Poly 3-hydroxybutyrate Depolymerase Research Articles

Abstract 1225 Bioplastic degradation by a polyhydroxybutyrate depolymerase from a thermophilic soil bacterium

Abstract 1225 Bioplastic degradation by a polyhydroxybutyrate depolymerase from a thermophilic soil bacterium

Biosynthesis of polyhydroxybutyrate by Methylorubrum extorquens DSM13060 is essential for intracellular colonization in plant endosymbiosis.

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, ΔphaR had a significantly increased number of granules, and ΔphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the ΔphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis.

Metagenome-Assembled Genomes of Four Southern Ocean Archaea Harbor Multiple Genes Linked to Polyethylene Terephthalate and Polyhydroxybutyrate Plastic Degradation.

Here, we present four archaeal metagenome-assembled genomes (MAGs) (three Thaumarchaeota MAGs and one Thermoplasmatota MAG) from a polar upwelling zone in the Southern Ocean. These archaea harbor putative genes encoding enzymes such as polyethylene terephthalate (PET) hydrolases (PETases) and polyhydroxybutyrate (PHB) depolymerases, which are associated with microbial degradation of PET and PHB plastics.

Bioplastic degradation by a polyhydroxybutyrate depolymerase from a thermophilic soil bacterium.

As the epidemic of single‐use plastic worsens, it has become critical to identify fully renewable plastics such as those that can be degraded using enzymes. Here we describe the structure and biochemistry of an alkaline poly[(R)‐3‐hydroxybutyric acid] (PHB) depolymerase from the soil thermophile Lihuaxuella thermophila. Like other PHB depolymerases or PHBases, the Lihuaxuella enzyme is active against several different polyhydroxyalkanoates, including homo‐ and heteropolymers, but L. thermophila PHB depolymerase (LtPHBase) is unique in that it also hydrolyzes polylactic acid and polycaprolactone. LtPHBase exhibits optimal activity at 70°C, and retains 88% of activity upon incubation at 65°C for 3 days. The 1.2 Å resolution crystal structure reveals an α/β‐hydrolase fold typical of PHBases, but with a shallow active site containing the catalytic Ser‐His‐Asp‐triad that appears poised for broad substrate specificity. LtPHBase holds promise for the depolymerization of PHB and related bioplastics at high temperature, as would be required in bioindustrial operations like recycling or landfill management.

Responses of Alcanivorax species to marine alkanes and polyhydroxybutyrate plastic pollution: Importance of the ocean hydrocarbon cycles

Understanding microbial responses to hydrocarbon and plastic pollution are crucial for limiting the detrimental impacts of environmental contaminants on marine ecosystems. Herein, we reported a new Alcanivorax species isolated from the North Atlantic Ocean capable of degrading alkanes and polyhydroxybutyrate (PHB) plastic (one of the emerging bioplastics that may capture the future plastic market). The whole-genome sequencing showed that the species harbors three types of alkane 1-monooxygenases (AlkB) and one PHB depolymerase (PhaZ) to initiate the degradation of alkanes and plastics. Growth profiling demonstrated that n-pentadecane (C15, the main alkane in the marine environment due to cyanobacterial production other than oil spills) and PHB could serve as preferential carbon sources. However, the cell membrane composition, PhaZ activity, and expression of three alkB genes were utterly different when grown on C15 and PHB. Further, Alcanivorax was a well-recognized alkane-degrader that participated in the ocean hydrocarbon cycles linking with hydrocarbon production and removal. Our discovery supported that the existing biogeochemical processes may add to the marine ecosystem's resilience to the impacts of plastics.

The Carbon Source Effect on the Production of Ralstonia eutropha H16 and Proteomic Response Underlying Targeting the Bioconversion with Solar Fuels.

