Carbohydrate Binding Module Domain Research Articles

A computational approach to optimising laccase-mediated polyethylene oxidation through carbohydrate-binding module fusion

Plastic pollution is a major global concern to the health and wellbeing of all terrestrial and marine life. However, no sustainable method for waste management is currently viable. This study addresses the optimisation of microbial enzymatic polyethylene oxidation through rational engineering of laccases with carbohydrate-binding module (CBM) domains. An explorative bioinformatic approach was taken for high-throughput screening of candidate laccases and CBM domains, representing an exemplar workflow for future engineering research. Molecular docking simulated polyethylene binding whilst a deep-learning algorithm predicted catalytic activity. Protein properties were examined to interpret the mechanisms behind laccase-polyethylene binding. The incorporation of flexible GGGGS(x3) hinges were found to improve putative polyethylene binding of laccases. Whilst CBM1 family domains were predicted to bind polyethylene, they were suggested to detriment laccase-polyethylene associations. In contrast, CBM2 domains reported improved polyethylene binding and may thus optimise laccase oxidation. Interactions between CBM domains, linkers, and polyethylene hydrocarbons were heavily reliant on hydrophobicity. Preliminary polyethylene oxidation is considered a necessity for consequent microbial uptake and assimilation. However, slow oxidation and depolymerisation rates inhibit the large-scale industrial implementation of bioremediation within waste management systems. The optimised polyethylene oxidation of CBM2-engineered laccases represents a significant advancement towards a sustainable method of complete plastic breakdown. Results of this study offer a rapid, accessible workflow for further research into exoenzyme optimisation whilst elucidating mechanisms behind the laccase-polyethylene interaction.

A thermostable and CBM2-linked GH10 xylanase from Thermobifida fusca for paper bleaching.

Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme’s temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.

Insights into substrate recognition and specificity for IgG by Endoglycosidase S2.

Antibodies bind foreign antigens with high affinity and specificity leading to their neutralization and/or clearance by the immune system. The conserved N-glycan on IgG has significant impact on antibody effector function, with the endoglycosidases of Streptococcus pyogenes deglycosylating the IgG to evade the immune system, a process catalyzed by the endoglycosidase EndoS2. Studies have shown that two of the four domains of EndoS2, the carbohydrate binding module (CBM) and the glycoside hydrolase (GH) domain are critical for catalytic activity. To yield structural insights into contributions of the CBM and the GH domains as well as the overall flexibility of EndoS2 to the proteins' catalytic activity, models of EndoS2-Fc complexes were generated through enhanced-sampling molecular-dynamics (MD) simulations and site-identification by ligand competitive saturation (SILCS) docking followed by reconstruction and multi-microsecond MD simulations. Modeling results predict that EndoS2 initially interacts with the IgG through its CBM followed by interactions with the GH yielding catalytically competent states. These may involve the CBM and GH of EndoS2 simultaneously interacting with either the same Fc CH2/CH3 domain or individually with the two Fc CH2/CH3 domains, with EndoS2 predicted to assume closed conformations in the former case and open conformations in the latter. Apo EndoS2 is predicted to sample both the open and closed states, suggesting that either complex can directly form following initial IgG-EndoS2 encounter. Interactions of the CBM and GH domains with the IgG are predicted to occur through both its glycan and protein regions. Simulations also predict that the Fc glycan can directly transfer from the CBM to the GH, facilitating formation of catalytically competent complexes and how the 734 to 751 loop on the CBM can facilitate extraction of the glycan away from the Fc CH2/CH3 domain. The predicted models are compared and consistent with Hydrogen/Deuterium Exchange data. In addition, the complex models are consistent with the high specificity of EndoS2 for the glycans on IgG supporting the validity of the predicted models.

