柠檬酸钠对脱氮假单胞杆菌发酵产维生素B12代谢过程的影响

摘要

以脱氮假单胞杆菌为生产菌,研究了柠檬酸钠对其发酵过程中产维生素B12的代谢调控作用.通过摇瓶培养的方式,在基础培养基中添加不同质量浓度的柠檬酸钠,利用分光光度法和HPLC法分别测定了关键酶活和有机酸等含量.结果表明,添加质量浓度为0.3%的柠檬酸钠最有利于维生素 B12的发酵,其产量高达45.73 μg/mL.在此基础上,结合有机酸和酶学方法,进一步研究发现,外源添加柠檬酸钠能显著降低菌体柠檬酸和琥珀酸的积累,并且加速苹果酸的消耗,降低EMP 途径中丙酮酸激酶和磷酸果糖激酶的活力,提高HMP途径中葡萄糖-6-磷酸脱氢酶的活力,从而避免发酵液pH急剧下降,减少EMP途径的通量,增加HMP途径的通量,为维生素B12的合成提供充足的还原力NADPH,提高维生素B12发酵产量.%In this study,the metabolism of vitamin B12in the process of fermentation was studied by using Pseudomonas aeruginosa as the producing strain.By shake flask method,sodium citrate in the basic culture me-dium supplemented with different concentrations,the key enzyme activity and organic acid content were deter-mined by spectrophotometry and HPLC respectively.The result showed that 0.3% sodium citrate was more fa-vorable for vitamin B12fermentation,and its yield was as high as 45.73 μg/mL.Based on this fact,combining with organic acids and enzyme methods,further research found that sodium citrate could significantly reduce the citric acid and succinic acid accumulation,accelerate the consumption of malic acid,reduce the EMP pathway of pyruvate kinase and phosphofructokinase activity,and increase the activity of glucose-6-phosphate dehydrogenase in HMP pathway.In order to avoid the rapid decline of pH,the flux of EMP pathway was reduced and the flux of HMP pathway was increased,which provided sufficient reducing power for vitamin B12synthesis.Thus,the fermentation yield of vitamin B12could be improved.