Fungal Melanin Research Articles

Post-aromatic deoxygenation in polyketide biosynthesis: reduction of aromatic rings in the biosyntheses of fungal melanin and anthraquinone.

Two enzyme reactions involved in the post-aromatic deoxygenation process of fungal melanin and pigments were studied from the viewpoint of enzymic reduction of aromatic rings. Hydroxynaphthalene reductase that catalyzes reduction of the aromatic rings of 1,3,6,8-tetrahydroxynaphthalene and 1,3,8-trihydroxynapthalene was partially purified from Phialophora lagerbergii and characterized. Emodin deoxygenase of Pyrenochaeta terrestris that catalyzes deoxygenation of emodin to afford chrysophanol was found to be resolved into two protein fractions with DEAE-cellulose column. The two protein fractions acted synergistically in regard to emodin deoxygenase activity.

ENZYMATIC BREAKDOWN AND DECOLOURISATION OF BLACK FUNGUS EMBEDDED IN ACRYLIC PAINT: A PROGRESS REPORT

Reported are the main findings of an investigation aimed at the breakdown and decolourisation of black micro-fungal debris embedded in the modem acrylic paint of a wooden Australian Aboriginal object. Results of experiments for the enzymatic breakdown of this fungal debris using gels containing chitinase, glucanase and protease enzymes are presented. Further testing of selected reagents for the decolourisation of melanin, the source of the black pigmentation in the fungi is described. The application of atypical enzymes to a convex acrylic paint surface and the resilience of the fungal pigment melanin is discussed.

Stability Constants of Copper(II)‐Humate Complexes Determined by Modified Potentiometric Titration

AbstractHumic substances form highly stable complexes with micronutrient cations. Little is known, however, regarding the nature of the complexes or of their stability constants. A ligand titration procedure using the Cu(II) ion‐selective electrode was investigated as a means of determining stability constants of the Cu(II) complexes with humic and fulvic acids. The procedure was superior to conventional titration (metal ion as titrant) because constants for binding at the strongest sites are measured. Furthermore, under the titration conditions employed (pH of 4 and 5; low ionic strength, 0.01 M; low Cu(II) concentration, 10−5 M), precipitation did not occur and thus did not adversely affect the binding measurements. A continuous‐distribution model showed promise for modeling the binding data. The binding affinity of some humates for Cu(II) followed the order: soil humic acid (SHA) > peat humic acid (PHA) > lignite humic acid (LHA) > soil fulvic acid (SFA) ≈ fungal melanin. Intrinsic stability constants (log Kint) at pH 4 and an ionic strength of 0.01 M for the five humate preparations were 8.3, 7.9, 7.4, 7.2, and 6.9, respectively; constants at pH 5 were 8.5, 8.4, 7.9, 7.6, and 7.6, respectively.

Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy

Humic acids from two Brazilian topsoils under savanna grassland and five soil fungal melanins were characterized by elemental, functional group and infrared analysis. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for several other fungal melanins and soil humic acids. Compared with the soil humic acids, the infrared spectra of the fungal melanins showed greater detail, indicative of higher aliphaticity. They were similar to the type III infrared spectra of humic acids, which are characteristically high in proteinaceous material and polysaccharides. The infrared spectra of the humic acids from the two Brazilian soils studied were classified as type I, which includes most soil humic acids. Notwithstanding the greater detail, in some areas the fungal melanin spectra were similar to those reported for other fungal melanins and humic acids of different origins. The probable contribution of the melanic fungi to the formation of soil humic polymers is discussed.

Studies on the Colouring Matter of Blue-stain Fungi. Part 3. Spectroscopic Studies on Fungal and Synthetic Melanins

In this study the variety of possibilities offered by spectroscopy was made use of for the characterization and identification of melanins. Melanins isolated from the blue-stain fungi Ceratocystis coerulescens and Alternaria alternata were compared with some synthetic melanins (tyrosine, dopa, catechol melanin)

Biodegradation of synthetic organo-metallic complexes of iron and aluminium with selected metal to carbon ratios

