Characterization of Chitinase from White-Rot Fungus Irpex lacteus NK-1

Enzymes are able to bind to their substrates specifically at the active site. The proximity and orientation of the substrates strongly increase the likelihood that productive E–S complexes will arise. Treated chitin (powder or flake) is more efficient than crystalline chitin. This is because the latter is less active due to its insolubility. The structure of treated chitin is opened; this facilitates its interaction with the enzyme. The purpose of this research was to create a kind of modified chitin and study the characterization of the different types of chitin including functional groups by IR spectrophotometer, pore size, surface area and crystallinity by X-Ray diffraction. Chitin from shrimp shell was modified into colloidal, bead, amorphous and superfine chitin. The results of the IR spectra of colloidal and bead chitin showed a similar pattern with chitin powder; they peaked at 3447 and 3113 cm-1 (OH and NH2 groups), 1645 cm-1 (amide groups N-H) and 1071 cm-1 (group C-O). Superfine and amorphous chitin had similar absorbance with powder chitin but appeared to peak in the fingerprint region. Characterization of physical properties based on the pore size and surface area of powder, colloidal, superfine, amorphous and bead chitin changed the pore radius of each type of chitin due to the treatment of swelling. Crystallinity showed that specific diffractogram pattern in the three main peaks 2q was 9.5, 19.5 and 26 with varying intensity. Chitinase activity assay using modified types of chitin substrate had higher values ​​than chitin powder. The highest activity was in amorphous chitin with values ​​of 1.858 U/mL. This is because it has chitin chain and the rearrangement of its structure was more open, facilitating its interaction with enzyme. Key words: Chitin modified, chitinase, substrate.

Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.

Over the last decade, fungal conservation has become an increasingly important topic, especially for species tied to forest ecosystems. Among these, wood decay fungi are a group of interesting species from ecological and applicative points of view. Culture collections represent an important tool for the conservation of species and research material. The aim of this study was to establish the first research culture collection of wood decay fungal strains, mainly corticioid and polyporoid species, at Salamanca University (Spain). From two areas of the Iberian Peninsula, a total of 120 basidiomata were collected and morphologically identified. From these, 55 strains were successfully isolated in pure culture and their identity was confirmed by DNA molecular analysis. The average growth rate of each strain was recorded and mycelium characteristics, such as colony morphology and microscopic features, were described. Notable strains in the collection included: (1) Botryobasidium asperulum and Phlebia rufa for taxonomical studies; (2) Hericium erinaceus, Grifola frondosa and Pleurotus species for medicinal properties; (3) Irpex lacteus, Phanerochaete sordida and Trametes versicolor for their degradation capabilities; (4) Stereum gausapatum and Stereum hirsutum for their applicative and enzymatic potential. The new fungal strain culture collection represents a valuable tool for the ex situ conservation of Mediterranean wood decay fungi.

Twenty-six isolates representing 17 species of aphyllophoraceous, wood-decaying Basidiomycetes and five species of agaricoid, turf-borne, thatch-decaying Basidiomycetes were screened for their abilities to degrade cellulose, lignin, and melanin by using colorimetric degradation assays on agar media. Selected ligninocellulolytic Basidiomycetes capable of degrading melanin were screened for antagonism of Botrytis cinerea Per.:Fr. The greatest inhibition of Botrytis colony and hyphal growth in vitro was observed in confrontations with Irpex lacteus (Fr.) Fr., Trametes versicolor (L.:Fr.) Pilat, and Chondrostereum purpureum (Pers.:Fr.) Pouzar. Hyphal interference and necrotrophic mycoparasitism by these ligninocellulolytic Basidiomycetes were recognized microscopically as coagulation and degeneration of Botrytis cytoplasm and as coiling and invasion of hyphae, conidiophores, and conidia, respectively. Sclerotia of B. cinerea were killed and parasitized in agar media, straw mulch, or moist sand infested separately with these three mycoparasites.

