Optimization Of Inosine Fermentation Process And Analysis Of Metabolic Mechanism By Bacillus Subtilis

As a typical primary metabolic,inosine is widely used in pharmaceutical,medical and food industries.For the past few years,domestic inosine fermentation industry was facing severe challenges with impact of foreign manufacturers.In order to increase its inosine production,the technology of shaking-flask fermentation was optimized by response surface methodology addly the best compound plan of adding accelerators was obtained by uniform design to increase inosine yield and reduce production cost.Finally,the whole metabolic flux distribution of inosine fermentation was analyzed to explain the metabolism.?1?Optimizing the medium and conditions for fermentation culture of inosine by the response surface analysis.The important parameters were screened by Plackett-Burman design from 9 factors among inosine shake flask fermentation process.The results showed that the yield of inosine could be significantly affected by concentration of glucose,concentration of coin liquid,liquid medium volume or inoculation amount.Then a steepest ascent experiment was used to approach to best region of variables,and the optimum values of parameters were obtained by Box-Behnken design.Expectedly,the inosine reached 8.26 g/L which increased49.9%than that under the initial conditions.?2?According to the regulating mechanism of inosine synthesis,some kinds of effective additives were added in two inosine-producing strains respectively.Then,the best compound plan of adding accelerators was obtained by uniform design based on the result of adding single accelerator test.The optimized combination increased inosine production by B.subtilis CICC20958 by 638%to 7.81 g/L when added by sodium citrate 7.9 g/L,NaF 0.001 g/L,CaCl₂0.032 g/L,hypoxanthine 2.1 g/L and MnSO₄ 0.2 g/L and B.subtilis JMUKC2 by 252%to 13.94g/L when added by sodium citrate 5.2 g/L,NaF 0.001 g/L,calcium gluconate 0.032 g/L,hypoxanthine 4.5 g/L and MnSO₄ 0.19 g/L.?3?The metabolic flux distribution of inosine fermentation was studied by mean of metabolic flux analysis.It revealed that the metabolic flux in the pathway of glucose catabolism and inosine synthesis were enhanced in three inosine-producing strains,which were screened from B.subtilis CICC20958 by the method of ultraviolet mutagenesis compared with B.subtilis CICC20958.The gene expression quantity of five key enzymes in inosine metabolic pathway was analyzed to the relationship between inosine production,expression quantity of key enzyme and result of metabolic flux.The results showed that the metabolic flux of the metabolic pathway which was catalyzed by phosphofructokinase which has a positive correlation with the gene expression quantity of phosphofructokinase.Meanwhile that of glucose-6-phosphate dehydrogenase has the same result.