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Lignin can affect the enzymatic hydrolysis efficiency of lignocellulose. In this study, the lignin isolated from sugarcane bagasse (SCB) pretreated with p-toluenesulfonic acid (PL) was firstly aminated, and then the effects of PL and aminated PL (APL) on the bagasse enzymatic hydrolysis efficiency (EHE) were investigated. The results showed that the addition of PL and APL promoted the EHE, and EHE with APL (73.82 %) was higher than PL (51.39 %). To explore the reason, the data were further analyzed including cellulase adsorption capacity, enzyme activity, cellulase-lignin interaction, and molecular docking. It was found that APL adsorbed more cellulase (27.83 mg protein/g lignin) than PL (4.96 mg protein/g lignin), resulting from the greater interaction force and lower binding free energy between APL and cellulase. The addition of APL more remarkably enhanced the cellobiohydrolase and endoglucanase activities than PL due to more effectively inducing cellulase conformation optimization. [Display omitted] • Enzymatic hydrolysis of cellulosic substrate was enhanced from 45.83 % to 73.82 % by aminated lignin. • Aminated lignin adsorbed more cellulase molecules. • Activities of cellobiohydrolase (CBH) and endoglucosidase (EG) were promoted by aminated lignin. • Aminated lignin owned lower binding energy with cellulase. [ABSTRACT FROM AUTHOR]

The production of cellulolytic enzymes in response to inducible carbon sources is mainly regulated at the transcriptional level in filamentous fungi. We have identified a cellobiose-response regulator (ClbR) controlling the expression of cellulolytic enzyme-encoding genes in Aspergillus aculeatus. However, the engineering potential of combining the deletion of transcriptional repressors with the overexpression of transcriptional activators to enhance enzyme production has not been analyzed. Here, we investigated the effect of the deletion of the transcriptional repressor creA and the overexpression of the transcriptional activator clbR in enzyme production in A. aculeatus. Here, we verified that a combination of creA deletion and clbR overexpression (Δc&OE) improved cellulase, β-1,4-xylanase, and β-glucosidase production. Cellulase and β-1,4-xylanase production increased 3.4- and 8.0-fold in Δc&OE compared with the host strain (MR12) at 96-h incubation, respectively. β-Glucosidase production in Δ creA and Δc&OE increased approximately 5.0-fold compared with that in MR12 at 240-h incubation. Transcriptional analysis revealed that the increase in enzyme production was due to increased expression of cellobiohydrolase, endo-β-1,4-glucanase, β-1,4-xylanase, and β-glucosidase 1 (bgl1). Interestingly, bgl1 expression in Δ creA increased in a dose-dependent manner in response to glucose. Thus, combinational manipulation of transcription factors improved cellulase, xylanase, and β-glucosidase production in A. aculeatus. [ABSTRACT FROM AUTHOR]

Methods of the pretreatment of reeds (grinding, hydrothermal treatment, treatment with acid or alkali solutions, organosolv, deep eutectic solvents) and their effect on the subsequent enzymatic hydrolysis by cellulase and hemicellulase complexes have been studied. Substrates with the highest reactivity were obtained by exposing the reeds to a weakly alkaline deep eutectic solvent (DES) and an alkali solution. The depth of enzymatic hydrolysis of these pretreated substrates was 63 and 59%, and the degree of conversion of polysaccharides (cellulose and hemicellulose) into monosaccharides taking into account the yield of the substrate at the preprocessing stage was 60 and 34%, respectively. After pretreatment of the reeds with acid DES, water steam, water, or a water/organic solution of sulfuric acid, the depth of enzymatic hydrolysis was 45, 25, 20, and 11% and the degree of polysaccharide conversion was 26, 18, 13, and 10%, respectively. The industrial enzyme preparation Agrocell Plus with a predominant content of cellobiohydrolases and endoglucanases was most effective in hydrolyzing the dust fraction of the reed, as was the reed pretreated with a solution of sulfuric acid or DES (acidic or alkaline). The industrial enzyme preparation Agroxil Plus, containing endoxylanase and cellobiohydrolases, was more effective in hydrolyzing the reed after hydrothermal pretreatment or alkali solution. The results of the hydrolysis of the reed pretreated with acidic or weakly alkaline DES under the action of individual (homogeneous) cellulases indicate that in both cases the key enzyme was cellobiohydrolase 1. [ABSTRACT FROM AUTHOR]

