p-Benzoquinone monoketals,novel degradation products of β-O-4 lignin model compounds by Coriolus versicolor and lignin peroxidase of Phanerochaete chrysosporium

2-(4-Ethoxy-3-methoxyphenyl)-3-hydroxymethyl-6,10-dimethoxy-1,4-dioxaspiro[4,5]deca-6,9-diene-8-one (III) and its isomer IV were identified as catabolites of 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I) by the culture of Coriolus versicolor. Compound III was also produced from 4-ethoxy-3-methoxyphenylglycerol-β-syringic acid ether (II) by lignin peroxidase of Phanerochaete chrysosporium. An isotopic experiment showed that molecular oxygen was incorporated into the quinone oxygen of III in the degradation of II by lignin peroxidase.     

Immunogold-cytochemical labelling of β-glucosidase in the white-rot fungus Coriolus versicolor

Summary Immunogold cytochemical labelling of hyphal sections of Coriolus versicolor showed that -glucosidase was localised in the extracellular mucilage, cell wall layers and cell interior in hyphae grown on glucose-rich malt extract medium whereas in hyphae grown with carboxymethylcellulose (CMC) as sole carbon source, most labelling was in the cell wall layers and cell interior. Little mucilage was visible around hyphae from these cultures. Hyphae from beechwood cultures showed gold labelling of -glucosidase in mucilage and fungal cell walls with some intracellular labelling. Biochemical studies of enzyme activity showed that similar amounts of enzyme were detected in the growth medium when cultures were grown on CMC medium, in agitated liquid cultures or in stationary cultures. In agitated cultures grown on glucose-rich malt extract, the activity of -glucosidase in the medium was 100 times less than that detected in stationary cultures on the same medium. However activity in the hyphae of stationary CMC-grown cultures was similar to that in hyphae from stationary glucose-rich cultures. These data confirm the patterns of gold labelling observed in hyphae from stationary cultures on glucose-rich malt extract when -glucosidase was immobilised in the extracellular mucilage layer around the hyphae. In this paper we propose that a primary function of the extracellular mucilage produced by hyphae of C. versicolor in vivo is to serve as a matrix for immobilisation of -glucosidase. Its substrate, cellobiose, which is released as a result of endo-and exoglucanase hydrolysis of cellulose, is absorbed and retained by the gel filtration properties of the mucilage, so encountering the immobilised -glucosidase. Glucose produced by this reaction is retained within the mucilage matrix around the hyphae before intracellular absorption.Offprint requests to: C. S. Evans     

Metabolism of non-phenolic β-O-4 lignin model compounds by the white-rot fungus Phlebia radiata

Summary The degradation of three non-phenolic -O-4 diarylpropane lignin model compounds was studied in cultures of the white-rot fungus Phlebia radiata. The degradation pattern of the model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) was also compared with that of Phanerochaete chrysosporium under conditions where both fungi were cultivated without agitation in an oxygen atmosphere. Compound I was readily degraded by both fungi, and qualitatively the degradation patterns were quite similar. The product, after C-C bond cleavage, was veratraldehyde (IV) which was almost stoichiometrically reduced to veratryl alcohol (V). However, large amounts of V were detected only in P. chrysosporium cultures. Experiments with the model compound 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (II) showed that in the presence of II, the total amount of veratryl compounds accounted for 15–33 m in standing cultures of Phlebia radiata. The model compound 1-(3,4-dimethoxyphenyl)-2-(4-methoxyphenoxy) propane-1,3-diol (III) was more readily degraded than I and II. The results indicated that, in P. radiata cultures, the acting enzymes were lignin peroxidases and IV reducing enzyme, while laccase was less important. Offprint requests to: A. Hatakka     

Photocatalytic Degradation of β-O-4 Lignin Model Compound by In<Subscript>2</Subscript>S<Subscript>3</Subscript> Nanoparticles Under Visible Light Irradiation

Cleaving the abundant β-O-4 linkages in lignin is a key issue for producing value-added products by controlled lignin depolymerization. Herein, hydrothermally synthesized In₂S₃ nanoparticles were primarily used to photodegrade guaiacylglycerol-β-guaiacyl ether, a β-O-4 lignin model compound, under visible light irradiation. The as-synthesized In₂S₃ nanoparticles are found to be typical β-In₂S₃ nanocrystals of cubic phase and composed of large plate-like particles and small granular particles by using X-ray diffraction technique and field-emission scanning electron microscopy. The bandgap energy of the In₂S₃ nanoparticles is estimated to be 1.78 eV using an UV-visible diffuse reflectance spectroscopy. The photodegradation and structure variation of lignin model compound were evaluated by the variation of its UV-vis absorption spectrum, Fourier transform infrared spectrum, and X-ray photoelectron spectroscopy, while its degradation products were identified by using the gas chromatography-mass spectrometry. The results show that the as-synthesized In₂S₃ nanoparticles can photocatalytically break the β-O-4 linkage and oxidize the hydroxyl/methoxyl groups of lignin model compound under visible light irradiation although the lignin model compound is photo-resistant even under UV irradiation. The photodegradation products of lignin model compound consist of various aromatic monomers including value-added acetovanillone, vanillin, and coniferyl aldehyde. A possible pathway is proposed for photodegrading lignin model compound in the presence of the as-synthesized In₂S₃ nanoparticles under visible light irradiation.     