Sequence Analysis of the Complete Gene for Endoglucanase from Strains of Thermotolerant Bacillus

Three thermotolerant Bacillus isolates from the Philippines – identified as Bacillus subtilis (1280),Bacillus velezensis (1573), and Bacillus amyloliquefaciens (10105) – exhibited cellulase activity in carboxymethylcellulose (CMC) agar plate. Moreover, the expected size (approximately 1,500 bp) putative endoglucanase gene was amplified from all three local isolates. The endoglucanase gene amplified from a Bacillus licheniformis isolate (1320) was also used in the gene sequence analysis. A colorimetric dinitrosalicylic (DNS) acid assay revealed that the total endoglucanase activity of B. velezensis 1573 at 0.168 µM reducing sugar/min is significantly higher than the endoglucanase activity of the B. subtilis 1280 at 0.0673 µM rs/min and B. amyloliquefaciens 10105 at 0.0579 µM rs/min. Endoglucanase gene sequence analysis showed the highest pairwise identity between the endoglucanase gene of B. velezensis 1573 and B. amyloliquefaciens 10105 at 97.4%. The endoglucanase gene of B. licheniformis 1320 has the lowest pairwise identity at 74.6%, 76.2%, and 75.4% with B. amyloliquefaciens 10105, B. subtilis 1280, and B. velezensis 1573, respectively. Predicted amino acid sequences of the endoglucanases revealed variations at the signal peptide, catalytic core (CC) domain, linker, and carbohydrate-binding module (CBM). The endoglucanase of B. subtilis 1280 has a 506D insertion at the CBM domain. None of these variations, however, are located in regions implicated in catalytic reaction and substrate binding. Although the predicted protein structures using MODELLER revealed endoglucanases with good fit and high homology, variations in amino acid sequences may have resulted in the differences in enzyme activity and stability. The study generated gene and protein sequences as well as amplified genes that can be used as a starting template for enzyme improvement by rational design and site-specific mutagenesis or directed evolution using DNA shuffling.

Inter domain interactions influence the substrate affinity and hydrolysis product specificity of xylanase from Streptomyces chartreusis L1105

PurposeThis study investigated the influence of inter-domain interactions on the substrate affinity and hydrolysis product specificity of xylanase.MethodsGenes encoding a GH10 endo-xylanase from Streptomyces chartreusis L1105 xynA and its truncated derivative were cloned and expressed in Escherichia coli. The catalytic activities of the enzyme (xynA) and the derivative xynADCBM, lacking the carbohydrate binding module (CBM), were assessed to evaluate the role of CBM in xynA.ResultsRecombinant xynA (44 kDa) was found to be optimally active on beechwood xylan at 65 °C with pH 7.7, while xynADCBM (34 kDa) exhibited optimal activity at 65 °C with pH 7.2. Additionally, xynA and xynADCBM were found to be highly thermostable at 40–60 °C, each retaining 80% of their original activity after 30 min. The xynADCBM without the CBM domain was highly efficient at hydrolyzing xylan to produce xylobiose (over 67%), which may be because the CBM domain facilitates substrate binding with xylanase. Meanwhile, the xylan hydrolysis efficiency of xynADCBM was higher than that of xynA.ConclusionThese findings showed that the CBM domain with non-catalytic activity has no significant effect on the characteristics of the enzyme at optimum pH and pH tolerance. It has also been suggested that the derivative xynADCBM without CBM components can promote hydrolysis of xylan to yield xylooligosaccharides, which has great potential economic benefits.

Improvement of GH10 family xylanase thermostability by introducing of an extra α-helix at the C-terminal

Xylanase is an important enzyme in industrial applications, which usually require the enzyme to maintain activity in high-temperature condition. In this study, a GH10 family xylanase XynAF0 from a thermophilic composting fungus, Aspergillus fumigatus Z5, was investigated to determine its thermostable mechanism. XynAF0 showed excellent thermostability, which could maintain 50% relative activity after incubation for 1 h at 70 °C. The homologous modeling structure of XynAF0 was constructed and an α-helix composed of poly-threonine has been found in the linker region between the catalytic domain and the carbohydrate-binding module domain. Both the molecular dynamics simulation and the biochemical experiments proved that the α-helix plays an important role in the thermostability of XynAF0. Introducing of this poly-threonine region to the C-terminus of another GH10 family xylanase improved its thermostability. Our results indicated that the poly-threonine α-helix at the C-terminus of the catalytic domain was important for improving the thermophilic of GH10 family xylanases, which provides a new strategy for the thermostability modification of xylanases.