The objective of this study was to assess the cellulase production of four fungi: Aspergillus terreus NIH2624, Aspergillus clavatus NRRL1, Aspergillus versicolor CBS583.65 and Aspergillus phoenicis ATCC3157, under submerged cultivation conditions. When these fungi were cultured in shake flasks using Mandels and Weber's minimal medium with 1% sugarcane bagasse as a carbon source and 1.8 g/L of rice bran extract as a nitrogen source, A. terreus showed maximum cellulase production (filter paper activity (FPase) 3.35 U/mL; carboxymethyl cellulase activity (CMCase) 1.69 U/mL). Consequently, A. terreus was selected for the optimization study for cellulase production. Among the different tested carbon sources, A. terreus showed higher CMCase activity when it was cultivated on delignified sugarcane bagasse (1.64 U/mL) and higher FPase activity on sugarcane straw (7.95 U/mL). Regarding the nitrogen sources, the maximum FPase activity was observed when using rice bran (FPase, 8.90 U/mL) and soybean meal (FPase, 9.63 U/mL). The optimized fermentation medium (minimal medium with delignified sugarcane bagasse and rice bran as carbon and nitrogen sources, respectively) resulted in an enzymatic cocktail mainly composed of xylanases, with a maximum activity of 1701.85 U/mL for beechwood xylan, 77.12 U/mL for endoglucanase and 21.02 U/mL for cellobiohydrolase. Additionally, the enzymatic cocktail showed efficient activities for β-glucosidase, β-xylanase, arabinofuranosidase and lytic polysaccharide monoxygenases (LPMOs). This cellulase enzyme solution has the potential to efficiently hydrolyze lignocellulosic biomass, producing second-generation sugars in biorefineries. [ABSTRACT FROM AUTHOR]

The escalating prevalence of drug-resistant pathogens not only jeopardizes the effectiveness of existing treatments but also increases the complexity and severity of infectious diseases. Escherichia coli is one the most common pathogens across all healthcare-associated infections. Enzymatic treatment of bacterial biofilms, targeting extracellular polymeric substances (EPS), can be used for EPS degradation and consequent increase in susceptibility of pathogenic bacteria to antibiotics. Here, we characterized three recombinant cellulases from Thermothelomyces thermophilus: a cellobiohydrolase I (TthCel7A), an endoglucanase (TthCel7B), and a cellobiohydrolase II (TthCel6A) as tools for hydrolysis of E. coli and Gluconacetobacter hansenii biofilms. Using a design mixture approach, we optimized the composition of cellulases, enhancing their synergistic activity to degrade the biofilms and significantly reducing the enzymatic dosage. In line with the crystalline and ordered structure of bacterial cellulose, the mixture of exo-glucanases (0.5 TthCel7A:0.5 TthCel6A) is effective in the hydrolysis of G. hansenii biofilm. Meanwhile, a mixture of exo- and endo-glucanases is required for the eradication of E. coli 042 and clinical E. coli biofilms with significantly different proportions of the enzymes (0.56 TthCel7B:0.44 TthCel6A and 0.6 TthCel7A:0.4 TthCel7B, respectively). X-ray diffraction pattern and crystallinity index of E. coli cellulose are comparable to those of carboxymethyl cellulose (CMC) substrate. Our results illustrate the complexity of E. coli biofilms and show that successful hydrolysis is achieved by a specific combination of cellulases, with consistent recurrence of TthCel7B endoglucanase. [ABSTRACT FROM AUTHOR]

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus , and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5–6.0 (CIB12) and 4.5–5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma. [ABSTRACT FROM AUTHOR]

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry. [ABSTRACT FROM AUTHOR]

Lignocellulose was directly used in itaconic acid production by a model filamentous fungus Neurospora crassa. The promoters of two clock control genes and cellobiohydrolase 1 gene were selected for heterologous genes expression by evaluating different types of promoters. The effect of overexpression of different cellulase was compared, and it was found that expression of cellobiohydrolase 2 from Trichoderma reesei increased the filter paper activity by 2 times, the cellobiohydrolase activity by 4.5 times, and that the itaconic acid titer was also significantly improved. A bidirectional cis-aconitic acid accumulation strategy was established by constructing the reverse glyoxylate shunt and expressing the transporter MTTA, which increased itaconic acid production to 637.2 mg/L. The simultaneous optimization of cellulase and metabolic pathway was more conducive to the improvement of cellulase activity than that of cellulase alone, so as to further increase itaconic acid production. Finally, through the combination of fermentation by optimized strains and medium optimization, the titers of itaconic acid using Avicel and corn stover as substrate were 1165.1 mg/L and 871.3 mg/L, respectively. The results prove the potential of the consolidated bioprocessing that directly converts lignocellulose to itaconic acid by a model cellulase synthesizing strain. [ABSTRACT FROM AUTHOR]

