Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

摘要: The extracellular poly(β-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. enzyme glycosylated, with molecular mass ≃40 KD, and exhibited novel self-aggregation behavior means hydrophobic interaction that resolved Triton X-100 (TX-100) pretreatment also TX-100 incorporation in native gel. apparent Km value for PHB 119 μg/mL 3-hydroxybutyrate detected as main endproduct hydrolysis. insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), para-chloromercuric benzoic (PCMB), but inactivated dithioerythritol (DTT) showed specificity short chain-length poly(β-hydroxyalkanoates) (PHAs) such PHB, poly(hydroxyvalerate) (PHV), copolymers (3HB) 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed get hydrolyzed. enzyme, however, strong cross reactivity Comamonas sp. antibodies, not PHV antibodies Pseudomonas lemoignei. Southern hybridization dot blot analysis A. genomic DNA alkaline phosphatase labeled probes P. lemoignei genes revealed no homology, although hydrolyzed both PHV.