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A method has been developed for the analysis of the induction of cbh1 gene transcription in the filamentous fungus Penicillium verruculosum after treatment with mono- and oligosaccharides. The method makes it possible to obtain mRNA, which encodes cellobiohydrolase 1 (CBH1), in an amount sufficient for RT-qPCR. Citrate, a compound that does not cause carbon catabolite repression, was used as a carbon source. It was shown for the first time that xylose, gentiobiose, and, mainly, cellobiose induce the expression of the cbh1 gene in P. verruculosum, while sophorose and a mixture of xylooligosaccharides with a degree of polymerization of 3–5 most likely serve as precursors of inducers. [ABSTRACT FROM AUTHOR]

A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4–12) and temperatures (40–100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL−1, 222.2 μmol mg−1 min−1, 2492 s−1 and 778.7 s−1 mg−1 mL−1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries. [ABSTRACT FROM AUTHOR]

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers. [ABSTRACT FROM AUTHOR]

is a genus of filamentous and cosmopolitan fungi that includes important species for medical mycology, food, basic research and agro-industry areas. Aspergillus section Nigri are efficient producers of hydrolytic enzymes such as cellulases that are employed in the cellulose conversion. Hence, the search of new cellulolytic isolates and their correct identification is important for carrying out safe biotechnological processes. This study aimed to characterise the cellulolytic potential of Aspergillus sp. LBM 134, isolated from the Paranaense rainforest (Argentina) and to identify the isolate through a polyphasic approach. The fungus was identified as Aspergillus niger and its cellulolytic potential was evaluated by using Congo red technique and fluorescence plate assays for carboxymethyl cellulase, β-glucosidase and cellobiohydrolase, respectively. All three cellulase activities were positive; this bio-prospective positioned A. niger LBM 134 as a promising alternative for industries that require organisms capable of carrying out cellulosic biomass processing. [ABSTRACT FROM AUTHOR]

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency. [ABSTRACT FROM AUTHOR]

In the present study, the cellulose from sugarcane tops (SCT) was separated and characterized for its purity. Approximately, 85% (w/w) of total cellulose present in raw SCT was recovered by using alkaline method. The monosaccharide analysis of SCT cellulose by HPLC showed 91% D-glucose, 7.5% D-xylose and 1.5% D-arabinose residues. Surface morphology study of dried cellulosic fibers by FESEM exhibited the fibrous structure. The FTIR analysis of separated cellulose displayed the peaks corresponding to the peaks obtained from commercial cellulose, confirming its purity. The crystallinity index (CrI) of separated cellulose increased to 49% after delignification and xylan extraction from 36% of raw SCT. The typical TGA curve of separated SCT cellulose showed decomposition and mass reduction at 327 °C resulting in single decomposition peak in TGA analysis, confirming its purity. CHNS analysis supported the purity of separated cellulose by confirming absence of nitrogen and sulfur. The separated cellulose was hydrolyzed by recombinant endo-β-1,4-glucanase (CtCel8A), cellobiohydrolase (CtCBH5A) from Clostridium themocellum and β-1,4-glucosidase (HtBgl) from Hungateiclostridium thermocellum at pH 5.8, 50 °C for 24 h, resulting in the production of 188 mg/g of total reducing sugar (TRS). The separated cellulose from SCT can be utilized as an alternative substrate for commercialization and for bioethanol production. 85% of total cellulose recovery yield was obtained from raw sugarcane top (SCT) SCT cellulose contains 91%, D-glucose, 7.5% D-xylose and 1.5% D-arabinose residues Crystallinity index (CrI) of separated cellulose increased from 36% to 49% of raw SCT TGA curve of separated SCT cellulose showed the single decomposition peak at 327 °C Separated cellulose hydrolyzed by recombinant enzymes gave 188 mg/g TRS yield [ABSTRACT FROM AUTHOR]

The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B‐type helix‐turn‐helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh‐binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B‐like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi. [ABSTRACT FROM AUTHOR]

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against p NPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w / v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector. • The increasing energy demand has intensified the need of renewable energy sources. • A novel cellulose-binding domain containing cellobiohydrolase (CbCBH) cloned from Caldicellulosiruptor bescii. • CbCBH exhibited great thermostability making it a promising candidate for biofuel. • Plackett-Burman model evaluated the saccharification conditions of sugarcane bagasse. • Optimized conditions yielded a remarkable 28.45% saccharification efficiency. [ABSTRACT FROM AUTHOR]

Glycosylation, a general post-translational modification for fungal cellulase, has been shown to affect cellulase binding to its substrate. However, the exact impact of glycosylation on cellulase-lignin interaction remain unclear. Here, we demonstrated that the lignin isolated from tetrahydrofuran-pretreated corn stover exhibits strong adsorption capability to cellulase due to its negatively charged and porous structure. For the cellulases with varying glycosylation levels, the less-glycosylated protein showed high adsorption capability to lignin, and that trend was observed for the main cellulase components secreted by Penicillium oxilicum , including endoglucanase PoCel5B, cellobiohydrolase PoCel7A-2, and β-glucosidase PoBgl1. Additionally, N-glycan sites and motifs were examined using mass spectrometry, and protein structures with N-glycans were constructed, where PoBgl1 and PoCel7A-2 contained 13 and 1 glycosylated sites respectively. The results of molecular dynamics simulations indicated that the N-glycans impacted on the solvent-accessible surface area and secondary structure of protein, and the binding conformation of lignin fragment on cellulase, resulting in a decrease in binding energy (14 kcal/mol for PoBgl1 and 13 kcal/mol for PoCel7A-2), particularly for van der Waals and electrostatic interaction. Those findings suggested that glycosylation negatively impacted the lignin-cellulase interaction, providing a theoretical basis for the rational engineering of enzymes to reduce lignin-enzyme interaction. [ABSTRACT FROM AUTHOR]

