Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19

An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.     

Fermentation process development for the production of medium-chain-length poly-3-hyroxyalkanoates

This paper presents a review of the existing fermentation processes for the production of medium-chain-length poly-3-hydroxyalkanoates (MCL-PHAs). These biodegradable polymers are usually produced most efficiently from structurally related carbon sources such as alkanes and alkanoic acids. Unlike alkanoic acids, alkanes exhibit little toxicity but their low aqueous solubility limits their use in high density culture. Alkanoic acids pose little mass transfer difficulty, but their toxicity requires that their concentration be well controlled. Using presently available technology, large-scale production of MCL-PHA from octane has been reported to cost from US $5 to 10 per kilogram, with expenditures almost evenly divided between carbon source, fermentation process, and the separation process. However, MCL-PHAs, even some with functional groups in their subunits, can also be produced from cheaper unrelated carbon sources, such as glucose. Metabolic engineering and other approaches should also allow increased PHA cellular content to be achieved. These approaches, as well as a better understanding of fermentation kinetics, will likely result in increased productivity and lower production costs.     

New FadB homologous enzymes and their use in enhanced biosynthesis of medium-chain-length polyhydroxyalkanoates in FadB mutant Escherichia coli

Recombinant Escherichia coli harboring the medium-chain-length (MCL) polyhydroxyalkanoate (PHA) synthase gene has been shown to accumulate MCL-PHAs from fatty acids when FadB is inactive. However, the enzymes in fadB mutant E. coli responsible for channeling the beta-oxidation intermediates to PHA biosynthesis have not been fully elucidated. Only recently, two enzymes encoded by yfcX and maoC have been found to be partially responsible for this. In this study, we identified five new FadB homologous enzymes in E. coli: PaaG, PaaF, BhbD, SceH, and YdbU, by protein database search, and examined their roles in the biosynthesis of MCL-PHAs in an fadB mutant E. coli strain. Coexpression of each of these genes along with the Pseudomonas sp. 61-3 phaC2 gene did not allow synthesis of MCL-PHA from fatty acid in recombinant E. coli W3110, which has a fully functional beta-oxidation pathway, but allowed MCL-PHA accumulation in an fadB mutant E. coli WB101. In particular, coexpression of the paaG, paaF, and ydbU genes resulted in a MCL-PHA production up to 0.37, 0.25, and 0.33 g/L, respectively, from 2 g/L of sodium decanoate, which is more than twice higher than that obtained with E. coli WB101 expressing only the phaC2 gene (0.16 g/L). These results suggest that the newly found FadB homologous enzymes, or at least the paaG, paaF, and ydbU genes, are involved in MCL-PHA biosynthesis in an fadB mutant E. coli strain and can be employed for the enhanced production of MCL-PHA.     

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72

A bacterial strain capable of degrading medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was isolated from a soil sample. This organism, which was identified as Streptomyces sp. KJ-72, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated on MCL-PHAs. The extracellular MCL-PHA depolymerase of the organism was purified to electrophoretic homogeneity by ion exchange column chromatography and gel filtration. The enzyme consisted of a monomeric subunit having a molecular mass of 27.1 kDa and isoelectric point of 4.7. The maximum activity was observed at pH 8.7 and 50 °C. The enzyme was sensitive to N-bromosuccinimide and acetic anhydride, indicating the presence of tryptophan and lysine residues in the catalytic domain. The enzyme was able to hydrolyze various chain-length p-nitrophenyl esters of fatty acids and polycaprolactone as well as various types of MCL-PHAs. However, lipase activity of the enzyme was not detected. The main hydrolysis product of poly(3-hydroxyheptanoate) was identified to be the dimer of 3-hydroxyheptanoate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fine control of polyester properties via epoxide ROP using monomers carrying diverse functional groups

Synthetic biodegradable polymers are important biomaterials. However, most of them are biologically inert. Free functional groups can allow easy biofunctionalization. Efficient introduction of functional groups to biodegradable polymers is still a challenge. Here, a practical strategy is presented to synthesize various functional polyesters with free hydroxyl groups polymerized via epoxide ring-opening polymerization between dicarboxylic acids and diglycidyl dicarboxylates without protection and deprotection. The polymers exhibit a wide range of physical, thermal, and mechanical properties, and good cytocompatibilities. This synthetic platform is expected to lead to functional polymers useful for a wide variety of biomedical applications.     

