Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH₂). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH₂ extended the reactions catalyzed by these enzymes to the production of H₂O₂, the oxidation of Mn²⁺, the reduction of Fe³⁺, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

A 24.7-kDa copper-containing oxidase, secreted by Thermobifida fusca, significantly increasing the xylanase/cellulase-catalyzed hydrolysis of sugarcane bagasse

Thermobifida fusca is a moderately thermophilic soil bacterium belonging to Actinobacteria. It has been known for its capability to degrade plant cell wall polymers except lignin and pectin. To know whether it can produce enzymes to facilitate lignin degradation, the extracellular proteins bound to sugarcane bagasse were harvested and identified by liquid chromatography tandem mass spectrometry. Among the identified proteins, a putative copper-containing polyphenol oxidase of 241 amino acids, encoded by the locus Tfu_1114, was thought to presumably play a role in lignin degradation. This protein (Tfu1114) was thus expressed in E. coli and characterized. Similarly to common laccases, Tfu1114 is able to catalyze the oxidation reaction of phenolic and nonphenolic lignin related compounds such as 2,6-dimethoxyphenol and veratryl alcohol. More interestingly, it can significantly enhance the enzymatic hydrolysis of bagasse by xylanase and cellulase. Tfu1114 is stable against heat, with a half-life of 4.7 h at 90 °C, and organic solvents. It is sensitive to ethylenediaminetetraacetic acid and reducing agents but resistant to sodium azide, a potent inhibitor of laccases. Atomic absorption spectroscopy indicated that the ratio of copper to the protein monomer is 1, instead of 4, a feature of classical laccases. All these data suggest that Tfu1114 is a novel oxidase with laccase-like activity, potentially useful in biotechnology application.     

A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity

Lignin degradation by the white rot basidiomycete Phanerochaete chrysosporium involves various extracellular oxidative enzymes, including lignin peroxidase, manganese peroxidase, and a peroxide-generating enzyme, glyoxal oxidase. Recent studies have suggested that laccases also may be produced by this fungus, but these conclusions have been controversial. We identified four sequences related to laccases and ferroxidases (Fet3) in a search of the publicly available P. chrysosporium database. One gene, designated mco1, has a typical eukaryotic secretion signal and is transcribed in defined media and in colonized wood. Structural analysis and multiple alignments identified residues common to laccase and Fet3 sequences. A recombinant MCO1 (rMCO1) protein expressed in Aspergillus nidulans had a molecular mass of 78 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the copper I-type center was confirmed by the UV-visible spectrum. rMCO1 oxidized various compounds, including 2,2′-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS) and aromatic amines, although phenolic compounds were poor substrates. The best substrate was Fe²⁺, with a K_m close to 2 μM. Collectively, these results suggest that the P. chrysosporium genome does not encode a typical laccase but rather encodes a unique extracellular multicopper oxidase with strong ferroxidase activity.     

Genome Sequence of Pseudomonas aeruginosa DQ8, an Efficient Degrader of n-Alkanes and Polycyclic Aromatic Hydrocarbons

Pseudomonas aeruginosa DQ8, which was isolated from the crude oil polluted soil in the Daqing oilfield of China, can efficiently degrade diesel, crude oil, n-alkanes, and polycyclic aromatic hydrocarbons (PAHs). Here, we present a 6.8-Mb assembly of its genome sequence. We have annotated 23 coding sequences (CDSs) responsible for catabolism of n-alkanes and PAHs.     

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20 % (v/v) tert-butanol.     

<Emphasis Type="Italic">Phoma herbarum</Emphasis>, a soil fungus able to grow on natural lignin and synthetic lignin (DHP) as sole carbon source and cause lignin degradation

The fungus Phoma herbarum isolated from soil showed growth on highly pure lignin extracted from spruce wood and on synthetic lignin (DHP). The lignin remaining after cultivation was shown to have a lower molecular weight. The reduction in the numbers of ether linkages of the extracted lignins was also observed by derivatization followed by reductive cleavage (DFRC) in combination with ³¹P NMR studies. The fungal strain showed an ability to degrade synthetic lignin by extracellular catalysts. GC–MS was applied to study the evolution of low molar mass adducts, e.g., monolignols and it was shown that a reduced coniferyl alcohol product was produced from DHP in a cell-free environment. The work has demonstrated the ability of soil microbes to grow on lignin as sole carbon source. The potential impact is in the production of low molar mass renewable phenols for material application.     

