Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Genus-wide analysis of Trichoderma antagonism toward Pythium and Globisporangium plant pathogens and the contribution of cellulases to the antagonism.

Parasitism is an important lifestyle in the Trichoderma genus but has not been studied in a genus-wide way toward Pythium and Globisporangium hosts. Our approach screened a genus-wide set of 30 Trichoderma species in dual culture assays with two soil-borne Pythium and three Globisporangium plant-parasitic species and used exo-proteomic analyses, with the aim to correlate Trichoderma antagonism with potential strategies for attacking Pythium and Globisporangium. The Trichoderma spp. showed a wide range of antagonism from strong to weak, but the same Trichoderma strain showed similar levels toward all the Pythium and Globisporangium species. The Trichoderma enzymes from strong (Trichoderma asperellum, Trichoderma atroviride, and Trichoderma virens), moderate (Trichoderma cf. guizhouense and Trichoderma reesei), and weak (Trichoderma parepimyces) antagonists were induced by the autoclaved mycelia of one of the screened Pythium species, Pythium myriotylum. The variable proportions of putative cellulases, proteases, and redox enzymes suggested diverse as well as shared strategies amongst the antagonists. There was a partial positive correlation between antagonism from microscopy and the cellulase activity induced by autoclaved P. myriotylum mycelia in different Trichoderma species. The deletion of the cellulase transcriptional activator XYR1 in T. reesei led to lower antagonism toward Pythium and Globisporangium. The antagonism of Pythium and Globisporangium appears to be a generic property of Trichoderma as most of the Trichoderma species were at least moderately antagonistic. While a role for cellulases in the antagonism was uncovered, cellulases did not appear to make a major contribution to T. reesei antagonism, and other factors are also likely contributing.IMPORTANCETrichoderma is an important genus widely distributed in nature with broad ecological impacts and applications in the biocontrol of plant diseases. The Pythium and Globisporangium genera of fungus-like water molds include many important soil-borne plant pathogens that cause various diseases. Most of the Trichoderma species showed at least a moderate ability to compete with or antagonize the Pythium and Globisporangium hosts, and microscopy showed examples of parasitism (a slow type of killing) and predation (a fast type of killing). Hydrolytic enzymes such as cellulases and proteases produced by Trichoderma likely contribute to the antagonism. A mutant deficient in cellulase activity had reduced antagonism. Interestingly, Pythium and Globisporangium species contain cellulose in their cell walls (unlike true fungi such as Trichoderma), and the cellulolytic ability of Trichoderma appears beneficial for antagonism of water molds.

An experimental and modeling approach to describe the deactivation of cellulases at the air-liquid interface.

Understanding the reaction mechanisms involved in the enzymatic hydrolysis of cellulose is important because it is kinetically the most limiting step of the bioethanol production process. The present work focuses on the enzymatic deactivation at the air-liquid interface, which is one of the aspects contributing to this global deactivation. This phenomenon has already been experimentally proven, but this is the first time that a model has been proposed to describe it. Experiments were performed by incubating Celluclast cocktail solutions on an orbital stirring system at different enzyme concentrations and different surface-to-volume ratios. A 5-day follow-up was carried out by measuring the global FPase activity of cellulases for each condition tested. The activity loss was proven to depend on both the air-liquid surface area and the enzyme concentration. Both observations suggest that the loss of activity takes place at the air-liquid surface, the total amount of enzymes varying with volume or enzyme concentration. Furthermore, tests performed using five individual enzymes purified from a Trichoderma reesei cocktail showed that the only cellulase that is deactivated at the air-liquid interface is cellobiohydrolase II. From the experimental data collected by varying the initial enzyme concentration and the ratio surface to volume, it was possible to develop, for the first time, a model that describes the loss of activity at the air-liquid interface for this configuration.

Methionine inducing carbohydrate esterase secretion of Trichoderma harzianum enhances the accessibility of substrate glycosidic bonds.

