A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production

A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production Peter T. Oluwasola, Oladipo O. Olaniyi, Olusola T. Lawal, Felix A. Akinyosoye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7933920/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The ruminant gut microbiome represents a valuable source of lignocellulolytic enzymes, particularly β-mannanases that hydrolyze mannan into fermentable sugars. However, few studies have characterized β-mannanases from Clostridium species with respect to their catalytic stability and potential for biofuel production from agro-industrial residues. Results β-Mannanase from Clostridium chauvoei was purified from the crude extract through ammonium sulfate precipitation, dialysis, and ion-exchange and size-exclusion chromatography, achieving a 36% yield and 9-fold purification. The enzyme exhibited optimal activity at pH 4.0 and 60°C and maintained stability over a broad pH range (2–12 for 6 h) and temperature range (30–80°C). Enzyme activity was enhanced by Mg²⁺, Zn²⁺, and Mn²⁺, while inhibited by EDTA, SDS, and cysteine; among organic solvents, only formaldehyde stabilized the enzyme. It showed a half-life of 216 min at 70°C, with thermodynamic parameters ΔG = 63 kJ/mol, ΔH = 23 kJ/mol, and ΔS = − 134.4 J/mol·K. Kinetic constants (Km = 30.7 mg/mL, Vmax = 7.88 µmol/mL/min) indicated strong substrate affinity and catalytic efficiency. Application of the purified enzyme to pretreated palm kernel substrate yielded substantial biobutanol (55 g/L), ethanol (60 g/L), and acetone (70 g/L) confirmed by GC–MS and FTIR analyses. Conclusion This study highlights a novel thermostable and pH-tolerant β-mannanase from C. chauvoei capable of efficiently hydrolyzing lignocellulosic biomass into fermentable sugars for acetone-butanol-ethanol (ABE) production. The enzyme’s robust catalytic properties and high saccharification efficiency position it as a promising biocatalyst for sustainable biofuel production and other industrial bioprocess applications. Ruminant gut microbiota thermostable and pH-stable β-mannanase palm kernel cake lignocellulosic biomass saccharification biobutanol production Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Mannanases are hydrolases that catalyze the hydrolysis of mannosidic bonds in mannan, a major component of plant cell walls (Kalyani et al., 2021). Efficient mannan degradation requires the coordinated action of β-mannanases (EC 3.2.1.78), β-mannosidases (EC 3.2.1.25), and β-glucosidases (EC 3.2.1.21) (Dhiman et al., 2019). β-Mannanases cleave glycosidic bonds to produce β-1,4-manno-oligosaccharides, whereas β-mannosidases release mannose from mannobiose (Tahir et al., 2024). Many bacterial and fungal species produce β-mannanase, which has industrial potential in processes such as hemicellulose extraction from pulp and paper (Dawood and Ma, 2020; Favaro et al., 2020). Mannan is a complex polysaccharide and a major hemicellulose component. Its primary forms include mannose, glucomannan, galactomannan, and galactoglucomannan, each with distinct structures and β-1,4 linkages (Badejo et al., 2021; Zuo et al., 2021; Olaniyi et al., 2023). Glucomannan, a heteropolymer of glucose and mannose, is abundant in hardwood and softwood, while galactomannan contains a β-1,4 D-mannose backbone with galactose side groups (Badejo et al., 2021). Plant and animal tissues contain inactive β-mannanases that are activated in the presence of mannan substrates, while microbial β-mannanases are secreted extracellularly upon induction (Dawood et al., 2020; Olaniyi et al., 2023). The rumen of ruminants serves as a reservoir of hemicellulolytic bacteria capable of efficiently metabolizing lignocellulosic materials due to symbiosis with the host (Badejo et al., 2021; Zhao et al., 2022). This makes ruminant guts natural bioreactors for lignocellulose degradation, producing enzymes such as xylanase, mannanase, and cellulase (Badejo et al., 2021; de Souza et al., 2021; Olaniyi et al., 2023). Despite this potential, the enzymatic capabilities of ruminant gut-derived bacteria, particularly Clostridium chauvoei, remain underexplored. The need for sustainable biofuels is driven by the environmental impact of fossil fuels, including greenhouse gas emissions and global warming (Jogdand, 2020; Aryal et al., 2022). β-Mannanase plays a pivotal role in converting hemicellulose into oligomers and mannose, which can be fermented into bioethanol or biobutanol (Mamo, 2020). Lignocellulosic biomass requires chemical pretreatment due to its recalcitrance, followed by enzymatic hydrolysis to release fermentable sugars (Bhatia et al., 2020; Zhang et al., 2022). While Saccharomyces cerevisiae is widely used for first-generation bioethanol production, thermophilic anaerobic bacteria can achieve higher biobutanol yields from inexpensive agricultural residues (Zabermawi et al., 2022). Given this context, this study addresses the gap in knowledge regarding the biochemical properties of β-mannanase from ruminant gut C. chauvoei and its potential for biofuel production. Specifically, we purified and characterized β-mannanase from C. chauvoei isolated from the cow rumen and evaluated its efficacy in producing biofuel from palm kernel substrate, highlighting its industrial relevance. Methods Culture-based identification of anaerobic bacteria from the ruminant gastrointestinal tract Anaerobic bacteria from the gut of ruminants were isolated by inoculating sterile tryptone-yeast extract-acetate (TYA) medium with 10% (v/v) filtered ruminal fluid, followed by anaerobic incubation for 18 hours, and were used as the source of inoculum. After incubation, 1 mL of the inoculum was pour-plated onto solidified TYA agar medium and incubated under anaerobic conditions at 30°C for 1–2 days. The composition of TYA medium was as follows (g/L): tryptone, 6; yeast extract, 2; ammonium acetate, 3; KH₂PO₄, 0.5; MgSO₄·7H₂O, 0.3; and FeSO₄·7H₂O, 0.01 (Al-Shorgani et al., 2014). Emergent colonies were enumerated and reported as colony-forming units per milliliter (CFU/mL). Pure isolates were obtained from the mixed cultures by repeated streaking on the same agar medium and incubated under the conditions described above. Unknown pure isolates were tentatively identified based on cultural and morphological characteristics, as well as selected biochemical tests, following standard bacteriological procedures (Sari et al., 2016). The morphological features and biochemical reactions of the isolates were compared with descriptions in standard bacteriological atlases. Screening and Assay of β-Mannanase-Producing Isolates Qualitative screening The bacterial isolates were screened for β-mannanase production using tryptone-yeast extract-acetate (TYA) agar supplemented with 1.5% (w/v) locust bean gum (LBG). The medium composition was as previously described, with the addition of 1.5 g/100 mL LBG and agar. Pure isolates were streaked individually onto the sterile TYA-LBG agar plates and incubated under anaerobic conditions. Colonies that developed visible clearance zones around their growth were considered mannolytic anaerobic bacteria. The diameters of the hydrolytic zones were measured in millimeters and recorded (Raita et al., 2016). Mannolytic isolates were sub-cultured on potato glucose (PG) medium and stored at 4°C for further analysis. The PG medium contained (g/L): potato, 150; glucose, 10; CaCO₃, 3; and (NH₄)₂SO₄, 0.5. Quantitative screening Quantitative β-mannanase activity was determined in TYA broth supplemented with 1.5% (w/v) LBG (without agar). The medium was inoculated with the isolates and incubated under the same anaerobic conditions (Shukor et al., 2016). After incubation, the culture broth was centrifuged at 6,000 rpm for 20 min, and the supernatant (crude enzyme extract) was collected and stored for enzymatic assay. Enzyme assay β-Mannanase activity was assayed using a reaction mixture containing 0.5 mL of crude enzyme and 0.5 mL of LBG substrate solution (1 g LBG dissolved in 50 mM potassium phosphate buffer, pH 6.8). The mixture was incubated at 45°C for 30 min in a water bath (Olaniyi et al., 2023). The reaction was terminated by adding 1 mL of dinitrosalicylic acid (DNSA) reagent, followed by boiling for 10 min. After cooling, absorbance was measured at 540 nm. One unit (U) of β-mannanase activity was defined as the amount of enzyme that liberates 1 µmol of mannose per minute under the assay conditions. Purification of β-Mannanase The crude enzyme extract was subjected to stepwise purification. Ammonium sulfate was gradually added to the extract at 4°C to induce protein precipitation. The mixture was left overnight at 4°C, after which the precipitate was collected by refrigerated centrifugation at 15,000 × g for 10 min. The pellet was dissolved in 100 mM potassium phosphate buffer (pH 6.8) and dialyzed extensively against the same buffer using a 3,500 Da molecular weight cut-off dialysis membrane to remove residual ammonium sulfate. The dialyzed enzyme solution was further purified by ion-exchange chromatography on a DEAE-Sephacel column equilibrated with 100 mM potassium phosphate buffer (pH 6.8). Bound proteins were eluted with a linear gradient of NaCl in the same buffer. Fractions showing absorbance at 280 nm were collected and assayed for β-mannanase activity. Active fractions were pooled and subjected to size-exclusion chromatography on a Sephadex G-100 column equilibrated with the same buffer. Fractions were monitored at 280 nm, and those exhibiting β-mannanase activity were combined. The purified β-mannanase fractions were concentrated and stored at 4°C until further use. Protein concentration at each purification step was determined by absorbance at 280 nm, and enzymatic activity was verified using the standard β-mannanase assay (Olaniyi et al., 2023). β-Mannanase molecular mass estimation The molecular mass of β-mannanase was determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using the Laemmli method (Laemmli, 1970). Resolving (12.5% acrylamide; Tris buffer, pH 8.8) and stacking (4% acrylamide; Tris buffer, pH 6.8) gels were prepared. The gel mixture contained SDS, ammonium persulfate, and N,N,N′,N′-tetramethylethylenediamine (TEMED) as polymerizing agents. Enzyme samples were mixed with 5× sample buffer containing SDS, β-mercaptoethanol, and Coomassie dye, followed by heating at 100°C for 60 s. Electrophoresis was carried out at 80 V using a vertical electrophoresis unit. After separation, the gels were stained with Coomassie Brilliant Blue R-250 (prepared in methanol:water:acetic acid, 5:5:1) and subsequently destained with an acetic acid solution (7% v/v in distilled water). The gels were then photographed to visualize protein bands. Purified β-mannanase: Physicochemical, thermodynamic, and kinetic properties Determination of optimum pH and pH stability of β-mannanase The effect of pH on β-mannanase activity was determined using different buffer systems: 0.1 M glycine/HCl (pH 2.0–3.0), 0.1 M sodium acetate buffer (pH 4.0–5.0), 0.1 M phosphate buffer (pH 6.0–7.0), 0.1 M Tris-HCl (pH 8.0–10.0), and 0.1 M Tris/NaOH (pH 11.0–12.0). Enzyme activity was measured under standard assay conditions in each buffer to determine the optimum pH. For pH stability, the enzyme was incubated in the same buffer systems without substrate, and aliquots were withdrawn at 30-minute intervals for up to 3 h. Residual activity was assayed under standard conditions to evaluate enzyme stability across the pH range. Determination of optimum temperature and thermal stability of purified β-mannanase The optimum temperature for β-mannanase activity was evaluated by performing the standard enzyme assay at temperatures ranging from 30 to 90°C, in 10°C increments, using 0.1 M phosphate buffer (pH 6.8) as the reaction medium. Enzyme activity at each temperature was determined following the standard β-mannanase assay. For thermal stability, the enzyme was incubated at the same temperature range without substrate, and aliquots were withdrawn at 30-minute intervals for up to 3 hours. Residual activity was measured under standard assay conditions to assess enzyme stability at different temperatures. Effect of metal ions, inhibitors, organic solvents, and surfactants on β-mannanase activity The influence of various additives on β-mannanase activity was investigated. The tested compounds included chloride salts of metal ions (K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Zn²⁺, and Cu²⁺), chelating agent (EDTA), chaotropic agents (urea and sodium dodecyl sulfate, SDS), sodium azide, organic solvents (acetic acid, acetone, formaldehyde, and dimethyl sulfoxide, DMSO), and surfactants (Tween-20 and Triton X-100). Each compound was tested at final concentrations of 1, 5, and 10 mM. The assay mixtures containing substrate (mannan), additives, and purified enzyme were prepared in 0.1 M phosphate buffer (pH 6.8) and incubated under standard assay conditions. Enzyme activity was determined using the β-mannanase assay, and the relative activity was calculated in comparison with the control (without additives). Estimation of Km and Vmax of β-mannanase The kinetic parameters of the purified enzyme, Km and Vmax, were determined using LBG as substrate at varying concentrations (0.1–1% w/v) in 50 mM phosphate buffer (pH 6.8). The initial reaction velocities were measured following the standard β-mannanase assay procedure. The reciprocal values of the initial velocities (1/V) and substrate concentrations (1/[S]) were plotted according to the Lineweaver–Burk method (Lineweaver and Burk, 1934). From the double reciprocal plot, the kinetic constants Km and Vmax were estimated. Thermodynamic studies of purified β-mannanase The thermal stability of purified β-mannanase was evaluated by incubating enzyme aliquots prepared in phosphate buffer (pH 6.8) at different temperatures, in 10°C intervals, using a water bath. For each temperature, samples were withdrawn at 0 min and subsequently every 30 min for up to 3 h. Enzyme activity was determined using the standard β-mannanase assay, and residual activity was expressed as a percentage of the initial (0 min) activity. The first-order inactivation constant (k) was calculated from the slope of the first-order inactivation equation: Residual activity (%) = C/C o ​×100 (i) ln( C/C o ) = − kt (ii) where C is the enzyme activity at time t , and C 0 is the initial enzyme activity (0 min). The rate constant k (min⁻¹) was obtained from the slope of the linear plot of ln( C/C o ) against incubation time. The enzyme half-life (t₁/₂) was calculated using: t₁ /₂= ln(2)/ k (iii) The decimal reduction time (D-value) was determined as: D = ln(10)/ k (iv) Plotting log(D) against temperature provided the Z-value, defined as the temperature change required to reduce the D-value by one log unit. The negative reciprocal of the slope was taken as the Z-value. The temperature dependence of the inactivation rate constant was evaluated using the Arrhenius equation: ln k = Ea/RT + C (v) Thermodynamic parameters, including Gibbs free energy change (Δ G ), enthalpy change (Δ H ), and entropy change (Δ S ), were calculated from the activation energy ( Ea ​) and the Arrhenius rate constant ( k ) using the following relationships: Δ H = Ea ​- RT (vi) Δ G = − RT ln kh / k B T (vii) Δ S = Δ H − Δ G/T (viii) where k B is Boltzmann’s constant (1.3806 × 10 − 23 J/K), h is Planck’s constant (6.6260 × 10 − 34 J·s), 𝑅 universal gas constant, and T is the absolute temperature (K). Saccharification and Bioethanol Production Preparation of Palm Kernel Cake and Corn Cob for Chemical Pretreatment Defatted palm kernel cake (PKC) was prepared using a Soxhlet extractor with petroleum ether, followed by drying, milling, and sieving. Approximately 172 g of the defatted PKC was soaked in 200 mL of 1% (v/v) H₂SO₄ and boiled at 160°C for 20 min in a covered stainless-steel pan. The mixture was subsequently autoclaved at 121°C for 60 min. Weight loss was determined before and after autoclaving. After cooling to room temperature, the pH of the hydrolysate was adjusted to 5.0 using 400 g/L NaOH. Purified β-mannanase (6 mL) was added to 1 L of the pretreated substrate, and the reaction mixture was incubated at 45°C for 72 h with agitation at 80 rpm to achieve enzymatic saccharification. The resulting mixture was centrifuged at 3,300 × g for 10 min, and the supernatant, containing the released fermentable sugars, was collected for subsequent bioethanol production (Shukor et al., 2016). Detoxification of Hydrolysates Inhibitory compounds present in the hydrolysates derived from pretreated palm kernel cake (PKC), corn cobs, and the enzyme production medium were neutralized following a modified chemical detoxification procedure. The pH of each hydrolysate was first adjusted to 10.0 using Ca(OH)₂, after which Na₂SO₃ (1 g/L) was added. The mixture was heated to 90°C with intermittent stirring, then allowed to cool to room temperature. The pH was subsequently readjusted to 7.0 using concentrated H₂SO₄. Activated charcoal (1 g/L) was added to adsorb residual inhibitory compounds, and the resulting mixture was filtered through a 0.2 mm membrane to obtain a clear supernatant. The concentrations of fermentable sugars, including glucose, mannose, xylose, and mannooligosaccharides (MOS) were determined using standard assay methods and expressed in mg/mL (De Barros et al., 2024). Biobutanol Production Fermentation was performed in 250 mL bottles with a working volume of 100 mL. The clarified supernatant was mixed with filtered hydrolysates obtained from the chemically pretreated samples. Clostridium chauvoei , previously cultured in TYA medium for 18 h, was aseptically inoculated into the fermentation medium and incubated anaerobically for 5 days. The concentrations of butanol, acetone, and ethanol were quantified using gas chromatography according to the method described by Shukor et al. (2016). The functional groups of alcohols present in the fermentation products were analyzed using Fourier-transform infrared (FTIR) spectroscopy (Phwan et al., 2019). Evaluation of Bioethanol Production by Colorimetric Method A standard stock solution of ethanol (1.6 mg/mL) was prepared in a 50 mL volumetric flask. To this, 5 mL each of sodium dichromate solution, pH 4.3 acetate buffer, and 1 N sulfuric acid were added sequentially. The resulting mixture produced a green-colored reaction product, which was gently shaken for 1 min and then allowed to stand at room temperature for 120 min. The absorbance of the mixture was measured at 578 nm using a UV–Visible spectrophotometer. Each sample was prepared and analyzed in triplicate. The ethanol concentration in the samples was determined from a standard calibration curve as described by Datta et al. (2018) and expressed as: Percentage of ethanol/acetone/butanol in sample (%) = ( Cs / Cu ) ( Au / As ) x 100 where Cs = Concentration of standard, Cu = Concentration of sample as per Labeled Claim, Au = Absorbance of standard, As = Absorbance of sample. Results Biochemical tests on bacterial isolates from the ruminant gut Results of biochemical tests on bacterial isolates are presented in Table S1 . Twenty (20) isolates were selected from culture plates and assigned unique codes. Biochemical characterization identified all isolates as belonging to the genus Clostridium . The predominant species detected were Clostridium sphenoide , C. clostridiforme , C. perfringens , C. chauvoei , and C. novyi type A. Screening of bacterial isolates for β-mannan-degrading ability The β-mannan-degrading ability of the bacterial isolates is shown in Fig. 1 . Isolate D3b ( C. chauvoei ) exhibited the highest activity, with β-mannanase activity of 92.8 U/mL and 73 mg/mL fermentable sugar released. Isolates B3 and B1 also demonstrated notable activity, with β-mannanase activities of 60 and 54.69 U/mL, and corresponding fermentable sugar concentrations of 60 and 64.59 mg/mL, respectively. Moderate β-mannan degradation was observed in isolates A2 ( C. clostridiforme ), B4a, E2 ( C. perfringens ), and A1 ( C. sphenoide ), exhibiting enzyme activities of 46, 42.7, 33, and 30 U/mL and fermentable sugar concentrations of 56.7, 59.6, 72, and 60 mg/mL, respectively. Minimal β-mannan-degrading activity was recorded for isolates A3a ( C. perfringens ), F2 ( C. perfringens ), and E1 ( C. chauvoei ), with enzyme activities of 3.5, 2.6, and 0.9 U/mL and fermentable sugar concentrations of 44, 15, and 9 mg/mL, respectively. Clostridium chauvoei β-mannanase purification profile The purification profile of β-mannanase from C. chauvoei is summarized in Table 1 . The crude enzyme exhibited a total activity of 1,400 U/mL and total protein concentration of 432.4 mg/mL, corresponding to a specific activity of 3.24 U/mg. Ammonium sulfate precipitation of the crude enzyme, followed by dialysis, increased the specific activity to 8.76 U/mg, representing a 2.7-fold purification with 62.7% recovery. Subsequent ion-exchange chromatography of the dialysate on DEAE-Sephadex resin yielded a single peak of enzymatic activity (Fig. 2 a), achieving 6.3-fold purification with approximately 38% recovery. Further purification by gel-filtration chromatography on Sephadex G-100 resin produced a single activity peak (Fig. 2 b), with 8.8-fold purification and 36% yield. Table 1 Purification summary of β-mannanase from Clostridium cnauvoei isolated from ruminant gut Purification step Vol. (mL) Enz. Activity (U/mL) Protein conc. (mg/mL) Total Activity (U) Total Protein (mg) Specific Activity (U/mg) Purification Fold Yield (%) Crude Extracts 400 3.5 1.081 1400 432.4 3.2 1 100 (NH 4 ) 2 SO 4 PPT 65 13.5 1.542 877.8 100.2 8.8 2.7 62.7 DEAE Sephacel 22 24.3 1.183 534.1 26.02 20.5 6.3 38.2 Sephadex G-100 25 20.1 0.704 503.6 17.6 28.6 8.8 35.97 Molecular mass of Clostridium chauvoei β-mannanase The molecular weight of the purified β-mannanase was estimated at 42 kDa, as shown in Figure S2 . Clostridium chauvoei β-mannanase properties Temperature optimum and stability The effect of temperature on the activity of purified β-mannanase from C. chauvoei is shown in Figure S3a . The enzyme exhibited maximum activity at 60°C, with activity increasing progressively up to this optimum. High relative activities of 88.6%, 81.5%, and 73% were observed at 70, 80, and 90°C, respectively. Thermal stability of the enzyme is presented in Fig. 3 a. The purified β-mannanase was maximally stable at 30 and 40°C, retaining 91% and 79% residual activity after 1 h and 3 h incubation, respectively. High residual activities of 77–69% were maintained between 50–70°C after 1 h, while 44% activity remained at 80°C. After 3 h, residual activities were 63% and 50% at 50 and 60°C, respectively, decreasing to 25% at 70°C and complete inactivation occurring between 80 and 90°C. pH optimum and stability The effect of pH on the activity of purified β-mannanase from C. chauvoei is shown in Figure S3b . The enzyme exhibited maximum activity at acidic pH 4, with activity sharply declining between pH 5 and 7. Relative activities of 74–58% were observed at alkaline pH 8–10, and 47% activity was retained at pH 11. The pH stability profile is presented in Fig. 3 b. The enzyme showed maximum stability at alkaline pH 9–11, retaining 95% and 87% of its original activity after 1 and 3 h, respectively. It also maintained 88–70% residual activity between pH 2–4 and 6–9 after 2 h. Lower residual activities of 63% were recorded at pH 2, 4, and 5, and 44–51% at pH 3 and 6–9. Effect of metal ions on β-mannanase activity The effect of metal ions on the activity of purified β-mannanase from C. chauvoei is shown in Fig. 4 a. Enzymatic activity was significantly enhanced by Mg²⁺, with a 41% increase at 5 mM and a 22% increase at 10 mM. Zn²⁺ and K⁺ also enhanced enzyme activity at 5 mM, while Mn²⁺ showed stimulatory effects at both 5 and 10 mM. In contrast, Ca²⁺ and Cu²⁺ exhibited inhibitory effects, reducing enzyme activity by 35% and 25%, respectively, at 1 mM. Both ions caused mild inhibition at higher concentrations of 5 and 10 mM. Effect of inhibitors on β-mannanase activity The effect of inhibitors on the activity of β-mannanase from C. chauvoei is shown in Fig. 4 b. Enzymatic activity was largely unaffected by EDTA, urea, and sodium azide at 5 mM. At 10 mM, EDTA caused slight inhibition, while urea and sodium azide resulted in 55% and 35% reductions in activity, respectively. SDS and cysteine strongly inhibited β-mannanase activity in a concentration-dependent manner. Effect of organic solvents and detergents on β-mannanase activity The effect of organic solvents and detergents on the activity of β-mannanase from C. chauvoei is shown in Fig. 5 . Formaldehyde did not affect enzyme activity at any of the concentrations tested. In contrast, acetic acid, acetone, and dimethyl sulfoxide (DMSO) strongly inhibited β-mannanase activity across all concentrations. Detergents, including Tween-20 and Triton X-100, also caused strong inhibition of enzymatic activity at all concentrations examined. Kinetic parameters of the purified β-mannanase The kinetic parameters of the purified β-mannanase from C. chauvoei are presented in Figure S4 . The K m and V max were estimated as 30.7 mM and 7.88 mg/mL/min respectively. Thermal inactivation of purified β-mannanase The thermal inactivation profile of purified β-mannanase is presented in Figure S5a . A plot of the logarithm of residual activity versus incubation time at different temperatures showed a linear relationship, consistent with first-order kinetics, with R² values ranging from 0.9157 to 0.9962. Kinetic parameters, half-life, and D-value of purified β-mannanase The kinetic parameters of the purified β-mannanase are presented in Table 2 a. Inactivation rate constants (k) were determined from the slopes of plots of residual activity versus incubation time, ranging from 5 × 10⁻⁴ to 3.2 × 10⁻² min⁻¹. Corresponding half-life (t₁/₂) values were estimated to range from 1,386 to 217 min, while D-values ranged from 4,605 to 720 min. Table 2 a : Kinetic values for the heat inactivation of β-mannanase Temp ( o C) Temp (K) Thermal inactivation, k − 1 (min) Half-life t 1/2 (min) D-value (min) R 2 log D 25 298 0.0005 1386.29 4605.17 0.9162 3.66 30 303 0.0006 1155.25 3837.64 0.9962 3.58 40 313 0.001 693.147 2302.59 0.0693 3.36 50 323 0.0017 407.734 1354.46 0.9221 3.13 60 333 0.0018 385.082 1279.21 0.9817 3.11 70 343 0.0032 216.608 719.558 0.9157 2.86 Table 2 b : Thermodynamic properties of the purified β-mannanase Temp ( o C) Temp (K) ΔH (KJ/mol) ΔS (J/mol/K) ΔG (KJ/mol) 25 298 20.536 -134.94 60.748 30 303 20.4944 -134.8 61.3392 40 313 20.4112 -134.53 62.5196 50 323 20.3281 -134.27 63.6973 60 333 20.245 -134.02 64.8725 70 343 20.1618 -133.77 66.0451 Average 20.3627 -134.39 63.2036 Arrhenius plot for β-mannanase inactivation The Arrhenius plot for the thermal inactivation of purified β-mannanase is shown in Figure S5b . The dependence of the inactivation rate constants on temperature was fitted to the Arrhenius equation, yielding a strong correlation with R² = 0.9735. Temperature dependence of decimal reduction (D-value) for β-mannanase inactivation The relationship between the D-value and inactivation temperature of purified β-mannanase is presented in Figure S5c . The activation energy (Eₐ) and Z-value (the temperature required to reduce the D-value by one logarithmic cycle) were calculated as 23 kJ/mol and 57°C, respectively. Thermodynamic parameters of β-mannanase The thermodynamic parameters of purified β-mannanase are presented in Table 2 b. The average values obtained were 63.2 kJ/mol for Gibbs free energy change (Δ G ), 20.3 kJ/mol for enthalpy change (Δ H ), and − 134.4 J/mol·K for entropy change (Δ S ). Bioethanol production by Clostridium chauvoei Quantification of bioethanol and other biofuels The production of butanol, ethanol, and acetone (ACE; acetone–ethanol–butanol) by C. chauvoei is summarized in Table 3 . The estimated quantities of biobutanol, ethanol, and ACE were 55.00%, 60.15%, and 70.10%, respectively. Table 3 Quantity of biofuel produced by Clostridium chauvoei isolated from the cow rumen Biofuel Quantity (g/L) Ethanol 60.15 ± 0.36 Butanol 55.00 ± 0.085 ABE 70.10 ± 0.41 Gas Chromatography and Fourier Transform Infrared (FTIR) Analyses The presence of butanol in the fermentation products was confirmed using gas chromatography (GC), as illustrated in Fig. 6 b and summarized in Table 4 . Several volatile compounds were detected, among which compounds 3, 7, 11, and 15-tetramethyl-2-hexadecen-1-ol corresponded to the expected biofuel product (chromatogram shown in Figure S6 ). Fourier Transform Infrared (FTIR) spectroscopy was employed to further characterize the functional groups present in the bioethanol sample ( Table S2 & Fig. 6 a). The characteristic absorption bands observed at 3,337 cm⁻¹ (O–H stretching) and 667 cm⁻¹ (C–O–H bending) confirmed the presence of hydroxyl functional groups typical of alcohols, corresponding to the fingerprint region of butanol (600–700 cm⁻¹). In addition, a band between 1,120 and 1,080 cm⁻¹, centered at 1,097 cm⁻¹, indicated C–C–CHO stretching vibrations associated with secondary or tertiary alcohols. Table 4 Gas chromatography analysis of biofuel produced from Clostridium cnauvoei S/N Retention Time Area (%) Name 1 7.933 4.35 alfa.-Copaene, Copaene, alpha.-Cubebene 2 8.511 16.59 Caryophyllene 3 8.958 6.28 Humulene, 1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z- Humulene 4 9.845 tetramethyl-, Z,Z,Z- Humulene, ,8a-hexahydronaphthalene 5 10.594 Caryophyllene oxide, 9-Isopropyl-1-methyl-2-methylene-5-oxatricyclo[5.4.0.0(3,8)]undecane 6 13.478 2.27 Neophytadiene, 3-Hexadecyne, 3-Tetradecyne 7 13.587 3.95 Caffeine 8 14.359 1.49 9-Hexadecenoic acid, methyl ester, (Z)- 7-Hexadecenoic acid, methyl ester 9 14.399 15.71 Hexadecanoic acid, methyl ester 10 15.452 1.77 geranyl-.alpha.-terpinene, p-Camphorene, Naphthalene 11 16.133 8.26 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 9,12,15-Octadecatrienoic acid 12 16.247 12.99 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Neophytadiene, 1,4-Eicosadiene 13 22.175 3.07 Cyclohexa-2,5-diene-1,4-dione, 2-methyl-5-(4-morpholinyl)- 4-Geranyloxy-3-hydroxy-5-methoxyphthalaldehyde 14 25.654 4.27 Cyclotrisiloxane, hexamethyl-1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)- Cyclotrisiloxane 15 25.946 3.92 Tris(tert-butyldimethylsilyloxy)arsane, Cyclotrisiloxane, hexamethyl- 16 26.341 7.16 1,4-Phthalazinedione, 2,3-dihydro-6-nitro- Methyltris(trimethylsiloxy)silane Discussion In this study, β-mannanase was produced from C. chauvoei , identified as the highest β-mannanase-producing bacterium among all isolates obtained from the gut of ruminant animals. The ruminant gut harbors a complex microbiome that facilitates efficient hydrolysis of plant materials consumed by the animals (Badejo et al., 2021). Consequently, the rumen is considered a natural and highly efficient bioreactor for the conversion of lignocellulosic materials into fermentable sugars (Selormey et al., 2021). Notably, mannanolytic bacteria have been observed in the rumen of cows, goats, and buffalo, highlighting their specialized role in the degradation of mannan-containing polysaccharides (Badejo et al., 2021; Selormey et al., 2021). The ability of these bacteria to convert mannan-rich compounds into absorbable sugars is particularly advantageous for the saccharification of agro-industrial residues for biofuel production. Moreover, since the bacterial species isolated in this study are anaerobic, there is potential for simultaneous saccharification and production of biofuels, including biobutanol and acetone–butanol–ethanol (ABE), demonstrating their dual functional role in bioconversion processes (Kaylani et al., 2017; Sharma et al., 2021). Despite the potential of β-mannanases in lignocellulosic biomass valorization, few studies have explored anaerobic rumen-derived bacteria that combine high enzymatic activity with dual functionality. The C. chauvoei isolated in this study exhibited remarkable thermostability, broad pH tolerance, and resilience to metal ions and certain inhibitors, while also efficiently producing biofuels. These findings address an existing research gap by identifying a rumen-derived anaerobe capable of both saccharification and biofuel production, providing a promising biocatalyst for integrated lignocellulosic biomass conversion and sustainable energy production. Although twenty bacterial isolates were obtained from the rumen of cow, biochemical identification revealed only five Clostridium species. Similar observations have been reported by Badejo et al. (2021) and Oyeleke and Okunsanmi (2008), with the highest bacterial counts observed in cows, while Deepa et al. (2019) also confirmed diverse rumen communities, albeit focusing on aerobes. The limited reports on anaerobes are likely due to their strict cultural requirements; however, Khattab et al. (2017) successfully isolated anaerobic bacteria from frozen rumen liquor, and notable rumen anaerobes include Clostridium thermocellum (Lamed and Bayer, 1988), C. cellulovorans (Yang et al., 2015), Ruminococcus albus (Ohara et al., 2000), R. flavefaciens (Suen et al., 2011), and Acetivibrio cellulolyticus (Dassa et al., 2012). The selection of anaerobic bacteria in this study was warranted because they are naturally adapted to the oxygen-limited rumen environment, efficiently degrading mannan-rich polysaccharides and enabling simultaneous saccharification and biofuel (acetone–butanol–ethanol) production, an advantage over many aerobic isolates (Kaylani et al., 2017; Sharma et al., 2021). A limitation of this study is the absence of molecular identification methods such as 16S rRNA gene sequencing. The bacterial isolates were identified solely based on morphological and biochemical characteristics, which may not provide precise taxonomic resolution, particularly among closely related genera such as Clostridium , Ruminococcus , and Acetivibrio . This limitation was primarily due to limited funding, self-sponsorship, and lack of access to molecular biology equipment. Future studies will incorporate molecular and genomic approaches to confirm strain identity and elucidate the genetic and enzymatic mechanisms underlying biobutanol production. β-Mannanase activity was detected in all twenty bacterial isolates, with isolate D3b, identified as C. chauvoei , exhibiting the highest enzyme activity. This corroborates findings by Badejo et al. (2021) and Olaniyi et al. (2023), who reported that rumen microbiota, particularly bacteria, possess substantial β-mannanase-producing capacity, with Providencia pulmonis and Alcaligenes sp. identified as the highest producers, respectively. Other potent β-mannanase producers include Bacillus amyloliquefaciens from horse feces (Cho, 2009), Bacillus clausii S10 from a soda lake (Zhou et al., 2018), and Bacillus cereus from Bani Salama lake (El-Sharouny et al., 2015). The remarkable ability of C. chauvoei to produce β-mannanase and hydrolyze β-mannan into fermentable sugars highlights its potential as a biocatalyst for diverse industrial applications, including biofuel production, paper pulp processing, and bleaching. The findings of this study underscore the rumen of ruminant animals as a rich reservoir of anaerobic bacteria capable of efficient lignocellulosic biomass conversion. The dual functionality of C. chauvoei , combining high β-mannanase activity with the ability to produce biofuels such as acetone–butanol–ethanol, presents a promising avenue for integrated bioprocessing of agro-industrial residues. These results provide a foundation for future studies employing molecular tools to confirm bacterial identity, optimize enzymatic performance, and explore the genetic and metabolic pathways underlying biofuel production. The 9-fold purification and ~ 36% yield achieved for C. chauvoei β-mannanase represent a substantial improvement over previously reported bacterial β-mannanases. For instance, Alcaligenes sp. β-mannanase exhibited only 2.6-fold purification with 1.75% yield (Olaniyi et al., 2023), while partially purified β-mannanases from Providencia vernicola and Psychrobacter pulmonis showed 2- and 6-fold purification with yields of 13% and 18%, respectively (Badejo et al., 2021). Although higher purification folds have been reported for fungal β-mannanases, such as 23.24-fold for Penicillium italicum (Olaniyi and Arotupin, 2014) and 32.9-fold by Cheng et al. (2016), the combination of relatively high yield and fold observed in this study highlights the efficiency of C. chauvoei β-mannanase purification from a bacterial source. The increase in specific activity during purification, as commonly observed (Cheng et al., 2016; Adeseko et al., 2021, 2022), confirms that the enzyme retained substantial catalytic potential. This notable retention of activity, coupled with high yield, underscores the enzyme’s novelty as a potent bacterial β-mannanase suitable for industrial applications, including biofuel production, saccharification of agro-residues, and bioconversion processes. The homogeneity of the purified C. chauvoei β-mannanase was confirmed by SDS-PAGE, which revealed a molecular weight of 43 kDa. This value falls within the range reported for bacterial and fungal β-mannanases, such as 18–39 kDa for Paenibacillus sp. (Dhawan, 2021), 32–53 kDa for Trichoderma sp. (Ferreira et al., 2004; Wang et al., 2014), and 40–100 kDa for Aspergillus sp. (Regalado et al., 2000; Naganagouda et al., 2009; Bhaturiwala et al., 2021). Lower molecular weights, such as 22 kDa, have been reported for Bacillus halodurans PPKS-2 (Vijayalaxmi et al., 2013), while higher molecular weights, including 130 kDa for Bacillus sp. JAMB-750 (Hatada et al., 2005), 162 kDa for Bacillus stearothermophilus ATCC 266 (Talbot and Sygusch, 1990), and 192 kDa for Bacteroides ovatus (Gherardini and Salyers, 1987), highlight the diversity of β-mannanase sizes. The intermediate molecular weight of 43 kDa observed for C. chauvoei β-mannanase, combined with its high catalytic efficiency and yield, underscores its unique biochemical features as a potent bacterial enzyme for industrial applications, bridging the gap between low- and high-molecular-weight β-mannanases. The optimum activity of C. chauvoei β-mannanase at pH 4 aligns with that reported for Providencia pulmonis (pH 4; Badejo et al., 2021) and Bacillus clausii strains (pH 4.5–5; Zhou et al., 2018). Other bacterial β-mannanases, including Bacillus subtilis E91, Bacillus SWU60, B. subtilis TBS2, and Alcaligenes sp., have been reported to exhibit optimum activity at mildly acidic pH 6 (Cheng et al., 2016; Seesom et al., 2017; Luo et al., 2017; Olaniyi et al., 2023). Notably, the enzyme maintained substantial activity at alkaline pH 8–10, retaining 47–71% of its relative activity, whereas other bacterial β-mannanases, such as Bacillus sp. JAMB-750, B. halodurans PPKS-2, and B. inaquosorum CSP31, exhibit optimum activity only at more extreme alkaline pH values of 10–12.5 (El-Sharouny et al., 2017; Vijayalaxmi et al., 2013; Regmi et al., 2017). This broad pH tolerance highlights the versatility and robustness of C. chauvoei β-mannanase, enabling it to function efficiently across both acidic and alkaline environments. Such flexibility enhances its potential for industrial applications, including the saccharification of agro-industrial residues, paper pulp processing, and biofuel production, where variable pH conditions are commonly encountered. The purified C. chauvoei β-mannanase exhibited remarkable stability across a broad pH range of 2–11 after 3 h of incubation, surpassing that of other reported β-mannanases. For comparison, β-mannanase from Acinetobacter sp. SP showed maximum stability between pH 3–10 (Titapoka et al., 2008), Alcaligen sp. was most stable at neutral pH 3–11 (Olaniyi et al., 2023), and P. pulmonis remained stable for only 50 min between pH 4–11 (Badejo et al., 2021). Fungal β-mannanase from Penicillium italicum was stable at pH 5 (Olaniyi and Arotupin, 2014), while a mutant Alcaligen sp. enzyme showed peak stability at acidic pH 5 and alkaline pH 8 (Olaniyi et al., 2023). Many β-mannanases reported in the literature exhibit either acidic or alkaline stability, with activity declining outside their optimum range (Cheng et al., 2016; Regmi et al., 2017). The broad pH stability of C. chauvoei β-mannanase highlights its distinctive functional versatility and suggests its suitability for diverse industrial applications, including feed supplementation, pulp bleaching, saccharification of agro-residues, and biofuel production. The purified C. chauvoei β-mannanase exhibited optimum activity at 60°C, consistent with β-mannanases from P. pulmonis (Badejo et al., 2021), Aspergillus foetidus (De Marco et al., 2015), and Pediococcus acidilactici (Nadaroglu et al., 2017). Remarkably, the enzyme maintained high activity between 70 and 90°C, indicating pronounced thermo-tolerance, a feature distinguishing it from most previously reported β-mannanases. In contrast, β-mannanase from P. italicum was optimal at 70°C (Olaniyi and Arotupin, 2014), while Bacillus circulans NT 6.7 and Trichoderma harzianum T7 exhibited optimum activity at 50°C (Phothichitto et al., 2006; Ferreira and Filho, 2004). The enzyme’s thermo-tolerant profile makes it highly suitable for industrial applications, where high-temperature processes are common. The thermal stability of C. chauvoei β-mannanase is further highlighted by its retention of substantial activity over extended incubation, comparable to Alcaligen sp., which maintained stability across 25–80°C after 1 h and retained high residual activity at 25–60°C after 3 h (Olaniyi et al., 2023). Other reported enzymes showed more limited stability: β-mannanases from Paenibacillus scooki and P. italicum were maximally stable at 40 and 50°C after 30–40 min (Yin et al., 2012; Olaniyi and Arotupin, 2014), while Malbranchea cinnamomea β-mannanase was stable at 60°C (Ahiwar et al., 2016), and Bacillus clausii β-mannanase exhibited a rapid decline in activity after 30–60 min (Zhou et al., 2018). The broad temperature tolerance and prolonged thermo-stability of C. chauvoei β-mannanase underscore its novel enzymatic properties and potential for high-temperature industrial processes, including biofuel production, pulp bleaching, and biomass saccharification. The purified C. chauvoei β-mannanase exhibited remarkable enhancement in activity in the presence of most metal ions tested, except Cu²⁺, highlighting its metal-ion promiscuity, a highly desirable feature for industrial applications. This behavior is comparable to β-mannanase from wild Alcaligen sp., which showed increased activity in the presence of Zn²⁺, Co²⁺, and Mg²⁺ but was inhibited by Na⁺, Ni²⁺, and Cu²⁺ (Olaniyi et al., 2023). Similarly, P. pulmonis β-mannanase was enhanced by Na⁺, Mn²⁺, and Co²⁺ but inhibited by K⁺, Cu²⁺, Mg²⁺, Pb²⁺, and Al³⁺ (Badejo et al., 2021), while P. cooki and A. foetidus enzymes were activated by Co²⁺ (Yin et al., 2012; De Marco et al., 2015). These variations in metal-ion responsiveness likely reflect environmental influences on the producing microbes (Yaashikaa et al., 2022), offering opportunities to optimize and enhance enzyme performance for biotechnological processes. Inhibitor studies revealed that SDS and cysteine were potent inhibitors of C. chauvoei β-mannanase across all concentrations tested. However, the enzyme displayed remarkable resilience to EDTA, urea, and sodium azide, even at high concentrations (10 mM), demonstrating its ability to withstand harsh conditions. These observations align with reports for β-mannanases from A. niger (Kote et al., 2009), Malbranchea cinnamomea (Ahirwar et al., 2016), P. pulmonis (Badejo et al., 2021), and Alcaligen sp. (Olaniyi et al., 2023), which tolerated 1–5 mM EDTA. The mild inhibition of Malbranchea cinnamomea β-mannanase by SDS and strong inhibition by urea (Ahirwar et al., 2016) further highlights the exceptional robustness of C. chauvoei β-mannanase. SDS inhibition is concentration-dependent: low concentrations bind the enzyme’s surface, while higher concentrations may denature the enzyme (Dhawal, 2021). The enhancement by metal ions and tolerance to inhibitors at high concentrations indicate that C. chauvoei β-mannanase is likely a metalloprotein and highly resilient, making it well-suited for industrial and biotechnological applications requiring stability under severe conditions. The activity of C. chauvoei β-mannanase was largely inhibited in the presence of surfactants (Tween-20 and Triton X-100) and most organic solvents, with the notable exception of formaldehyde, which enhanced enzymatic activity. This contrasts with recombinant β-mannanase AoMan134 from Aspergillus oryzae , whose activity decreased in the presence of organic solvents (methanol, ethanol, isopropanol, and acetone) and detergents (SDS, Triton X-100, and Tween-20) (Wang et al., 2012). Similarly, Bacillus licheniformis HDYM-04 β-mannanase retained maximal stability in dimethyl sulfoxide and hexane, was moderately inhibited by acetone and chloroform, and strongly inhibited by cetyl trimethyl ammonium bromide (Ge et al., 2016). Paenibacillus thiaminolyticus β-mannanase displayed stability against most organic solvents, except methanol, ethanol, propanol, and propanone, which caused varying degrees of inhibition (Dhawan, 2021). Remarkably, among the polar, water-miscible solvents tested, formaldehyde uniquely enhanced C. chauvoei β-mannanase activity, suggesting it may play a stabilizing role, either during enzymatic reactions or storage (Dhawan, 2021). This feature highlights the enzyme’s potential for industrial applications where solvent tolerance or stabilization is advantageous, such as in biofuel production, paper pulp processing, and biocatalysis under non-aqueous conditions. The kinetic parameters of purified C. chauvoei β-mannanase were determined, with a Km of 30.7 mM and Vmax of 7.88 mg/mL/min, indicating strong substrate affinity and high catalytic efficiency. Comparable studies report varied kinetic behaviors for β-mannanases from different sources: Aspergillus terrus exhibited a Km of 5.9 mg/mL and Vmax of 39.42 µmol/mL/min (Soni et al., 2016), Bacillus licheniformis 2.69 mg/mL and 251.41 U/mg (Ge et al., 2016), and Paenibacillus thiaminolyticus 5 mg and 1.1 × 10³ U/mg (Dhawan, 2021). Lower Km and Vmax values have been reported for P. italicum (Km 0.26 mg/mL; Vmax 0.12 mol/min/mL) (Olaniyi and Arotupin, 2014), P. pulmonis (Km 0.73 mg/mL) (Badejo et al., 2021), and Alcaligen sp. (Km 5.2 × 10⁻⁴ mg/mL) (Olaniyi et al., 2023). The comparatively low Km and high Vmax observed for C. chauvoei β-mannanase suggest an excellent substrate specificity, strong affinity, and efficient catalysis, highlighting its potential for industrial applications requiring efficient hydrolysis of β-mannan-rich polysaccharides, such as biofuel production, pulp and paper processing, and feedstock saccharification. The thermal inactivation of purified C. chauvoei β-mannanase followed first-order kinetics, consistent with β-mannanases from Enterobacter asburiae (Dhiman et al., 2019) and Klebsiella pneumoniae SS11 (Singh et al., 2019). The inactivation rate constant (ki) was temperature-dependent, increasing with rising temperatures, and similarly, the deactivation rate (kd) increased with temperature (Dhiman et al., 2019; Singh et al., 2019; Sadaqat et al., 2024). Both ki and kd reflect the enzyme’s time-dependent denaturation at specific temperatures and serve as indicators of thermostability (Singh et al., 2019). The enzyme retained high half-life (t1/2) and D-values even at 70°C, indicating remarkable thermal stability. Specifically, at 70°C, the t1/2 was 216.6 min, which is superior to reported β-mannanases from A. awamori (t1/2 > 30 min at 66°C) (Neustroev et al., 1991), Bacillus subtilis (t1/2 106.4 min at 65°C) (Liu et al., 2008), Caldocellum saccharolyticum (t1/2 45 min at 80°C), and Klebsiella pneumoniae (t1/2 135.91 min at 70°C) (Singh et al., 2019). The Z-value of 57°C and activation energy (Ea) of 23 kJ/mol further highlighted the enzyme’s thermostable nature. High Z-values indicate lower sensitivity to heat, while the linear Arrhenius plot reflects the temperature dependence of the inactivation rate and the unfolding mechanism of the enzyme (Bedel et al., 2020). The low Ea value indicates that C. chauvoei β-mannanase can efficiently hydrolyze mannan to fermentable sugars, a desirable feature for industrial applications such as biofuel production, pulp bleaching, and feed processing. Thermodynamic parameters further supported the enzyme’s stability. The free energy of activation (ΔG) was 63 kJ/mol, indicating non-spontaneity and structural stability, comparable to β-mannanases from Enterobacter asburiae (73.66 kJ/mol) (Dhiman et al., 2019) and Thermotoga maritima (74.82–82.8 kJ/mol) (Sadaqat et al., 2022), but lower than Penicillium humicola (107.8–111.41 kJ/mol) (Ismail et al., 2019). The enthalpy change (ΔH) of 20 kJ/mol aligns with Bacillus sp. CSB-39 (24 kJ/mol) (Dhiman et al., 2019) and reflects conformational changes due to heat (Ortega et al., 2019). The negative entropy change (ΔS = -134.4 J/mol/K) suggests the enzyme maintains structural rigidity with low disorder during activation, consistent with observations in Penicillium humicola (-118.99 to -133.67 J/mol/K) (Ismail et al., 2019). Together, the combination of high t1/2, D-values, Z-value, low Ea, and favorable thermodynamic parameters indicates that C. chauvoei β-mannanase is highly thermostable and capable of retaining activity under harsh thermal conditions. This remarkable thermal resilience, coupled with its substrate specificity, broad pH range, and metal ion tolerance, underscores its potential for diverse industrial bioprocesses, particularly in biofuel production, feed supplementation, and lignocellulosic biomass conversion. The saccharification of palm-kernel by purified β-mannanase from C. chauvoei and