Purification And Characterization Of 3-Ketovalidoxylamine C-N Lyase And Other Related Enzymes

Valienamine was obtained from microbial degradation of validamycin A. It is a cyclitol, with strong inhibitory effect onα-glucosidase. It is a very important chemical intermediate because many antihyperglycemic agents can be synthesized with valienamine as a starting material, such as acarviosin, voglibose and so on. 3-Ketovalidoxylamine A C-N lyase was first purified from E saccharophilum in 1984. Little research on glucoside 3-dehydrogenase has been done. Researchs on enzyme degradation of validamycin A to valienamine, could advance economic benefit of validamycin A, and achieve the conversion from biopesticide to biomedicine.Stenotrophomonas maltrophilia CCTCC M 204024 was isolated by our lab from the local soil. It can use validamycin A as sole carbon source. Elucidation of the degradation pathway of validamycins A by resting cells of S. maltrophilia revealed that validamycin A was firstly hydrolyzed to validoxylamine A byβ-glucosidase. Validoxylamine A was then degraded through 3-ketovalidoxylamine A by glucoside 3-dehydrogenase （G3DH） and 3-ketovalidoxylamine A C-N lyase to validamine, valienamine and unsaturated ketocyclitols.N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C-N lyase, was firstly prepared from N-p-nitrophenyl-validamine with free cells of S. maltrophilia. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylene diamine tetraacetic acid （EDTA）. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was 6.0 and 30℃. N-p-nitrophenyl-3-keto-validamine was formatted with Y₃-KpNPV of 0.68 from the first batch. Culture conditions of enzyme formation was optimized. The optimal carbon source was 0.5% validamycin A, which had some inducement for enzyme formation. The highest three key enzyme activity was obtained in a 500-mL conical flask containing 100 mL of medium, with natural pH and 10% inoculation volume, at 30℃for 36 h.The three key enzymes were purified and characterized in this paper. 3-Ketovalidoxylamine A C-N lyase was purified to 367 fold with a yield of 16.4% through High S IEX, HIC, High Q IEX and gel filtration. The enzyme was estimated to be a single band with molecular weight of 31.4-kDa by SDS-PAGE. The optimal reaction pH of 3-ketovalidoxylamine A C-N lyase was 7.0, and the optimal reaction temperature was 40℃. The enzyme was stable at a pH range of 7.0-10.0 and was sensitive to heat. EDTA inhibited the activity of the enzyme. Calcium was needed for the enzyme activity of 3-ketovalidoxylamine A C-N lyase. The apparent K_m value for N-p-nitrophenyl-3-ketovalidamine was 0.15 mM. Amino acid analyses of this enzyme showed that N-terminal was closed.A soluble glucoside 3-dehydrogenase （G3DH） was purified to 37.4 fold with a yield of 24.7% through High Q IEX, HIC High S IEX, and was estimated by SDS-PAGE with molecular mass of 66-kDa. The optimal pH of G3DH was 6.5 in the presence of DCPIP. The enzyme was stable in the pH range of 4.4-10.6 and was sensitive to heat. G3DH exhibited extremely broad substrate specificity by converting many sugars to their corresponding 3-ketoglucosides. The apparent K_m values for validoxylamine A and D-glucose were 8.3 mM and 1.1 mM, respectively. Cu²⁺, Ag²⁺and Hg₂Cl₂ inhibited the activity of G3DH. Amino acid analyses of this enzyme showed that N-terminal was closed. Tryptic Peptide Mapping of G3DH showed that there was no protein could match this enzyme, deduced that it was probably a new enzyme.A solubleβ-glucosidase was purified to 270 fold with a yield of 6.9% through High S IEX, HIC High Q IEX and gel filtration, and was estimated by SDS-PAGE with a molecular mass of 93.4-kDa. The optimal pH of β-glucosidase was about 6.0 and the optimal reaction temperature was 40℃. The enzyme was sensitive to heat. Cu²⁺and Hg₂Cl₂ inhibitedβ-glucosidase activity completely, and Ag²⁺, Co²⁺and Ni²⁺ inhibited it partly. The apparent K_m value for pNPG was 0.79 mM.