Purification and properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.     

Lignin degradeation by white rot fungi

Abstract. The wood-degrading white-rot fungus Phanerochaete chrysosporium , has been the subject of intensive research in recent years and, based upon isolation of the extracellular enzyme ligninase, major advances have now been made toward elucidating the mechanism by which this fungus degrades lignin. From these developments, a model emerges which could explain the process by which wood-degrading fungi in general, attack lignin.     

Southern blot screening for lignin peroxidase and aryl-alcohol oxidase genes in 30 fungal species

Screening to detect genes encoding lignin peroxidase (LiP) and aryl-alcohol oxidase (AAO) has been carried out with 30 fungal strain using DNA probes from genes lpo of Phanerochaete chrysosporium (encoding LiP isoenzyme H8) and aao of Pleurotus eryngii. Evidence for the presence of genes closely related to lpo was found in Bjerkandera adusta, Fomes fomentarius, Ganoderma applanatum, Ganoderma australe, Lentinula degener, Peniophora gigantea, P. chrysosporium, Phanerochaete flavido-alba and Trametes tersicolor, whereas the gene aao was detected in Pleurotus species and B. adusta. The presence of both genes was only detected in B. adusta. These results suggest that different enzymatic system, formed by enzymes encoded by different genes, are responsible for lignin degradation by white-rot fungi.     

Production and characterization of recombinant Phanerochaete chrysosporium beta-glucosidase in the methylotrophic yeast Pichia pastoris

The extracellular beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium was expressed heterologously in the methylotrophic yeast Pichia pastoris. After 7 days' cultivation in an induction medium containing 1% (v/v) methanol, the expression level of the recombinant enzyme was 28,500 U/l, 38 times that of the wild-type enzyme. The specific activity of the crude recombinant enzyme for p-nitrophenyl-beta-D-glucoside was 52 U/mg, 37 times that of the wild-type enzyme; this difference made the purification of the enzyme simple. On a SDS-PAGE, the molecular mass of the recombinant enzyme was 133 kDa, and that of the wild-type enzyme was 116 kDa, but the difference had no effect on the hydrolysis of cellobiose or p-nitrophenyl-beta-D-glucoside. We concluded that the recombinant enzyme produced by Pichia pastoris retains the catalytic properties of the wild-type enzyme from Phanerochaete chrysosporium.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

黄孢原毛平革菌乙醇脱氢酶基因的克隆和表达

乙醇是黄孢原毛平革菌(Phanerochaete chrysosporium)在限氧培养条件下重要的代谢物之一, 为了更好的理解P. chrysosporium在低氧条件下的代谢机制, 文章从P. chrysosporium中克隆到一个长1071 bp的乙醇脱氢酶基因PCAdh1 cDNA, 该基因编码一个由356个氨基酸组成的蛋白质, 它与其他生物的乙醇脱氢酶的氨基酸序列的相似性很低, 但酶催化活性位点序列却高度保守。将PCAdh1在大肠杆菌中表达, 并获得有酶活性的重组蛋白。纯化的蛋白质用于制备抗体。半定量RT-PCR和Western blot分析结果显示, 在限氧条件的培养过程中, PCAdh1基因在mRNA水平和蛋白水平上都保持相对稳定, 表明该基因的表达是组成型的; 但从菌丝体提取的粗蛋白中的乙醇脱氢酶活性却随着培养时间的增加及氧气含量的持续降低而逐渐升高, 这暗示P. chrysospo-rium中存在其他低氧诱导型乙醇脱氢酶基因的表达。     

Lignin peroxidase is involved in the biobleaching of manganese-less oxygen-delignified hardwood kraft pulp by white-rot fungi in the solid-fermentation system

Biobleaching of manganese-less oxygen-delignified hardwood kraft pulp (E-OKP) by the white-rot fungi Phanerochaete sordida YK-624 and P. chrysosporium was examined in the solid-state fermentation system. P. sordida YK-624 possessed a higher brightening activity than P. chrysosporium, increasing pulp brightness by 13.4 points after seven days of treatment. In these fermentation systems, lignin peroxidase (LiP) activity was detected as the principle ligninolytic enzyme, and manganese peroxidase and laccase activities were scarcely detected over the course of treatment of E-OKP by either fungus. Moreover, a linear relationship between brightness increase and cumulative LiP activity was observed under all tested culture conditions with P. sordida YK-624 and P. chrysosporium. These results indicated that LiP is involved in the brightening of E-OKP by both white-rot fungi.     

