Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite -2. Furthermore, the protein has a larger ß5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase.

Recombinant cellobiose dehydrogenase from Thermothelomyces thermophilus: Its functional characterization and applicability in cellobionic acid production.

The fungus Thermothelomyces thermophilus is a thermotolerant microorganism that has been explored as a reservoir for enzymes (hydrolytic enzymes and oxidoreductases). The functional analysis of a recombinant cellobiose dehydrogenase (MtCDHB) from T. thermophilus demonstrated a thermophilic behavior, an optimal pH in alkaline conditions for inter-domain electron transfer, and catalytic activity on cellooligosaccharides with different degree of polymerization. Its applicability was evaluated to the sustainable production of cellobionic acid (CBA), a potential pharmaceutical and cosmetic ingredient rarely commercialized. Dissolving pulp was used as a disaccharide source for MtCDHB. Initially, recombinant exoglucanases (MtCBHI and MtCBHII) from T. thermophilus hydrolyzed the dissolving pulp, resulting in 87% cellobiose yield, which was subsequently converted into CBA by MtCDHB, achieving a 66% CBA yield after 24 h. These findings highlight the potential of MtCDHB as a novel approach to obtaining CBA through the bioconversion of a plant-based source.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9.

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Recombinant Prevotella melaninogenica α-1,3 glucanase and Capnocytophaga ochracea α-1,6 glucanase as enzymatic tools for in vitro degradation of S. mutans biofilms.

Dental biofilms represent a serious oral health problem playing a key role in the development of caries and other oral diseases. In the present work, we cloned and expressed in E. coli two glucanases, Prevotella melaninogenica mutanase (PmGH87) and Capnocytophaga ochracea dextranase (CoGH66), and characterized them biochemically and biophysically. Their three-dimensional structures were elucidated and discussed. Furthermore, we tested the capacity of the enzymes to hydrolyze mutan and dextran to prevent formation of Streptococcus mutans biofilms, as well as to degrade pre- formed biofilms in low and abundant sugar conditions. The percentage of residual biofilm was calculated for each treatment group in relation to the control, as well as the degree of synergism. Our results suggest that both PmGH87 and CoGH66 are capable of inhibiting biofilm formation grown under limited or abundant sucrose conditions. Degradation of pre-formed biofilms experiments reveal a time-dependent effect for the treatment with each enzyme alone. In addition, a synergistic and dose-dependent effects of the combined enzymatic treatment with the enzymes were observed. For instance, the highest biomass degradation was 95.5% after 30 min treatment for the biofilm grown in low sucrose concentration, and 93.8% after 2 h treatment for the biofilm grown in sugar abundant condition. Strong synergistic effects were observed, with calculated degree of synergism of 5.54 and 3.18, respectively and their structural basis was discussed. Jointly, these data can pave the ground for the development of biomedical applications of the enzymes for controlling growth and promoting degradation of established oral biofilms.

Staphylococcus aureus microbial biofilms degradation using cellobiose dehydrogenase from Thermothelomyces thermophilus M77.

This work reports biochemical characterization of Thermothelomyces thermophilus cellobiose dehydrogenase (TthCDHIIa) and its application as an antimicrobial and antibiofilm agent. We demonstrate that TthCDHIIa is thermostable in different ionic solutions and is capable of oxidizing multiple mono and oligosaccharide substrates and to continuously produce H2O2. Kinetics measurements depict the enzyme catalytic characteristics consistent with an Ascomycota class II CDH. Our structural analyses show that TthCDHIIa substrate binding pocket is spacious enough to accommodate larger cello and xylooligosaccharides. We also reveal that TthCDHIIa supplemented with cellobiose reduces the viability of S. aureus ATCC 25923 up to 32 % in a planktonic growth model and also inhibits its biofilm growth on 62.5 %. Furthermore, TthCDHIIa eradicates preformed S. aureus biofilms via H2O2 oxidative degradation of the biofilm matrix, making these bacteria considerably more susceptible to gentamicin and tetracycline.

Enzymatic production of xylooligosaccharides from corn cobs: Assessment of two different pretreatment strategies.

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via "green" and sustainable XOS production.

Biochemical and biophysical properties of a recombinant serine peptidase from Purpureocillium lilacinum.

The industrial uses of peptidases have already been consolidated; however, their range of applications is increasing. Thus, the biochemical characterization of new peptidases could increase the range of their biotechnological applications. In silico analysis identified a gene encoding a putative serine peptidase from Purpureocillium lilacinum (Pl_SerPep), annotated as a cuticle-degrading enzyme. The Pl_SerPep gene product was expressed as a recombinant in a Komagataella phaffii (previously Pichia pastoris) expression system. The enzyme (rPl_SerPep) showed optimal pH and temperature of 8.0 and 60 °C, respectively. Moreover, rPl_SerPep has a higher thermal stability than the cuticle-degrading enzymes described elsewhere. The structural analysis indicated a conformational change in the rPl_SerPep secondary structure, which would allow an increase in catalytic activity at 60 °C. Komagataella phaffii secretes rPl_SerPep with the pro peptide in its inactive form. Low-resolution small-angle X-ray scattering (SAXS) analysis showed little mobility of the pro peptide portion, which indicates the apparent stability of the inactive form of the enzyme. The presence of 20 mM guanidine in the reaction resulted in the maintenance of activity, which was apparently a consequence of pro peptide structure flexibilization.

