Production, purification, and kinetics of the poly-β-hydroxybutyrate depolymerase fromMicrobacterium paraoxydansRZS6: A novel biopolymer-degrading organism isolated from a dumping yard

Poly-β-hydroxybutyrate (PHB) depolymerase can decompose biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, few reports have described PHB depolymerases based on isolates obtained from plastic-contaminated sites that reflect the potential of the source organism. In this study, we evaluated Microbacterium paraoxydans RZS6 as a producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using the polyphasic approach, i.e., 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters, and BIOLOG identification, and was found to hydrolyze PHB on minimal salt medium containing PHB as the only source of carbon. Both isolates produced PHB depolymerase at 30°C within 2 days and at 45°C within 4 days. The enzyme was purified most efficiently using an octyl-sepharose CL - 4B column, with the highest purification yield of 6.675 U/mg/mL. The enzyme required Ce 2+ and Mg 2+ ions but was inhibited by Fe 2+ ions and mercaptoethanol. Moreover, enzyme kinetic analysis revealed that the enzyme was a metalloenzyme requiring Mg 2+ ions, with optimum enzyme activity at 45°C (thermophilic) and under neutrophilic conditions (optimum pH = 7). The presence of Fe 2+ ions (1 mM) and mercaptoethanol (1000 ppm) completely inhibited the enzyme activity. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled that of PHB depolymerase from Aureobacterium saperdae . Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra, India.