validamycin-a and valienamine

A simple and rapid CZE method was established for the simultaneous determination of valienamine, acarbose and validamycin A, using a 20-kV CZE with the detection wavelength of 193 nm and 50 mM phosphoric acid-20 mM Tris (pH 5.3) as a running buffer. The calibration curves of valienamine, acarbose, and validamycin A showed a good linear relationship at a concentration range of 5-1000 microg/mL. The detection limits of valienamine, acarbose, and validamycin A were 0.3, 0.6, and 0.6 microg/mL, respectively, and the average recoveries of each of the above were 99.9, 99.5, and 100.3%. The method has been successfully applied for simultaneous determination of substrate and product in the process of preparation of valienamine.

Topics: Acarbose; Cyclohexenes; Electrophoresis, Capillary; Hexosamines; Inositol; Reproducibility of Results

To isolate, identify and assess valienamine production by a soil bacterial isolate from a wheat field in Hangzhou, China.. A validamycin A-degrading bacterial strain, numbered ZJB-041, was isolated and identified as Stenotrophomonas maltophilia, based on morphology, physiological tests, ATB system (ID32 GN), and 16S rDNA analysis. The strain was capable of producing valienamine by decomposing validamycin A. After fermentation in shaking flasks at 30 degrees C for 7 days, 96.0% of 34.49 mmol l(-1) of validamycin A was degraded and 2.65 mmol l(-1) of valienamine was obtained. The resting cells of this strain also produced valienamine by degrading validamycin A. After 72 h of incubation in 0.2 mol l(-1) of phosphate buffer (pH 7.5), 90.2% of 17.16 mmol l(-1) of validamycin A was degraded, and 1.77 mmol l(-1) of valienamine was obtained.. Our data suggested that S. maltophilia ZJB-041, a bacterial isolate, has the potential for validamycin A degradation and valienamine production.. The validamycin A-degrading bacterium could potentially be utilized in the disposal of validamycin residues and in the production of valienamine.

Topics: China; Cyclohexenes; DNA, Bacterial; DNA, Ribosomal; Fermentation; Hexosamines; Inositol; Phenotype; Phylogeny; Soil Microbiology; Stenotrophomonas maltophilia; Triticum

The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin A biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length valC restored the production of validamycin A, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone--but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose--to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin A.

Topics: Amino Acid Sequence; Antifungal Agents; Carbohydrate Sequence; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cloning, Molecular; Cyclins; Cyclohexenes; Hexosamines; Inositol; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Phosphotransferases; Plasmids; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Spectrophotometry, Ultraviolet; Streptomyces

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.

Topics: Bacteria; Biotransformation; Calibration; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Cyclohexenes; Fluorobenzenes; Hexosamines; Inositol; Nitrobenzenes; Reproducibility of Results; Soil Microbiology; Temperature; Time Factors