Orthogonal Active-Site Labels for Mixed-Linkage endo-ß-Glucanases.

Small molecule irreversible inhibitors are valuable tools for determining catalytically important active-site residues and revealing key details of the specificity, structure, and function of glycoside hydrolases (GHs). ß-glucans that contain backbone ß(1,3) linkages are widespread in nature, e.g., mixed-linkage ß(1,3)/ß(1,4)-glucans in the cell walls of higher plants and ß(1,3)glucans in yeasts and algae. Commensurate with this ubiquity, a large diversity of mixed-linkage endoglucanases (MLGases, EC 3.2.1.73) and endo-ß(1,3)-glucanases (laminarinases, EC 3.2.1.39 and EC 3.2.1.6) have evolved to specifically hydrolyze these polysaccharides, respectively, in environmental niches including the human gut. To facilitate biochemical and structural analysis of these GHs, with a focus on MLGases, we present here the facile chemo-enzymatic synthesis of a library of active-site-directed enzyme inhibitors based on mixed-linkage oligosaccharide scaffolds and N-bromoacetylglycosylamine or 2-fluoro-2-deoxyglycoside warheads. The effectiveness and irreversibility of these inhibitors were tested with exemplar MLGases and an endo-ß(1,3)-glucanase. Notably, determination of inhibitor-bound crystal structures of a human-gut microbial MLGase from Glycoside Hydrolase Family 16 revealed the orthogonal labeling of the nucleophile and catalytic acid/base residues with homologous 2-fluoro-2-deoxyglycoside and N-bromoacetylglycosylamine inhibitors, respectively. We anticipate that the selectivity of these inhibitors will continue to enable the structural and mechanistic analyses of ß-glucanases from diverse sources and protein families.

Non-productive celluase binding onto deep eutectic solvent (DES) extracted lignin from willow and corn stover with inhibitory effects on enzymatic hydrolysis of cellulose.

In this work, deep eutectic solvent (DES) was prepared by mixing choline chloride (ChCl) with lactic acid (LA), and effects of cellulase non-productive binding onto DES-extracted lignin from willow and corn stover on enzymatic hydrolysis of cellulose was investigated. The correlation between hydrolysis yield of cellulose and chemical features of lignin was evaluated, and a potential inhibitory mechanism was proposed. Condensation of lignin was observed during DES treatment, and these condensed aromatic structures had an increased tendency to adsorb enzymes through hydrophobic interactions. As well as hydrophobic interactions mediated by lignin condensation, an increase in phenolic hydroxyl groups resulted in a greater amount of hydrogen bonds between cellulases and lignin that appeared to inhibit enzymatic hydrolysis yields of cellulose (39.96-42.86 % to 31.96-32.68 %). Although large amounts of COOHs were generated, the elevated electrostatic repulsion as a result of ionic groups was insufficient to decrease non-productive adsorption.

Study on the Synthesis and Biological Activities of N-Alkylated Deoxynojirimycin Derivatives with a Terminal Tertiary Amine.

A series of N-alkylated deoxynojirimycin (DNJ) derivatives connected to a terminal tertiary amine at the alkyl chains of various lengths were prepared. These novel synthetic compounds were assessed for preliminary glucosidase inhibition and anticancer activities in vitro. Potent and selective inhibition was observed among them. Compound 7d (IC50 = 0.052 mM) showed improved and selective inhibitory activity against ?-glucosidase compared to DNJ (IC50 = 0.65 mM). In addition, analysis of the kinetics of enzyme inhibition by using Lineweaver-Burk plots indicated that 7d inhibited ?-glu-cosidase in a competitive manner, suggesting that 7d was expected to bind to the active site of ?-glucosidase. Compounds 8b and 8c were found to be moderate and selective inhibitors of ?-glucosidase. Nevertheless, none of compounds inhib-ited the growth of B16F10 melanoma cells.

Catalytic and thermodynamic properties of ß-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis.

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of ß-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of ß-glucosidase produced by L. corymbifera was 39 U/g dry substrate (or 3.9 U/mL), and that by B. spectabilis was 77 U/g (or 7.7 U/mL). The optimum pH and temperature were 4.5 and 55 °C and 4.0 and 50 °C for the enzyme from L. corymbifera and B. spectabilis, respectively. ß-Glucosidase produced by L. corymbifera was stable at pH 4.0-7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0-6.0. Regarding the thermostability, ß-glucosidase produced by B. spectabilis remained stable for 1 h at 50 °C, and that from L. corymbifera was active for 1 h at 45 °C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (Ea), and half-life t(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.

