丙氨酸消旋酶C-端结构域重组体的构建及其功能初探

Study on the construction and function of C-terminal domain recombinant of alanine racemase

【目的】将嗜碱芽孢杆菌丙氨酸消旋酶OF4DadX的N-端结构域分别与多个不同种属的丙氨酸消旋酶C-端结构域重组,探究丙氨酸消旋酶C-端结构域功能。【方法】利用基因拼接构建丙氨酸消旋酶重组基因,通过镍亲和层析纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测重组酶蛋白的酶学特性,借助分子筛和HPLC液相色谱分析其聚合状态及动力学参数。【结果】通过基因拼接构建了12个重组基因,经检测,表达、纯化获得的重组酶蛋白中只有OF4TtDadX240c具有催化活性,其活性仅为OF4DadX的60.54%,酶催化动力学结果显示OF4TtDadX240c催化反应速率Vmax/Km下降约10倍,但其稳定性大幅提高,半衰期比OF4DadX延长约5倍,耐热性提高较明显;聚合状态表明OF4DadX、OF4TMDadX226c和OF4TtDadX240c为二聚体结构,其他酶蛋白均为单体,但OF4TMDadX226c未检测到活性,推测可能是酶催化活性中心移位,未能形成质子转移而失去活性。【结论】丙氨酸消旋酶C-端折叠结构域对消旋酶低聚化、稳定性和酶催化功能具有重要作用。

[Objective]The N-terminal domain of alanine racemase from Bacillus pseudofirmus OF4 was recombined with the alanine racemase C-terminal domains of many different species to explore the function of C-terminal domain.[Methods]The recombinant genes of alanine racemase were constructed by the gene splicing and expressed in E.coli BL21(DE3).The recombinant proteins were purified by affinity chromatography.D-amino acid oxidase coupling method was used to detect the enzymatic properties of the proteins,while the polymerization states and kinetic parameters of recombinant enzymes were analyzed by the molecular sieve and high-performance liquid chromatography(HPLC).[Results]The recombinant proteins were expressed and purified successfully.OF4TtDadX240c had a catalytic activity which was 60.54%of OF4DadX,whereas other recombinant enzymes lost their activities.The catalytic kinetics showed that the catalytic rate(Vmax/Km)of OF4TtDadX240c decreased about 10-fold,but its stability improved significantly.The half-life of OF4TtDadX240c had about a 5-fold prolongation than OF4DadX,and a significantly improved heat resistance.The results of the molecular sieve showed that OF4DadX,OF4TMDadX226c and OF4TtDadX240c were dimers and other proteins were monomers.However,OF4TMDadX226c lost its activity which could be attributed to the shift of the catalytic active center that failed to form the proton transfer.[Conclusion]The C-terminal folding domain of alanine racemase plays an important role in racemase dimerization,stability and catalytic function.