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.

Current perspective in research and industrial applications of microbial cellulases.

The natural interactions between various bacteria, fungi, and other cellulolytic microorganisms destroy lignocellulosic polymers. The efficacy of this process is determined by the combined action of three main enzymes: endoglucanases, exo-glucanases, and ß-glucosidase. The enzyme attacks the polymeric structure's ß-1,4-linkages during the cellulose breakdown reaction. This mechanism is crucial for the environment as it recycles cellulose in the biosphere. However, there are problems with enzymatic cellulose breakdown, including complex cellulase structure, insufficient degradation efficacy, high production costs, and post-translational alterations, many of which are closely related to certain unidentified cellulase properties. These issues impede the practical use of cellulases. A developing area of research is the application of this similar paradigm for industrial objectives. Cellulase enzyme exhibits greater promise in many critical industries, including biofuel manufacture, textile smoothing and finishing, paper and pulp manufacturing, and farming. However, the study on cellulolytic enzymes must move forward in various directions, including increasing the activity of cellulase as well as designing peptides to give biocatalysts their desired attributes. This manuscript includes an overview of current research on different sources of cellulases, their production, and biochemical characterization.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to µM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

Glycosylation of cellulase: a novel strategy for improving cellulase.

Protein glycosylation is the most complex posttranslational modification process. Most cellulases from filamentous fungi contain N-glycosylation and O-glycosylation. Here, we discuss the potential roles of glycosylation on the characteristics and function of cellulases. The use of certain cultivation, inducer, and alteration of engineering glycosylation pathway can enable the rational control of cellulase glycosylation. Glycosylation does not occur arbitrarily and may tend to modify the 3D structure of cellulases by using specially distributed glycans. Therefore, glycoengineering should be considered comprehensively along with the spatial structure of cellulases. Cellulase glycosylation may be an evolution phenomenon, which has been considered as an economical way for providing different functions from identical proteins. In addition to gene and transcription regulations, glycosylation may be another regulation on the protein expression level. Enhanced understanding of the potential regulatory role of cellulase glycosylation will enable synthetic biology approaches for the development of commercial cellulase.

A critical process variable-regulated, parameter-balancing auxostat, performed using disposed COVID-19 personal protective equipment-based substrate mixture, yields sustained and improved endoglucanase titers.

Fifty percent of the overall operational expenses of biorefineries are incurred during enzymatic-saccharification processes. Cellulases have a global-market value of $1621 USD. Dearth of conventional lignocelluloses have led to the exploration of their waste stream-based, unconventional sources. Native fungus-employing cellulase-production batches fail to yield sustained enzyme titers. It could be attributed to variations in the enzyme-production broth's quasi-dilatant behavior, its fluid and flow properties; heat and oxygen transfer regimes; kinetics of fungal growth; and nutrient utilization. The current investigation presents one of the first-time usages of a substrate mixture, majorly comprising disposed COVID-19 personal protective-equipment (PPE). To devise a sustainable and scalable cellulase-production process, various variable-regulated, continuous-culture auxostats were performed. The glucose concentration-maintaining auxostat recorded consistent endoglucanase titers throughout its feeding-cum-harvest cycles; furthermore, it enhanced oxygen transfer, heat transfer co-efficient, and mass transfer co-efficient by 91.5, 36, and 77%, respectively. Substrate-characterization revealed that an unintended, autoclave-based organsolv pretreatment caused unanticipated increases in endoglucanase titers. The cumulative lab-scale cellulase-production cost was found to be $16.3. The proposed approach is economical, and it offers a pollution-free waste management process, thereby generating carbon credits.

Testing and improving the performance of protein thermostability predictors for the engineering of cellulases.

Thermostability of cellulases can be increased through amino acid substitutions and by protein engineering with predictors of protein thermostability. We have carried out a systematic analysis of the performance of 18 predictors for the engineering of cellulases. The predictors were PoPMuSiC, HoTMuSiC, I-Mutant 2.0, I-Mutant Suite, PremPS, Hotspot, Maestroweb, DynaMut, ENCoM ([Formula: see text] and [Formula: see text], mCSM, SDM, DUET, RosettaDesign, Cupsat (thermal and denaturant approaches), ConSurf, and Voronoia. The highest values of accuracy, F-measure, and MCC were obtained for DynaMut, SDM, RosettaDesign, and PremPS. A combination of the predictors provided an improvement in the performance. F-measure and MCC were improved by 14% and 28%, respectively. Accuracy and sensitivity were also improved by 9% and 20%, respectively, compared to the maximal values of single predictors. The reported values of the performance of the predictors and their combination may aid research in the engineering of thermostable cellulases as well as the further development of thermostability predictors.

