Enzymatic synthesis and various properties of poly(catechol)

Poly(catechol) was prepared by using peroxidase as a catalyst in two types of solvent systems: an aqueous dioxane solution and a reverse micellar solution. Peroxidases derived from two sources, horseradish (HRP) and soybean (SBP), were employed as catalyst. The structure of the prepared polymer was elucidated by infrared analysis. Enzymatically prepared film of poly(catechol) at a Pt electrode was subjected to cyclic voltammetric studies in aqueous HCl medium and phosphate buffer pH 6.5. Thermal studies of the polymer were performed by thermogravimetric analysis. The iodine-labelled polymers showed low electrical conductivity in the range of 10⁻⁶ to 10⁻⁹ S cm⁻¹. The magnetic susceptibility and surface morphological property of the polymer were also studied.     

Amplification of inhibitory activity of catechin against disease-related enzymes by conjugation on poly(epsilon-lysine)

A new inhibitor against disease-related enzymes, collagenase, hyaluronidase, and xanthine oxidase, has been developed by the laccase-catalyzed conjugation of catechin on poly(epsilon-lysine). The resulting poly(epsilon-lysine)-catechin conjugate showed greatly improved inhibition effects on activity of these enzymes, whereas the catechin monomer showed very low inhibition activity. The kinetic analysis on the inhibition of collagenase exhibited that the conjugate was a mixed-type inhibitor. The amplified activities might offer high potential as a therapeutic agent for prevention of various enzyme-related diseases.     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

The production of poly-(gamma-glutamic acid) from microorganisms and its various applications

This review article deals with the chemistry and biosynthesis of poly-(gamma-glutamic acid) (gamma-PGA) produced by various strains of Bacillus. Potential applications of gamma-PGA as thickener, cryoprotectant, humectant, drug carrier, biological adhesive, flocculant, or heavy metal absorbent, etc. with biodegradability in the fields of food, cosmetics, medicine and water treatments are also reviewed.     

Microbial degradation of poly(amino acid)s

Natural poly(amino acid)s are a group of poly(ionic) molecules (ionomers) with various biological functions and putative technical applications and play, therefore, an important role both in nature and in human life. Because of their biocompatibility and their synthesis from renewable resources, poly(amino acid)s may be employed for many different purposes covering a broad spectrum of medical, pharmaceutical, and personal care applications as well as the domains of agriculture and of environmental applications. Biodegradability is one important advantage of naturally occurring poly(amino acid)s over many synthetic polymers. The intention of this review is to give an overview about the enzyme systems catalyzing the initial steps in poly(amino acid) degradation. The focus is on the naturally occurring poly(amino acid)s cyanophycin, poly(epsilon-L-lysine) and poly(gamma-glutamic acid); but biodegradation of structurally related synthetic polyamides such as poly(aspartic acid) and nylons, which are known from various technical applications, is also included.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils

[This corrects the article on p. 3236 in vol. 59.].     

Microbial synthesis of biodiesel and its prospects

An analytical review of the current data on the microbial synthesis of biodiesel and prospects of its usage is presented. The technological, biochemical, and genetic aspects of biodiesel production using microorganisms are discussed.     

Novel synthesis of functionalized poly(3-hydroxybutanoic acid) and its copolymers.

Novel feasibility of fuctionalized poly(3-hydroxybutanoic acid), PHB, and its copolymers synthesis via ring-opening of beta-butyrolactone (ROP) mediated by activated anionic initiators or enzymes in vitro is presented. Using these new synthetic approaches, PHB with defined chemical structure of the end groups as well as block, graft and random copolymers have been obtained and characterized by IR, NMR, ESI-MS and GPC techniques. The relationship between the structure and properties of the novel polymeric materials prepared is discussed.     

Microbial proteases in peptide synthesis: approaches and applications

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the environment is often limited due to unfavorable nutrient conditions for the bacteria that use these PAHs as sole source of carbon and energy. Mycobacterium and Sphingomonas are 2 PAH-degrading specialists commonly present in PAH-polluted soil, but not much is known about their specific nutrient requirements. By adding different inorganic supplements of nitrogen (N) and phosphorus (P), affecting the overall carbon/nitrogen/phosphorus ratio of soil in soil slurry degradation tests, we investigated the impact of soil inorganic N and P nutrient conditions on PAH degradation by PAH-degrading Sphingomonas and Mycobacterium strains. The general theoretically calculated C/N/P ratio of 100/10/1 (expressed in moles) allowed rapid PAH metabolization by Sphingomonas and Mycobacterium strains without limitation. In addition, PAH-degradation rate and extent was not affected when ca. ten times lower concentrations of N and P were provided, indicating that Sphingomonas and Mycobacterium strains are capable of metabolizing PAHs under low nutrient conditions. Nor does PAH-degradation seem to be affected by excesses of N and P creating an imbalanced C/N/P ratio. However, supplements of N and P salts increased the salinity of soil slurry solutions and seriously limited or even completely blocked biodegradation.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils.