Separation and sensing based on molecular recognition using molecularly imprinted polymers

Molecular recognition-based separation and sensing systems have received much attention in various fields because of their high selectivity for target molecules. Molecular imprinting has been recognized as a promising technique for the development of such systems, where the molecule to be recognized is added to a reaction mixture of a cross-linker(s), a solvent(s), and a functional monomer(s) that possesses a functional groups(s) capable of interacting with the target molecule. Binding sites in the resultant polymers involve functional groups originating from the added functional monomer(s), which can be constructed according to the shape and chemical properties of the target molecules. After removal of the target molecules, these molecularly imprinted complementary binding sites exhibit high selectivity and affinity for the template molecule. In this article, recent developments in molecularly imprinted polymers are described with their applications as separation media in liquid chromatography, capillary electrophoresis, solid-phase extraction, and membranes. Examples of binding assays and sensing systems using molecularly imprinted polymers are also presented.     

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.     

Amphipols for Each Season

Amphipols (APols) are short amphipathic polymers that can substitute for detergents at the transmembrane surface of membrane proteins (MPs) and, thereby, keep them soluble in detergent free aqueous solutions. APol-trapped MPs are, as a rule, more stable biochemically than their detergent-solubilized counterparts. APols have proven useful to produce MPs, most noticeably by assisting their folding from the denatured state obtained after solubilizing MP inclusion bodies in either SDS or urea. They facilitate the handling in aqueous solution of fragile MPs for the purpose of proteomics, structural and functional studies, and therapeutics. Because APols can be chemically labeled or functionalized, and they form very stable complexes with MPs, they can also be used to functionalize those indirectly, which opens onto many novel applications. Following a brief recall of the properties of APols and MP/APol complexes, an update is provided of recent progress in these various fields.     

Polypeptoid polymers: Synthesis,characterization, and properties

Polypeptoids, a class of peptidomimetic polymers, have emerged at the forefront of macromolecular and supramolecular science and engineering as the technological relevance of these polymers continues to be demonstrated. The chemical and structural diversity of polypeptoids have enabled access to and adjustment of a variety of physicochemical and biological properties (eg, solubility, charge characteristics, chain conformation, HLB, thermal processability, degradability, cytotoxicity and immunogenicity). These attributes have made this synthetic polymer platform a potential candidate for various biomedical and biotechnological applications. This review will provide an overview of recent development in synthetic methods to access polypeptoid polymers with well‐defined structures and highlight some of the fundamental physicochemical and biological properties of polypeptoids that are pertinent to the future development of functional materials based on polypeptoids.     

Antimicrobial polymers: mechanism of action,factors of activity,and applications

Complex epidemiological situation, nosocomial infections, microbial contamination, and infection risks in hospital and dental equipment have led to an ever-growing need for prevention of microbial infection in these various areas. Macromolecular systems, due to their properties, allow one to efficiently use them in various fields, including the creation of polymers with the antimicrobial activity. In the past decade, the intensive development of a large class of antimicrobial macromolecular systems, polymers, and copolymers, either quaternized or functionalized with bioactive groups, has been continued, and they have been successfully used as biocides. Various permanent microbicidal surfaces with non-leaching polymer antimicrobial coatings have been designed. Along with these trends, new moderately hydrophobic polymer structures have been synthesized and studied, which contain protonated primary or secondary/tertiary amine groups that exhibited rather high antimicrobial activity, often unlike their quaternary analogues. This mini-review briefly highlights and summarizes the results of studies during the past decade and especially in recent years, which concern the mechanism of action of different antimicrobial polymers and non-leaching microbicidal surfaces, and factors influencing their activity and toxicity, as well as major applications of antimicrobial polymers.     

