Cellobiohydrolase Cel7A Research Articles

The S-S bridge mutation between the A2 and A4 loops (T416C-I432C) of Cel7A of Aspergillus fumigatus enhances catalytic activity and thermostability.

Disulfide bonds are important for maintaining the structural conformation and stability of the protein. The introduction of the disulfide bond is a promising strategy to increase the thermostability of the protein. In this report, cysteine residues are introduced to form disulfide bonds in the Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of Aspergillus fumigatus. Disulfide by Design 2.0 (DbD2), an online tool is used for the detection of the mutation sites. Mutations are created (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) inside and outside of the peripheral loops but, not in the catalytic region. The introduction of cysteine in the A2 and A4 loop of DSB3 mutant showed higher thermostability (70% activity at 70°C), higher substrate affinity (Km = 0.081 mM) and higher catalytic activity (Kcat = 9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild-type AfCel7A (50% activity at 70°C; Km = 0.128 mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min-1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3: 13.6 Å; Wt: 5.3 Å) at the product exit site, giving flexibility in the entrance region or mobility of the substrate in the exit region. It may facilitate substrate entry into the catalytic tunnel and release the product faster than the wild type, whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop.IMPORTANCEBioethanol is one of the cleanest renewable energy and alternatives to fossil fuels. Cost efficient bioethanol production can be achieved through simultaneous saccharification and co-fermentation that needs active polysaccharide degrading enzymes. Cellulase enzyme complex is a crucial enzyme for second-generation bioethanol production from lignocellulosic biomass. Cellobiohydrolase (Cel7A) is an important member of this complex. In this work, we engineered (disulfide bond engineering) the Cel7A to increase its thermostability and catalytic activity which is required for its industrial application.

Chiral recognition mechanism of cellobiohydrolase Cel7A for ligands based on the β‐blocker propranolol: The effect of explicit water molecules on binding and selectivities

AbstractProteins are useful chiral selectors. In order to understand the recognition mechanism and chiral discrimination, the binding of the (R)‐ and (S)‐enantiomers of a series of designed amino alcohol inhibitors based on propranolol to cellobiohydrolase Cel7A (Trichoderma reesei) has been studied more closely. X‐ray crystal structures were determined of the protein complex with the (R)‐ and (S)‐enantiomers of the strongest binding propranolol analogue. The combination of the structural data, thermodynamic data from capillary electrophoresis and microcalorimetry experiments and computational modelling give a clearer insight into the origin of the enantioselectivity and its opposite thermodynamic signature. The new crystal structures were used in computational molecular flexible dockings of the propranolol analogues using the program Glide. The results indicated that several water molecules in the active site were essential for the docking of the (R)‐enantiomers but not for the (S)‐enantiomers. The results are discussed in relation to the enantiomeric discrimination of the enzyme. Both dissociation constants (Kd values) and thermodynamical data are included to show the effects of the structural modifications in the ligand on enthalpy and entropy in relation to the enantioselectivity.

Comparative Biochemical and Structural Properties of an Industrially Important Biocatalyst Cellobiohydrolase Cel7A from Thermophilic Aspergillus fumigatus

Comparative Biochemical and Structural Properties of an Industrially Important Biocatalyst Cellobiohydrolase Cel7A from Thermophilic Aspergillus fumigatus

Amoeba-inspired magnetic microgel assembly assisted by engineered dextran-binding protein for vaccination against life-threatening systemic infection.

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.Electronic Supplementary MaterialSupplementary material (AFM image and zeta potential of MG; TEM, FT-IR, DLS, and zeta potential of MNP−DexBP; zeta potential of MG+CaAg and MG+MNP−DexBP+CaAg; antigen release profile of MG+CaAg and MG+MNP−DexBP+CaAg; aggregation and dispersion of dendritic cells induced by MG+MNP−DexBP+CaAg; uptake of FITC-labeled CaAg (fCaAg) and intracellular distribution of fCaAg in the dendritic cells; antigen retention and dendritic cell activation in lymph nodes; and serum anti-CaAg antibody levels on day 3 after C. albicans infection in the mice pre-immunized by PBS (control), CaAg, MG+CaAg, and MG+MNP−DexBP+CaAg) is available in the online version of this article at 10.1007/s12274-022-4809-1.

Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design.

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 (pH5-7). 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.

Promoting and Impeding Effects of Lytic Polysaccharide Monooxygenases on Glycoside Hydrolase Activity

Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestris (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.

Removal of N-linked glycans in cellobiohydrolase Cel7A from Trichoderma reesei reveals higher activity and binding affinity on crystalline cellulose

BackgroundCellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure–function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible, O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme’s stability and kinetics.ResultsTwo expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs’ value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30–60% higher for the single deficient variants compared to the WT, and about twofold larger for the triple deficient enzyme. Molecular dynamic simulation of the N-glycan mutants TrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384.ConclusionsThe kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.

Rational design and structure insights for thermostability improvement of Penicillium verruculosum Cel7A cellobiohydrolase

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.

Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

BackgroundMolecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy.ResultsBinding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized.ConclusionsCel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

Molecular recognition in the product site of cellobiohydrolase Cel7A regulates processive step length.

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

Overexpression of endogenous stress-tolerance related genes in Saccharomyces cerevisiae improved strain robustness and production of heterologous cellobiohydrolase.

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.

Systematic deletions in the cellobiohydrolase (CBH) Cel7A from the fungus Trichoderma reesei reveal flexible loops critical for CBH activity

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A.

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady‐state expressions that can be used to describe the pseudo‐steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme‐substrate affinity at which the pseudo‐steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.

Enantioselective Binding of Propranolol and Analogues Thereof to Cellobiohydrolase Cel7A.

At the catalytic site for the hydrolysis of cellulose the enzyme cellobiohydrolase Cel7A binds the enantiomers of the adrenergic beta-blocker propranolol with different selectivity. Methyl-to-hydroxymethyl group modifications of propranolol, which result in higher affinity and improved selectivity, were herein studied by 1 H,1 H and 1 H,13 C scalar spin-spin coupling constants as well as utilizing the nuclear Overhauser effect (NOE) in conjunction with molecular dynamics simulations of the ligands per se, which showed the presence of all-antiperiplanar conformations, except for the one containing a vicinal oxygen-oxygen arrangement governed by the gauche effect. For the ligand-protein complexes investigated by NMR spectroscopy using, inter alia, transferred NOESY and saturation-transfer difference (STD) NMR experiments the S-isomers were shown to bind with a higher affinity and a conformation similar to that preferred in solution, in contrast to the R-isomer. The fact that the S-form of the propranolol enantiomer is pre-arranged for binding to the protein is also observed for a crystal structure of dihydroxy-(S)-propranolol and Cel7A presented herein. Whereas the binding of propranolol is entropy driven, the complexation with the dihydroxy analogue is anticipated to be favored also by an enthalpic term, such as for its enantiomer, that is, dihydroxy-(R)-propranolol, because hydrogen-bond donation replaces the corresponding bonding from hydroxyl groups in glucosyl residues of the natural substrate. In addition to a favorable entropy component, albeit lesser in magnitude, this represents an effect of enthalpy-to-entropy compensation in ligand-protein interactions.

Rate-limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichodermareesei.

The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichodermareesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T.reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T.reesei.

The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei.

The ascomycete Trichoderma reesei is one of the main fungal producers of cellulases and xylanases based on its high production capacity. Its enzymes are applied in food, feed, and textile industry or in lignocellulose hydrolysis in biofuel and biorefinery industry. Over the last years, the demand to expand the molecular toolbox for T. reesei to facilitate genetic engineering and improve the production of heterologous proteins grew. An important instrument to modify the expression of key genes are promoters to initiate and control their transcription. To date, the most commonly used promoter for T. reesei is the strong inducible promoter of the main cellobiohydrolase cel7a. Beside this one, there is a number of alternative inducible promoters derived from other cellulase- and xylanase encoding genes and a few constitutive promoters. With the advances in genomics and transcriptomics the identification of new constitutive and tunable promoters with different expression strength was simplified. In this review, we will discuss new developments in the field of promoters and compare their advantages and disadvantages. Synthetic expression systems constitute a new option to control gene expression and build up complex gene circuits. Therefore, we will address common structural features of promoters and describe options for promoter engineering and synthetic design of promoters. The availability of well-characterized gene expression control tools is essential for the analysis of gene function, detection of bottlenecks in gene networks and yield increase for biotechnology applications.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65°C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Visualizing cellulase adsorption and quantitatively determining cellulose accessibility with an updated fungal cellulose-binding module-based fluorescent probe protein