Intraspecific interactions among wood-decay fungi alter decay rates and dynamics of interspecific interactions

In this paper, the growth rate of five wood-rotting fungi, i.e., Coriolus versicolor, Irpex lacteus, Fomes fomentarius, Piptoporus betulinus and Pholiota adiposa, in solid medium and their biomass in liquid culture medium were compared by measuring mycelium length and dry mass. The activity of three main ligninolytic enzymes in those fungi, namely LiP, MnP and Lac, were also tested by colorimetry. At the same time, these fungi were used to decay the wood samples from 300 natural trees of white birch, to study their wood-decaying ability by measuring wood mass loss. The result showed that the growth rate, biomass, ligninolytic enzyme activity, and wood-decaying ability of the fungi were incompletely correlated. The growth rates of C. versicolor and I. lacteus were faster than those of P. betulinus and F. fomentarius; P. adiposa was the slowest in growth. The biomass of P. betulinus was the highest; C. versicolor, I. lacteus and F. fomentarius were in the middle, and P. adiposa was the lowest. There existed LiP, MnP and Lac activities in all fungi except P. betulinus, and the enzyme activities induced by wood powder were all higher than those of the control. The Lac of I. lacteus and the LiP of F. fomentarius and P. adiposa were only expressed in wood powder medium; the longer the fungi were cultured, the higher activity the enzyme had. The decomposition ability of C. versicolor to wood samples was the highest, followed by F. fomentarius and P. betulinus; I. lacteus and P. adiposa were the lowest.

In this study, four species of wood-rot fungi—Piptoporus betulinus, Fomes fomentarius, Irpex lacteus, and Coriolus versicolor—were compared regarding their ability to degrade the wood of white birch and used to assess the degradation mechanisms. Chemical analyses were conducted following the Chinese national standard methods and included Fourier transform infrared spectroscopy (FTIR). The wood samples were inoculated with the four wood-rot fungi for a predetermined duration in the wood-decaying test. In the wood weight loss test, both F. fomentarius and P. betulinus showed the greatest reduction, but through different mechanisms: F. fomentarius mainly decomposed lignin, whereas P. betulinus mainly acted on cellulose. F. fomentarius, I. lacteus, and C. versicolor exhibited a shift at 3417 cm−1 related to O-H stretching in hydroxyl groups, along with decreased absorption at 3410, 3406, and 3405 cm−1, most likely due to the degradation of the related functional groups of lignin side chains. The wood decayed by P. betulinus displayed a change in the relative position of cellulose-associated bands at 1161 and 898 cm−1. F. fomentarius can be considered a potential agent for the biopulping of white birch because of its high ability to degrade lignin, high holocellulose content, low content of 1% NaOH, and ethanol-benzene extractives.

Characterization of a high performance α-galactosidase from Irpex lacteus and its usage in removal of raffinose family oligosaccharides from soymilk.

Heartwood samples from three species of Juniperus (i.e., J. virginiana, J. occidentalis, and J. ashei) were extracted with hexane, ethanol, and methanol. The hexane and ethanol extracts were tested for antifungal activity against four species of wood-rot fungi (i.e, Gloeophyllum trabeum, Postia placenta, Trametes versicolor, and Irpex lacteus). Ashe juniper (AJ) gave the highest extract yields (6.60 to 11.27%), followed by Eastern red cedar (ERC) (4.78 to 9.56%), and then Western juniper (WJ) (4.26 to 7.32%). WJ contained the highest level of cedrol (over 60%), while AJ contained the highest level of thujopsene (over 30%). Methanol and ethanol gave the highest extract yields as well as slightly higher percentages of cedrol and widdrol. The juniper extracts were more effective against white-rot fungi than brown-rot fungi. The ethanol extracts had higher antifungal activity than the hexane extracts. The AJ extracts had the greatest bioactivity against the wood-rot fungi.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60°C and over a broad pH range between 5.0 and 9.0, and was stable at 50°C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of β-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

ABSTRACTTwo formulations of wood preservative one containing water-dispersible copper naphthenate (CuN) alone and in combination with sodium fluoride (NaF) were used to treat southern pine sapwood blocks to determine the effect of the addition of sodium fluoride in the QNS on the protection against wood decay fungi using laboratory soil block tests following AWPA (American Wood protection Association) standard E10-16 [American Wood Protection Association AWPA-E10. (2016) Laboratory Method for Evaluating the Decay Resistance of Wood-based Materials Against Pure Basidiomycete Cultures: Soil/Block Test]. The standard consists in exposing treated blocks to pure monoculture of white rot (Irpex lacteus (FP-105915) and Trametes versicolor (R-105)) and brown rot fungi (Gloeophyllum trabeum (Madison 617) and Postia placenta (Madison 698) during a specific period. The weight loss (WL) in percentage of treated blocks was used as an indicator of the biological performance of treated blocks against basidiomycetes at specific retentions. Minimum WL was obtained with blocks treated with the formulation containing mixture of QNS and sodium fluoride with the retention of 0.29 kg/m3 Cu and 0.51 kg/m3 of fluorine in treated blocks. A copper retention of 1.65 kg/m3 from blocks treated with formulations containing only QNS was needed to obtain similar WL to blocks containing blocks Cu++ and F− (fluorine). The lower Cu retention is attributed to a combination of the presence of the fluorine with some toxic activities against bacteria and decay and the interaction of anions fluorine and ionic Cu (II) in the aqueous treating solutions to form more soluble biological toxic hydrate of copper fluoride ((H2O Cu (II) F2) in treated blocks compared to copper complexes less biological available in water-dispersible CuN-treated bocks.

