Valine dehydrogenase (VDH) was purified to homogeneity from cell-free extract of Streptomycesfradk, which produces t y h i a . The egzyme was purified 1508-fold in a 17.7% yield using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The M r of the native enzyme was determined to be 218000 and 215000, by equilibrium uitracentrifugation and sizeexclusion high-pedomance liquid Chromatography, respectively. The enzyme is composed of 12 subunits of M, 18000. Using analytical isoelectric focusing the isoelectric point of VDH was found to be 4.7. Oxidative deamination of L-valine was optimal at pH 10.6. Reductive amination of 2-oxoisovalerate was optimal at pH 8.8. TheMichaelis constants (Km) were 1 m~ for L-valine and 0.029 m~ for NAD+. K, values for reductive amination were 0.80 m~ for 2-oxoisovderate, 0-050 m~ for NADH and 22 m M for NHJ.
Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of a-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for a-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4C1.
Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomycrs fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210000 or 205000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of M , 51 000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The K,,, were 10.0 mM for ~-alanine and 0.18 mM for NAD'. K, values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH: and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD' binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH. Regulation of alanine dehydrogenase synthesis in streptomycetes has been studied in connection with assimilation of NH,f and regulation of synthesis of secondary metabolites by nitrogen sources [16, 171. Since biosynthesis of ammo and fatty acids and the production of the oligoketide antibiotic tylosin in Streptomyces fradiae are regulated by NH,f [21-251, some molecular and catalytic properties of alanine dehydrogenase isolated from this organism are described. MATERIALS AND METHODS MaterialsSephadex G-200 SF and Mono Q HR 515 prepacked fast protein liquid chromatography (FPLC) column were from Pharmacia Fine Chemicals, Uppsala, Sweden. All other chemicals were of the highest purity available. Microorganism and growth conditionsThe microorganism used was Strrptomyces fradiae 3013 obtained from the collection of the Research Institute of Biofactors and Veterinary Drugs in Koui-im near Prague [24]. The first generation was cultivated for 48 h in a medium containing (mass/vol.): 2% sucrose, 0.3% Casamino acids (Difco), 0.5% NaC1, 0.3% CaC03, 0.05% MgS04.7H20, 0.1% K2HP04, 0.2% L-valine, 0.1% yeast extract, 0.17% (NH4)2S04. The second generation was inoculated with 5% (by vol.) of the first generation and cultivated in a medium as above containing L-valine (0.47%) and yeast extract (0.050/,); (NH4)2S04 was omitted. Both media were adjusted to pH 7.3 before thermal sterilization. The cultivation was carried out in 50o-ni1 flasks containing 70ml medium on a reciprocal shaker (1.7 Hz, 29°C).
Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.
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