In this study, CRISPR/Cas9 genome editing was used to knockout the bgl2 gene encoding intracellular β-glucosidase filamentous fungus Penicillium verruculosum. This resulted in a dramatic reduction of secretion of cellulolytic enzymes. The study of P. verruculosum Δbgl2 found that the transcription of the cbh1 gene, which encodes cellobiohydrolase 1, was impaired when induced by cellobiose and cellotriose. However, the transcription of the cbh1 gene remains at level of the host strain when induced by gentiobiose. This implies that gentiobiose is the true inducer of the cellulolytic response in P. verruculosum , in contrast to Neurospora crassa where cellobiose acts as an inducer. [ABSTRACT FROM AUTHOR]

• Atmospheric nitrogen (N) deposition enhanced soil heterotrophic respiration (R H). • Biochar mitigated the stimulatory effect of N deposition on soil R H. • R H was positively related to β-glucosidase and cellobiohydrolase (CBH) activities. • Reduced R H by biochar was linked to decreased CBH activity and cbh I gene abundance. Both atmospheric nitrogen (N) deposition and biochar application can markedly impact soil heterotrophic respiration (R H), an important component of the global carbon cycle. However, the interactive effects of N deposition and biochar application on soil R H in subtropical forest ecosystems remain unclear. Here, we conducted a three-year (2019–2022) field trial within a bamboo forest in subtropical China to examine the responses of soil physicochemical and microbial properties to N deposition and biochar application, and to elucidate how biochar regulates N deposition-induced change in soil R H. Nitrogen deposition stimulated soil R H by 8.1–9.8 % annually over three years compared to the control, and this stimulation was mitigated (by 8.1–8.9 % annually) with biochar addition. In the context of N deposition, the decrease of soil R H by biochar application was not through changing soil temperature, moisture or labile organic carbon content. Biochar treatment reduced the abundances of bacterial glycoside hydrolase family 48 gene (GH48) and fungal glycoside hydrolase family 7 cellobiohydrolase I gene (cbh I) and the activities of β-glucosidase and cellobiohydrolase (CBH), but increased the abundance of cbbL gene and activity of RubisCO enzyme. Furthermore, the R H was correlated positively (P < 0.01) with β-glucosidase and CBH activities and negatively (P < 0.01) with RubisCO enzyme activity. Structural equation modeling revealed that the biochar-induced reduction of soil R H under N deposition was associated with decreases in the abundance of cbh I gene and the activity of CBH in soils. We highlight that management practices can mitigate soil carbon loss in forests through modulating soil microbial functions under atmospheric N deposition, and that biochar application in Moso bamboo forests has the potential to reduce R H by approximately 7.6 × 106 t CO 2 yr−1 under atmospheric N deposition. [Display omitted] [ABSTRACT FROM AUTHOR]

A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0–10.4, and 60.2 % Avicelase activity in the range of 30–90 °C. After incubation at 70–90 °C for 1 h, the Avicelase activity retained 60.5 % of its initial activity. The presence of Zn2+, Ca2+ or Cd2+ enhanced the Avicelase activity of the CBH2, of which Cd2+ at 10 mM causing the highest increase. The recombinant CBH2 was used to enhance the Avicel hydrolysis by improving the exo-exo-synergism between CBH2 and CBH1 in N.crassa cellulase. The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBH2 and CBH1, and the yield was increased by 215.4 % when the temperature is raised to 70 °C. This work provided a CBH2 with broader pH range and better heat resistance, which is a potential enzyme candidate in food, textile, pulp and paper industries, and other industrial fields. • A cellobiohydrolase of Neurospora crassa CBH2 was expressed in Pichia pastoris. • The recombinant CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0–10.4. • The recombinant CBH2 maintained 60.5 % Avicelase activity at 70–90 °C for 1 h. • The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBHs. [ABSTRACT FROM AUTHOR]

• Various polymer-binding domains were fused to a PET hydrolase variant Is PETaseEHA. • Is PETaseEHA_CBM fused with a cellulose-binding domain showed enhanced PET hydrolysis. • Dose-dependent effects of Is PETaseEHA_CBM on PET hydrolysis were observed. The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (Is PETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of Is PETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered Is PETaseS121E/D186H/R280A (Is PETaseEHA) variant. We found that fusing a cellulose–binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of Is PETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, Is PETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of Is PETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 μg/mL to 20 μg/mL. These findings suggest that the fusion of polymer-binding module to Is PETase is a promising strategy to stimulate the enzymatic hydrolysis of PET. [ABSTRACT FROM AUTHOR]

