Selective delignification of wood by white-rot fungi

The white-rot fungi,Cerrena unicolor, Ganoderma applanatum, G. tsugae,Ischnoderma resinosum, andPerenniporia medullapanis, caused two distinct types of decay. Large areas of decayed wood were selectively delignified and a typical white-rot causing a simultaneous removal of all cell wall components was present. Preferential lignin degradation was intermittently dispersed throughout the decayed wood. Scanning and transmission electron microscopy were used to identify the micromorphological and ultrastructural changes that occurred in the cells during degradation. In delignified areas the compound middle lamella was extensively degraded without substantial alteration of the secondary wall. The S₂ layer of the secondary wall was least affected. The loss of middle lamellae resulted in extensive defibration of the cells. Sulfuric acid lignin determinations indicated that 95–98% of the lignin was removed. Wood sugar analyses using high pressure liquid chromatography demonstrated that hemicelluloses were removed in preference to cellulose when lignin was degraded. The results suggest that a highly diffusible ligninolytic system was responsible for the selective degradation of the wood. In simultaneously white-rotted wood, all cell wall layers were progressively removed from the cell lumen toward the middle lamella, causing erosion troughs or holes to form. Large voids filled with fungal mycelia resulted from a coalition of degraded areas. Chemical analyses of white-rotted wood indicated lignin, cellulose, and hemicellulose were removed in approximately the same amounts. Degradation was confined to areas around fungal hyphae.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium under solid-state fermentation

Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28±1 °C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.     

Syringic acid metabolism by some white-rot,soft-rot,and brown-rot fungi

Syringic acid metabolism by four white-rot, two soft-rot, and two brown-rot fungi has been studied. The pathways for syringic acid metabolism have been studied in detail for the white-rot fungus,Sporotrichum pulverulentum, and a reaction sequence proposed. According to identified metabolites, the following reactions occur: reduction of the carboxyl group, hydroxylation and simultaneous decarboxylation, demethylation, and methylation of thep-hydroxyl group. In the case of the two soft-rot fungi,Petriellidium boydii andPhialophora mutabilis, rapid metabolism of syringic acid was observed, and the medium was depleted of the acid within 12 h. The formation of trimethoxybenzoic acid indicates an ability within the soft-rot fungi also to methylate the p-hydroxylic group. The two brown-rot fungi,Daedalea quercina andFomes pinicola, were poor metabolizers of syringic acid. However, demethylation was observed withF. pinicola and an unidentified product appeared in the culture solution ofD. quercina.     

Mass spectrometry in the study of extracellular enzymes produced by filamentous fungi

The use of MALDI-TOF and other types of mass spectrometry for the identification and investigation of extracellular enzymes (carbohydrases, proteinases, esterases, etc.) produced by filamentous fungi belonging to the genera Aspergillus, Trichoderma, Penicilium, Chrysosporium, etc. is discussed. The method of mass spectrometric peptide fingerprinting combined with the use of Internet software for on line data analysis (MASCOT, Aldente, FindPept, FindMod, GlycoMod) allows the fast and reliable identification of both individual enzymes and the components of crude multienzyme preparations without their fractionation. The method was also applied to the discrimination of the fungal genes encoding enzymes with similar substrate specificity. Other enzyme-based applications of mass spectrometry, such as the revelation of the presence or absence of structural domains in the molecules of carbohydrases, the detection of posttranslational and artificial modifications in the enzymes, and the use of tandem mass spectrometry for de novo peptide sequencing are described.     

Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production

The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, β-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly β-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29°C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.     

Natural products of filamentous fungi: enzymes, genes, and their regulation

We review the literature on the enzymes, genes, and whole gene clusters underlying natural product biosyntheses and their regulation in filamentous fungi. We have included literature references from 1958, yet the majority of citations are between 1995 and the present. A total of 295 references are cited.     

Ethanol production from cellulose by two lignocellulolytic soil fungi

Applied Biochemistry and Biotechnology - The present work examines the production of ethanol via direct fermentation of pure celluloses and lignocellulosic wastes by two soil fungi isolated under...     

