Depolymerase Enzyme Research Articles

More than PHB – the PHAmily of Copolymers: Justifications for Broader Use and Summary of Biodegradation Facts on Polyhydroxyalkanoates (PHA) – A Review

Globally, a significant portion of plastics are mismanaged, ultimately ending up in <br /> the environment. While reducing, reusing, and recycling plastics should be the preferred <br /> end-of-life scenarios, biodegradable plastics are compatible with all of these routes and <br /> represent a promising alternative. Polyhydroxyalkanoates (PHAs), a family of polyesters <br /> synthesized by various microorganisms, stand out in this regard. The most common type <br /> of PHA, poly(3-hydroxybutyrate) (PHB), is particularly relevant as it is both biobased <br /> and biodegradable. Additionally, there is substantial knowledge about this thermoplastic <br /> polyester, its applications, and its commercial availability. However, over 150 other PHA <br /> building blocks have been identified, with some having been tested and developed for <br /> various promising applications. Despite this, not all PHA copolymers have been fully <br /> evaluated for their biodegradability across diverse environmental conditions. Nevertheless, due to their biosynthesis in living microorganisms, a good degree of biodegradability is expected. Moreover, PHAs can also be chemically recycled into valuable chemicals, positioning them as the biobased polymer with the most versatile end-of-life options. <br /> This review summarizes the current state of knowledge on PHA copolymers, their cur<br /> rent end-of-life pathways, the most relevant biodegradability standards, and their degradability by depolymerase enzymes.

Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

A new Rogue-like Escherichia phage UDF157lw to control Escherichia coli O157:H7.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is one of the notorious foodborne pathogens causing high mortality through the consumption of contaminated food items. The food safety risk from STEC pathogens could escalate when a group of bacterial cells aggregates to form a biofilm. Bacterial biofilm can diminish the effects of various antimicrobial interventions and enhance the pathogenicity of the pathogens. Therefore, there is an urgent need to have effective control measurements. Bacteriophages can kill the target bacterial cells through lytic infection, and some enzymes produced during the infection have the capability to penetrate the biofilm for mitigation compared to traditional interventions. This study aimed to characterize a new Escherichia phage vB_EcoS-UDF157lw (or UDF157lw) and determine its antimicrobial efficacy against E. coli O157:H7. Phage characterization included biological approaches, including phage morphology, one-step growth curve, stability tests (pH and temperature), and genomic approaches (whole-genome sequencing). Later, antimicrobial activity tests, including productive infection against susceptible bacterial strains, in vitro antimicrobial activity, and anti-biofilm, were conducted. UDF157lw is a new member of the phages belonging to the Rogunavirus genus, comprising a long and non-contractile tail, isolated from bovine feces and shares close genomic evolutionary similarities with Escherichia phages vB_EcoS-BECP10 and bV_EcoS_AKS96. When used against E. coli O157:H7 (ATCC35150), phage UDF157lw exhibited a latent period of 14 min and a burst size of 110 PFU per infected cell. The phage remained viable in a wide range of pH values (pH 4-11) and temperatures (4-60°C). No virulence genes, such as stx, lysogenic genes, and antibiotic resistance genes, were found. Phage UDF157lw demonstrated high infection efficiencies against different E. coli O157:H7 and generic E. coli strains. In addition, UDF157lw encoded a unique major tail protein (ORF_26) with prominent depolymerase enzyme activity against various E. coli O157:H7 strains, causing large plaque sizes. In contrast to the phage without encoding depolymerase gene, UDF157lw was able to reduce the 24-h and 48-h E. coli O157:H7 biofilm after 1-h phage treatment. The findings of this study provide insights into a new member of the Rogunavirus phages and demonstrate its antimicrobial potential against E. coli O157:H7 in vitro.

Phage-encoded depolymerases as a strategy for combating multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii, a predominant nosocomial pathogen, represents a grave threat to public health due to its multiple antimicrobial resistance. Managing patients afflicted with severe infections caused by multiple drug-resistant A. baumannii is particularly challenging, given the associated high mortality rates and unfavorable prognoses. The diminishing efficacy of antibiotics against this superbug underscores the urgent necessity for novel treatments or strategies to address this formidable issue. Bacteriophage-derived polysaccharide depolymerase enzymes present a potential approach to combating this pathogen. These enzymes target and degrade the bacterial cell's exopolysaccharide, capsular polysaccharide, and lipopolysaccharide, thereby disrupting biofilm formation and impairing the bacteria's defense mechanisms. Nonetheless, the narrow host range of phage depolymerases limits their therapeutic efficacy. Despite the benefits of these enzymes, phage-resistant strains have been identified, highlighting the complexity of phage-host interactions and the need for further investigation. While preliminary findings are encouraging, current investigations are limited, and clinical trials are imperative to advance this treatment approach for broader clinical applications. This review explores the potential of phage-derived depolymerase enzymes against A. baumannii infections.

