| Inosine is an important flavor enhancer in the food industry, a precursor for antiviral drug of curing coronary heart disease, hepatitis and leucopenia, and the raw material of antibiotic production in the medicine field. However, with the impact of foreign manufactures on our market, the domestic nucleoside fermentation industry faces a severe challenge in recent years. So, how to enhance inosine productive yield has become a top priority to reduce the cost of commercial products.An inosine-producing strain Bacillus subtilis JMUKC2was screened from Bacillus subtilis CICC20958by the method of ultraviolet mutagenesis in the earlier research. In order to increase its inosine production, the technology of shaking-flask fermentation was optimized and the effects of different additives on inosine accumulation for Bacillus subtilis JMUKC2were studied in the paper. In addition, the whole metabolic process of inosine fermentation was analysed to explain the mechanism of additives on inosine accumulation.Firstly, the optimization of fermentation medium and fermentation conditions for inosine-producing strain in the shake was carried out by single factor experiment and orthogonal experiment. The optimized fermentation medium was composed of140g/L glucose,20g/L corn liquor,17g/L yeast,7g/L urea,20g/L ammonium sulfate,1g/L MgSO4-7H2O,7g/L KH2PO4,20g/L CaCCb. Under the optimized medium, cell biomass was improved by41%and inosine production was increased by47%compared with that of pre-optimized medium. The optimized fermentation conditions was composed of vaccination time18h, inoculum size7%, liquid volume15mL in250mL flask, initial pH of the medium6.5, culture temperature37℃. Under the optimized conditions, cell biomass and inosine production reached24.6g/L and10.02g/L respectively, which was51%and222%higher than that at initial conditions.Secondly, according to the regulating mechanism of inosine synthesis, five kinds of effective additives (sodium citrate, calcium gluconate, sodium fluoride, hypoxanthine and calcium chloride) which had a significant promotion effect on cell growth and inosine production were chosen and exhibited the optimum concentration added. Moreover, addition of effective additives with different combinations would have a greater promotion effect on inosine accumulation than adding single additive. Especially, when0.15%hypoxanthine,2mg/L sodium fluoride,10mg/L calcium chloride and15mg/L calcium gluconate were added into the media, inosine production achieved to14.01g/L which was about4.39times higher than the control group without adding effective additives.Finally, based on the law of mass conservation and the assumption of pseudo-steady-state for the intermediates in the metabolic pathways, the effects of different additives on metabolic flux distribution of inosine synthesis were studied by means of metabolic flux analysis. It was revealed that addition of several effective additives caused metabolic flux migration and then remarkablely enhanced the metabolic flux of inosine synthesis. Moreover, compared with the effect of single-additive addition, synergistic effects of multi-additive addition on inosine accumulation were revealed by means of metabolic flux analysis. |
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