Molecularly imprinted polymers from nicotinamide and its positional isomers

Imprinted polymers were prepared for nicotinamide and its positional isomers. The influence of porogenic solvent and functional monomer on recognition properties of the polymer was compared. The results indicated that two functional groups, the heterocyclic nitrogen and the amide group, in the nicotinamide or isonicotinamide molecule have a synergistic effect in binding to the polymer. The polymers prepared with nicotinamide and isonicotinamide can be used as HPLC stationary phase for the separation of positional isomers of nicotinamide or isonicotinamide, while the polymer prepared with picolinamide showed no specificity toward the template. The mechanisms for the differences in recognition are discussed. In addition to the retention of polymers to their templates the polymers also displayed excellent retention to nicotinic acid and isonicotinic acid, compounds structurally similar to the template. This dual recognition property of the polymer may be useful in circumstances where the preparation of a polymer for a specific template may be problematic because of poor stability or solubility.     

Metabolic engineering of Escherichia coli for the production of medium-chain-length polyhydroxyalkanoates rich in specific monomers

The Escherichia coli fabG(Ec) gene and the Pseudomonas aeruginosa rhlG(Pa) gene, which encode 3-ketoacyl-acyl carrier protein reductase, were expressed in E. coli W3110 and its fadA mutant strain WA101 to examine their roles in medium-chain-length (MCL) polyhydroxyalkanoate (PHA) biosynthesis from fatty acids. When one of these 3-ketoacyl-acyl carrier protein reductase genes was co-expressed with the Pseudomonas sp. 61-3 PHA synthase gene (phaC2(Ps)) in E. coli W3110, MCL-PHA composed mainly of 3-hydroxyoctanoate and 3-hydroxydecanoate was synthesized from sodium decanoate. When the fabG(Ec) gene and the phaC2(Ps) gene were co-expressed in the fadA mutant E. coli strain WA101, MCL-PHA rich in 3-hydroxydecanoate monomer up to 93 mol% was accumulated from sodium decanoate. This was possible by efficiently redirecting 3-ketoacyl-coenzymes A from the beta-oxidation pathway to the PHA biosynthesis pathway without losing two carbon units, the strategy of which can be extended for the production of MCL-PHAs rich in other specific monomers.     

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.     

海藻酸盐裂解酶研究进展

海藻酸盐裂解酶是一类降解褐藻中海藻酸盐的酶。此酶已经在多种有机体中得到分离。对海藻酸盐裂解酶的生物特性、研究方法及其生物学功能进行了介绍。在酶学特性研究的基础上 ,通过酶解构建新型海藻酸盐多聚物 ,可增强和扩展海藻酸盐裂解酶在工业、农业、医药领域中的应用 ,使其在海藻多糖的高值化应用中发挥重要的作用。概述了海藻酸盐和海藻酸盐裂解酶过去和现在的研究状况 ,展望了海藻酸盐和海藻酸盐裂解酶将来的应用前景。     

Mannanases: microbial sources,production, properties and potential biotechnological applications

Mannans are the major constituents of the hemicellulose fraction in softwoods and show widespread distribution in plant tissues. The major mannan-degrading enzymes are β-mannanases, β-mannosidases and β-glucosidases. In addition to these, other enzymes such as α-galactosidases and acetyl mannan esterases, are required to remove the side chain substituents. The mannanases are known to be produced by a variety of bacteria, fungi, actinomycetes, plants and animals. Microbial mannanases are mainly extracellular and can act in wide range of pH and temperature because of which they have found applications in pulp and paper, pharmaceutical, food, feed, oil and textile industries. This review summarizes the studies on mannanases reported in recent years in terms of important microbial sources, production conditions, enzyme properties, heterologous expression and potential industrial applications.     

Screening for MCL-PHA-Producing Fluorescent Pseudomonads and Comparison of MCL-PHA Production Under Iso-osmotic Conditions Induced by PEG and NaCl