Chemoautotrophic bacterium Ralstonia eutropha H16 can fix CO2 to bioplastic and is potentially useful for CO2 neutralization. Targeting the solar fuel-based plastic biomanufactory, the polyhydroxybutyrate (PHB) production between heterotrophy and chemoautotrophy conditions was evaluated and the proteomic responses of the R. eutropha H16 cells to different carbon and energy sources were investigated. The results show that the chemoautotrophic mode hardly affected the cellular PHB accumulation capacity. Benefited from the high coverage proteome data, the global response of R. eutropha H16 to different carbon and energy sources was presented with a 95% KEGG pathway annotation, and the genome-wide location-related protein expression pattern was also identified. PHB depolymerase Q0K9H3 was found as a key protein responding to the low carbon input while CO2 and H2 were used, and will be a new regulation target for further high PHB production based on solar fuels.

Polyhydroxybutyrate degradation by biocatalyst of municipal sludge water and degradation efficacy in sequencing batch biofilm reactor

The main aim of the study was to degrade poly-β-hydroxybutyrate (P(3HB)) in the sequencing batch biofilm reactor (SBBR) using biocatalyst. Enrichment method was used for the isolation of P(3HB) degrading bacteria. These bacterial strains were isolated from the wastewater sludge sample treated with P(3HB) sheets. A total of 75 bacteria were isolated after 60 days of incubation. The zone of clearance varied between 12 ± 1 mm and 19 ± 2 mm. Two bacterial strains (Nitrobacter vulgaris SW1 and Pseudomonas aeruginosa KS10) showed rapid PHB degradation activity on agar plates. Plate screening experiments confirmed PHB degrading ability of P. aeruginosa KS10 and N. vulgaris SW1. Biodegrading potential improved after 72 h fermentation period. The bacteria produced depolymerase and enzyme activity was maximum after 72 h. The sequencing batch biofilm reactor (SBBR) co-cultured with N. vulgaris SW1 and P. aeruginosa KS10 was operated to remove PHB from the wastewater. Biofilm in the reactor degraded PHB and the production of polyhydroxybutyrate depolymerase influenced on PHB degradation. Polyhydroxybutyrate degradation improved continuously and maximum degradation (95.6%) was achieved after 8 days. The degradation of biopolymers help to reduce environmental pollution associated with the petroleum based polymers.

Improvement of polyhydroxybutyrate (PHB) plate-based screening method for PHB degrading bacteria using cell-grown amorphous PHB and recovered by sodium dodecyl sulfate (SDS)

Poly(3-hydroxybutyrate) (PHB) is a biobased and biodegradable plastic. Considering the environmental issues of petroleum-based plastics, PHB is promising as it can be degraded in a relatively short time by bacteria to water and carbon dioxide. Substantial efforts have been made to identify PHB-degrading bacteria. To identify PHB-degrading bacteria, solid-based growth or clear zone assays using PHB as the sole carbon source are the easiest methods; however, PHB is difficult to dissolve and distribute evenly, and bacteria grow slowly on PHB plates. Here, we suggest an improved PHB plate assay using cell-grown PHB produced by Halomonas sp. and recovered by sodium dodecyl sulfate (SDS). Preparation using SDS resulted in evenly distributed PHB plates that could be used for sensitive depolymerase activity screening in less time compared with solvent-melted pellet or cell-grown PHB. With this method, we identified 15 new strains. One strain, Cutibacterium sp. SOL05 (98.4% 16S rRNA similarity to Cutibacterium acne), showed high PHB depolymerase activity in solid and liquid conditions. PHB degradation was confirmed by clear zone size, liquid culture, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The results indicate this method can be used to easily identify PHB-degrading bacteria from various sources to strengthen the benefits of bioplastics.

Streamlined production, purification, and characterization of recombinant extracellular polyhydroxybutyrate depolymerases.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation.

Coordinated Regulation of the Size and Number of Polyhydroxybutyrate Granules by Core and Accessory Phasins in the Facultative Microsymbiont Sinorhizobium fredii NGR234.

