A sensitive pH indicator-based spectrophotometric assay for PHB depolymerase activity on microtiter plates

A continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed.

Production, purification and evaluation of biodegrading potential of PHB depolymerase of Stenotrophomonas sp. RZS 7

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/bioplastic degradation. 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. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-β-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U mg-1 ml-1) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae. Experiments on assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS 7. The results obtained in FTIR analysis, HPLC study and GC-MS analysis confirmed the biodegradation attempt in liquid medium by Stenotrophomonas sp. RZS 7. Changes in surface morphology of PHB film in soil burial as observed in FE SEM analysis confirmed the biodegradation of PHB. The isolate was capable of degrading PHB and resulted in 87.74% degradation. Higher rate of degradation under natural soil condition is the result of activity of soil microbes that complemented the degradation by Stenotrophomonas sp. RZS 7.

Thermophilic PHB depolymerase of Stenotrophomonas sp., an isolate from the plastic contaminated site is best purified on Octyl-Sepharose CL-4B

There are numerous reports on PHB depolymerases produced by a wide variety of microorganisms isolated from various habitats, however, reports on PHB depolymerase isolated from plastic contaminated sites are scares. Thermophilic PHB polymerase produced by isolates obtained from plastic contaminated sites is expected to have better relevance for its application in plastic/ bioplastic degradation. 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. RZS 7 isolated from a plastic contaminated site. The isolate produced extracellular poly-β-hydroxybutyrate (PHB) depolymerase in the mineral salt medium at 30oC during 4 days of incubation under shake flask condition. Purification of the enzyme was carried out by three different methods using PHB as a substrate. Purification of PHB depolymerase by ammonium salt precipitation, column chromatography, and solvent purification method was successfully carried out. Among the purification method tested, the enzyme was best purified by column chromatography on Octyl-Sepharose CL-4B column with maximum (0.7993 U/mg/ml) purification yield. The molecular weight of purified PHB depolymerase (40 kDa) closely resembled with PHB depolymerase of Aureobacterium saperdae.

Production, purification, and kinetics of the poly-β-hydroxybutyrate depolymerase from Microbacterium paraoxydans RZS6: 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 Ce2+ and Mg2+ ions but was inhibited by Fe2+ ions and mercaptoethanol. Moreover, enzyme kinetic analysis revealed that the enzyme was a metalloenzyme requiring Mg2+ ions, with optimum enzyme activity at 45°C (thermophilic) and under neutrophilic conditions (optimum pH = 7). The presence of Fe2+ 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.

A novel, thermotolerant, extracellular PHB depolymerase producer Paenibacillus alvei PHB28 for bioremediation of biodegradable plastics

Background Poly-β-hydroxybutyrate (PHB) is the most important and versatile class of biodegradable polymers used successfully in the medical, agricultural and industrial field. Idea is to find the novel isolate for degradation of biodegradable plastics that can enhance the bioremediation. Materials and methods Thirty-one PHB and PHB depolymerase enzyme producing isolates out of 80 mesophilic bacteria from Lucknow region were further screened for PHB degradation capability by secreting extracellular PHB depolymerase enzyme in minimal salt media supplemented with PHB (0.15%). Various biodegradable plastic films were tested by soil burial method for weight loss determination. Result 37.3% weight loss has been observed in PHB films when buried under the soil for 45 days in the presence of a novel PHB degrader identified as Paenibacillus alvei PHB28 by 16S rRNA sequencing (GenBank accession number KX886342). These Gram-negative, spore-forming, thermotolerant bacteria produce maximum PHB depolymerase (5.03 U/mL) at 45°C, pH 8.0, with 0.15% substrate concentration when incubated for 96 h with starch (0.1%) and yeast extract (0.01%) as an additional nutrient supplements. Conclusion To the best of our knowledge this is the first report of PHB depolymerase production by P. alvei PHB28 which may contribute successfully to combat plastic pollution and to sustain the green environment.

Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 ofRalstonia eutrophaAre PhosphorylatedIn Vivo

ABSTRACTIn this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 ofRalstonia eutrophafor the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability ofR. eutrophato mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCEPoly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules inRalstonia eutropha(alternative designation,Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conductedin vitroandin vivoanalyses of PHB synthase activity and PHB contents.