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

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

A fluorescence-based screening assay for DNA damage induced by genotoxic industrial chemicals

A rapid screening assay to detect chemically-induced DNA damage resulting from exposure of surrogate DNA to genotoxic compounds is reported. This assay is based on changes in the melting and annealing behavior observed for damaged DNA. Exposure of calf thymus DNA to genotoxic industrial chemicals reduced the extent to which the DNA annealed as measured using a double strand DNA selective fluorescent indicator dye. Formaldehyde, acrolein, crotonaldehyde and bromoethane showed the most prominent effects, chloroacetone and allylamine exhibited lesser effects, and acryrlonitrile showed no statistically significant assay response. The assay response for formaldehyde and crotonaldehyde were measured over the concentration range of 10-100 mM and 50-300 mM, respectively. This assay showed little response for the cytotoxic compounds phenol, cyclohexane and toluene but was sensitive to the effects of DNA damaging compounds such as mitomycin C and glutaraldehyde.