Fungi are important decomposers in terrestrial ecosystems, so their responses to climate change might influence carbon (C) and nitrogen (N) dynamics. We investigated whether growth and activity of fungi under drought conditions were structured by trade-offs among traits in 15 fungal isolates from a Mediterranean Southern California grassland. We inoculated fungi onto sterilized litter that was incubated at three moisture levels (4, 27, and 50% water holding capacity, WHC). For each isolate, we characterized traits that described three potential lifestyles within the newly proposed "YAS" framework: growth yield, resource acquisition, and stress tolerance. Specifically, we measured fungal hyphal length per unit litter decomposition for growth yield; the potential activities of the extracellular enzymes cellobiohydrolase (CBH), ! β -glucosidase (BG), β -xylosidase (BX), and N-acetyl- β - D -glucosaminidase (NAG) for resource acquisition; and ability to grow in drought vs. higher moisture levels for drought stress tolerance. Although, we had hypothesized that evolutionary and physiological trade-offs would elicit negative relationships among traits, we found no supporting evidence for this hypothesis. Across isolates, growth yield, drought stress tolerance, and extracellular enzyme activities were not significantly related to each other. Thus, it is possible that drought-induced shifts in fungal community composition may not necessarily lead to changes in fungal biomass or decomposer ability in this arid grassland. [ABSTRACT FROM AUTHOR]

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, β-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of β-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5. [ABSTRACT FROM AUTHOR]

The present study examined microbial substrate utilization by community level physiological profiling and carbon-cycling enzyme assays (cellobiohydrolase, β-xylosidase, and oxidases) in three Japanese larch forests. The forests differed in their locations, topographies, and soil microclimates, and each covered three treatments, namely 20 (IT) and 35% basal area thinning (HT) without intensive residue harvests and an un-thinned control (UTC). Microbial substrate utilization and soil properties (temperature, moisture, total carbon and nitrogen, inorganic nitrogen, and pH) were analyzed at 0–10 cm depth, six years after thinning. Microbial utilization of carbohydrate group under IT was 27 and 62% higher than that under UTC and HT, respectively, in only one of the forests. This might occur because this forest featured a steeper slope, rockier soil texture, and cooler and drier soil surface than the other two forests, where no thinning effect was observed. However, neither microbial utilization of any other substrate groups nor enzyme activity changed by thinning across all forests. It could result from the exclusion of intensive residue harvests or the lack of changes in soil inorganic nitrogen and pH. These results indicate that the thinning effects on microbial substrate utilization might be inconsistent across multiple sites, and at least, not decline the associated forest ecosystem functions and sustainability. [ABSTRACT FROM AUTHOR]

Inducers play an important role in fungus cellulase production. In this study, a combination of Avicel and laver powder, which contains cellulose, hemicellulose, other polysaccharides, and crude lipid, was used to induce lignocellulolytic enzyme production in Penicillium piceum H16. When Avicel was supplemented with various amounts of laver powder, cellulase activities or extracellular protein yields increased by 11.4%–102.5%, relative to those observed when Avicel alone was used. In particular, filter paper and cellobiohydrolase activities increased by as much as 39.3% and 102.5%, respectively, after supplementation with laver powder. Furthermore, laver powder supplementation enhanced the efficiency of cellulose and hemicellulose hydrolysis in crude corn stover by 39.3% and 22.2%, respectively. The results suggest that laver powder and Avicel synergistically induce lignocellulytic enzyme production and P. piceum H16 growth. iTRAQ‐based quantitative proteomics analysis results further demonstrate that laver powder supplementation optimized the synergistic lignocellulytic enzyme cocktail from P. piceum H16 by upregulating the production of cellulase‐enhancing factors and lignocellulolytic enzymes. The oligosaccharides released by laver powder may account for the synergistic induction, which may help reduce the cost of producing lignocellulytic enzymes by filamentous fungi. [ABSTRACT FROM AUTHOR]

Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49 , finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley β-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo -β-1,3-1,4-glucanase (EC 3.2.1.73) and exo -cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass. • We isolated and characterized two novel glucanases, Cel5A-h38 and Cel5A-h49, from rumen uncultured bacterium. • Cel5A-h38 exclusively generated glucose and cellobiose as end products from glucan. • Cel5A-h38 is a novel bifunctional enzyme with both endo-β-1,3-1,4-glucanase and exo-cellobiohydrolase activities. • Cel5A-h38 was highly effective at releasing glucose and cellobiose from natural lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]

Soil labile organic carbon (LOC) responds rapidly to environmental changes and plays an important role in carbon cycle. In this study, the seasonal fluctuations in LOC, the activities of carbon-cycle related enzymes, and the bacterial and fungal communities were analyzed for soils collected from two forests, namely Betula albosinensis (Ba) and Picea asperata Mast. (Pa), in the Qinling Mountains of China. Results revealed that the seasonal average contents of microbial biomass carbon (MBC), easily oxidized organic carbon (EOC), and dissolved organic carbon (DOC) of Pa forest soil were 13.5%, 30.0% and 15.7% less than those in Ba soil. The seasonal average enzyme activities of β-1,4-glucosidase (βG), and β-1,4-xylosidase (βX) of Ba forest soils were 30.0% and 32.3% higher than those of Pa soil while the enzyme activity of cellobiohydrolase (CBH) was 19.7% lower. Furthermore, the relative abundance of Acidobacteria was significantly higher in summer than in winter, whereas the relative abundance of Bacteroidetes was higher in winter. Regarding the fungal communities, the relative abundance of Basidiomycota was lowest in winter, whereas Ascomycota predominated in the same season. In addition, the soil LOC was significantly positively correlated with the CBH, βG and βX activities. Changes in LOC were significantly correlated with Acidobacteria, Bacteroidetes and Basidiomycota. We conclude that the seasonal fluctuations in forest soil LOC fractions relied on carbon cycle-associated enzymatic activities and microorganisms, which in turn were affected by climatic conditions. [ABSTRACT FROM AUTHOR]

