[Metabolism of phosphate-limited Streptomyces cultures. I. Purification and characterization of alkaline phosphatase produced by Streptomyces hygroscopicus].
Alkaline phosphatase from Streptomyces hygroscopicus strain JA 5999-R 27-158 was purified and characterized. The enzyme was found in the culture filtrate and in the mycelium. The phosphatase was extracted from the mycelium and purified by adsorption to DEAE-cellulose. To separate impurities, the crude enzyme solution was heated and the phosphatase purified by chromatography through CM-Sepharose and Sephadex G 100. The specific activity of the resulting enzyme was 1000 microMol/min/mg at 25 degrees C. The molecular weight determined by SDS gel electrophoresis was found to be 56 000. The Michaelis-Menten constant determined with p-nitrophenylphosphate as substrate was Km = 1.25 X 10(-3) M. Phosphatase activity was dependent on the presence of Ca++ and the maximum activity of enzyme with p-nitrophenylphosphate as substrate was found at pH 9.2. The pI as detected by isoelectric focusing was at pH 5.6. Temperatures from 30 degrees to 75 degrees C did not affect the stability of the enzyme. The alkaline phosphatase exhibited high substrate specificity; of various phosphomonoesters tested, only p-nitrophenylphosphate, methylumbelliferyl-phosphate, phosphoenolpyruvate, ADP, ATP and tyrosine-O-phosphate was hydrolysed. The activity was inhibited by NAF, Na2P2O7 and EDTA. The involvement of the alkaline phosphatase in the regulation of secondary metabolism was discussed.
D000469:Alkaline Phosphatase / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004794:Enzyme Repression; D005285:Fermentation; D007525:Isoelectric Focusing; D007933:Leucomycins / Q000096:biosynthesis; D008970:Molecular Weight; D012969:Sodium Fluoride / Q000494:pharmacology; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D007933:Leucomycins; C011138:turimycin; D012969:Sodium Fluoride; D000469:Alkaline Phosphatase
Untersuchungen zum Stoffwechsel phosphatlimitierter Streptomycetenkulturen. I. Mitteilung: Reinigung und Charakterisierung einer alkalischen Phosphatase aus Streptomyces hygroscopicus.