Purification and characterization of extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) and their biodegradation studies with polymer films.

PHB depolymerase enzymes are able to breakdown the PHB polymers and thereby get significant economic value in the bioplastics industry and for bioremediation as well. This study shows the purification of novel extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) using dialysis followed by gel filtration and HPLC. The purification fold and yield after HPLC were 45.92 and 27.04%, respectively. HPLC data showed a single peak with a retention time of 1.937min. GC-MS analysis reveals the presence of three compounds, of which 1-Dodecanol was found to be most significant with 54.48% area and 8.623-min retention time (RT). The molecular weight of the purified enzyme was obtained as 35kDa with Km and apparent Vmax values of 0.769mg/mL and 1.89 U/mL, respectively. The enzyme was moderately active at an optimum temperature of 35 °C and at pH 8.0. The stability was detected at pH 7.0-9.0 and 35-45 °C. Complete activity loss was observed with EDTA, SDS, Tween-20 at 5 mM and with 0.1% Triton X 100. A biodegradation study of commercially available biodegradable polymer films was carried out in a liquid medium and in soil separately with pure microbial culture and with purified enzyme for 7, 14, 28, and 49 consecutive days. In a liquid medium, with a pure strain of Aeromonas caviae Kuk1-(34), the maximum degradation (89%) was achieved on the PHB film, while no changes were observed with other polymer films. With purified enzyme in the soil, 71% degradation of the PHB film was noticed, and it was only 18% in the liquid medium. All such weight analysis were confirmed by SEM images where several holes, pits, grooves, crest, and surface roughness are clearly observed. Our results demonstrated the potential utility of Aeromonas caviae Kuk1-(34) as a source of extracellular PHB depolymerase capable of degrading PHB under a wide range of natural/ lab conditions.

Characterization of a Lytic Bacteriophage and Demonstration of Its Combined Lytic Effect with a K2 Depolymerase on the Hypervirulent Klebsiella pneumoniae Strain 52145

Klebsiella pneumoniae is a nosocomial pathogen. Among its virulence factors is the capsule with a prominent role in defense and biofilm formation. Bacteriophages (phages) can evoke the lysis of bacterial cells. Due to the mode of action of their polysaccharide depolymerase enzymes, phages are typically specific for one bacterial strain and its capsule type. In this study, we characterized a bacteriophage against the capsule-defective mutant of the nosocomial K. pneumoniae 52145 strain, which lacks K2 capsule. The phage showed a relatively narrow host range but evoked lysis on a few strains with capsular serotypes K33, K21, and K24. Phylogenetic analysis showed that the newly isolated Klebsiella phage 731 belongs to the Webervirus genus in the Drexlerviridae family; it has a 31.084 MDa double-stranded, linear DNA with a length of 50,306 base pairs and a G + C content of 50.9%. Out of the 79 open reading frames (ORFs), we performed the identification of orf22, coding for a trimeric tail fiber protein with putative capsule depolymerase activity, along with the mapping of other putative depolymerases of phage 731 and homologous phages. Efficacy of a previously described recombinant K2 depolymerase (B1dep) was tested by co-spotting phage 731 on K. pneumoniae strains, and it was demonstrated that the B1dep-phage 731 combination allows the lysis of the wild type 52145 strain, originally resistant to the phage 731. With phage 731, we showed that B1dep is a promising candidate for use as a possible antimicrobial agent, as it renders the virulent strain defenseless against other phages. Phage 731 alone is also important due to its efficacy on K. pneumoniae strains possessing epidemiologically important serotypes.

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium.

A high-throughput expression and screening platform for applications-driven PETase engineering.

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.