Background: Penicillium funiculosum NCIM1228 is a non-model filamentous fungus that produces high-quality secretome for lignocellulosic biomass saccharification. Despite having desirable traits to be an industrial workhorse, P. funiculosum has been underestimated due to a lack of reliable genetic engineering tools. Tolerance towards common fungal antibiotics had been one of the major hindrances towards development of reliable transformation tools against the non-model fungi. In this study, we sought to understand the mechanism of drug tolerance of P. funiculosum and the provision to counter it. We then attempted to identify a robust method of transformation for genome engineering of this fungus. Results: Penicillium funiculosum showed a high degree of drug tolerance towards hygromycin, zeocin and nourseothricin, thereby hindering their use as selectable markers to obtain recombinant transformants. Transcriptome analysis suggested a high level expression of efflux pumps belonging to ABC and MFS family, especially when complex carbon was used in growth media. Antibiotic selection medium was optimized using a combination of efflux pump inhibitors and suitable carbon source to prevent drug tolerability. Protoplast-mediated and Agrobacterium-mediated transformation were attempted for identifying efficiencies of linear and circular DNA in performing genetic manipulation. After finding Ti-plasmid-based Agrobacterium-mediated transformation more suitable for P. funiculosum, we improvised the system to achieve random and homologous recombination-based gene integration and deletion, respectively. We found single-copy random integration of the T-DNA cassette and could achieve 60% efficiency in homologous recombination-based gene deletions. A faster, plasmid-free, and protoplast-based CRISPR/Cas9 gene-editing system was also developed for P. funiculosum. To show its utility in P. funiculosum, we deleted the gene coding for the most abundant cellulase Cellobiohydrolase I (CBH1) using a pair of sgRNA directed towards both ends of cbh1 open reading frame. Functional analysis of ∆cbh1 strain revealed its essentiality for the cellulolytic trait of P. funiculosum secretome. Conclusions: In this study, we addressed drug tolerability of P. funiculosum and developed an optimized toolkit for its genome modification. Hence, we set the foundation for gene function analysis and further genetic improvements of P. funiculosum using both traditional and advanced methods. [ABSTRACT FROM AUTHOR]

Functional screening of metagenomic libraries is an effective approach for identification of novel enzymes. A Caatinga biome goat rumen metagenomic library was screened using esculin as a substrate, and a gene from an unknown bacterium encoding a novel GH3 enzyme, BGL11, was identified. None of the BGL11 closely related genes have been previously characterized. Recombinant BGL11 was obtained and kinetically characterized. Substrate specificity of the purified protein was assessed using seven synthetic aryl substrates. Activity towards nitrophenyl-β-D-glucopyranoside (pNPG), 4-nitrophenyl-β-D-xylopyranoside (pNPX) and 4-nitrophenyl-β-D-cellobioside (pNPC) suggested that BGL11 is a multifunctional enzyme with β-glucosidase, β-xylosidase, and cellobiohydrolase activities. However, further testing with five natural substrates revealed that, although BGL11 has multiple substrate specificity, it is most active towards xylobiose. Thus, in its native goat rumen environment, BGL11 most likely functions as an extracellular β-xylosidase acting on hemicellulose. Biochemical characterization of BGL11 showed an optimal pH of 5.6, and an optimal temperature of 50°C. Enzyme stability, an important parameter for industrial application, was also investigated. At 40°C purified BGL11 remained active for more than 15 hours without reduction in activity, and at 50°C, after 7 hours of incubation, BGL11 remained 60% active. The enzyme kinetic parameters of Km and Vmax using xylobiose were determined to be 3.88 mM and 38.53 μmol.min-1.mg-1, respectively, and the Kcat was 57.79 s-1. In contrast to BLG11, most β-xylosidases kinetically studied belong to the GH43 family and have been characterized only using synthetic substrates. In industry, β-xylosidases can be used for plant biomass deconstruction, and the released sugars can be fermented into valuable bio-products, ranging from the biofuel ethanol to the sugar substitute xylitol. [ABSTRACT FROM AUTHOR]

Sequential pretreatments for sugarcane bagasse (scb) by NaOH followed by organosolv under mild conditions were evaluated for cellulose recovery and dilignification. The best-optimized sequential pretreatment of scb was obtained at 10% (w/v) of raw scb loading at 1% (w/v) NaOH (50 °C, 2 h) followed by treatment with organosolv (85%, v/v phosphoric acid, 50 °C, 1 h) with chilled acetone. This sequentially pretreated scb showed cellulose recovery, 66.1% (w/w) and delignification, 83.2% (w/w). NaOH or organosolv pretreated scb showed lower cellulose recovery 47.4% (w/w) or 54.5% (w/w) with lower delignification, 61% (w/w) or 56% (w/w), respectively. Pretreated solid residue of sequentially pretreated scb was enzymatically saccharified by chimera (β-glucosidase and endoglucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) cloned from Clostridium thermocellum. Enzymatic hydrolysate of best sequentially pretreated scb gave total reducing sugar (TRS) yield, 230 mg/g and glucose yield, 137 mg/g pretreated scb. Only organosolv pretreated scb gave TRS yield, 112.5 mg/g and glucose yield, 72 mg/g of pretreated scb. Thus, sequentially pretreated scb resulted in 37% higher enzymatic digestibility than only orgnaosolv pretreated scb. Higher enzymatic digestibility was supported by higher crystallinity index CrI (45%) than those obtained with only organosolv pretreated (38%) or raw scb (25%). Field Emission Scanning Electron Microscope (FESEM) and Fourier-transform infrared (FT-IR) analyses showed enhanced cellulose exposure in sequentially pretreated scb. Preliminary investigation of bioethanol production at small scale by separate hydrolysis and fermentation (SHF) of enzymatic hydrolysate from best sequentially pretreated scb by Saccharomyces cerevisiae gave maximum ethanol yield of 0.42 g/g of glucose. [ABSTRACT FROM AUTHOR]

