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Glycoengineering ultimately allows control over glycosylation patterns to generate new glycoprotein variants with desired properties. A common challenge is glycan heterogeneity, which may affect protein function and limit the use of key techniques such as mass spectrometry. Moreover, heterologous protein expression can introduce nonnative glycan chains that may not fulfill the requirement for therapeutic proteins. One strategy to address these challenges is partial trimming or complete removal of glycan chains, which can be obtained through selective application of exoglycosidases. Here, we demonstrate an enzymatic O-deglycosylation toolbox of a GH92 α-1,2-mannosidase from Neobacillus novalis, a GH2 β-galactofuranosidase from Amesia atrobrunnea and the jack bean α-mannosidase. The extent of enzymatic O-deglycosylation was mapped against a full glycosyl linkage analysis of the O-glycosylated linker of cellobiohydrolase I from Trichoderma reesei (TrCel7A). Furthermore, the influence of deglycosylation on TrCel7A functionality was evaluated by kinetic characterization of native and O-deglycosylated forms of TrCel7A. This study expands structural knowledge on fungal O-glycosylation and presents a ready-to-use enzymatic approach for controlled O-glycan engineering in glycoproteins expressed in filamentous fungi.

Exoglucanase (CBH) is the rate limiting enzyme in the process of cellulose degradation. The carbohydrate binding module (CBM) can improve the accessibility of cellulase to substrate, thereby promoting the enzymatic hydrolysis of cellulase. In this study, the influence of CBM on the properties of GH6 exoglucanase from Chaetomium thermophilum (CtCBH) is systematically explored from three perspectives: the fusion of exogenous CBM, the exogenous CBM replacement of its own CBM, and the deletion of its own CBM. The parental and reconstructed CtCBH presented the same optimum pH (6.0) and temperature (60 °C) for maximum activity. Fusion of exogenous CBM increased the binding capacity of CtCBH to Avicel by 8% and 9%, respectively, but it had no significant effect on its catalytic activity. The exogenous CBM replacement of its own CBM resulted in a 12% reduction in the binding ability of CtCBH to Avicel, and a 26% reduction in the catalytic activity of Avicel. The deletion of its own CBM significantly reduced the binding ability of CtCBH to Avicel by approximately 53%, but its catalytic activity was not obviously reduced. These observations suggest that binding ability of CBM is not necessary for the catalysis of CtCBH.

This study presents a novel fully enzymatic process for the controlled depolymerisation of fungal and shrimp chitosan, and their subsequent use in the synthesis of lignin cross-linked chitosan (CTS) hydrogels. Cellobiosehydrolase (CBH) was used to depolymerize CTS resulting in decrease in average molecular weight (Mw) of shrimp CTS from 140 kDa and degree of deacetylation (DD %) from 91.3% to an average MW of 15 kDa and 16% DD. Similarly, fungal chitosan average molecular weight decreased from 92 kDa and the degree of deacetylation (DD) of 48.3% to 12 kDa and a DD of 13%. The depolymerized CTS were completely soluble in water and miscible with lignosulfonates without encountering the usual problem of formation of flocs. Introduction of laccase into a lignosulfonate-chitosan mixture resulted in the oxidation and generation of lignin reactive phenoxyl radicals that cross-linked with CTS-NH2 reactive groups resulting in the increase of Mw from 20 kDa to >500 kDa and viscosity from 20 mPa to >500 mPa. This resulted in the formation of stable lignin-cross-linked hydrogels with elongation at break of 111% and tensile strength of 7 mPa. The produced functional hydrogels have potential application in food and biomedical industries as e.g. as oxygen barriers in packaging or as functional wound dressing or tissue engineering platforms.

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.

Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

Mortierella alpina has gained remarkable interest due to its high capacity for arachidonic acid (AA) production and potential for eicosapentaenoic acid (EPA) production recently. However, the development of genetically modified strains is limited by lacking inducible promoters, which can express genes conditionally. Here the inducible promoter of cellobiohydrolase (Pcbh1) was utilized in M. alpina and the gene oPpFADS17 encoding ω-3 fatty acid desaturase was selected as the reporter gene. Under conditions with inducer, expression of this gene enables M. alpina to produce EPA at room temperature, while no EPA was detected without inducer. We then optimized the induction conditions. The results demonstrated that the optimal induction condition was broth medium with 1% avicel as the inducer and 5% glucose as extra carbon source and the transcription level of the reporter gene was increasing with the extension of induction time. Successful application of Pcbh1 in M. alpina would significantly contribute to the steerable system to construct engineered strains for industrial production of microbial oils. SIGNIFICANCE AND IMPACT OF THE STUDY: Mortierella alpina is a commercial strain for production of polyunsaturated fatty acids. Genetic engineering strategies based on M. alpina require the development of inducible promoters to regulate gene expression conditionally at specific times. However, available inducible promoters for M. alpina were limited. In this study, we explore the feasibility of inducible cbh1 promoter in M. alpina and determined the optimal induction condition, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of the reporter gene under the control of Pcbh1 showed that Pcbh1 is a strong inducible promoter for M. alpina.

