A novel biological recovery approach for PHA employing selective digestion of bacterial biomass in animals

Applied Microbiology and Biotechnology - Polyhydroxyalkanoate (PHA) is a family of microbial polyesters that is completely biodegradable and possesses the mechanical and thermal properties of some...     

Biodegradation of polyesters containing aromatic constituents

Polymers, which undergo a controlled biological degradation by micro-organisms came to remarkable interest during the last years. Composting for instance could so be established as an alternative waste management system for parts of the plastic waste. Within this group of innovative polymer, polyesters play a predominant role, due to their potentially hydrolyzable ester bonds. While aromatic polyesters such as poly(ethylene terephthalate) exhibit excellent material properties but proved to be almost resistant to microbial attack, many aliphatic polyesters turned out to be biodegradable but lack in properties, which are important for application. To combine good material properties with biodegradability, aliphatic-aromatic copolyesters have been developed as biodegradable polymers for many years. This article reviews the attempts to combine aromatic and aliphatic structures in biodegradable plastics and work, which has been done to evaluate the degradation behaviour and environmental safety of biodegradable polyesters, containing aromatic constituents.     

Biotechnological production of (R)-3-hydroxybutyric acid monomer

The escalating problems regarding the treatment of plastic waste materials have led to development of biodegradable plastics. At present, a number of aliphatic polyesters; such as poly[(R)-3-hydroxybutyrate] (PHB), poly(l-lactide), polycaplolactone, poly(ethylene succinate) and poly(butylene succinate) have been developed. Among these aliphatic polyesters, PHB is one of the most attractive since it can undergo biodegradation at various environmental conditions and has properties similar to polypropylene. Although much effort has been made to produce PHB and its copolyesters from renewable resources or through microbial processes, their commercialization and widespread application are still not economically attractive compared to conventional non-biodegradable plastic. Moreover, wide application of PHB and its copolyesters as biodegradable plastic have not only been limited by the cost of production but also by their stinky smell during industrial processing. However, (R)-3-hydroxybutyric acid, a monomer of PHB has wide industrial and medical applications. (R)-3-hydroxybutyric acid can also serve as chiral precursor for synthesis of pure biodegradable PHB and its copolyesters. A number of options are available for production of (R)-3-hydroxybutyric acid. This review discusses each of these options to assess the alternatives that exist for production of pure biodegradable PHB and its copolyesters with good properties.     

Recent developments in ring opening polymerization of lactones for biomedical applications

Aliphatic polyesters prepared by ring-opening polymerization of lactones are now used worldwide as bioresorbable devices in surgery (orthopaedic devices, sutures, stents, tissue engineering, and adhesion barriers) and in pharmacology (control drug delivery). This review presents the various methods of the synthesis of polyesters and tailoring the properties by proper control of molecular weight, composition, and architecture so as to meet the stringent requirements of devices in the medical field. The effect of structure on properties and degradation has been discussed. The applications of these polymers in the biomedical field are described in detail.     

Biocatalytic polyester synthesis: analysis of the evolution of molecular weight and end group functionality

The enzymatic synthesis of polyesters from activated diesters and diols has been investigated. Differences between enzymatic synthesis and traditional chemical condensation processes are discussed. The disappearance of monomers during the initial phase of reaction indicates that enzyme has a higher specificity for transesterification of ester-terminated oligomers. During the intermediate phase, enzymatic polymerization involves a competition between diol and enzyme-bound water for the nucleophilic attack of the acyl enzyme intermediate. Competition between enzymatic transesterification and hydrolysis at different stages of polymerization in nonaqueous media is responsible for termination of polyesters with acid end-groups and also for limiting the polymer molecular weight. The resulting oligoester consists of chains that are either terminated with - OH groups and/or - COOH groups. We have used Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass spectroscopy (MALDI-TOF) along with colorimetric titration techniques to determine the acidity of enzyme-synthesized polyesters. This paper addresses how the enzymatic polymerization proceeds, and compares our results to the growing literature in this field. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 227-239, 1997.     

Biodegradation of polyhydroxyalkanoic acids

Stimulated by the commercial availability of bacteriologically produced polyesters such as poly[(R)-3-hydroxybutyric acid], and encouraged by the discovery of new constituents of polyhydroxyalkanoic acids (PHA), a considerable body of knowledge on the metabolism of PHA in microorganisms has accumulated. The objective of this essay is to give an overview on the biodegradation of PHA. The following topics are discussed: (i) general considerations of PHA degradation, (ii) methods for identification and isolation of PHA-degrading microorganisms, (iii) characterization of PHA-degrading microorganisms, (iv) biochemical properties of PHA depolymerases, (v) mechanisms of PHA hydrolysis, (vi) regulation of PHA depolymerase synthesis, (vii) molecular biology of PHA depolymerases, (viii) influence of the physicochemical properties of PHA on its biodegradability, (ix) degradation of polyesters related to PHA, (x) biotechnological aspects of PHA and PHA depolymerases. Received: 28 May 1996 / Received revision: 5 August 1996 / Accepted: 12 August 1996     

Partial purification of antibacterial proteinaceous factors from erythrocytes of Oncorhynchus mykiss

Antimicrobial peptides are natural antibiotics known to be present in both myeloid cells and epithelial surfaces of vertebrates. Nevertheless, the reports of antimicrobial peptides isolated from blood cells of teleosts are scarce. In this paper we show that acid-soluble erythrocyte extracts from rainbow trout, Oncorhynchus mykiss, display antibacterial activity against Planococcus citreus on a radial diffusion assay. Following tC18 solid phase extraction, cationic exchange chromatography and C18 reversed phase HPLC, two groups of fractions with antibacterial properties were obtained. This antibacterial activity is thermostable and susceptible to digestion by proteinase K, thus showing that the antibacterial agents have a proteinaceous nature. The factors eluted from a C18 column with circa 33% acetonitrile are active against P. citreus and Escherichia coli, with minimal inhibitory concentrations in the range 7-14 microg ml(-1) and 14-28 microg ml(-1), respectively; the ones eluted with approximately 44% acetonitrile on the same column only displayed activity against P. citreus, with a minimal inhibitory concentration of 1-2 microg ml(-1). These results raise the possibility that trout erythrocytes may contain antimicrobial factors not previously considered to be part of the innate immune system.     

Sequence analysis and structure prediction of type II Pseudomonas sp. USM 4–55 PHA synthase and an insight into its catalytic mechanism

Background  

Polyhydroxyalkanoates (PHA), are biodegradable polyesters derived from many microorganisms such as the pseudomonads. These polyesters are in great demand especially in the packaging industries, the medical line as well as the paint industries. The enzyme responsible in catalyzing the formation of PHA is PHA synthase. Due to the limited structural information, its functional properties including catalysis are lacking. Therefore, this study seeks to investigate the structural properties as well as its catalytic mechanism by predicting the three-dimensional (3D) model of the Type II Pseudomonas sp. USM 4–55 PHA synthase 1 (PhaC1_{P.sp USM 4–55}).     

Lipase/esterase-catalyzed synthesis of aliphatic polyesters via polycondensation: A review

Over the last decade, there has been an increasing interest in lipase/esterase-catalyzed polycondensation as an alternative to metal-based catalytic process, because the former can proceed under mild reaction conditions and does not cause undesirable side reactions or produce trace metallic residues. In this review, the in vitro synthesis of aliphatic polyesters by polycondensation using lipases or esterases is systematically summarized, especially for the synthesis of complex and well-defined polyesters. The polycondensation of diols with diacids or their activated esters, including alkyl, haloalkyl and vinyl esters, through esterification and transesterification polycondensation reactions is discussed. In addition, three or more monomers can also be polymerized simultaneously, which provides a new route for preparing functional polymers. Self-polycondensation with respect to hydroxyl and mercapto acids or their esters is another reaction mode discussed in the review. Finally, concurrent enzymatic ring-opening polymerization and polycondensation has been developed to construct novel polyesters with tailor-made structures and properties. Overall, the review demonstrates that lipase/esterase-catalyzed synthesis of polyesters via polycondensation provides an effective platform for conducting “eco-friendly polymer chemistry”.     

Carbohydrate-based polyesters made from bicyclic acetalized galactaric acid

The dimethyl ester of 2,3:4,5-di-O-methylene-galactaric acid (Galx) was made to react in the melt with 1,n-alkanediols HO(CH(2))(n)OH containing even numbers of methylenes (n from 6 to 12) to produce linear polycyclic polyesters. Two sets of poly(alkylene 2,3:4,5-di-O-methylene-galactarate) polyesters (PE-nGalx) with weight-average molecular weights in the ～ 5000-10000 and ～ 35000-45000 ranges were obtained using TBT and DBTO catalysts, respectively. For comparative purposes a set of poly(alkylene adipate) polyesters (PE-nAd) was also synthesized with molecular weights in the higher range using a similar procedure. The thermal stability of PE-nGalx was greater than that of PE-nAd although it notably decayed as molecular weight decreased. The replacement of Ad by Galx in the polyesters caused increases in T(g) of up to 70 °C, and almost doubled the tensile mechanical parameters. All PE-nGalx were semicrystalline but only those made from 1,12-dodecanediol were able to crystallize from the melt with a crystallization rate that diminished as the molecular weight increased. In general, the galactarate containing polyesters displayed higher solubility and wettability than polyadipates, they hydrolyzed faster and exhibited comparable sensitivity to the action of lipases.     

聚羟基脂肪酸酯的应用展望

聚羟基脂肪酸酯(Polyhydroxyalkanoates,简称PHA)是由微生物合成的天然高分子基材料,作为微生物碳源和能源的储备物质。目前,PHA的单体种类有150多种,致使PHA的品种繁多、材料学性质各不相同。PHA具有材料多变性、非线性光学性能、压电性能、气体阻隔性能、热塑性、生物可降解性、良好的生物相容性等特点,使其在塑料包装、化工、医药、农业、生物能源等诸多领域的具有很大的应用前景。文中系统介绍了目前PHA的应用和未来的发展。     

Biodegradation of microbial and synthetic polyesters by fungi

A variety of biodegradable polyesters have been developed in order to obtain useful biomaterials and to reduce the impact of environmental pollution caused by the large-scale accumulation of non-degradable waste plastics. Polyhydroxyalkanoates, poly(epsilon-caprolactone), poly( l-lactide), and both aliphatic and aromatic polyalkylene dicarboxylic acids are examples of biodegradable polyesters. In general, most aliphatic polyesters are readily mineralized by a number of aerobic and anaerobic microorganisms that are widely distributed in nature. However, aromatic polyesters are more resistant to microbial attack than aliphatic polyesters. The fungal biomass in soils generally exceeds the bacterial biomass and thus it is likely that fungi may play a considerable role in degrading polyesters, just as they predominantly perform the decomposition of organic matter in the soil ecosystem. However, in contrast to bacterial polyester degradation, which has been extensively investigated, the microbiological and environmental aspects of fungal degradation of polyesters are unclear. This review reports recent advances in our knowledge of the fungal degradation of microbial and synthetic polyesters and discusses the ecological importance and contribution of fungi in the biological recycling of waste polymeric materials in the biosphere.     

Antibacterial fatty acids: An update of possible mechanisms of action and implications in the development of the next-generation of antibacterial agents

The antibacterial activity of fatty acids (FA) is well known in the literature and represents a promising option for developing the next-generation of antibacterial agents to treat a broad spectrum of bacterial infections. FA are highly involved in living organisms' defense system against numerous pathogens, including multidrug-resistant bacteria. When combined with other antibacterial agents, the remarkable ability of FA to enhance their bactericidal properties is a critical feature that is not commonly observed in other naturally-occurring compounds. More reviews focusing on FA antibacterial activity, traditional and non-traditional mechanisms and biomedical applications are needed. This review is intended to update the reader on the antibacterial properties of recent FA and how their chemical structures influence their antibacterial activity. This review also aims to better understand both traditional and non-traditional mechanisms involved in these recently explored FA antibacterial activities.     

Requirements on bacterial polyesters as future substitute for conventional plastics for consumer goods

Bacterial polyesters such as polyhydroxybutyrate (PHB) or polyhydroxyalkanoates (PHAs) have to pass the following requirements to be accepted on a large scale: (i) they have to fulfil an urgent market need: (ii) they require that new and efficient composting systems are installed in urban areas; (iii) they have to complete with the present plastics as far as quality and processing performance are concerned; (iv) they have to meet the requirement for the registration as food packages; and (v) they have to meet competitive price limits. (i) Some 30% of the plastics in the municipal waste originates from goods which are less than 1 year in use and tend to be heavily soiled by food and feed residues. This part is difficult and expensive to dispose of. Biodegradable alternatives could replace a large part of it. The waste could be diverted from landfills and incineration to composting sites near the end user. The savings in costs and frustrations are the source of a pressing demand for biopolymers, especially for producing goods which do not demand longevity and which are likely to end up soiled with organic matters. (ii) Composting infrastructures exist in rural areas. In urban areas new systems for collecting and composting ‘garden and kitchen wastes’ are being installed for reducing landfill problems, especially in Austria, Denmark, Germany and the Netherlands. These installations give biopolymers a competitive edge in the disposal discussion. (iii) Bacterial polyesters meet various quality and processing performances. They are water-resistant, and goods made of the polyesters are water-tight. The material can be processed by injection and by blow moulding. However, the esters are not flexible enough for forming films or foils. They also tend to become brittle and to lose their vapour barrier properties. It is expected that these limits will be overcome by improving blend formulations. (iv) Bacterial polyesters are not yet allowed for use as food package material. Since the esters represent a novel product, the procedure for the registration poses serious, but no insoluble problems. They require long and costly tests. There are no indications that bacterial polyesters would not attain the requirements. However, they are not expected to serve as food packages in the near future. (v) The present prices for bacterial polyesters are far too high to be accepted on a large scale by the processing and packaging industry. Costs are high mainly because of the raw material prices and to the small-scale production units. They can be lowered to accepted levels by investing in larger units in countries where inexpensive raw materials are available. Thus they will be able to meet the price limits. Since bacterial polyesters increasingly meet the requirements for the penetration of a mass market and since more and more consumers accept composting as an environmentally sound way of recycling organic materials, the polyesters are expected to penetrate a significant part of the short-lived and contaminated plastic products markets by the turn of the century.     

FabG mediates polyhydroxyalkanoate production from both related and nonrelated carbon sources in recombinant Escherichia coli LS5218

Polyhydroxyalkanoates (PHAs) composed of a mixture of short-chain-length-medium-chain-length (SCL-MCL) hydroxyacyl monomers are biologically produced polyesters that have properties ranging from thermoplastic to elastomeric, dependent on the molar ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of properties and applications for SCL-MCL PHA copolymers, it is important to develop and characterize novel metabolic pathways for SCL-MCL PHA production. The current study shows that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes enhances the production of SCL-MCL PHA copolymer from both related and nonrelated carbon sources in Escherichia coli LS5218, indicating the flexibility of FabG as a monomer-supplying enzyme for biological PHA production.     

Synthesis of amphiphilic alternating polyesters with oligo(ethylene glycol) side chains and potential use for sustained release drug delivery

Novel amphiphilic alternating polyesters, poly((N-phthaloyl-l-glutamic anhydride)-co-(2-(2-(2-methoxyethoxy)ethoxy)methyl)oxirane) (P(PGA-co-ME(2)MO)), were synthesized by alternating copolymerization of PGA and ME(2)MO. The structures of the synthesized polyesters were characterized by (1)H NMR, (13)C NMR, FT-IR, and GPC analyses. Because of the presence of oligo(ethylene glycol) (OEG) side chains, the polyesters could self-assemble into thermosensitive micelles. Dynamic light scattering (DLS) showed that these micelles underwent thermoinduced size decrease without intermicellar aggregation. In vitro methyl thiazolyl tetrazolium (MTT) assay demonstrated that the polyesters were biocompatible to Henrietta Lacks (HeLa) cells, rendering their potential for drug delivery applications. Two hydrophobic drugs, rifampin and doxorubicin (DOX), were loaded into the polyester micelles and observed to be released in a zero-order sustained manner. The sustained release could be accelerated in lower pH or in the presence of proteinase K, due to the degradation of the polyester under these conditions. Remarkably, in vitro cell experiments showed that the polyester micelles accomplished fast release of DOX inside cells and higher anticancer efficacy as compared with the free DOX. With enhanced stability during circulation condition and accelerated drug release at the target sites (e.g., low pH or enzyme presence), these novel polyesters with amphiphilic structures are promising to be used in sustained release drug delivery systems.     

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis.     

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.     

Review Degradation of microbial polyesters

Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics. Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA. Microorganisms degrading these polyesters are widely distributed in various environments. Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review. We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB.     

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.