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase I from Trichoderma reesei (TrCel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of TrCel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of TrCel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of TrCel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of TrCel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake (kon for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel. [ABSTRACT FROM AUTHOR]

Cellobiose is one of the most abundant carbohydrates available in nature which can be used as biofuel in various applications such as food industry, energy production, paper production, etc. with no harm to the environment. One way for a safe decomposition the cellobiose is enzymatic hydrolysis. Cellobiohydrolase enzyme, which is naturally present in some yeasts and bacteria, is capable of decomposing carbohydrates. Because of its low production in its original organism and also high volumes of requirements to this enzyme for industrial usage, there is strong desired to mass-produce of this enzyme using biotechnological approaches. In this study, several signal peptide sequences with Lactococcus originality, with the aim of binding to the cellobiohydrolase gene, were evaluated. In this regard, signalP server, a valid and accurate online tool, was used to determine the most important details including N-terminal (n), hydrophobic (h) and carboxy-terminal (c) regions of signal peptides and their probability. Thereafter, Portparam and Solpro online tools were used to investigate the physico-chemical properties of each signal peptide. Finally, the best peptide signal with the best specificity and the highest D-score was selected for binding to the cellobiohydrolase gene sequence aimed at expression in the Gram-positive bacterium Lactococcus lactis. Final frame of target gen was synthetized and ligated in pBU003 expression vector using double digestion by SalI and XhoI restriction enzymes. Recombinant expression vector was transferred to MC1061 for replication and then transferred into the MG1363 as expressing strain of Lacticoccus lactis. Recombinant proteins were purified from M17 medium using the Ni-NTA Agarose column. Their quantification and qualification examined through Bradford assay and 12% SDS-PAGE respectively. Phosphoric acid-treated cotton as a substrate was used to determine the Ce16B cellobiohydrolase activity at 450 nm absorbance. In silico investigating the signal peptides have led to the choice of the USP45 signal peptide as a powerful secretory signal. Recombinant proteins were successfully expressed in Lactococcus lactis. The concentration of each recombinant protein was determined 0.7 g/l (R2 = 0.989). In vitro assessment of enzymatic activity or recombinant proteins on PC showed 1.89 U (R2: 9941). [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolitederepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with -200% and -66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading. [ABSTRACT FROM AUTHOR]

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Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover. [ABSTRACT FROM AUTHOR]

Thermostability is a fundamental characteristic of enzymes that is of high importance for industrial implementation of enzymatic catalysis. Cellobiohydrolases are enzymes capable to hydrolyze the most abundant natural polysaccharide – cellulose. These enzymes are widely applied in industry for processing of cellulose containing materials. However, structural and functional engineering of cellobiohydrolases for improving their properties is a challenging task. In this study, the thermostability of Penicillium verruculosum Cel7A cellobiohydrolase was increased through rational design of substitutions with proline. The stabilizing substitution G415P resulted in 3.4-fold increase in half-life time at 60 °C compared to wild-type enzyme. Molecular dynamics simulations indicated a clear effect of the stabilizing substitution G415P and the destabilizing substitutions D62P, S191P, and S273P on the stability of the enzyme tertiary structure. The stabilizing substitution G415P decreased flexibility of the lateral sides of the enzyme active site tunnel, while the considered destabilizing substitutions increased their flexibility. Image 1 • Protein rational design was performed to improve the enzyme thermostability. • Rational design was based on the structure analysis and substitutions with proline. • 3.4-Fold improvement in half-life time at 60 °C was archived. • Molecular dynamics simulations revealed effects on the enzyme structure stability. • Stabilizing substitution G415P decreased flexibility of active site tunnel sides. [ABSTRACT FROM AUTHOR]

Saprobic soil fungi drive many important ecosystem processes, including decomposition, and many of their effects are related to growth rate and enzymatic ability. In mycology, there has long been the implicit assumption of a trade-off between growth and enzymatic investment, which we test here using a set of filamentous fungi from the same soil. For these fungi we measured growth rate (as colony radial extension) and enzymatic repertoire (activities of four enzymes: laccase, cellobiohydrolase, leucine aminopeptidase and acid phosphatase), and explored the interaction between the traits based on phylogenetically corrected methods. Our results support the existence of a trade-off, however only for the enzymes presumably representing a larger metabolic cost (laccase and cellobiohydrolase). Our study offers new insights into potential functional complementarity within the soil fungal community in ecosystem processes, and experimentally supports an enzymatic investment/growth rate trade-off underpinning phenomena including substrate succession. [ABSTRACT FROM AUTHOR]