In a previous study, we reported an alkaliphilic and thermostable endoglucanase (BsGH7-3) of glycoside hydrolase family 7 (GH7) from the hemibiotrophic plant pathogen Bipolaris sorokiniana. However, the catalytic efficiency of the enzyme was lower than for some other endoglucanases of the GH7 family reported in the literature. To engineer a more active enzyme, we identified conserved residues in the substrate-binding tunnel and on the surface of the protein that could play a role in charge-charge interaction and stabilize the structure. The mutants D257W and Q225H in the substrate-binding tunnel and Y222R and Q401N on the protein surface showed a 2-fold increase in specific activity and a 1.5-fold increase in turnover number and were active over a broader range of pH. The mutants also showed a higher tolerance to NaCl. The rational design of the BsGH7-3 mutants helped in increasing the catalytic efficiency of the thermostable enzyme and may be useful in combination with other cellulases like cellobiohydrolase and β-glucosidase towards complete saccharification of cellulose into glucose.

A metagenomic library of mangrove soil samples consisting of approximately 11,000 clones was constructed, and a rare bifunctional cellobiohydrolase/β-xylosidase Cbh2124 was identified by functional screening. Cbh2124 displayed the highest homology (56.43%) with a protein of the glycoside hydrolase 10 (GH10) family from Proteobacteria. Phylogenetic analysis confirmed that Cbh2124 belongs to the GH10 family. The recombinant enzyme showed a strong cellobiohydrolase activity and a relatively high β-xylosidase activity, and its catalytic efficiency to the cellobiose substrate was as high as 1.27 × 10

Soluble compounds produced during the enzymatic hydrolysis of lignocelluloses hampers cellulose conversion. Cellobiose and vanillin most severely inhibited the effect of cellobiohydrolase I. A concentration-dependent negative cooperative effect was found between cellobiose and vanillin. The combined inhibitory effect was about 83.5% of the cellobiose and 88.1% of the vanillin when their concentration was 20 mg/ml. However, the negative synergy could be eliminated by excessive enzyme loading. Differences in their binding sites on the catalytic domain of cellobiohydrolase I lead to negative synergistic inhibition, which should be considered in devising strategies to alleviate this effect. Combined β-glucosidase and PEG addition at an appropriate dose was feasible to balance cost and hydrolytic efficiency. To achieve efficient hydrolysis, especially at high solid concentrations, it is important to understand the synergistic inhibition between these inhibitors.

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 MtLPMO9L. 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 MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

Searching for viable strategies to accelerate the catalytic cycle of glycoside hydrolase family 7 (GH7) cellobiohydrolase I (CBHI)-the workhorse cellulose-degrading enzymes, we have performed a total of 12-μs molecular dynamics simulations on GH7 CBHI, which brought to light a new mechanism for cellobiose expulsion, coined "claw-arm" action. The loop flanking the product binding site plays the role of a flexible "arm" extending toward cellobiose, and residue Thr389 of this loop acts as a "claw" that captures cellobiose. Five mutations of residue Thr389 were considered to enhance the loop-cellobiose interaction. The lysine mutant was found to significantly accelerate cellobiose expulsion and facilitate polysaccharide-chain translocation. Lysine mutation of Thr393 in Talaromyces emersonii CBHI (TeCel7A) performed similarly. Lysine approaches the catalytic area and stabilizes the Michaelis complex, potentially affecting glycosylation, the rate-limiting step of the catalytic cycle. QM/MM calculations indicate that lysine replacement diminishes the barrier against proton transfer, the crucial step of glycosylation, by 2.3 kcal/mol. Experimental validation was performed using the full-length wild-type (WT) of TeCel7A and its mutants, recombinantly expressed in Pichia pastoris, to degrade the substrates. Compared with the WT, the lysine mutant revealed an associated higher enzymatic reaction rate. Furthermore, cellobiose yield was also increased by lysine mutation, indicating that dissociation of the enzyme from cellulose was accelerated, which largely stems from the enhanced flexibility of the "arm". The present work is envisioned to help design strategies for improving enzymatic activity, while decreasing enzyme cost.