Exploration of the rumen microbial diversity and carbohydrate active enzyme profile of black Bengal goat using metagenomic approach

Black Bengal goats possess a rich source of rumen microbiota that helps them to adapt for the better utilization of plant biomaterial into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiota. Therefore the study was designed in order to explore the taxonomic profile of rumen microbial communities and potential biomass degradation enzymes present in the rumen of back Bengal goat using Illumina Nextseq-500 platform. A total of 83.18 million high-quality reads were generated and bioinformatics analysis was performed using various tools and subsequently, the predicted ORFs along with the rRNA containing contigs were then uploaded to MG-RAST to analyze taxonomic and functional profiling. The results highlighted that Bacteriodetes (41.38–59.74%) were the most abundant phyla followed by Firmicutes (30.59–39.96%), Proteobacteria (5.07–7.61%), Euryarcheaota (0.71–7.41%), Actinobacteria (2.05–2.75%). Genes that encode glycoside hydrolases (GHs) had the highest number of CAZymes, and accounted for (39.73–37.88%) of all CAZymes in goat rumen. The GT families were the second-most abundant in CAZymes (23.73–23.11%) and followed by Carbohydrate Binding module Domain (17.65–15.61%), Carbohydrate Esterase (12.90–11.95%). This study indicated that goat rumen had complex functional microorganisms produce numerous CAZymes, and that can be further effectively utilised for applied ruminant research and industry based applications.

Novel Endotype Xanthanase from Xanthan-Degrading Microbacterium sp. Strain XT11.

Under general aqueous conditions, xanthan appears in an ordered conformation, which makes its backbone largely resistant to degradation by known cellulases. Therefore, the xanthan degradation mechanism is still unclear because of the lack of an efficient hydrolase. Here, we report the catalytic properties of MiXen, a xanthan-degrading enzyme identified from the genus Microbacterium MiXen is a 952-amino-acid protein that is unique to strain XT11. Both the sequence and structural features suggested that MiXen belongs to a new branch of the GH9 family and has a multimodular structure in which a catalytic (α/α)6 barrel is flanked by an N-terminal Ig-like domain and by a C-terminal domain that has very few homologues in sequence databases and functions as a carbohydrate-binding module (CBM). Based on circular dichroism, shear-dependent viscosity, and reducing sugar and gel permeation chromatography analysis, we demonstrated that recombinant MiXen efficiently and randomly cleaved glucosidic bonds within the highly ordered xanthan substrate. A MiXen mutant free of the C-terminal CBM domain partially lost its xanthan-hydrolyzing ability because of decreased affinity toward xanthan, indicating the CBM domain assisted MiXen in hydrolyzing highly ordered xanthan via recognizing and binding to the substrate. Furthermore, side chain substituents and the terminal mannosyl residue significantly influenced the activity of MiXen via the formation of barriers to enzymolysis. Overall, the results of this study provide insight into the hydrolysis mechanism and enzymatic properties of a novel endotype xanthanase that will benefit future applications.IMPORTANCE This work characterized a novel endotype xanthanase, MiXen, and elucidated that the C-terminal carbohydrate-binding module of MiXen could drastically enhance the hydrolysis activity of the enzyme toward highly ordered xanthan. Both the sequence and structural analysis demonstrated that the catalytic domain and carbohydrate-binding module of MiXen belong to the novel branch of the GH9 family and CBMs, respectively. This xanthan cleaver can help further reveal the enzymolysis mechanism of xanthan and provide an efficient tool for the production of molecular modified xanthan with new physicochemical and physiological functions.