An annotated checklist of the Amphipoda (Crustacea) of Kuwait, north-western Arabian Gulf.

Kuwaits intertidal and subtidal habitats were surveyed from 2013 to 2016 for amphipods, and earlier records from Kuwait and the rest of the Gulf were compared to provide the first annotated checklist of Amphipoda in Kuwait. A total of 82 sites in mainland and islands were surveyed including intertidal and subtidal zones. The resulting data identified 86 amphipod taxa belonging to 54 genera, 32 families and 3 suborders, including 20 new records to science that are currently being described. A total of 46 amphipod species, 34 genera and 19 families constituted new regional records either for Kuwait or the entire Gulf area. Information on local distribution, occurrence, and habitat are provided for each species. Within the tidal zonation, 50 species inhabited both the intertidal and subtidal areas, 19 were intertidal, 12 subtidal, and 3 species were found in the supratidal zone. Sandy and rocky shores supported the most diverse amphipod assemblages. A gradient of increasing amphipod species richness from north to south along Kuwaits mainland coastline was revealed. The high level of regional endemism supported notable taxonomic distinction of the amphipod fauna of Kuwait from the other Gulf areas. Compositional dissimilarity among amphipod faunas increased with distance within a wide longitudinal gradient across the Gulf, Sea of Oman, and north-western Arabian Sea.

A GH115 α-glucuronidase structure reveals dimerization-mediated substrate binding and a proton wire potentially important for catalysis.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the ß-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Šresolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca2+ ions were identified in the wtsAgu115A structures. One Ca2+ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg2+ or Mn2+ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca2+ resulted in a decrease in activity.

SAXSMoW 3.0: New advances in the determination of the molecular weight of proteins in dilute solutions from SAXS intensity data on a relative scale.

SAXSMoW (SAXS Molecular Weight) is an online platform widely used over the past few years for determination of molecular weights of proteins in dilute solutions. The scattering intensity retrieved from small-angle X-ray scattering (SAXS) raw data is the sole input to SAXSMoW for determination of molecular weights of proteins in liquid. The current updated SAXSMoW version 3.0 determines the linear dependence of the true protein volume on their apparent protein volume, based on SAXS curves calculated for 67,000 protein structures selected from the Protein Data Bank. SAXSMoW 3.0 was tested against 43 experimental SAXS scattering curves from proteins with known molecular weights. Our results demonstrate that most of the molecular weights determined for the nonglycosylated and also for the glycosylated proteins are in good agreement with their expected molecular weights. Additionally, the average discrepancies between the calculated molecular weights and their nominal values for glycosylated proteins are similar to those for nonglycosylated ones.

Polymer ultrastructure governs AA9 lytic polysaccharide monooxygenases functionalization and deconstruction efficacy on cellulose nano-crystals.

Lytic Polysaccharide MonoOxygenases display great variability towards cellulose ultrastructure while performing oxidative functionalization of the polymers. Aiming at employing AA9-LPMOs for isolation of cellulose nano-crystals (CNCs), the ratio between functionalization/crystalline degradation became a crucial parameter. Here are reported the constraints posed by the substrate ultrastructure on the activity of seven different AA9 LPMOs representative of various regioselectivity and substrate affinity: TtAA9E, TaAA9A, PcAA9D, MtAA9A, MtAA9D, MtAA9I-CBM and MtAA9J. The substrate crystallinity and dry matter loading greatly affected the seven AA9s in an enzyme-specific manner, impacting their efficiency for CNCs functionalization purposes. X-ray diffraction pattern analyses were used to assess the cracking efficacy of the enzymatic treatment to de-crystallize CNCs, revealing that those AA9s with minor efficiency in releasing oligosaccharides resulted instead the most disruptive towards the crystal lattice and in reducing the particle sizes. These non-catalytic effects were found comparable with the one caused by the expansin BsEXLX1 enzyme.

Paludibacter propionicigenes GH10 xylanase as a tool for enzymatic xylooligosaccharides production from heteroxylans.

Bioconversion of lignocellulosic biomass into value-added products relies on polysaccharides depolymerization by carbohydrate active enzymes. This work reports biochemical characterization of Paludibacter propionicigenes xylanase from GH10 (PpXyn10A) and its application for enzymatic xylooligosaccharides (XOS) production from commercial heteroxylans and liquor of hydrothermally pretreated corn cobs (PCC). PpXyn10A is tolerant to ethanol and NaCl, and releases xylobiose (X2) and xylotriose (X3) as the main hydrolytic products. The conversion rate of complex substrates into short XOS was approximately 30% for glucuronoxylan and 8.8% for rye arabinoxylan, after only 4 h; while for PCC, PpXyn10A greatly increased unbranched XOS yields. B. adolescentis fermentation with XOS from beechwood glucuronoxylan produced mainly acetic and lactic acids. Structural analysis shows that while the glycone region of PpXyn10A active site is well preserved, the aglycone region has aromatic interactions in the +2 subsite that may explain why PpXyn10A does not release xylose.