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.

The gene lpmo1 encoding Penicillium verruculosum lytic polysaccharide monooxygenase (PvLPMO9A) was sequenced and homologously overexpressed in P. verruculosum B1-537 (ΔniaD) auxotrophic strain under the control of the cbh1 gene promoter in combination with either the cbh1 signal sequence (sCBH1-X series of samples) or the native lpmo1 signal sequence (sLPMO1-X series). Three enzyme samples of the sCBH1-X series were characterized by a lower overall content of cellobiohydrolases (CBHs: 26-45%) but slightly higher content of endoglucanases (EGs: 17-23%) relative to the reference B1-537 preparation (60% of CBHs and 14% of EGs), while the PvLPMO9A content in them made up 9-21% of the total secreted protein. The PvLPMO9A content in four enzyme preparations of the sLPMO1-X series was much higher (30-57%), however the portion of CBHs in most of them (except for sLPMO1-8) decreased even to a greater extent (to 21-42%) than in the samples of the sCBH1-X series. Two enzyme preparations (sCBH1-8 and sLPMO1-8), in which the content of cellulases was substantially retained and the portion of PvLPMO9A was 9-30%, demonstrated the increased yields of reducing sugars in 48-h saccharification of Avicel and milled aspen wood: 19-31 and 11-26%, respectively, compared to the reference cellulase cocktail.

Cellulases are glycosylated enzymes that have wide applications in fields like biofuels. It has been widely accepted that glycosylation of cellulases impact their performance. Trichoderma reesei is the most important cellulase-producer and cellobiohydrolase I (CBHI) is the most important cellulase from T. reesei. Therefore, the glycosylation of T. reesei CBHI has been a focus of research. However, investigations have been focused on N-glycosylation of three of the four potential glycosylation sites, as well as O-glycosylation on the linker region, while a full picture of glycosylation of T. reesei CBHI is still needed. In this work, with extensive mass spectrometric investigations on CBHI from two T. reesei strains grown under three conditions, several new discoveries were made: 1) N45 and N64 are N-glycosylated with high mannose type glycans; 2) the catalytic domain of CBHI is extensively O-glycosylated with hexoses and N-acetylhexosamines; 3) experimental evidence on the mannosylation of carbohydrate binding domain (other than the linker adjacent region) was found. With structural analysis, we found several glycosylation sites (such as T383, S8, and S46) are located at the openings of the substrate-binding tunnel, and potentially involve in the binding of cellulose. These investigations provide a full and comprehensive picture on the glycosylation of CBHI from T. reesei, which benefits the engineering of CBHI by raising potential sites for modification.

Fungus-derived GH-7 family cellobiohydrolase I (CBHI, EC 3.2.1.91) is one of the most important industrial enzymes for cellulosic biomass saccharification. Talaromyces cellulolyticus is well known as a mesophilic fungus producing a high amount of CBHI. Thermostability enhances the economic value of enzymes by making them more robust. However, CBHI has proven difficult to engineer, a fact that stems in part from its low expression in heterozygous hosts and its complex structure. Here, we report the successful improvement of the thermostability of CBHI from T. cellulolyticus using our homologous expression system and protein engineering method. We examined the key structures that seem to contribute to its thermostability using the 3D structural information of CBHI. Some parts of the structure of the Talaromyces emersonii CBHI were grafted into T. cellulolyticus CBHI and thermostable mutant CBHIs were constructed. The thermostability was primarily because of the improvement in the loop structures, and the positive effects of the mutations for thermostability were additive. By combing the mutations, the constructed thermophilic CBHI exhibits high hydrolytic activity toward crystalline cellulose with an optimum temperature at over 70°C. In addition, the strategy can be applied to the construction of the other thermostable CBHIs.

