Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19.
An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.
Kim
Do Young
DY
Department of Microbiology, Chungnam National University, Daejeon 305-764, Korea.
Nam
Jin Sik
JS
Rhee
Young Ha
YH
D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase