Characterization of Neoagarooligosaccharide Hydrolase BpGH117 from a Human Gut Bacterium Bacteroides plebeius.

α-Neoagarobiose (NAB)/neoagarooligosaccharide (NAO) hydrolase plays an important role as an exo-acting 3,6-anhydro-α-(1,3)-L-galactosidase in agarose utilization. Agarose is an abundant polysaccharide found in red seaweeds, comprising 3,6-anhydro-L-galactose (AHG) and D-galactose residues. Unlike agarose degradation, which has been reported in marine microbes, recent metagenomic analysis of Bacteroides plebeius, a human gut bacterium, revealed the presence of genes encoding enzymes involved in agarose degradation, including α-NAB/NAO hydrolase. Among the agarolytic enzymes, BpGH117 has been partially characterized. Here, we characterized the exo-acting α-NAB/NAO hydrolase BpGH117, originating from B. plebeius. The optimal temperature and pH for His-tagged BpGH117 activity were 35 °C and 9.0, respectively, indicative of its unique origin. His-tagged BpGH117 was thermostable up to 35 °C, and the enzyme activity was maintained at 80% of the initial activity at a pre-incubation temperature of 40 °C for 120 min. Km and Vmax values for NAB were 30.22 mM and 54.84 U/mg, respectively, and kcat/Km was 2.65 s-1 mM-1. These results suggest that His-tagged BpGH117 can be used for producing bioactive products such as AHG and agarotriose from agarose efficiently.

Different Levels of Skin Whitening Activity among 3,6-Anhydro-l-galactose, Agarooligosaccharides, and Neoagarooligosaccharides.

3,6-Anhydro-l-galactose (AHG), a major monomeric constituent of red macroalgae (Rhodophyta), was recently reported to possess skin whitening activity. Moreover, AHG-containing oligosaccharides, such as agarooligosaccharides (AOSs) and neoagarooligosaccharides (NAOSs), have various physiological activities, including anti-inflammatory, antioxidant, and skin moisturizing effects. In this study, AHG and NAOSs were produced from agarose by enzymatic reactions catalyzed by an endo-type ß-agarase, an exo-type ß-agarase, and a neoagarobiose hydrolase. In a cell proliferation assay, AHG, AOSs, and NAOSs at 12.5, 25, and 50 µg/mL concentrations did not exhibit cytotoxicity toward murine B16 melanoma cells or human epidermal melanocytes. In an in vitro skin whitening activity assay of AHG, AOSs, and NAOSs at 50 µg/mL, AHG showed the highest skin whitening activity in both murine B16 melanoma cells and human epidermal melanocytes; this activity was mediated by the inhibition of melanogenesis. Neoagarotetraose and neoagarohexaose also exhibited in vitro skin whitening activity, whereas neoagarobiose and AOSs with degrees of polymerization of 3 (agarotriose), 5 (agaropentaose), and 7 (agaroheptaose) did not. Therefore, AHG is responsible for the skin whitening activity of agar-derived sugars, and the structural differences among the AHG-containing oligosaccharides may be responsible for their different skin whitening activities.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(ß-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-ß-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Acetonic Extract from the Feijoa sellowiana Berg. Fruit Exerts Antioxidant Properties and Modulates Disaccharidases Activities in Human Intestinal Epithelial Cells.

Feijoa sellowiana fruit has been shown to possess various biological activities, such as anti-bacterial and anti-cancer properties, in a variety of cellular models, but its activity on human intestinal epithelial cells has never been tested. The purpose of this study was to investigate the effects of the acetonic extract of F. sellowiana fruits on the viability, membrane peroxidation, disaccharidases activities and proliferation of in vitro models of human intestinal epithelial cells. To obtain this goal, Caco-2 and HT-29 cells were exposed to the acetonic extract for 24 h. Cell proliferation, viability, lactase and sucrase-isomaltase activity and H2 O2 -induced membrane lipid peroxidation were tested. We found that, compared to control conditions, the acetonic extract significantly increased lactase and sucrase-isomaltase activity in Caco-2, but not HT-29, cells, decreased proliferation, had no effects on viability and restored lipid peroxidation in both cell models. This study suggests that the acetonic extract improves lactase and sucrase-isomaltase activity, inhibits cell proliferation, have no cytotoxic effects and prevent lipid peroxidation of intestinal epithelial cells. These effects may be exploited in case of disaccharidases deficit and also as an adjuvant treatment of diseases related to oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd.

Solution structure of the monovalent lectin microvirin in complex with Man(alpha)(1-2)Man provides a basis for anti-HIV activity with low toxicity.