Bacteriophage DW-EC with the capability to destruct and inhibit biofilm formed by several pathogenic bacteria

Biofilm formation by pathogenic bacteria is a major challenge in the food industry. Once a biofilm is established, such as on food processing equipment, it becomes more difficult to eradicate. Although physical and chemical treatments are often used to control biofilm formation, these treatments can have significant drawbacks. Alternative biofilm treatments are needed. Phage DW-EC was isolated from dawet, an Indonesian traditional Ready-To-Eat food, which has high specificity for Enterohaemorrhagic Escherichia coli (EHEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC). Phage DW-EC produces several enzymes that can prevent the development of biofilm and biofilm eradication. Depolymerase enzymes break down the polysaccharides layer on the biofilms can lead to biofilm damage. On the other hand, endolysin and putative like-T4 lysozyme will lyse and kill a bacterial cell, thereby preventing biofilm growth. This research aims to determine the capability of previously identified phage DW-EC to inhibit and destroy biofilms produced by several foodborne pathogens. Phage DW-EC formed plaques on the bacterial lawns of EHEC, EPEC, and ETEC. The efficiency of plating (EOP) values for EHEC, EPEC, ETEC, and Bacillus cereus were 1.06, 0.78. 0.70, and 0.00, demonstrating that DW-EC was effective in controlling pathogenic E. coli populations. Furthermore, phage DW-EC showed anti-biofilm activity against foodborne pathogenic bacteria on polystyrene and stainless-steel substrates. DW-EC biofilm inhibition and destruction activities against pathogenic E. coli were significantly higher than against B. cereus biofilms, which was indicated by a lower density of the biofilm than B. cereus. Microscopic visualization verified that bacteriophage DW-EC effectively controlled EHEC, EPEC, and ETEC biofilms. The results showed that DW-EC could inhibit and destroy biofilm, making it promising to be used as an anti-biofilm candidate for polystyrene and stainless steel equipment in the food industry.

Production of Fungal Pectin Lyase and Polygalacturonase from Fruit Wastes by Solid State Fermentation

Polygalacturonase (PG or PGase) and Pectin lyase (PL) are depolymerase enzymes that split the α-1, 4-glycosidic linkages in the backbone of homogalacturonans. They are produced by microorganisms degrading pectin-containing substrates. PG and PL were produced in the solid-state fermentation (SSF) of beans testa (BT), mango peels (MP), Plantain peels (PP), and BT: MP: PP (1:1:1). Substrates were seeded with individual fungal strains namely Aspergillus tamarii, Aspergillus terreus, Aspergillus piperis, Aspergillus parasiticus and Mucor piriformis. PG production ranged from 0.0377 - 141.0095 U/g with A. tamarii, A. terreus and A. piperis producing the highest on mango peels. PL production ranged from 50.50 - 10,852.50 U/g with Mucor piriformis producing the highest on plantain peels. The pH of the fermentation medium changed during growth, metabolism, and pectinase production. The best pH for pectinase production falls within the acidic range. Unconventional substrates such as PP are viable for pectinase production, and PL yield in SSF is improved by compositing substrates.

Isolation and optimization of extracellular PHB depolymerase producer Aeromonas caviae Kuk1-(34) for sustainable solid waste management of biodegradable polymers

Bioplastics, synthesized by several microbes, accumulates inside cells under stress conditions as a storage material. Several microbial enzymes play a crucial role in their degradation. This research was carried to test the biodegradability of poly-β-hydroxybutyrate (PHB) utilizing PHB depolymerase, produced by bacteria isolated from sewage waste soil samples. Potent PHB degrader was screened based on the highest zone of hydrolysis followed by PHB depolymerase activity. Soil burial method was employed to check their degradation ability at different incubation periods of 15, 30, and 45 days at 37±2°C, pH 7.0 at 60% moisture with 1% microbial inoculum of Aeromonas caviae Kuk1-(34) (MN414252). Without optimized conditions, 85.76% of the total weight of the PHB film was degraded after 45 days. This degradation was confirmed with Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscope (SEM) analysis. The presence of bacterial colonies on the surface of the degraded film, along with crest, holes, surface erosion, and roughness, were visible. Media optimization was carried out in statistical mode using Plackett Burman (PB) and Central Composite Design (CCD) of Response Surface Methodology (RSM) by considering ten different factors. Analysis of Variance (ANOVA), Pareto chart, response surface plots, and F-value of 3.82 implies that the above statistical model was significant. The best production of PHB depolymerase enzyme (14.98 U/mL) was observed when strain Kuk1-(34) was grown in a media containing 0.1% PHB, K2HPO4 (1.6 gm/L) at 27 ℃ for seven days. Exploiting these statistically optimized conditions, the culture was found to be a suitable candidate for the management of solid waste, where 94.4% of the total weight of the PHB film was degraded after 45 days of incubation.