Microbial cellulase production and stability investigations via graphene like carbon nanostructure derived from paddy straw.

Cellulases are among the most in-demand bioprocess enzymes, and the high cost of production, combined with their low enzymatic activity, is the main constraint, particularly in the biofuels industry. As a result, low-cost enzyme production modes with high activity and stability have emerged as the primary focus of research. Here, a method for producing a graphene like carbon nanostructure (GLCNs) has been investigated utilizing paddy straw (Ps), and its physicochemical characteristics have been examined using a variety of techniques including XRD, FT-IR, SEM and TEM. Further, the pretreatment of Ps feedstock for cellulase production was done using diluted waste KOH liquid collected during the preparation of the GLCNs. To increase the production and stability of the enzyme, newly prepared GLCNs is utilized as a nanocatalyst. Using 15 mg of GLCNs, 35 IU/gds FP activity was seen after 72 h, followed by 158 IU/gds EG and 114 IU/gds BGL activity in 96 h. This nanocatalyst supported enzyme was thermally stable at 70 °C up to 15 h and exhibited stability at pH 7.0 for 10 h by holding 66 % of its half-life.

Engineering cellulases for conversion of lignocellulosic biomass.

Lignocellulosic biomass is a renewable source of energy, chemicals and materials. Many applications of this resource require the depolymerization of one or more of its polymeric constituents. Efficient enzymatic depolymerization of cellulose to glucose by cellulases and accessory enzymes such as lytic polysaccharide monooxygenases is a prerequisite for economically viable exploitation of this biomass. Microbes produce a remarkably diverse range of cellulases, which consist of glycoside hydrolase (GH) catalytic domains and, although not in all cases, substrate-binding carbohydrate-binding modules (CBMs). As enzymes are a considerable cost factor, there is great interest in finding or engineering improved and robust cellulases, with higher activity and stability, easy expression, and minimal product inhibition. This review addresses relevant engineering targets for cellulases, discusses a few notable cellulase engineering studies of the past decades and provides an overview of recent work in the field.

Supplementation of recombinant cellulases with LPMOs and CDHs improves consolidated bioprocessing of cellulose.

The increased demand for energy has sparked a global search for renewable energy sources that could partly replace fossil fuel resources and help mitigate climate change. Cellulosic biomass is an ideal feedstock for renewable bioethanol production, but the process is not currently economically feasible due to the high cost of pretreatment and enzyme cocktails to release fermentable sugars. Lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenases (CDHs) are auxiliary enzymes that can enhance cellulose hydrolysis. In this study, four LPMO and two CDH genes were subcloned and expressed in the Saccharomyces cerevisiae Y294 laboratory strain. SDS-PAGE analysis confirmed the extracellular production of the LPMOs and CDHs in the laboratory S. cerevisiae Y294 strain. A rudimentary cellulase cocktail (cellobiohydrolase 1 and 2, endoglucanase and ß-glucosidase) was expressed in the commercial CelluX™ 4 strain and extracellular production of the individual cellulases was confirmed by SDS-PAGE analysis. In vitro cooperation of the CDHs and LPMOs with the rudimentary cellulases produced by strain CelluX™ 4[F4-1] was demonstrated on Whatman filter paper. The significant levels of soluble sugars released from this crystalline cellulose substrate indicated that these auxiliary enzymes could be important components of the CBP yeast cellulolytic system.

Model of Processive Catalysis with Site Clustering and Blocking and Its Application to Cellulose Hydrolysis.