Background: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. Results: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in β -glucosidase activity compared to batch processes using the same type and amount of carbon sources. Conclusion: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology. [ABSTRACT FROM AUTHOR]

Forest productivity usually declines with the increase in the age of stand, which may be associated with the disturbance in the balance of nitrogen (N) and phosphorus (P) nutrients. However, the effects of stand age on plantation nutrient balance and productivity in plantation is still unclear. Therefore, an experiment was conducted in this study to investigate the influence of the increase in the age of stand (6‐, 10‐, 15‐, 25‐, 30‐, and 34‐year‐old stands) on plant growth and the dynamics of N and P in Castanopsis hystrix plantations of Southern China. The increase in the age of stand reduced plantation productivity and leaf N:P. Moreover, productivity was found to be positively correlated with leaf N:P, reflecting that the decrease in productivity was related to changes in N and P contents in leaves. Reduction in leaf N:P was associated with declining N content and stable P content in leaves. The decrease in leaf N content may be due to the declining NO3−–N and microbial biomass N contents in soil, although activities of N‐acetylglucosaminidase, cellobiohydrolase, and polyphenol oxidase decreased with the increase in stand age. The stability of leaf P content may be associated with the increase in soil total P and microbial biomass P, boosting P supply to plants. In contrast, decreased soil phosphatase activity with the increase in the age of stand likely suggests that no additional P was needed through P mineralization. The findings indicate that increasing stand age would improve N consumption but alleviate relative P limitations. Thus, more attention needs to be paid to N fertilizer management (e.g., 10–15‐year stages) to sustain C. hystrix productivity with stand age. [ABSTRACT FROM AUTHOR]

Biochar (BC) was used in Trichoderma sp. culture to produce high-activity cellulase on a laboratory scale. The biochar was added into the flask before being applied to the fermenter to identify the enhancement effect and to determine the best amount of addition and the most suitable incubation period. Cellulase production was performed with a working volume of 4 L, and enzymatic hydrolysis was conducted to evaluate the saccharification ability of the enzyme. During incubation, the activities of three enzymes (Endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH)) were measured for three days, and the cellulase activity was determined using a filter paper unit (FPU). In flask scale, EG, BGL, and CBH activities were increased by 1.4, 2.1, and 1.8 folds, respectively, and the incubation period was shortened by adding BC. In the fermenter scale, EG, BGL, and CBH activities were noticeably enhanced by 12.1, 5.8, and 7.2 folds, respectively, and FPU was 42.1 (9.8 folds). Additionally, the conversion rates of cellulose and steam exploded softwood and hardwood were 109.4%, 75.4%, and 87.3%, which were similar to a commercial enzyme (Cellic CTec II). This study demonstrated that biochar could be used to produce high-activity cellulase in a shorter period and suggests a novel method for effective cellulase production. [ABSTRACT FROM AUTHOR]

