Study On High-throughput Assay Of Metabolites In Fermentation Broth Based On Enzyme Catalysis

Objective:Adenosine and cytidine are very important pharmaceutiacal raw materials and the rapid high-throughput method for detecting adenosine and cytidine in fermentation broth based on adenosine deaminase(ADA) and cytidine deaminase(CD) are established. A preliminary study is done on screening cytidine-producing strain in bacillus subtilis by laser mutation breeding.Methods:Using sodium carbonate-sodium bicarbonate, sodium nitroprussid, sodium hypochlorite and thymol as detection reagents, we optimized them by response surface model analysis. The standard curve of adenosine was drawn and the adenosine of detection was determined based on adenosine deaminase(ADA). Similarly, the standard curve of cytidine was drawn and the cytidine of detection was determined based on cytidine deaminase(CD). We screened cytidine-producing strain in bacillus subtilis by laser mutation breeding and determined the optimal conditions initially.Results:Using response surface model analysis, the results showed sodium carbonate-sodium bicarbonate 34.20 mmol/L-27.40 mmol/L, sodium nitroprussid 3.35 g/L, the effective chlorine of sodium hypochlorite 0.1725%, thymol 6.64 g/L. The regression equation of standard curve of adenosine was y=0.36045x+0.06313, R2=0.99702 and the limit of detection was between 0.025 mmol/L and 6 mmol/L. The regression equation of standard curve of cytidine was y=0.36045x+0.06313, R2=0.99702 and the limit of detection was between 0.1mM-6mM. The initial results of cytidine-producing strain in bacillus subtilis screened by laser mutation breeding showed the optimal concentration of screening agent was 1.2 g/L, the optimal time of laser mutation breeding was 1.5 min and the optimum concentration of cytidine produced by bacillus subtilis was 74.04 mg/L.Conclusions:The method is sensitive and can meet the needs of the fermentation detection and high-throughput screening to adenosine and cytidine. Our study will be the initial step of further production of cytidine by laser mutation breeding.