An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

Chemical synthesis of lactones from cycloalkanes is a multi‐step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by‐products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in‐vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp. CHX100. Resting (non‐growing) cells of the recombinant host P. taiwanensis VLB120 converted cyclohexane, cyclohexanol, and cyclohexanone to ?‐caprolactone at 22, 80–100, and 170 U g_CDW^?1, respectively. Cyclohexane (5 mM) was completely converted with a selectivity of 65% for ?‐caprolactone formation in 2 hr without accumulation of intermediate products. Promiscuity of the whole‐cell biocatalyst gave access to analogous lactones from cyclooctane and cyclodecane. A total product concentration of 2.3 g L^?1 and a total turnover number of 36,720 was achieved over 5 hr with a biocatalyst concentration of 6.8 g_CDW L^?1.

     

Ambulant 24‐h glucose rhythms mark calendar and biological age in apparently healthy individuals

Glucose metabolism marks health and disease and is causally inferred in the aging process. Ambulant continuous glucose monitoring provides 24‐h glucose rhythms under daily life conditions. We aimed to describe ambulant 24‐h glucose rhythms measured under daily life condition in relation to calendar and biological age in apparently healthy individuals. In the general population and families with propensity for longevity, we studied parameters from 24‐h glucose rhythms; glucose levels; and its variability, obtained by continuous glucose monitoring. Participants were 21 young (aged 22–37 years), 37 middle‐aged (aged 44–72 years) individuals from the general population, and 26 middle‐aged (aged 52–74 years) individuals with propensity for longevity. All were free of diabetes. Compared with young individuals, middle‐aged individuals from the general population had higher mean glucose levels (5.3 vs. 4.7 mmol L^?1, P < 0.001), both diurnally (P < 0.001) and nocturnally (P = 0.002). Glucose variability was higher in the middle‐aged compared with the young (standard deviation 0.70 vs. 0.57 mmol L^?1, P = 0.025). Compared with middle‐aged individuals from the general population, middle‐aged individuals with propensity for longevity had lower overall mean glucose levels (5.2 vs. 5.4 mmol L^?1, P = 0.047), which were more different nocturnally (4.8 vs. 5.2 mmol L^?1, P = 0.003) than diurnally (5.3 vs. 5.5 mmol L^?1, P = 0.14). There were no differences in glucose variability between these groups. Results were independent of body mass index. Among individuals without diabetes, we observed significantly different 24‐h glucose rhythms depending on calendar and biological age.     

Computational analysis of the CB1 carboxyl‐terminus in the receptor‐G protein complex

Despite the important role of the carboxyl‐terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1‐Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449–32465) to incorporate a complete CB1 Ct (Glu416^Ct–Leu472^Ct). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1‐Gi complex. The resulting models were consistent with the NMR‐determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site‐directed mutagenesis studies of Glu416^Ct, Asp423^Ct, Asp428^Ct, and Arg444^Ct of CB1 Ct suggested that the CB1 Ct can influence receptor‐G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. Proteins 2016; 84:532–543. © 2016 Wiley Periodicals, Inc.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Structural mechanism of ATP‐dependent DNA binding and DNA end bridging by eukaryotic Rad50

The Mre11–Rad50–Nbs1 (MRN) complex is a central factor in the repair of DNA double‐strand breaks (DSBs). The ATP‐dependent mechanisms of how MRN detects and endonucleolytically processes DNA ends for the repair by microhomology‐mediated end‐joining or further resection in homologous recombination are still unclear. Here, we report the crystal structures of the ATPγS‐bound dimer of the Rad50^NBD (nucleotide‐binding domain) from the thermophilic eukaryote Chaetomium thermophilum (Ct) in complex with either DNA or CtMre11^RBD (Rad50‐binding domain) along with small‐angle X‐ray scattering and cross‐linking studies. The structure and DNA binding motifs were validated by DNA binding experiments in vitro and mutational analyses in Saccharomyces cerevisiae in vivo. Our analyses provide a structural framework for the architecture of the eukaryotic Mre11–Rad50 complex. They show that a Rad50 dimer binds approximately 18 base pairs of DNA along the dimer interface in an ATP‐dependent fashion or bridges two DNA ends with a preference for 3′ overhangs. Finally, our results may provide a general framework for the interaction of ABC ATPase domains of the Rad50/SMC/RecN protein family with DNA.     