A novel cellobiohydrolase (CBH)-generating fungi have been isolated and categorized as Schizophyllum commune KMJ820 based on morphology and rDNA gene sequence. Cellulose powder was used as carbon source, the total enzyme activity was 11.51 U/ml is noted; which is among the highest amounts of CBH-generating microbes studied. CBH have been purified to homogenize, with pursual of serial chromatography using S. commune supernatants and two different CBHs were found; CBH 1 and 2. The filtered CBHs showed greater activity (Vmax = 51.4 and 20.8 U/mg) in contrast to CBHs from earlier studies. The MW (molecular weights) of S. commune CBH 1 and 2 were verified to be approximately 50 kDa and 150 kDa, respectively, by size exclusion chromatography. Even though CBHs have been evaluated from other sources, but S. commune CBH is prominent in comparison to other CBHs by its high enzyme activity. [ABSTRACT FROM AUTHOR]

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks. Image 1 • Successful expression of seven secreted lignocellulolytic enzymes in second-generation industrial yeast strain. • Combined secreted activity of nearly 100 FPU/g CDW. • Direct fermentation of multiple lignocellulosic polymers into bioethanol. • No significant decline in glucose or xylose fermentation capacity. [ABSTRACT FROM AUTHOR]

Hydrothermal pre-treatments decrease lignocellulose recalcitrance against enzymatic hydrolysis by removing the majority of the hemicellulose, thus increasing cellulase accessibility. However, a small amount of the hemicellulose may remain and become adsorbed to the cellulose, leading to cellulase inhibition. Here, we produced hemicellulose bound cellulose, using glucuronoxylan and galactomannan, to simulate hydrothermally pre-treated hardwoods and softwoods, respectively, and evaluated how this can affect cellulose hydrolysis by Trichoderma reesei derived cellobiohydrolase I (Cel7A). Based on X-ray powder diffraction (XRD), histochemistry, scanning electron microscopy and Simon's staining, hemicellulose binding onto cellulose affected the physical properties of the biomass, which subsequently affected its hydrolysis rate. As a result of hemicellulose binding onto cellulose, the adsorption of Cel7A was significantly impacted (up to 45%), leading to lowered activities (a 40% reduction), especially for glucuronoxylan. The bound hemicellulose may be released from the cellulose during agitation and hydrolysis. We therefore evaluated the effect of free hemicellulose on Cel7A. Free xylan was more inhibitory to Cel7A than free mannan, demonstrating non-competitive inhibition, while mannan exhibited uncompetitive inhibition. The recalcitrant effect of both bound and free hemicellulose could be relieved by the addition of hemicellulolytic enzymes (i.e. XT6 and Man26A) during cellulose hydrolysis. During the degradation of cellulose in "realistic" woody biomasses by Cel7A, the addition of hemicellulases led to a significant improvement in cellulose hydrolysis. This study showed that hemicellulose remains a critical factor regarding biomass recalcitrance and that the addition of hemicellulolytic activities in commercial enzyme cocktails is required (especially the mannanolytic activities lacking from most commercial enzyme cocktails), in order to realise high sugar yields at low enzyme protein loadings for low-cost biofuel production. [ABSTRACT FROM AUTHOR]

Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (Af Cel6A). Structure-based variants of Af Cel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5–7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase Cel7A is an industrial important enzyme that breaks down cellulose by a complex processive mechanism. The enzyme threads the reducing end of a cellulose strand into its tunnel-shaped catalytic domain and progresses along the strand while sequentially releasing the disaccharide cellobiose. While some molecular details of this intricate process have emerged, general structure-function relationships for Cel7A remain poorly elucidated. One interesting aspect is the occurrence of particularly strong ligand interactions in the product binding site. In this work, we analyze these interactions in Cel7A from Trichoderma reesei with special emphasis on the Arg251 and Arg394 residues. We made extensive biochemical characterization of enzymes that were mutated in these two positions and showed that the arginine residues contributed strongly to product binding. Specifically, ∼50% of the total standard free energy of product binding could be ascribed to four hydrogen bonds to Arg251 and Arg394, which had previously been identified in crystal structures. Mutation of either Arg251 or Arg394 lowered production inhibition of Cel7A, but at the same time altered the enzyme product profile and resulted in ∼50% reduction in both processivity and hydrolytic activity. The position of the two arginine residues closely matches the two-fold screw axis symmetry of the substrate, and this energetically favors the productive enzyme-substrate complex. Our results indicate that the strong and specific ligand interactions of Arg251 and Arg394 provide a simple proofreading system that controls the step length during consecutive hydrolysis and minimizes dead time associated with transient, non-productive complexes. [ABSTRACT FROM AUTHOR]

Peatlands store a large proportion of global soil carbon in the form of peat because decomposition of plant organic matter is slow. In Sphagnum-dominated peatlands, dissolved organic matter (DOM) is traditionally considered an important inhibitor of hydrolytic enzymes due to the polyphenolic polymers it contains. Interestingly, the acidic character of the polymers in such DOM has never been tested for its enzyme-inhibitory properties. We raised two principal hypotheses: (1) not only the polyphenolic but also the acidic character of DOM inhibits the activity of extracellular enzymes in Sphagnum-dominated peatlands; (2) environmental, peat-extracted enzymes will show adaptation to their environment. We tested the inhibition of commercial acid phosphatase and cellobiohydrolase, and environmental phosphatase and β-glucosidase by following dissolved substances: (1) polyphenol-free polycarboxylates from Sphagnum cell walls, i.e. sphagnan, (2) environmental DOM (peat-DOM) containing polymers of polyphenolic-polycarboxylate nature, (3) tannic acid (carboxyl-free polyphenolic oligomer) and (4) monomeric phenolic acids. Sphagnan strongly inhibited commercial acid phosphatase, to a similar extent as peat-DOM and more strongly than tannic acid and a polycarboxylate from another moss (Leucobryum glaucum). Monomeric phenolic acids were weak inhibitors. Commercial cellobiohydrolase was only partially inhibited by sphagnan or peat-DOM. Environmental phosphatase and β-glucosidase were consistently slightly inhibited by tannic acid, but not by sphagnan or peat-DOM. Inhibition of commercial phosphatase by sphagnan and peat-DOM was counteracted by a polycation chitosan, indicating the electrostatic nature of carboxylate-mediated inhibition. Our results question the polyphenol-mediated enzyme inhibition in Sphagnum-dominated peatlands as (1) the DOM had a strong inhibitory potential due to its polycarboxylates; nevertheless, (2) the peat microbial communities exhibited enzyme resistance to both polyphenol and polycarboxylate polymers in peat-DOM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

