Anti-α-Glucosidase and Antiglycation Activities of α-Mangostin and New Xanthenone Derivatives: Enzymatic Kinetics and Mechanistic Insights through In Vitro Studies.

Diabetes mellitus is characterized by chronic hyperglycemia that promotes ROS formation, causing severe oxidative stress. Furthermore, prolonged hyperglycemia leads to glycation reactions with formation of AGEs that contribute to a chronic inflammatory state. This research aims to evaluate the inhibitory activity of α-mangostin and four synthetic xanthenone derivatives against glycation and oxidative processes and on α-glucosidase, an intestinal hydrolase that catalyzes the cleavage of oligosaccharides into glucose molecules, promoting the postprandial glycemic peak. Antiglycation activity was evaluated using the BSA assay, while antioxidant capacity was detected with the ORAC assay. The inhibition of α-glucosidase activity was studied with multispectroscopic methods along with inhibitory kinetic analysis. α-Mangostin and synthetic compounds at 25 µM reduced the production of AGEs, whereas the α-glucosidase activity was inhibited only by the natural compound. α-Mangostin decreased enzymatic activity in a concentration-dependent manner in the micromolar range by a reversible mixed-type antagonism. Circular dichroism revealed a rearrangement of the secondary structure of α-glucosidase with an increase in the contents of α-helix and random coils and a decrease in ß-sheet and ß-turn components. The data highlighted the anti-α-glucosidase activity of α-mangostin together with its protective effects on protein glycation and oxidation damage.

Inhibitory Activity and Mechanism Investigation of Hypericin as a Novel α-Glucosidase Inhibitor.

α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10-5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.

Co-evolution of ß-glucosidase activity and product tolerance for increasing cellulosic ethanol yield.

ß-Glucosidase (BGL) plays a key role in cellulose hydrolysis. However, it is still a great challenge to enhance product tolerance and enzyme activity of BGL simultaneously. Here, we utilized one round error-prone PCR to engineer the Penicillium oxalicum 16 BGL (16BGL) for improving the cellulosic ethanol yield. We identified a new variant (L-6C), a triple mutant (M280T/V484L/D589E), with enhanced catalytic efficiency ([Formula: see text]) for hydrolyzing pNPG and reduced strength of inhibition ([Formula: see text]) by glucose. To be specific, L-6C achieved a [Formula: see text] of 0.35 at a glucose concentration of 20 mM, which was 3.63 times lower than that attained by 16BGL. The catalytic efficiency for L-6C to hydrolyze pNPG was determined to be 983.68 mM-1 s-1, which was 22% higher than that for 16BGL. However, experiments showed that L-6C had reduced binding affinity (2.88 mM) to pNGP compared with 16BGL (1.69 mM). L-6C produced 6.15 g/L ethanol whose yield increased by about 10% than 16BGL. We performed molecular docking and molecular dynamics (MD) simulation, and binding free energy calculation using the Molecular Mechanics/Poisson Boltzmann surface area (MM/PBSA) method. MD simulation together with the MM/PBSA calculation suggested that L-6C had reduced binding free energy to pNPG, which was consistent with the experimental data.

Quillaja bark saponin effects on Kluyveromyces lactis ß-galactosidase activity and structure.

Saponins are known for their bioactive and surfactant properties, showing applicability to the food, cosmetic and pharmaceutical industries. This work evaluated the saponins effects on Kluyveromyces lactis ß-galactosidase activity and correlated these changes to the protein structure. Enzyme kinetic was evaluated by catalytic assay, protein structure was studied by circular dichroism and fluorescence, and isothermal titration calorimetry was used to evaluate the interactions forces. In vitro enzymatic activity assays indicated an increase in the protein activity due to the saponin-protein interaction. Circular dichroism shows that saponin changes the ß-galactosidase secondary structure, favoring its protein-substrate interaction. Besides, changes in protein microenvironment due to the presence of saponin was observed by fluorescence spectroscopy. Isothermal titration calorimetry analyses suggested that saponins increased the affinity of ß-galactosidase with the artificial substrate o-nitrophenyl-ß-galactoside. The increase in the enzyme activity by saponins, demonstrated here, is important to new products development in food, cosmetic, and pharmaceutical industries.

Dual substrate/solvent- roles of water and mixed reaction-diffusion control of ß-Galactosidase catalyzed reactions in PEG-induced macromolecular crowding conditions.