Interactions between particles moving on a linear track and their possible blocking by obstacles can lead to crowding, impeding the particles' transport kinetics. When the particles are enzymes processively catalyzing a reaction along a linear polymeric substrate, these crowding and blocking effects may substantially reduce the overall catalytic rate. Cellulose hydrolysis by exocellulases processively moving along cellulose chains assembled into insoluble cellulose particles is an example of such a catalytic transport process. The details of the kinetics of cellulose hydrolysis and the causes of the often observed reduction of hydrolysis rate over time are not yet fully understood. Crowding and blocking of enzyme particles are thought to be one of the important factors affecting the cellulose hydrolysis, but its exact role and mechanism are not clear. Here, we introduce a simple model based on an elementary transport process that incorporates the crowding and blocking effects in a straightforward way. This is achieved by making a distinction between binding and non-binding sites on the chain. The model reproduces a range of experimental results, mainly related to the early phase of cellulose hydrolysis. Our results indicate that the combined effects of clustering of binding sites together with the occupancy pattern of these sites by the enzyme molecules play a decisive role in the overall kinetics of cellulose hydrolysis. It is suggested that periodic desorption and rebinding of enzyme molecules could be a basis of a strategy to partially counter the clustering of and blocking by the binding sites and so enhance the rate of cellulose hydrolysis. The general nature of the model means that it could be applicable also to other transport processes that make a distinction between binding and non-binding sites, where crowding and blocking are expected to be relevant.

Enhancing cellular sulfane sulfur through ß-glycosidase-activated persulfide donors: mechanistic insights and oxidative stress mitigation.

Sulfane sulfur species such as persulfides and polysulfides along with hydrogen sulfide protect cells from oxidative stress and are key members of the cellular antioxidant pool. Here, we report perthiocarbamate-based prodrugs that are cleaved by ß-glycosidases to produce persulfide and relatively innocuous byproducts. The ß-glucosidase-activated persulfide donor enhances cellular sulfane sulfur and protects cells against lethality induced by elevated reactive oxygen species (ROS).

Understanding the toxicity of lignin-derived phenolics towards enzymatic saccharification of lignocellulose for rationally developing effective in-situ mitigation strategies to maximize sugar production from lignocellulosic biorefinery.

The lignin-derived phenolics are highly inhibitory toward lignocellulose enzymatic hydrolysis, while the relationship between phenolic structure and inhibitory effect is still not fully understood. In this study, the compositions of phenolics from dilute acid pretreated wheat straw were analyzed and their impact on cellulose hydrolysis was studied. With increase of pretreatment severity, more toxic phenolics were produced from lignin degradation reactions, which were the major contributor to the increased inhibitory effect of pretreatment hydrolysate towards cellulases. Through analyzing the relationship of phenolic structure and their inhibitory effect, a useful model was developed to predict the phenolics-caused inhibition by combining the indexes of electrophilicity and hydrophobicity. Further, through understanding the interactions between phenolics and cellulases, a novel biocomponent alleviator was rationally designed to block the phenolics-cellulase interactions, the degree of improvement of enzymatic hydrolysis reached as high as 135.8%. This study provides directions for developing more effective pretreatment and detoxification methods.

Comparative molecular dynamics simulations identify a salt-sensitive loop responsible for the halotolerant activity of GH5 cellulases.

Halotolerant glycoside hydrolases (GH) have broad application potentials in biorefinery industries. Elucidating the structure-activity relationship underlying the halotolerant catalysis is essential to design superior biocatalysts. Here, we performed molecular dynamics simulations to investigate the structural dynamics of two GH5 cellulases, namely the halotolerant Cel5R and non-halotolerant TfCel5A. Through characterizing the physical properties at different salt concentrations, the results revealed that the overall structures of Cel5R and TfCel5A were marginally affected by the increase in salt concentrations. However, a salt-sensitive loop was identified from both Cel5R and TfCel5A based on its significantly increased flexibility at high salt concentrations. Importantly, compared to TfCel5A the salt-sensitive loop of Cel5R engaged more sodium ions and water molecules around the active site of the enzyme. Besides, the unique residue motif of the salt-sensitive loop in Cel5R formed more intramolecular hydrogen bonds, stabilizing the active architecture of Cel5R at high salt concentrations. Collectively, the structural and dynamic differences may contribute to the various catalytic halotolerance of Cel5R and TfCel5A. These findings provide mechanistic insight into the halotolerant catalysis and will guide the ration design of GH5 cellulases with improved catalytic properties.Communicated by Ramaswamy H. Samy.

Fungus-originated glucanase and monooxygenase genes in creeping bent grass (Agrostis stolonifera L.).