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or a copolymer of 90% 3-hydroxybutyric acid and 10% 3-hydroxyvaleric acid was studied in soils incubated at a constant temperature of 15, 28, or 40 degrees C for up to 200 days. In addition, hydrolytic degradation in sterile buffer at temperatures ranging from 4 to 55 degrees C was monitored for 98 days. Degradation was measured through loss of weight (surface erosion), molecular weight, and mechanical strength. While no weight loss was recorded in sterile buffer, samples incubated in soils were degraded at an erosion rate of 0.03 to 0.64% weight loss per day, depending on the polymer, the soil, and the incubation temperature. The erosion rate was enhanced by incubation at higher temperatures, and in most cases the copolymer lost weight at a higher rate than the homopolymer. The molecular weights of samples incubated at 40 degrees C in soils and those incubated at 40 degrees C in sterile buffer decreased at similar rates, while the molecular weights of samples incubated at lower temperatures remained almost unaffected, indicating that molecular weight decrease is due to simple hydrolysis and not to the action of biodegrading microorganisms. The degradation resulted in loss of mechanical properties. From the samples used in the biodegradation studies, 295 dominant microbial strains capable of degrading P (3HB) and the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer in vitro were isolated and identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial alginate production,modification and its applications

Alginate is an important polysaccharide used widely in the food, textile, printing and pharmaceutical industries for its viscosifying, and gelling properties. All commercially produced alginates are isolated from farmed brown seaweeds. These algal alginates suffer from heterogeneity in composition and material properties. Here, we will discuss alginates produced by bacteria; the molecular mechanisms involved in their biosynthesis; and the potential to utilize these bacterially produced or modified alginates for high-value applications where defined material properties are required.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

Microbial biosynthesis and secretion of l-malic acid and its applications

l-Malic acid has many uses in food, beverage, pharmaceutical, chemical and medical industries. It can be produced by one-step fermentation, enzymatic transformation of fumaric acid to l-malate and acid hydrolysis of polymalic acid. However, the process for one-step fermentation is preferred as it has many advantages over any other process. The pathways of l-malic acid biosynthesis in microorganisms are partially clear and three metabolic pathways including non-oxidative pathway, oxidative pathway and glyoxylate cycle for the production of l-malic acid from glucose have been identified. Usually, high levels of l-malate are produced under the nitrogen starvation conditions, l-malate, as a calcium salt, is secreted from microbial cells and CaCO₃ can play an important role in calcium malate biosynthesis and regulation. However, it is still unclear how it is secreted into the medium. To enhance l-malate biosynthesis and secretion by microbial cells, it is very important to study the mechanisms of l-malic acid biosynthesis and secretion at enzymatic and molecular levels.     

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone)

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.     

Enzymatic synthesis and antioxidant properties of poly(rutin)

Rutin, quercetin-3-rutinoside, is one of the most famous glycosides of flavonoid and widely present in many plants. In this study, we performed an oxidative polymerization of rutin using Myceliophthora laccase as catalyst in a mixture of methanol and buffer to produce a flavonoid polymer and evaluated antioxidant properties of the resultant polymer. Under selected conditions, the polymer with molecular weight of several thousands was obtained in good yields. The resulting polymer was readily soluble in water, DMF, and DMSO, although rutin monomer showed very low water solubility. UV measurement showed that the polymer had broad transition peaks around 255 and 350 nm in water, which were red-shifted in an alkaline solution. Electron spin resonance (ESR) measurement showed the presence of a radical in the polymer. The polymer showed greatly improved superoxide scavenging activity and inhibition effects on human low-density lipoprotein (LDL) oxidation initiated by 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), compared with the rutin monomer. The polymer also protected endothelial cells from oxidative injury induced by AAPH as a radical generator with a much greater effect than the rutin monomer.     

Microbial CO conversions with applications in synthesis gas purification and bio-desulfurization

Recent advances in the field of microbial physiology demonstrate that carbon monoxide is a readily used substrate by a wide variety of anaerobic micro-organisms, and may be employed in novel biotechnological processes for production of bulk and fine chemicals or in biological treatment of waste streams. Synthesis gas produced from fossil fuels or biomass is rich in hydrogen and carbon monoxide. Conversion of carbon monoxide to hydrogen allows use of synthesis gas in existing hydrogen utilizing processes and is interesting in view of a transition from hydrogen production from fossil fuels to sustainable (CO2-neutral) biomass. The conversion of CO with H2O to CO2 and H2 is catalyzed by a rapidly increasing group of micro-organisms. Hydrogen is a preferred electron donor in biotechnological desulfurization ofwastewaters and flue gases. Additionally, CO is a good alternative electron donor considering the recent isolation of a CO oxidizing, sulfate reducing bacterium. Here we review CO utilization by various anaerobic micro-organisms and their possible role in biotechnological processes, with a focus on hydrogen production and bio-desulfurization.     

Solid-phase synthesis and applications of N-(S-acetylmercaptoacetyl) peptides

The reagent pentafluorophenyl S-acetylmercaptoacetate was used to modify the N-terminus of resin-bound side-chain-protected peptides. The modification was carried out in an automated cycle in the final stage of fluorenylmethoxycarbonyl (Fmoc)/polyamide-mediated solid-phase synthesis. Side-chain deprotection and cleavage from the resin with aqueous trifluoroacetic acid gave the N-(S-acetylmercaptoacetyl) peptides. The S-acetylmercaptoacetyl peptides were transformed into reactive thiol-containing peptides by incubation with hydroxylamine at neutral pH. The S-deacetylation was performed in the presence of a sulfhydryl-reactive compound (or intramolecular group) to enable immediate capture of the sensitive thiol. Three applications were investigated. An S-acetylmercaptoacetyl peptide, containing a sequence of a meningococcal membrane protein, was incubated with hydroxylamine in the presence of 5-(iodoacetamido)fluorescein to give the corresponding fluorescein-labeled peptide in 62% yield. The same peptide was also S-deacetylated in the presence of bromoacetylated poly-L-lysine to afford a peptide/polylysine conjugate. Finally, a peptide corresponding to a sequence of herpes simplex virus glycoprotein D was prepared. This peptide, containing an N-terminal-S-acetylmercaptoacetyl group and an additional C-terminal S-(3-nitro-2-pyridinesulfenyl)cysteine residue, was converted into a cyclic disulfide peptide (20%).     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.