Here we studied the effect of molecular crowding on the hydrolysis of ortho- and para-nitrophenyl-ß-D-galactopyranosides (ONPG, PNPG) catalysed by Escherichia coli ß-Galactosidase in the presence of 0-35%w/v 6kD polyethyleneglycol (PEG6000). The Eadie-Hofstee data analysis exhibited single straight lines for PNPG at all [PEG6000] as well as for ONPG in the absence of PEG6000 so a Michaelian model was applied to calculate the kinetic parameters KM and kcat (catalytic rate constant) values. However, for ONPG hydrolysis in the presence of PEG6000, the two slopes visualized in Eadie-Hofstee plots leaded to apply a biphasic kinetic model to fit initial rate vs. [ONPG] plots hence calculating two apparent KM and two kcat values. Since the rate limiting-step of the enzymatic hydrolysis mechanism of ONPG, but not of PNPG, is the water-dependent one, the existence of several molecular water populations differing in their energy and/or their availability as reactants may explain the biphasic kinetics in the presence of PEG6000. With PNPG, KM as well as kcat varied with [PEG6000] like a parabola opening upward with a minimum at 15 %w/v [PEG6000]. In the case of ONPG, one of the components became constant while the other component exhibited a slight increasing tendency in kcat plus high and [PEG6000]-dependent increasing KM values. Sedimentation velocity analysis demonstrated that PEG6000 impaired the diffusion of ß-Gal but not that of substrates. In conjunction, kinetic data reflected complex combinations of PEG6000-induced effects on enzyme structure, water structure, thermodynamic activities of all the chemical species participating in the reaction and protein diffusion.

Effect of tannic acid on the structure and activity of Kluyveromyces lactis ß-galactosidase.

Tannins are compounds with antinutrient properties that hinder food digestibility, prejudicing human and animal nutrition. This work aimed to evaluate the negative effects of tannic acid on Kluyveromyces lactis ß-galactosidase catalytic activity and correlate these changes with the protein structure. ß-Galactosidase activity decreased in the presence of tannins, which caused changes to the structure of the enzyme, as demonstrated by circular dichroism. It was verified that tannin binds to the protein by a static mechanism. Additionally, isothermal titration calorimetry suggested that tannic acid modified the molecular interaction between ß-galactosidase and o-nitrophenyl-ß-d-galactoside, reducing their affinity and prejudicing the protein activity. This study helps to understand the effects of tannins on the ß-galactosidase structure and how they are related to the enzyme catalytic activity. The alterations in the conformation and activity of the enzyme should be taken into consideration when dairy products are consumed with tannin-rich food.

Is divalent magnesium cation the best cofactor for bacterial ß-galactosidase?

ß-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of ß-galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight ~95 kDa exhibited specific activity of 137.7 U mg-1 protein with a purification of 11.22-fold and yield 12.42%. The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg+2 as a cofactor of ß-galactosidase. In this present investigation, we have checked the kinetic behavior of the purified ß-galactosidase in presence of several bivalent metals. Lowest Km with highest substrate (orthonitrophenyl- ß-galactoside or ONPG) affinity was measured in presence of Ca2+ (42.45 µM ONPG). However, our results demonstrated that Vmax was maximum in presence of Mn+2 (55.98 µM ONP produced mg-1 protein min-1), followed by Fe=2, Zn+2, Mg+2, Cu+2 and Ca+2. A large number of investigations reported Mg+2 as potential co factor for bgalacosidase. However, ß-galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn+2 or Fe2+.

Characterization of an acidic α-galactosidase from hemp (Cannabis sativa L.) seeds and its application in removal of raffinose family oligosaccharides (RFOs).

An acidic α-galactosidase designated as hemp seed α-galactosidase (HSG) was purified from hemp (Cannabis sativa L.) seeds. By means of chromatographic procedures which involved chromatography on the cation-exchangers CM-cellulose and SP-Sepharose, chromatography on the anion-exchangers DEAE-cellulose and Q-Sepharose, and gel filtration on Superdex 75 using fast protein liquid chromatography, HSG was purified to electrophoretic homogeneity. Results of SDS-PAGE and gel filtration on FPLC Superdex 75 revealed that the enzyme was a monomeric protein with a molecular weight of 38 kDa. Sequences of the inner peptides of the α-galactosidase obtained by MALDI-TOF-MS showed that HSG was a novel α-galactosidase since there was a little similarity to the majority of α-galactosidases recorded in the literature. A pH of 3.0 and a temperature of 50°C were optimal for the activity of the enzyme. The activity of HSG was inhibited by the chemical modification with N-bromosuccinimide (NBS) reagent. HSG contained 16 tryptophan residues and two tryptophan residues on the surface, which were crucial to the α-galactosidase activity. The heavy metal ions Cd2+, Cu2+, Hg2+ and Zn2+ inhibited its activity. The Km and Vmax for the hydrolysis of pNPGal (4-nitrophenyl α-D-galactopyranoside) were respectively 0.008 mM and 68 µM min-1 mg-1. HSG also catalyzed the hydrolysis of raffinose and other natural substrates. Hence the α-galactosidase possesses a tremendous potential for food and feed industries in the elimination of indigestible oligosaccharides from leguminous products.