Genus-Wide Assessment of Lignocellulose Utilization in the Extremely Thermophilic Genus Caldicellulosiruptor by Genomic, Pangenomic, and Metagenomic Analyses.

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter, Fervidobacterium, Caloramator, and Clostridium). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator, had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic.

Effect of β-mannanase domain from Trichoderma reesei on its biochemical characters and synergistic hydrolysis of sugarcane bagasse.

β-mannanase is a key enzyme for hydrolyzing mannan, a major constituent of hemicellulose, which is the second most abundant polysaccharide in nature. Different structural domains greatly affect its biochemical characters and catalytic efficiency. However, the effects of linker and carbohydrate-binding module (CBM) on β-mannanase from Trichoderma reesei (Man1) have not yet been fully described. The present study aimed to determine the influence of different domains on the expression efficiency, biochemical characteristics and hemicellulosic deconstruction of Man1. The expression efficiency was improved after truncating CBM. Activities of Man1 and Man1ΔCBM (CBM) in the culture supernatant after 168 h of induction were 34.5 and 42.9 IU mL-1 , although a value of only 0.36 IU mL-1 was detected for Man1ΔLCBM (lacking CBM and linker). Man1 showed higher thermostability than Man1ΔCBM at low temperature, whereas Man1ΔCBM had a higher specificity for galactomannan (Km = 2.5 mg mL-1 ) than Man1 (Km = 4.0 mg mL-1 ). Both Man1 and Man1ΔCBM could synergistically improve the hydrolysis of cellulose, galactomannan and pretreated sugarcane bagasse, with a 10-30% improvement of the reducing sugar yield. Linker and CBM domains were vital for mannanase activity and expression efficiency. CBM affected the thermostability and adsorption ability of Man1. The results obtained in the present study should help guide the rational design and directional modification of Man with respect to improving its catalytic efficiency. © 2017 Society of Chemical Industry.

Domain evolution in enzymes of the neopullulanase subfamily.

Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZyme (CAZy) database, the α-amylase family GH13 containing ~30 different enzyme specificities and more than 37 000 sequences represents one of the largest GH families. Earlier, based on a characteristic sequence motif in their fifth conserved sequence region, the two closely related subfamilies, the so-called oligo-1,6-glucosidase and neopullulanase subfamilies, were described. Currently, the two subfamilies cover several CAZy-defined GH13 subfamilies because the α-amylase family GH13 has officially been divided into 41 subfamilies. The subfamily GH13_20 also contains, in addition to neopullulanase, cyclomaltodextrinase and maltogenic amylase. These usually possess the N-terminal starch-binding domain (SBD) classified as the carbohydrate-binding module family CBM34. The present in silico study has been focused on the neopullulanase subfamily in an effort to shed some light on the evolution of its modular arrangement. The main goal was to reveal the evolutionary relationships between the catalytic domain representing the enzyme specificity and the non-catalytic SBDs. The studied set based on the CAZy subfamily GH13_20 and family CBM34 was completed by related amylolytic enzymes, such as α-amylases, glycogen debranching enzymes and amylopullulanases. It finally consisted of 74 mostly biochemically characterized GH13 enzymes. The analysed sequences were divided into nine groups based on the presence of various carbohydrate-binding module domains (CBM20 and CBM48 in addition to CBM34). A special unique domain arrangement was revealed in the the α-amylase from Bacillus sp. AAH-31, in which the three consecutive SBDs (i.e. CBM20, CBM48 and CBM34, in that order) are present at its N-terminus.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Determining the Role of Lafora Disease Patient Mutations on Glycogen Dephosphorylation.