• Mutant C6 of Cel12A obtained after four rounds of ep PCR showed nearly 5-fold increase in activity. • The mutation E57 K in C6 produced conformational changes promoting binding affinity with the ligand and thus enhancing activity. • Analysis of the hydrolytic products showed that Cel12A should be a cellobiohydrolase instead of an endoglucanase as previously reported. Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7−, 5− and 4.8−fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported. [ABSTRACT FROM AUTHOR]

The catalytic domain (residues 128–449) of the Orpinomyces sp. Y102 CelC7 enzyme (Orp CelC7) exhibits cellobiohydrolase and cellotriohydrolase activities. Crystal structures of Orp CelC7 and its cellobiose‐bound complex have been solved at resolutions of 1.80 and 2.78 Å, respectively. Cellobiose occupies subsites +1 and +2 within the active site of Orp CelC7 and forms hydrogen bonds to two key residues: Asp248 and Asp409. Furthermore, its substrate‐binding sites have both tunnel‐like and open‐cleft conformations, suggesting that the glycoside hydrolase family 6 (GH6) Orp CelC7 enzyme may perform enzymatic hydrolysis in the same way as endoglucanases and cellobiohydrolases. LC‐MS/MS analysis revealed cellobiose (major) and cellotriose (minor) to be the respective products of endo and exo activity of the GH6 Orp CelC7. [ABSTRACT FROM AUTHOR]

A cellobiohydrolase gene was cloned and analyzed from Auricularia heimuer, and named A-cbh. The cDNA sequence was submitted to GenBank under the accession number MK620906. Sequence analysis showed that the cDNA comprised 1551 bp and encoded a putative polypeptide of 516 amino acids. The molecular weight of this protein was predicted as 54.44 kDa, and the theoretical isoelectric point was 5.24. The recombinant prokaryotic expression vector pET32a-A-cbh was constructed and transformed into Escherichia coli BL21. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated that the recombinant protein had molecular size between 66.2 kDa and 94.0 kDa, which was consistent with our expectations because a tag protein of approximately 20 kDa was present in the vector of pET32a. The transcription levels of A-cbh in the mycelium were lower than those in the fruiting bodies. The highest level of A-cbh expression was observed in the primordium, which was 8.65-fold higher than that in the control (mycelia at 6 days). We speculated that A-cbh might be involved in the formation of primordia and the development of fruiting bodies. The results of the present study will form the basis for further research into the function of the cellobiohydrolase in A. heimuer. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila , denoted Mt LPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since Mt LPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that Mt LPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used Mt LPMO9L and CBHI. Changing the incubation time and concentration ratio of Mt LPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between Mt LPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration. [ABSTRACT FROM AUTHOR]

An experiment was conducted to determine the effects of recombinant cellobiohydrolase on the hydrolysis and in vitro rumen microbial fermentation of agricultural straws including rice straw, wheat straw, and corn straw. The cellobiohydrolase from Lentinula edodes (LeCel7A) was produced in Pichia pastoris. The optimal temperature and pH for LeCel7A were 60 °C and 5.0, respectively. The recombinant protein enhanced the hydrolysis of three straws. During in vitro rumen fermentation of three straws, the fiber digestibility, concentration of acetate and total volatile fatty acids, and fermentation liquid microbial protein were increased by LeCel7A. High throughput sequencing and real-time PCR data showed that the effects of LeCel7A on ruminal microbial community depended on the fermentation substrates. The relative abundances of Prevotellaceae_UCG_003 and Saccharofermentans were increased by LeCel7A regardless of agricultural straws. With rice straw, LeCel7A increased the relative abundances of Desulfovibrio , Ruminococcaceae and its some genus. With wheat straw, LeCel7A increased the relative abundances of Succiniclasticum , Ruminococcus flavefaciens , and Ruminococcus albus. With corn straw, Succiniclasticum , Christensenellaceae_R_7_group and Desulfovibrio were increased by LeCel7A. This study demonstrates that LeCel7A could enhance the hydrolysis and in vitro ruminal fermentation of agricultural straws, showing the potential of LeCel7A for improving the utilization of agricultural straws in ruminants. [ABSTRACT FROM AUTHOR]

Summary: Plant‐tissue‐colonizing fungi fine‐tune the deconstruction of plant‐cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant‐tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131‐coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131‐family expansions and horizontal‐gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant‐tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic‐plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β‐1,4, β‐1,3 and mixed β‐1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes. [ABSTRACT FROM AUTHOR]

