An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage fo...An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.展开更多
The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of th...The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of the marine photosynthetic bacterium Rhodovulum sulfidophilum P5 under aerobic-dark conditions. The results show that the accumulation of PHB in strain P5 is a growth-associated process. Strain P5 had maximum biomass and PHB accumulation at 2%-3% NaCl, suggesting that the bacterium can maintain growth and potentially produce PHB at natural seawater salinity. In the nitrogen source test, the maximum biomass accumulation (8.10±0.09 g/L) and PHB production (1.11±0.13 g/L and 14.62%±2.25% of the cell dry weight) were observed when peptone and ammonium chloride were used as the sole nitrogen source. NH 4 + -N was better for PHB production than other nitrogen sources. In the carbon source test, the maximum biomass concentration (7.65±0.05 g/L) was obtained with malic acid as the sole carbon source, whereas the maximum yield of PHB (5.03±0.18 g/L and 66.93%±1.69% of the cell dry weight) was obtained with sodium pyruvate as the sole carbon source. In the carbon to nitrogen ratios test, sodium pyruvate and ammonium chloride were selected as the carbon and nitrogen sources, respectively. The best carbon to nitrogen molar ratio for biomass accumulation (8.77±0.58 g/L) and PHB production (6.07±0.25 g/L and 69.25%±2.05% of the cell dry weight) was 25. The results provide valuable data on the production of PHB by R. sulfidophilum P5 and further studies are on-going for best cell growth and PHB yield.展开更多
基金Supported by the National Natural Science Foundation of China (Nos.31100046,31100099)
文摘An extracellular polyhydroxybutyrate（PHB） depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） was ap- proximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20--50 ℃ and sensitive to pH value within a pH range of 8.0-9.5. PHB depo- lymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate（P3/4HB） and poly-3-hydroxybutyrate-co-3- hydroxyvalerate（PHBV） but did not degrade poly（lactic acid）（PLA）, poly（butylene succinate）（PBS） or poly- （caprolactone）（PCL）. PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride（PMSF）, H202 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer（3HB） by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis ofphaZpm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA（dPHAscL, PHA=polyhydroxyalkanoate） depolymerase containing catalytic domain, linker and substrate-binding domain.
基金Supported by the National Natural Science Foundation of China (Nos.30830015, 40806063)the Key Natural Science Foundation of Tianjin,China (No. 12JC2DJC22200)+2 种基金the Natural Science Foundation of Guangxi,China (No. 1000050096)the Foundation of Tianjin Key Laboratory of Marine Resources and Chemistry (Tianjin University of Science & Technology) (No. 200913)the Introduced Talents Scientific Research Initiating Foundation of Tianjin University of Science and Technology (No.20100410)
文摘The effects of different NaCl concentrations, nitrogen sources, carbon sources, and carbon to nitrogen molar ratios on biomass accumulation and polyhydroxybutyrate (PHB) production were studied in batch cultures of the marine photosynthetic bacterium Rhodovulum sulfidophilum P5 under aerobic-dark conditions. The results show that the accumulation of PHB in strain P5 is a growth-associated process. Strain P5 had maximum biomass and PHB accumulation at 2%-3% NaCl, suggesting that the bacterium can maintain growth and potentially produce PHB at natural seawater salinity. In the nitrogen source test, the maximum biomass accumulation (8.10±0.09 g/L) and PHB production (1.11±0.13 g/L and 14.62%±2.25% of the cell dry weight) were observed when peptone and ammonium chloride were used as the sole nitrogen source. NH 4 + -N was better for PHB production than other nitrogen sources. In the carbon source test, the maximum biomass concentration (7.65±0.05 g/L) was obtained with malic acid as the sole carbon source, whereas the maximum yield of PHB (5.03±0.18 g/L and 66.93%±1.69% of the cell dry weight) was obtained with sodium pyruvate as the sole carbon source. In the carbon to nitrogen ratios test, sodium pyruvate and ammonium chloride were selected as the carbon and nitrogen sources, respectively. The best carbon to nitrogen molar ratio for biomass accumulation (8.77±0.58 g/L) and PHB production (6.07±0.25 g/L and 69.25%±2.05% of the cell dry weight) was 25. The results provide valuable data on the production of PHB by R. sulfidophilum P5 and further studies are on-going for best cell growth and PHB yield.