BACKGROUND: The conversion of plant biomass into biochemicals is a promising way to alleviate energy shortage, which depends on efficient microbial saccharification and cellular metabolism. Trichoderma spp. have plentiful CAZymes systems that can utilize all-components of lignocellulose. Acetylation of polysaccharides causes nanostructure densification and hydrophobicity enhancement, which is an obstacle for glycoside hydrolases to hydrolyze glycosidic bonds. The improvement of deacetylation ability can effectively release the potential for polysaccharide degradation. RESULTS: Ammonium sulfate addition facilitated the deacetylation of xylan by inducing the up-regulation of multiple carbohydrate esterases (CE3/CE4/CE15/CE16) of Trichoderma harzianum. Mainly, the pathway of ammonium-sulfate's cellular assimilates inducing up-regulation of the deacetylase gene (Thce3) was revealed. The intracellular metabolite changes were revealed through metabonomic analysis. Whole genome bisulfite sequencing identified a novel differentially methylated region (DMR) that existed in the ThgsfR2 promoter, and the DMR was closely related to lignocellulolytic response. ThGsfR2 was identified as a negative regulatory factor of Thce3, and methylation in ThgsfR2 promoter released the expression of Thce3. The up-regulation of CEs facilitated the substrate deacetylation. CONCLUSION: Ammonium sulfate increased the polysaccharide deacetylation capacity by inducing the up-regulation of multiple carbohydrate esterases of T. harzianum, which removed the spatial barrier of the glycosidic bond and improved hydrophilicity, and ultimately increased the accessibility of glycosidic bond to glycoside hydrolases.

Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal.

BACKGROUND: Azo dyes represent a common textile dye preferred for its high stability on fabrics in various harsh conditions. Although these dyes pose high-risk levels for all biological forms, fungal laccase is known as a green catalyst for its ability to oxidize numerous dyes. METHODS: Trichoderma isolates were identified and tested for laccase production. Laccase production was optimized using Plackett-Burman Design. Laccase molecular weight and the kinetic properties of the enzyme, including Km and Vmax, pH, temperature, and ionic strength, were detected. Azo dye removal efficiency by laccase enzyme was detected for Congo red, methylene blue, and methyl orange. RESULTS: Eight out of nine Trichoderma isolates were laccase producers. Laccase production efficiency was optimized by the superior strain T. harzianum PP389612, increasing production from 1.6 to 2.89 U/ml. In SDS-PAGE, purified laccases appear as a single protein band with a molecular weight of 41.00 kDa. Km and Vmax values were 146.12 µmol guaiacol and 3.82 µmol guaiacol/min. Its activity was stable in the pH range of 5-7, with an optimum temperature range of 40 to 50 °C, optimum ionic strength of 50 mM NaCl, and thermostability properties up to 90 °C. The decolorization efficiency of laccase was increased by increasing the time and reached its maximum after 72 h. The highest efficiency was achieved in Congo red decolorization, which reached 99% after 72 h, followed by methylene blue at 72%, while methyl orange decolorization efficiency was 68.5%. CONCLUSION: Trichoderma laccase can be used as an effective natural bio-agent for dye removal because it is stable and removes colors very well.

Optimization, purification and characterization of laccase from a new endophytic Trichoderma harzianum AUMC14897 isolated from Opuntia ficus-indica and its applications in dye decolorization and wastewater treatment.

BACKGROUND: Hazardous synthetic dye wastes have become a growing threat to the environment and public health. Fungal enzymes are eco-friendly, compatible and cost-effective approach for diversity of applications. Therefore, this study aimed to screen, optimize fermentation conditions, and characterize laccase from fungal endophyte with elucidating its ability to decolorize several wastewater dyes. RESULTS: A new fungal endophyte capable of laccase-producing was firstly isolated from cladodes of Opuntia ficus-indica and identified as T. harzianum AUMC14897 using ITS-rRNA sequencing analysis. Furthermore, the response surface methodology (RSM) was utilized to optimize several fermentation parameters that increase laccase production. The isolated laccase was purified to 13.79-fold. GFC, SDS-PAGE revealed laccase molecular weight at 72 kDa and zymogram analysis elucidated a single band without any isozymes. The peak activity of the pure laccase was detected at 50 °C, pH 4.5, with thermal stability up to 50 °C and half life span for 4 h even after 24 h retained 30% of its activity. The Km and Vmax values were 0.1 mM, 22.22 µmol/min and activation energy (Ea) equal to 5.71 kcal/mol. Furthermore, the purified laccase effectively decolorized various synthetic and real wastewater dyes. CONCLUSION: Subsequently, the new endophytic strain produces high laccase activity that possesses a unique characteristic, it could be an appealing candidate for both environmental and industrial applications.

Recombinant expression and characterization of the endochitinase Chit36-TA from Trichoderma asperellum in Komagataella phaffii for chitin degradation of black soldier fly exuviae.