Recent studies have revealed presence of fungus-originated genes in genomes of cool-season grasses, suggesting occurrence of multiple ancestral gene transfer events between the two distant lineages. The current article describes identification of glucanase-like and monooxygenase-like genes from creeping bent grass, as lateral gene transfer candidates. An in silico analysis suggested presence of the glucanase-like gene in Agrostis, Deyeuxia, and Polypogon genera, but not in other species belonging to the clade 1 of the Poeae tribe. Similarly, the monooxygenase-like gene was confined to Agrostis and Deyeuxia genera. A consistent result was obtained from PCR-based screening. The glucanase-like gene was revealed to be ubiquitously expressed in young seedlings of creeping bent grass. Although expression of the monooxygenase-like gene was suggested in plant tissues, the levels were considerably lower than those of the glucanase-like gene. A phylogenetic analysis revealed close relationships of the two genes between the corresponding genes in fungal endophyte species of the Epichloë genus, suggesting that the genes originated from the Epichloë lineage.

Thermostable cellulose saccharifying microbial enzymes: Characteristics, recent advances and biotechnological applications.

Cellulases play a promising role in the bioconversion of renewable lignocellulosic biomass into fermentable sugars which are subsequently fermented to biofuels and other value-added chemicals. Besides biofuel industries, they are also in huge demand in textile, detergent, and paper and pulp industries. Low titres of cellulase production and processing are the main issues that contribute to high enzyme cost. The success of ethanol-based biorefinery depends on high production titres and the catalytic efficiency of cellulases functional at elevated temperatures with acid/alkali tolerance and the low cost. In view of their wider application in various industrial processes, stable cellulases that are active at elevated temperatures in the acidic-alkaline pH ranges, and organic solvents and salt tolerance would be useful. This review provides a recent update on the advances made in thermostable cellulases. Developments in their sources, characteristics and mechanisms are updated. Various methods such as rational design, directed evolution, synthetic & system biology and immobilization techniques adopted in evolving cellulases with ameliorated thermostability and characteristics are also discussed. The wide range of applications of thermostable cellulases in various industrial sectors is described.

Factors Affecting the Catalytic Efficiency and Synergism of Xylanase and Cellulase During Enzymatic Hydrolysis of Birch Wood.

Understanding factors that affect the catalytic efficiency and synergism of enzymes is helpful to enhance the process of bioconversion. In this study, birch wood (BW) was sequentially treated by delignification (DL), deacetylation (DA), and decrystallization (DC) treatments. The physiochemical structures of treated BW were characterized. Moreover, the influences of sequential treatments on the catalytic efficiency and synergism of xylanase and cellulase were studied. DL treatments efficiently improved the conversion of cellulose and xylan. A high degree of synergy (DS) between xylanase and cellulase was produced during hydrolysis of DL-treated BW. DA treatments enhanced xylan conversion but reduced the DS between xylanase and cellulase for xylan hydrolysis, whereas DC treatments enhanced cellulose conversion but reduced the DS between xylanase and cellulase for cellulose hydrolysis. The cellulose conversion of lithium chloride/N,N-dimethylacetamide (LiCl/DMAc)-treated BW (89.69%) was higher than the cellulose conversion of ball milling (BM)-treated BW (81.63%), whereas the xylan conversion of LiCl/DMAc-treated BW (83.77%) was lower than the xylan conversion of BM-treated BW (87.21%). This study showed that the catalytic efficiency and synergism of xylanase and cellulase are markedly affected by lignin hindrance, hemicellulose acetylation, and cellulose crystallization.

Current Status of Mining, Modification, and Application of Cellulases in Bioactive Substance Extraction.

Cellulases have been used to extract bioactive ingredients from medical plants; however, the poor enzymatic properties of current cellulases significantly limit their application. Two strategies are expected to address this concern: (1) new cellulase gene mining strategies have been promoted, optimized, and integrated, thanks to the improvement of gene sequencing, genomic data, and algorithm optimization, and (2) known cellulases are being modified, thanks to the development of protein engineering, crystal structure data, and computing power. Here, we focus on mining strategies and provide a systemic overview of two approaches based on sequencing and function. Strategies based on protein structure modification, such as introducing disulfide bonds, proline, salt bridges, N-glycosylation modification, and truncation of loop structures, have already been summarized. This review discusses four aspects of cellulase-assisted extraction. Initially, cellulase alone was used to extract bioactive substances, and later, mixed enzyme systems were developed. Physical methods such as ultrasound, microwave, and high hydrostatic pressure have assisted in improving extraction efficiency. Cellulase changes the structure of biomolecules during the extraction process to convert them into effective ingredients with better activity and bioavailability. The combination of cellulase with other enzymes and physical technologies is a promising strategy for future extraction applications.

Physical constraints and functional plasticity of cellulases.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.

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.

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general.