the subsequent production of acetone, butanol, and ethanol (ABE) were highly successful, yielding 55.0 g/L biobutanol, 60.2 g/L ethanol, and 70.1 g/L acetone. These yields are notably higher than those reported for other Clostridium strains, such as Clostridium sp. A53, which produced 10.5 g/L ABE and 0.11 g/L biobutanol (Johnravindar et al., 2019), and Clostridium saccharoperbutylacetonium N1-4, which produced 3.27 g/L biobutanol (Shukor et al., 2016). Comparatively, Serratia marcescens yielded 10 g/L bioethanol, and engineered Lactobacillus diolivorans increased production to 13.4 g/L (Russmayer et al., 2019). A common limitation in Clostridium -based fermentations is low yield due to solvent accumulation, which inhibits growth and sugar consumption (Al-Shorgani et al., 2014). High biobutanol concentrations disrupt membrane fluidity and transport processes, leading to leakage of intracellular proteins and metabolites. The exceptionally high biobutanol production observed in this study may reflect the inherent solvent tolerance of C. chauvoei , enabling sustained growth and efficient sugar conversion even under solvent stress. FTIR analysis further confirmed the production of biobutanol and related biofuels. Characteristic functional groups, including 3337 cm⁻¹ (OH stretch) and 667 cm⁻¹ (C–OH), were observed, consistent with previous reports on Clostridium sp. (Johnravindar et al., 2019). Additional peaks corresponding to secondary or tertiary alcohols (1120–1080 cm⁻¹) aligned with fingerprint regions for butanol. These observations are consistent with acetone-butanol-ethanol (ABE) spectral signatures reported by Huang et al. (2015) (3352–3246 cm⁻¹), confirming the successful fermentation and co-production of acetone, butanol, and ethanol. The FTIR analysis provides direct structural evidence of biofuel formation and highlights the potential of C. chauvoei for industrial-scale ABE fermentation from lignocellulosic substrates. Conclusion This study demonstrates the novel characteristics of purified β-mannanase from C. chauvoei and its efficient conversion of palm kernel cake into fermentable sugars, subsequently transformed into acetone, butanol, and ethanol (ABE) by the bacterium. The enzyme displayed exceptional thermo-stability and pH versatility, with optimum activity at 60°C and pH 4, stability across 30–80°C for 1 h, and a broad pH range of 2–12 over 6 h. Activity was enhanced by metal ions and tolerated inhibitors such as sodium azide and urea, while partial inhibition by EDTA indicated metal ion dependence. Formaldehyde uniquely stabilized the enzyme among organic solvents. Thermodynamic and kinetic analyses confirmed its robustness, with ΔG = 63 kJ/mol, ΔH = 23 kJ/mol, Z-value = 57°C, and half-life = 216 min. The high ABE yields highlight the potential of both the enzyme and C. chauvoei for biofuel production. Collectively, these properties position this β-mannanase as a versatile biocatalyst for industrial applications, including feed supplementation, agro-residue saccharification, pulp and paper processing, and sustainable biofuel production. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was self-sponsored. This research received no external funding Acknowledgements We are grateful to Mr. A. Emmanuel and Mr. O. Christopher, both of Bowen University, Iwo, Osun State, Nigeria, for their assistance in the provision and operation of the FTIR machine Authors’ contributions PTO: Investigation, Methodology, Funding Acquisition, Formal Analysis, Writing–Original Draft; OOO: Conceptualization, Supervision, Validation, Visualization, Project Administration, OTL: Investigation, Methodology, Resources, Formal Analysis, Validation, Visualization, Writing–Original Draft, Writing – Review and Editing, FAA: Supervision, Validation, Visualization. All authors read and approved the final manuscript. References Adeseko, C. J., Sanni, D. M., & Lawal, O. T. (2022). Biochemical studies of enzyme-induced browning of African bush mango (Irvingia gabonensis) fruit pulp. Preparative Biochemistry & Biotechnology , 52 (7), 835-844. Adeseko, C. J., Sanni, D. M., Salawu, S. O., Kade, I. J., Bamidele, S. O., & Lawal, O. T. (2021). 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Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients. Scientific reports , 11 (1), 1580. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.docx Graphicalabstract.jpg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":111860,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of bacterial isolates for β-mannan-degrading ability.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/6f92a140f56a0d4a3aa7e35d.jpg"},{"id":95640013,"identity":"0ea09507-d7fb-4c96-9962-b81546f34007","added_by":"auto","created_at":"2025-11-11 13:12:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":100918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u0026amp;b: \u003c/strong\u003ePurification and Subunit molecular weight estimation of β\u003cem\u003e Clostridium cnauvoei\u003c/em\u003e -mannanase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Ion exchange chromatography of β-mannanase from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e using DEAE Sephacel resin (Column: 2.5 × 15 cm; flow rate: 60 mL/h). The pooled fractions are presented by thick red line\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e \u0026nbsp;Gel-filtration chromatography of β-mannanase from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e using Sephadex G-100 (2.5 × 70 cm; flow rate 25 mL/h). The pooled fractions are presented by thick red line\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/34ec2a49f220f45b5856edc4.jpg"},{"id":95640016,"identity":"a4567f33-a860-40c4-b8ba-520344e81ffd","added_by":"auto","created_at":"2025-11-11 13:12:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":104171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u0026amp;b: \u003c/strong\u003eEffect of temperature and pH on the stability of β-mannanase from\u003cem\u003e Clostridium cnauvoei\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea. Effect of temperature on the stability of purified β-mannanase from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eb. Effect of pH on the stability of purified β-mannanase from \u003cem\u003eClostridium cnauvoe\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/b5647f3ace165b24443d5dc2.jpg"},{"id":95640015,"identity":"14b14a2c-d010-4891-b0b5-f2f26fee7abb","added_by":"auto","created_at":"2025-11-11 13:12:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79443,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u0026amp;b: \u003c/strong\u003eEffect of metal ions and inhibitors on the activity of β-mannanase from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ea. Effect of inhibitors on the activity of purified β-mannanase\u003c/p\u003e\n\u003cp\u003eb. Effect of metal ions on the activity of purified β-mannanase\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/d89f78f1eaca4fd94cbd2218.jpg"},{"id":95640019,"identity":"3b008396-0456-45fe-9266-e05d29b05e9b","added_by":"auto","created_at":"2025-11-11 13:12:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58912,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of organic solvents and surfactants on the activity of purified β-mannanase\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/4724e860582b96e262bc5eff.jpg"},{"id":95640017,"identity":"1003c097-655f-4859-91c4-0d25261bcbe2","added_by":"auto","created_at":"2025-11-11 13:12:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":93417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u0026amp;b: \u003c/strong\u003eStructural analysis of Biobutanol produced by \u003cem\u003eClostridium chauvoei\u003c/em\u003eisolated from the gut of ruminant animal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e FTIR showing acetone-butanol-ethanol (ABE) production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Gas chromatography revealing bioethanol production.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/6dbd6e1c93d6bb17ff328c48.jpg"},{"id":96060253,"identity":"1d4b99a6-aa17-4812-9b14-cd11321e5fe7","added_by":"auto","created_at":"2025-11-17 08:25:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2511373,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/0b2acc27-31e3-4682-ad16-d2af589e2eb4.pdf"},{"id":95656999,"identity":"00cf5575-b38a-4dbb-9129-1966ecbd7fbb","added_by":"auto","created_at":"2025-11-11 16:19:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":210976,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/b300ed6d1acfa71257d93806.docx"},{"id":95640014,"identity":"c8edce05-3961-496a-a1ae-c11f05ca38ce","added_by":"auto","created_at":"2025-11-11 13:12:21","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":86155,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7933920/v1/97fd359fae1739a3d687dc1b.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production","fulltext":[{"header":"Background","content":"\u003cp\u003eMannanases are hydrolases that catalyze the hydrolysis of mannosidic bonds in mannan, a major component of plant cell walls (Kalyani et al., 2021). Efficient mannan degradation requires the coordinated action of β-mannanases (EC 3.2.1.78), β-mannosidases (EC 3.2.1.25), and β-glucosidases (EC 3.2.1.21) (Dhiman et al., 2019). β-Mannanases cleave glycosidic bonds to produce β-1,4-manno-oligosaccharides, whereas β-mannosidases release mannose from mannobiose (Tahir et al., 2024). Many bacterial and fungal species produce β-mannanase, which has industrial potential in processes such as hemicellulose extraction from pulp and paper (Dawood and Ma, 2020; Favaro et al., 2020).\u003c/p\u003e\u003cp\u003eMannan is a complex polysaccharide and a major hemicellulose component. Its primary forms include mannose, glucomannan, galactomannan, and galactoglucomannan, each with distinct structures and β-1,4 linkages (Badejo et al., 2021; Zuo et al., 2021; Olaniyi et al., 2023). Glucomannan, a heteropolymer of glucose and mannose, is abundant in hardwood and softwood, while galactomannan contains a β-1,4 D-mannose backbone with galactose side groups (Badejo et al., 2021). Plant and animal tissues contain inactive β-mannanases that are activated in the presence of mannan substrates, while microbial β-mannanases are secreted extracellularly upon induction (Dawood et al., 2020; Olaniyi et al., 2023).\u003c/p\u003e\u003cp\u003eThe rumen of ruminants serves as a reservoir of hemicellulolytic bacteria capable of efficiently metabolizing lignocellulosic materials due to symbiosis with the host (Badejo et al., 2021; Zhao et al., 2022). This makes ruminant guts natural bioreactors for lignocellulose degradation, producing enzymes such as xylanase, mannanase, and cellulase (Badejo et al., 2021; de Souza et al., 2021; Olaniyi et al., 2023). Despite this potential, the enzymatic capabilities of ruminant gut-derived bacteria, particularly Clostridium chauvoei, remain underexplored.\u003c/p\u003e\u003cp\u003eThe need for sustainable biofuels is driven by the environmental impact of fossil fuels, including greenhouse gas emissions and global warming (Jogdand, 2020; Aryal et al., 2022). β-Mannanase plays a pivotal role in converting hemicellulose into oligomers and mannose, which can be fermented into bioethanol or biobutanol (Mamo, 2020). Lignocellulosic biomass requires chemical pretreatment due to its recalcitrance, followed by enzymatic hydrolysis to release fermentable sugars (Bhatia et al., 2020; Zhang et al., 2022). While \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e is widely used for first-generation bioethanol production, thermophilic anaerobic bacteria can achieve higher biobutanol yields from inexpensive agricultural residues (Zabermawi et al., 2022).\u003c/p\u003e\u003cp\u003eGiven this context, this study addresses the gap in knowledge regarding the biochemical properties of β-mannanase from ruminant gut \u003cem\u003eC. chauvoei\u003c/em\u003e and its potential for biofuel production. Specifically, we purified and characterized β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e isolated from the cow rumen and evaluated its efficacy in producing biofuel from palm kernel substrate, highlighting its industrial relevance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCulture-based identification of anaerobic bacteria from the ruminant gastrointestinal tract\u003c/h2\u003e\u003cp\u003eAnaerobic bacteria from the gut of ruminants were isolated by inoculating sterile tryptone-yeast extract-acetate (TYA) medium with 10% (v/v) filtered ruminal fluid, followed by anaerobic incubation for 18 hours, and were used as the source of inoculum. After incubation, 1 mL of the inoculum was pour-plated onto solidified TYA agar medium and incubated under anaerobic conditions at 30\u0026deg;C for 1\u0026ndash;2 days. The composition of TYA medium was as follows (g/L): tryptone, 6; yeast extract, 2; ammonium acetate, 3; KH₂PO₄, 0.5; MgSO₄\u0026middot;7H₂O, 0.3; and FeSO₄\u0026middot;7H₂O, 0.01 (Al-Shorgani et al., 2014).\u003c/p\u003e\u003cp\u003eEmergent colonies were enumerated and reported as colony-forming units per milliliter (CFU/mL). Pure isolates were obtained from the mixed cultures by repeated streaking on the same agar medium and incubated under the conditions described above. Unknown pure isolates were tentatively identified based on cultural and morphological characteristics, as well as selected biochemical tests, following standard bacteriological procedures (Sari et al., 2016). The morphological features and biochemical reactions of the isolates were compared with descriptions in standard bacteriological atlases.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eScreening and Assay of β-Mannanase-Producing Isolates\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eQualitative screening\u003c/h2\u003e\u003cp\u003eThe bacterial isolates were screened for β-mannanase production using tryptone-yeast extract-acetate (TYA) agar supplemented with 1.5% (w/v) locust bean gum (LBG). The medium composition was as previously described, with the addition of 1.5 g/100 mL LBG and agar. Pure isolates were streaked individually onto the sterile TYA-LBG agar plates and incubated under anaerobic conditions. Colonies that developed visible clearance zones around their growth were considered mannolytic anaerobic bacteria. The diameters of the hydrolytic zones were measured in millimeters and recorded (Raita et al., 2016). Mannolytic isolates were sub-cultured on potato glucose (PG) medium and stored at 4\u0026deg;C for further analysis. The PG medium contained (g/L): potato, 150; glucose, 10; CaCO₃, 3; and (NH₄)₂SO₄, 0.5.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative screening\u003c/h3\u003e\n\u003cp\u003eQuantitative β-mannanase activity was determined in TYA broth supplemented with 1.5% (w/v) LBG (without agar). The medium was inoculated with the isolates and incubated under the same anaerobic conditions (Shukor et al., 2016). After incubation, the culture broth was centrifuged at 6,000 rpm for 20 min, and the supernatant (crude enzyme extract) was collected and stored for enzymatic assay.\u003c/p\u003e\n\u003ch3\u003eEnzyme assay\u003c/h3\u003e\n\u003cp\u003eβ-Mannanase activity was assayed using a reaction mixture containing 0.5 mL of crude enzyme and 0.5 mL of LBG substrate solution (1 g LBG dissolved in 50 mM potassium phosphate buffer, pH 6.8). The mixture was incubated at 45\u0026deg;C for 30 min in a water bath (Olaniyi et al., 2023). The reaction was terminated by adding 1 mL of dinitrosalicylic acid (DNSA) reagent, followed by boiling for 10 min. After cooling, absorbance was measured at 540 nm. One unit (U) of β-mannanase activity was defined as the amount of enzyme that liberates 1 \u0026micro;mol of mannose per minute under the assay conditions.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePurification of β-Mannanase\u003c/h2\u003e\u003cp\u003eThe crude enzyme extract was subjected to stepwise purification. Ammonium sulfate was gradually added to the extract at 4\u0026deg;C to induce protein precipitation. The mixture was left overnight at 4\u0026deg;C, after which the precipitate was collected by refrigerated centrifugation at 15,000 \u0026times; g for 10 min. The pellet was dissolved in 100 mM potassium phosphate buffer (pH 6.8) and dialyzed extensively against the same buffer using a 3,500 Da molecular weight cut-off dialysis membrane to remove residual ammonium sulfate.