Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard
| Autor: | Asad Syed, S. J. Wani, Hesham A. El-Enshasy, Abdulaziz Alqasim, R. Z. Sayyed, Abdullah A. Alyousef |
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| Rok vydání: | 2019 |
| Předmět: |
Molecular biology
Microbacterium Gene Sequencing Hydroxybutyrates Fractional Precipitation Biochemistry 01 natural sciences Sequencing techniques Magnesium DNA sequencing Ammonium Sulfate Precipitation Gel electrophoresis 0303 health sciences Multidisciplinary biology Salting Out Organic Compounds Chemistry Hydrolysis Fatty Acids Chemical Reactions Temperature Hydrogen-Ion Concentration Lipids Precipitation Techniques Nucleic acids Actinobacteria Biodegradation Environmental Ribosomal RNA Physical Sciences Medicine lipids (amino acids peptides and proteins) Research Article Mesophile Cell biology Cellular structures and organelles Microbacterium paraoxydans Science Polyesters India macromolecular substances Research and Analysis Methods 03 medical and health sciences Enzyme kinetics Non-coding RNA Ammonium sulfate precipitation 030304 developmental biology Enzyme Kinetics Biology and life sciences Bacteria Ethanol 010405 organic chemistry Organic Chemistry Organisms Chemical Compounds technology industry and agriculture biology.organism_classification Enzyme assay 0104 chemical sciences Molecular Weight Kinetics Molecular biology techniques Alcohols Enzymology biology.protein RNA Ribosomes Carboxylic Ester Hydrolases |
| Zdroj: | PLoS ONE PLoS ONE, Vol 14, Iss 6, p e0212324 (2019) |
| ISSN: | 1932-6203 |
| Popis: | Poly-β-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymerases from isolates obtained from plastic-contaminated sites that reflect the potential of the source organism is scarce. In this study, we evaluated the production of extracellular PHB depolymerase from Microbacterium paraoxydans RZS6 isolated from the plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters (GC-FAME), and BIOLOG method. Ithydrolyzed PHB on minimal salt medium (MSM) containing PHB as the only source of carbon. The isolate produced PHB depolymerase at 45°C during 48 h of incubation. The enzyme was purified most efficiently using octyl-sepharose CL-4B column, with the highest purification yield of 6.675 Umg-1mL-1. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ ions but inhibited by Fe2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg2+ ions, that showed optimum enzyme activity at 30°C (mesophilic) and under neutrophilic (pH 7) conditions. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield by 0.809 UmL-1. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled the PHB depolymerase of Aureobacterium saperdae. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase having potential of degrading PHB under diverse conditions. |
| Databáze: | OpenAIRE |
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