Background Cellulase is the one of the largest contributors to the high production costs of the lignocellulose-based biorefineries. As the most widely used cellulase producer, Trichoderma reesei has two weaknesses, deficiencies in β-glucosidase and cellobiohydrolase II. This work aimed at solving this problem by simultaneous enhancement of the beta–exo synergism and exo–exo synergism in T. reesei cellulase to increase the cellulose degrading capability, i.e. enhanced co-expression of the β-glucosidase gene the cellobiohydrolase II gene of T. reesei. Results Enhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 was found successful in overcoming the two weaknesses. Filter paper activities of T. reesei cellulase were greatly elevated, which were 7.21 ± 0.45 (E7, Aabgl1 and Trcbh2) and 7.69 ± 0.42 (F6, Anbgl1 and Trcbh2) FPIU/mL. They were much higher than that of the parental strain Rut-C30, 2.45 ± 0.36 FPIU/mL. Enzymatic hydrolysis yields were also improved, from 67.22 ± 1.61% by Rut-C30 cellulase to 87.98 ± 0.65% by E7 cellulase and 86.50 ± 1.01% by F6 cellulase. The substrate loading for 1 g glucose release from SECS were decreased, from 2.9637 g SECS using Rut-C30 cellulase to 2.0291 g SECS using E7 cellulase and 2.0573 g SECS using F6 cellulase. As a result, the efficiency of the process from SECS to glucose was substantially improved. Conclusions Enhanced co-expression of the β-glucosidase gene and the cellobiohydrolase II gene in T. reesei using the strong promoter Pcbh1 in T. reesei was proven triumphal in the simultaneous enhancement of the beta–exo synergism and exo–exo synergism in T. reesei cellulase. This strategy also improved the cellulase production, enzymatic hydrolysis yield and the efficiency of the process from SECS to glucose in the context of on-site cellulase production. This work is a commendable attempt in the cellulase composition optimization at the transcriptional level.

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 (IsPETase) 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 IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose–binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_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 IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.

Lytic polysaccharide monooxygenases (LPMOs) are known to act synergistically with glycoside hydrolases in industrial cellulolytic cocktails. However, a few studies have reported severe impeding effects of C1-oxidizing LPMOs on the activity of reducing-end cellobiohydrolases. The mechanism for this effect remains unknown, but it may have important implications as reducing-end cellobiohydrolases make up a significant part of such cocktails. To elucidate whether the impeding effect is general for different reducing-end cellobiohydrolases and study the underlying mechanism, we conducted a comparative biochemical investigation of the cooperation between a C1-oxidizing LPMO from Thielavia terrestris and three reducing-end cellobiohydrolases; Trichoderma reesei (TrCel7A), Thielavia terrestris (TtCel7A), and Myceliophthora heterothallica (MhCel7A). The enzymes were heterologously expressed in the same organism and thoroughly characterized biochemically. The data showed distinct differences in synergistic effects between the LPMO and the cellobiohydrolases; TrCel7A was severely impeded, TtCel7A was moderately impeded, while MhCel7A was slightly boosted by the LPMO. We investigated effects of C1-oxidations on cellulose chains on the activity of the cellobiohydrolases and found reduced activity against oxidized cellulose in steady-state and pre-steady-state experiments. The oxidations led to reduced maximal velocity of the cellobiohydrolases and reduced rates of substrate-complexation. The extend of these effects differed for the cellobiohydrolases and scaled with the extent of the impeding effect observed in the synergy experiments. Based on these results, we suggest that C1-oxidized chain ends are poor attack-sites for reducing-end cellobiohydrolases. The severity of the impeding effects varied considerably among the cellobiohydrolases, which may be relevant to consider for optimization of industrial cocktails.

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.

Soil enzymes mediate key processes and functions of the soils, such as organic matter decomposition and nutrient cycling in both natural and agricultural ecosystems. Here, we studied the activity of five extracellular soil enzymes involved in the C, N, and P-mineralizing process in both litter and surface soil layer of rainforest in the northwest region of the Colombian Amazon and the response of those soil enzymes to land use change. The experimental study design included six study sites for comparing long-term pasture systems to native forest and regeneration practices after pasture, within the main landscapes of the region, mountain and hill landscapes separately. Results showed considerable enzymatic activity in the litter layer of the forest, highlighting the vital role of this compartment in the nutrient cycling of low fertility soils from tropical regions. With the land use transition to pastures, changes in soil enzymatic activities were driven by the management of pastures, with SOC and N losses and reduced absolute activity of soil enzymes in long-term pastures under continuous grazing (25 years). However, the enzyme activities expressed per unit of SOC did not show changes in C and N-acquiring enzymes, suggesting a higher mineralization potential in pastures. Enzymatic stoichiometry analysis indicated a microbial P limitation that could lead to a high catabolic activity with a potential increase in the use of SOC by microbial communities in the search for P, thus affecting soil C sequestration, soil quality and the provision of soil-related ecosystem services.

