Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp RY-1

A novel bacterial strain capable of growing in a medium containing a medium -chain-length polyhydroxyalkanoate (MCL-PHA) as the sole carbon source was isolated from a soil sample, The isolate, which was identified as Pseudomon as sp. RY-1, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated in a medium containing a MCL-PHA, such as polyhydroxyoct anoate (PBO) or polyhydroxynonanoate (PHN). The extracellular MCL-PHA depol ymerase of this organism was purified to electrophoretic homogeneity. The e nzyme was a tetramer with identical subunits and a total molecular mass of 115 kDa. The isoelectric point of this enzyme was estimated to be 5.9 by is oelectric focusing. The maximal activity was observed. at pH 8.5 and 35 deg rees C. The enzyme was insensitive to phenylmethylsulfonyl fluoride and dit hiothreitol, unlike other short-chain-length (SCL) PRA depolymerases, The K -m values for PHO and PHN were 0.86 and 1.47 mg/ml, respectively. The enzym e could not hydrolyze SCL-PHAs and p-nitropbenyl esters of fatty acids.