NDP-sugars, floridoside and floridean starch levels in relation to activities of UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase in Solieria chorda l is (Rhodophyceae) under experimental conditions

Under limited nutrient availability (i.e. unenriched sea‐water) and under 75 mol photons m^–2 s^–1 irradiance 12:12 LD, thalli of Solieria chordalis J. Agardh accumulated floridean starch and floridoside. When they were transferred into nutrient‐enriched seawater (150 umol L^?1 NO3¹‐ and 7 umol L^?1 P0₄³i at 35 umol photons m^?2 s^?1 in irradiance 12:12 LD, starch and floridoside levels decreased. The main nucleotide diphosphate (NDP) sugars (i.e. UDP‐glucose, UDP‐galactose and ADP‐glucose) and the activities of UDP‐glucose pyrophosphorylase [Enzyme Code (EC) 2.7.7.9] and UDP‐glucose‐4‐epimerase (EC 5.1.3.2) were measured under these controlled culture conditions. Both UDP‐glucose and UDP‐galactose in the thal l i increased under conditions known to favor the accumulation of floridean starch and floridoside, whereas they decreased under conditions leading to floridean starch and floridoside breakdown. On the other hand, ADP‐glucose level only varied slightly. Although UDP‐glucose pyrophosphorylase activity rose under conditions of floridean starch synthesis, little variation was observed in UDP‐glucose‐4‐epimerase activity. These results suggest a possible enzymatic regulation of the NDP‐sugar and carbohydrate pool in which UDP‐glucose pyrophosphorylase would play a major role.     

Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5–15 g L^?1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^?1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^?1 and 45.98 U mL^?1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^?1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.     

Engineering the productivity of recombinant Escherichia coli for limonene formation from glycerol in minimal media

The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)‐limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)‐limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth‐dependent production of (S)‐limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two‐liquid phase fed‐batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L_org^–1 (S)‐limonene. Specific activities of 75 mU g_cdw^–1 were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g_cdw^–1) leading to a final (S)‐limonene concentration of 2,700 mg L_org^–1. Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)‐limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)‐limonene production. The two‐liquid phase fed‐batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.     

Simultaneous real‐time assay of copper and cadmium ions by infrared photo diode electrode implanted in the muscle of live fish

The electrical circuit of an infrared photodiode electrode (IPE) was used in the simultaneous assay of copper and cadmium ions. The electrode's cyclic voltammetry (CV), chronoamperometry and square‐wave (SW) stripping voltammetric optimum conditions were examined. Results for 0–160 mg L^?1 and 50–400 μg L^?1 SW Cu(II) Cd(II), the relative standard deviation of 0.158 Cu(II), 0.077 Cd(II) (n = 15) using 20.0 mg L^?1 have been obtained at optimum conditions. The low detection limit (S/N) was attained to be at 14.71 μg L^?1(2.31 × 10^?7 mol L^?1) Cu(II) and 18.42 μg L^?1(1.63 × 10^?7 mol L^?1) Cd(II). The handmade electrode was implanted deep in the muscle of live fish and interfaced with an electrochemical workstation. Real‐time analytical application was performed on the online assay of living tissue as the specimen was moving. The methods are deemed useful in interfaced assay for physiological control, nanodiode fabrication, and in the production of laboratory on a biochip. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:256–262, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20287     

Reference values for selected hematological and serum biochemical parameters of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles under intensive aquaculture conditions

