Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Shah, Aamer Ali; Hasan, Fariha; Hameed, Abdul; Ahmed, Safia

doi:10.1007/bf03175359

Cited by 23 publications

(10 citation statements)

References 45 publications

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“…PHBV depolymerase of Bacillus sp. AF3 and Streptoverticillium kashmirense AF1 have also been purified on Sephadex G-75 [30]. Kim et al [31] have also purified PHB depolymerase of Emericellopsis minima W2 and Streptomyces sp.…”

Section: Production Of Phb Depolymerasementioning

confidence: 99%

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7

et al. 2020

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There are numerous reports on poly-β-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30˚C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg -1 mL -1 ). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the PLOS ONE | https://doi.org/10.1371/journal.pone.0220095 January 7, 2020 1 / 18 OPEN ACCESS Citation: Sayyed RZ, Wani SJ, Alarfaj AA, Syed A, El-Enshasy HA (2020) Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7. PLoS ONE 15(1): e0220095. https://doi.org/ 10.

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Section: Production Of Phb Depolymerasementioning

confidence: 99%

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7

et al. 2020

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“…Thus, column chromatography using an octyl-sepharose CL - 4B column resulted in the most efficient purification. Purification of PHB depolymerase from various organisms has been carried out using Sephadex columns [33–34]. Wang et al [29], DEAE-Sepharose column and using column chromatography [6–8].…”

Section: Resultsmentioning

confidence: 99%

Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard

et al. 2019

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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 -1 mL -1 . The activity of the enzyme was enhanced in the presence of Ca 2+ and Mg 2+ ions but inhibited by Fe 2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg 2+ 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.

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“…Purification of PHB depolymerase from various organisms has been carried out using Sephadex columns, e.g., PHB depolymerase of Bacillus sp. (1.79 U/mg) (Shah et al 2007) (32) Streptoverticillium kashmirense AF1, and Streptomyces ascomycinicus (Garcia et al 2013) (33). Wang et al (2012) (28) reported the purification of an extracellular PHB depolymerase from Pseudomonas mendocina DSWY0601 using a DEAE-Sepharose column.…”

Section: Resultsmentioning

confidence: 99%

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

Sayyed

Wani

Alyousef

et al. 2019

Preprint

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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...

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Isolation and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading actinomycetes and purification of PHBV depolymerase from newly isolatedStreptoverticillium kashmirense AF1

Cited by 23 publications

References 45 publications

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7

Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7

Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard

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

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