Search Results (7)

Multi-layer cell constructs produced in vitro are an innovative treatment option to support the growing demand for therapy in regenerative medicine. Our research introduces a novel construct integrating organ-derived decellularised extracellular matrix (dECM) hydrogels and 3D-printed biodegradable polymer meshes composed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) to support and maintain multiple layers of different cell types. We achieved that by integrating the mechanical stability of PHBV+P34HB, commonly used in the food storage industry, with a dECM hydrogel, which replicates organ stiffness and supports cellular survival and function. The construct was customised by adjusting the fibre arrangement and pore sizes, making it a suitable candidate for a personalised design. We showed that the polymer is degradable after precoating it with PHB depolymerase (PhaZ), with complete degradation achieved in 3–5 days and delayed by adding the hydrogel to 10 days, enabling tuneable degradation for regenerative medicine applications. Finally, as a proof of concept, we composed a three-layered tissue in vitro; each layer represented a different tissue type: epidermal, vascular, and subcutaneous layers. Possible future applications include wound healing and diabetic ulcer paths, personalised drug delivery systems, and personalised tissue implants. Full article

Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants of Pseudomonas sp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by ¹H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production. Full article

Pseudomonas sp. SG4502 screened from biodiesel fuel by-products can synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) using glycerol as a substrate. It contains a typical PHA class II synthase gene cluster. This study revealed two genetic engineering methods for improving the mcl-PHA accumulation capacity of Pseudomonas sp. SG4502. One way was to knock out the PHA-depolymerase phaZ gene, the other way was to insert a tac enhancer into the upstream of the phaC1/phaC2 genes. Yields of mcl-PHAs produced from 1% sodium octanoate by +(tac-phaC2) and ∆phaZ strains were enhanced by 53.8% and 23.1%, respectively, compared with those produced by the wild-type strain. The increase in mcl-PHA yield from +(tac-phaC2) and ∆phaZ was due to the transcriptional level of the phaC2 and phaZ genes, as determined by RT-qPCR (the carbon source was sodium octanoate). ¹H-NMR results showed that the synthesized products contained 3-hydroxyoctanoic acid (3HO), 3-hydroxydecanoic acid (3HD) and 3-hydroxydodecanoic acid (3HDD) units, which is consistent with those synthesized by the wild-type strain. The size-exclusion chromatography by GPC of mcl-PHAs from the (∆phaZ), +(tac-phaC1) and +(tac-phaC2) strains were 2.67, 2.52 and 2.60, respectively, all of which were lower than that of the wild-type strain (4.56). DSC analysis showed that the melting temperature of mcl-PHAs produced by recombinant strains ranged from 60 °C to 65 °C, which was lower than that of the wild-type strain. Finally, TG analysis showed that the decomposition temperature of mcl-PHAs synthesized by the (∆phaZ), +(tac-phaC1) and +(tac-phaC2) strains was 8.4 °C, 14.7 °C and 10.1 °C higher than that of the wild-type strain, respectively. Full article

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). Full article

Biodegradable and compostable plastics are getting more attention as the environmental impacts of fossil-fuel-based plastics are revealed. Microbes can consume these plastics and biodegrade them within weeks to months under the proper conditions. The biobased polyhydroxyalkanoate (PHA) polymer family is an attractive alternative due to its physicochemical properties and biodegradability in soil, aquatic, and composting environments. Standard test methods are available for biodegradation that employ either natural inocula or defined communities, the latter being preferred for standardization and comparability. The original marine biodegradation standard test method ASTM D6691 employed such a defined consortium for testing PHA biodegradation. However, the taxonomic composition and metabolic potential of this consortium have never been confirmed using DNA sequencing technologies. To this end, we revived available members of this consortium and determined their phylogenetic placement, genomic sequence content, and metabolic potential. The revived members belonged to the Bacillaceae, Rhodobacteraceae, and Vibrionaceae families. Using a comparative genomics approach, we found all the necessary enzymes for both PHA production and utilization in most of the members. In a clearing-zone assay, three isolates also showed extracellular depolymerase activity. However, we did not find classical PHA depolymerases, but identified two potentially new extracellular depolymerases that resemble triacylglycerol lipases. Full article

Polyhydroxybutyrate (PHB) is one of the polyhydroxyalkanoates (PHAs) which has biodegradable and biocompatible properties. They are adopted in the biomedical field, in, for example, medical implants and drug delivery carriers. This study seeks to promote the production of PHB by Vibrio sp. BM-1, isolated from a marine environment by improving constituents of medium and implementing an appropriate fermentation strategy. This study successfully developed a glycerol-yeast extract-tryptone (GYT) medium that can facilitate the growth of Vibrio sp. BM-1 and lead to the production of 1.4 g/L PHB at 20 h cultivation. This study also shows that 1.57 g/L PHB concentration and 16% PHB content were achieved, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium (mineral salts-supplemented GYT medium) for 12 h. Both cell dry weight (CDW) and residual CDW remained constant at around 8.2 g/L and 8.0 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. The results suggest that the secretion of PHB depolymerase that might be caused by the addition of mineral salts reduced PHB after 12 h of cultivation. However, work will be done to explain the effect of adding mineral salts on the production of PHB by Vibrio sp. BM-1 in the near future. Full article

Poly(hydroxyalkanoate) (PHA), which is produced from renewable carbon resources by many microorganisms, is an environmentally compatible polymeric material and can be processed into films and fibers. Biodegradation of PHA material occurs due to the action of extracellular PHA depolymerase secreted from microorganisms in various natural environments. A key step in determining the overall enzymatic or environmental degradation rate of PHA material is the degradation of PHA lamellar crystals in materials; hence, the degradation mechanism of PHA lamellar crystals has been studied in detail over the last two decades. In this review, the relationship between crystal structure and enzymatic degradation behavior, in particular degradation rates, of films and fibers for PHA is described. Full article