Screening Of Validamycins-biotransformation Strains And The Structural Identification Of Products

Validamycins, a agricultural antibiotic for treatment of sheath blight of rice, is a secondary metabolite of Streptomyces.hygroscopicus var.jinggangensis, which was discovered by Chinese scientist SHEN Yinchu. Validamycins has great potential in producing its derivatives. The derivatives of validamycins contain validoxylamine A, validamine, valienamine and valiolamine. Validoxylamine A has strong inhibition activity on trehalase and it is a new promising insecticide. Valienamine and validamine are competitive inhibitors against glycosidase such asα-amylase, glucoamylase, invertase and maltase, which can also be developed as drugs for treatment of type II Diabetes Mellitus, or as intermediates for synthesis of hypoglycemic drug, such as voglibose and acarbose. This research focuses on exploration a way to produce valienamine and its derivatives by microbial transformation of validamycins with the advantages of high efficiency, cleanness and low cost.Through the method of mixing microbes culture, three fungi have been screened out with the strong activity of transformation validamycins to validoxylamine A. They are named as Y07F-1287, Y07F-1248 and Y07F-1239 separately. Based on the characteristics of colony morphology, the mycelium morphology and ITS-5.8S sequence, the stain Y07F-1287 has been identified as Fusarium oxysporum, strain Y07F-1248 has been identified as Fusarium equiseti and strain Y07F-1239 has been identified as Fusarium incarnatum. Through the method of enrichment culture, a bacteria strain Y07B-0134 has been screened out, which can transform validamycins to validamine. According to its physiological and biochemical characteristics and the 16S rDNA sequence, stain Y07F-0134 has been identified as Sphingobacterium multivorum.The preliminary optimization of strain Y07B-0134 transformation process are as follows: 2.5% Validamycin as the only carbon source, 0.5% ammonium nitrate as nitrogen source, initial medium pH6.5, cultivate time 5d. The transformation results showed that under the optimization condition, the yield of validamine reached 3.18mg·mL^-1, the mol conversion percentage reached 28.89% which almost improved 19.37% compared with the base condition.In the isolation and purification process of validoxylamine A, cationic resin was used in separation of validoxylamine A, anion resin was used to remove pigments and ethanol was used to precipitate final product. The purity of validoxylamine A reached 95% by the detection of HPLC.In the isolation and purification process of validamine, cationic resin was used two times to remove impurities, anion resin was used to remove pigments, and ethanol was used to precipitate final product.