\u003c/p\u003e\u003cp\u003eThe dialyzed enzyme solution was further purified by ion-exchange chromatography on a DEAE-Sephacel column equilibrated with 100 mM potassium phosphate buffer (pH 6.8). Bound proteins were eluted with a linear gradient of NaCl in the same buffer. Fractions showing absorbance at 280 nm were collected and assayed for β-mannanase activity. Active fractions were pooled and subjected to size-exclusion chromatography on a Sephadex G-100 column equilibrated with the same buffer. Fractions were monitored at 280 nm, and those exhibiting β-mannanase activity were combined. The purified β-mannanase fractions were concentrated and stored at 4\u0026deg;C until further use. Protein concentration at each purification step was determined by absorbance at 280 nm, and enzymatic activity was verified using the standard β-mannanase assay (Olaniyi et al., 2023).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eβ-Mannanase molecular mass estimation\u003c/h3\u003e\n\u003cp\u003eThe molecular mass of β-mannanase was determined by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using the Laemmli method (Laemmli, 1970). Resolving (12.5% acrylamide; Tris buffer, pH 8.8) and stacking (4% acrylamide; Tris buffer, pH 6.8) gels were prepared. The gel mixture contained SDS, ammonium persulfate, and N,N,N\u0026prime;,N\u0026prime;-tetramethylethylenediamine (TEMED) as polymerizing agents.\u003c/p\u003e\u003cp\u003eEnzyme samples were mixed with 5\u0026times; sample buffer containing SDS, β-mercaptoethanol, and Coomassie dye, followed by heating at 100\u0026deg;C for 60 s. Electrophoresis was carried out at 80 V using a vertical electrophoresis unit. After separation, the gels were stained with Coomassie Brilliant Blue R-250 (prepared in methanol:water:acetic acid, 5:5:1) and subsequently destained with an acetic acid solution (7% v/v in distilled water). The gels were then photographed to visualize protein bands.\u003c/p\u003e\n\u003ch3\u003ePurified β-mannanase: Physicochemical, thermodynamic, and kinetic properties\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of optimum pH and pH stability of β-mannanase\u003c/h2\u003e\u003cp\u003eThe effect of pH on β-mannanase activity was determined using different buffer systems: 0.1 M glycine/HCl (pH 2.0\u0026ndash;3.0), 0.1 M sodium acetate buffer (pH 4.0\u0026ndash;5.0), 0.1 M phosphate buffer (pH 6.0\u0026ndash;7.0), 0.1 M Tris-HCl (pH 8.0\u0026ndash;10.0), and 0.1 M Tris/NaOH (pH 11.0\u0026ndash;12.0). Enzyme activity was measured under standard assay conditions in each buffer to determine the optimum pH. For pH stability, the enzyme was incubated in the same buffer systems without substrate, and aliquots were withdrawn at 30-minute intervals for up to 3 h. Residual activity was assayed under standard conditions to evaluate enzyme stability across the pH range.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of optimum temperature and thermal stability of purified β-mannanase\u003c/h2\u003e\u003cp\u003eThe optimum temperature for β-mannanase activity was evaluated by performing the standard enzyme assay at temperatures ranging from 30 to 90\u0026deg;C, in 10\u0026deg;C increments, using 0.1 M phosphate buffer (pH 6.8) as the reaction medium. Enzyme activity at each temperature was determined following the standard β-mannanase assay. For thermal stability, the enzyme was incubated at the same temperature range without substrate, and aliquots were withdrawn at 30-minute intervals for up to 3 hours. Residual activity was measured under standard assay conditions to assess enzyme stability at different temperatures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEffect of metal ions, inhibitors, organic solvents, and surfactants on β-mannanase activity\u003c/h2\u003e\u003cp\u003eThe influence of various additives on β-mannanase activity was investigated. The tested compounds included chloride salts of metal ions (K⁺, Ca\u0026sup2;⁺, Mg\u0026sup2;⁺, Mn\u0026sup2;⁺, Zn\u0026sup2;⁺, and Cu\u0026sup2;⁺), chelating agent (EDTA), chaotropic agents (urea and sodium dodecyl sulfate, SDS), sodium azide, organic solvents (acetic acid, acetone, formaldehyde, and dimethyl sulfoxide, DMSO), and surfactants (Tween-20 and Triton X-100). Each compound was tested at final concentrations of 1, 5, and 10 mM. The assay mixtures containing substrate (mannan), additives, and purified enzyme were prepared in 0.1 M phosphate buffer (pH 6.8) and incubated under standard assay conditions. Enzyme activity was determined using the β-mannanase assay, and the relative activity was calculated in comparison with the control (without additives).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEstimation of Km and Vmax of β-mannanase\u003c/h2\u003e\u003cp\u003eThe kinetic parameters of the purified enzyme, Km and Vmax, were determined using LBG as substrate at varying concentrations (0.1\u0026ndash;1% w/v) in 50 mM phosphate buffer (pH 6.8). The initial reaction velocities were measured following the standard β-mannanase assay procedure. The reciprocal values of the initial velocities (1/V) and substrate concentrations (1/[S]) were plotted according to the Lineweaver\u0026ndash;Burk method (Lineweaver and Burk, 1934). From the double reciprocal plot, the kinetic constants Km and Vmax were estimated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eThermodynamic studies of purified β-mannanase\u003c/h2\u003e\u003cp\u003eThe thermal stability of purified β-mannanase was evaluated by incubating enzyme aliquots prepared in phosphate buffer (pH 6.8) at different temperatures, in 10\u0026deg;C intervals, using a water bath. For each temperature, samples were withdrawn at 0 min and subsequently every 30 min for up to 3 h. Enzyme activity was determined using the standard β-mannanase assay, and residual activity was expressed as a percentage of the initial (0 min) activity.\u003c/p\u003e\u003cp\u003eThe first-order inactivation constant (k) was calculated from the slope of the first-order inactivation equation:\u003c/p\u003e\u003cp\u003eResidual activity (%)\u0026thinsp;=\u0026thinsp;\u003cem\u003eC/C\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e​\u0026times;100 (i)\u003c/p\u003e\u003cp\u003eln(\u003cem\u003eC/C\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003ekt\u003c/em\u003e (ii)\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e is the enzyme activity at time \u003cem\u003et\u003c/em\u003e, and \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e is the initial enzyme activity (0 min). The rate constant \u003cem\u003ek\u003c/em\u003e (min⁻\u0026sup1;) was obtained from the slope of the linear plot of ln(\u003cem\u003eC/C\u003c/em\u003e\u003csub\u003e\u003cem\u003eo\u003c/em\u003e\u003c/sub\u003e) against incubation time.\u003c/p\u003e\u003cp\u003eThe enzyme half-life (t₁/₂) was calculated using:\u003c/p\u003e\u003cp\u003e\u003cem\u003et₁\u003c/em\u003e/₂= ln(2)/\u003cem\u003ek\u003c/em\u003e (iii)\u003c/p\u003e\u003cp\u003eThe decimal reduction time (D-value) was determined as:\u003c/p\u003e\u003cp\u003e\u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;ln(10)/\u003cem\u003ek\u003c/em\u003e (iv)\u003c/p\u003e\u003cp\u003ePlotting log(D) against temperature provided the Z-value, defined as the temperature change required to reduce the D-value by one log unit. The negative reciprocal of the slope was taken as the Z-value.\u003c/p\u003e\u003cp\u003eThe temperature dependence of the inactivation rate constant was evaluated using the Arrhenius equation:\u003c/p\u003e\u003cp\u003eln\u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eEa/RT\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eC\u003c/em\u003e (v)\u003c/p\u003e\u003cp\u003eThermodynamic parameters, including Gibbs free energy change (Δ\u003cem\u003eG\u003c/em\u003e), enthalpy change (Δ\u003cem\u003eH\u003c/em\u003e), and entropy change (Δ\u003cem\u003eS\u003c/em\u003e), were calculated from the activation energy (\u003cem\u003eEa\u003c/em\u003e​) and the Arrhenius rate constant (\u003cem\u003ek\u003c/em\u003e) using the following relationships:\u003c/p\u003e\u003cp\u003eΔ\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003eEa\u003c/em\u003e​-\u003cem\u003eRT\u003c/em\u003e (vi)\u003c/p\u003e\u003cp\u003eΔ\u003cem\u003eG\u003c/em\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003eRT\u003c/em\u003e ln \u003cem\u003ekh\u003c/em\u003e/\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eT\u003c/em\u003e (vii)\u003c/p\u003e\u003cp\u003eΔ\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;Δ\u003cem\u003eH\u0026thinsp;\u0026minus;\u003c/em\u003e\u0026thinsp;Δ\u003cem\u003eG/T\u003c/em\u003e (viii)\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003eB\u003c/em\u003e\u003c/sub\u003e is Boltzmann\u0026rsquo;s constant (1.3806 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;23\u003c/sup\u003e J/K), \u003cem\u003eh\u003c/em\u003e is Planck\u0026rsquo;s constant (6.6260 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;34\u003c/sup\u003e J\u0026middot;s),\u003c/p\u003e\u003cp\u003e\u0026#119877; universal gas constant, and \u003cem\u003eT\u003c/em\u003e is the absolute temperature (K).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSaccharification and Bioethanol Production\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003ePreparation of Palm Kernel Cake and Corn Cob for Chemical Pretreatment\u003c/h2\u003e\u003cp\u003eDefatted palm kernel cake (PKC) was prepared using a Soxhlet extractor with petroleum ether, followed by drying, milling, and sieving. Approximately 172 g of the defatted PKC was soaked in 200 mL of 1% (v/v) H₂SO₄ and boiled at 160\u0026deg;C for 20 min in a covered stainless-steel pan. The mixture was subsequently autoclaved at 121\u0026deg;C for 60 min. Weight loss was determined before and after autoclaving. After cooling to room temperature, the pH of the hydrolysate was adjusted to 5.0 using 400 g/L NaOH.\u003c/p\u003e\u003cp\u003ePurified β-mannanase (6 mL) was added to 1 L of the pretreated substrate, and the reaction mixture was incubated at 45\u0026deg;C for 72 h with agitation at 80 rpm to achieve enzymatic saccharification. The resulting mixture was centrifuged at 3,300 \u0026times; g for 10 min, and the supernatant, containing the released fermentable sugars, was collected for subsequent bioethanol production (Shukor et al., 2016).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDetoxification of Hydrolysates\u003c/h2\u003e\u003cp\u003eInhibitory compounds present in the hydrolysates derived from pretreated palm kernel cake (PKC), corn cobs, and the enzyme production medium were neutralized following a modified chemical detoxification procedure. The pH of each hydrolysate was first adjusted to 10.0 using Ca(OH)₂, after which Na₂SO₃ (1 g/L) was added. The mixture was heated to 90\u0026deg;C with intermittent stirring, then allowed to cool to room temperature. The pH was subsequently readjusted to 7.0 using concentrated H₂SO₄.\u003c/p\u003e\u003cp\u003eActivated charcoal (1 g/L) was added to adsorb residual inhibitory compounds, and the resulting mixture was filtered through a 0.2 mm membrane to obtain a clear supernatant. The concentrations of fermentable sugars, including glucose, mannose, xylose, and mannooligosaccharides (MOS) were determined using standard assay methods and expressed in mg/mL (De Barros et al., 2024).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eBiobutanol Production\u003c/h2\u003e\u003cp\u003eFermentation was performed in 250 mL bottles with a working volume of 100 mL. The clarified supernatant was mixed with filtered hydrolysates obtained from the chemically pretreated samples. \u003cem\u003eClostridium chauvoei\u003c/em\u003e, previously cultured in TYA medium for 18 h, was aseptically inoculated into the fermentation medium and incubated anaerobically for 5 days. The concentrations of butanol, acetone, and ethanol were quantified using gas chromatography according to the method described by Shukor et al. (2016). The functional groups of alcohols present in the fermentation products were analyzed using Fourier-transform infrared (FTIR) spectroscopy (Phwan et al.,\u003c/p\u003e\u003cp\u003e2019).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of Bioethanol Production by Colorimetric Method\u003c/h2\u003e\u003cp\u003eA standard stock solution of ethanol (1.6 mg/mL) was prepared in a 50 mL volumetric flask. To this, 5 mL each of sodium dichromate solution, pH 4.3 acetate buffer, and 1 N sulfuric acid were added sequentially. The resulting mixture produced a green-colored reaction product, which was gently shaken for 1 min and then allowed to stand at room temperature for 120 min.\u003c/p\u003e\u003cp\u003eThe absorbance of the mixture was measured at 578 nm using a UV\u0026ndash;Visible spectrophotometer. Each sample was prepared and analyzed in triplicate. The ethanol concentration in the samples was determined from a standard calibration curve as described by Datta et al. (2018) and expressed as:\u003c/p\u003e\u003cp\u003ePercentage of ethanol/acetone/butanol in sample (%) = (\u003cem\u003eCs\u003c/em\u003e/\u003cem\u003eCu\u003c/em\u003e) (\u003cem\u003eAu\u003c/em\u003e/\u003cem\u003eAs\u003c/em\u003e) x 100\u003c/p\u003e\u003cp\u003ewhere \u003cem\u003eCs\u003c/em\u003e\u0026thinsp;=\u0026thinsp;Concentration of standard, \u003cem\u003eCu\u003c/em\u003e\u0026thinsp;=\u0026thinsp;Concentration of sample as per Labeled Claim, \u003cem\u003eAu\u003c/em\u003e\u0026thinsp;=\u0026thinsp;Absorbance of standard, \u003cem\u003eAs\u003c/em\u003e =\u0026thinsp;Absorbance of sample.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eBiochemical tests on bacterial isolates from the ruminant gut\u003c/h2\u003e\u003cp\u003eResults of biochemical tests on bacterial isolates are presented in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Twenty (20) isolates were selected from culture plates and assigned unique codes. Biochemical characterization identified all isolates as belonging to the genus \u003cem\u003eClostridium\u003c/em\u003e. The predominant species detected were \u003cem\u003eClostridium sphenoide\u003c/em\u003e, \u003cem\u003eC. clostridiforme\u003c/em\u003e, \u003cem\u003eC. perfringens\u003c/em\u003e, \u003cem\u003eC. chauvoei\u003c/em\u003e, and \u003cem\u003eC. novyi\u003c/em\u003e type A.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eScreening of bacterial isolates for β-mannan-degrading ability\u003c/h2\u003e\u003cp\u003eThe β-mannan-degrading ability of the bacterial isolates is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Isolate D3b (\u003cem\u003eC. chauvoei\u003c/em\u003e) exhibited the highest activity, with β-mannanase activity of 92.8 U/mL and 73 mg/mL fermentable sugar released. Isolates B3 and B1 also demonstrated notable activity, with β-mannanase activities of 60 and 54.69 U/mL, and corresponding fermentable sugar concentrations of 60 and 64.59 mg/mL, respectively. Moderate β-mannan degradation was observed in isolates A2 (\u003cem\u003eC. clostridiforme\u003c/em\u003e), B4a, E2 (\u003cem\u003eC. perfringens\u003c/em\u003e), and A1 (\u003cem\u003eC. sphenoide\u003c/em\u003e), exhibiting enzyme activities of 46, 42.7, 33, and 30 U/mL and fermentable sugar concentrations of 56.7, 59.6, 72, and 60 mg/mL, respectively. Minimal β-mannan-degrading activity was recorded for isolates A3a (\u003cem\u003eC. perfringens\u003c/em\u003e), F2 (\u003cem\u003eC. perfringens\u003c/em\u003e), and E1 (\u003cem\u003eC. chauvoei\u003c/em\u003e), with enzyme activities of 3.5, 2.6, and 0.9 U/mL and fermentable sugar concentrations of 44, 15, and 9 mg/mL, respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClostridium chauvoei\u003c/b\u003e \u003cb\u003eβ-mannanase purification profile\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe purification profile of β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The crude enzyme exhibited a total activity of 1,400 U/mL and total protein concentration of 432.4 mg/mL, corresponding to a specific activity of 3.24 U/mg. Ammonium sulfate precipitation of the crude enzyme, followed by dialysis, increased the specific activity to 8.76 U/mg, representing a 2.7-fold purification with 62.7% recovery. Subsequent ion-exchange chromatography of the dialysate on DEAE-Sephadex resin yielded a single peak of enzymatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), achieving 6.3-fold purification with approximately 38% recovery. Further purification by gel-filtration chromatography on Sephadex G-100 resin produced a single activity peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), with 8.8-fold purification and 36% yield.