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.
Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of β-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.
The composition of fatty acids and the spectrum of macrolide antibiotics produced in 7 mutant strains of Streptomyces fradiae, a tylosin producer, were investigated. The strains under investigation differed in the production level and representation of individual tylosin-like compounds. The composition of fatty acids in the mycelium did not depend on the total production. However, the strains producing relomycin in addition to tylosin produced a significantly higher fraction of fatty acids with a higher melting point, and, on the contrary, the strains producing only tylosin or tylosin and desmycosin synthesized a significantly lower proportion of these acids. The results obtained indicate that in addition to the activity and substrate specificity of secondary metabolism enzymes, the composition of the tylosin-like compounds produced can be influenced by the cell membrane and its function.
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