Real-Time Adsorption of Exo- and Endoglucanases on Cellulose: Effect of pH, Temperature, and Inhibitors.

Effective regulation of cellulase adsorption is key to improving the efficiencies of the two major bottlenecks of lignocellulose hydrolysis and cellulase recovery. In this work, we investigated the effect of inhibitors, pH, and temperature on the adsorption of exo- and endoglucanases (Cel7A and Cel7B, respectively) on cellulose using quartz crystal microgravimetry with dissipation. The addition of glucose and cellobiose can both inhibit the hydrolysis activity of Cel7A, whereas only cellobiose can inhibit that of Cel7B. Notably, the adsorption was favored by acidic conditions (pH ≤ 4.8) and low temperature, whereas alkaline conditions (pH 9 and 10) facilitated enzyme desorption, which is useful to guide the process of cellulase recovery. The adsorption and hydrolysis activity of Cel7A and Cel7B were both higher at 45 °C than at 25 °C. These findings pave the way to effective regulation of cellulase adsorption and thus improve lignocellulose conversion and cellulase recovery.

Inhibition of the Exoglucanase Cel7A by a Douglas-Fir-Condensed Tannin.

Douglas-fir forestry residues are a potential feedstock for saccharification-based biofuels, and condensed tannins are expected to make up ∼3% of the dry mass of this feedstock. Condensed tannins are well-known for their ability to interact with proteins and can bind and inhibit cellulase enzymes used in saccharification. In this study, we use molecular docking and classical molecular dynamics simulations to investigate how a characterized condensed tannin from Douglas-fir bark binds to the exoglucanase Cel7A from Trichoderma reesei. Through looking at the "occupancy" and "residency" of specific amino acid residue-tannin interactions, we find that the binding sites are characterized by many simultaneous tannin-enzyme interactions with the strongest occurring on the catalytic module as opposed to the carbohydrate-binding module. The simulations indicate that tannin inhibition can result from binding at or near the catalytic tunnel's entrance and exit. The analyzed tannin further prefers to bind to loops around the catalytic region and has affinity for aromatic and charged amino acid residues. These insights provide direction for the rational design of tannin-resistant cellulases.

Inhibition and kinetic studies of lignin degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds.

AIM: Lignolytic (lignin degrading) enzyme, from oil palm pathogen Ganoderma boninense Pat. (Syn G. orbiforme (Ryvarden)), is involved in the detoxification and the degradation of lignin in the oil palm and is the rate-limiting step in the infection process of this fungus. Active inhibition of lignin-degrading enzymes secreted by G. boninense by various naturally occurring phenolic compounds and estimation of efficiency on pathogen suppression was aimed at. METHODS AND RESULTS: In our work, 10 naturally occurring phenolic compounds were evaluated for their inhibitory potential towards the lignolytic enzymes of G. boninense. Additionally, the lignin-degrading enzymes were characterized. Most of the peholic compounds exhibited an uncompetitive inhibition towards the lignin-degrading enzymes. Benzoic acid was the superior inhibitor to the production of lignin-degrading enzymes, when compared between the 10 phenolic compounds. The inhibitory potential of the phenolic compounds towards the lignin-degrading enzymes are higher than that of the conventional metal ion inhibitor. The lignin-degrading enzymes were stable in a wide range of pH but were sensitive to higher temperature. CONCLUSION: The study demonstrated the inhibitor potential of 10 naturally occurring phenolic compounds towards the lignin-degrading enzymes of G. boninense with different efficacies. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has shed a light towards a new management strategy to control basal stem rot disease in oil palm. It serves as a replacement for the existing chemical control.

Inhibition and kinetic studies of cellulose- and hemicellulose-degrading enzymes of Ganoderma boninense by naturally occurring phenolic compounds.

AIM: Ganoderma sp, the causal pathogen of the basal stem rot (BSR) disease of oil palm, secretes extracellular hydrolytic enzymes. These play an important role in the pathogenesis of BSR by nourishing the pathogen through the digestion of cellulose and hemicellulose of the host tissue. Active suppression of hydrolytic enzymes secreted by Ganoderma boninense by various naturally occurring phenolic compounds and estimation of their efficacy on pathogen suppression is focused in this study. METHODS AND RESULTS: Ten naturally occurring phenolic compounds were assessed for their inhibitory effect on the hydrolytic enzymes of G. boninense. The enzyme kinetics (Vmax and Km ) and the stability of the hydrolytic enzymes were also characterized. The selected compounds had shown inhibitory effect at various concentrations. Two types of inhibitions namely uncompetitive and noncompetitive were observed in the presence of phenolic compounds. Among all the phenolic compounds tested, benzoic acid was the most effective compound suppressive to the growth and production of hydrolytic enzymes secreted by G. boninense. The phenolic compounds as inhibitory agents can be a better replacement for the metal ions which are known as conventional inhibitors till date. The three hydrolytic enzymes were stable in a wide range of pH and temperature. CONCLUSION: These findings highlight the efficacy of the applications of phenolic compounds to control Ganoderma. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has proved a replacement for chemical controls of G. boninense with naturally occurring phenolic compounds.

Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics.

Non-productive enzyme binding onto lignin is the major inhibitory mechanism, which reduces hydrolysis rates and yields and prevents efficient enzyme recycling in the hydrolysis of lignocellulosics. The detailed mechanisms of binding are still poorly understood. Enzyme-lignin interactions were investigated by comparing the structural properties and binding behaviour of fungal monocomponent enzymes, cellobiohydrolases TrCel7A and TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn11 and a ß-glucosidase AnCel3A, onto lignins isolated from steam pretreated spruce and wheat straw. The enzymes exhibited decreasing affinity onto lignin model films in the following order: TrCel7B>TrCel6A>TrCel5A>AnCel3A>TrCel7A>TrXyn11. As analysed in Avicel hydrolysis, TrCel6A and TrCel7B were most inhibited by lignin isolated from pretreated spruce. This could be partially explained by adsorption of the enzyme onto the lignin surface. Enzyme properties, such as enzyme surface charge, thermal stability or surface hydrophobicity could not alone explain the adsorption behaviour.

Selenoureido-iminosugars: A new family of multitarget drugs.

Herein we report the synthesis of N-alkylated deoxynojirimycin derivatives decorated with a selenoureido motif at the hydrocarbon tether as an example of unprecedented multitarget agents. Title compounds were designed as dual drugs for tackling simultaneously the Gaucher disease (by selective inhibition of ß-glucosidase, Ki = 1.6-5.5 µM, with improved potency and selectivity compared to deoxynojirimycin) and its neurological complications (by inhibiting AChE, Ki up to 5.8 µM). Moreover, an excellent mimicry of the selenoenzyme glutathione peroxidase was also found for the catalytic scavenging of H2O2 (Kcat/Kuncat up to 640) using PhSH as a cofactor, with improved activity compared to known positive controls, like (PhSe)2 and ebselen; therefore, such compounds are also excellent scavengers of peroxides, an example of reactive oxygen species present at high concentrations in patients of Gaucher disease and neurological disorders.

Identification and Characterization of Trichoderma Species Damaging Shiitake Mushroom Bed-Logs Infested by Camptomyia Pest.

The shiitake mushroom industry has suffered from Camptomyia (gall midges) pest, which feeds on the mycelium of shiitake mushroom during its cultivation. It has been postulated that fungal damage of shiitake bed-logs is associated with infestation by the insect pest, but this is not well understood. To understand the fungal damage associated with Camptomyia pest, various Trichoderma species were isolated, identified, and characterized. In addition to two previously known Trichoderma species, T. citrinoviride and T. deliquescens, two other Trichoderma species, T. harzianum and T. atroviride, were newly identified from the pestinfested bed-log samples obtained at three mushroom farms in Cheonan, Korea. Among these four species, T. harzianum was the most evident. The results of a chromogenic media-based assay for extracellular enzymes showed that these four species have the ability to produce amylase, carboxyl-methyl cellulase, avicelase, pectinase, and ß-glucosidase, thus indicating that they can degrade wood components. A dual culture assay on PDA indicated that T. harzianum, T. atroviride, and T. citrinoviride were antagonistic against the mycelial growth of a shiitake strain (Lentinula edodes). Inoculation tests on shiitake bed-logs revealed that all four species were able to damage the wood of bed-logs. Our results provide evidence that the four green mold species are the causal agents involved in fungal damage of shiitake bed-logs infested by Camptomyia pest.

Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance.

In this study, we monitored the inhibition and deactivation effects of various compounds associated with lignocellulosic hydrolysates on individual and combinations of cellulases. Tannic acid representing polymeric lignin residues strongly inhibited cellobiohydrolase 1 (CBH1) and ß-glucosidase 1 (BGL1), but had a moderate inhibitory effect on endoglucanase 2 (EG2). Individual monomeric lignin residues had little or no inhibitory effect on hydrolytic enzymes. However, coniferyl aldehyde and syringaldehyde substantially decreased the activity of CBH1 and deactivated BGL1. Acetic and formic acids also showed strong inhibition of BGL1 but not CBH1 and EG2, whereas tannic, acetic and formic acid strongly inhibited a combination of CBH1 and EG2 during Avicel hydrolysis. Diminishing enzymatic hydrolysis is largely a function of inhibitor concentration and the enzyme-inhibitor relationship, rather than contact time during the hydrolysis process (i.e. deactivation). This suggests that decreased rates of hydrolysis during the enzymatic depolymerisation of lignocellulosic hydrolysates may be imparted by other factors related to substrate crystallinity and accessibility.

Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity.

Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24-28% of the total observed enzyme inhibition while a 3 kDa MWCO membrane removed 60-65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34-43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44-56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69-70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption.

Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis.

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted.

Carbohydrate derived-pseudo-lignin can retard cellulose biological conversion.

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo-lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo-lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT-IR, and SEM imaging. It was found that hemicelluloses (xylan) derived-pseudo-lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ~ 1.94) dilute acid pretreatment of a xylan-Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo-lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions.

Novel activity-based probes for broad-spectrum profiling of retaining ß-exoglucosidases in†situ and in†vivo.

A high-end label: Cyclophellitol aziridine-type activity-based probes allow for ultra-sensitive visualization of mammalian ß-glucosidases (GBA1, GBA2, GBA3, and LPH) as well as several non-mammalian ß-glucosidases (see picture). These probes offer new ways to study ß-exoglucosidases, and configurational isomers of the cyclophellitol aziridine core may give activity-based probes targeting other retaining glycosidase families.

Competitive sorption kinetics of inhibited endo- and exoglucanases on a model cellulose substrate.

For the first time, the competitive adsorption of inhibited cellobiohydrolase I (Cel7A, an exoglucanase) and endoglucanase I (Cel7B) from T. longibrachiatum is studied on cellulose. Using quartz crystal microgravimetry (QCM), sorption histories are measured for individual types of cellulases and their mixtures adsorbing to and desorbing from a model cellulose surface. We find that Cel7A has a higher adsorptive affinity for cellulose than does Cel7B. The adsorption of both cellulases becomes irreversible on time scales of 30-60 min, which are much shorter than those typically used for industrial cellulose hydrolysis. A multicomponent Langmuir kinetic model including first-order irreversible binding is proposed. Although adsorption and desorption rate constants differ between the two enzymes, the rate at which each surface enzyme irreversibly binds is identical. Because of the higher affinity of Cel7A for the cellulose surface, when Cel7A and Cel7B compete for surface sites, a significantly higher bulk concentration of Cel7B is required to achieve comparable surface enzyme concentrations. Because cellulose deconstruction benefits significantly from the cooperative activity of endoglucanases and cellobiohydrolases on the cellulose surface, accounting for competitive adsorption is crucial to developing effective cellulase mixtures.

Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases.

Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase.

The crystallization and structural analysis of cellulases (and other glycoside hydrolases): strategies and tactics.

The three-dimensional (3-D) structures of cellulases, and other glycoside hydrolases, are a central feature of research in carbohydrate chemistry and biochemistry. 3-D structure is used to inform protein engineering campaigns, both academic and industrial, which are typically used to improve the stability or activity of an enzyme. Examples of classical protein engineering goals include higher thermal stability, reduced metal-ion dependency, detergent and protease resistance, decreased product inhibition, and altered specificity. 3-D structure may also be used to interpret the behavior of enzyme variants that are derived from screening or random mutagenesis approaches, with a view to establishing an iterative design process. In other areas, 3-D structure is used as one of the many tools to probe enzymatic catalysis, typically dovetailing with physical organic chemistry approaches to provide complete reaction mechanisms for enzymes by visualizing catalytic site interactions at different stages of the reaction. Such mechanistic insight is not only fundamentally important, impacting on inhibitor and drug design approaches with ramifications way beyond cellulose hydrolysis, but also provides the framework for the design of enzyme variants to use as biocatalysts for the synthesis of bespoke oligosaccharides. Here we review some of the strategies and tactics that may be applied to the X-ray structure solution of cellulases (and other carbohydrate-active enzymes). The general approach is first to decide why you are doing the work, then to establish correct domain boundaries for truncated constructs (typically the catalytic domain only), and finally to pursue crystallization of pure, homogeneous, and monodisperse protein with appropriate ligand and additive combinations. Cellulase-specific strategies are important for the delineation of domain boundaries, while glycoside hydrolases generally also present challenges and opportunities for the selection and optimization of ligands to both aid crystallization, and also provide structural and mechanistic insight. As the many roles for plant cell wall degrading enzymes increase, so does the need for rapid high-quality structure determination to provide a sound structural foundation for understanding mechanism and specificity, and for future protein engineering strategies.

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization.

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.