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePurification summary of β-mannanase from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e isolated from ruminant gut\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePurification step\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVol. (mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEnz. Activity (U/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eProtein conc. (mg/mL)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal Activity (U)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTotal Protein (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSpecific Activity (U/mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePurification Fold\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eYield (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCrude Extracts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.081\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1400\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e432.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e PPT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e13.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.542\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e877.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e100.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e62.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDEAE Sephacel\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e534.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e26.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e20.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e38.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSephadex G-100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.704\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e503.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e17.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e28.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e35.97\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMolecular mass of \u003cem\u003eClostridium chauvoei\u003c/em\u003e β-mannanase\u003c/p\u003e\u003cp\u003eThe molecular weight of the purified β-mannanase was estimated at 42 kDa, as shown in Figure \u003cb\u003eS2\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eClostridium chauvoei\u003c/b\u003e \u003cb\u003eβ-mannanase properties\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eTemperature optimum and stability\u003c/h2\u003e\u003cp\u003eThe effect of temperature on the activity of purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is shown in \u003cb\u003eFigure S3a\u003c/b\u003e. The enzyme exhibited maximum activity at 60\u0026deg;C, with activity increasing progressively up to this optimum. High relative activities of 88.6%, 81.5%, and 73% were observed at 70, 80, and 90\u0026deg;C, respectively. Thermal stability of the enzyme is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The purified β-mannanase was maximally stable at 30 and 40\u0026deg;C, retaining 91% and 79% residual activity after 1 h and 3 h incubation, respectively. High residual activities of 77\u0026ndash;69% were maintained between 50\u0026ndash;70\u0026deg;C after 1 h, while 44% activity remained at 80\u0026deg;C. After 3 h, residual activities were 63% and 50% at 50 and 60\u0026deg;C, respectively, decreasing to 25% at 70\u0026deg;C and complete inactivation occurring between 80 and 90\u0026deg;C.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003epH optimum and stability\u003c/h2\u003e\u003cp\u003eThe effect of pH on the activity of purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is shown in \u003cb\u003eFigure S3b\u003c/b\u003e. The enzyme exhibited maximum activity at acidic pH 4, with activity sharply declining between pH 5 and 7. Relative activities of 74\u0026ndash;58% were observed at alkaline pH 8\u0026ndash;10, and 47% activity was retained at pH 11. The pH stability profile is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. The enzyme showed maximum stability at alkaline pH 9\u0026ndash;11, retaining 95% and 87% of its original activity after 1 and 3 h, respectively. It also maintained 88\u0026ndash;70% residual activity between pH 2\u0026ndash;4 and 6\u0026ndash;9 after 2 h. Lower residual activities of 63% were recorded at pH 2, 4, and 5, and 44\u0026ndash;51% at pH 3 and 6\u0026ndash;9.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eEffect of metal ions on β-mannanase activity\u003c/h2\u003e\u003cp\u003eThe effect of metal ions on the activity of purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Enzymatic activity was significantly enhanced by Mg\u0026sup2;⁺, with a 41% increase at 5 mM and a 22% increase at 10 mM. Zn\u0026sup2;⁺ and K⁺ also enhanced enzyme activity at 5 mM, while Mn\u0026sup2;⁺ showed stimulatory effects at both 5 and 10 mM. In contrast, Ca\u0026sup2;⁺ and Cu\u0026sup2;⁺ exhibited inhibitory effects, reducing enzyme activity by 35% and 25%, respectively, at 1 mM. Both ions caused mild inhibition at higher concentrations of 5 and 10 mM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eEffect of inhibitors on β-mannanase activity\u003c/h2\u003e\u003cp\u003eThe effect of inhibitors on the activity of β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Enzymatic activity was largely unaffected by EDTA, urea, and sodium azide at 5 mM. At 10 mM, EDTA caused slight inhibition, while urea and sodium azide resulted in 55% and 35% reductions in activity, respectively. SDS and cysteine strongly inhibited β-mannanase activity in a concentration-dependent manner.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eEffect of organic solvents and detergents on β-mannanase activity\u003c/h2\u003e\u003cp\u003eThe effect of organic solvents and detergents on the activity of β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Formaldehyde did not affect enzyme activity at any of the concentrations tested. In contrast, acetic acid, acetone, and dimethyl sulfoxide (DMSO) strongly inhibited β-mannanase activity across all concentrations. Detergents, including Tween-20 and Triton X-100, also caused strong inhibition of enzymatic activity at all concentrations examined.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eKinetic parameters of the purified β-mannanase\u003c/h2\u003e\u003cp\u003eThe kinetic parameters of the purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e are presented in Figure \u003cb\u003eS4\u003c/b\u003e. The K\u003csub\u003em\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e were estimated as 30.7 mM and 7.88 mg/mL/min respectively.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThermal inactivation of purified β-mannanase\u003c/h3\u003e\n\u003cp\u003eThe thermal inactivation profile of purified β-mannanase is presented in Figure \u003cb\u003eS5a\u003c/b\u003e. A plot of the logarithm of residual activity versus incubation time at different temperatures showed a linear relationship, consistent with first-order kinetics, with R\u0026sup2; values ranging from 0.9157 to 0.9962.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eKinetic parameters, half-life, and D-value of purified β-mannanase\u003c/h2\u003e\u003cp\u003eThe kinetic parameters of the purified β-mannanase are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Inactivation rate constants (k) were determined from the slopes of plots of residual activity versus incubation time, ranging from 5 \u0026times; 10⁻⁴ to 3.2 \u0026times; 10⁻\u0026sup2; min⁻\u0026sup1;. Corresponding half-life (t₁/₂) values were estimated to range from 1,386 to 217 min, while D-values ranged from 4,605 to 720 min.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003ea\u003c/b\u003e: Kinetic values for the heat inactivation of β-mannanase\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemp (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemp (K)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eThermal inactivation, k\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHalf-life t\u003csub\u003e1/2\u003c/sub\u003e (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eD-value (min)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003elog D\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1386.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4605.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9162\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1155.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3837.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9962\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e313\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e693.147\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2302.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.0693\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0017\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e407.734\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1354.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e385.082\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1279.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9817\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3.11\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.0032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e216.608\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e719.558\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.9157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eb\u003c/b\u003e: Thermodynamic properties of the purified β-mannanase\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemp (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemp (K)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eΔH (KJ/mol)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eΔS (J/mol/K)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eΔG (KJ/mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e298\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.536\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e60.748\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e303\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.4944\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e61.3392\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e313\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.4112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e62.5196\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.3281\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63.6973\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e333\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.245\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e64.8725\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.1618\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-133.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e66.0451\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAverage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e20.3627\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e-134.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63.2036\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eArrhenius plot for β-mannanase inactivation\u003c/h2\u003e\u003cp\u003eThe Arrhenius plot for the thermal inactivation of purified β-mannanase is shown in Figure \u003cb\u003eS5b\u003c/b\u003e. The dependence of the inactivation rate constants on temperature was fitted to the Arrhenius equation, yielding a strong correlation with R\u0026sup2; = 0.9735.\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003eTemperature dependence of decimal reduction (D-value) for β-mannanase inactivation\u003c/h2\u003e\u003cp\u003eThe relationship between the D-value and inactivation temperature of purified β-mannanase is presented in Figure \u003cb\u003eS5c\u003c/b\u003e. The activation energy (Eₐ) and Z-value (the temperature required to reduce the D-value by one logarithmic cycle) were calculated as 23 kJ/mol and 57\u0026deg;C, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eThermodynamic parameters of β-mannanase\u003c/h2\u003e\u003cp\u003eThe thermodynamic parameters of purified β-mannanase are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. The average values obtained were 63.2 kJ/mol for Gibbs free energy change (Δ\u003cem\u003eG\u003c/em\u003e), 20.3 kJ/mol for enthalpy change (Δ\u003cem\u003eH\u003c/em\u003e), and \u0026minus;\u0026thinsp;134.4 J/mol\u0026middot;K for entropy change (Δ\u003cem\u003eS\u003c/em\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eBioethanol production by\u003c/b\u003e \u003cb\u003eClostridium chauvoei\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eQuantification of bioethanol and other biofuels\u003c/h3\u003e\n\u003cp\u003eThe production of butanol, ethanol, and acetone (ACE; acetone\u0026ndash;ethanol\u0026ndash;butanol) by \u003cem\u003eC. chauvoei\u003c/em\u003e is summarized in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The estimated quantities of biobutanol, ethanol, and ACE were 55.00%, 60.15%, and 70.10%, respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantity of biofuel produced by \u003cem\u003eClostridium chauvoei\u003c/em\u003e isolated from the cow rumen\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiofuel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuantity (g/L)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEthanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e60.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eButanol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e55.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.085\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e70.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eGas Chromatography and Fourier Transform Infrared (FTIR) Analyses\u003c/h3\u003e\n\u003cp\u003eThe presence of butanol in the fermentation products was confirmed using gas chromatography (GC), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Several volatile compounds were detected, among which compounds 3, 7, 11, and 15-tetramethyl-2-hexadecen-1-ol corresponded to the expected biofuel product (chromatogram shown in \u003cb\u003eFigure S6\u003c/b\u003e). Fourier Transform Infrared (FTIR) spectroscopy was employed to further characterize the functional groups present in the bioethanol sample (\u003cb\u003eTable S2 \u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The characteristic absorption bands observed at 3,337 cm⁻\u0026sup1; (O\u0026ndash;H stretching) and 667 cm⁻\u0026sup1; (C\u0026ndash;O\u0026ndash;H bending) confirmed the presence of hydroxyl functional groups typical of alcohols, corresponding to the fingerprint region of butanol (600\u0026ndash;700 cm⁻\u0026sup1;). In addition, a band between 1,120 and 1,080 cm⁻\u0026sup1;, centered at 1,097 cm⁻\u0026sup1;, indicated C\u0026ndash;C\u0026ndash;CHO stretching vibrations associated with secondary or tertiary alcohols.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGas chromatography analysis of biofuel produced from \u003cem\u003eClostridium cnauvoei\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS/N\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRetention Time\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eArea (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7.933\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ealfa.-Copaene, Copaene, alpha.-Cubebene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e16.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCaryophyllene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.958\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHumulene, 1,4,7,-Cycloundecatriene, 1,5,9,9-tetramethyl-, Z,Z,Z-\u003c/p\u003e\u003cp\u003eHumulene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.845\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003etetramethyl-, Z,Z,Z-\u003c/p\u003e\u003cp\u003eHumulene, ,8a-hexahydronaphthalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e10.594\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCaryophyllene oxide, 9-Isopropyl-1-methyl-2-methylene-5-oxatricyclo[5.4.0.0(3,8)]undecane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.478\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNeophytadiene, 3-Hexadecyne, 3-Tetradecyne\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.587\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCaffeine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.359\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9-Hexadecenoic acid, methyl ester, (Z)- 7-Hexadecenoic acid, methyl ester\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14.399\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e15.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHexadecanoic acid, methyl ester\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e15.452\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003egeranyl-.alpha.