This work aims to establish normal reference intervals for selected hemato‐biochemical parameters, based on their potential clinical relevance, and which may contribute to evaluating the health, nutritional and welfare status of Senegalese sole (Solea senegalensis Kaup, 1858) juveniles. Thirty‐one healthy Senegalese sole juveniles grown under intensive aquaculture conditions were used in the study. Based on the robust method with Box–Cox transformation data the established reference intervals for hematological parameters were: hematocrit 12–26%, hemoglobin 2.8–6 g dl^?1, erythrocytes 90–97.0% total, leucocytes 4–10% total; erythrocyte indices and differential leucocytes counts were also evaluated. Reference intervals for biochemical parameters were (g dl^?1) glucose 19–86 mg dl^?1, total protein 2.6–6.3, albumin 1–2.34, globulins 1.8–4.1, lipids 0.7–1.3, triglycerides 0.3–1.8, total cholesterol 0.1–0.9 g dl^?1, HDL‐cholesterol 4–65 mg dl^?1, LDL‐cholesterol 7–532 mg dl^?1, sodium 124–202 mmol L^?1), potassium 1.1–4.6 mmol L^?1, calcium 7.6–13.2 mg L^?1, magnesium 1.8–4.8 mg L^?1, inorganic phosphorus 3.4–9.5 mg L^?1, alkaline phosphatase 93–598 U L^?1, aspartate aminotransferase 118–605 U L^?1, lactate dehydrogenase 8.7–782 U L^?1, and creatine phosphokinase 31.5–552 U L^?1. This data is expected to provide a valuable tool to monitor the stress, health and nutritional conditions of Senegalese sole juveniles under aquaculture production.     

Transpiration rate relates to within‐ and across‐species variations in effective path length in a leaf water model of oxygen isotope enrichment

Stable oxygen isotope ratio of leaf water (δ¹⁸O_L) yields valuable information on many aspects of plant–environment interactions. However, current understanding of the mechanistic controls on δ¹⁸O_L does not provide complete characterization of effective path length (L) of the Péclet effect, – a key component of the leaf water model. In this study, we collected diurnal and seasonal series of leaf water enrichment and estimated L in six field‐grown angiosperm and gymnosperm tree species. Our results suggest a pivotal role of leaf transpiration rate (E) in driving both within‐ and across‐species variations in L. Our observation of the common presence of an inverse scaling of L with E in the different species therefore cautions against (1) the conventional treatment of L as a species‐specific constant in leaf water or cellulose isotope (δ¹⁸O_p) modelling; and (2) the use of δ¹⁸O_p as a proxy for g_s or E under low E conditions. Further, we show that incorporation of a multi‐species L‐E scaling into the leaf water model has the potential to both improve the prediction accuracy and simplify parameterization of the model when compared with the conventional approach. This has important implications for future modelling of oxygen isotope ratios.     

Production of recombinant beta-amylase of Bacillus aryabhattai

In this study, the effects of carbon source, nitrogen source, and metal ions on cell growth and Bacillus aryabhattai β-amylase production in recombinant Brevibacillus choshinensis were investigated. The optimal medium for β-amylase production, containing glucose (7.5?g·L^?1), pig bone peptone (40.0?g·L^?1), Mg²⁺ (0.05?mol·L^?1), and trace metal elements, was determined through single-factor experiments in shake flasks. When cultured in the optimized medium, the β-amylase yield reached 925.4?U mL^?1, which was 7.2-fold higher than that obtained in the initial medium. Besides, a modified feeding strategy was proposed and applied in a 3-L fermentor fed with glucose, which achieved a dry cell weight of 15.4?g L^?1. Through this cultivation approached 30?°C with 0?g·L^?1 initial glucose concentration, the maximum β-amylase activity reached 5371.8?U mL^?1, which was 41.7-fold higher than that obtained with the initial medium in shake flask.     

Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy

Para‐maleimidophenyl (p‐MP) modified gold surfaces have been prepared by one‐step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N‐terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p‐MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read‐out for a broad variety of biomolecular interactions on the same chip. Copyright © 2014 John Wiley & Sons, Ltd.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Chemiluminescence assay for Listeria monocytogenes based on Cu/Co/Ni ternary nanocatalyst coupled with penicillin as generic capturing agent

