Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

• Published in: Journal of fermentation and bioengineering Vol. 81; no. 1; pp. 72 - 75
• Main Authors: Nojima, Saiko, Mineki, Shigeru, Iida, Mitsugi
• Format: Journal Article
• Language: English
• Published: Osaka Elsevier B.V 1996; Society for Fermentation and Bioengineering
• Subjects: 3HB dimer methyl ester; AGROBACTERIUM; Agrobacterium sp; BIODEGRADACION; BIODEGRADATION; Biological and medical sciences; Biotechnology; BUTIRATOS (ESTERES); BUTYRATE (ESTER); BUTYRATES (ESTERS); ENZIMAS; ENZYME; Enzyme engineering; ENZYMES; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Methods. Procedures. Technologies; PHB depolymerase; POLIMEROS; POLYMERE; POLYMERS; PURIFICACION; PURIFICATION; PHB depolymerase; 3HB dimer methyl ester; Agrobacterium sp; Temperature; Isozyme; Environmental factor; Characterization; Agrobacterium; Enzymatic activity; Substrate specificity; Production; pH; Bacteria; Biodegradation; Purification; Stability; Thermoplastics; Enzyme; Molecular weight determination; Ester polymer; Extracellular; Pollutant; Microorganism culture; Gel filtration; Hydrolases; Rhizobiaceae; Column chromatography; Physicochemical properties
• ISSN: 0922-338X
• DOI: 10.1016/0922-338X(96)83124-2

A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.