfitting term P is 0.0634,indicating that the regression equation was credible.The absolute values of the coefficients of the primary term of the regression equation were successively X4（EctD concentration）>X3（Ect concentration）>X1（temperature）>X2（pH）,indicating that X4（EctD concentration）was a significant factor affecting the synthesis of 5-HE by EctD,while X2（pH）has little influence on it.At the same time, the interaction between X1（temperature）and X4（EctD concentration）also has significant influence on the enzyme-catalyzed reaction.Conclusion The optimal reaction conditions for the synthesis of 5-HE by EctD in vitro are as follows: temperature 35℃,pH7,Ect 1 mM,EctD 0.6 U/mL,Fe²⁺ 1 mM and α-ketoglutarate 10 mM.Temperature is the main external factor affecting enzyme catalysis.更多还原