The necrotrophic fungus, Sclerotinia sclerotiorum, employs an array of cell wall-degrading enzymes (CWDEs), including cellulase, to dismantle host cell walls. However, the molecular mechanisms through which S. sclerotiorum degrades cellulose remain elusive. Here, we unveil a novel secretory cellobiohydrolase, SsdchA, characterized by a signal peptide and a Glyco_hydro_7 (GH7) domain. SsdchA exhibits a robust expression of during early infection stages. Interestingly, colony morphology and growth rates remain unaffected across the wild-type, SsdchA deletion strains and SsdchA overexpression strains on potato dextrose agar (PDA) medium. Nevertheless, the pathogenicity and cellobiohydrolase activity decreased in the SsdchA deletion strains, but enhanced in the SsdchA overexpression strains. Moreover, the heterologous expression of SsdchA in Arabidopsis thaliana leads to reduced cellulose content and heightened susceptibility to S. sclerotiorum. Collectively, our data underscore the pivotal role of the novel cellobiohydrolase SsdchA in the pathogenicity of S. sclerotiorum. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 6 cellobiohydrolase (GH6 CBH) is a group of cellulases capable of hydrolyzing crystalline cellulose. However, the synergistic reaction of GH6 CBH with other cellulases is hindered by its relatively low thermotolerance. We previously obtained a thermotolerant double mutant, C240S/C393S, of GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCelôA) by replacing the two free cysteine (Cys) residues, C240 and C393, with serine (Yamaguchi et al., J Appl Glycosci. 2020; 67;79-86). In the accompanying paper (Part I; Yamaguchi et al., J Appl Glycosci. 2024; 71: 55-62), we measured the temperature dependence of the activity and folding of C240S/C393S and its single mutants, C240S and C393S, and found that replacement of C393 was the major contributor to the increased thermotolerance of C240S/C393S. Here, in order to investigate the mechanism involved, we crystallized the wild-type and the mutant enzymes and compared their X-ray crystal structures. The overall structures of the wild-type and the three mutant enzymes were similar. However, C240S/C393S had the lowest relative B-factor at both the N-terminal loop (residues 172-177) and the C-terminal loop (residues 390-425). This result suggests that reduced structural fluctuation of the substrate-enclosing loops, possibly due to stronger hydrogen bonding involving C393, could account for the increased thermotolerance of C240S/C393S. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase (CBH), belonging to glycoside hydrolase family 6 (GH6), plays an essential role in cellulose saccharification, but its low thermotolerance presents a challenge in improving the reaction efficiency. Based on a report that chimeric CBH II (GH6) engineered to remove non-disulfide-bonded free Cys shows increased thermotolerance, we previously mutated the two free Cys residues to Ser in GH6 CBH from the basidiomycete Phanerochaete chrysosporium (PcCelöA) and obtained a thermotolerant double mutant, C240S/C393S (Yamaguchi et al., J. Appl. Glycosci. 2020; 67: 79~86). Here, characterization of the double mutant revealed that its activity towards both amorphous and crystalline cellulose was higher than that of the wild-type enzyme at elevated temperature, suggesting that the catalytic domain is the major contributor to the increased thermotolerance. To investigate the role of each free Cys residue, we prepared both single mutants, C240S and C393S, of the catalytic domain of PcCelôA and examined their residual activity at high temperature and the temperature-dependent changes of folding by means of circular dichroism measurements and thermal shift assay. The results indicate that the C393S mutation is the main contributor to both the increased thermotolerance of C240S/C393S and the increased activity of the catalytic domain at high temperature. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo‐acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60–80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non‐reducing end of the cellulose‐binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass holds promise as a renewable feedstock for various applications, but its efficient conversion requires cost-effective degradation strategies. The main objective of this study was to investigate the effect of the growth conditions of Cellulomonas phragmiteti in the production of (hemi)cellulosic supernatants. To meet this aim, different lignocellulosic residues were used as carbon sources for growth using defined mineral or nutritive culture media. Cell-free culture supernatants with xylanolytic activity were produced in all the conditions evaluated, but the highest xylanase activity (15.3 U/mL) was achieved in Luria–Bertani (LB) medium containing 1% waste paper. Under these conditions, almost negligible β-glucosidase, cellobiohydrolase, β-xylosidase, and α-arabinofuranosidase activity was detected. The xylanolytic supernatant showed tolerance to salt and displayed maximal catalytic efficiency at pH 6 and 45 °C, along with good activity in the ranges of 45–55 °C and pH 5–8. As it showed good stability at 45 °C, the supernatant was employed for the hydrolysis of birchwood xylan (50 g/L) under optimal conditions, releasing 10.7 g/L xylose in 72 h. Thus, C. phragmiteti was found to produce a xylanolytic enzymatic supernatant efficiently by utilizing the cheap and abundant lignocellulosic residue of waste paper, and the produced supernatant has promising attributes for industrial applications. [ABSTRACT FROM AUTHOR]