The natural polymer chitin is an abundant source for valuable N-acetylchitooligosaccharides and N-acetylglucosamine applicable in several industries. The endochitinase Chit36-TA from Trichoderma asperellum was recombinantly expressed in Komagataella phaffii for the enzymatic degradation of chitin from unused insect exuviae into N-acetylchitooligosaccharides. Chit36-TA was purified by Ni-NTA affinity chromatography and subsequently biochemically characterized. After deglycosylation, the endochitinase had a molecular weight of 36 kDa. The optimum pH for Chit36-TA was 4.5. The temperature maximum of Chit36-TA was determined to be 50 °C, while it maintained > 93% activity up to 60 °C. The chitinase was thermostable up to 45 °C and exhibited ~ 50% activity after a 15 min incubation at 57 °C. Chit36-TA had a maximum specific enzyme activity of 50 nkat/mg with a Km value of 289 µM with 4-methylumbelliferyl-N,N',N″-triacetyl-ß-chitotrioside as substrate. Most tested cations, organic solvents and reagents were well-tolerated by the endochitinase, except for SDS (1 mM), Cu2+ (10 mM) and Mn2+ (10 mM), which had stronger inhibitory effects with residual activities of 3, 41 and 28%, respectively. With a degree of hydrolysis of 32% applying colloidal shrimp chitin (1% (w/v)) and 12% on insect larvae (1% (w/v)) after 24 h, the endochitinase was found to be suitable for the conversion of colloidal chitin as well as chitin from black soldier fly larvae into water-soluble N-acetylchitooligosaccharides. To prove scalability, a bioreactor process was developed in which a 55-fold higher enzyme activity of 49 µkat/l and a tenfold higher protein expression of 1258 mg/l were achieved.

Characteristics of a Novel Zearalenone Lactone Hydrolase ZHRnZ and Its Thermostability Modification.

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed.

Rice straw hydrolysis using in-situ produced enzymes: Feedstock influences fungal enzyme composition and hydrolytic efficiency.

Trichoderma reesei RUT-C30 was cultivated on differentially pretreated rice straw and pure cellulose as a carbon source/inducer for cellulase production, and the enzymes were evaluated for hydrolysis of sequential acid and alkali pretreated rice straw. Growth on pretreated rice straw enhanced protein secretion and cellulase activities compared to pure cellulose as a carbon source. The yield of cellulolytic enzymes was higher for alkali pretreated rice straw (ALP-RS), while H2O2-treated (HP-RS) could not induce cellulases to a larger level compared to pure cellulose. Protein concentration was 3.5-fold higher on ALP-RS as compared to pure cellulose, with a maximum filter-paper cellulase (FPase) activity of 1.76 IU/ml and carboxy-methyl cellulase (CMCase) activity of 40.16 IU/ml (2.18 fold higher). Beta-glucosidase (BGL) activity was more or less the same with the different substrates and supplementation of heterologous BGL could result in a quantum jump in hydrolytic efficiencies, which in the case of ALP-RS induced enzymes was 34% (increased from 69.26% to 92.51%). The use of lignocellulosic biomass (LCB) itself as a substrate for the production of cellulase is advantageous not only in terms of raw material costs but also for obtaining a more suitable enzyme profile for biomass hydrolysis.

Cloning and sequence analysis of 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase from Sarocladium kiliense U4520.

A novel enzyme, 4-O-α-d-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), was previously discovered from Sarocladium kiliense U4520. In order to identify the factors underlying the unique substrate specificity of IMM-4IH, we endeavored to determine the amino acid sequence of the enzyme. By comparing the partial amino acid sequence of the enzyme to whole genome sequencing data of S. kiliense U4520, the IMM-4IH gene was estimated. The putative gene was expressed in Pichia pastoris, and its activity and properties were found to be consistent with those of the native enzyme. Comparing the amino acid sequence of IMM-4IH with those in the CAZy database led to classification in the glycoside hydrolase family 49 (GH49). Several amino acids important for catalysis (Asp406, Asp425, and Asp426) and substrate recognition at subsites + 1 and -3 were estimated by multiple sequence alignment analysis. These results provide important information for characterizing IMM-4IH and other GH49 enzymes.

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei.

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency [Formula: see text] of the cellulose peroxygenase reaction (kcat = 8.5 s-1, and [Formula: see text] ) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H2O2 in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.

Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A.

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.

A comparative biochemical investigation of the impeding effect of C1-oxidizing LPMOs on cellobiohydrolases.

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), T. 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 extent 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.

Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III.

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.

Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Polyextremophilic Chitinolytic Activity by a Marine Strain (IG119) of Clonostachys rosea.