-terpinene, p-Camphorene, Naphthalene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.133\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-\u003c/p\u003e\u003cp\u003e9,12,15-Octadecatrienoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16.247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3,7,11,15-Tetramethyl-2-hexadecen-1-ol, Neophytadiene, 1,4-Eicosadiene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e22.175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCyclohexa-2,5-diene-1,4-dione, 2-methyl-5-(4-morpholinyl)-\u003c/p\u003e\u003cp\u003e4-Geranyloxy-3-hydroxy-5-methoxyphthalaldehyde\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25.654\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCyclotrisiloxane, hexamethyl-1,2-Benzenediol, 3,5-bis(1,1-dimethylethyl)-\u003c/p\u003e\u003cp\u003eCyclotrisiloxane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25.946\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTris(tert-butyldimethylsilyloxy)arsane, Cyclotrisiloxane, hexamethyl-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26.341\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1,4-Phthalazinedione, 2,3-dihydro-6-nitro-\u003c/p\u003e\u003cp\u003eMethyltris(trimethylsiloxy)silane\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, β-mannanase was produced from \u003cem\u003eC. chauvoei\u003c/em\u003e, identified as the highest β-mannanase-producing bacterium among all isolates obtained from the gut of ruminant animals. The ruminant gut harbors a complex microbiome that facilitates efficient hydrolysis of plant materials consumed by the animals (Badejo et al., 2021). Consequently, the rumen is considered a natural and highly efficient bioreactor for the conversion of lignocellulosic materials into fermentable sugars (Selormey et al., 2021). Notably, mannanolytic bacteria have been observed in the rumen of cows, goats, and buffalo, highlighting their specialized role in the degradation of mannan-containing polysaccharides (Badejo et al., 2021; Selormey et al., 2021).\u003c/p\u003e\u003cp\u003eThe ability of these bacteria to convert mannan-rich compounds into absorbable sugars is particularly advantageous for the saccharification of agro-industrial residues for biofuel production. Moreover, since the bacterial species isolated in this study are anaerobic, there is potential for simultaneous saccharification and production of biofuels, including biobutanol and acetone\u0026ndash;butanol\u0026ndash;ethanol (ABE), demonstrating their dual functional role in bioconversion processes (Kaylani et al., 2017; Sharma et al., 2021).\u003c/p\u003e\u003cp\u003eDespite the potential of β-mannanases in lignocellulosic biomass valorization, few studies have explored anaerobic rumen-derived bacteria that combine high enzymatic activity with dual functionality. The \u003cem\u003eC. chauvoei\u003c/em\u003e isolated in this study exhibited remarkable thermostability, broad pH tolerance, and resilience to metal ions and certain inhibitors, while also efficiently producing biofuels. These findings address an existing research gap by identifying a rumen-derived anaerobe capable of both saccharification and biofuel production, providing a promising biocatalyst for integrated lignocellulosic biomass conversion and sustainable energy production.\u003c/p\u003e\u003cp\u003eAlthough twenty bacterial isolates were obtained from the rumen of cow, biochemical identification revealed only five \u003cem\u003eClostridium\u003c/em\u003e species. Similar observations have been reported by Badejo et al. (2021) and Oyeleke and Okunsanmi (2008), with the highest bacterial counts observed in cows, while Deepa et al. (2019) also confirmed diverse rumen communities, albeit focusing on aerobes. The limited reports on anaerobes are likely due to their strict cultural requirements; however, Khattab et al. (2017) successfully isolated anaerobic bacteria from frozen rumen liquor, and notable rumen anaerobes include \u003cem\u003eClostridium thermocellum\u003c/em\u003e (Lamed and Bayer, 1988), \u003cem\u003eC. cellulovorans\u003c/em\u003e (Yang et al., 2015), \u003cem\u003eRuminococcus albus\u003c/em\u003e (Ohara et al., 2000), \u003cem\u003eR. flavefaciens\u003c/em\u003e (Suen et al., 2011), and \u003cem\u003eAcetivibrio cellulolyticus\u003c/em\u003e (Dassa et al., 2012). The selection of anaerobic bacteria in this study was warranted because they are naturally adapted to the oxygen-limited rumen environment, efficiently degrading mannan-rich polysaccharides and enabling simultaneous saccharification and biofuel (acetone\u0026ndash;butanol\u0026ndash;ethanol) production, an advantage over many aerobic isolates (Kaylani et al., 2017; Sharma et al., 2021).\u003c/p\u003e\u003cp\u003eA limitation of this study is the absence of molecular identification methods such as 16S rRNA gene sequencing. The bacterial isolates were identified solely based on morphological and biochemical characteristics, which may not provide precise taxonomic resolution, particularly among closely related genera such as \u003cem\u003eClostridium\u003c/em\u003e, \u003cem\u003eRuminococcus\u003c/em\u003e, and \u003cem\u003eAcetivibrio\u003c/em\u003e. This limitation was primarily due to limited funding, self-sponsorship, and lack of access to molecular biology equipment. Future studies will incorporate molecular and genomic approaches to confirm strain identity and elucidate the genetic and enzymatic mechanisms underlying biobutanol production.\u003c/p\u003e\u003cp\u003eβ-Mannanase activity was detected in all twenty bacterial isolates, with isolate D3b, identified as \u003cem\u003eC. chauvoei\u003c/em\u003e, exhibiting the highest enzyme activity. This corroborates findings by Badejo et al. (2021) and Olaniyi et al. (2023), who reported that rumen microbiota, particularly bacteria, possess substantial β-mannanase-producing capacity, with \u003cem\u003eProvidencia pulmonis\u003c/em\u003e and \u003cem\u003eAlcaligenes\u003c/em\u003e sp. identified as the highest producers, respectively. Other potent β-mannanase producers include \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e from horse feces (Cho, 2009), \u003cem\u003eBacillus clausii\u003c/em\u003e S10 from a soda lake (Zhou et al., 2018), and \u003cem\u003eBacillus cereus\u003c/em\u003e from Bani Salama lake (El-Sharouny et al., 2015). The remarkable ability of \u003cem\u003eC. chauvoei\u003c/em\u003e to produce β-mannanase and hydrolyze β-mannan into fermentable sugars highlights its potential as a biocatalyst for diverse industrial applications, including biofuel production, paper pulp processing, and bleaching.\u003c/p\u003e\u003cp\u003eThe findings of this study underscore the rumen of ruminant animals as a rich reservoir of anaerobic bacteria capable of efficient lignocellulosic biomass conversion. The dual functionality of \u003cem\u003eC. chauvoei\u003c/em\u003e, combining high β-mannanase activity with the ability to produce biofuels such as acetone\u0026ndash;butanol\u0026ndash;ethanol, presents a promising avenue for integrated bioprocessing of agro-industrial residues. These results provide a foundation for future studies employing molecular tools to confirm bacterial identity, optimize enzymatic performance, and explore the genetic and metabolic pathways underlying biofuel production.\u003c/p\u003e\u003cp\u003eThe 9-fold purification and ~\u0026thinsp;36% yield achieved for \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase represent a substantial improvement over previously reported bacterial β-mannanases. For instance, \u003cem\u003eAlcaligenes\u003c/em\u003e sp. β-mannanase exhibited only 2.6-fold purification with 1.75% yield (Olaniyi et al., 2023), while partially purified β-mannanases from \u003cem\u003eProvidencia vernicola\u003c/em\u003e and \u003cem\u003ePsychrobacter pulmonis\u003c/em\u003e showed 2- and 6-fold purification with yields of 13% and 18%, respectively (Badejo et al., 2021). Although higher purification folds have been reported for fungal β-mannanases, such as 23.24-fold for \u003cem\u003ePenicillium italicum\u003c/em\u003e (Olaniyi and Arotupin, 2014) and 32.9-fold by Cheng et al. (2016), the combination of relatively high yield and fold observed in this study highlights the efficiency of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase purification from a bacterial source. The increase in specific activity during purification, as commonly observed (Cheng et al., 2016; Adeseko et al., 2021, 2022), confirms that the enzyme retained substantial catalytic potential. This notable retention of activity, coupled with high yield, underscores the enzyme\u0026rsquo;s novelty as a potent bacterial β-mannanase suitable for industrial applications, including biofuel production, saccharification of agro-residues, and bioconversion processes.\u003c/p\u003e\u003cp\u003eThe homogeneity of the purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase was confirmed by SDS-PAGE, which revealed a molecular weight of 43 kDa. This value falls within the range reported for bacterial and fungal β-mannanases, such as 18\u0026ndash;39 kDa for \u003cem\u003ePaenibacillus\u003c/em\u003e sp. (Dhawan, 2021), 32\u0026ndash;53 kDa for \u003cem\u003eTrichoderma\u003c/em\u003e sp. (Ferreira et al., 2004; Wang et al., 2014), and 40\u0026ndash;100 kDa for \u003cem\u003eAspergillus\u003c/em\u003e sp. (Regalado et al., 2000; Naganagouda et al., 2009; Bhaturiwala et al., 2021). Lower molecular weights, such as 22 kDa, have been reported for \u003cem\u003eBacillus halodurans\u003c/em\u003e PPKS-2 (Vijayalaxmi et al., 2013), while higher molecular weights, including 130 kDa for \u003cem\u003eBacillus\u003c/em\u003e sp. JAMB-750 (Hatada et al., 2005), 162 kDa for \u003cem\u003eBacillus stearothermophilus\u003c/em\u003e ATCC 266 (Talbot and Sygusch, 1990), and 192 kDa for \u003cem\u003eBacteroides ovatus\u003c/em\u003e (Gherardini and Salyers, 1987), highlight the diversity of β-mannanase sizes. The intermediate molecular weight of 43 kDa observed for \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase, combined with its high catalytic efficiency and yield, underscores its unique biochemical features as a potent bacterial enzyme for industrial applications, bridging the gap between low- and high-molecular-weight β-mannanases.\u003c/p\u003e\u003cp\u003eThe optimum activity of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase at pH 4 aligns with that reported for \u003cem\u003eProvidencia pulmonis\u003c/em\u003e (pH 4; Badejo et al., 2021) and \u003cem\u003eBacillus clausii\u003c/em\u003e strains (pH 4.5\u0026ndash;5; Zhou et al., 2018). Other bacterial β-mannanases, including \u003cem\u003eBacillus subtilis\u003c/em\u003e E91, \u003cem\u003eBacillus\u003c/em\u003e SWU60, \u003cem\u003eB. subtilis\u003c/em\u003e TBS2, and \u003cem\u003eAlcaligenes\u003c/em\u003e sp., have been reported to exhibit optimum activity at mildly acidic pH 6 (Cheng et al., 2016; Seesom et al., 2017; Luo et al., 2017; Olaniyi et al., 2023). Notably, the enzyme maintained substantial activity at alkaline pH 8\u0026ndash;10, retaining 47\u0026ndash;71% of its relative activity, whereas other bacterial β-mannanases, such as \u003cem\u003eBacillus\u003c/em\u003e sp. JAMB-750, \u003cem\u003eB. halodurans\u003c/em\u003e PPKS-2, and \u003cem\u003eB. inaquosorum\u003c/em\u003e CSP31, exhibit optimum activity only at more extreme alkaline pH values of 10\u0026ndash;12.5 (El-Sharouny et al., 2017; Vijayalaxmi et al., 2013; Regmi et al., 2017). This broad pH tolerance highlights the versatility and robustness of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase, enabling it to function efficiently across both acidic and alkaline environments. Such flexibility enhances its potential for industrial applications, including the saccharification of agro-industrial residues, paper pulp processing, and biofuel production, where variable pH conditions are commonly encountered.\u003c/p\u003e\u003cp\u003eThe purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase exhibited remarkable stability across a broad pH range of 2\u0026ndash;11 after 3 h of incubation, surpassing that of other reported β-mannanases. For comparison, β-mannanase from \u003cem\u003eAcinetobacter\u003c/em\u003e sp. SP showed maximum stability between pH 3\u0026ndash;10 (Titapoka et al., 2008), \u003cem\u003eAlcaligen\u003c/em\u003e sp. was most stable at neutral pH 3\u0026ndash;11 (Olaniyi et al., 2023), and \u003cem\u003eP. pulmonis\u003c/em\u003e remained stable for only 50 min between pH 4\u0026ndash;11 (Badejo et al., 2021). Fungal β-mannanase from \u003cem\u003ePenicillium italicum\u003c/em\u003e was stable at pH 5 (Olaniyi and Arotupin, 2014), while a mutant \u003cem\u003eAlcaligen\u003c/em\u003e sp. enzyme showed peak stability at acidic pH 5 and alkaline pH 8 (Olaniyi et al., 2023). Many β-mannanases reported in the literature exhibit either acidic or alkaline stability, with activity declining outside their optimum range (Cheng et al., 2016; Regmi et al., 2017). The broad pH stability of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase highlights its distinctive functional versatility and suggests its suitability for diverse industrial applications, including feed supplementation, pulp bleaching, saccharification of agro-residues, and biofuel production.\u003c/p\u003e\u003cp\u003eThe purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase exhibited optimum activity at 60\u0026deg;C, consistent with β-mannanases from \u003cem\u003eP. pulmonis\u003c/em\u003e (Badejo et al., 2021), \u003cem\u003eAspergillus foetidus\u003c/em\u003e (De Marco et al., 2015), and \u003cem\u003ePediococcus acidilactici\u003c/em\u003e (Nadaroglu et al., 2017). Remarkably, the enzyme maintained high activity between 70 and 90\u0026deg;C, indicating pronounced thermo-tolerance, a feature distinguishing it from most previously reported β-mannanases. In contrast, β-mannanase from \u003cem\u003eP. italicum\u003c/em\u003e was optimal at 70\u0026deg;C (Olaniyi and Arotupin, 2014), while \u003cem\u003eBacillus circulans\u003c/em\u003e NT 6.7 and \u003cem\u003eTrichoderma harzianum\u003c/em\u003e T7 exhibited optimum activity at 50\u0026deg;C (Phothichitto et al., 2006; Ferreira and Filho, 2004). The enzyme\u0026rsquo;s thermo-tolerant profile makes it highly suitable for industrial applications, where high-temperature processes are common.\u003c/p\u003e\u003cp\u003eThe thermal stability of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase is further highlighted by its retention of substantial activity over extended incubation, comparable to \u003cem\u003eAlcaligen\u003c/em\u003e sp., which maintained stability across 25\u0026ndash;80\u0026deg;C after 1 h and retained high residual activity at 25\u0026ndash;60\u0026deg;C after 3 h (Olaniyi et al., 2023). Other reported enzymes showed more limited stability: β-mannanases from \u003cem\u003ePaenibacillus scooki\u003c/em\u003e and \u003cem\u003eP. italicum\u003c/em\u003e were maximally stable at 40 and 50\u0026deg;C after 30\u0026ndash;40 min (Yin et al., 2012; Olaniyi and Arotupin, 2014), while \u003cem\u003eMalbranchea cinnamomea\u003c/em\u003e β-mannanase was stable at 60\u0026deg;C (Ahiwar et al., 2016), and \u003cem\u003eBacillus clausii\u003c/em\u003e β-mannanase exhibited a rapid decline in activity after 30\u0026ndash;60 min (Zhou et al., 2018). The broad temperature tolerance and prolonged thermo-stability of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase underscore its novel enzymatic properties and potential for high-temperature industrial processes, including biofuel production, pulp bleaching, and biomass saccharification.\u003c/p\u003e\u003cp\u003eThe purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase exhibited remarkable enhancement in activity in the presence of most metal ions tested, except Cu\u0026sup2;⁺, highlighting its metal-ion promiscuity, a highly desirable feature for industrial applications. This behavior is comparable to β-mannanase from wild \u003cem\u003eAlcaligen\u003c/em\u003e sp., which showed increased activity in the presence of Zn\u0026sup2;⁺, Co\u0026sup2;⁺, and Mg\u0026sup2;⁺ but was inhibited by Na⁺, Ni\u0026sup2;⁺, and Cu\u0026sup2;⁺ (Olaniyi et al., 2023). Similarly, \u003cem\u003eP. pulmonis\u003c/em\u003e β-mannanase was enhanced by Na⁺, Mn\u0026sup2;⁺, and Co\u0026sup2;⁺ but inhibited by K⁺, Cu\u0026sup2;⁺, Mg\u0026sup2;⁺, Pb\u0026sup2;⁺, and Al\u0026sup3;⁺ (Badejo et al., 2021), while \u003cem\u003eP. cooki\u003c/em\u003e and \u003cem\u003eA. foetidus\u003c/em\u003e enzymes were activated by Co\u0026sup2;⁺ (Yin et al., 2012; De Marco et al., 2015). These variations in metal-ion responsiveness likely reflect environmental influences on the producing microbes (Yaashikaa et al., 2022), offering opportunities to optimize and enhance enzyme performance for biotechnological processes.