Study On Intracellular NADPH Regeneration With Bacillus Pumilus And Optimization Fermentation Of Hydroxy Fatty Acids

ω-1,ω-2,ω-3-hydroxy fatty acids of C18 long-chain hydroxy fatty acids can be applied to the surface-active agent, cosmetic ingredients, lubricants and anti-rust agent in machinery industry, and anti-bacterial agents in the food industry. They also work as a monomer in the synthesis of polymers to provide a maximum carbon chain, which is one of the best monomers in research and development of green polymers. In this paper, a mutant strain of Bacillus pumilus M-F641 used as a starting strain, in order to further improve the conversion rate of oleic acid and the hydroxy fatty acid production rate, the cell growth and its metabolism were studied, and on the basis of single factors(incubation temperature, initial pH and liquid volume)experiments, the optimal fermentation conditions were obtained by response surface methodology, the oleic acid conversion rate were used as indicator.Response surface optimization method was used to determine the BP M-F641 optimal fermentation conditions, further experiment was conducted under the optimal culture condition. On the basis of carbon metabolism study, the optimum carbon source was glucose, and combined with the microbial fermentation metabolism pathway of glucose, a new efficient and economical cofactor NADPH regeneration method was established after using D-xylose as the co-substrate in Bacillus pumilus MF-641.The main research results were as follows:(1)Alkaline and acid methyl ester method were compared, and acid methyl ester method was determined. By orthogonal experiment(reaction time, temperature, sulfuric acid-methanol volume ratio), a method of sulfuric acid-methanol(2% sulfuric acid-methanol,70℃water bath for 1h)for the fatty acids methyl was established.(2)By studying the change of its growth and metabolism, its optimum carbon source of glucose which is needed for growth, was determined, optimum glucose concentration was 20g/L. Combined with the microbial fermentation metabolism pathway of glucose, a new efficient and economical cofactor NADPH regeneration method was established after using D-xylose (D-xylose addition of 10g/L) as the cosubstrate in Bacillus pumilus MF-641. Compared with the control group (the addition of D-xylose was 0g/L), NADPH content increased 0.0345mmol/L, an increase rate of 40.68%, and the conversion rate of oleic acid(75.31%)and the yield ofω-hydroxy fatty acids(22.61%) also had a greater increase.(with the control group, the conversion rate of oleic acid was 70.04%, the yield ofω-hydroxy fatty acids was 18.90%) .(3)Determination of BP M-F641 optimum fermentation conditions on the basis of single factors (incubation temperature, initial pH and liquid volume)experiments, the optimal fermentation condition was obtained by response surface methodology as follows, medium volume 60mL(approximate)in 250mL, temperature 30℃, initial pH7.4, under these conditions, the maxmium NADPH concentration was 0.0268mmol/L, the conversion rate of oleic acid was 74.58%.