Bacterial pathogen control is important in seafood production. In this study, a Cu/Co/Ni ternary nanoalloy (Cu/Co/Ni TNA) was synthesized using the oleylamine reducing method. It was found that Cu/Co/Ni TNA greatly enhanced the chemiluminescence (CL) signal of the hydroxylamine‐O‐sulfonic acid (HOSA)–luminol system. The CL properties of Cu/Co/Ni TNA were investigated systemically. The possible CL mechanism also was intensively investigated. Based on the enhanced CL phenomenon of Cu/Co/Ni TNA, a Cu/Co/Ni TNA, penicillin, and anti‐L. monocytogenes (Listeria monocytogenes) antibody‐based sandwich complex assay for detection of L. monocytogenes was established. In this sandwich CL assay, penicillin was employed to capture and enrich pathogenic bacteria with penicillin‐binding proteins (PBPs) while anti‐L. monocytogenes antibody was adopted as the specific recognition molecule to recognize L. monocytogenes. L. monocytogenes was detected sensitively based on this new Cu/Co/Ni TNA–HOSA–luminol CL system. The CL intensity was proportional to the L. monocytogenes concentration ranging from 2.0 × 10² CFU ml^?1 to 3.0 × 10⁷ CFU ml^?1 and the limit of detection wa 70 CFU ml^?1. The reliability and potential applications of our method was verified by comparison with official methods and recovery tests in environment and food samples.     

Influence of carbon and nitrogen concentration on itaconic acid production by the smut fungus Ustilago maydis

Itaconic acid is a valuable platform compound for the production of bio‐based polymers, chemicals, and fuels. Ustilago maydis is a promising host for the production of itaconic acid from biomass‐derived substrates due to its unicellular growth pattern and its potential to utilize biomass‐derived sugar monomers and polymers. The potential of U. maydis for industrial itaconate production was assessed in pH‐controlled batch fermentations with varying medium compositions. Using 200 g/L glucose and 75 mM ammonium, 44.5 g/L of itaconate was produced at a maximum rate of 0.74 g L^?1 h^?1. By decreasing the substrate concentrations to 50 g/L glucose and 30 mM ammonium, a yield of 0.34 g/g (47 mol%) could be achieved. Itaconate production from xylose was also feasible. These results indicate that high itaconic acid titers can be achieved with U. maydis. However, further optimization of the biocatalyst itself through metabolic engineering is still needed in order to achieve an economically feasible process, which can be used to advance the development of a bio‐based economy.     

Optimization and effect of dairy industrial waste as media components in the production of hyaluronic acid by Streptococcus thermophilus

Hyaluronic acid (HA) production using a dairy industrial waste is a more cost-efficient strategy than using an expensive synthetic medium. In this study, we investigated the production of HA using Streptococcus thermophilus under shake flask conditions using dairy industrial waste as nutritional supplements, namely whey permeate (WP) and whey protein hydrolysate (WPH). Preliminary screening using Plackett–Burman design exhibited WP, WPH, initial pH, and inoculum size as significant factors influencing HA titer. Response surface methodology design of four factors was formulated at three levels for enhanced production of HA. Shake flask HA fermentation by S. thermophilus was performed under global optimized process conditions and the optimal HA titer (342.93?mg?L^?1) corroborates with Box–Behnken design prediction. The molecular weight of HA was elucidated as 9.22–9.46?kDa. The ultralow-molecular weight HA reported in this study has a potential role in drug and gene delivery applications.     

The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ions

Abstract

The gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase (Ct43Araf) was cloned in pET-21a(+) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography (IMAC). The recombinant CtCBM6B showed a molecular size close to 15 kDa by SDS-PAGE analysis, which was close to the expected size of 14.74 kDa. The ligand-binding affinity of CtCBM6B was assessed against ligands for which the catalytic enzyme, Ct43Araf showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arabinoxylan showed lower equilibrium association constant (K_a, 4.0% C^{? 1}), whereas, it exhibited higher affinity (K_a, 19.6% C^{? 1}) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low K_a (3.26% C^{? 1}) with rye arabinoxylan and higher affinity for oat spelt xylan (K_a, 17.9% C^{? 1}) which was corroborated by greater blue-shift in case of oat spelt xylan binding. The CtCBM6B binding with insoluble wheat arabinoxylan by adsorption isotherm analysis showed significant binding affinity as reflected by the equilibrium association constant (K_a), 9.4 × 10³ M^{? 1}. The qualitative analysis by SDS-PAGE also corroborated the CtCBM6B binding with insoluble wheat arabinoxylan. The protein-melting curve of CtCBM6B displayed the peak shift from 53°C to 59°C in the presence of Ca²⁺ ions indicating that Ca²⁺ ions impart thermal stability to the CtCBM6B structure.