Research On The Deacetylation Of 7-ACA By Biocatalysis

D-7-ACA is an important intermediate of the synthetic or semi-synthetic cephalosporin antibiotics, previously mainly produced by chemical hydrolysis of 7-ACA. Due to various shortcomings of chemical methods, the product of D-7-ACA by enzymatic hydrolysis of 7-ACA gradually attracts widespread attention because of its unique advantages. In this research paper, we have screened a strain which can produce cephalosporin C deacetylase. We also studied its enzymatic properties, immobilization conditions and culture conditions.In this paper, we have screened a microorganism with CAH (Cephalosporin-C deacetylase) activity from soil by using 7-ACA as the single carbon source in the enrichment culture. It was intially identified as Rhodotorula glutinis, named Rhodotorula glutinis LHS3072 according to the microscope observations and colony morphology compared with the result previously reported in some literatures. The Rhodotorula glutinis LHS3072 is more attractive because its intact cells can be used directly, and the extraction and purification steps of the enzyme can be avoided.The enzymatic properties of Rhodotorula glutinis LHS3072 had been studied. We determined the optimal pH, temperature, thermal stability, pH stability of the enzyme. We also determined the substrate concentration and buffer system for the biocatalysis system.15% of 7-ACA may be completely converted by the wet biomass of 30%(w/v) in 3h at pH 7.0, temperature 25℃.Rhodotorula glutinis cell was immobilized by entrapping within polyacylamide gel. The level of total gel concentration, cross-linking degree and cell loading concentration that significantlly influence the immobilization in polyacrylamide gel was analyzed and optimized using single factor methodology. The optimal total gel concentration is 12%, the optimal croos-linking degree is 6% and the cell loading concentration is 30 g/L. The activity yield of immobilized cells was approximately 60% of the free cells. The optimum pH range of the immobilized cells was broader than that for free cells. The immobilized cells were more stable than the free cells at higher temperature. Moreover, the immobilized cells maintained 79.7% of the initial activity after 50 cycles while the free cells only maintained 79.4% after 7 cycles.Finally, the enzyme production conditions of Rhodotorula glutinis LHS3072 were optimized. It was potential to be used as an industrial biocatalyst with reduced costs. Culture conditions were as follows:glucose 30g/L, cheap yeast extract 20g/L, KH2PO4 5g/L, pH...