Microbial Degradation of Alkyl Carbazoles in Norman Wells Crude Oil

Norman Wells crude oil was fractionated by sequential alumina and silicic acid column chromatography methods. The resulting nitrogen-rich fraction was analyzed by gas chromatography-mass spectrometry and showed 26 alkyl (C₁ to C₅) carbazoles to be the predominant compounds. An oil-degrading mixed bacterial culture was enriched on carbazole to enhance its ability to degrade nitrogen heterocycles. This culture was used to inoculate a series of flasks of mineral medium and Norman Wells crude oil. Residual oil was recovered from these cultures after incubation at 25°C for various times. The nitrogen-rich fraction was analyzed by capillary gas chromatography, using a nitrogen-specific detector. Most of the C₁-, C₂-, and C₃- carbazoles and one of the C₄-isomers were degraded within 8 days. No further degradation occurred when incubation was extended to 28 days. The general order of susceptibility of the isomers to biodegradation was C₁ > C₂ > C₃ > C₄. The carbazole-enriched culture was still able to degrade n-alkanes, isoprenoids, aromatic hydrocarbons, and sulfur heterocycles in the crude soil.     

<Emphasis Type="Italic">Fusarium equiseti</Emphasis> LPSC 1166 and its in vitro role in the decay of <Emphasis Type="Italic">Heterostachys ritteriana</Emphasis> leaf litter

The role of microorganisms in litter degradation in arid and semi-arid zones, where soil and water salinization is one of the main factors limiting carbon turnover and decay, remains obscure. Heterostachys ritteriana (Amaranthaceae), a halophyte shrub growing in arid environments such as “Salinas Grandes” (Córdoba, Argentina), appears to be the main source of organic matter in the area. Little is known regarding the microorganisms associated with H. ritteriana, although they are a potential source of enzymes such as cellulolytic ones, which might be important in biotechnological fields such as bioethanol production using ionic liquids. In the present study, by studying the microbiota growing on H. ritteriana leaf litter in “Salinas Grandes,” we isolated the cellulolytic fungus Fusarium equiseti LPSC 1166, which grew and degraded leaf litter under salt stress. The growth of this fungus was a function of the C substrate and the presence of NaCl. Although in vitro the fungus used both soluble and polymeric compounds from H. ritteriana litter and synthesized extracellular β-1,4 endoglucanases, its activity was reduced by 10% NaCl. Based on these results, F. equiseti LPSC 1166 can be described as a halotolerant cellulolytic fungus most probably playing a key role in the decay of H. ritteriana leaf litter in “Salinas Grandes.”     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

Degradation of Lignin by Cyathus Species

The ability of 12 Cyathus species to degrade ¹⁴C-labeled lignin in kenaf was studied. The sum of ¹⁴C released into solution plus ¹⁴C released into the gas phase over a 32-day fermentation period was used to determine average daily rates of lignin biodegradation. Cyathus pallidus. C. africanus, and C. berkeleyanus delignified kenaf most rapidly. C. canna showed the greatest preference for lignin degradation over other plant components, and its rate of lignin degradation was only slightly lower than the three most active species. The apparent ability of fungi to metabolize low-molecular-weight lignin breakdown products correlated well with their overall delignification rates. C. stercoreus metabolized degradation products of lignin from wheat straw better than those from kenaf lignin, based on the amount of low-molecular-weight products left in solution.     

鸡枞菌转录组分析揭示其对木质纤维素的降解功能

【目的】探究鸡枞菌是否能降解木质纤维素成分,并理解其与共生白蚁之间的共生关系。【方法】本研究是应用新一代高通量测序技术454 GS FLX Titanium对鸡枞菌的转录组进行测序,挖掘鸡枞菌中能参与降解纤维素和木质素等成分的多样性酶系。【结果】八分之一的RUN测序总共得到了82386条表达序列标签,去除引物和载体等序列后,剩余的54410条序列被拼接成3301条contigs以及3193条singletons。根据序列相似性,将这些unigenes与三大蛋白数据库(Nr数据库、SwissProt数据库、CDD数据库)中的蛋白序列进行BLAST比较,发现有2681条基因与其他生物的已知基因有不同程度的相似性。在鸡枞菌的这些转录产物中,有33条编码可能参与降解纤维素或半纤维素的酶基因,其中包括5种纤维素酶以及28种水解半纤维素、淀粉或几丁质等物质的酶类。更重要的是,还发现了4种漆酶以及一种芳基乙醇氧化酶基因,这些都是能有效降解木质素的酶类。这些结果揭示了鸡枞菌中存在漆酶并可能有效降解植物残渣中的酚化合物。【结论】这些基因的发现说明了鸡枞菌能降解木质素,并能与共生白蚁分泌的纤维素酶协同作用有效降解纤维素。     