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

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.

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.

Enzymes involved in β-glucan breakdown in plants include endoglucanases, exoglucanases and β-glucosidases. Glycoside hydrolase family 3 (GH3) exoglucanases from barley and maize and a few plant GH3 β-glucosidases have been characterized, but none from rice. A few of these enzymes have been expressed in recombinant yeast and plant systems, but bacterial expression of plant GH3 enzymes has not been successful. We expressed the rice GH3 exoglucanase OsExo2 in Escherichia coli as a thioredoxin fusion protein, while other active plant GH3 enzymes could not be produced in this system. The protein was purified over 2000-fold in three chromatographic steps. The enzyme hydrolyzed β-1,3- and β-1,4-linked oligosaccharides and polysaccharides, consistent with a role in cell wall remodeling. Of the oligosaccharides tested, it had highest catalytic efficiency toward laminaritriose, (apparent kcat/Km = 37.7 mM−1s−1). Among polysaccharides, OsExoII hydrolyzed barley mixed β-glucan and laminarin with similar efficiencies (apparent kcat/Km = 3.7 and 3.4 mL mg−1 s−1, respectively), but achieved its highest apparent kcat with lichenan (2.9 s−1). OsExoII was found to be stimulated by ethylene glycol, which increased the apparent kcat and decreased the Km and was transglycosylated. These results imply that E. coli expression may be successful for certain plant GH3 enzymes and OsExoII may be a useful enzyme for application to glycoside production.

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 (AfCel6A). Structure-based variants of AfCel6A 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.

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.

Glycoside hydrolase cellulase family 6 from Trichoderma reesei (TrCel6A) is an important cellobiohydrolase to hydrolyze cellooligosaccharide into cellobiose. The knowledge of enzymatic mechanisms is critical for improving the conversion efficiency of cellulose into ethanol or other chemicals. However, the process of product expulsion, a key component of enzymatic depolymerization, from TrCel6A has not yet been described in detail. Here, conventional molecular dynamics and steered molecular dynamics (SMD) were applied to study product expulsion from TrCel6A. Tyr103 may be a crucial residue in product expulsion given that it exhibits two different posthydrolytic conformations. In one conformation, Tyr103 rotates to open the −3 subsite. However, Tyr103 does not rotate in the other conformation. Three different routes for product expulsion were proposed on the basis of the two different conformations. The total energy barriers of the three routes were calculated through SMD simulations. The total energy barrier of product expulsion through Route 1, in which Tyr103 does not rotate, was 22.2 kcal·mol−1. The total energy barriers of product expulsion through Routes 2 and 3, in which Tyr103 rotates to open the −3 subsite, were 10.3 and 14.4 kcal·mol−1, respectively. Therefore, Routes 2 and 3 have lower energy barriers than Route 1, and Route 2 is the thermodynamically optimal route for product expulsion. Consequently, the rotation of Tyr103 may be crucial for product release from TrCel6A. Results of this work have potential applications in cellulase engineering.

Lignocellulose degradation is a challenging step for value added products and biofuels production. Cellulomonas fimi secretes complex mixtures of carbohydrate active enzymes (CAZymes) which synergistically degrade cellulose and hemicelluloses. Their characterization may provide new insights for enzymatic cocktails implementation. Bioinformatic analysis highlighted 1127 secreted proteins, constituting the in silico secretome, graphically represented in a 2DE map. According to Blast2GO functional annotation, many of these are involved in carbohydrates metabolism. In vivo secretomes were obtained, growing C. fimi on glucose, CMC or wheat straw for 24 h. Zymography revealed degradative activity on carbohydrates and proteomic analysis identified some CAZymes, only in secretomes obtained with CMC and wheat straw. An interaction between cellobiohydrolases is proposed as a strategy adopted by soluble multimodular cellulases. Such approach can be crucial for a better characterization and industrial exploitation of the synergism among C. fimi enzymes.

The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM , for cellotetraose at pH 5.0 and 50 °C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.