Lafora Disease (LD) is fatal, congenital neurodegenerative epilepsy caused by mutations in the gene that encodes the protein laforin. These mutations disrupt glycogen metabolism in cells causing accumulations of malformed glycogen inclusions called Lafora Bodies (LB). LBs inhibit normal neuronal functions, leading to epilepsy, dementia, and death by age 25. Laforin contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. To date, more than 40 laforin point mutations have been identified in LD patients. However, how each of these mutations leads to LB formation and/or how they impact glycogen dephosphorylation is not known. Our lab recently determined the 3‐dimensional structure of laforin using X‐ray crystallography. The laforin X‐ray crystal structure reveals that these patient point mutations localize in four different regions within the DSP and CBM domains suggesting multiple possible disease mechanisms. To determine the role of each of these mutations on glycogen dephosphorylation, we employed a generic pNPP phosphatase activity assay and a biologically relevant assay utilizing glycogen to measure glycogen phosphatase activity. The glycogen phosphatase assay was very specific and faithfully reported both wildtype and mutant laforin phosphatase activity. Mutations that are located in the CBM‐DSP interface and the DSP‐DSP dimer interface resulted in 50‐80% decrease, suggesting the importance of these regions in optimal laforin activity. We found a 70‐90% decrease in glycogen phosphatase activity for mutations that are localized in the CBM binding site and the DSP active site.

Characterisation and functional importance of β-1,4-endoglucanases from the potato rot nematode, Ditylenchus destructor

Plant-parasitic nematodes have developed a series of enzymes to degrade the rigid plant cell wall; β-1,4-endoglucanase is a very important component. Ditylenchus destructor is a migratory endoparasite for which few molecular data have been published. Two novel β-1,4-endoglucanases (Dd-eng-1a and Dd-eng-2) were cloned and characterised from D. destructor. The DD-ENG-1A putative protein consists of a signal peptide, a catalytic domain and a carbohydrate-binding module (CBM). By contrast, the CBM domain is absent from DD-ENG-2. The exon/intron structure and phylogenetic tree indicate that both cellulase genes could have evolved from common ancestral genes. Southern blotting confirmed that the β-1,4-endoglucanases were of nematode origin and a member of a small multi-gene family. In situ hybridisation localised the expression of Dd-eng-1a and Dd-eng-2 to the subventral pharyngeal glands. RT-PCR showed that both genes were expressed in the adult female and second-stage juvenile. The stylet secretions of D. destructor showed clear cellulase activity in carboxymethylcellulose (CMC) plate assay, and similar results were observed in total homogenates and DD-ENG-1A and DD-ENG-2 recombinant proteins. These results demonstrated that D. destructor can produce and secrete functional cellulases. Silencing the putative β-1,4-endoglucanases by double-stranded RNA (dsRNA) resulted in an average decrease in infection of 50%. Successful RNAi in vitro was demonstrated in this study, which confirmed that Dd-eng-1a and Dd-eng-2 play important roles in nematode parasitism.

Exploring the Structural Insights on Human Laforin Mutation K87A in Lafora Disease—A Molecular Dynamics Study

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

A kinetic model for the enzymatic action of cellulase.

We develop a mechanochemical model for the dynamics of cellulase, a two-domain enzyme connected by a peptide linker, as it extracts and hydrolyzes a cellulose polymer from a crystalline substrate. We consider two random walkers, representing the catalytic domain (CD) and the carbohydrate binding module (CBM), whose rates for stepping are biased by the coupling through the linker and the energy required to lift the cellulose polymer from the crystalline surface. Our results show that the linker length and stiffness play a critical role in the cooperative action of the CD and CBM domains and that, for a given linker length, the steady-state hydrolysis shows a maximum at some intermediate linker stiffness. The maximum hydrolysis rate corresponds to a transition of the linker from a compressed to an extended conformation, where the system exhibits maximum fluctuation, as measured by the variance of the separation distance between the two domains and the dispersion around the mean hydrolysis speed. In the range of experimentally known values of the parameters of our model, improving the intrinsic hydrolytic activity of the CD leads to a proportional increase in the overall hydrolysis rate.