Using Asymmetric Degradation Principle Of Microorganism Isolate D-Alanine Strains And Fermentation Process

D-alanine is very important organic chiral source. Its mainly applications are as the chiral source in the process of chiral synthesis in food and pharmaceutical industry. It is applicable to chiral additive, chiral auxiliaries, chiral drugs and so on. Currently D-alanine is all have important applications to the production of D-alaninol, new broad-spectrum antibiotics, vitamin B6, food additives, alanine protective agent of peptide synthesis process, synthesis of new sweetener Alitame, cosmetics and other fields. In recent years, D-alanine was more and more applications in scientific research and production. The demand is also increasing year by year and it has potential market value.In this thesis, the test use the asymmetric degradation principle of microorganism to screening microorganisms from the nature. The microorganisms can asymmetric degradation L-alanine from the DL-alanine and obtain D-alanine product. Through laboratory shake flask experiments to obtain the best seed and fermentation culture medium formula. Through the research of automatic control tank fermentation conditions to obtain the optimum fermentation processes. In order to obtain microorganisms required for the experiment, each of L-alanine and D-alanine as the sole source of organic carbon substrates. The soil samples were collected from vineyards. After the primary and secondary screening, finally got one can quickly and thoroughly degradation L-alanine for D-alanine almost no degradation strain of Ala-D45. The ratio of the strain medium and culture conditions were optimized to determine the best formula for the seed medium as follow:DL-Alanine 10.0g/L, glucose 5.0g/L, peptone 10.0g/L, yeast extract 10.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 0.5g/L. The best conditions for the seed culture was 30 ℃, initial pH6.0, 100r/min, shaking culture 22h. Through single factor experiment determine the best formula for the fermentation medium as follow:DL-alanine 100.0g/L, glucose 5.0g/L, peptone 10.0g/L, yeast extract 10.0g/L, potassium dihydrogen phosphate 5.0g/L, dipotassium hydrogen phosphate 2.5g/L, magnesium sulfate 1.0g/L, calcium chloride 1.0g/L, ferric chloride 5.6mg/L, zinc sulfate 5.0mg/L. The optimum fermentation conditions were seed volume 15%, fermentation temperature 30℃, fermentation optimum pH6.0, optimum shaking speed 180r/min. Meanwhile verified by experiments, the substrate DL-alanine concentration was too high will inhibit the degradation of L-alanine in the mixed spin matter. Therefore, by the method of flowing feed when adjusting acid and use the automatic control fermenter to achieve the D-alanine pilot to expand production.Finally, the extraction of the fermentation broth was studied to determine the optimum extraction process route. The fermentation broth is filtered through the ceramic membrane, calcium salt precipitated, decompressed drive ammonia, activated carbon decolored, concentrated, crystallized, centrifuged, separated and convection dried steps to obtain D-alanine product. Through single factor test and orthogonal test, the optimal decolorization condition was determined as follows:activated carbon dosage 7.5g/L, temperature 95 ℃, time 70min, stirring speed 200r/min. For the recovery and quality of the product was analysed that the total recovery ratio of D-alanine was 67.72%, specific rotation of -14.67°, purity of 99.86%, the product quality accorded with Japan’s Ajinomoto Company(AJI97) standards.