Roles of various enzymes in the biotransformation of 6:2 fluorotelomer alcohol (6:2 FTOH) by a white-rot fungus.

Six-carbon-chained polyfluoroalkyl substances, such as 6:2 fluorotelomer alcohol (6:2 FTOH), are being used to replace longer chained compounds in the manufacture of various commercial products. This study examined the effects of growth substrates and nutrients on specific intracellular and extracellular enzymes mediating 6:2 FTOH aerobic biotransformation by the white-rot fungus, Phanerochaete chrysosporium. Cellulolytic conditions with limited glucose were a suitable composition, resulting in high 5:3 FTCA yield (37 mol%), which is a key intermediate in 6:2 FTOH degradation without forming significant amounts of terminal perfluorocarboxylic acids (PFCAs). Sulfate and ethylenediaminetetraacetic acid (EDTA) were also essential for 5:3 FTCA production, but, at lower levels, resulted in the buildup of 5:2 sFTOH (52 mol%) and 6:2 FTUCA (20 mol%), respectively. In non-ligninolytic nutrient-rich medium, 45 mol% 6:2 FTOH was transformed but produced only 12.7 mol% 5:3 FTCA. Enzyme activity studies imply that cellulolytic conditions induce the intracellular cytochrome P450 system. In contrast, extracellular peroxidase synthesis is independent of 6:2 FTOH exposure. Gene expression studies further verified that peroxidases were relevant in catalyzing the downstream transformations from 5:3 FTCA. Collectively, the identification of nutrients and enzymatic systems will help elucidate underlying mechanisms and biogeochemical conditions favorable for fungal transformation of PFCA precursors in the environment.

Exogenous microbial antagonism affects the bioaugmentation of humus formation under different inoculation using Trichoderma reesei and Phanerochaete chrysosporium.

This study was aimed at exploring the effect of antagonism of Trichoderma reesei (T.r) and Phanerochaete chrysosporium (P.c) on humification during fermentation of rice (RS) and canola straw (CS). Results showed that exogeneous fungi accelerated straw degradation and enzyme activities of CMCase, xylanase and LiP. P.c inhibited the activity of LiP when co-existing with T.r beginning, it promoted the degradation of lignin and further increased the production of humus-like substances (HLS) and humic-like acid (HLA) in later fermentation when nutrients were insufficient. The HLS of RTP was 54.9 g/kg RS, higher than the other treatments, and displayed more complex structure and higher thermostability. Brucella and Bacillus were the main HLA bacterial producers. P.c was the HLA fungal producer, while T.r assisted FLA and polyphenol transformation. Therefore, RTP was recommended to advance technologies converting crop straw into humus resources.

Insights into characteristics of white rot fungus during environmental plastics adhesion and degradation mechanism of plastics.

Since the 1980s, plastic waste in the environment has been accumulating, and little is known about fungi biodegradation, especially in dry environments. Therefore, the research on plastic degradation technology is urgent. In this study, we demonstrated that Phanerochaete chrysosporium (P. chrysposporium), a typical species of white rot fungi, could react as a highly efficient biodegrader of polylactic acid (PLA), and 34.35 % of PLA degradation was obtained during 35-day incubation. A similar mass loss of 19.71 % could be achieved for polystyrene (PS) degradation. Here, we presented the visualization of the plastic deterioration process and their negative reciprocal on cell development, which may be caused by the challenge of using PS as a substrate. The RNA-seq analysis indicated that adaptations in energy metabolism and cellular defense were downregulated in the PS group, while lipid synthesis was upregulated in the PLA-treated group. Possible differentially expressed genes (DEG) of plastic degradation, such as hydrophobic proteins, lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac), Cytochrome P450 (CYP450), and genes involved in styrene or benzoic acid degradation pathways have been recorded, and we proposed a PS degradation pathway.

Loosenin-Like Proteins from Phanerochaete carnosa Impact Both Cellulose and Chitin Fiber Networks.