Recent advances in the enzymatic production and applications of xylooligosaccharides.

The majority of lignocellulosic biomass on the planet originates from plant cell walls, which are complex structures build up mainly by cellulose, hemicellulose and lignin. The largest part of hemicellulose, xylan, is a polymer with a ß-(1→4)-linked xylose residues backbone decorated with α-D-glucopyranosyl uronic acids and/or L-arabinofuranose residues. Xylan is the second most abundant biopolymer in nature, which can be sustainably and efficiently degraded into decorated and undecorated xylooligosaccharides (XOS) using combinations of thermochemical pretreatments and enzymatic hydrolyses, that have broad applications in the food, feed, pharmaceutical and cosmetic industries. Endo-xylanases from different complex carbohydrate-active enzyme (CAZyme) families can be used to cleave the backbone of arabino(glucurono)xylans and xylooligosaccharides and degrade them into short XOS. It has been shown that XOS with a low degree of polymerization have enhanced prebiotic effects conferring health benefits to humans and animals. In this review we describe recent advances in the enzymatic production of XOS from lignocellulosic biomass arabino- and glucuronoxylans and their applications as food and feed additives and health-promoting ingredients. Comparative advantages of xylanases from different CAZy families in XOS production are discussed and potential health benefits of different XOS are presented.

Differences in Gluco and Galacto Substrate-Binding Interactions in a Dual 6Pß-Glucosidase/6Pß-Galactosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis.

Bacterial glycoside hydrolase 1 (GH1) enzymes with 6-phospho-ß-galactosidase and 6-phospho-ß-glucosidase activities have the important task of releasing phosphorylated and nonphosphorylated monosaccharides into the cytoplasm. Curiously, dual 6-phospho-ß-galactosidase/6-phospho-ß-glucosidase (dual-phospho) enzymes have broad specificity and are able to hydrolyze galacto- and gluco-derived substrates. This study investigates the structure and substrate specificity of a GH family 1 enzyme from Bacillus licheniformis, hereafter known as BlBglC. The enzyme structure has been solved, and sequence analysis, molecular dynamics simulations, and binding free energy calculations offered evidence of dual-phospho activity. Both test ligands p-nitrophenyl-ß-d-galactoside-6-phosphate (PNP6Pgal) and p-nitrophenyl-ß-d-glucoside-6-phosphate (PNP6Pglc) demonstrated strong binding to BlBglC although the pose and interactions of the PNP6Pglc triplicates were slightly more consistent. Interestingly, known specificity-inducing residues, Gln23 and Trp433, bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-BlBglC complex and to the ligand O4 hydroxyl group in the PNP6Pglc-BlBglC complex. Additionally, the BlBglC-His124 residue is a major contributor of hydrogen bonds to the PNP6Pgal O3 hydroxyl group but does not form any hydrogen bonds with PNP6Pglc. On the other hand, BlBglC residues Tyr173, Tyr301, Gln302, and Thr321 form hydrogen bonds with PNP6Pglc but not PNP6Pgal. These findings provide important details of the broad specificity of dual-phospho activity GH1 enzymes.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Unlocking the structural features for the xylobiohydrolase activity of an unusual GH11 member identified in a compost-derived consortium.

The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a diverse set of enzymes including endo-ß-1,4-xylanases, ß-xylosidases, acetylxylan esterases, α-l-arabinofuranosidases, and α-glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo-ß-1,4- and ß-1,3-xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose-bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.

Phytoplankton bloom detection during the COVID-19 lockdown with remote sensing data: Using Copernicus Sentinel-3 for north-western Arabian/Persian Gulf case study.

To examine whether a country-wide COVID-19 lockdown affected phytoplankton development, variability of chlorophyll-a concentrations in the north-western Arabian/Persian Gulf (Kuwait Bay) was investigated using remote sensing instruments Sentinel OLCI between 2018 and 2020 and compared to available in situ collected data. Satellite-retrieved chlorophyll concentrations considerably increased in inshore waters of Kuwait Bay, 1-2 months following the initiation of the 24/7 curfew. The extremely high concentrations of dissolved inorganic nutrients, especially ammonia, and coincided phytoplankton bloom were revealed in June-July 2020 by opportunity field sampling, supporting the satellite-derived bloom detection. Remote sensing operational monitoring with high spatial resolution sensors provides an exceptional opportunity for emergency analysis and decision making in conditions of natural or anthropogenic crises, which forces the development of regional remote sensing algorithms for the shallow marine environment of the Gulf.

Structural and molecular dynamics investigations of ligand stabilization via secondary binding site interactions in Paenibacillus xylanivorans GH11 xylanase.

Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-ß-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

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.