Synthetic organo-ferric and organo-aluminous complexes with various metal :C ratios were prepared and incubated for 44 days in an Eutrochrept A 1 horizon. Citric acid was used as a model of a natural soil acid. Fungal melanins synthesized by Epicoccum purpurescens were separated in fulvic acid-like and humic acid-like polymers and used as models for natural transformed soil organic matter. Under certain conditions, the biodegradation of such metal-organic complexes was slower than that of the free forms of the corresponding organic compounds. The intensity of this protective effect depended mainly on the metal to complexing functional groups molar ratio (M/CG) of the complexes and was also related to their aqueous solubility. For some organics, the protective effect was observed at ratios exceeding the saturation of their complexing sites, and sometimes needed a metal content exceeding more than twice their complexing capacity. In such cases, the formation of a flocculated metallic hydroxide trapping and wrapping the organic molecules is suggested to account better for the observed protective effect than chemical binding per se. As the solubility of the “humic” acids decreased faster than that of the “fulvic” ones when their metallic charge increased, the biodegradability of the former decreased more rapidly than that of the latter by metal complexing. For the same reasons, citric acid was more protected by complexing Al than Fe. The protective effect of both aluminium and iron against the biodegradation of all types of organic matter was high and of a comparable intensity at high M/CG molar ratios. These data are interpreted in terms of pedological processes.

Biosorption of copper by fungal melanin

Melanin obtained from Aureobasidium pullulans and Cladosporium resinae was an efficient biosorbent for copper. Copper uptake could be expressed using various adsorption isotherms; melanin from A. pullulans obeyed Freundlich and Langmuir isotherms whereas C. resinae melanin followed the BET isotherm indicating a more complex type of adsorption than in A. pullulans. In general, uptake capacities of melanin were greater than for intact biomass and the higher uptake by pigmented rather than albino biomass could be correlated with the presence of melanin. Cu2+ was less readily desorbed from melanin by dilute mineral acids than from intact biomass and again, the relative ease of Cu2+ desorption from pre-loaded pigmented or albino biomass was correlated with the presence or absence of melanin. Mg2+ and Zn2+ appeared to be the most effective cations for desorption with Na+ and K+ the least effective. The addition of melanin to a coppercontaining culture of the albino strain of A. pullulans resulted in some reduction of toxicity.

Sugars in hydrolysates of fungal melanins and soil humic acids

Humic acids from four Brazilian topsoils of different origin and four fungal melanins, synthesized under two cultural conditions were subjected to a two step hydrolysis procedure and the released monosaccharides qualitatively and quantitatively determined by gas-liquid chromatography. The neutral sugars, glucose, galactose, mannose, arabinose, xylose, fucose, rhamnose and the alcohol sugar inositol, were detected in most of the soil humic acid samples. The fungal melanins showed the presence of glucose, galactose, mannose and arabinose. Ribose was present in two out of the eight samples tested. Some quantitative differences in the two types of humic polymers were noted and expected considering their origins. However, similarities were more apparent than differences and give further indication that melanic fungi may play a significant role in the formation of soil humic acids.

Incorporation of newly immobilized 15N into stable organic forms in soil

Studies were conducted on an 15N-enriched Illinois Mollisol to determine the extractability and distribution of newly immobilized N in the classical humic fractions. Only about 50% of the added 15N was recovered by sequential extraction with 150 mM Na 4P 2O 7 and 100 mM KOH; another 25 to 30% N was solubilized by further extractions with a variety of inorganic and organic solvents in sequence. In comparison to the native soil N, higher proportions of the applied 15N were accounted for in the humic acid (24 vs 16%) and fulvic acid (25 vs 22%) fractions. For the fulvic acid fraction, a higher proportion of the applied 15 (30%) was recovered as low molecular weight compounds, as compared to the native soil N (20%). The findings support the concept that a significant fraction of the recently immobilized N in soil occurs as insoluble components of microbial tissues, such as fungal melanins.

Biosynthesis and Functions of Fungal Melanins

Melanins are dark-brown to black pigments found in animals, plants, and microorganisms. These pigments are not essential for growth and develop­ ment, but rather they enhance the survival and competitive abilities of species in certain environments. The association of melanins with immune responses has been noted for plants (13) and invertebrates (180, 227, 239, 250). Melanin biosynthesis and function have been studied most extensively in animals, primarily because of the association of melanins with skin disorders (25,53, 188) and malignant melanomas (119). Melanins in the eye and inner ear have also received considerable attention because of their interactions with drugs (84). The synthesis of animal melanin from tyrosine by the action of tyrosinase is proven (52, 175, 270). Tyrosinase, the key enzyme in animal melanin synthesis, also occurs and is best characterized in the common mushroom (Agaricus bisporus) and Neurospora crassa (136). Consequently, some researchers have been hasty to conclude that fungal melanins also are derived from tyrosine. The importance of melanins for survival and longevity of fungal propagules has been recognized for many years (126, 235). During the past ten years three additional discoveries have increased interest among plant pathologists in fungal melanins. First, unique pathways for melanin biosynthesis were discovered in both Ascomycotina (16) and Basidiomycotina (234). Second, investigators discovered a new class of fungicides that prevent direct penetra­ tion of plant tissue by inhibiting melanin biosynthesis in appressorial cells (221, 223). Third, phytotoxins were discovered that are shunt products of