Novel Drexlerviridae bacteriophage KMI8 with specific lytic activity against Klebsiella michiganensis and its biofilms.

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Isolation and Characterization of a Novel Lytic Bacteriophage against the K2 Capsule-Expressing Hypervirulent Klebsiella pneumoniae Strain 52145, and Identification of Its Functional Depolymerase.

Klebsiella pneumoniae is among the leading bacteria that cause nosocomial infections. The capsule of this Gram-negative bacterium is a dominant virulence factor, with a prominent role in defense and biofilm formation. Bacteriophages, which are specific for one bacterial strain and its capsule type, can evoke the lysis of bacterial cells, aided by polysaccharide depolymerase enzymes. In this study, we isolated and characterized a bacteriophage against the nosocomial K. pneumoniae 52145 strain with K2 capsular serotype. The phage showed a narrow host range and stable lytic activity, even when exposed to different temperatures or detergents. Preventive effect of the phage in a nasal colonization model was investigated in vivo. Phlyogenetic analysis showed that the newly isolated Klebsiella phage B1 belongs to the Webervirus genus in Drexlerviridae family. We identified the location of the capsule depolymerase gene of the new phage, which was amplified, cloned, expressed, and purified. The efficacy of the recombinant B1dep depolymerase was tested by spotting on K. pneumoniae strains and it was confirmed that the extract lowers the thickness of the bacterium lawn as it degrades the protective capsule on bacterial cells. As K. pneumoniae strains possessing the K2 serotype have epidemiological importance, the B1 phage and its depolymerase are promising candidates for use as possible antimicrobial agents.

Biodegradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Newly Isolated Penicillium oxalicum SS2 in Soil Microcosms and Partial Characterization of Extracellular Depolymerase.

A fungus, designated as strain SS2 able to degrade aliphatic polyesters, poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), was isolated from soil. Strain SS2 was identified through rDNA gene sequencing and showed maximum closeness to Penicillium oxalicum. The newly isolated P. oxalicum strain SS2 had completely degraded PHB and PHBV both in emulsion and films form within 36-48h at 30°C. Furthermore, P. oxalicum SS2 degraded PHB and PHBV films in soil environment in lab-built soil microcosms within 1 week. The polymer films were evaluated for changes after degradation through scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and Fourier Transform Infrared spectroscopy (FTIR). The PHBV depolymerase enzyme was purified to homogeneity through column chromatography and molecular mass was found approximately 36kDa. The depolymerase was stable over a wide range of temperature (15-60°C) and pH (3.0-8.0) with optimum 40°C and pH 5.0. The enzyme activity was significantly affected by various metal ions and surfactants. The enzyme activity was strongly enhanced in the presence of divalent cationic metal Cu2+ while inhibited by Zn2+ and non-polar detergents Tween 20 and Tween 60. Finally, it is concluded that P. oxalicum strain SS2 has profound degradation capabilities, and can be applied for the treatment of plastic-contaminated environments.

Relation between Surface Area and Surface Potential Change during (co)Polyesters Degradation as Langmuir Monolayer

ABSTRACTPolyhydroxyalkanoates (PHAs) are degradable (co)polyesters synthesized by microorganisms with a variety of side-chains and co-monomer ratios. PHAs can be efficiently hydrolyzed under alkaline conditions and by PHA depolymerase enzymes, altering their physicochemical properties. Using 2D Langmuir monolayers as model system to study the degradation behavior of macromolecules, we aim to describe the the interdependency between the degradation of two PHAs and the surface potential, which influences material-proteins interaction and cell response. We hypothesize that the mechanism of hydrolysis of the labile ester bonds in (co)polyesters defines the evolution of the surface potential, owing to the rate of accumulation of charged insoluble degradation products. The alkaline hydrolysis and the enzymatically catalyzed hydrolysis of PHAs were previously defined as chain-end scission and random-scission mechanisms, respectively. In this study, these two distinct scenarios are used to validate our model. The surface potential change during the chain-end scission of poly(3-R-hydroxybutyrate) (PHB) under alkaline conditions was compared to that of the enzymatically catalyzed hydrolysis (random-scission) of poly[(3-R-hydroxyoctanoate)-co-(3-R-hydroxyhexanoate)] (PHOHHx), using the Langmuir monolayer technique. In the random-scission mechanism the dissolution of degradation products, measured as a decrease in the area per molecule, was preceded by a substantial change of the surface potential, provoked by the negative charge of the broken ester bonds accumulated in the air-water interface. In contrast, when chains degraded via the chain-ends, the surface potential changed in line with the dissolution of the material, presenting a kinetic dependent on the surface area of the monolayers. These results provide a basis for understanding PHAs degradation mechanism. Future research on (co)polymers with different main-chain lengths might extend the elucidation of the surface potential development of (co)polyesters as Langmuir monolayer.

