Medium Chain Length Polyhydroxyalkanoates Research Articles

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Production of Medium Chain Length Polyhydroxyalkanoate from Waste Cannabis sativa Biomass

AbstractThis works aims to evaluate a process to convert residual medical Cannabis sativa stalks into medium chain length polyhydroxyalkanoate (mcl-PHA), using chemical pretreatment and enzymatic hydrolysis to obtain sugars as a carbon source for a fermentation process with Pseudomonas aeruginosa. Chemical composition analysis revealed a content of structural polysaccharides of 57.64%. Thermochemical pretreatments with 2% sulfuric acid or 2% sodium hydroxide were capable of partially removing hemicellulose and lignin, as well as increasing cellulose crystallinity. Pretreated biomass was subjected to hydrolysis using commercial cellulase cocktails Celluclast® 1.5 L and Cellic® CTec3. Acid pretreatment showed an adverse effect on hydrolysis yield of holocellulose, decreasing to 39.5%; compared to 44.4% of untreated biomass. Alkaline pretreatment increased degree of hydrolysis up to 73.3%. Shake flask fermentation of hydrolysate with Pseudomonas aeruginosa produced cell growth of 1.65 g/L and a mcl-PHA titer of 0.41 g/L. Extracted polymer presented characteristic FTIR bands for PHAs, glass transition temperature of − 50.8 °C, melting temperature of 48.9 °C, possibly allowing its use in the biomedical industry. The developed process represents a potential way to valorize Cannabis waste stalks, using alkaline pretreatment, enzymatic hydrolysis with Celluclast® 1.5 L and fermentation with Pseudomonas aeruginosa. Further work should focus on improving yield of the obtained polyhydroxyalkanoate in fermentation, in order to improve industrial feasibility of the entire process and, in turn, increasing revenue of the medical Cannabis industry. Graphical Abstract

Degradation kinetics of medium chain length Polyhydroxyalkanoate degrading enzyme: a quartz crystal microbalance study.

This study investigates the enzymatic degradation processes of different classes of polyhydroxyalkanoates (PHAs), a group of biopolymers naturally synthesized by various microorganisms. Medium chain length PHAs (mcl-PHAs) are distinguished biopolymers due to their biodegradability and diverse material properties. Using quartz crystal microbalance measurements as a valuable tool for accurate real-time monitoring of the enzymatic degradation process, the research provides detailed kinetic data, describing the interaction between enzymes and substrates during the enzymatic degradation process. Thin films of poly-3-hydroxybutyrate (PHB) and polyhydroxyoctanoate copolymer (PHO), containing molar fractions of about 84% 3-hydroxyoctanoate and 16% 3-hydroxyhexanoate, were exposed to scl-depolymerases from Pseudomonas lemoignei LMG 2207 and recombinant mcl-depolymerase produced in Escherichia coli DH5α harboring the plasmid pMAD8, respectively. Analyses based on a heterogeneous kinetic model for the polymer degradation indicated a six-fold stronger adsorption equilibrium constant of mcl-depolymerase to PHO. Conversely, the degradation rate constant was approximately twice as high for scl-depolymerases acting on PHB. Finally, the study highlights the differences in enzyme-substrate interactions and degradation mechanisms between the investigated scl- and mcl-PHAs.

Fabrication and biological properties of artificial tendon composite from medium chain length polyhydroxyalkanoate

Medium chain length polyhydroxyalkanoate (MCL-PHA), a biodegradable and biocompatible material, has a mechanical characteristic of hyper-elasticity, comparable to elastomeric material with similar properties to human tendon flexibility. These MCL-PHA properties gave rise to applying this material as an artificial tendon or ligament implant. In this study, the material was solution-casted in cylinder and rectangular shapes in the molds with the designated small holes. A portion of the torn human tendon was threaded into the holes as a suture to generate a composite tendon graft. The tensile testing of the three types of MCL-PHA/tendon composite shows that the cylinder material shape with the zigzag threaded three holes has the highest value of maximum tensile strength at 56 MPa, closing to the ultimate tendon tensile stress (50–100 MPa). Fibroblast cells collected from patients were employed as primary tendon cells for growing to attach to the surface of the MCL-PHA material to prove the concept of the composite tendon graft. The cells could attach and proliferate with substantial viability and generate collagen, leading to chondrogenic induction of tendon cells. An in vivo biocompatibility was also conducted in a rat subcutaneous model in comparison with medical-grade silicone. The MCL-PHA material was found to be biocompatible with the surrounding tissues. For surgical application, after the MCL-PHA material is decomposed, tendon cells should develop into an attached tendon and co-generated as a tendon graft.

Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H−1, where the rate of PHA production was 0.28 g L−1. H−1 and PHA concentration was 4.94 g. L−1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Biosynthesis of 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDd) in the polyhydroxyalkanoate copolymeric ester chain by Pseudomonas aeruginosa MCC 5300 using oleic acid as a carbon source

The application of non-biodegradable plastics has raised severe environmental concerns over the past few decades. The medium chain length polyhydroxyalkanoates (mcl-PHA) are biodegradable elastomeric polymers produced by the bacteria Pseudomonas, that can potentially solve this crisis. The industrial production of mcl-PHA is limited by low PHA yield of wild bacterial strains and the increased time the bacteria takes to achieve maximum PHA yield. Hence the present study focuses on the early PHA production in Pseudomonas aeruginosa MCC 5300. Pseudomonas aeruginosa MCC 5300 can grow to a high biomass and produce PHA within a short duration of 16 h of growth, reaching up to 75.6 % cell dry weight in media containing only oleic acid as a carbon source. A high amount of PHA production in such a short time was not reported in any wild-type bacteria cultured in nutrient-defined media. The mcl-PHA was a copolymer of 3-hydroxydecanoate and 3-hydroxydodecanoate.

Scaffolds based on β tricalcium phosphate and polyhydroxyalkanoates as biodegradable and bioactive bone substitutes with enhanced physicochemical properties

Polyhydroxyalkanoates (PHAs) are biocompatible and biodegradable bacterial-origin polyesters, which have recently emerged as a potential prosperous coating for bioceramic scaffolds. However, biopolymers often have properties inferior to commercially available polymers, and thus blending seems to be an efficient method to tailor their characteristics. In our study poly(3-hydroxybutyrate) (P(3HB)) and medium chain length PHA (mcl-PHA) blends were used as coatings on β tricalcium phosphate (βTCP) scaffolds. The influence of the coating type on the physicochemical properties of composites was investigated using various techniques such as X-ray diffraction (XRD), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis with differential scanning calorimetry (TG/DSC), scanning electron microscopy (SEM), atomic force microscopy (AFM), wettability measurements, ultra high-performance liquid chromatography with and mass spectrometry (UHPLC-MS) and in vitro studies. It has been demonstrated that blends with various amounts of brittle P(3HB) and viscous mcl-PHA can serve as coatings on βTCP scaffolds, modulating their physicochemical properties. Macroporous scaffolds covered with blends possessed high open porosity (~ 65 vol%) and improved compressive strength (up to 4.9 ± 0.9 MPa). It has been shown that their wettability can be tailored by modifying composition of blends, making the surface more hydrophobic with increasing amount of mcl-PHA. The highest amount of mcl-PHA was shown to significantly influence the degradation of the composites which may be a valuable feature in the case of customized scaffolds for the controlled release of bioactive substances. Moreover, UHPLC-MS analysis revealed that PHAs degrade to hydroxy acids and their oligomers, which may serve as potentially nourishing compounds for surrounding tissues. According to in vitro biocompatibility tests on mouse preosteoblasts, all evaluated scaffolds were nontoxic. The greater the surface hydrophilicity, the better cell adhesion and proliferation were observed.

Pseudomonas umsongensis GO16 as a platform for the in vivo synthesis of short and medium chain length polyhydroxyalkanoate blends

Polyhydroxyalkanoates (PHAs) are biological polyesters, viewed as a replacement for petrochemical plastic. However, they suffer from suboptimal physical and mechanical properties. Here, it was shown that a metabolically versatile Pseudomonas umsongensis GO16 can synthesise a blend of short chain length (scl) and medium chain length (mcl)-PHA. A defined mix of butyric (BA) and octanoic acid (OA) in different ratios was used. The PHA monomer composition varied depending on the feeding strategy. When OA and BA were fed at 80:20 ratio it showed 14, 8, 77 and 1 mol% of (R)-3-hydroxybutyrate, (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate and (R)-3-hydroxydecanoate respectively. The polymer characterisation clearly shows that polyhydroxybutyrate (PHB) and mcl-PHA are produced individually. The two polymers are blended on the PHA granule level, as demonstrated by fluorescence microscopy and yeast two-hybrid assay. The resulting blend has a specific viscoelasticity compared to PHB and PHO. Mcl-PHA acts as a plasticiser and reduces PHB brittleness.

