灵菌红素缩合酶PigC在大肠杆菌中表达条件优化

Optimization of Inducing Conditions for the Production of Prodigiosin Synthetase PigC from Recombinant Escherichia coli

灵菌红素及其类似物具有良好的抗菌性能,在新型抗菌药物开发领域具有广阔的应用前景。灵菌红素缩合酶PigC是酶法合成灵菌红素及其类似物的关键酶,为了提高重组大肠杆菌Escherichia coli BL21（DE3）/pET-32a-PigC中pigC基因的表达水平,本研究对其诱导条件进行优化。在单因素实验结果的基础上,依据Box-Behnken中心组合原则设计培养基初始pH、乳糖浓度和诱导温度的3因素3水平响应面实验,以Pig C酶活为响应值优化重组菌的诱导条件。结果表明,在培养基初始pH7.1,乳糖浓度4.2 g/L,诱导温度为16.2℃的最佳条件下,该重组菌所产PigC的最高酶活达62.4 U/mL,是优化前的4.9倍,实际值与响应面预测值拟合良好,说明通过响应面试验设计对该重组菌诱导条件的优化是有效的。本研究为PigC的制备及其酶学特性研究提供了基础资料,为酶法合成灵菌红素及其类似物提供了一定的基础。

Prodigiosin and its analogues have broad application prospects in the development of new antimicro- bial agents due to their excellent antibacterial activities. Prodigiosin synthetase PigC is the key enzyme in the prodigiosin biosynthesis. In order to enhance the production of PigC in Escherichia coli BL21 （DE3）/pET-32a- PigC, the inducing conditions were optimized in this study. On the basis of single factor tests, Box-Behnken response surface methodology was employed to optimize 3 key factors including the initial pH of medium, the con- centration of lactose, and the induced temperature. The highest activity of PigC reached 62.4 U/mL when the initial pH of medium was 7. 1, the concentration of lactose was 4.2 g/L, and the induced temperature was 16.2℃, which was 4.9 times of the production before optimization. The experimental value was agree with the predicted one, which suggested that the prediction model is reliable and suitable for the optimization of PigC inducing conditions. This study can provide the basis for the PigC manufacture and its enzymatic properties research, it also provide a certain basis for the enzymatic synthesis ofprodigiosin and its analogues.