The exact roles of various granule-associated proteins (GAPs) of polyhydroxybutyrate (PHB) are poorly investigated, particularly for bacteria associated with plants. In this study, four structural GAPs, named phasins PhaP1 to PhaP4, were identified and demonstrated as true phasins colocalized with PHB granules in Sinorhizobium fredii NGR234, a facultative microsymbiont of Vigna unguiculata and many other legumes. The conserved PhaP2 dominated in regulation of granule size under both free-living and symbiotic conditions. PhaP1, another conserved phasin, made a higher contribution than accessory phasins PhaP4 and PhaP3 to PHB biosynthesis at stationary phase. PhaP3, with limited phyletic distribution on the symbiosis plasmid of Sinorhizobium, was more important than PhaP1 in regulating PHB biosynthesis in V. unguiculata nodules. Under the test conditions, no significant symbiotic defects were observed for mutants lacking individual or multiple phaP genes. The mutant lacking two PHB synthases showed impaired symbiotic performance, while mutations in individual PHB synthases or a PHB depolymerase yielded no symbiotic defects. This phenomenon is not related to either the number or size of PHB granules in test mutants within nodules. Distinct metabolic profiles and cocktail pools of GAPs of different phaP mutants imply that core and accessory phasins can be differentially involved in regulating other cellular processes in the facultative microsymbiont S. fredii NGR234.IMPORTANCE Polyhydroxybutyrate (PHB) granules are a store of carbon and energy in bacteria and archaea and play an important role in stress adaptation. Recent studies have highlighted distinct roles of several granule-associated proteins (GAPs) in regulating the size, number, and localization of PHB granules in free-living bacteria, though our knowledge of the role of GAPs in bacteria associated with plants is still limited. Here we report distinct roles of core and accessory phasins associated with PHB granules of Sinorhizobium fredii NGR234, a broad-host-range microsymbiont of diverse legumes. Core phasins PhaP2 and PhaP1 are conserved major phasins in free-living cells. PhaP2 and accessory phasin PhaP3, encoded by an auxiliary gene on the symbiosis plasmid, are major phasins in nitrogen-fixing bacteroids in cowpea nodules. GAPs and metabolic profiles can vary in different phaP mutants. Contrasting symbiotic performances between mutants lacking PHB synthases, depolymerase, or phasins were revealed.

Biodegradation of polyhydroxybutyrate by Pseudomonas sp. DSDY0501 and purification and characterization of polyhydroxybutyrate depolymerase.

Strain DSDY0501 with polyhydroxybutyrate (PHB)-degrading ability was isolated from activated sludge. The morphological, physiological, and biochemical properties of the strain and phylogenetic analysis indicated that the strain belongs to Pseudomonas sp. The strain used PHB as the sole carbon source and degraded PHB films completely in 21h in liquid culture. An extracellular PHB depolymerase was purified from the supernatant of the culture by ultrafiltration and Sephacryl S-200 gel filtration. The specific activity of the purified enzyme increased 24.2-fold, and the recovery yield was 16.61%. Extracellular PHB depolymerase, a monomeric enzyme with a molecular weight of approximately 57.9kDa, showed optimum activity at 60°C and pH 9.0, and was stable in the temperature range of 10-60°C and a pH range of 6.0-10.0. The secondary structure of the enzyme contained approximately 60% α-helix and 40% β-pleated sheet according to the circular dichroism spectrum. Mass spectrum analysis showed that the main degradation product of the enzyme was PHB monomer, indicating the exo-type action of this PHB depolymerase.

Shotgun Metagenomics Reveals the Benthic Microbial Community Response to Plastic and Bioplastic in a Coastal Marine Environment.

Plastic is incredibly abundant in marine environments but little is known about its effects on benthic microbiota and biogeochemical cycling. This study reports the shotgun metagenomic sequencing of biofilms fouling plastic and bioplastic microcosms staged at the sediment–water interface of a coastal lagoon. Community composition analysis revealed that plastic biofilms were indistinguishable in comparison to a ceramic biofilm control. By contrast, bioplastic biofilms were distinct and dominated by sulfate-reducing microorganisms (SRM). Analysis of bioplastic gene pools revealed the enrichment of esterases, depolymerases, adenylyl sulfate reductases (aprBA), and dissimilatory sulfite reductases (dsrAB). The nearly 20-fold enrichment of a phylogenetically diverse polyhydroxybutyrate (PHB) depolymerase suggests this gene was distributed across a mixed microbial assemblage. The metagenomic reconstruction of genomes identified novel species of Desulfovibrio, Desulfobacteraceae, and Desulfobulbaceae among the abundant SRM, and these genomes contained genes integral to both bioplastic degradation and sulfate reduction. Findings indicate that bioplastic promoted a rapid and significant shift in benthic microbial diversity and gene pools, selecting for microbes that participate in bioplastic degradation and sulfate reduction. If plastic pollution is traded for bioplastic pollution and sedimentary inputs are large, the microbial response could unintentionally affect benthic biogeochemical activities through the stimulation of sulfate reducers.