BackgroundCellulose accessibility to cellulases (CAC) is a direct factor determining the enzymatic digestibility of lignocellulosic cellulose. Improving CAC by pretreatment is a prerequisite step for the efficient release of fermentable sugars from biomass cell wall. However, conventional methods to study the porosimetry of solid materials showed some limitations to be used for investigating CAC. In this work, an updated novel fusion protein comprising a fungal cellulose-binding module (CBM) from Cel7A cellobiohydrolase I (CBH I) of Trichoderma reesei QM6 and a di-green fluorescent protein (GFP2) was constructed for quantitative determination of CAC.ResultsThe obtained probe protein had similar molecular size (e.g., weight) with that of Cel7A and could give detectable signal for quantitative analysis. Several construction strategies were compared with regard to the site of His-tag and order of CBM and GFP2 modules in the protein sequence, in order to achieve good expression quantity and usability of the probe protein. His6–CBM–GFP2 has been identified as the best probe protein for investigating the effects of structural features of cellulosic substrates on cellulose accessibility. Substrate samples with different contents of xylan, lignin, and degree of substitution of cellulose –OH by formyl group were obtained, respectively, by mild H2SO4 pre-hydrolysis, NaClO2 selective delignification, and treatment of filter paper cellulose with concentrated formic acid. The determined CAC was in a wide range of 0.6–20.4 m2/g depending on the contents of hemicelluloses, lignin, and formyl group as well as cellulose degree of crystallization.ConclusionsThe obtained fusion probe protein could be used as a versatile tool to quantitatively investigate the impacts of biomass structural features on CAC and hydrolyzability of cellulose substrates, as well as nonproductive adsorption of cellulase enzymes on lignin.

Characterization of a novel sugar transporter involved in sugarcane bagasse degradation in Trichoderma reesei

BackgroundTrichoderma reesei is a saprophytic fungus implicated in the degradation of polysaccharides present in the cell wall of plants. T. reesei has been recognized as the most important industrial fungus that secretes and produces cellulase enzymes that are employed in the production of second generation bioethanol. A few of the molecular mechanisms involved in the process of biomass deconstruction by T. reesei; in particular, the effect of sugar transporters and induction of xylanases and cellulases expression are yet to be known.ResultsIn our study, we characterized a novel sugar transporter, which was previously identified by our group through in silico analysis of RNA-seq data. The novel T. reesei 69957-sugar transport system (Tr69957) is capable of transporting xylose, mannose, and cellobiose using a T. reesei 69957-sugar transport system in Saccharomyces cerevisiae. The deletion of Tr69957 in T. reesei affected the fungal growth and biomass accumulation, and the sugar uptake in the presence of mannose, cellobiose, and xylose. Molecular docking studies revealed that Tr69957 shows reduced protein–ligand binding energy for interactions towards disaccharides in comparison with monosaccharides. Furthermore, the deletion of Tr69957 affected the gene expression of cellobiohydrolases (cel7a and cel6a), β-glucosidases (cel3a and cel1a), and xylanases (xyn1 and xyn2) in the cultures of parental and mutant strains in the presence of cellobiose and sugarcane bagasse (SCB).ConclusionThe transporter Tr69957 of T. reesei can transport cellobiose, xylose, and mannose, and can affect the expression of a few genes encoding enzymes, such as cellulases and xylanases, in the presence of SCB. We showed for the first time that a filamentous fungus (T. reesei) contains a potential mannose transporter that may be involved in the degradation of cellulose.

Insight into the process of product expulsion in cellobiohydrolase Cel6A from Trichoderma reesei by computational modeling

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.