Polyhydroxyalkanoate production in Pseudomonas putida from alkanoic acids of varying lengths.

Many studies have been conducted to produce microbial polyhydroxyalkanoates (PHA), a biopolymer, from Pseudomonas sp. fed with various alkanoic acids. Because this previous data was collected using methodologies that varied in critical aspects, such as culture media and size range of alkanoic acids, it has been difficult to compare the results for a thorough understanding of the relationship between feedstock and PHA production. Therefore, this study utilized consistent culture media with a wide range of alkanoic acids (C7-C14) to produce medium chain length PHAs. Three strains of Pseudomonas putida (NRRL B-14875, KT2440, and GN112) were used, and growth, cell dry weight, PHA titer, monomer distribution, and molecular weights were all examined. It was determined that although all the strains produced similar PHA titers using C7-C9 alkanoic acids, significant differences were observed with the use of longer chain feedstocks. Specifically, KT2440 and its derivative GN112 produced higher PHA titers compared to B-14875 when fed longer chain alkanoates. We also compared several analytical techniques for determining amounts of PHA and found they produced different results. In addition, the use of an internal standard had a higher risk of calculating inaccurate concentrations compared to an external standard. These observations highlight the importance of considering this aspect of analysis when evaluating different studies.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48g/l biomass and 0.32g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73g/l biomass and 0.33g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Fractionation of medium-chain-length polyhydroxyalkanoate biosynthesized by pilot-scale production for improving material properties

Medium-chain-length polyhydroxyalkanoate (MCL-PHA), a biodegradable and biocompatible material, is a biopolyester that obtains great attention, especially in the medical area. By focusing on a range of fractions of MCL-PHA, a clear understanding of the thermal behavior and the crystalline structure, especially the tensile properties, have been gained. In this study, we successfully biosynthesized the MCL-PHA in 300 L pilot-scale fermentation by Pseudomonas putida, and all the chemomechanical properties have been characterized. The obtained MCL-PHA (A-original) was fractionated by using a chloroform/methanol mixture into several fractions (A-1, A-2, A-3, and A-4) with different comonomer compositions consisting of 3-hydroxydecanoate (3HD), 3-hydroxyoctanoate (3HO), and 3-hydroxyhexanoate (3HHx). It is crucial to demonstrate the mechanical characteristics of the MCL-PHA fraction in this area of application, which has not previously been studied. We found that the fractions of natural MCL-PHA exhibited distinctive thermal properties with different crystalline patterns, giving rise to the overall behavior of the as-produced MCL-PHA blend primarily dependent on a fraction of high 3HD content. It can be concluded that there are 2 fractions (A-2 and A-3) that have more strength and flexibility than the as-produced MCL-PHA (A-original), indicating that these fractions are suitable for use as medical elastomers.

Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99.

High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.

Medium chain length polyhydroxyalkanoate produced from ethanol by Pseudomonas putida grown in liquid obtained from acidogenic digestion of organic municipal solid waste

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) up to about 6 g.L−1 was obtained by feeding ethanol to Pseudomonas putida growing in liquid obtained from acidogenic digestion of organic municipal solid waste. Washing the wet, heat-inactivated Pseudomonas cells at the end of the fermentation with ethanol obviated the need of drying the biomass and enabled the removal of contaminating lipids before solvent-mediated extraction of PHA. Using ‘green’ solvents, 90 to near 100% of the mcl-PHA was extracted and purities of 71–78% mcl-PHA were reached already by centrifugation and decantation without further filtration for biomass removal. The mcl-PHA produced in this way consists of 10–18% C8, 72–78% C10 and 8–12% C12 chains (entirely medium chain length), has a crystallinity and melting temperature of ∼13% and ∼49 °C, respectively, and is a stiff rubberlike, colourless material at room temperature.

Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences.

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).

Medium chain length polyhydroxyalkanoates as potential matrix materials for peripheral nerve regeneration.

