米根霉As3.2686丙酮酸分支点代谢通量分析及相关酶活性研究

摘要

The metabolic flux equations of Rhizopus oryzae As3.2686 were established and the metabolic fluxes of different ventilations in the steady-state in a fermentor were calculated by metabolic flux analysis (MFA) method. The changes of the enzyme activities at pyruvate branch point including PDC (pyruvate decarboxylase), PC (pyruvate carboxylase), PDH (pyruvate dehydrogenase), and LDH (lactate dehydrogenase) were analyzed by enzyme-linked immunosorbent assay (ELISA) and spectrophotometry. These results illustrated that the flux of fumarate and ethanol were relatively higher at the ventilations of 0.5 and 1.0 L⋅(L⋅min)-1. At the ventilations of 1.5 and 2.0 L⋅(L⋅min)-1, over half of flux at pyruvate branch point flowed to L-lactate. When the ventilation was 2.5 L⋅(L⋅min)-1, more metabolic flux in the steady-state flowed to TCA cycle. According to the comparison of the activities of the same enzyme at different ventilations, the enzyme activities of PDC, PC, and PDH increased at beginning then declined with increasing ventilations, and inconsistent with ethanol, oxaloacetate and acetyl coenzyme fluxes. The activities of LDH and ADH increased gradually with the raise of ventilation, but ADH enzyme activity inconsistent with ethanol flux. More flux flowed to L-lactic acid at 2.0 L⋅(L⋅min)-1 than 1.5 L⋅(L⋅min)-1, but less flux of L-lactic acid outside the cells were obtained. Conclusion: the function of single enzyme to regulate the pyruvate flux is limited, several enzymes around the pyruvate branch point corporately control the metabolic flux, there are other enzymes affecting pyruvate to transform to L-lactic acid.%利用代谢通量分析(MFA)研究建立米根霉As3.2686的代谢通量方程，计算发酵罐不同通气条件下丙酮酸分支点的稳态代谢通量，并分别使用酶联免疫吸附法(ELISA)和分光光度法测定丙酮酸分支酶 PDC(丙酮酸脱羧酶)、PC(丙酮酸羧化酶)、PDH(丙酮酸脱氢酶)和LDH(乳酸脱氢酶)活性变化情况。结果表明，通气量为0.5及1.0 L⋅(L⋅min)-1时苹果酸、富马酸和乙醇的通量相对较高；通气量为1.5和2.0 L⋅(L⋅min)-1时，丙酮酸分支点超过一半的流量流向L-乳酸；通气量达到2.5 L⋅(L⋅min)-1时，丙酮酸分支点流向TCA循环的稳态通量增多。比较不同通气条件下同一酶活性变化， PDC、PC和PDH的酶活性总体上都随通气量的增大先增加后减小，并且与乙醇、草酰乙酸和乙酰辅酶A的通量变化不一致；LDH 和 ADH 变化随通气量的增加而逐渐增大，但 ADH 酶活性也与乙醇通量变化不一致。通气量为2.0 L⋅(L⋅min)-1比1.5 L⋅(L⋅min)-1时丙酮酸分支点流向L-乳酸的通量多，但得到的胞外L-乳酸产量却较少。结果说明米根霉产L-乳酸丙酮酸分支点处单个酶的作用是有限的，多个酶都对物流起控制作用，存在其它因素间接影响丙酮酸转化L-乳酸。