Microbial expansin-related proteins are ubiquitous across bacterial and fungal organisms and reportedly play a role in the modification and deconstruction of cell wall polysaccharides, including lignocellulose. So far, very few microbial expansin-related proteins, including loosenins and loosenin-like (LOOL) proteins, have been functionally characterized. Herein, four LOOLs encoded by Phanerochaete carnosa and belonging to different subfamilies (i.e., PcaLOOL7 and PcaLOOL9 from subfamily A and PcaLOOL2 and PcaLOOL12 from subfamily B) were recombinantly produced and the purified proteins were characterized using diverse cellulose and chitin substrates. The purified PcaLOOLs weakened cellulose filter paper and cellulose nanofibril networks (CNF); however, none significantly boosted cellulase activity on the selected cellulose substrates (Avicel and Whatman paper). Although fusing the family 63 carbohydrate-binding module (CBM63) of BsEXLX1 encoded by Bacillus subtilis to PcaLOOLs increased their binding to cellulose, the CBM63 fusion appeared to reduce the cellulose filter paper weakening observed using wild-type proteins. Binding of PcaLOOLs to alpha-chitin was considerably higher than that to cellulose (Avicel) and was pH dependent, with the highest binding at pH 5.0. Amendment of certain PcaLOOLs in fungal liquid cultivations also impacted the density of the cultivated mycelia. The present study reveals the potential of fungal expansin-related proteins to impact both cellulose and chitin networks and points to a possible biological role in fungal cell wall processing. IMPORTANCE The present study deepens investigations of microbial expansin-related proteins and their applied significance by (i) reporting a detailed comparison of diverse loosenins encoded by the same organism, (ii) considering both cellulosic and chitin-containing materials as targeted substrates, and (iii) investigating the impact of the C-terminal carbohydrate binding module (CBM) present in other expansin-related proteins on loosenin function. By revealing the potential of fungal loosenins to impact both cellulose and chitin-containing networks, our study reveals a possible biological and applied role of loosenins in fungal cell wall processing.

Effect of Phanerochaete chrysosporium inoculation on manganese passivation and microbial community succession during electrical manganese residue composting.

Electrolytic manganese residue poses potentially threats to the environment and therefore needs eco-friendly treatment. Composting has been reported to effectively passivate heavy metals and alleviate their ecotoxicity. Observation of the Mn concentration during composting indicated that the mobility of Mn was significantly reduced, with the easily extraction fraction (acid extractable and easily reduction fraction) of Mn in the control pile (pile 1 without Phanerochaete chrysosporium inoculation) and treat pile (pile 2 with Phanerochaete chrysosporium inoculation) decreasing by 17% and 29%, respectively. The inoculation of Phanerochaete chrysosporium prompted the passivation of manganese, prolonged the thermophilic period, and enriched the microbial community structure, which was attributed to the rapid growth and reproduction of thermophilic bacteria. Moreover, Phanerochaete chrysosporium inoculation promoted the effect of pH on the stabilization of Mn, but the opposite contribution of organic matter. This study would provide a new perspective for remediating EMR contaminated soil via composting.

Particulate toxicity of metal-organic framework UiO-66 to white rot fungus Phanerochaete chrysosporium.

