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

Abstract

27Poly-β-hydroxybutyrate (PHB) depolymerase can decompose biodegradable polymers and 28 therefore has great commercial significance in the bioplastic sector. However, few reports 29 have described PHB depolymerases based on isolates obtained from plastic-contaminated 30 sites that reflect the potential of the source organism. In this study, we evaluated 31 Microbacterium paraoxydans RZS6 as a producer of extracellular PHB depolymerase 32 isolated from a plastic-contaminated site in the municipal area of Shahada, Maharashtra, 33 India, for the first time. The isolate was identified using the polyphasic approach, i.e., 16S 34 rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters, and 35 BIOLOG identification, and was found to hydrolyze PHB on minimal salt medium 36 containing PHB as the only source of carbon. Both isolates produced PHB depolymerase at 37 30°C within 2 days and at 45°C within 4 days. The enzyme was purified most efficiently 38 using an octyl-sepharose CL-4B column, with the highest purification yield of 6.675 39 U/mg/mL. The enzyme required Ce 2+ and Mg 2+ ions but was inhibited by Fe 2+ ions and 40 mercaptoethanol. Moreover, enzyme kinetic analysis revealed that the enzyme was a 41 metalloenzyme requiring Mg 2+ ions, with optimum enzyme activity at 45°C (thermophilic) 42 and under neutrophilic conditions (optimum pH = 7). The presence of Fe 2+ ions (1 mM) and 43 mercaptoethanol (1000 ppm) completely inhibited the enzyme activity. The molecular weight 44 of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel 45 electrophoresis, closely resembled that of PHB depolymerase from Aureobacterium 46 saperdae. Scale-up from the shake-flask level to a laboratory-scale bioreactor further 47 enhanced the enzyme yield. Our findings highlighted the applicability of M. paraoxydans as a 48 producer of extracellular PHB depolymerase isolated from a plastic-contaminated site in the 49 municipal area of Shahada, Maharashtra, India. Introduction 54 Poly-β-hydroxy alkanoates (PHAs) and poly-β-hydroxybutyrate (PHB) are stored as food and 55 energy sources in bacteria under a carbon-rich environment and are catabolized during 56 nutrient stress conditions under the influence of PHB depolymerase. PHB is a biocompatible, 57 thermoplastic, nontoxic, completely biodegradable molecule exhibiting the properties of 58 synthetic plastics. Moreover, PHB is easily degraded by PHB depolymerases and hence is an 59 eco-friendly alternative to recalcitrant synthetic plastics (1-5)(Sayyed and Chincholkar 2009; 60 Gangure et al. 2017; Wani et al. 2016; Wani and Sayyed 2016; Soam et al. 2012). 61 PHB is degraded under natural conditions by the actions of PHB depolymerases produced 62 by a wide variety of microorganisms (6, 7) (Papaneophytou et al. 2009; Hsu et al. 2012). 63Although PHB has commercial applications, identification of potent PHB degraders from 64 relevant habitats and evaluation of their production, scale-up purification, and enzyme kinetic 65 must be carried out. Repor...