| Mandelic acid is an important pharmaceutical intermediates and fine chemical intermediates. Since 90 years, the development of chiral drug has become boom,and the study on the single-configuration of mandelic acid derivatives has a good development prospect and huge space. Now, the technology route of chiral chemistry resolution has been used to the preparation craft of R-(-)-mandelic acids,with racemic modification as the substrate. As the expensive price of chiral resolution reagent,the resources waste and the environmental pollution, now, people shift the vision to the research of microorganism preparation gradually.We obtain a new strain (Pseudomonas aeruginosa) which can selective degrade the S-mandelic acid to the R-mandelic acid effectively, with the racemization mandelic acid as the only carbon source. Based on the study of the nature of the strain, in this paper, medium and culture conditions were optimizated systematically, to obtain large yields and high optical purity of R-mandelic acid. Concrete works are as followed:1. The prupose strain has been isolated using racemic mandelic acid as sole carbon source. The physiological and biochemical characteristics of strains have been sdutied, and identified the 16S rDNA also. By means of domestic experiments, the vitality and substrate tolerance of Pseudomonas sp.L49-S14 have been improved.2. Preliminaryily, we study the metabolic pathways of the Pseudomonas sp.L49-S14, make use of mandelic acid. Determine the S-(=)-MA is oxidated by the dehydrogenase into phenyl gyoxilic acid, then decarboxylated into benzaldehyde by decarboxylase, and oxidized to benzoic acid, finally, enter the metabolism ofβ-keto adipic acid, thus leaving R-MA as the purpose product.3. We studied on the induction conditions detailly. And determined that the racemic mandelic acid can use as the optimal inducer, adding amount of induction is 5 g/L, Pre-induction time of 12h, adding substrate directly is the best way to Fermentation.4. With single-factor method and statistical experimental design, the fermentation medium components and fermentation conditions of P.aeruginosa L49-S14 were optimized, to obtain high yield and optical purity (ee value) of R-mandelic acid,Finally, Nitrogen concentration> pH value> Mg2+ concentration were significant factors which affect the fermentation conditions.At last,we determined the optimizated medium and culture conditions were that: MA 5g/L, Yeast extract 4.4 g/L, pepton 4.4 g/L, MgSO4 0.58 g/L, MnSO4 0.05 g/L, K2HPO4 1 g/L, KH2PO41g/L, NaCl 1 g/L, Inoculum 5%, temperature 35℃, pH7.8, speeding 160r/min,concentration of substrate up to 20g/L, Fermentation completed in 72h,the e.e value of fementation products canbe achieved (99.2±0.5)%。5 Using with the air-lift fermentor made by ourselves, aeration of the fermentation tank, method of additional substrate and the final concentration of substrate were studied.Finally, we determined thatPre-fermentation with low ventilation, and Post-fermentation (24h later) with high ventilation by the means of additonal batches of substrate, additional interval time is 12h,the first two amountion were 10g/L,later three added 5/L. Final concentration of substrate could up to 40g/L, e.e value of production greater than 99.7%. and could achieve the fermentation in 98h.In summary, after the optimization experiments, the substrate concentration was upgraded 4 times, and the ability of.P.aeruginosa L49-S14 which prepared for R-MA in fermentation, was 4-fold increased,compared to original conditions without optimization.
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