Relationship between the peel strength and microdomain orientation in the interfacial region for a block copolymerbased pressure-sensitive adhesive

Abstract Aspergillus fumigatus strain 76T-3 formed clear zones on agar plates containing emulsified polyhydroxybutyrate (PHB), polyethylene succinate (PES), polybutylene succinate (PBS), polycaprolactone (PCL), or polylactide (PLA). The strain grew well at 40 °C in Sabouraud Dextrose Broth. Solution-casted PHB films were almost completely degraded after incubation with 76T-3 at 45 °C for 17 h. An extracellular polyester-degrading enzyme was purified from the supernatant of 76T-3 cultures in basal medium containing PHB as the sole carbon source. Zymography results portrayed that the purified enzyme degraded PHB, PES, and PBS but not PCL or PLA. The amino acid sequence obtained from LC-MS/MS identified this enzyme to be a PHB depolymerase with a molecular mass of 57 kDa. The optimal reaction condition for the enzyme was pH 6.4 at 55 °C. The recombinant PHB depolymerase (rPhaZ) expressed in E. coli showed the enzyme can act on PHB only and not on PES or PBS.

Polyhydroxybutyrate (PHB) biodegradation using bacterial strains with demonstrated and predicted PHB depolymerase activity

The biodegradation of polyhydroxybutyrate (PHB) has been broadly investigated, but studies typically focus on a single strain or enzyme and little attention has been paid to comparing the interaction of different PHB depolymerase (PhaZ)-producing strains with this biopolymer. In this work, we selected nine bacterial strains-five with demonstrated and four with predicted PhaZ activity-to compare their effectiveness at degrading PHB film provided as sole carbon source. Each of the strains with demonstrated activity were able to use the PHB film (maximum mass losses ranging from 12% after 2days for Paucimonas lemoignei to 90% after 4days for Cupriavidus sp.), and to a lower extent Marinobacter algicola DG893 (with a predicted PhaZ) achieved PHB film mass loss of 11% after 2weeks of exposure. Among the strains with proven PhaZ activity, Ralstonia sp. showed the highest specific activity since less biomass was required to degrade the polymer in comparison to the other strains. In the case of Ralstonia sp., PHB continued to be degraded at pH values as low as pH3.3-3.7. In addition, analysis of the extracellular fractions of the strains with demonstrated activity showed that Comamonas testosteroni, Cupriavidus sp., and Ralstonia sp. readily degraded both PHB film and PHB particles in agar suspensions. This study highlights that whole cell cultures and enzymatic (extracellular) fractions display different levels of activity, an important factor in the development of PHB-based applications and in understanding the fate of PHB and other PHAs released in the environment. Furthermore, predictions of PhaZ functionality from genome sequencing analyses remain to be validated by experimental results; PHB-degrading ability could not be proven for three of four investigated species predicted by the polyhydroxyalkanoates (PHA) depolymerase engineering database.

Purification, characterization, and gene cloning of an Aspergillus fumigatus polyhydroxybutyrate depolymerase used for degradation of polyhydroxybutyrate, polyethylene succinate, and polybutylene succinate

Aspergillus fumigatus strain 76T-3 formed clear zones on agar plates containing emulsified polyhydroxybutyrate (PHB), polyethylene succinate (PES), polybutylene succinate (PBS), polycaprolactone (PCL), or polylactide (PLA). The strain grew well at 40 °C in Sabouraud Dextrose Broth. Solution-casted PHB films were almost completely degraded after incubation with 76T-3 at 45 °C for 17 h. An extracellular polyester-degrading enzyme was purified from the supernatant of 76T-3 cultures in basal medium containing PHB as the sole carbon source. Zymography results portrayed that the purified enzyme degraded PHB, PES, and PBS but not PCL or PLA. The amino acid sequence obtained from LC-MS/MS identified this enzyme to be a PHB depolymerase with a molecular mass of 57 kDa. The optimal reaction condition for the enzyme was pH 6.4 at 55 °C. The recombinant PHB depolymerase (rPhaZ) expressed in E. coli showed the enzyme can act on PHB only and not on PES or PBS.