In this study, extended artificial scaffoldins possessing multiple cohesin modules were created in vivo by employing split-intein-mediated protein ligation. Artificial scaffoldins having one Clostridium thermocellum cohesin (Coht), one carbohydrate binding module (CBM) from Clostridium cellulolyticum scaffolding protein CipC, and one to five cohesins (Cohc) derived from CipC, were assembled. These scaffoldins were used to assemble cellulosomal enzyme complexes for investigating the interplay among endoglucanase, exoglucanase, and scaffoldin-borne CBM, on the hydrolysis of a model microcrystalline cellulose substrate, Avicel. The cellulosomal complexes were assembled in vitro by incubating recombinant C. thermocellum endoglucanase (At) and C. cellulolyticum exoglucanase (Ec), with the various artificial scaffoldins. Under a fixed total cellulase concentration, improved hydrolysis is noted by recruiting both Ec and At on the same scaffoldin, for all scaffoldins tested, compared with free cellulases. The improvement is more profound with scaffoldins having a higher Cohc/Coht ratio (i.e., increased Ec/At ratio). Furthermore, among scaffoldins having the same Cohc/Coht ratio, highest rates of Avicel hydrolysis are noted when Coht, and hence an endoglucanase, is situated next to the CBM and not flanked by Cohc. These results point to the importance of using scaffoldins with sufficiently high numbers of cohesin units to achieve an optimal exo-/endo-glucanase ratio to create efficient designer cellulosomes. Furthermore, intein-trans-splicing is proven here to be an effective method for assembling complex scaffoldins and more intricate cellulosomes.

Enzymatic hydrolysis is considered an efficient and environmental strategy for the degradation of organic waste materials. Compared to mesophilic cellulases, thermostable cellulases with considerable activity are more advantageous in waste paper hydrolysis, particularly in terms of their participation in synergistic action. In this study, the synergistic effect of two different types of thermostable Chaetomium thermophilum cellulases, the endoglucanase CTendo45 and the cellobiohydrolase CtCel6, on five common kinds of waste papers was investigated. CtCel6 significantly enhanced the bioconversion process, and CTendo45 synergistically increased the degradation, with a maximum degree of synergistic effect of 1.67 when the mass ratio of CTendo45/CtCel6 was 5:3. The synergistic degradation products of each paper material were also determined. Additionally, the activities of CTendo45 and CtCel6 were found to be insensitive to various metals at 2 mM and 10 mM ion concentrations. This study gives an initial insight into a satisfactory synergistic effect of C. thermophilum thermostable cellulases for the hydrolysis of different paper materials, which provides a potential combination of enzymes for industrial applications, including environmentally friendly waste management and cellulosic ethanol production.

Cellulolytic microorganisms play important roles in global carbon cycling and have evolved diverse strategies to digest cellulose. Some are 'generous,' releasing soluble sugars from cellulose extracellularly to feed both themselves and their neighbors. The gliding soil bacterium Cytophaga hutchinsonii exhibits a more 'selfish' strategy. It digests crystalline cellulose using cell-associated cellulases and releases little soluble sugar outside of the cell. The mechanism of C. hutchinsonii cellulose utilization is still poorly understood. In this review, we discuss novel aspects of the C. hutchinsonii cellulolytic system. Recently developed genetic manipulation tools allowed the identification of proteins involved in C. hutchinsonii cellulose utilization. These include periplasmic and cell-surface endoglucanases and novel cellulose-binding proteins. The recently discovered type IX secretion system is needed for cellulose utilization and appears to deliver some of the cellulolytic enzymes and other proteins to the cell surface. The requirement for periplasmic endoglucanases for cellulose utilization is unusual and suggests that cello-oligomers must be imported across the outer membrane before being further digested. Cellobiohydrolases or other predicted processive cellulases that play important roles in many other cellulolytic bacteria appear to be absent in C. hutchinsonii. Cells of C. hutchinsonii attach to and glide along cellulose fibers, which may allow them to find sites most amenable to attack. A model of C. hutchinsonii cellulose utilization summarizing recent progress is proposed.

Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far, the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide are valuable steps toward being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule's physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.