Metal-organic frameworks (MOF) are emerging materials with fantastic properties and wide applications. The release of metal ions from MOF materials is usually regarded as the origin of soluble MOF toxicity. However, whether the stable MOF particulates would induce environmental hazards is not clear. Herein, we aimed to reveal the particulate toxicity of MOF materials using the insoluble UiO-66 as the representative MOF and Phanerochaete chrysosporium as the model microorganism. UiO-66 nanoparticles (NPs) were synthesized by solvothermal method and their diameter was 68.4 ± 8.5 nm. UiO-66 NPs were stable in the culture system and the dissolution rate of 500 mg/L group was 0.26% after 14 d incubation. UiO-66 NPs did not affect the fungus growth according to the fresh weight increases and unchanged dry weights. Fungus mycelia kept even at concentrations up to 500 mg/L. Ultrastructural observation showed that UiO-66 NPs did not enter the fungal cells, but slightly destroyed the cell wall. UiO-66 NPs inhibited the laccase activity and promoted the activity of manganese peroxidase. The overall impact on the decomposition activity of P. chrysosporium was low in dye coloration test and sawdust degradation assay. Meaningful oxidative stress was aroused by UiO-66 NPs, as indicated by the decreases of catalase, glutathione, and total superoxide dismutase, and the increases of H2O2. Our results collectively suggested that the MOF particulates could induce mild mechanical damage to fungi and the toxicity was low comparing to other instable MOF materials.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate. As expression of fungal enzymes remains challenging, our ABPP-mediated approach represents a preselection procedure for focusing experimental efforts on the most promising biocatalysts. Furthermore, this approach may also allow the functional annotation of domains-of-unknown functions (DUFs). The ABPP-based biocatalyst screening described here may thus allow the identification of active enzymes in a process of interest and the elucidation of novel biocatalysts that share no sequence similarity to known counterparts.

Remediation of chromium, zinc, arsenic, lead and antimony contaminated acidic mine soil based on Phanerochaete chrysosporium induced phosphate precipitation.

Microbial induced phosphate precipitation (MIPP) is an advanced bioremediation technology to reduce the mobility and bioavailability of heavy metals (HMs), but the high level of HMs would inhibit the growth of phosphate solubilizing microbes. This study proposed a new combination system for the remediation of multiple HMs contaminated acidic mine soil, which included hydroxyapatite (HAP) and Phanerochaete chrysosporium (P. chrysosporium, PC) that had high phosphate solubilizing ability and HMs tolerance. Experimental data suggested that in HAP/PC treatment after 35 d of remediation, labile Cr, Zn and As could be transformed into the stable fraction with the maximum immobilization efficiencies increased by 53.01 %, 22.43 %, and 35.65 %, respectively. The secretion of organic acids by P. chrysosporium was proved to promote the dissolution of HAP. Besides, the pH value, available phosphorus (AP) and organic matter (OM) increased in treated soil than in original soil, which also indicated the related dissolution-precipitation mechanism of HMs immobilization. Additionally, characterization results revealed that adsorption and ion exchange also played an important role in the remediation process. The overall results suggested that applying P. chrysosporium coupled with HAP could be considered as an efficient strategy for the remediation of multiple HMs contaminated mine soil and laid a foundation for the future exploration of soil microenvironment response during the remediation process.

Biotransformation of bisphenol F by white-rot fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Environmental bisphenol F (BPF) has a cyclic endocrine disruption effect, seriously threatening animal and human health. It is frequently detected in environmental samples worldwide. For BPF remediation, biological methods are more environmentally friendly than physicochemical methods. White-rot fungi have been increasingly studied due to their potential capability to degrade environmental pollutants. Phanerochaete sordida YK-624 has been shown to degrade BPF by ligninolytic enzymes under ligninolytic conditions. In the present study, degradation of BPF under non-ligninolytic conditions (no production of ligninolytic enzymes) was investigated. Our results showed that BPF could be completely removed after 7-d incubation. A metabolite of BPF, 4,4'-dihydroxybenzophenone (DHBP) was identified by mass spectrometry and nuclear magnetic resonance, and DHBP was further degraded by this fungus to form 4-hydroxyphenyl 4-hydroxybenzoate (HPHB). DHBP and HPHB were the intermediate metabolites of BPF and would be further degraded by P. sordida YK-624. We also found that cytochrome P450s played an important role in BPF degradation. Additionally, transcriptomic analysis further supported the involvement of these enzymes in the action of BPF degradation. Therefore, BPF is transformed to DHBP and then to HPHB likely oxidized by cytochrome P450s in P. sordida YK-624. Furthermore, the toxicological studies demonstrated that the order of endocrine-disrupting activity for BPF and its metabolites was HPHB > BPF > DHBP. KEY POINTS: • White-rot fungus Phanerochaete sordida YK-624 could degrade BPF. • Cytochrome P450s were involved in the BPF degradation. • The order of endocrine disrupting activity was: HPHB > BPF > DHBP.