Polyhydroxyalkanoates are natural, biodegradable, thermoplastic and sustainable polymers with a huge potential in fabrication of bioresorbable implantable devices for tissue engineering. We describe a comparative evaluation of three medium chain length polyhydroxyalkanoates (mcl-PHAs), namely poly(3-hydroxyoctanoate), poly(3-hydroxyoctanoate-co-3-hydoxydecanoate) and poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate), one short chain length polyhydroxyalkanoate, poly(3-hydroxybutyrate), P(3HB) and synthetic aliphatic polyesters (polycaprolactone and polylactide) with a specific focus on nerve regeneration, due to mechanical properties of mcl-PHAs closely matching nerve tissues. In vitro biological studies with NG108-15 neuronal cell and primary Schwann cells did not show a cytotoxic effect of the materials on both cell types. All mcl-PHAs supported cell adhesion and viability. Among the three mcl-PHAs, P(3HO-co-3HD) exhibited superior properties with regards to numbers of cells adhered and viable cells for both cell types, number of neurite extensions from NG108-15 cells, average length of neurite extensions and Schwann cells. Although, similar characteristics were observed for flat P(3HB) surfaces, high rigidity of this biomaterial, and FDA-approved polymers such as PLLA, limits their applications in peripheral nerve regeneration. Therefore, we have designed, synthesized and evaluated these materials for nerve tissue engineering and regenerative medicine, the interaction of mcl-PHAs with neuronal and Schwann cells, identifying mcl-PHAs as excellent materials to enhance nerve regeneration and potentially their clinical application in peripheral nerve repair.

Customized valorization of waste streams by Pseudomonas putida: State-of-the-art, challenges, and future trends

Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using engineered microbes has emerged as a long-term eco-friendly solution for addressing the global climate crisis and transitioning to clean energy. Notably, Pseudomonas putida can valorize industry-derived synthetic wastes including plastics, oils, food, and agricultural waste into products of interest, and it has been extensively explored for establishing a fully circular bioeconomy through the conversion of waste into bio-based products, including platform chemicals (e.g., cis,cis-muconic and adipic acid) and biopolymers (e.g., medium-chain length polyhydroxyalkanoate). However, the efficiency of waste pretreatment technologies, capability of microbial cell factories, and practicability of synthetic biology tools remain low, posing a challenge to the industrial application of P. putida. The present review discusses the state-of-the-art, challenges, and future prospects for divergent biosynthesis of versatile products from waste-derived feedstocks using P. putida.

Genomic insights into the metabolic potential of a novel lignin-degrading and polyhydroxyalkanoates producing bacterium Pseudomonas sp. Hu109A

Lignin is the most abundant heterogeneous aromatic polymer present on planet Earth and is recalcitrant to degradation due to its complex structure, therefore, imposing a challenge to biorefinery procedures. Identifying new microbial strains with the potential to valorize lignin into useful compounds is indispensable to achieving green sustainable consumption. In this study, a novel Pseudomonas strain designated as Hu109A was isolated from the termite gut and the genome was sequenced and analyzed further. The genome contains a circular chromosome with the size of 5,131,917 bp having a GC content of 62.6% and 4698 genes. Genome annotation reveals that the strain possesses lignin-oxidizing enzymes such as DyP-type peroxidases, laccase, dioxygenase, and aromatic degradation gene clusters. The genome also contains O-methyltransferases which function in accelerating the lignin degradation by methylating the free hydroxyl phenolic compounds which in high concentration can inhibit the lignin peroxidase. Furthermore, the genome exhibits two gene clusters encoding the enzymes related to polyhydroxyalkanoates (PHA) synthesis. Pseudomonas strains are generally assumed to produce medium chain length PHAs (mcl-PHAs) only, however, strain Hu109A contains both Class II PHA synthase genes involved in mcl-PHAs and Class III PHA synthase gene involved in short-chain length PHAs (scl-PHAs). Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that using 1 g/L lignin as the sole carbon source, the maximum production of PHA observed was 103.68 mg/L, which increased to 186 mg/L with an increase in lignin concentration to 3 g/L. However, PHA production while using glucose as the sole carbon source was significantly lower than the lignin source, and maximum production was 125.6 mg/L with 3 g/L glucose. The strain Hu109A can tolerate a broad range of solvents including methanol, isopropanol, dimethylformamide, and ethanol, revealing its potential for industrial applications.