Lectins that bind surface envelope glycoprotein gp120 of HIV with high avidity can potently inhibit viral entry. Yet properties such as multivalency that facilitate strong interactions can also cause nonspecific binding and toxicity. The cyanobacterial lectin microvirin (MVN) is unusual as it potently inhibits HIV-1 with negligible toxicity compared with cyanovirin-N (CVN), its well studied antiviral homolog. To understand the structural and mechanistic basis for these differences, we solved the solution structure of MVN free and in complex with its ligand Manα(1-2)Man, and we compared specificity and time windows of inhibition with CVN and Manα(1-2)Man-specific mAb 2G12. We show by NMR and analytical ultracentrifugation that MVN is monomeric in solution, and we demonstrate by NMR that Manα(1-2)Man-terminating carbohydrates interact with a single carbohydrate-binding site. Synchronized infectivity assays show that 2G12, MVN, and CVN inhibit entry with distinct kinetics. Despite shared specificity for Manα(1-2)Man termini, combinations of the inhibitors are synergistic suggesting they recognize discrete glycans and/or dynamic glycan conformations on gp120. Entry assays employing amphotropic viruses show that MVN is inactive, whereas CVN potently inhibits both. In addition to demonstrating that HIV-1 can be inhibited through monovalent interactions, given the similarity of the carbohydrate-binding site common to MVN and CVN, these data suggest that gp120 behaves as a clustered glycan epitope and that multivalent-protein interactions achievable with CVN but not MVN are required for inhibition of some viruses.

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40.

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2-40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed ß-propeller catalytic domain. The structure of the enzyme-ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.

Carbohydrate specificity of chicken and human tandem-repeat-type galectins-8 in composition of cells.

The network of adhesion/growth-regulatory galectins in chicken (chicken galectin, CG) has only one tandem-repeat-type protein, CG8. Using a cell-based assay and probing galectin reactivity with a panel of fluorescent neoglycoconjugates (glycoprobes), its glycan-binding profile was determined. For internal validation, human galectin-8 (HG8) was tested. In comparison to HG8, CG8 showed a rather similar specificity: both galectins displayed high affinity to blood group ABH antigens as well as to 3'-sialylated and 3'-sulfated lactosamine chains. The most remarkable difference was found to be an ability of HG8 (but not CG8) to bind the disaccharide Galß1-3GlcNAc (Le(c)) as well as branched and linear oligolactosamines. The glycan-binding profile was shown to be influenced by glycocalix of the cell, where the galectin is anchored. Particularly, glycosidase treatment of galectin-loaded cells led to the change of the profile. Thus, we suppose the involvement of cis-glycans in the interaction of cell-anchored galectins with external glycoconjugates.

Development of a Novel Cell Surface Attachment System to Display Multi-Protein Complex Using the Cohesin-Dockerin Binding Pair.

Autodisplay of a multimeric protein complex on a cell surface is limited by intrinsic factors such as the types and orientations of anchor modules. Moreover, improper folding of proteins to be displayed often hinders functional cell surface display. While overcoming these drawbacks, we ultimately extended the applicability of the autodisplay platform to the display of a protein complex. We designed and constructed a cell surface attachment (CSA) system that uses a noncovalent protein-protein interaction. We employed the high-affinity interaction mediated by an orthogonal cohesin-dockerin (Coh-Doc) pair from Archaeoglobus fulgidus to build the CSA system. Then, we validated the orthogonal Coh-Doc binding by attaching a monomeric red fluorescent protein to the cell surface. In addition, we evaluated the functional anchoring of proteins fused with the Doc module to the autodisplayed Coh module on the surface of Escherichia coli. The designed CSA system was applied to create a functional attachment of dimeric α-neoagarobiose hydrolase to the surface of E. coli cells.

Structure-guided minimalist redesign of the L-fuculose-1-phosphate aldolase active site: expedient synthesis of novel polyhydroxylated pyrrolizidines and their inhibitory properties against glycosidases and intestinal disaccharidases.

A minimalist active site redesign of the L-fuculose-1-phosphate aldolase from E. coli FucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N-Cbz-amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N-Cbz-amino aldehydes including N-Cbz-prolinal derivatives. FucA F131A showed an aldol activity of 62 µmol h(-1) mg(-1) with (R)-N-Cbz-prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)-amino aldehydes gave exclusively the anti configured aldol adducts whereas their S counterparts gave variable ratios of anti/syn diastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)- and with (S)-N-Cbz-prolinal, exclusively producing the anti and syn aldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N-Cbz-prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π-cation interaction with the residue K205' and to efficient contact between the substrate and the mechanistically important Y113' and Y209' residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4-dideoxy-1,4-imino-L-arabinitol (LAB) and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.

Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40.

Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl-(1-->3)-D-galactose] using various beta-agarases. Neoagarobiose hydrolase is an enzyme that acts on the alpha-1,3 linkage in neoagarobiose to yield D-galactose and 3,6-anhydro-L-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 A, beta = 101.86 degrees . The crystals diffracted to 1.98 A resolution and possibly contains two molecules in the asymmetric unit.

Novel steroidal saponin isolated from Trillium tschonoskii maxim. exhibits anti-oxidative effect via autophagy induction in cellular and Caenorhabditis elegans models.

BACKGROUND: Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration. OBJECTIVE: In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans. METHODS: The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H2O2-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H2DCFDA probe. RESULTS: The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H2O2-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect. CONCLUSION: Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo.

The sim operon facilitates the transport and metabolism of sucrose isomers in Lactobacillus casei ATCC 334.

Inspection of the genome sequence of Lactobacillus casei ATCC 334 revealed two operons that might dissimilate the five isomers of sucrose. To test this hypothesis, cells of L. casei ATCC 334 were grown in a defined medium supplemented with various sugars, including each of the five isomeric disaccharides. Extracts prepared from cells grown on the sucrose isomers contained high levels of two polypeptides with M(r)s of approximately 50,000 and approximately 17,500. Neither protein was present in cells grown on glucose, maltose or sucrose. Proteomic, enzymatic, and Western blot analyses identified the approximately 50-kDa protein as an NAD(+)- and metal ion-dependent phospho-alpha-glucosidase. The oligomeric enzyme was purified, and a catalytic mechanism is proposed. The smaller polypeptide represented an EIIA component of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Phospho-alpha-glucosidase and EIIA are encoded by genes at the LSEI_0369 (simA) and LSEI_0374 (simF) loci, respectively, in a block of seven genes comprising the sucrose isomer metabolism (sim) operon. Northern blot analyses provided evidence that three mRNA transcripts were up-regulated during logarithmic growth of L. casei ATCC 334 on sucrose isomers. Internal simA and simF gene probes hybridized to approximately 1.5- and approximately 1.3-kb transcripts, respectively. A 6.8-kb mRNA transcript was detected by both probes, which was indicative of cotranscription of the entire sim operon.

Development of a 1H NMR structural-reporter-group concept for the primary structural characterisation of alpha-D-glucans.

An NMR study of proton chemical shift patterns of known linear alpha-D-glucopyranose di- and trisaccharide structures was carried out. Chemical shift patterns for (alpha1-->2)-, (alpha1-->3)-, (alpha1-->4)- and (alpha1-->6)-linked D-glucose residues were analysed and compared to literature data. Using these data, a 1H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of alpha-D-glucans.

Coimmobilization of ß-Agarase and α-Neoagarobiose Hydrolase for Enhancing the Production of 3,6-Anhydro-l-galactose.

Here we report a simple and efficient method to produce 3,6-anhydro-l-galactose (l-AHG) and agarotriose (AO3) in one step by a multienzyme system with the coimmobilized ß-agarase AgWH50B and α-neoagarobiose hydrolase K134D. K134D was obtained by AgaWH117 mutagenesis and showed improved thermal stability when immobilized via covalent bonds on functionalized magnetic nanoparticles. The obtained multienzyme biocatalyst was characterized by Fourier transform infrared spectroscopy (FTIR). Compared with free agarases, the coimmobilized agarases exhibited a relatively higher agarose-to-l-AHG conversion efficiency. The yield of l-AHG obtained with the coimmobilized agarases was 40.6%, which was 6.5% higher than that obtained with free agarases. After eight cycles, the multienzyme biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge, this is the first report where two different agarases were coimmobilized. These results demonstrated the feasibility of the new method to fabricate a new multienzyme system onto magnetic nanoparticles via covalent bonds to produce l-AHG.

Cyclic nigerosyl-1,6-nigerose-based nanosponges: An innovative pH and time-controlled nanocarrier for improving cancer treatment.

The design and structural optimisation of a novel polysaccharide-based nanomaterial for the controlled and sustained release of doxorubicin are here reported. A cross-linked polymer was obtained by reacting a tetraglucose, named cyclic nigerosyl-1-6-nigerose (CNN), with pyromellitic dianhydride. The cross-linking reaction formed solid nanoparticles, named nanosponges, able to swell as a function of the pH. Nanoparticle sizes were reduced using High Pressure Homogenization, to obtain uniform nanosuspensions. Doxorubicin was incorporated into the CNN-nanosponges in a good extent. DSC and solid state NMR analyses proved the drug interaction with the polymer matrix. In vitro studies demonstrated pH-dependent slow and prolonged release kinetics of the drug from the nanoformulation. Doxorubicin-loaded CNN-nanosponges were easily internalized in A2780 cell line. They might considered an intracellular doxorubicin reservoir, able to slowly release the drug over time. CNN-nanosponges may be promising biocompatible nanocarriers for the sustained delivery of doxorubicin with potential localised application in cancer treatments.