Quantitative binding of azure A to melanin of the black yeast Phaeococcomyces

Existing assays of melanins are time-consuming extraction and alkaline digestion methods. The dye azure A has a high affinity for fungal and synthetic melanins. A rapid (30 minutes), simple, sensitive, and nondestructive spectroscopic assay for fungal melanins is described, using azure A. The azure A assay was compared with a modified extraction and digestion method, using melanin produced by the black yeast Phaeococcomyces.

HPLC Analysis of Fungal Melanin Intermediates and Related Metabolites

Abstract A convenient method to concentrate and separate intermediates and related secondary products of the pentaketide pathway leading to melanin biosynthes is has been developed. Twelve of the fourteen known compounds were separated using reverse phase high performance liquid chromatography with a 12 to 42% linear gradient of acetonitrile in 2% acetic acid. The remaining two, highly water soluble isomers, were isocraticly separated on the same column with 5% acetonitrile in 2% acetic acid. Solid phase extraction and concentration of representitive compounds in acidified aqueous brine was accomplished with reverse phase Sep-Pak cartridges. The effectiveness of the method was evaluated by determining the pentaketides produced by cell-free homogenates of Verticillium dahliae supplied with scytalone as the substrate and by cultures of two V. dahliae mutants. The method has the potential for quantifying all known pentaketide melanin metabolites and thus will allow for more comprehensive studies of fungal me...

Structural Characteristics of a Fungal Melanin and a Soil Humic Acid

AbstractA melanin produced by Hendersonula toruloidea and a soil humic acid were characterized by a number of chemical (elemental and functional group analyses, carbohydrate content, amino acid, amino sugar, and NH3 analyses) and spectroscopic methods (IR and 13C NMR) before and after 6 M HCl hydrolysis. The following distinct differences were observed between the two materials: (i) phenolic components were prominent in the untreated and hydrolyzed melanins but not in the corresponding soil humic acids (HA); (ii) the melanin contained more complex unidentified N than did the soil HA; and (iii) following hot acid hydrolysis, the melanin was more aliphatic but considerably less aromatic than the soil HA. Hot acid hydrolysis, by removing practically all of the proteinaceous materials and carbohydrates from the two materials, made it possible to focus on the structural chemistry of the residual materials.

Biosynthesis and Functions of Fungal Melanins

Biosynthesis and Functions of Fungal Melanins

Amino acid distribution in some fungal melanins and of soil humic acids from Brazil

Humic acids from four Brazilian topsoils of different origins and four soil fungal melanins, synthesized under two cultural conditions, were subjected to 6N HCl hydrolysis and their amino acid distribution patterns qualitatively and quantitatively determined. Both soil and fungal polymers showed similar patterns with aspartic acid, glutamic acid, glycine and alanine as the dominant amino acids. Some variations noted were more quantitative than qualitative, the similarities were more pronounced than differences, indicating that the fungal melanins may play a significant role in the formation of soil humic acid polymers. The humic acids of Brazilian soils had amino acid distribution patterns similar to those reported for humic acids of other tropical and temperate soils.

ELECTRON SPIN RESONANCE SPECTROMETRY OF FUNGAL MELANINS

We investigated electron spin resonance (ESR) parameters (free radical concentrations, g values, and linewidths) of a variety of fungal melanins. The influences of oxygen, culturing conditions, methylation, solvent extractipn, aCld hydrolysis, and persulfate oxidation on ESR parameters were also studied. Our major objective was to gain a better knowledge of the similarities of fungal melanins with soil humic aClds. We suggest that these biopolymers are different from soil humic aClds with regard to ESR parameters.