The medium chain length polyhydroxyalkanoates (MCL-PHA) have attracted much attention from academic and industrial communities for their interesting applications in medical field. The aim of this study was to screen high MCL-PHA-producing fluorescent pseudomonads, and to compare the effect of osmotic stress generated by NaCl (ionic) and polyethylene glycol (PEG, non-ionic inert polymer) on PHA production. A total of 50 fluorescent pseudomonads isolated from rhizospheric soil were screened for PHA production by Sudan Black staining. Out of all the PHA-producing isolates only five were MCL-PHA producers as detected by MCL-PCR. Isolate Bar1 identified as Pseudomonas fluorescens by 16S rRNA gene sequencing was selected for further analysis due to its high MCL-PHA production ability. The iso-osmotic stress generated by NaCl and PEG-6000 showed 5.75- and 3.19-fold enhanced production of PHA at ?2 bar osmotic potential, over control (0 bar), respectively. There was 1.8-fold enhanced production of PHA at ?2 bar osmotic stress induced by NaCl over PEG. PEG reduces availability of water to microorganisms without reducing exogenously provided nutrients which appear to be responsible for its down performance over NaCl. The FTIR analysis of PHA sample purified from cells showed strong marker bands near 1742, 2870, 1170, 1099, and 2926 cm^?1, corresponding to MCL-PHA. The study reported that supplementation of NaCl (electrolyte) in growth media enhances the production of MCL-PHA which can be very useful for its industrial production.     

Applications of natural silk protein sericin in biomaterials

Silk sericin is a natural macromolecular protein derived from silkworm Bombyx mori. During the various stages of producing raw silk and textile, sericin can be recovered for other uses. Also, sericin recovery reduces the environmental impact of silk manufacture. Sericin protein is useful because of its properties. The protein resists oxidation, is antibacterial, UV resistant, and absorbs and releases moisture easily. Sericin protein can be cross-linked, copolymerized, and blended with other macromolecular materials, especially artificial polymers, to produce materials with improved properties. The protein is also used as an improving reagent or a coating material for natural and artificial fibers, fabrics, and articles. The materials modified with sericin and sericin composites are useful as degradable biomaterials, biomedical materials, polymers for forming articles, functional membranes, fibers, and fabrics.     

Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid

The potential use of activated sludge for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was investigated. The enrichment of bacterial populations capable of producing MCL-PHAs was achieved by periodic feeding with nonanoic acid in a sequencing batch reactor (SBR). Denaturing gradient gel electrophoresis analysis revealed Pseudomonas aeruginosa strains to be predominant in the bacterial community during the SBR process. The composition of PHA synthesized by the enriched biomass from nonanoic acid consisted of a large concentration (>89 mol%) of MCL monomer units and a small amount of short-chain-length monomer units. Under fed-batch fermentation with continuous feeding of nonanoic acid at a flow rate of 0.225 g/L/h and a C/N ratio of 40, a maximum PHA content of 48.6% dry cell weight and a conversion yield (Y_p/s) of 0.94 g/g were achieved. These results indicate that MCL-PHA production by activated sludge is a promising alternative to typical pure culture approaches.     

Biosynthesis of polyesters and polyamide building blocks using microbial fermentation and biotransformation

Biopolymers can be a green alternative to fossil-based polymers and can contribute to environmental protection because they are produced using renewable raw materials. Biopolymers are composed of various small subunits (building blocks) that are the intermediates or end products of major metabolic pathways. Most building blocks are secreted directly outside of cells, making downstream processes easier and more economic. These molecules can be extracted from fermentation broth and polymerized to produce a variety of biopolymers, e.g., polybutylene terephthalate, polyethylene terephthalate, polytrimethylene terephthalate, nylon-5,4 and nylon-4,6, with applications in medicine, pharmaceuticals, and textiles. Microbes are unable to naturally produce these types of polymers; thus, the production of building blocks and their polymerization is a fascinating approach for the production of these polymers. In comparison to naturally occurring biopolymers, synthesized polymers have improved and controlled structures and higher purity. The production of monomer units provides a new direction for polymer science because new classes of polymers with unique properties that were not previously possible can be prepared. Furthermore, the engineering of microbes for building-block production is an easy process compared to engineering an entire biopolymer synthesis pathway in a single microbe. Polyesters and polyamide polymers have become an important part of human life, and their demand is increasing daily. In this review, recent approaches and technology are discussed for the production of polyester/polyamide building blocks, i.e., 2-hydroxyisobutyric acid, 3-hydroxypropionic acid, mandelic acid, itaconic acid, adipic acid, terephthalic acid, succinic acid, 1,3-propanediol, 2,3-butanediol, 1,4-butanediol, 1,3-butanediol, cadaverine, and putrescine.