The crystal structure of the catalytic domain of a glycoside hydrolase family 6 cellobiohydrolase from the basidiomycete P. chrysosporium was solved in apo and cellobiose-liganded forms at 1.2 and 2.1 Å resolution, respectively., Cellobiohydrolases belonging to glycoside hydrolase family 6 (CBH II, Cel6A) play key roles in the hydrolysis of crystalline cellulose. CBH II from the white-rot fungus Phanerochaete chrysosporium (PcCel6A) consists of a catalytic domain (CD) and a carbohydrate-binding module connected by a linker peptide, like other known fungal cellobiohydrolases. In the present study, the CD of PcCel6A was crystallized without ligands, and p-nitrophenyl β-d-cellotrioside (pNPG3) was soaked into the crystals. The determined structures of the ligand-free and pNPG3-soaked crystals revealed that binding of cellobiose at substrate subsites +1 and +2 induces a conformational change of the N-terminal and C-terminal loops, switching the tunnel-shaped active site from the open to the closed form.

The saprophytic fungus Trichoderma reesei has long been used as a model to study microbial degradation of lignocellulosic biomass. The major cellulolytic enzymes of T. reesei are the cellobiohydrolases CBH1 and CBH2, which constitute more than 70% of total proteins secreted by the fungus. However, their physiological functions and effects on enzymatic hydrolysis of cellulose substrates are not sufficiently elucidated. Here, the cellobiohydrolase-encoding genes cbh1 and cbh2 were deleted, individually or combinatively, by using an auxotrophic marker-recycling technique in T. reesei. When cultured on media with different soluble carbon sources, all three deletion strains (Δcbh1, Δcbh2, and Δcbh1Δcbh2) exhibited no dramatic variation in morphological phenotypes, but their growth rates increased apparently when cultured on soluble cellulase-inducing carbon sources. In addition, Δcbh1 showed dramatically reduced growth and Δcbh1Δcbh2 could hardly grew on microcrystalline cellulose (MCC), whereas all strains grew equally on sodium carboxymethyl cellulose (CMC-Na), suggesting that the influence of the CBHs on growth was carbon source-dependent. Moreover, five representative cellulose substrates were used to analyse the influence of the absence of CBHs on saccharification efficiency. CBH1 deficiency significantly affected the enzymatic hydrolysis rates of various cellulose substrates, where acid pre-treated corn stover (PCS) was influenced the least. CBH2 deficiency reduced the hydrolysis of MCC, PCS, and acid pre-treated and delignified corncob but improved the hydrolysis ability of filter paper. These results demonstrate the specific contributions of CBHs to the hydrolysis of different types of biomass, which could facilitate the development of tailor-made strains with highly efficient hydrolysis enzymes for certain biomass types in the biofuel industry.

A new cellulase producer strain of Penicillium digitatum (RV 06) was previously obtained from rotten maize grains. This work aim was to optimize the production and characterize this microorganism produced cellulase. A CMCase maximum production (1.6 U/mL) was obtained in stationary liquid culture, with an initial pH of 5.0, at 25 °C, with 1% lactose as carbon source, and cultured for 5 days. The produced enzyme was purified by ammonium sulfate precipitation and exclusion chromatography. The purified enzyme optimal temperature and pH were 60 °C and 5.2, respectively. The experimental Tm of thermal inactivation was 63.68 °C, and full activity was recovered after incubation of 7 h at 50 °C. The purified 74 kDa CMCase presented KM for CMC of 11.2 mg/mL, Vmax of 0.13 μmol/min, kcat of 52 s−1, and kcat/KM of 4.7 (mg/mL)−1 s−1. The purified enzyme had a high specificity for CMC and p-nitrophenyl cellobioside and released glucose and cellobiose as final products of the CMC hydrolysis. The enzyme trypsin digestion produced peptides whose masses were obtained by MALDI-TOF/TOF mass spectrometry, which was also used to obtain two peptide sequences. These peptide sequences and the mass peak profile retrieved a CBHI within the annotated genome of P. digitatum PD1. Sequence alignments and phylogenetic analysis confirmed this enzyme as a CBHI of the glycoside hydrolase family 7. The P. digitatum PD1 protein in silico structural model revealed a coil and β-conformation predominance, which was confirmed by circular dichroism of the P. digitatum RV 06 purified enzyme.

