Breeding Of Vancomycin-Producing Strain

High productive strain was obtained by UV irradiation. Based on the study of strain's fermentation ability, the medium was optimized. Also, the determination methods of vancomycin was studied.Both HPLC and agar diffusion method were studied. After comparision, HPLC was assumed as better one to assay vancomycin. The operating conditions were Phenomenex prodigy ODS3 100A, at room temperature, triethylamine buffer solution (pH was adjusted to 3.3 with H₃PO₄)-acetonitrile-tetrahydrofuran (92:7:1,V/V) as mobile phase with a flow rate of 1.5mL.min^-1 and UV detection at 216nm. Linear response was found in the range between 80-600μg.mL^-1.Strain A.Orientalis-4 was screened by agar block method because of its excellent fermentation capability and was treated as original strain .The vancomycin yield of AO-4 reached 0.794g/L.After the monospore suspension of AO-4 was mutanted by UV, the mutants were screened on agar medium containing streptomycin., a high-yield mutant AO-29 was obtained, and its productivity was increased by 167% over the patent strain. Submerged culture indicated that the hereditary character of high productivity of AO-29 was stable.Based on the study of its fermentation ability, the optimum medium for vancomycin production was demonstrated (g/L): beef extract 18, Cottonseed flour 12, cane sugar 26, maltose 38, potassium nitrate 6,potassium dihydrogen phosphate 0.05,and magnesium chloride 0.3.Inoculum volume was 7%. A 250mL flask containing 25mL medium was cultivated at 28℃ for 132h and the speed was 170r/min. Adding 0.2% after36 hour in the medium.The yield of vancomycin under this condition was 3.66 g/L.