Iron and copper binding by fungal phenolic polymers: an electron spin resonance study

In an electron spin resonance (ESR) survey of fungal phenolic polymers and melanins, it was found that the phenolic polymers fromEpicoccum purpurascens andPenicillium funiculosum displayed hyperfine splitting, indicating the presence of transition metal bonding. The ESR parameters agree well with those of iron and copper organic complexes. Further, the copper-complex ESR parameters were similar to those reported for mixed complexes of copper, bipyridyl and dicarboxylic acids; this suggests that nitrogen-containing groups from the proteinaceous moiety are involved in the process. Acid hydrolysis removed the proteins and metals, but the hydrolyzed polymers were still able to remove 72% of the copper present in a 0.5× 10−4M solution. It was concluded that fungal phenolic polymers, which constitute a part of the soil humus, may play an important role in the translocation of metal ions and their availability to biological systems.

The Persulfate Oxidation of Fungal Melanins

AbstractFour fungal melanins were degraded by potassium persulfate, a new, mild chemical oxidation method. The percentage of degradation varied from 15 to 50% calculated by weight loss. Analysis of the degradation products by gas chromatography showed numerous fatty acids, both straight and branched chain, in widely varying amounts. The n‐C16 and n‐C18 acids were present in greatest abundance. By two‐dimensional thin‐layer chromatography, several phenols and anthraquinones were noted.

Biodegradation, Stabilization in Humus, and Incorporation into Soil Biomass of 2,4‐D and Chlorocatechol Carbons

Abstract14C‐labeled catechol, 4‐chlorocatechol, and 4,5‐dichlorocatechol readily linked into model peroxidase humic acid‐type polymers, with 84 to 96% recovery of the 14C‐activity in the reaction mixtures. The 4,5‐Dichlorocatechol was the least reactive. 2,4‐D did not link into the polymers. The biodegradation of labeled catechol, 4‐chlorocatechol, and 4,5‐dichlorocatechol, free and linked into model polymers and of 2,4‐D, a fungal melanin, ferulic acid, wheat straw, and glucose were followed over 6‐month and 1‐year incubation periods in four soils, and the distribution of the residual activity in biomass, new humus, and 6N HCl hydrolyzable substrates was determined. The rate and extent of degradation depended upon the substrate, the concentration, and the soil. The 2,4‐D was rapidly degraded with 86 to 94% of the side chain and 73 to 85% of the ring‐14C evolved as CO2 over 1 year. Only 38 to 50% of the catechol, 22 to 63% of the 4‐chlorocatechol, and 22 to 62% of the 4,5‐dichlorocatechol carbons were lost over 1 year indicating substantial stabilization of these compounds, probably by enzymatic polymerization reactions. Catechol, 4‐chlorocatechol, and 4,5‐dichlorocatechol incorporated into model polymers were still more stable with 6 to 22, 8 to 42, and 13 to 43%, respectively, of the 14C evolved as CO2.After 1 year, about 3 to 8, 2 to 14, 1 to 7, 0 to 8, and 2 to 15% of the residual 14C activity from catechol, 4‐chlorocatechol, 4,5‐dichlorocatechol, the ring portion, and the side chain of 2,4‐D, respectively, was present in the biomass. The amount of residual C in the biomass from the catechols incorporated into the model polymers was very low and varied from 0 to 3%. The amounts of the residual 14C solubilized upon 6N HCl hydrolysis were related to the extent of biodegradation of the substrates. With highly degraded substrates, such as side chain 2,4‐D, glucose and wheat straw carbons the major portion of the residual C was lost upon hydrolysis. With highly resistant substrates such as catechol, the model polymers, and the fungal melanin, the major portion was not lost upon hydrolysis. The other substrates gave intermediate values.

Carbon‐13 Nuclear Magnetic Resonance Spectra of some Fungal Melanins and Humic Acids

AbstractA study was made to compare the 13C‐NMR (Carbon‐13 nuclear magnetic resonance) spectra of a variety of fungal melanins with that of soil and Leonardite peat humic acids. The spectra were made in 5% wt/wt solution in 0.1N NaOD in D2O using the Fourier transform technique. All of the polymers gave complex 13C NMR spectra with strong signals, shoulders, or plateaus in the area of resonance for aliphatic, peptide, polysaccharide, aromatic, and carboxylic acid groups. The soil humic acid and all the original fungal melanins except that from Stachybotrys atra showed strong peaks or shoulders at about 20, 25, 28, 35, 40, 58, 75, 80, 110, 120, 130, 136, 175, and 180 ppm. The study gives further evidence that many fungal melanins are similar to soil humic acids in their chemical structure.