Efficient production of mannan-degrading enzymes by the basidiomyceteSclerotium rolfsii

Sclerotium rolfsii CBS 191.62 was cultivated on a number of carbon (C) sources, including mono- and disaccharides, as well as on polysaccharides, to study the formation of different mannan-degrading enzyme activities. Highest levels of mannanase activity were obtained when α-cellulose-based media were used for growth, but formation of mannanase could not be enhanced by employing galactomannan as the only carbon source. Although both xylanase and cellulase formation was almost completely repressed whenS. rolfsii was grown on more readily metabolizable carbohydrates, including glucose or mannose, considerable amounts of mannanase activity were secreted under these growth conditions. Enhanced mannanase production only commenced when glucose was depleted in the medium. The maximal mannanase activity of 240 IU/mL obtained in a laboratory fermentation is remarkable. Mannanase activity formed under these derepressed conditions could be mainly attributed to one major, acidic mannanase isoenzyme with a pI value of 2.75.     

Increase of biogas production from pretreated hay and leaves using wood-rotting fungi

Wood-decaying mushrooms can be applied for the pretreatment of lignocellulosic substrates such as leaves, hay and straw. The use of wood-decaying fungus Auricularia auricula-judae for the decomposition of sweet chestnut (Castanea sativa) leaves and hay is discussed in the proposed paper. Such pretreated substrate was employed in the anaerobic processes for biogas production. Comparison of pretreated and non-pretreated substrate revealed that an increase of 15 % in the biogas production can be achieved using the pretreated substrate. Composition of organic compounds in the sludge during the anaerobic process was identified by HPLC. The obtained results show that the utilization of pretreated leaves and hay leads to a gradual increase of the concentration of formic, acetic, and volatile fatty acids as well as to the formation of some aldehydes, ketones, and alcohols.     

微生物法生产1,3-丙二醇的代谢及关键酶研究进展

微生物法生产1,3-丙二醇具有条件温和、环境友好的特点,是目前研究的热点。本文着重介绍了微生物法生产1,3-丙二醇所涉及的菌种和代谢过程,系统阐述了1,3-丙二醇代谢过程中关键酶的性质特点及有关基因,并展望了未来的发展趋势。     

Action of white-rot fungus Panus tigrinus on sugarcane bagasse

Biological pretreatments with three selected strains of Panus tigrinus were used for delignification of sugarcane bagasse. The fungi with potential for delignification were analyzed by determining the chemical composition of the decayed bagasse samples, and the selectivity in terms of weight loss of the different components was evaluated. All the strains grow abundantly on bagasse as unique carbon source. After determining the chemical composition of degraded bagasse, P. tigrinus FTPT-4745 was selected as the most efficient strain on a 6-g scale, since the carbohydrates were preserved. P. tigrinus FTPT-4741 and FTPT-4742 were the most efficient strains on a large scale (100 g).     

Transformation of textile dyes by white-rot fungus Trametes versicolor

We have investigated transformation of eight industrial dyes by a whiterot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red, Reactive Violet 5, Reactive Blue 28, and Ponceau Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H₂O₂-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced by Mn²⁺ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor.     

Esterification with solid enzymes

Water activation of the enzyme (an esterase of Mucor miehei) is studied during oleic acid/1-decanol esterification with or without solvent. The activation is rapid, but not instantaneous. Reaction water and water added before the beginning of the reaction do not have the same influence. The activation of the enzyme is effected by its swelling with water. When the initial quantity of water is sufficient, the reaction order is zero. But from a certain conversion, the rate decreases very suddenly or very slowly. This observation is attributed to the partitioning of an aqueous phase around the enzyme.

