Degradation of keratin substrates by fungi isolated from sewage sludge

Four fungal species including two dermatophytes and two saprophytes were isolated from sewage sludge samples at Basrah (Iraq) they were tested for their degradative ability towards three types of keratin substrates (human hair, chicken feathers and wool). The rate of keratin degradation was expressed as weight loss over three weeks of incubation using a liquid culture medium. Human hair had the highest degradation rate by colonization of Chrysosporium pannicola and Microsporum gypseum at a rate of 62% and 4% respectively. Chicken feathers were highly degraded by Aspergillus flavus (32%) while wool degradation was highest by C. pannicola (45.5%) and Trichophyton mentagrophytes var. erinacei (38%). There was a significant difference (p < 0.00l) in keratin substrate degradation rates by the examined fungi. Keratinase activity was highest for C. pannicola and M. gypseum in the culture medium baited with human hair. Aspergillus flavus revealed the highest activity of this enzyme in cultures amended with chicken feathers while T. mentagrophytes var. erinacei showed highest keratinase activity in cultures with wool substrate. The amount of protein released into the culture medium varied among the tested fungi. The medium's alkalinity increased over incubation time from 6.5 to 7.8. Microscopic examination showed maceration of the keratin substrates by the fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Degradation of keratinous materials by the plant pathogenic fungus <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis>

In this paper it is described for the first time the capability of Myrothecium verrucaria to grow in submerged and solid state cultures using poultry feathers as the only substrate. The fungus produced a protease with an unusual keratinolytic activity among plant pathogenic fungi. Its crude protease hydrolyzed keratinous substrates at pH 9.0 and 40 °C in the following order: poultry feather keratin > sheep wool keratin > human nail keratin > human hair keratin. Protease activity was highly sensitive to phenylmethyl sulphonyl fluoride (PMSF) indicating that the enzyme belonged to the serine protease family.     

Production of extracellular keratinases by keratinophilic fungal species inhabiting feathers of living poultry birds (Gallus domesticus): A comparison

Fourteen species of keratinophilic fungi belonging to ten genera (Chrysoporium, Malbranchea, Chaetomium,Sepedonium, Microascus, Scopulariopsis, Curvularia, Fusarium, Aspergillus, Penicillium) were isolated from feathers of about one hundred living poultry birds. The isolated fungi were compared for their keratinase activity after growing them on two different media: (1) basal salts solution containing natural keratin (human hair) as the only source of carbon and nitrogen; (2) the medium was supplemented with a minor amount of readily assimilable source of carbon along with natural keratin. All the test fungi could grow on keratinous material, degrading it and releasing sulphydryl containing compounds detected as cysteine, total proteins and extracellular keratinase. Maximum enzyme release by these fungi occurred in the broth supplemented with glucose and vitamins, thereby indicating a correlation between the mycelial biomass and production of proteolytic keratinases. This revised version was published online in June 2006 with corrections to the Cover Date.     

Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil

Soil samples from an agricultural field contaminated with 10 ppm¹⁴C-benz(a)anthracene in glass tubes were brought into contact with cultures of wood-rotting fungi, precultivated on wheat straw substrate. Forty-five strains of white-rot fungi and four brown-rot fungi were tested for their ability to colonize the soil and to mineralize¹⁴C-benz(a)anthracene to¹⁴CO₂ within a 20-week incubation time. Twenty-two white-rot fungi and all brown-rot fungi were unable to colonize the soil. Twenty-three strains of white-rot fungi, all belonging to the genusPleurotus, colonized the soil. During the experiment the noncolonizing fungi and their substrate disintegrated more and more to a nonstructured pulp from which water diffused into the soil. The same phenomenon was observed in the control which contained only straw without fungus and contaminated soil. In samples with colonizing fungi the substrate as well as the mycelia in the soil remained visibly unchanged during the entire experiment. Surprisingly, most samples with fungi not colonizing the soil and the control without fungus liberated between 40 and 58 % of the applied radioactivity as¹⁴CO₂ whereas the samples with the colonizing fungi respired only 15–25 % as¹⁴CO₂. This was 3–5 times more¹⁴CO₂ than that liberated from the control (4.9 %) which contained only contaminated soil without straw and fungus. A similar result was obtained with selected colonizing and noncolonizing fungi and soil contaminated with 10 ppm¹⁴C-pyrene. However, in pure culture studies in which¹⁴C-pyrene was added to the straw substrate,Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3 % of the added radioactivity to¹⁴CO₂. The noncolonizing fungiDichomitus squalens andFlammulina velutipes liberated only 17.2 or 1.7 %, respectively, as¹⁴CO₂. These results lead to the hypothesis that the native soil microflora stimulated by the formed products of straw lysis is responsible for high degradation rates found with noncolonizing fungi.     