A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity.

A novel α-galactosidase of glycoside hydrolase family 36 was cloned from Bacillus coagulans, overexpressed in Escherichia coli, and characterized. The purified enzyme Aga-BC7050 was 85 kDa according to SDS-PAGE and 168 kDa according to gel filtration, indicating that its native structure is a dimer. With p-nitrophenyl-α-d- galactopyranoside (pNPGal) as the substrate, optimal temperature and pH were 55 °C and 6.0, respectively. At 60 °C for 30 min, it retained > 50% of its activity. It was stable at pH 5.0-10.0, and showed remarkable resistance to proteinase K, subtilisin A, α-chymotrypsin, and trypsin. Its activity was not inhibited by glucose, sucrose, xylose, or fructose, but was slightly inhibited at galactose concentrations up to 100 mM. Aga-BC7050 was highly active toward pNPGal, melibiose, raffinose, and stachyose. It completely hydrolyzed melibiose, raffinose, and stachyose in < 30 min. These characteristics suggest that Aga-BC7050 could be used in feed and food industries and sugar processing.

Lectin-mediated protocell crosslinking to mimic cell-cell junctions and adhesion.

Cell adhesion is a crucial feature of all multicellular organisms, as it allows cells to organise themselves into tissues to carry out specific functions. Here, we present a mimetic approach that uses multivalent lectins with opposing binding sites to crosslink glycan-functionalised giant unilamellar vesicles. The crosslinking process drives the progression from contact puncta into elongated protocellular junctions, which form the vesicles into polygonal clusters resembling tissues. Due to their carbohydrate specificity, different lectins can be engaged in parallel with both natural and synthetic glycoconjugates to generate complex interfaces with distinct lectin domains. In addition, the formation of protocellular junctions can be combined with adhesion to a functionalised support by other ligand-receptor interactions to render increased stability against fluid flow. Furthermore, we consider that adhesion is a complex process of attraction and repulsion by doping the vesicles with a PEG-modified lipid, and demonstrate a dose-dependent decrease of lectin binding and formation of protocellular junctions. We suggest that the engineering of prototissues through lectin-glycan interactions is an important step towards synthetic minimal tissues and in designing artificial systems to reconstruct the fundamental functions of biology.

Design and characterization of new ß-glucuronidase active site variants with altered substrate specificity.

OBJECTIVE: To isolate and characterize the kinetics of variants of E. coli ß-glucuronidase (GUS) having altered substrate specificity. RESULTS: Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-ß-D-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved kcat/KM value towards pNP-gal compared to wild-type GUS, up to 9 × 108-fold shift in specificity from p-nitrophenyl-ß-D-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 103-fold increase in specificity shift compared to a previously evolved GUS variant. CONCLUSIONS: The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic ß-galactosidase.

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or ß-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

In vivo assay to identify bacteria with ß-glucosidase activity

Background: ß-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with ß-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo ß-glucosidase assay as a fast method to find a ß-glucosidase producer strain. Results: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-ß-glucopyranoside (pNPG). The presence of ß-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks ß-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows ß-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The ß-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher ß-glucosidase activity among several lactobacillus species. Conclusion: This in vivo ß-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with ß-glucosidase activity but also with high ß-glucosidase activity.

Biochemical and structural characterization of Penicillium purpurogenum α-D galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition.

The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The α-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of α-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 °C, respectively. The KM for p-nitrophenyl-α-D-galactopyranoside (GalαpNP) is 0.138 mM. The enzyme is inhibited by GalαpNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an α-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications.

Active-Site His85 of Pasteurella dagmatis Sialyltransferase Facilitates Productive Sialyl Transfer and So Prevents Futile Hydrolysis of CMP-Neu5Ac.