\u003c/p\u003e\u003cp\u003eInhibitor studies revealed that SDS and cysteine were potent inhibitors of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase across all concentrations tested. However, the enzyme displayed remarkable resilience to EDTA, urea, and sodium azide, even at high concentrations (10 mM), demonstrating its ability to withstand harsh conditions. These observations align with reports for β-mannanases from \u003cem\u003eA. niger\u003c/em\u003e (Kote et al., 2009), \u003cem\u003eMalbranchea cinnamomea\u003c/em\u003e (Ahirwar et al., 2016), \u003cem\u003eP. pulmonis\u003c/em\u003e (Badejo et al., 2021), and \u003cem\u003eAlcaligen\u003c/em\u003e sp. (Olaniyi et al., 2023), which tolerated 1\u0026ndash;5 mM EDTA. The mild inhibition of \u003cem\u003eMalbranchea cinnamomea\u003c/em\u003e β-mannanase by SDS and strong inhibition by urea (Ahirwar et al., 2016) further highlights the exceptional robustness of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase. SDS inhibition is concentration-dependent: low concentrations bind the enzyme\u0026rsquo;s surface, while higher concentrations may denature the enzyme (Dhawal, 2021). The enhancement by metal ions and tolerance to inhibitors at high concentrations indicate that \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase is likely a metalloprotein and highly resilient, making it well-suited for industrial and biotechnological applications requiring stability under severe conditions.\u003c/p\u003e\u003cp\u003eThe activity of \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase was largely inhibited in the presence of surfactants (Tween-20 and Triton X-100) and most organic solvents, with the notable exception of formaldehyde, which enhanced enzymatic activity. This contrasts with recombinant β-mannanase AoMan134 from \u003cem\u003eAspergillus oryzae\u003c/em\u003e, whose activity decreased in the presence of organic solvents (methanol, ethanol, isopropanol, and acetone) and detergents (SDS, Triton X-100, and Tween-20) (Wang et al., 2012). Similarly, \u003cem\u003eBacillus licheniformis\u003c/em\u003e HDYM-04 β-mannanase retained maximal stability in dimethyl sulfoxide and hexane, was moderately inhibited by acetone and chloroform, and strongly inhibited by cetyl trimethyl ammonium bromide (Ge et al., 2016). \u003cem\u003ePaenibacillus thiaminolyticus\u003c/em\u003e β-mannanase displayed stability against most organic solvents, except methanol, ethanol, propanol, and propanone, which caused varying degrees of inhibition (Dhawan, 2021). Remarkably, among the polar, water-miscible solvents tested, formaldehyde uniquely enhanced \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase activity, suggesting it may play a stabilizing role, either during enzymatic reactions or storage (Dhawan, 2021). This feature highlights the enzyme\u0026rsquo;s potential for industrial applications where solvent tolerance or stabilization is advantageous, such as in biofuel production, paper pulp processing, and biocatalysis under non-aqueous conditions.\u003c/p\u003e\u003cp\u003eThe kinetic parameters of purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase were determined, with a Km of 30.7 mM and Vmax of 7.88 mg/mL/min, indicating strong substrate affinity and high catalytic efficiency. Comparable studies report varied kinetic behaviors for β-mannanases from different sources: \u003cem\u003eAspergillus terrus\u003c/em\u003e exhibited a Km of 5.9 mg/mL and Vmax of 39.42 \u0026micro;mol/mL/min (Soni et al., 2016), \u003cem\u003eBacillus licheniformis\u003c/em\u003e 2.69 mg/mL and 251.41 U/mg (Ge et al., 2016), and \u003cem\u003ePaenibacillus thiaminolyticus\u003c/em\u003e 5 mg and 1.1 \u0026times; 10\u0026sup3; U/mg (Dhawan, 2021). Lower Km and Vmax values have been reported for \u003cem\u003eP. italicum\u003c/em\u003e (Km 0.26 mg/mL; Vmax 0.12 mol/min/mL) (Olaniyi and Arotupin, 2014), \u003cem\u003eP. pulmonis\u003c/em\u003e (Km 0.73 mg/mL) (Badejo et al., 2021), and \u003cem\u003eAlcaligen\u003c/em\u003e sp. (Km 5.2 \u0026times; 10⁻⁴ mg/mL) (Olaniyi et al., 2023). The comparatively low Km and high Vmax observed for \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase suggest an excellent substrate specificity, strong affinity, and efficient catalysis, highlighting its potential for industrial applications requiring efficient hydrolysis of β-mannan-rich polysaccharides, such as biofuel production, pulp and paper processing, and feedstock saccharification.\u003c/p\u003e\u003cp\u003eThe thermal inactivation of purified \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase followed first-order kinetics, consistent with β-mannanases from \u003cem\u003eEnterobacter asburiae\u003c/em\u003e (Dhiman et al., 2019) and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e SS11 (Singh et al., 2019). The inactivation rate constant (ki) was temperature-dependent, increasing with rising temperatures, and similarly, the deactivation rate (kd) increased with temperature (Dhiman et al., 2019; Singh et al., 2019; Sadaqat et al., 2024). Both ki and kd reflect the enzyme\u0026rsquo;s time-dependent denaturation at specific temperatures and serve as indicators of thermostability (Singh et al., 2019).\u003c/p\u003e\u003cp\u003eThe enzyme retained high half-life (t1/2) and D-values even at 70\u0026deg;C, indicating remarkable thermal stability. Specifically, at 70\u0026deg;C, the t1/2 was 216.6 min, which is superior to reported β-mannanases from \u003cem\u003eA. awamori\u003c/em\u003e (t1/2\u0026thinsp;\u0026gt;\u0026thinsp;30 min at 66\u0026deg;C) (Neustroev et al., 1991), \u003cem\u003eBacillus subtilis\u003c/em\u003e (t1/2 106.4 min at 65\u0026deg;C) (Liu et al., 2008), \u003cem\u003eCaldocellum saccharolyticum\u003c/em\u003e (t1/2 45 min at 80\u0026deg;C), and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (t1/2 135.91 min at 70\u0026deg;C) (Singh et al., 2019).\u003c/p\u003e\u003cp\u003eThe Z-value of 57\u0026deg;C and activation energy (Ea) of 23 kJ/mol further highlighted the enzyme\u0026rsquo;s thermostable nature. High Z-values indicate lower sensitivity to heat, while the linear Arrhenius plot reflects the temperature dependence of the inactivation rate and the unfolding mechanism of the enzyme (Bedel et al., 2020). The low Ea value indicates that \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase can efficiently hydrolyze mannan to fermentable sugars, a desirable feature for industrial applications such as biofuel production, pulp bleaching, and feed processing.\u003c/p\u003e\u003cp\u003eThermodynamic parameters further supported the enzyme\u0026rsquo;s stability. The free energy of activation (ΔG) was 63 kJ/mol, indicating non-spontaneity and structural stability, comparable to β-mannanases from \u003cem\u003eEnterobacter asburiae\u003c/em\u003e (73.66 kJ/mol) (Dhiman et al., 2019) and \u003cem\u003eThermotoga maritima\u003c/em\u003e (74.82\u0026ndash;82.8 kJ/mol) (Sadaqat et al., 2022), but lower than \u003cem\u003ePenicillium humicola\u003c/em\u003e (107.8\u0026ndash;111.41 kJ/mol) (Ismail et al., 2019). The enthalpy change (ΔH) of 20 kJ/mol aligns with \u003cem\u003eBacillus\u003c/em\u003e sp. CSB-39 (24 kJ/mol) (Dhiman et al., 2019) and reflects conformational changes due to heat (Ortega et al., 2019). The negative entropy change (ΔS = -134.4 J/mol/K) suggests the enzyme maintains structural rigidity with low disorder during activation, consistent with observations in \u003cem\u003ePenicillium humicola\u003c/em\u003e (-118.99 to -133.67 J/mol/K) (Ismail et al., 2019).\u003c/p\u003e\u003cp\u003eTogether, the combination of high t1/2, D-values, Z-value, low Ea, and favorable thermodynamic parameters indicates that \u003cem\u003eC. chauvoei\u003c/em\u003e β-mannanase is highly thermostable and capable of retaining activity under harsh thermal conditions. This remarkable thermal resilience, coupled with its substrate specificity, broad pH range, and metal ion tolerance, underscores its potential for diverse industrial bioprocesses, particularly in biofuel production, feed supplementation, and lignocellulosic biomass conversion.\u003c/p\u003e\u003cp\u003eThe saccharification of palm-kernel by purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e and the subsequent production of acetone, butanol, and ethanol (ABE) were highly successful, yielding 55.0 g/L biobutanol, 60.2 g/L ethanol, and 70.1 g/L acetone. These yields are notably higher than those reported for other \u003cem\u003eClostridium\u003c/em\u003e strains, such as \u003cem\u003eClostridium\u003c/em\u003e sp. A53, which produced 10.5 g/L ABE and 0.11 g/L biobutanol (Johnravindar et al., 2019), and \u003cem\u003eClostridium saccharoperbutylacetonium\u003c/em\u003e N1-4, which produced 3.27 g/L biobutanol (Shukor et al., 2016). Comparatively, \u003cem\u003eSerratia marcescens\u003c/em\u003e yielded 10 g/L bioethanol, and engineered \u003cem\u003eLactobacillus diolivorans\u003c/em\u003e increased production to 13.4 g/L (Russmayer et al., 2019). A common limitation in \u003cem\u003eClostridium\u003c/em\u003e-based fermentations is low yield due to solvent accumulation, which inhibits growth and sugar consumption (Al-Shorgani et al., 2014). High biobutanol concentrations disrupt membrane fluidity and transport processes, leading to leakage of intracellular proteins and metabolites. The exceptionally high biobutanol production observed in this study may reflect the inherent solvent tolerance of \u003cem\u003eC. chauvoei\u003c/em\u003e, enabling sustained growth and efficient sugar conversion even under solvent stress.\u003c/p\u003e\u003cp\u003eFTIR analysis further confirmed the production of biobutanol and related biofuels. Characteristic functional groups, including 3337 cm⁻\u0026sup1; (OH stretch) and 667 cm⁻\u0026sup1; (C\u0026ndash;OH), were observed, consistent with previous reports on \u003cem\u003eClostridium\u003c/em\u003e sp. (Johnravindar et al., 2019). Additional peaks corresponding to secondary or tertiary alcohols (1120\u0026ndash;1080 cm⁻\u0026sup1;) aligned with fingerprint regions for butanol. These observations are consistent with acetone-butanol-ethanol (ABE) spectral signatures reported by Huang et al. (2015) (3352\u0026ndash;3246 cm⁻\u0026sup1;), confirming the successful fermentation and co-production of acetone, butanol, and ethanol. The FTIR analysis provides direct structural evidence of biofuel formation and highlights the potential of \u003cem\u003eC. chauvoei\u003c/em\u003e for industrial-scale ABE fermentation from lignocellulosic substrates.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates the novel characteristics of purified β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e and its efficient conversion of palm kernel cake into fermentable sugars, subsequently transformed into acetone, butanol, and ethanol (ABE) by the bacterium. The enzyme displayed exceptional thermo-stability and pH versatility, with optimum activity at 60\u0026deg;C and pH 4, stability across 30\u0026ndash;80\u0026deg;C for 1 h, and a broad pH range of 2\u0026ndash;12 over 6 h. Activity was enhanced by metal ions and tolerated inhibitors such as sodium azide and urea, while partial inhibition by EDTA indicated metal ion dependence. Formaldehyde uniquely stabilized the enzyme among organic solvents. Thermodynamic and kinetic analyses confirmed its robustness, with ΔG\u0026thinsp;=\u0026thinsp;63 kJ/mol, ΔH\u0026thinsp;=\u0026thinsp;23 kJ/mol, Z-value\u0026thinsp;=\u0026thinsp;57\u0026deg;C, and half-life\u0026thinsp;=\u0026thinsp;216 min. The high ABE yields highlight the potential of both the enzyme and \u003cem\u003eC. chauvoei\u003c/em\u003e for biofuel production. Collectively, these properties position this β-mannanase as a versatile biocatalyst for industrial applications, including feed supplementation, agro-residue saccharification, pulp and paper processing, and sustainable biofuel production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was self-sponsored. This research received no external funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Mr. A. Emmanuel and Mr. O. Christopher, both of Bowen University, Iwo, Osun State, Nigeria, for their assistance in the provision and operation of the FTIR machine\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePTO:\u003c/strong\u003e Investigation, Methodology, Funding Acquisition, Formal Analysis, Writing\u0026ndash;Original Draft;\u0026nbsp;\u003cstrong\u003eOOO:\u003c/strong\u003e Conceptualization, Supervision, Validation, Visualization, Project Administration,\u003cbr\u003e\u003cstrong\u003eOTL:\u003c/strong\u003e Investigation, Methodology, Resources, Formal Analysis, Validation, Visualization, Writing\u0026ndash;Original Draft, Writing \u0026ndash; Review and Editing, \u003cstrong\u003eFAA:\u003c/strong\u003e Supervision, Validation, Visualization. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdeseko, C. J., Sanni, D. 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Plasma tissue plasminogen activator and plasminogen activator inhibitor-1 in hospitalized COVID-19 patients. \u003cem\u003eScientific reports\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(1), 1580.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ruminant gut microbiota, thermostable and pH-stable β-mannanase, palm kernel cake, lignocellulosic biomass saccharification, biobutanol production","lastPublishedDoi":"10.21203/rs.3.rs-7933920/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7933920/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe ruminant gut microbiome represents a valuable source of lignocellulolytic enzymes, particularly β-mannanases that hydrolyze mannan into fermentable sugars. However, few studies have characterized β-mannanases from \u003cem\u003eClostridium\u003c/em\u003e species with respect to their catalytic stability and potential for biofuel production from agro-industrial residues.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eβ-Mannanase from \u003cem\u003eClostridium chauvoei\u003c/em\u003e was purified from the crude extract through ammonium sulfate precipitation, dialysis, and ion-exchange and size-exclusion chromatography, achieving a 36% yield and 9-fold purification. The enzyme exhibited optimal activity at pH 4.0 and 60\u0026deg;C and maintained stability over a broad pH range (2\u0026ndash;12 for 6 h) and temperature range (30\u0026ndash;80\u0026deg;C). Enzyme activity was enhanced by Mg\u0026sup2;⁺, Zn\u0026sup2;⁺, and Mn\u0026sup2;⁺, while inhibited by EDTA, SDS, and cysteine; among organic solvents, only formaldehyde stabilized the enzyme. It showed a half-life of 216 min at 70\u0026deg;C, with thermodynamic parameters ΔG\u0026thinsp;=\u0026thinsp;63 kJ/mol, ΔH\u0026thinsp;=\u0026thinsp;23 kJ/mol, and ΔS\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;134.4 J/mol\u0026middot;K. Kinetic constants (Km\u0026thinsp;=\u0026thinsp;30.7 mg/mL, Vmax\u0026thinsp;=\u0026thinsp;7.88 \u0026micro;mol/mL/min) indicated strong substrate affinity and catalytic efficiency. Application of the purified enzyme to pretreated palm kernel substrate yielded substantial biobutanol (55 g/L), ethanol (60 g/L), and acetone (70 g/L) confirmed by GC\u0026ndash;MS and FTIR analyses.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study highlights a novel thermostable and pH-tolerant β-mannanase from \u003cem\u003eC. chauvoei\u003c/em\u003e capable of efficiently hydrolyzing lignocellulosic biomass into fermentable sugars for acetone-butanol-ethanol (ABE) production. The enzyme\u0026rsquo;s robust catalytic properties and high saccharification efficiency position it as a promising biocatalyst for sustainable biofuel production and other industrial bioprocess applications.\u003c/p\u003e","manuscriptTitle":"A thermostable and alkaline β-mannanase from Clostridium chauvoei isolated from the ruminant gut exhibits potential for bioethanol production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 13:12:17","doi":"10.21203/rs.3.rs-7933920/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"afde0746-c2ff-4354-84b9-48805d40d610","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T08:24:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 13:12:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7933920","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7933920","identity":"rs-7933920","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}