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Breeding Of Pseudomonas Sp.DSDY0501 To Degrade PHB And The Research Of PHB Depolymerase

Posted on:2009-12-27Degree:MasterType:Thesis
Country:ChinaCandidate:Y DiFull Text:PDF
GTID:2120360245454539Subject:Microbiology
Abstract/Summary:PDF Full Text Request
The strain DSDY0501 which can degrade PHB efficiently was isolated from the soil which collected from different areas and ecological environments. According to morphological observation,the physiological and bio-chemical characteristics and 16S rDNA analysis, the strain was identified to belong to Pseudomonas.The study of the strain growth regulation on the culture medium which use PHB as the sole carbon and energy showed as follows: DSDY0501 can degrade PHB granule and films completely in one day. The PHB depolymerase is synthesized and secreted when the strain was at logarithmic phase, and its secretion decreased when the strain was at stationary phase.An extracellular PHB depolymerase was purified according to gel filtration technique by Sephacryl S-200. The specific activity of the purified enzyme was 24.2-folds and the recovery yield was 16.61%.The extracellular PHB depolymerase belongs to monomeric enzyme. The molecular weight is 57.9kDa approximately. The optimum activity was observed when the temperature was 60℃and pH was 9.0. The half life of PHB depolymerase at the optimum temperature was 2.5 h. Ca2+,Mg2+,Zn2+ and CO2+ can increase the enzyme activity, but the enzyme was inhibited by Cu2+,Fe3+,Mn2+. 5mM H2O2 and 0.5% (v/v) mercaptoethyl alcohol also can increase the enzyme activity, whereas 0.1% (w/v) SDS, 0.5% (w/v) EDTA and 4mol/L Urea can inhibite it. The apparent Km of the enzyme was determined as 0.182mg/ml. The secondary structure of the enzyme was determined by circular dichroism (CD) spectrum.α-Helix andβ-pleated sheet were 60% and 40% respectively. The mass spectrum analysis showed that the main degraded product of enzyme was PHB monomer.
Keywords/Search Tags:Poly(3-hydroxybutyrate)(PHB), Biodegradation, PHB depolymerase, strain DSDY0501
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