Research Of Fermention And Properties Of Poly β-hydroxybutyrate Depolymerase

Poly(3-hydroxybutyrate)(PHB) is polyster that is synthesized and accumulated intracellularlyduring unbalanced growth by a large of microorganisim.As new kind of biodegradable andbiocompatible thermoplastic polymers, it has attracted much commercial and academic interestwhich can be widely used in wrapper and medicine.An extracellular PHB depolymerase was separated and purified from the culture supernatant ofa fungus which can degrade PHB efficiently . Investigate its enzyme and protein properties.Themain results obtained from this work are as follows:1 Using the fungus (Penicillium sp.DS9713a) determined its depolymerase activity andprotein content . And it showed the most activity was in 96h by shaking culture. The proteasewas purified from the cell-free supernatant by ultrafiltration, ammonium sulfate precipitationand gel filtration chromatography. The specific activity of the purified enzyme was increasedby 59.1 folds over crude extract ,and the recovery yield was 20%.2 Enzyme properties: The high performance liquid chromatography had been used to testthe protein;SDS-PAGE analyses revealed a single band of 18 Kda;pI was 7.6;the optimumactivity of enzyme was observed at pH 8.6;The protease activity was inhibited by EDTA,SDSand mercaptoethanol.From the result that EDTA showed obvious inhibiting effect on theenzyme ,it can be suggested that metal ions played an important role in the conservation ofenzyme conformation for the catalytic activity.3 The result of amino acid composition showed that besides Met ,it contained 15 kinds ofnormal amino acids;The secondary structure of the protein was determined by circulardichroism spectrum, Helix:47.5%,Turn:24.3%, Random:28.2%;The mass spectrometeranalysis of water–soluble products after enzymatic degradation revealed that the primaryproduct was the monomer 3-hydroxybutyrate acid.