Nutritional evaluation of the white-rot fungus Sporotrichum pulverulentum as a feedstuff to rats,pigs, and sheep

The production of single-cell protein (SCP) based on cheap carbon sources such as spent liquor from paper mills is of interest for different reasons. The White-rot fungus (Sporotrichum pulverulentum) has earlier been shown to degrade cellulose and lignin. The nutritive value of this fungus was investigated with rats, pigs, and sheep. The effect of different drying process was evaluated on rats. Experiments with piglets, growing pigs, and sheep were aimed at getting primary information on nutritive parameters with domestic animal species, Chemical analysis of S. pulverulentum showed that the sum of the amino acids corresponded to 70% and ammonia, GABA, and glucosamine to 20% of its crude protein content. Differences between drying treatments in their effect on protein digestibility were not noted. From a protein quality viewpoint, a tendency toward superiority was noted for two of the drying processes. The amino acid digestibility of S. pulverulentum was inferior to values for soybean oil meal given in textbooks. The piglet experiment confirmed the lower nutritive value of S. pulverulentum compared with soybean oil meal. in the piglet stage a content of metabolizable energy of S. pulverulentum was found which corresponded to 60% of that for soybean oil meal. With increasing age the ability of pigs to utilize the fungus increased. The limited nutritive value for monogastric animals is most certainly caused by the cell-wall structure of S. pulverulentum with poor digestibility of the carbohydrates. The experiment with sheep showed more satisfactory results than with monogastric species, with digestibility of crude protein of 82% and a content of metabolizable energy of 70% of soybean oil meal.     

Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil

Pseudomonas putida strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida strain BNF1. The nucleotide base sequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Km^r) to get a novel transposon vector pUT/mini-Tn5-C23O. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O was introduced into one alkanes degrading strain Acinetobacter sp. BS3 by triparental conjugation, and then the C23O gene was integrated into the chromosome of Acinetobacter sp. BS3. And the recombinant BS3-C23O, which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O was a potential candidate used for the biodegradation of crude oil.     

Lignin-Degrading Enzymes of the Commercial Button Mushroom, Agaricus bisporus

Agaricus bisporus, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by A. bisporus. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in A. bisporus suggests significant roles for these two enzymes in lignin degradation by this fungus.     

Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes

Laccases are oxidoreductases which oxidize a variety of aromatic compounds using oxygen as the electron acceptor and producing water as by-product. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnological applicability. The presence of redox mediators is required for a number of biotechnological applications, providing the oxidation of complex substrates not oxidized by the enzyme alone. The efficiency of laccase–mediator systems to degrade recalcitrant compounds has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compounds related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries.     

Degradation of aromatic compounds through the β‐ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β‐ketoadipate pathway. In this study, we investigated the role of 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (CMLE), an enzyme of the β‐ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β‐ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β‐ketoadipate pathway in plant‐pathogenic, soil‐borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.     

Degradation of hydrocarbons and their derivatives by a microbial association on the base of Canadian pondweed

The degrading action of an aquatic plant-microbial association on the base of Canadian pondweed (Elodea canadensis) and its components (sterilized plant and two periphytonic strains, Pseudomonas fluorescens El-2.1 and Brevundimonas diminuta El-3.1) on crude oil, the water-soluble crude oil fraction, and individual test compounds (phenol, toluene, benzene, decalin, and naphthalene) was studied. It was found that the native association had a wider range and higher degree of degrading activity than individual species. Bacterial strains were significantly more active only towards naphthalene. The ability of the sterilized plant to degrade crude oil and phenol was no less than that of microorganisms and much more for toluene. Enzymatic activity towards the pollutants studied was found in E. canadensis exudates and buffer extracts of its cells.     

Polycyclic Aromatic Hydrocarbon Degradation of Phytoplankton-Associated Arenibacter spp. and Description of Arenibacter algicola sp. nov., an Aromatic Hydrocarbon-Degrading Bacterium

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409^T) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409^T represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409^T emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton.     

Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of “alpeorujo” (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.     

Inducer and culture medium dependent properties of extracellular laccase from Botrytis cinerea

Both the composition of the culture medium and the nature of the phenolic inducer determine the amount, the rate of formation and the molecular properties of extracellular laccase formed by Botrytis cinerea. Coumaric acid is shown to act as inducer in addition to gallic acid and grape juice. It is suggested that the fungus adapts to different environments by excreting different laccases. These laccases differ in pK, heat stability and substrate specificity but not in K_m values to quinol and oxygen.