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme’s ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

To enable Saccharomyces cerevisiae to produce renewable fuels from lignocellulose in a consolidated bioprocess, a heterologous cellulase system must be engineered into this yeast. In addition, inherently low secretion titers and sensitivity to adverse environmental conditions must be overcome. Here, two native S. cerevisiae genes related to yeast stress tolerance, YHB1 and SET5, were overexpressed under transcriptional control of the constitutive PGK1 promoter and their effects on heterologous secretion of Talaromyces emersonii cel7A cellobiohydrolase was investigated. Transformants showed increased secreted enzyme activity that ranged from 22% to 55% higher compared to the parental strains and this did not lead to deleterious growth effects. The recombinant strains overexpressing either YHB1 or SET5 also demonstrated multi-tolerant characteristics desirable in bioethanol production, i.e. improved tolerance to osmotic and heat stress. Quantitative reverse transcriptase PCR analysis in these strains showed decreased transcription of secretion pathway genes. However, decreased unfolded protein response was also observed, suggesting novel mechanisms for enhancing enzyme production through stress modulation. Overexpression of YHB1 in an unrelated diploid strain also enhanced stress tolerance and improved ethanol productivity in medium containing acetic acid. To our knowledge, this is the first demonstration that improved heterologous secretion and environmental stress tolerance could be engineered into yeast simultaneously.

Cellobiohydrolases (CBHs) from glycoside hydrolase family 6 (GH6) make up an important part of the secretome in many cellulolytic fungi. They are also of technical interest, particularly because they are part of the enzyme cocktails that are used for the industrial breakdown of lignocellulosic biomass. Nevertheless, functional studies of GH6 CBHs are scarce and focused on a few model enzymes. To elucidate functional breadth among GH6 CBHs, we conducted a comparative biochemical study of seven GH6 CBHs originating from fungi living in different habitats, in addition to one enzyme variant. The enzyme sequences were investigated by phylogenetic analyses to ensure that they were not closely related phylogenetically. The selected enzymes were all heterologously expressed in Aspergillus oryzae, purified and thoroughly characterized biochemically. This approach allowed direct comparisons of functional data, and the results revealed substantial variability. For example, the adsorption capacity on cellulose spanned two orders of magnitude and kinetic parameters, derived from two independent steady-state methods also varied significantly. While the different functional parameters covered wide ranges, they were not independent since they changed in parallel between two poles. One pole was characterized by strong substrate interactions, high adsorption capacity and low turnover number while the other showed weak substrate interactions, poor adsorption and high turnover. The investigated enzymes essentially defined a continuum between these two opposites, and this scaling of functional parameters raises interesting questions regarding functional plasticity and evolution of GH6 CBHs.

The microscopic kinetics of enzymes at the single-molecule level often deviate considerably from those expected from bulk biochemical experiments. Here, we propose a coarse-grained-model approach to bridge this gap, focusing on the unexpectedly slow bulk hydrolysis of crystalline cellulose by cellulase, which constitutes a major obstacle to mass production of biofuels and biochemicals. Building on our previous success in tracking the movements of single molecules of cellulase on crystalline cellulose, we develop a mathematical description of the collective motion and function of enzyme molecules hydrolyzing the surface of cellulose. Model simulations robustly explained the experimental findings at both the microscopic and macroscopic levels and revealed a hitherto-unknown mechanism causing a considerable slowdown of the reaction, which we call the crowding-out effect. The size of the cellulase molecule impacted significantly on the collective dynamics, whereas the rate of molecular motion on the surface did not.

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM-cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1-cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

BACKGROUND Because of the insensitivity of renal cell carcinoma (RCC) to both chemotherapy and radiotherapy, surgery remains the primary approach for anticancer treatment. However, patients who do not receive timely diagnoses may not be suitable for surgery, especially in the late phase of tumor development. Thus, the discovery of novel effective treatment is of great importance. Allyl isothiocyanate (AITC) can inhibit the proliferation and induce apoptosis in many cancer cells. In this paper, we report on an in vitro study to determine the effect of AITC on proliferation and apoptosis of RCC line GRC-1. MATERIAL AND METHODS CCK8 assay was used to detect cell proliferation under gradient concentrations of AITC. Flow cytometry was employed to evaluate cell apoptosis. Real-time fluorescent polymerase chain reaction quantified mRNA levels of Bax and Bcl-2 genes. Western blotting was further employed for protein expression assay. RESULTS AITC inhibited GRC-1 cell proliferation and induced cell apoptosis in a dose-dependent manner; it also elevated Bax while suppressing Bcl-2 gene expression at both mRNA and protein levels. In general, increasing concentration of AITC decreased Bcl-2/Bax ratio. CONCLUSIONS The inhibitory effect of AITC on GRC-1 cells is exerted via cell apoptosis, in which the imbalance of Bcl-2/Bax plays a significant role.