Analyses of carbohydrate binding property of lectin-chaperone calreticulin.

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum. It is a lectin that promotes the folding of proteins carrying N-linked glycans. Recent investigations have revealed that glucosylated high-mannose-type glycans are employed as key elements in this process. Here, we performed quantitative analyses of the interaction of CRT with various disaccharides, including fluorine-substituted analogues using a quartz-crystal microbalance (QCM). These experiments revealed the weak affinity of 2- and 3-fluoroglucose derivatives. On the other hand, 6-fluoroglucose derivatives exhibited a significant affinity, indicating that the role of 6-position of OH is less significant for binding to CRT. We also characterized binding epitope of the Glcalpha1-3Man(alpha)Me to CRT by saturation transfer difference (STD) NMR spectroscopy. It is proposed that 2-, 3-, and 4-positions of Glc and 3-, 4-, and 6-positions of Man are in close contact with CRT binding pocket, while 6-position of Glc and 2-position of Man are not. These finding are in excellent agreement with our QCM experiment.

Characterization of Mucosal Disaccharidases from Human Intestine.

In this study, we used a brush border membrane (BBM) preparation from human small intestine to analyze the proportion and the activity of major intestinal disaccharidases, including sucrase-isomaltase (SI), maltase-glucoamylase (MGAM) and lactase-phlorizin hydrolase (LPH). SI, MGAM and LPH respectively constituted 8.2%, 2.7% and 1.4% of total BBM protein. The activity of SI and LPH decreased threefold after purification from the brush border membrane, which highlights the effect of membrane microdomains on the functional capacity of these enzymes. All of the disaccharidases showed optimal activity at pH 6, over 50% residual activity between pH 5 to pH 7, and increasing activity with rising temperatures up to 45 °C, along with a stable functional structure. Therefore the enzymes can withstand mild intraluminal pH alterations with adequate function, and are able to increase their activity with elevated core body temperature. Our data provide a functional measure for characterization of intestinal disaccharidases under different physiological and pathological conditions.

Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity.

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.

Determination Trial of Nondigestible Oligosaccharide in Processed Foods by Improved AOAC Method 2009.01 Using Porcine Small Intestinal Enzyme.

We have previously shown that the Association of Official Analytical Chemists' (AOAC) methods 2001.03 and 2009.01 were not able to measure accurately nondigestible oligosaccharide because they are incapable of hydrolyzing digestible oligosaccharide, leading to overestimation of nondigestible oligosaccharide. Subsequently, we have proposed improved AOAC methods 2001.03 and 2009.01 using porcine small intestinal disaccharidases instead of amyloglucosidase. In the present study, we tried to determine nondigestible oligosaccharide in marketed processed foods using the improved AOAC method (improved method), and the results were compared with those by AOAC method 2009.01. In the improved method, the percentages of recovery of fructooligosaccharide, galactooligosaccharide, and raffinose to the label of processed food were 103.0, 89.9, and 102.1%, respectively. However, the AOAC method 2009.01 overestimated >30% of the quantity of nondigestible oligosaccharide in processed foods, because the margin of error was accepted ±20% on the contents of nondigestible oligosaccharides in processed foods for Japanese nutrition labeling, the improved method thus provided accurate quantification of nondigestible oligosaccharides in processed food and allows a comprehensive determination of nondigestible oligosaccharides.

Beneficial effect of sugar osmolytes on the refolding of guanidine hydrochloride-denatured trehalose-6-phosphate hydrolase from Bacillus licheniformis.

The influence of three sugar osmolytes on the refolding of guanidine hydrochloride- (GdnHCl-) denatured trehalose-6-phosphate hydrolase of Bacillus licheniformis (BlTreA) was studied by circular dichroism (CD) spectra, fluorescence emission spectra, and the recovery of enzymatic activity. These experimental results clearly indicated that sorbitol, sucrose, and trehalose at a concentration of 0.75 M improved the refolding yields of GdnHCl-denatured BlTreA, probably due to the fact that these sugars favored the formation of tertiary architectures. Far-UV CD measurements demonstrated the ability of sugar osmolytes to shift the secondary structure of GdnHCl-denatured enzyme towards near-native conformations. ANS fluorescence intensity measurements revealed a reduction of exposed hydrophobic surfaces upon the treatment of denatured enzyme with sugar osmolytes. These observations suggest that sugar osmolytes possibly play a chaperone role in the refolding of chemically denatured BlTreA.