Characterization of medium chain length polyhydroxyalkanoates from Priestia megaterium POD1

Polyhydroxyalkanoate (PHA) is the most promising solution to the major ecological problem of plastic accumulation. It is synthesized by bacteria when there is limited availability of nutrients as intracellular granules. The biodegradable and biocompatible properties of PHA make it highly demanding in the biomedical and agricultural field. Among PHA, medium chain length (mcl) PHA has shown more interest because of its favorable properties like having more viscous material, low crystallinity degrees, low melting temperature and low glass transition temperatures for many applications which require flexible material. Therefore, we have isolated a bacterial strain, Priestia megaterium POD1 which can produce mcl PHA very efficiently. The isolate was confirmed to have phaC gene responsible for PHA synthesis. The PHA extracted from the isolate was characterized by FTIR, NMR and GC-MS which revealed the extracted polymer to be a mixture of different mcl PHA. Hence, the present study would provide a potential mcl PHA producer which can be explored for its properties which are demanding in present bioplastic market.

Characterization of Polymer Degrading Lipases, LIP1 and LIP2 From Pseudomonas chlororaphis PA23.

The outstanding metabolic and bioprotective properties of the bacterial genus Pseudomonas make these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacterium Pseudomonas chlororaphis PA23 and subsequently performed cloning and expression in Escherichia coli. The lip1 gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas the lip2 consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyanion pocket, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with an oxyanion pocket of GGX-type, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. Our three-dimensional models of LIP1 and LIP2 complexed with a 3-hydroxyoctanoate dimer revealed the core α/β hydrolase-type domain with an exposed substrate binding pocket in LIP1 and an active-site capped with a closing lid domain in LIP2. The recombinant LIP1 was optimally active at 45°C and pH 9.0, and the activity improved in the presence of Ca2+. LIP2 exhibited maximum activity at 40°C and pH 8.0, and was unaffected by Ca2+. Despite different properties, the enzymes exhibited broadsubstrate specificity and were able to hydrolyze short chain length and medium chain length polyhydroxyalkanoates (PHAs), polylactic acid (PLA), and para-nitrophenyl (pNP) alkanoates. Gel Permeation Chromatography (GPC) analysis showed a decrease in the molecular weight of the polymers after incubation with LIP1 and LIP2. The enzymes also manifested some polymer-degrading activity on petroleum-based polymers such as poly(ε-caprolactone) (PCL) and polyethylene succinate (PES), suggesting that these enzymes could be useful for biodegradation of synthetic polyester plastics. The study will be the first report of the complete characterization of intracellular lipases from bacterial and/or Pseudomonas species. The lipases, LIP1 and LIP2 are different from other bacterial lipases/esterases in having broad substrate specificity for polyesters.

Polyenes in Medium Chain Length Polyhydroxyalkanoate (mcl-PHA) Biopolymer Microspheres with Reduced Toxicity and Improved Therapeutic Effect against Candida Infection in Zebrafish Model.

Immobilizing antifungal polyenes such as nystatin (Nys) and amphotericin B (AmB) into biodegradable formulations is advantageous compared to free drug administration providing sustained release, reduced dosing due to localized targeting and overall reduced systemic drug toxicity. In this study, we encapsulated Nys and AmB in medium chain length polyhydroxyalkanoates (mcl-PHA) microspheres (7–8 µm in diameter). The obtained formulations have been validated for antifungal activity in vitro against a panel of pathogenic fungi including species of Candida, Aspergillus, Microsporum and Trichophyton genera and toxicity and efficacy in vivo using the zebrafish model of disseminated candidiasis. While free polyenes, especially AmB, were highly toxic to zebrafish embryos at the effective (MIC) doses, after their loading into mcl-PHA microspheres, inner organ toxicity and teratogenicity associated with both drugs were not observed, even at 100 × MIC doses. The obtained mcl-PHA/polyene formulations have successfully eradicated C. albicans infection and showed an improved therapeutic profile in zebrafish by enhancing infected embryos survival. This approach is contributing to the antifungal arsenal as polyenes, although the first broad-spectrum antifungals on the market are still the gold standard for treatment of fungal infections.