Optimization of medium composition for 3-hydroxycarboxylic acid production by Pseudomonas mendocina-biodegraded polyhydroxybutyrate.

We optimized the culture medium for 3-hydroxycarboxylic acid production by Pseudomonas mendocina DS-04-T-biodegraded polyhydroxybutyrate (PHB) using the Plackett-Burman design, steepest ascent method, and Box-Behnken design. The optimized concentrations of the constituents of the culture medium were as follows: PHB (7.57 g/L), NH4 Cl (5.0 g/L), KH2 PO4 (2.64 g/L), Na2 HPO4 ·12H2 O (12 g/L), MgSO4 ·7H2 O (0.5 g/L), and CaCl2 ·2H2 O (5 mg/L). The yield of 3-hydroxycarboxylic acid obtained using the optimized culture medium was 56.8 ± 1.64%, which was 2.5-fold higher than that obtained when the unoptimized culture medium was used.

Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina.

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na(+) and K(+) slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H2O2 also inhibited the activities of both enzymes.

Purification and characterization of an extracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Acidovorax sp. HB01.

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading strain Acidovorax sp. HB01 was isolated from an activated sludge sample. A novel PHBV depolymerase with a molecular weight of 43.4 kDa was purified to homogeneity from the culture supernatant of the HB01 strain. The optimum pH and temperature of the PHBV depolymerase were 7.0 and 50 °C, respectively. The PHBV depolymerase can also degrade polyhydroxybutyrate, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(caprolactone); however, the PHBV degradation activity of the depolymerase is higher than its activity against the other polymers. Effect of metal ions and various inhibitors on the PHBV depolymerase activity was examined. The addition of Na(+), K(+), and Ca(2+) markedly increased the hydrolysis rate, whereas the enzyme activity was inhibited by Zn(2+), Mg(2+), Mn(2+), and particularly by Cu(2+) and Fe(2+). Ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of depolymerase was identified as the 3-hydroxybutyric acid monomer and 3-hydroxyvaleric acid monomers via mass spectrometry.