Biodeterioration of low-density polyethylene by mangrove-associated endolichenic fungi and their enzymatic regimes.

Fungal involvement in the biodeterioration of low-density polyethylene (LDPE) has received great attention in recent years. Among diverse groups of fungi, endolichenic fungi (ELF) are adapted to thrive in resource-limited conditions. The present study was designed to investigate the potential of mangrove-associated ELF, in the biodeterioration of LDPE and to quantify key-depolymerizing enzymes. A total of 31 ELF species, isolated from 22 lichens of mangrove ecosystems in Negombo lagoon, Sri Lanka were identified using DNA barcoding techniques. ELF were inoculated into a mineral salt medium, containing LDPE strips and incubated at 28 ± 2°C, for 21 days, under laboratory conditions. After incubation, biodeterioration was monitored based on percent reductions in weights and tensile properties, increments in the degree of water absorption, changes in peaks of infrared spectra and surface erosions using scanning electron microscopy. Out of 31 species, Chaetomium globosum, Daldinia eschscholtzii, Neofusicoccum occulatum, Phanerochaete chrysosporium, Schizophyllum commune and Xylaria feejeensis showed significant changes. Production of depolymerizing enzymes by these species was assayed qualitatively using plate-based methods and quantitatively by mass-level enzyme production. Among them, Phanerochaete chrysosporium showed the highest enzyme activities as (9·69 ± 0·04) × 10-3 , (1·96 ± 0·01) × 10-3 , (5·73 ± 0·03) × 10-3 , (0·88 ± 0·01), (0·64 ± 0·06), (1·43 ± 0·01) U ml-1 for laccase, lignin peroxidase, manganese peroxidase, amylase, lipase and esterase, respectively.

Effect of pretreatment with Phanerochaete chrysosporium on physicochemical properties and pyrolysis behaviors of corn stover.

Fungal pretreatment can selectively degrade partial biomass components, which undoubtedly exerts a significant influence on biomass pyrolysis behavior. The corn stover was pretreated with Phanerochaete chrysosporium, and its influence on the physicochemical properties and pyrolysis behaviors of biomass together with the product characteristics were investigated. The Phanerochaete chrysosporium was more active to degrade hemicellulose and lignin. The hemicellulose and lignin contents in corn stover were decreased by 35.14 % and 31.80 %, respectively, after five weeks pretreatment, compared to the untreated sample. The reaction activation energy decreased from 52.89 kJ·mol-1 for the untreated sample to 40.88 kJ·mol-1 for the sample pretreated for five weeks. The Phanerochaete chrysosporium pretreatment was beneficial to the biochar production but exerted an unfavorable effect on the texture structure. The Phanerochaete chrysosporium also had an obvious influence on the bio-oil compositions. This study can provide a scientific reference for the application of biological pretreatment for biomass pyrolysis technology.

Reprint of: Purification and Characterization of an Extracellular Mn(ll)-Dependent Peroxidase from the Lignin-Degrading Basidiomycete, Phanerochaete chrysosporium.

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N3- readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 µM. The enzyme is also stimulated to varying degrees by α-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl- 6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism. © 1985 Academic Press, Inc.

Characterization of two 1,2,4-trihydroxybenzene 1,2-dioxygenases from Phanerochaete chrysosporium.