嗜角蛋白真菌的界定、研究方法及其应用价值

嗜角蛋白真菌(keratinophilic fungi)是指能降解角蛋白并能利用其作为唯一营养源的真菌类群,大部分隶属于爪甲团囊菌目(Onygenales)、散囊菌目(Eurotiales)和肉座菌目(Hypocreales).由于该类群分布广、种类多、营养方式多样,其研究方法和技术手段也在不断进步,加深了其研究的深度...     

Microbial hydroxylation of non-activated carbon atoms in racemic 2-dodeceno-1-yl-succinic anhydride by <Emphasis Type="Italic">Mortierella isabellina 212</Emphasis>

Microbial hydroxylation of 2-dodecen-1-yl-succinic anhydride at non-activated carbon atoms has been described. A screening with fourteen fungi strains was carried out. Most of the microorganisms transformed the substrate into new products. Biotransformation conditions with Mortierella isabellina 212 were optimized. Optically active products of hydroxylation at C–10 and C–11 were isolated. Chemical structures of the unreacted optically active substrate and the products were determined by spectroscopic methods (¹H NMR, ¹³C NMR, GCMS, IR). Non-enzymatic hydrolysis of the anhydride at different pH was studied and methods of detection of the anhydride and the forming acid were discussed.     

A novel metabolic pathway for biodegradation of DDT by the white rot fungi, <Emphasis Type="Italic">Phlebia lindtneri</Emphasis> and <Emphasis Type="Italic">Phlebia brevispora</Emphasis>

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was used as the substrate for a degradation experiment with the white rot fungi Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596, which are capable of degrading polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated biphenyls (PCBs). Pure culture of P. lindtneri and P. brevispora with DDT (25 μmol l⁻¹) showed that 70 and 30% of DDT, respectively, disappeared in a low-nitrogen medium after a 21-day incubation period. The metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS). Both fungi metabolized DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2-bis(4-chlorophenyl)acetic acid (DDA) and 4,4-dichlorobenzophenone (DBP). Additionally, DDD was converted to DDA and DBP. DDA was converted to DBP and 4,4-dichlorobenzhydrol (DBH). While DBP was treated as substrate, DBH and three hydroxylated metabolites, including one dihydroxylated DBP and two different isomers of monohydroxylated DBH, were produced from fungal cultures, and these hydroxylated metabolites were efficiently inhibited by the addition of a cytochrome P-450 inhibitor, piperonyl butoxide. These results indicate that the white rot fungi P. lindtneri and P. brevispora can degrade DBP/DBH through hydroxylation of the aromatic ring. Moreover, the single-ring aromatic metabolites, such as 4-chlorobenzaldehyde, 4-chlorobenzyl alcohol and 4-chlorobenzoic acid, were found as metabolic products of all substrate, demonstrating that the cleavage reaction of the aliphatic-aryl carbon bond occurs in the biodegradation process of DDT by white rot fungi.     

Isolation of keratinolytic fungi from a coal mine dump

During the study of fungal succesion in the coal mine dump in Brzezinka (Poland), soil samples were examined for keratinolytic fungi. These micro-organisms were rather poorly represented in the area studied. Out of 300 soil samples examined, only 48 (16%) were positive for keratinolytic fungi.Trichophyton ajelloi andArthroderma curreyi were the prevailing species. These species occurred practically at two locations, i.e. on the naked carbon rocks inhabited by algae crops (chiefly byCyanophyta) and in the pine litter. It can be supposed that the occurrence of keratinolytic fungi was more dependent on the favourable general conditions such as increasing organic matter content, microflora, and humidity than on the presence of keratin remains in the soil. Because of the lack of potentially pathogenic fungi, the coal mine dump examined cannot be considered as an important source of fungal infection.     

