Identification Of Poly (3－Hydroxybutyrate) Degrading Strain And Characterization Of Its Depolymerase

In this study, we isolated and identified a PHB-degrading strain from naturalenvironments. Comparative morphology, physiological and biochemical characteristics and16SrDNA sequene analyses demonstrated that the PHB-degrading stain was the mostsimilar with Pseudomonas mendocina. The16SrDNA sequence homology was100%. Thus,the PHB-degrading strain was named Pseudomonas mendocina DSWY0601. Strain P.mendocina DSWY0601could form clear zone on agar plates of PHB as the sole carbonsource. The PHB films were eroded by the strain, and they cracked and breaked so thatthey were gradually degraded to3-hydroxbutyrate monomer （3HB） in liquid medium.Based on single factor experiment and orthogonal test, the optimized culture mediumsand culture conditions of strain P. mendocina DSWY0601were studied, such as carbonsource, nitrogen source, phosphorus source, pH, cultural temperature, time, rotation rate,volume of culture. The optimized culture media showed as follows: the optimal carbonsource was0.15%; the optimal nitrogen source was0.15%and the optimal phosphorussource was0.554%KH₂PO₄/1.108%Na₂HPO₄·12H₂O. The optimal culture condition waschosen as follows: the optimal culture temperature was35°C; the optimal culture pH was8.5; the optimal culture time was36h and rotation rate was200r/min. An extracellular PHBdepolymerase was purified to homogeneity by centrifugation, ultrafiltration,DEAE-Sepharose fast flow column and a Phenyl Sepharose6fast flow column. Thepurified PHB depolymerase activity increased by4.2fold, and the yield was5.7%. Themolecular weight of PHB depolymerase was approximately59.8kDa by SDS-PAGE. Theoptimal temperature and pH value were50°C and8.5, respectively. The enzyme was stablein a temperature range of20⁵0°C and a pH range of8.0⁹.5. Mn2+and Fe2+had slightlyinbibited effect on the PHB-degrading activity. PHB-degrading enzyme was stronglyinhibited by PMSF、H₂O₂and SDS. Tween20and Triton X-100had no obvious inhibitedeffect. The apparent Km of PHB depolymerase was0.172mg/mL. Furthermore, PHBdepolymerase had esterase activity, and could degrade poly（3-hydroxybutyricacid-co-3-hydroxyvaleric acid）（PHBV） and poly（3-hydroxybutyrate-co-4-hydroxybutyrate）（P3/4HB） but did not degrade polylactide （PLA）, poly（ε-caprolactone）（PCL） orpoly（butylene succinate）（PBS）. To analysis degradation characteristics, the degradationproducts of PHB depolymerase showed that the main degradation products were3-hydroxbutyrate monomers （3HB） by mass spectrometric analysis, suggesting that PHBdepolymerase acted as an exo-type hydrose, releasing one monmer unit at a time. According to peptide mass fingerprinting analysis, the PHB depolymerase had highhomology with the putative PHB depolymerase from P. mendocina ymp. PHBdepolymerase gene fragment （phaZpm） was cloned and successfully expressed. Therecombinant PHB depolymerase was purified on a Ni-NTA resin column. The molecularweight of the recombinant PHB depolymerase was approximately62.1kDa by SDS-PAGE.The optimum pH and temperature of the recombinant PHB depolymerase were8.5and50°C, respectively. The structure research of PHB depolymerase was based on summaryand analysis. We had concluded that the PHB depolymerase of Pseudomonas mendocinaDSWY0601consisted of576amino acids. The amino acids sequence contained a signalpeptide of38-amino-acid polypeptide at the N-terminal, a catalytic domain, a linker regionand two SBDs at the C-terminal. The catalytic domain contained conserved amino acids ofthe catalytic triad and the lipase box.