Lignin-modifying enzymes from selected white-rot fungi: production and role from in lignin degradation

Abstract: White-rot fungi produce extracellular lignin-modifying enzymes, the best characterized of which are laccase (EC 1.10.3.2), lignin peroxidases (EC 1.11.1.7) and manganese peroxidases (EC 1.11.1.7). Lignin biodegradation studies have been carried out mostly using the white-rot fungus Phanerochaete chrysosporium which produces multiple isoenzymes of lignin peroxidase and manganese peroxidase but does not produce laccase. Many other white-rot fungi produce laccase in addition to lignin and manganese peroxidases and in varying combinations. Based on the enzyme production patterns of an array of white-rot fungi, three categories of fungi are suggested: (i) lignin-manganese peroxidase group (e.g. P. chrysosporium and Phlebia radiata ), (ii) manganese peroxidase-laccase group (e.g. Dichomitus squalens and Rigidoporus lignosus ), and (iii) lignin peroxidase-laccase group (e.g. Phlebia ochraceofulva and Junghuhnia separabilima ). The most efficient lignin degraders, estimated by ¹⁴CO₂ evolution from ¹⁴C-[Ring]-labelled synthetic lignin (DHP), belong to the first group, whereas many of the most selective lignin-degrading fungi belong to the second, although only moderate to good [¹⁴C]DHP mineralization is obtained using fungi from this group. The lignin peroxidase-laccase fungi only poorly degrade [¹⁴C]DHP.     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

Cloning and characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor

Six putative lignin peroxidase (LIP) genes were isolated from a lambda EMBL3 phage library of the white-rot fungus, Trametes versicolor, using the Phanerochaete chrysosporium LIP cDNA CLG5 as the probe. Sequence analysis of one of the genes, VLG1, showed that its coding region is interrupted by six small introns (49-64 bp) and that it encodes a mature LIP protein (341 aa; Mr: 36,714) that is preceded by a 25 aa signal sequence. This protein has a relatively high degree of aa homology to the N-termini of the LIP proteins purified from T. versicolor and has an aa homology of 55-60% to the LIP proteins of P. chrysosporium, which is comparable to that found between P. chrysosporium and Phlebia radiata LIP proteins.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.     

Kinetic and antigenic similarities for cellobiose dehydrogenase from the brown rot fungus Coniophora puteana and the white rot fungus Phanerochaete chrysosporium

Abstract Cellobiose dehydrogenase was purified from the brown rot fungus Coniophora puteana . Strong cross-reaction was observed with antibodies to cellobiose:quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium . Kinetic measurements were made with cellobiose as electron donor. Ferricyanide and DCPIP both showed a pH optimum close to pH 4, but activity with ferricyanide declined more rapidly when the pH was raised. Dioxygen reduction to hydrogen peroxide was observed, but at a much lower rate than for other acceptors. These properties are similar to those of cellobiose dehydrogenase from P. chrysosporium , despite differences between brown and white rot modes of decay.     

Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid

Intracellular processes of the white-rot basidiomycete Phanerochaete chrysosporium involved in the metabolism of benzoic acid (BA) were investigated at the proteome and metabolome level. Up-regulation of aryl-alcohol dehydrogenase, arylaldehyde dehydrogenase, and cytochrome P450s was observed upon addition of exogenous BA, suggesting that these enzymes play key roles in its metabolism. Intracellular metabolic shifts from the short-cut TCA/glyoxylate bicycle system to the TCA cycle and an increased flux in the TCA cycle indicated activation of the heme biosynthetic pathway and the production of NAD(P)H. In addition, combined analyses of proteome and metabolome clearly indicated the role of trehalose as a storage disaccharide and that the mannitol cycle plays a role in an alternative energy-producing pathway.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Extracellular mannose-6-phosphatase of Phanerochaete chrysosporium: a lignin peroxidase-modifying enzyme