Cupressus macrocarpa (Monterey cypress) heartwood has natural durability. The heartwood oil was prepared by steam distillation and its anti-fungal activity was tested against four wood decay fungi, i.e. Trametes versicolor, Irpex lacteus, Gloeophyllum trabeum, and Postia placenta with a filter paper disc method. The oil was active against all these fungi at the concentration of 8 mg.mL-1 or greater, but Postia placenta was the most difficult for the oil to inhibit. GC-MS was adopted to analyze the components of the heartwood oil. Two components, 4-terpineol and carvacrol were identified and carvacrol represented 94.428% of the oil. The oil had anti-fungal activity mostly because of carvacrol in the high content

The effectiveness of radio frequency (RF) treatment in the control of wood decay fungi (Gloeophyllum trabeum, Ganoderma lucidum, and Irpex lacteus) and sapstain fungus (Ceratocystis fimbriata) in red oak (Quercus spp.), poplar (Populus alba), and southern yellow pine (Pinus spp.) was evaluated in the laboratory as an alternative to methyl bromide (MB) treatment. Wood samples (15.5 x 10 x 10 cm) were inoculated with fungi from a 7-day culture by dipping them to a depth of one face deep (2 cm) into inoculum and incubating them at 25°C for 14 days. Identical wood samples were left uninoculated as controls. Subsequent to incubation, the wood blocks were exposed to RF radiation in an industrial 40-kW dielectric oven at temperatures between 60° and 70°C for 2 min. The test fungi were recovered and reisolated from all of the control wood blocks but not from RF-treated wood blocks. RF treatment resulted in complete inhibition of the fungus in 98%-100% of the wood samples. Moisture content loss (≥1%) was noted after wood had been exposed to RF treatment. Moisture content may be an important factor to consider with RF treatments. RF treatment can, therefore, potentially provide an effective and rapid quarantine treatment as an alternative to MB fumigation for certain pathogen-wood combinations.

Wood from trunks of Canary Island date palm (Phoenix canariensis) was decayed for 12 weeks with white-rot fungi (Ganoderma colossum, G. zonatum, Phanerochaete chrysosporium, Scytinostroma galactinum, or Trametes versicolor) or brown-rot fungi (Wolfiporia cocos, Gloeophyllum trabeum, or Fomitopsis pinicola). Using the vermiculite-block assay, white-rot fungi caused significantly more weight loss (63%) than brown-rot fungi (32%). Of the white-rot fungi, G. colossum caused the greatest weight loss (81%), while S. galactinum caused the least (36%). In contrast, weight loss caused by the brown-rot fungi was similar. Chemical analyses indicated that both white-rot and brown-rot fungi caused losses of starch, holocellulose, and lignin. White-rot fungi, however, removed greater amounts of lignin than the brown-rot fungi with three species, S. galactinum, P. chrysosporium, and G. zonatum, causing selective delignification. Scanning and transmission electron microscopy showed that phloem and parenchyma cells were more susceptible to decay than xylem and fiber cells. Starch grains were degraded by all fungi and were nearly removed in wood decayed by G. colossum. In wood decayed by white-rot fungi, cell walls were eroded and middle lamellae were degraded. Selective delignification was observed in fibers adjacent to vascular tissue in wood decayed by the three white-rot fungi. In wood decayed by brown-rot fungi, walls of ground parenchyma and vascular bundle cells were swollen and fragmented when physically disrupted. In wood decayed by F. pinicola, some cell walls were nearly disintegrated. Key words: selective delignification, simultaneous decay, ultrastructure.

BackgroundThe white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet.ResultsBased on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system.ConclusionsThe results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.