The investigation for novel unique extremozymes is a valuable business for which the marine environment has been overlooked. The marine fungus Clonostachys rosea IG119 was tested for growth and chitinolytic enzyme production at different combinations of salinity and pH using response surface methodology. RSM modelling predicted best growth in-between pH 3.0 and 9.0 and at salinity of 0-40‱, and maximum enzyme activity (411.137 IU/L) at pH 6.4 and salinity 0‱; however, quite high production (>390 IU/L) was still predicted at pH 4.5-8.5. The highest growth and activity were obtained, respectively, at pH 4.0 and 8.0, in absence of salt. The crude enzyme was tested at different salinities (0-120‱) and pHs (2.0-13.0). The best activity was achieved at pH 4.0, but it was still high (in-between 3.0 and 12.0) at pH 2.0 and 13.0. Salinity did not affect the activity in all tested conditions. Overall, C. rosea IG119 was able to grow and produce chitinolytic enzymes under polyextremophilic conditions, and its crude enzyme solution showed more evident polyextremophilic features. The promising chitinolytic activity of IG119 and the peculiar characteristics of its chitinolytic enzymes could be suitable for several biotechnological applications (i.e., degradation of salty chitin-rich materials and biocontrol of spoiling organisms, possibly solving some relevant environmental issues).

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

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.

The fungal NADPH oxidase is an essential element for the molecular dialog between Trichoderma and Arabidopsis.

Members of the fungal genus Trichoderma stimulate growth and reinforce plant immunity. Nevertheless, how fungal signaling elements mediate the establishment of a successful Trichoderma-plant interaction is largely unknown. In this work, we analyzed growth, root architecture and defense in an Arabidopsis-Trichoderma co-cultivation system, including the wild-type (WT) strain of the fungus and mutants affected in NADPH oxidase. Global gene expression profiles were assessed in both the plant and the fungus during the establishment of the interaction. Trichoderma atroviride WT improved root branching and growth of seedling as previously reported. This effect diminished in co-cultivation with the ∆nox1, ∆nox2 and ∆noxR null mutants. The data gathered of the Arabidopsis interaction with the ∆noxR strain showed that the seedlings had a heightened immune response linked to jasmonic acid in roots and shoots. In the fungus, we observed repression of genes involved in complex carbohydrate degradation in the presence of the plant before contact. However, in the absence of NoxR, such repression was lost, apparently due to a poor ability to adequately utilize simple carbon sources such as sucrose, a typical plant exudate. Our results unveiled the critical role played by the Trichoderma NoxR in the establishment of a fine-tuned communication between the plant and the fungus even before physical contact. In this dialog, the fungus appears to respond to the plant by adjusting its metabolism, while in the plant, fungal perception determines a delicate growth-defense balance.

Heterologous expression of Phanerochaete chrysosporium cellobiose dehydrogenase in Trichoderma reesei.

BACKGROUND: Cellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp). RESULTS: PcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-ß-D-glucosaminidase in the supernatant. CONCLUSIONS: Heterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Speciation of Transition-Metal-Substituted Keggin-Type Silicotungstates Affected by the Co-crystallization Conditions with Proteinase K.

We report on the synthesis of the tetrasubstituted sandwich-type Keggin silicotungstates as the pure Na salts Na14[(A-α-SiW10O37)2{Co4(OH)2(H2O)2}]·37H2O (Na{SiW10Co2}2) and Na14[(A-α-SiW10O37)2{Ni4(OH)2(H2O)2}]·77.5H2O (Na{SiW10Ni2}2), which were prepared by applying a new synthesis protocol and characterized thoroughly in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis. Proteinase K was applied as a model protein and the polyoxotungstate (POT)-protein interactions of Na{SiW10Co2}2 and Na{SiW10Ni2}2 were studied side by side with the literature-known K5Na3[A-α-SiW9O34(OH)3{Co4(OAc)3}]·28.5H2O ({SiW9Co4}) featuring the same number of transition metals. Testing the solution behavior of applied POTs under the crystallization conditions (sodium acetate buffer, pH 5.5) by time-dependent UV/vis spectroscopy and electrospray ionization mass spectrometry speciation studies revealed an initial dissociation of the sandwich POTs to the disubstituted Keggin anions HxNa5-x[SiW10Co2O38]3- and HxNa5-x[SiW10Ni2O38]3- ({SiW10M2}, M = CoII and NiII) followed by partial rearrangement to the monosubstituted compounds (α-{SiW11Co} and α-{SiW11Ni}) after 1 week of aging. The protein crystal structure analysis revealed monosubstituted α-Keggin POTs in two conserved binding positions for all three investigated compounds, with one of these positions featuring a covalent attachment of the POT anion to an aspartate carboxylate. Despite the presence of both mono- and disubstituted anions in a crystallization mixture, proteinase K selectively binds to monosubstituted anions because of their preferred charge density for POT-protein interaction.