The lignin peroxidase (LIP) isozyme profile of the white-rot fungus Phanerochaete chrysosporium changes markedly with culture age. This change occurs extracellularly and results from enzymatic dephosphorylation of LIP isozymes. In this study, a novel mannose 6-phosphatase (M6Pase) from extracellular culture fluid filtrate of P. chrysosporium, shown to be responsible for the extracellular postranslational modification of LIP, was purified and characterized. In vitro incubation of the purified M6Pase with purified LIP isozyme H2 resulted in its conversion to isozyme H1, with an equimolar release of orthophosphate. Using different sugar phosphates as substrate, the enzyme exhibited narrow specificity, showing activity mostly for mannose 6-phosphate (K(m) = 0.483 mM). The enzyme displayed a molecular mass of 82 kDa, as determined by gel filtration, and 40.4 and 39.1 kDa, on SDS-PAGE, suggesting that the native form is a dimer. The N-terminal sequence of the enzyme has no homology with that of other reported phosphatases. M6Pase is a metalloprotein with manganese and cobalt as the preferred metal ions. It is N-glycosylated proteins with an isoelectric point of 4. 7-4.8 and a pH optimum of 5. Based on its characteristics, M6Pase from P. chrysosporium seems to be a unique phosphatase responsible for posttranslation modification of LIP isozymes.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.     

Relative stability of recombinant versus native peroxidases from Phanerochaete chrysosporium.

Two types of glycosylated peroxidases are secreted by the white-rot fungus Phanerochaete chrysosporium, lignin peroxidase (LiP) and manganese peroxidase (MnP). The thermal stabilities of recombinant LiPH2, LiPH8, and MnPH4, which were expressed without glycosylation in Escherichia coli, were lower than those of corresponding native peroxidases isolated from P. chrysosporium. Recovery of thermally inactivated recombinant enzyme activities was higher than with that of the thermally inactivated native peroxidases. Removal of N-linked glycans from native LiPH8 and MnPH4 did not affect enzyme activities or thermal stabilities of the enzymes. Although LiPH2, LiPH8, and MnPH4 contained O-linked glycans, only the O-linked glycans from MnPH4 could be removed by O-glycosidase, and the glycan-depleted MnPH4 exhibited essentially the same activity as nondeglycosylated MnPH4, but thermal stability decreased. Periodate-treated MnPH4 exhibited even lower thermal stability than O-glycosidase treated MnPH4. The role of O-linked glycans in protein stability was also evidenced with LiPH2 and LiPH8. Based on these data, we propose that neither N- nor O-linked glycans are likely to have a direct role in enzyme activity of native LiPH2, LiPH8, and MnPH4 and that only O-linked glycans may play a crucial role in protein stability of native peroxidases.     

三种白腐菌及其组合菌种木质素降解酶比较研究

朱红栓菌Trametes cinnabarina、糙皮侧耳Pleurotus ostreatus、黄孢原毛平革菌Phanerochaete chrysosporium是产生木质素降解酶能力强的菌株。对三种白腐菌及其组合菌种产生木质素降解酶能力和行为进行了比较分析和研究。结果表明,最佳培养方式为液体振荡培养;最佳培养基为酵母膏液体培养基。在产漆酶(laccases,lacs)方面,Pleurotus ostreatus和Phanerochaete chrysosporium的组合菌种的酶活最强,在第6天出现峰值,酶活达到450U/L;在产锰过氧化物酶(manganese peroxidases,mnps)方面,Trametes cinnabarina和Pleurotus ostreatus的组合菌种的酶活最强,在第10天出现峰值,酶活达到1050U/L;在产木质素过氧化物酶(lignin peroxidases,lips)方面,Trametes cinnabarina和Phanerochaete chrysosporium的组合菌种的酶活最强,在第8天出现产酶峰值,酶活达到2990U/L。筛选结果表明,组合菌种比单菌种产生的三种主要木质素降解酶的活性强,这为白腐菌高效产酶提供了一条新的途径,并为白腐菌研究领域的后续工作奠定基础。     

Comparative genomics of the white-rot fungi, Phanerochaete carnosa and P. chrysosporium, to elucidate the genetic basis of the distinct wood types they colonize

ABSTRACT: BACKGROUND: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome. RESULTS: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood. CONCLUSIONS: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.