Abstract A low-level secretion of hydrolase and low conversion efficiency represent two major challenges in production of second-generation bioethanol. In the study, the co-cultivation system of Penicillium oxalicum 16 and Trichoderma reesei RUT-C30 optimized by response surface methodology under solid state fermentation produced 38.0 IU/gds, 352.9 IU/gds, 713.2 IU/gds, 15.7 IU/gds and 188.6 IU/gds for FPase, xylanase, amylase, cellobiohydrolase and β-1, 4-glucosidase, respectively, which was corresponding to 4.2 fold, 2.9 fold, 2.03 fold, 1.08 fold and 1.96 fold higher than that without optimization. Moreover, unpretreated wheat bran which was hydrolyzed by the optimized co-cultivation system and fermented by Saccharomyces cerevisiae UV-20 was converted into 26.8 g/L ethanol corresponding to 98.41% of the conversion rate, and produced much more ethanol than milled rice straw. The study provided a feasible method which can enhance hydrolase yield and very efficiently produce second-generation bioethanol. Highlights • PO16 and RUT-C30 were co-cultured for the first time under SSF. • Co-48 optimized by RSM generated a significant enhancement of the hydrolase yield. • Unpretreated WB was completely hydrolyzed by the optimized Co-48. • Unpretreated WB was almost completely turned into ethanol. [ABSTRACT FROM AUTHOR]

Abstract Roots increase microbial activities depending on exudate composition, especially on the ratios of sugars, carboxylic and amino acids, and thus structure enzyme activities in the rhizosphere. We introduce a new approach combining soil zymography and simulated exudates released from Rhizon® samplers to stimulate microbial activities but avoid the direct release of enzymes by living roots. This enabled visualizing, localizing and analyzing the effects of simulated root exudates on activity of five microbial enzymes involved in carbon (C) (β-glucosidase, cellobiohydrolase), nitrogen (N) (leucine aminopeptidase), phosphorus (P) (phosphatase) and sulfur (S) (sulfatase) cycles. We tested the hypotheses that 1) artificial exudates stimulate microorganisms for enzyme production and form spatial gradients around roots, and 2) the extent of microbial enzyme activities in the rhizosphere is component-specific. In line with these hypotheses, the activities of P-, N- and S-related enzymes were higher near the artificial root and gradually decreased as a function of distance from the root. The pattern for C-cycle enzymes was uniform and independent of the exudate composition. Among all components, alanine increased the rhizosphere extent much stronger than other substances, while methionine had no effect on the spatial distribution of enzyme activities. V max of all enzymes increased with alanine addition, but decreased after adding citrate. The ratios of enzyme activities demonstrated that rhizosphere microorganisms release more leucine aminopeptidase than other enzymes to meet their N demand. Glucose increased the K m of cellobiohydrolase and β-glucosidase, while alanine had the greatest effect on the K m of leucine aminopeptidase and sulfatase. Phosphatase is the enzyme most sensitive to the composition of root exudates; consequently, any factor influencing root exudate composition can strongly affect the P cycle. We conclude that the rhizosphere extent of microbial-derived enzyme activities is component- and enzyme-specific and that this extent depends on the substrate stoichiometry and microbial nutrient demand. Graphical abstract Image 1 Highlights • We introduce a new approach to avoid direct release of enzymes by roots. • Distributions of microbial enzyme activities were enzyme- and component-specific. • Alanine increased the rhizosphere extent stronger than other substances. • Phosphatase had the most sensitive enzyme system to exudate composition. [ABSTRACT FROM AUTHOR]

Abstract Earthworms in interaction with soil microorganisms play a key role in litter decomposition. Moreover, as soil engineers, earthworms modify microbial communities and their enzymatic activities. Most studies focusing on earthworms and soil enzymatic activities compare distinct ecological categories of earthworms whereas their contributions and interactions within a given ecological category remain largely unknown. In this context, the aims of the present study were to determine and compare the contribution of (1) three strict-anecic earthworm species, (2) three epi-anecic earthworm species and (3) the pairwise interactions between these different species on Lolium perenne leaf litter decomposition and soil microbial activity. After 30 days of incubation, the surface litter mass loss and five soil enzymatic activities (FDAse, β-D-glucosidase, cellobiohydrolase, leucine amino-peptidase and acid phosphatase) were measured in both earthworm burrows and middens. In mono-specific assemblages, leaf litter mass loss and enzymatic activities were significantly higher in the presence of epi-anecic compared to strict-anecic species, whatever the species identity. These differences were higher for the β-D-glucosidase, leucine amino-peptidase and FDAse (+78%, +57% and +34%, respectively). Earthworm species interactions at both intra- and inter-ecological sub-categories did not enhance either leaf litter mass loss or enzymatic activities. Interestingly, FDAse activity was higher in earthworm burrows whereas acid phosphatase activity was higher in earthworm middens. These results indicate that the two anecic ecological sub-categories have different impacts on soil functioning and each of them regroups earthworm species with similar behaviour. This functional distinction highlights the key role of epi-anecic earthworms in fresh surface litter burial and decomposition, featuring their importance on nutrient cycling in soil and for microbial activities stimulation through resource availability. Highlights • Epi-anecic earthworms enhanced surface litter mass loss and soil enzymatic activities. • Strict-anecic earthworms only enhanced β-D-glucosidase activity. • Stimulation intensity depended on both the soil microsites and the enzymatic activity considered. • Earthworm species interactions did not enhance litter mass loss and soil enzymatic activities. [ABSTRACT FROM AUTHOR]

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