Improvement Of Guanosine Producer Bacillus Subtilis JSIM-G518 And Studies On Optimization Of Its Fermentation Conditions

Guanosine as known as 9-13-D-ribofuranosyl-guanine, is an important intermediate of foods and medicines. It can be used to synthesis 5'-disodium guanylate, disodium nucleotide and nucleoside antiviral drugs such as ribavirin, acyclovir. And it is the main material to manufacture Sodium Guanosine Triphosphate. As more and more enterprises are going to produce acyclovir, ribavirin and guanosine 5'-acid, the demand of guanosine is increasing quickly.With the goal of increasing the yield of guanosine, this study shows a mutant JSIM-GU-124-19 selected from Bacillus subtilis JSIM-G518, obtained from a production strain with protoplast mutation, which produced guanosine in a high yield after optimizing the fermentation condition. And the fed-batch fermentation and regulation of guanosine were studied. The main research and results are as follows:1. A fast and reliable method of obtaining protoplasts from JSIM-G518 which can produce guanosine is studied. The conditions of protoplast formation and the regeneration were determined through the analysis of enzyme concentration and temperature. The high-yield of protoplast is in 40 minute, 2.4 mg/mL (the final lysozyme concentration) at 36℃. The method of protoplasts mutagenesis is simple and effective, it can improve the production capacity and performance of bacteria in a short time. The paper electrophoresis display that fermentation both contain guanosine and guanine, so in order to obtain a high-yield guanosine strain we have to gain a mutant which lacks nucleoside hydrolyase through protoplasts mutagenesis. At the same time a mutant with SGr, Psir was obtained, the mutant could produce guanosine 24.0 g/L, which increased by 30% of the original strain.2. The optimizations of medium and fermentation conditions for the production of guanosine by the mutant JSIM-GU-124-19 were carred out on the basis of single factor experiments and response surface methodology. The optimal conditions were: pH 6.4, temperature 36℃, dissolved oxygen 40%, inoculation time 10 h. The optimized medium was: glucose 12.5%, yeast powder 1.4%, (NH4)2SO4 1.5%, MgSO4 0.5%, KH2PO4 0.4%, Sodium Glutamate (MSG) 1.0%, CaCl2 0.2%, soybean digest 4.0 mL/dL, corn steep liquor1.7 mL/dL, CaCO3 2.0%. The yield of guanosine was increased to 28.3 g/L after optimization.3. The fed-batch fermentation and regulation of guanosine were studied and the flow mode and concentration of Sodium Glutamate and Dioxane were established. The experiments show that adding 0.25%Sodium Glutamate can reduce the cost of raw materials. In 30 h adding the final concentration of 100 mg/L Dioxane can short the fermentation period.