Inactivation of an intracellular poly-3-hydroxybutyrate depolymerase of Azotobacter vinelandii allows to obtain a polymer of uniform high molecular mass

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.

Isolation and Characterization of Bacteria that Produce Polyhydroxybutyrate Depolymerases.

Isolation of environmental bacterial strains with diverse metabolic properties such as antibiotic production, sulfide oxidation, and the ability to use different nutrient sources is a common undergraduate lab experience. This article describes a useful protocol for the detection and isolation of bacteria that can degrade the common carbon storage biopolymer poly (3-hydroxybutyrate) (PHB). The procedure utilizes a low nutrient media supplemented with insoluble PHB that allows for the easy, visible detection of strains that produce extracellular PHB degrading enzymes. This method can be used to determine the percent of culturable bacteria from various environments that can degrade PHB. Further, PHB degrading exoenzyme activity can be monitored from pure cultures by: (1) Growing cells in broth, (2) inducing with PHB, and (3) measuring activity of supernatant with a UV/Vis spectrophotometer. The enzymatic breakdown of PHB presents an opportunity to expose students to the concept of using biodegradable plastics as a solution to the global plastic waste problem.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16.

In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutrophaIMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

Developing an efficient strategy for immobilization of PHB depolymerase on magnetite-based nanoparticles for degrading polyhydroxybutyrate in acidic conditions

Several approaches towards the efficient immobilization of poly-3-hydroxybutyrate (PHB) depolymerase from Streptomyces ascomycinicus (PhaZSa) on magnetic-silanized nanoparticles have been explored in order to accomplish the preparation of an active immobilized biocatalyst in PHB hydrolysis in acid conditions. A simple non-covalent adsorption of PhaZSa to silanized magnetic particles at acidic pH during a short incubation time allowed the preparation of immobilized biocatalysts with increased specific activity. In contrast, covalent binding of PhaZSa to silanized magnetite by different methods led to immobilized biocatalysts whose retained activity were low or void.

Biodegradation of different formulations of polyhydroxybutyrate films in soil.

BackgroundPetroleum polymers contribute to non-degradable waste materials and it would therefore be desirable to produce ecofriendly degradable materials. Biodegradation of polyhydroxybutyrate (PHB) in the presence of oligomer hydrolase and PHB depolymerase gave 3-hydroxybutyric acid which could be oxidized to acetyl acetate. Several bacteria and fungi can degrade PHB in the soil.ResultsBiodegradation of PHB showed a significant decrease in the molecular weight (Mw), number-average molecular weight (Mn) and the dispersity (Mw/Mn) for all the film formulations. Nanofibers of PHB and its composites showed faster degradation compared to other films and displayed complete degradation after 3 weeks. The SEM micrographs showed various surface morphology changes including alterations in appearance of pores, cavity, grooves, incisions, slots and pointers. Such changes were due to the growth of microorganisms that secreted PHB depolymerase enzyme which lead to the biopolymer films degradation. However, PHB nanofibers and its composites films in the presence of TiO2 demonstrated more surface changes with rupture of most nanofibers in which there was a drop in fibres diameter.ConclusionsThe degradation of biopolymers help to overcome some of the pollution problems associated with the use of petroleum polymers. PHB nanofiber and its TiO2 composite were degraded faster compared to other PHB film types due to their three dimensional and high surface area structures. The presence of TiO2 nanoparticles in the composite films slowdown the degradation process compared to PHB films. Additionally, the PHB and its composite films that were prepared from UV treated PHB films led to acceleration of the degradation.Graphical abstractBiodegradation of polyhydroxybutyrate films in soil