Lignin is the most abundant aromatic compound in nature, and it plays an important role in the carbon cycle. White-rot fungi are microbes that are capable of efficiently degrading lignin. Enzymes from these fungi possess exceptional oxidative potential and have gained increasing importance for improving bioprocesses, such as the degradation of organic pollutants. The aim of this study was to identify the enzymes involved in the ring cleavage of the lignin-derived aromatic 1,2,4-trihydroxybenzene (THB) in Phanerochaete chrysosporium, a lignin-degrading basidiomycete. Two intradiol dioxygenases (IDDs), PcIDD1 and PcIDD2, were identified and produced as recombinant proteins in Escherichia coli. In the presence of O2, PcIDD1 and PcIDD2 acted on eight and two THB derivatives, respectively, as substrates. PcIDD1 and PcIDD2 catalyze the ring cleavage of lignin-derived fragments, such as 6-methoxy-1,2,4-trihydroxybenzene (6-MeOTHB) and 3-methoxy-1,2-catechol. The current study also revealed that syringic acid (SA) was converted to 5-hydroxyvanillic acid, 2,6-dimethoxyhydroquinone, and 6-MeOTHB by fungal cells, suggesting that PcIDD1 and PcIDD2 may be involved in aromatic ring fission of 6-MeOTHB for SA degradation. This is the first study to show 6-MeOTHB dioxygenase activity of an IDD superfamily member. These findings highlight the unique and broad substrate spectra of PcIDDs, rendering it an attractive candidate for biotechnological application. KEY POINTS: • Novel intradiol dioxygenases (IDD) in lignin degradation were characterized. • PcIDDs acted on lignin-derived fragments and catechol derivatives. • Dioxygenase activity on 6-MeOTHB was identified in IDD superfamily enzymes.

Fluorescent Imaging of Extracellular Fungal Enzymes Bound onto Plant Cell Walls.

Lignocelluloytic enzymes are industrially applied as biocatalysts for the deconstruction of recalcitrant plant biomass. To study their biocatalytic and physiological function, the assessment of their binding behavior and spatial distribution on lignocellulosic material is a crucial prerequisite. In this study, selected hydrolases and oxidoreductases from the white rot fungus Phanerochaete chrysosporium were localized on model substrates as well as poplar wood by confocal laser scanning microscopy. Two different detection approaches were investigated: direct tagging of the enzymes and tagging specific antibodies generated against the enzymes. Site-directed mutagenesis was employed to introduce a single surface-exposed cysteine residue for the maleimide site-specific conjugation. Specific polyclonal antibodies were produced against the enzymes and were labeled using N-hydroxysuccinimide (NHS) ester as a cross-linker. Both methods allowed the visualization of cell wall-bound enzymes but showed slightly different fluorescent yields. Using native poplar thin sections, we identified the innermost secondary cell wall layer as the preferential attack point for cellulose-degrading enzymes. Alkali pretreatment resulted in a partial delignification and promoted substrate accessibility and enzyme binding. The methods presented in this study are suitable for the visualization of enzymes during catalytic biomass degradation and can be further exploited for interaction studies of lignocellulolytic enzymes in biorefineries.

On 'Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium' by Jeffrey K. Glenn and Michael H. Gold.

This paper by Jeffrey K. Glenn and Michael H. Gold (Department of Chemical, Biological, and Environmental Sciences, Oregon Graduate Center) reported for the first time the purification and characterization of a manganese peroxidase. It was shown that the extracellular heme b-containing oxidoreductase purified from the basidiomycete Phanerochaete chrysosporium requires hydrogen peroxide and Mn(II) for the oxidation of a variety of different compounds. This discovery in 1985 was the prelude to countless research papers on structure-function relationships of manganese peroxidases, their ecological role(s) in the degradation of lignocellulose and lignin and on their relevance for industrial and commercial applications. This paper has been cited 575 times in Scopus. A Scopus search for the term manganese peroxidase yielded 6163 results (April 2022).

Latent potentials of the white-rot basidiomycete Phanerochaete chrysosporium responsible for sesquiterpene metabolism: CYP5158A1 and CYP5144C8 decorate (E)-α-bisabolene.