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

Abstract 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.

Purification and characterization of new bio-plastic degrading enzyme from Burkholderia cepacia DP1

Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0–10 and at 35–55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.

Isolation and Characterization of Polyester-Based Plastics-Degrading Bacteria from Compost Soils

Four potential polyester-degrading bacterial strains were isolated from compost soils in Thailand. These bacteria exhibited strong degradation activity for polyester biodegradable plastics, such as polylactic acid (PLA), polycaprolactone (PCL), poly-(butylene succinate) (PBS) and polybutylene succinate-co-adipate (PBSA) as substrates. The strains, classified according to phenotypic characteristics and 16S rDNA sequence, belonging to the genera Actinomadura, Streptomyces and Laceyella, demonstrated the best polyester- degrading activities. All strains utilized polyesters as a carbon source, and yeast extract with ammonium sulphate was utilized as a nitrogen source for enzyme production. Optimization for polyester-degrading enzyme production by Actinomadura sp. S14, Actinomadura sp. TF1, Streptomyces sp. APL3 and Laceyella sp. TP4 revealed the highest polyester-degrading activity in culture broth when 1% (w/v) PCL (18 U/mL), 0.5% (w/v) PLA (22.3 U/mL), 1% (w/v) PBS (19.4 U/mL) and 0.5% (w/v) PBSA (6.3 U/mL) were used as carbon sources, respectively. All strains exhibited the highest depolymerase activities between pH 6.0–8.0 and temperature 40–60°C. Partial nucleotides of the polyester depolymerase gene from strain S14, TF1 and APL3 were studied. We determined the amino acids making up the depolymerase enzymes had a highly conserved pentapeptide catalytic triad (Gly-His-Ser-Met-Gly), which has been shown to be part of the esterase-lipase superfamily (serine hydrolase).

Enzymatic degradation of poly-[(R)-3-hydroxybutyrate]: Mechanism, kinetics, consequences

Poly-[(R)-3-hydroxybutyrate] (PHB) films prepared by compression molding and solvent casting, respectively, were degraded with the intracellular depolymerase enzyme natively synthetized by the strain Bacillus megaterium. Quantitative analysis proved that practically only (R)-3-hydroxybutyric acid (3-HBA) forms in the enzyme catalyzed reaction, the amount of other metabolites or side products is negligible. The purity of the product was verified by several methods (UV–VIS spectroscopy, liquid chromatography, mass spectroscopy). Degradation was followed as a function of time to determine the rate of enzymatic degradation. Based on the Michaelis-Menten equation a completely new kinetic model has been derived which takes into consideration the heterogeneous nature of the enzymatic reaction. Degradation proceeds in two steps, the adsorption of the enzyme onto the surface of the PHB film and the subsequent degradation reaction. The rate of both steps depend on the preparation method of the samples, degradation proceed almost twice as fast in compression molded films than in solvent cast samples. The model can describe and predict the formation of the reaction product as a function of time. The approach can be used even for the commercial production of 3-HBA, the chemical synthesis of which is complicated and expensive.

Therapeutic Application of Phage Capsule Depolymerases against K1, K5, and K30 Capsulated E. coli in Mice.

Capsule depolymerase enzymes offer a promising class of new antibiotics. In vivo studies are encouraging but it is unclear how well this type of phage product will generalize in therapeutics, or whether different depolymerases against the same capsule function similarly. Here, in vivo efficacy was tested using cloned bacteriophage depolymerases against Escherichia coli strains with three different capsule types: K1, K5, and K30. When treating infections with the cognate capsule type in a mouse thigh model, the previously studied K1E depolymerase rescued poorly, whereas K1F, K1H, K5, and K30 depolymerases rescued well. K30 gp41 was identified as the catalytically active protein. In contrast to the in vivo studies, K1E enzyme actively degraded K1 capsule polysaccharide in vitro and sensitized K1 bacteria to serum killing. The only in vitro correlate of poor K1E performance in vivo was that the purified enzyme did not form the expected trimer. K1E appeared as an 18-mer which might limit its in vivo distribution. Overall, depolymerases were easily identified, cloned from phage genomes, and as purified proteins they proved generally effective.