Isolation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading bacteria and purification of PHBV depolymerase from newly isolated Bacillus sp. AF3

Abstract

The contribution of bacteria, isolated from sewage sludge, towards the microbial degradation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was studied. A microbial consortium was inoculated on mineral salt agar plates containing different concentrations of PHBV as a substrate. The PHBV degraders showed optimum depolymerase production at pH 7.0, 37 °C, and 0.4% substrate concentration during 2–5 weeks of incubation. Additional carbon sources like glucose, fructose, sucrose, lactose and nitrogen sources including yeast extract were used as co-metabolites. The highest degree of degradation of PHBV was observed in the presence of glucose. Among the surfactants, Tween 80, was found to be a good inducer for the release of extracellular depolymerase enzyme as compared to Tween 20. When the PHBV depolymerase activity of isolates present in the consortium was determined separately, it was found that the enzyme activity was higher in case of Bacillus sp. AF3. The purification steps included ammonium sulfate fractionation and gel permeation chromatography on Sephadex G-75. The enzyme has been purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular weight was found to be approximately 37 kDa.