Cellulose, a sustainable source of fermentable glucose and nanomaterials, exists as highly-ordered, solvent-inaccessible fibrils held by intra- and intermolecular hydrogen bonds. Cellulose hydrolysis rates and extents are limited by the availability and accessibility of productive cellulase binding sites at the water–cellulose interface. There is a need to understand how spatial heterogeneity of celluloses impacts hydrolysis kinetics. We report a real-time, in situ infrared spectromicroscopy study of enzymatic cellulose hydrolysis in buffer with micrometer-scale spatial mapping. Algal cellulose depletion by a purified cellobiohydrolase Cel7A was tracked as time-resolved decreases in the absorption peak intensities of the glycosidic bond (1161 cm−1), C2–O2 (1112 cm−1), C3–O3 (1059 cm−1), and C6–O6 rotamers (1034 cm−1, 1013 cm−1, and 997 cm−1). Depletion kinetics varied spatially, with peak intensities decreasing to zero in some areas and plateauing in others. Hydration impacted cellulose ordering where C6–O6 and C3–O3 peaks were sharp, narrow, and centered at higher frequencies when hydrated, but broader, less well-defined and centered at lower frequencies when dried. Temporal trends of the Lateral Order Index (LOI), Total Crystallinity Index (TCI), and Hydrogen Bonding Index (HBI) indicated that hydrolysis by Cel7A preferentially removed less extensively hydrogen bonded cellulose, without significantly affecting the overall crystallinity of highly ordered cellulose. [ABSTRACT FROM AUTHOR]

It has been demonstrated that LPMO reactions can be driven by light, using the photosynthetic pigment chlorophyllin to achieve efficient oxidative degradation of cellulose. However, the effect of chlorophyllin on cellulases remains unclear. This study discovered that chlorophyllin does not affect the hydrolytic activity of cellulases under dark conditions. However, under light exposure, chlorophyllin-derived reactive oxygen species (ROS) exhibit a strong inhibitory effect on cellulases. These ROS primarily inhibit the hydrolytic action of endoglucanase II (Cel5A) and cellobiohydrolase II (Cel6A), while the action of cellobiohydrolase I and β-glucosidase remains unaffected. Scavenger studies revealed that singlet oxygen (1O₂) is the key inhibitory ROS responsible for the inhibition of Cel5A and Cel6A. The removal of 1O₂ by sodium azide effectively mitigates this inhibition, increasing the conversion yield of cellulose to glucose by 25.9 % when using the light-driven LPMO system in conjunction with cellulases. This study provides new insights into the role of chlorophyllin-derived 1O₂ in hindering hydrolytic action of cellulases and demonstrates the successful mitigation of this inhibition by sodium azide, thereby enhancing the cooperative degradation of cellulose to glucose by the light-driven LPMO system and cellulases. • The effect of chlorophyllin on the hydrolytic action of cellulases was studied. • Chlorophyllin-derived ROS inhibited the activities of Cel5A and Cel6A. • 1O₂ was identified as the primary inhibitory ROS for Cel5A and Cel6A. • The inhibition was effectively mitigated by the removal of 1O₂ using sodium azide. [ABSTRACT FROM AUTHOR]

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (Le GH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7–5 and GH7–6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with β-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the Le GH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops. [Display omitted] [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis is important for lignocellulosic biomass conversion into fermentable sugars. However, the nonproductive adsorption of enzyme on lignin was major hinderance for the enzymatic hydrolysis efficiency. In this study, non-productive adsorption mechanism of cellulase component cellobiohydrolase (CBH) onto lignin was specific investigated. Research revealed that the adsorption behavior of CBH on eucalyptus alkali lignin (E uA) was affected by reaction conditions. As study on the adsorption kinetic, it was indicated that the adsorption cellulose binding domain (CBD) of CBH onto E uA well fitted with Langmuir adsorption model and pseudo second-order adsorption kinetics model. And the tyrosine site related to the adsorption of CBD onto lignin was proved by the fluorescence and UV spectra analysis. The results of this work provide a theoretical guidance to understanding the nonproductive adsorption mechanism and building method to reduce the adsorption of cellulase on the lignin. [Display omitted] • CBD was separated to study the inhibition mechanism of E uA on enzymatic hydrolysis. • The effect of reaction conditions on the adsorption of CBD-E uA was investigated. • The adsorption of CBD-E uA suited Langmuir and pseudo second-order kinetics model. • Both physical and chemical adsorption exist in adsorption of lignin with CBD. • The tyrosine site on CBD was adsorption site. [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]

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.)