Properties of the β-d-glucosidase (cellobiase) from the wood-rotting fungus,Coriolus versicolor

Summary Structural and kinetic parameters of the -d-glucosidase (cellobiase, -d-glucoside glucohydrolase) from Coriolus versicolor have been determined. It is a high molecular weight glycoprotein (300,000 d) composed 10% by weight of protein, 90% by weight of carbohydrate in which glucose is the primary hexose sugar. The K_m for 4-nitrophenyl--d-glucopyranoside (4 NPG) and cellobiose are 0.276 and 2.94 mM respectively at pH 4.5 and 40°. d-Glucose is a competitive inhibitor with a K_i of 1.8 mM with 4 NPG as substrate, and at high concentrations, cellobiose exhibits a substrate inhibition effect on the enzyme, so negating attempts to overcome the competitive inhibition of glucose by increasing the concentration of the substrate.     

Mechanism of aromatic ring cleavage of β-O-4 lignin substructure models by lignin peroxidase

This investigation examined the aromatic ring cleavage of β-O-4 lignin substructure model compounds by lignin peroxidase of Phanerochaete chrysosporium. Based on tracer experiments using H₂¹⁸O and ¹⁸O₂, mechanisms of the aromatic ring cleavage of the β-O-4 lignin models were proposed. The mechanisms involve one-electron oxidation of the β-O-4 lignin models by the enzyme followed by attack of nucleophiles and radical coupling with O₂.     

Enzymatic synthesis of capsaicin 4-O-β-xylooligosaccharides by β-xylosidase from Aspergillus sp

Capsaicin 4-O-β-xylooligosaccharides were synthesized by a biocatalytic xylosylation using Aspergillus sp. β-xylosidase. Capsaicin was converted into three new capsaicin glycosides, i.e. capsaicin 4-O-β-xyloside, capsaicin 4-O-β-xylobioside, and capsaicin 4-O-β-xylotrioside in 15, 12 and 10% yield, respectively. All products were isolated from the reaction mixtures by preparative HPLC. The structures of the products were determined by NMR spectroscopic method.     

Thermal mobility of β-O-4-type artificial lignin

Several lignin model polymers and their derivatives comprised exclusively of β-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.     

Microbial and biochemical characterization of a bacterial consortium isolated from decaying wood by growth on a β-O-4 lignin-related dimeric compound

As an approach to evaluate the contribution of bacteria to lignin degradation in wood, we have chosen to study these microorganisms in the natural wood decay ecosystem known as Palo Podrido. Initially, the characterization of bacteria able to metabolize lignin-related compounds present in samples of Palo Podrido was undertaken. For their isolation, minimal salt media containing lignin dimers of either the arylglycerol--aryl ether (-O-4) or 1,2-diarylpropane (-1) types as the only source of carbon and energy were inoculated with various wood samples exhibiting different degrees of decay. The -1 dimers used failed to support bacterial growth. However, three bacterial consortia able to consume quantitatively the -O-4 model 1-[3,4-dimethoxyphenyl]-2-[2-methoxyphenoxy]-3-hydroxypropanone (compound 1) were isolated. One of these was further characterized. It is composed of eight strains belonging to the families of Streptomycetaceae, Dermatophilaceae and Actinoplanaceae. HPLC and GC-MS analyses revealed that the consortium utilizes two pathways to degrade -O-4 dimers, both involving direct cleavage of the ether linkage. The formation of a novel C₆–C₃ degradation intermediate is described. Some metabolic properties of each strain, as well as those of the intact consortium, are also reported.     

Metabolism of the Lathyrus sativus L. Neurotoxin, β-N-Oxalyl-L-α, β-diaminopropionic Acid,by a Pure Culture of a Soil-borne Microbe

Pure cultures of bacteria capable of utilizing the Lathyrus sativus L. neurotoxin, β-N-oxalyl-L-α, β-diaminopropionic acid (ODAP), as their sole carbon and nitrogen source have been isolated from soil-sludge filtrates. Three independent isolates, designated BYA1, BYT1, and BYK1, were selected by repetitive growth on the neurotoxin and purified based upon their antibiotic resistance. Of the three Isolates, strain BYA1 demonstrated the highest capacity for ODAP utilization, degrading greater than 98% of the ODAP present In the culture media within 12 h. Using a variety of morphological and biochemical criteria BYA1 was Identified as an Enterobacter cloacae. The bacterium harbors a single large plasmid (designated pBYA1) approximately 40–50 kb in size that contains the genetic Information for ODAP utilization and antibiotic resistance. Transformation experiments with E. coli recipient strains were used to further define the location of the sequences involved in ODAP metabolism.     