Sialyltransferases of the GT-80 glycosyltransferase family are considered multifunctional because of the array of activities detected. They exhibit glycosyl transfer, trans-sialylation, and hydrolysis activities. How these enzymes utilize their active-site residues in balancing the different enzymatic activities is not well understood. In this study of Pasteurella dagmatis α2,3sialyltransferase, we show that the conserved His85 controls efficiency and selectivity of the sialyl transfer. A His85→Asn variant was 200 times less efficient than wild-type for sialylation of lactose, and exhibited relaxed site selectivity to form not only the α2,3- but also the α2,6-sialylated product (21 %). The H85N variant was virtually inactive in trans-sialylation but showed almost the same CMP-Neu5Ac hydrolase activity as wild-type. The competition between sialyl transfer and hydrolysis in the conversion of CMP-Neu5Ac was dependent on the lactose concentration; this was characterized by a kinetic partition ratio of 85 m-1 for the H85N variant, compared to 17 000 m-1 for the wild-type enzyme. His85 promotes the productive sialyl transfer to lactose and so prevents hydrolysis of CMP-Neu5Ac in the reaction.

pKa of Glu325 in LacY.

Lactose permease (LacY), a paradigm for the largest family of membrane transport proteins, catalyzes the coupled translocation of a galactoside and a H+ across the cytoplasmic membrane of Escherichia coli (galactoside/H+ symport). One of the most important aspects of the mechanism is the relationship between protonation and binding of the cargo galactopyranoside. In this regard, it has been shown that protonation is required for binding. Furthermore when galactoside affinity is measured as a function of pH, an apparent pK (pKapp) of ∼10.5 is obtained. Strikingly, when Glu325, a residue long known to be involved in coupling between H+ and sugar translocation, is replaced with a neutral side chain, the pH effect is abolished, and high-affinity binding is observed until LacY is destabilized at alkaline pH. In this paper, infrared spectroscopy is used to identify Glu325 in situ. Moreover, it is demonstrated that this residue exhibits a pKa of 10.5 ± 0.1 that is insensitive to the presence of galactopyranoside. Thus, it is apparent that protonation of Glu325 specifically is required for effective sugar binding to LacY.

Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY.

On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation.

A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Efficient and regioselective synthesis of globotriose by a novel α-galactosidase from Bacteroides fragilis.

Globotriose (Galα1-4Galß1-4Glc) is an important cell surface epitope that acts as the receptor for Shiga-like toxins, and it is also the core structure of Globo H and SSEA4 that are tumor-associated glycans. Hence, the enzymatic synthesis of globotriose would be necessary for the development of carbohydrate-based therapeutics for bacterial infections and cancers. Here, a novel GH27 α-galactosidase gene (agaBf3S), a 1521-bp DNA encoding 506 amino acids with a calculated molecular mass of 57.7 kDa, from Bacteroides fragilis NCTC9343 was cloned and heterogeneously expressed in Escherichia coli. The recombinant enzyme AgaBf3S preferentially hydrolyzed p-nitrophenyl-α-D-galactopyranoside (pNPαGal) in all tested nitrophenyl glycosides. It showed maximum activity at pH 4.5 and 40 °C, and it was stable at pH 4.0-11.0 below 40 °C and metal-independent. The K m and k cat values for pNPαGal, melibiose, and globotriose were 1.27 mM and 172.97 S(-1), 62.76 mM and 17.74 S(-1), and 4.62 mM and 388.45 S(-1), respectively. AgaBf3S could transfer galactosyl residue from pNPαGal to lactose (Galß1-4Glc) with high efficiency and strict α1-4 regioselectivity. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation reaction catalyzed by AgaBf3S were studied in detail. AgaBf3S could synthesize globotriose as a single transglycosylation product with a maximum yield of 32.4 % from 20 mM pNPαGal and 500 mM lactose (pH 4.5) at 40 °C for 30 min. This new one-enzyme one-step synthetic reaction is simple, fast, and low cost, which provides a promising alternative to the current synthetic methods for access to pharmaceutically important Galα1-4-linked oligosaccharides.

Anti-diabetic Activity.

The hyperglycaemia continues to be a major health problem in India and other developing countries. This imbalance of blood glucose causes serious health problems such as damages to the blood vessel, poor healing of wounds, retinal damage, renal damage--kidney failure. The in vitro enzyme models and evaluation of hypoglycaemic effect of sample on normal and glucose-loaded rats has been used as a prediction experiment in this chapter before going for anti-diabetic experiment using animal models.