Basidiomycetes produce various sesquiterpenoids and their relevance for pharmaceutical and agricultural applications and understanding their biosynthetic machinery to produce these secondary metabolites have attracted significant interest. Because sesquiterpene synthases (STSs) and cytochrome P450 monooxygenases (P450s) play pivotal roles in the production of sesquiterpenoids, functional characterization of these enzymes is fundamentally essential. In this study, we found 11 possible STSs from the white-rot basidiomycete Phanerochaete chrysosporium (PcSTSs) and isolated nine of these as full-length cDNAs encoding a mature open reading frame. Using the isolated cDNAs, we performed heterologous expression of PcSTSs in Saccharomyces cerevisiae. Metabolic studies revealed that seven of the PcSTSs produce a series of sesquiterpene scaffolds, including (E)-α-bisabolene. Furthermore, we constructed a co-expression system of (E)-α-bisabolene synthase and P450 from P. chrysosporium (PcCYP). Semi-comprehensive screening using 120 isoforms of PcCYPs resulted in the identification of CYP5158A1 and CYP5144C8, two P450s capable of decorating (E)-α-bisabolene.

Enhanced aerobic granular sludge formation by applying Phanerochaete chrysosporium pellets as induced nucleus.

The long start-up period is a major challenging issue for the widespread application of aerobic granular sludge (AGS). In this study, a novel rapid start-up strategy was developed by inoculating Phanerochaete chrysosporium (P. chrysosporium) pellets as the induced nucleus in a sequencing batch airlift reactor (SBAR) to enhance activated sludge granulation. The results demonstrated that P. chrysosporium pellets could effectively shorten the aerobic granulation time from 32 to 20 days. The AGS promoted by P. chrysosporium pellets had a larger average diameter (2.60-2.74 mm) than that without P. chrysosporium pellets (1.78-1.88 mm) and had better biomass retention capacity and sedimentation properties; its mixed liquor suspended solids (MLSS) and sludge volume index (SVI30) reached approximately 5.2 g/L and 45 mL/g, respectively. The addition of P. chrysosporium pellets promoted the secretion of extracellular polymeric substances (EPS), especially protein (PN). The removal efficiencies of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), total nitrogen (TN), and total phosphorus (TP) in P. chrysosporium pellets reactor were 98.91%, 89.17%, 64.73%, and 94.42%, respectively, which were higher than those in the reactor without P. chrysosporium pellets (88.73%, 82.09%, 55.75%, and 88.92%). High throughput sequencing analysis indicated that several functional genera that were responsible for the formation of aerobic granules and the removal of pollutants, such as Acinetobacter, Pseudomonas, Janthinobacterium, and Enterobacter, were found to be predominant in the mature sludge granules promoted by P. chrysosporium pellets.

The microbial mechanisms of enhanced humification by inoculation with Phanerochaete chrysosporium and Trichoderma longibrachiatum during biogas residues composting.

This study investigated effects of composite microbes (CMs) (Phanerochaete chrysosporium and Trichoderma longibrachiatum) on humification during co-composting of biogas residue, spent mushroom substrate and rice straw. Results showed that CMs inoculation elevated degradation ratios of cellulose, hemicellulose and lignin by 7.86%, 8.87% and 6.45%, and contents of humus and humic acid were correspondingly promoted by 15.5% and 23.6%, respectively. Relative abundances of bacteria associated with refractory macromolecules degradation (Flavobacterium, Anseongella and Actinomadura) and cellulolytic fungi (Hypocreales_Incertae_sedis, Hypocreaceae and Psathyrellaceae) were raised by CMs addition. Redundancy analysis demonstrated a positive correlation between microbial communities and temperature, fulvic acid and lignocellulose contents. Moreover, CMs inoculation promoted pathways of xenobiotics biodegradation and metabolism, and biosynthesis of other secondary metabolites, which was closely associated with lignocellulose degradation and humus formation. These results suggested that biological inoculation could enhance composting efficiency and improve compost quality, benefiting biogas residues composting.

Comparison of glycoside hydrolase family 3 ß-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) ß-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A.

To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing ß-1,4-glucans rather than the previously reported ß-1,3/1,4-glucans lichenan and ß-glucan. The dominating products from ß-1,4-glucans were C3-C6 oligosaccharides, whereas from ß-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.