Synthesis of oligosaccharides by reversal of a fungal β-glucanase

Summary A crude commercial preparation of -glucanase fromPenicillium emersonii was used to synthesise glucose-containing oligosaccharides by condensation reactions in high concentrations of glucose at elevated temperature. Gentiobiose, laminaribiose, cellobiose, isomaltose and trehalose were identified as products. Heterooligosaccharides were produced by enzyme in some mixtures of glucose and an acceptor sugar. High performance ion-exchange chromatography was used to analyse synthetic products.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Investigation of apigenin-7-O-β-glucoside hydrolysis by β-glucosidase from almonds

Summary The enzymatic transformation of apigenin-7-O--glucoside into apigenin is effected using -glucosidase from almonds. Kinetic studies gave the following values K_m=7.63×10^–4mol/dm³, v_max=10.37 mol mg^–1 min^–1 and E₁=65.21 kJ/mol when the substrate was pure apigenin-7-O--glucoside, while in case of apigenin-7-O--glucoside from dry extract of camomile ligulate flowers E₂ = 65.57 kJ/mol.Nomenclature S_o initial substrate concentration (g/100 cm³) - P product concentration (g/100 cm³) - k reaction rate constant (min^–1) - t reaction time (min)     

Mathematical Model of Metabolism and Electrophysiology of Amino Acid and Glucose Stimulated Insulin Secretion: In Vitro Validation Using a β-Cell Line

We integrated biological experimental data with mathematical modelling to gain insights into the role played by L-alanine in amino acid-stimulated insulin secretion (AASIS) and in D-glucose-stimulated insulin secretion (GSIS), details important to the understanding of complex β-cell metabolic coupling relationships. We present an ordinary differential equations (ODEs) based simplified kinetic model of core metabolic processes leading to ATP production (glycolysis, TCA cycle, L-alanine-specific reactions, respiratory chain, ATPase and proton leak) and Ca²⁺ handling (essential channels and pumps in the plasma membrane) in pancreatic β-cells and relate these to insulin secretion. Experimental work was performed using a clonal rat insulin-secreting cell line (BRIN-BD11) to measure the consumption or production of a range of important biochemical parameters (D-glucose, L-alanine, ATP, insulin secretion) and Ca²⁺ levels. These measurements were then used to validate the theoretical model and fine-tune the parameters. Mathematical modelling was used to predict L-lactate and L-glutamate concentrations following D-glucose and/or L-alanine challenge and Ca²⁺ levels upon stimulation with a non metabolizable L-alanine analogue. Experimental data and mathematical model simulations combined suggest that L-alanine produces a potent insulinotropic effect via both a stimulatory impact on β-cell metabolism and as a direct result of the membrane depolarization due to Ca²⁺ influx triggered by L-alanine/Na⁺ co-transport. Our simulations indicate that both high intracellular ATP and Ca²⁺ concentrations are required in order to develop full insulin secretory responses. The model confirmed that K⁺_ATP channel independent mechanisms of stimulation of intracellular Ca²⁺ levels, via generation of mitochondrial coupling messengers, are essential for promotion of the full and sustained insulin secretion response in β-cells.     

Purification and Some Properties of Lignostilbene-a,/i-dioxygenase Responsible for the Ca—Cp Cleavage of a Diarylpropane Type Lignin Model Compound from Pseudomonas sp. TMY1009

A novel dioxygenase, lignostilbene-a,β-dioxygenase (LSD), which catalyzes cleavage of the interphenyl double bond of lignin-derived stilbenes, was isolated. Four isozymes of LSD were separated from cell-free extracts of Pseudomonas sp. TMY1009 by ion-exchange chromatography on a DEAE- Toyopearl column. The major isozyme, LSD-I, was purified to electrophoretic homogeneity and characterized.

LSD-I cleaved the interphenyl double bond of l,2-bis(4′-hydroxy-3′-methoxyphenyl)ethylene with the optimum pH at 8.5. The Km of LSD-I was 11 μm for the stilbene and 110/iM for oxygen. The molecular weight of LSD-I, which is composed of two identical subunits, was estimated to be 94,000. LSD-I contained 1 g atom of iron per 1 mol of enzyme protein.     

Metabolism of Phosphatidylinositol 4-Kinase III��-Dependent PI4P Is Subverted by HCV and Is Targeted by a 4-Anilino Quinazoline with Antiviral Activity

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV – by recruiting PI4KIIIα in the RNA replication complex – hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product.     

Intracellular Metabolism of β-L-ddAMP-bis(tbutylSATE), a Potent Inhibitor of Hepatitis B Virus Replication

Abstract

β-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC₅₀ = 0.1 μM. Following a 0-to72-hrs exposure of human hepatocytes to a 10 μM [2′,3′^?3H] β-L-ddAMP-bis(tbutylSATE), the pharmacologically active β-L-ddATP was the predominant metabolite attaining a concentration of 268.53 ± 107.97 pmoles/10⁶ cells at 2 hrs. In Hep-G2 cell, β-L-ddATP accounted for 146.8 ± 29.8 pmoles/10⁶ cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of β-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.