Leaf-eating invertebrates as competitors of aquatic hyphomycetes

Summary Leaf-eating invertebrates selectively ingest leaf areas rich in fungal cells. The effect of this process on coincident and cumulative species diversity (species numbers and evenness) of the fungi was studied on 3 substrates (oak leaves, larch and spruce needles) in 2 hardwater and 2 softwater streams. Cumulative species number of colonizing fungi follows the equation S=k·A ^z(A=area below decay curve of the substrate, k=substrate-specific constant, Z=0.47). Higher feeding activity means faster weight loss of the substrate which leads to lower species richness of the fungi. The opposite is true for early successional stages on larch needles. Evenness of the fungi (distribution of individuals among species) is negatively correlated with feeding intensity by invertebrates, as measured by increased decay rates. The overall effect of leaf-eating invertebrates on aquatic hyphomycetes resembles that of potent competitors preempting substrate otherwise used by a late successional tail of relatively rare fungi.     

Effect of keratin on the lipid composition of Scopulariopsis brevicaulis (Bainier)

Lipid content and composition of Scopulariopsis brevicaulis cultured with or without its natural substrate keratin was investigated. Lipid content was found to be lower in the presence of keratin (maximum 12% against 19% in the absence of keratin), with differences in levels of linoleic acid (C₁₈₌₂), palmitic (C₆₀) and to a lesser extent palmitoleic (C₁₆₌₁) and -linolenic (C₁₈₌₃) acids. The degree of unsaturation was correspondingly lower in the organisms cultured with keratin as substrate.     

Degradation of Raspberry Suberin by Fusarium solani f. sp. Pisi and Armillaria mellea

When submers cultures of Fusarium solani f. sp. pisi and Armillaria mellea were grown in a medium supplemented with 0.5 % suberin isolated from raspberry periderm, hydrolytic enzymes were produced and measured by a spectrophotometric assay using p-nitrophenyl butyrate as substrate. The enzymatic activity in the culture fluids reached its peak after 32 to 44 days of incubation. In a gas-chromatographic assay of the enzymatic degradation of suberin, concentrated culture fluids of suberin-grown fungi were incubated with raspberry suberin. The culture fluids of F. solani and A. mellea catalyzed the release of chloroform-soluble products, which were analyzed by gas-liquid chromatography. Suberin monomers like fatty alcohols and acids with chain-lengths from C₁₆ to C₂₆ as well as C₁₆ and C₁₈ω-hy-droxyacids could be identified as products. The suberin-induced enzymes showed catalytic properties similar to cutin-hydrolyzing enzymes previously isolated from different fungi.     

Some effects of time, temperature of treatment and fumigant concentration on the fungicidal properties of methyl bromide

Survival of fungi after fumigation with methyl bromide was affected by fumigant concentration, duration of exposure and treatment temperature. The concentration x time (ct) products required to kill slow growing species were lower than those required for fast growing fungi; in addition, for some species the ct products required also increased with increasing treatment times. In all fungi studied, ct products required decreased with increasing treatment temperature.     

降解鸡毛真菌板蜡蚧的鉴定及产酶培养优化

鉴定降解鸡毛真菌并通过单因素和正交实验优化其产角蛋白酶发酵培养条件.从加入鸡毛粉钓饵的医院花坛土中分离筛选获得3株角蛋白高效降解真菌,利用形态学和分子系统学鉴定均为板蜡蚧(Lecani-cillium testudineum).单因素实验表明,对优选菌株1Y2-12产酶能力具促进作用的碳源为乳糖,氮源为酵母膏,无机离子...     

Keratinophilic fungi inhabiting floor dusts of student houses at the South Valley University in Egypt

Keratinophilic fungi include true fungi that vigorously degrade keratin as well as a number of important human pathogenic dermatophytes. We identified 41 species and one variety belonging to 19 genera in 50 floor dust samples following culture on Sabouraud’s dextrose agar medium at 28°C. Dermatophytes and closely related fungi were represented by six species—Aphanoascus fulvescens, Aphanoascus sp., Arthroderma cuniculi, Chrysosporium lucknowense, Gymnoascus uncinatus and Trichophyton rubrum. There were 35 species and one variety of other fungal species, with members of Aspergillus and Penicllium being the most prevalent. Twenty-seven species and 1 variety belonging to 14 genera were identified from 24 dust samples gathered at 2-week intervals from male student housing at El-Kenose during January–December 2005 that had been cultured on Sabauraud’s dextrose agar at 28°C. The monthly counts of keratinophilic fungi showed irregular fluctuations, with a peak in April and the lowest point in November. Dermatophytes and closely related fungi were represented by Aphanoascus fulvescens, Aphanoascus sp., Chrysosporium lucknowense, Gymnoascus uncinatus, and Trichophyton rubrum. In conclusion, the study reports the incidences of some dermatophytes found in the floor dust of student houses of South Valley University, Egypt.     

Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory,Jabalpur, India

Chrysosporium queenslandicum, Graphium penicilloideus andScopulariopsis brevicaulis were grown on various supplemented basal salts media to compare keratinase induction, activity and repression. All three fungi can utilize keratin as a sole source of carbon, nitrogen and sulfur. Total keratinase activity inC. queenslandicum andS. brevicaulis, was not repressed by supplementation of keratin-containing medium with glucose, ammonium or sulfate. The production of keratinase activity was not derepressible in keratin-free media. Keratin utilization commenced before the detection of significant extracellular keratinase activity which was always associated with mycelial growth.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.     

Keratinolytic Activity of Aspergillus fumigatus Fresenius

Aspergillus fumigatus can utilize chicken feather keratin as its sole carbon and nitrogen source. Because enzymatic conversion of native keratin into readily usable products is of economic interest, this fungus was studied for its capacity to produce and secrete keratin-hydrolyzing proteinases. Substantial keratin-azure hydrolyzing activity was present in the culture fluid of keratin-containing media. Considerably lower activity was present in cultures containing glucose and nitrate as the carbon and nitrogen sources, or keratin plus glucose and nitrate. Secretion of keratin-hydrolyzing activity in A. fumigatus was induced by keratin but repressed by low-molecular-weight carbon and nitrogen sources. The amount of keratinolytic enzyme present in the culture fluid was dependent on the initial pH of the culture medium. The crude enzyme also hydrolyzed native keratin and casein in vitro. Hydrolysis was optimal at pH 9 and 45°C. The crude enzyme was remarkably thermostable. At 70°C, it retained about 90% of its original activity for 1.5 h. The obtained results indicated that the A. fumigatus keratinolytic enzyme may be suitable for enzymatic improvement of feather meal. Received: 25 April 1996 / Accepted: 18 June 1996     

Oxidation of Polyamines by Fungal Enzymes

Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen. The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate. The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H₂O₂. Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.     

Fungal colonization of synthetic substrates for use in space craft

Summary Materials being used or considered for use in space flight were examined for their susceptibility to fungal colonization. The materials included soft goods (clothing) and insulation and fabrication products such as Velcro^® attachments and elastic cord binders. Materials were exposed for at least 28 days in a highhumidity chamber colonized with over 50 species of fungi, including those species recommended for determining recalcitrance of materials to fungal biodegradation. At least nine of 25 products demonstrated extensive microscopic colonization by fungi, mostly byAcremonium obclavatum. Challenge procedures that rely on observations with the unaided eye, or 40×magnification of growth by a restricted number of fungal species with a cellulosic substrate as a positive control, are insufficient for determining the resistance of synthetic substrates to fungal colonization.     

Degradation and oligomerization of syringic acid by distinctive ecological groups of fungi

Forty-four terrestrial and aeroaquatic and aquatic fungi, including fifteen species causing white-rot, four species causing brown-rot, and some species causing soft-rot of wood, were tested for their ability to degrade the monomer syringic acid, which is released during decay of angiosperm lignin. None of the white- or brown-rot species caused any detectable degradation of syringic acid under the test conditions; however, six typical white-rot fungi strongly oligomerized syringic acid, both with and without cosubstrate. The main polymerization product was identified as a 1,3-dimethylpyrogallol oligomer by¹³C-NMR. Other minor metabolic products were methylated and hydroxylated derivatives. Oligomerization depended on the presence of 1 or 2 methoxy groups in ortho position to the hydroxy group of the substrate. Among the remaining fungi,Exophiala jeanselmei, Fusarium eumartii, andPaecilomyces variotii completely and rapidly degraded syringic acid (5 g/liter) within 48 to 100 hours. A further seven species were able to degrade syringic acid to some extent when glucose was added. Methylated and demethylated metabolic intermediates were identified by GC/MS.