     

Induced production of novel prenyldepside and coumarins in endophytic fungi Pestalotiopsis acaciae

Concomitant supplementation of a histone deacetylase inhibitor, suberoylanilide hydroxamic acid, and a DNA methyltransferase inhibitor, 5-azacytidine, to the culture medium of a plant endophytic fungus, Pestalotiopsis acaciae, dramatically altered its metabolic profiles. As a result, three novel aromatic compounds, 2′-hydroxy-6′-hydroxymethyl-4′-methylphenyl-2,6-dihydroxy-3-(2-isopentenyl)benzoate (1), 4,6-dihydroxy-7-hydroxymethyl-3-methylcoumarin (2) and 4,6-dihydroxy-3,7-dimethylcoumarin (3), were isolated, along with five known polyketides, endocrocin (4), pestalotiollide B (5), pestalotiopyrone G (6), scirpyrone A (7) and 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (8).     

Recent advances in production of lignocellulolytic enzymes by solid-state fermentation of agro-industrial wastes

1. Download : Download high-res image (254KB)
2. Download : Download full-size image

     

Solid-state fermentation of new substrates for production of cellulase and other biopolymer-hydrolyzing enzymes

Applied Biochemistry and Biotechnology -     

Production of cellulolytic and xylanolytic enzymes during growth of anaerobic fungi from ruminant and nonruminant herbivores on different substrates

Applied Biochemistry and Biotechnology - Three anaerobic fungi, twoNeocallimastix strains isolated from a ruminant (sheep) and onePiromyces strain isolated from a nonruminant (black rhinoceros),...     

Dioxygenases, key enzymes to determine the aglycon structures of fusicoccin and brassicicene, diterpene compounds produced by fungi

Fusicoccin A and cotylenin A are structurally related diterpene glucosides and show a phytohormone-like activity. However, only cotylenin A induces the differentiation of human myeloid leukemia cells. Since the cotylenin A producer lost its ability to proliferate during preservation, a study on the relationship between structure and activity was carried out and a modified fusicoccin A with hydroxyl group at the 3-position showed a similar biological activity with that of cotylenin A. We then searched for an enzyme source that catalyzes the introduction of a hydroxyl group into the 3-position and found that brassicicene C, which is structurally related to fusicoccin A with hydroxyl group at the 3-position, was produced by Alternaria brassicicola ATCC96836. We recently cloned a brassicicene C biosynthetic gene cluster including the genes encoding fusicocca-2,10(14)-diene synthase and two cytochrome P450s, which were responsible for the formation of fusicocca-2,10(14)-diene-8β,16-diol. In this study, we report that a α-ketoglutarate dependent dioxygenase, the gene coding for which was located in the cluster, catalyzed a hydroxylation at the 3-position of fusicocca-2,10(14)-diene-8β,16-diol. On the other hand, a α-ketoglutarate-dependent dioxygenase, which had been identified in a fusicoccin A biosynthetic gene cluster, catalyzed the 16-oxidation of fusicocca-2,10(14)-diene-8β,16-diol to yield an aldehyde (8β-hydroxyfusicocca-1,10(14)-dien-16-al), although both dioxygenases had 51% amino acid sequence identity. These findings suggested that the dioxygenases played critical roles for the formation of the fusicoccin A-type and cotylenin A-/brassicicene C-type aglycons. Moreover, we showed that short-chain dehydrogenase/reductase located in the fusicoccin A biosynthetic gene cluster catalyzed the reduction of the aldehyde to yield fusicocca-1,10(14)-diene-8β,16-diol.     

Siderophore production by a marine Pseudomonas aeruginosa and its antagonistic action against phytopathogenic fungi

A marine isolate of fluorescent Pseudomonas sp. having the ability to produce the pyoverdine type of siderophores under low iron stress (up to 10 μM iron in the succinate medium) was identified as Pseudomonas aeruginosa by using BIOLOG Breathprint and siderotyping. Pyoverdine production was optimum at 0.2% (w/v) succinate, pH 6.0, in an iron-deficient medium. Studies carried out in vitro revealed that purified siderophores and Pseudomonas culture have good antifungal activity against the plant deleterious fungi, namely, Aspergillus niger, Aspergillus flavus, Aspergillus oryzae, Fusarium oxysporum, and Sclerotium rolfsii. Siderophore-based maximum inhibition was observed against A. niger. These in vitro antagonistic actions of marine Pseudomonas against phytopathogens suggest the potential of the organism to serve as a biocontrol agent.