Hyperthermophilic Alpha-Glucosidase Gene and its Use

Recombinant, thermostable alpha-glucosidases from archaeal micro-organisms and isolated DNA encoding for such alpha-glucosidases are provided. The isolated DNA is obtained by use of DNA or antibody probes prepared from the DNA encoding S. sulfataricus alpha-glucosidase. Also provided are methods for producing recombinant archaeal thermostable alpha-glucosidase and transformants incorporating thermostable alpha-glucosidase. Autoprocessing of plant tissue through the use of transgenic thermostable glycosyl hydrolases is described

Crystal Structure of Thermotoga maritima α-Glucosidase AglA Defines a New Clan of NAD+-dependent Glycosidases

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD(+), divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD(+) and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD(+) and maltose, refined at a resolution of 1.9 Angstrom. The NAD(+) is bound to a typical Rossman fold NAD(+)-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme

Isolation And Characterization Of Natural Alpha-Glucosidase Inhibitors From Antioxidant Rich Red Wine Grapes (vitis Vinifera)

Background: Diabetes is currently a global public health problem affecting people at all ages. Dietary antioxidants have been associated with a reduced risk of type 2 diabetes. Grape pomace contains considerable amounts of polyphenols and it has been reported to exhibit an inhibitory activity against alpha- glucosidases. Alpha-glucosidases, in turn, play a major role in controlling starch digestion and therefore postprandial blood glucose, a target for diabetes management. Objective: This study aims to investigate the anti-diabetes potential of a selection of six grape pomaces and prepare and purify active components in the active variety that specifically inhibit intestinal alpha-glucosidases. The study was also designed to evaluate the applicability of the isolated active components as natural inhibitors of alpha-glucosidases. Methods: Chambourcin, Merlot, Norton, Petit Verdot, Syrah and Tinta Cao red wine grape pomace extracts were assessed for their rat intestinal alpha-glucosidase inhibiting activity and antioxidant properties via biochemical assays and UV detection. Then, the grape pomace variety shown to potently inhibit the enzyme was subjected to bioactivity-guided fractionation and the isolated active component was identified via analytical chemistry techniques. The characterized compound was then tested for functional food applicability via stability, enzyme specificity and cytotoxicity testing. Results: Tinta Cao grape pomace extract was the most potent alpha-glucosidase inhibiting variety and possessed a remarkable antioxidant activity, both properties of which appeared to be correlated. HPLC analysis did not yield an antioxidant responsible for the observed trend. Hence, bioactivity-guided fractionation of the extract was pursued, yielding a pure active compound that was determined to be D-Glucopyranose 6-{(2E)-3-(4-Hydroxyphenyl)prop-2-enoate}, which also exhibited a strong antioxidant activity. Further testing indicated that the compound inhibits alpha-glucosidase and not alpha-amylase, and specifically inhibits the maltase and isomaltase moieties of alpha-glucosidase, in a dose-dependent fashion. The compound was fairly stable under different environmental and storage conditions, and it was also not cytotoxic to animal cells. Conclusion: Red grape pomace, namely Tinta Cao, is a promising bioresource for the future development of a food-derived antidiabetic agent. At least one component, D-Glucopyranose 6-{(2E)-3-(4-Hydroxyphenyl)prop-2-enoate}, isolated from Tinta Cao grape pomace appears to potently and specifically inhibit mammalian intestinal alpha-glucosidases while exhibiting a notable ability to quench free radicals. It may thus represent an alternative future strategy for diabetes management and a novel antioxidant compound. Pre-clinical and clinical testing will validate the obtained results in vivo

Microbial β-Glucosidases: screening, characterization, cloning and applications

Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass. Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases

Microbial β-Glucosidase: sources, production and applications

Cellulose is the most abundant biopolymer in biosphere and the major constituent of plant biomass. Cellulose polymer is made up of β-glucose units linked by β-glucosidic bonds. Cellulase is an enzymatic system that catalyzes the hydrolysis of cellulose polymer to glucose monomers. This enzymatic system consists of three individual enzymes namely endoglucanase, exoglucanase and β-glucosidase which act synergistically to degrade cellulose molecules into glucose. Cellulases are produced by bacteria, fungi, plants, and animals and used in many industrial applications such as textile industries, laundry and detergent industries, paper and pulp industry, animal feeds, and biofuels production. β-Glucosidase is a diverse group of enzymes with wide distribution in bacteria, fungi, plants and animals and has the potential to be utilized in various biotechnological processes such as biofuel production, isoflavone hydrolysis, flavor enhancement and alkyl/aryl β-D-glucoside and oligosaccharides synthesis. Thus, there is increased demand of β-glucosidase production from microbial sources under profitable industrial conditions. In this review, β-glucosidase classification, localization, and mechanism of action will be described. Subsequently, the various sources of β-glucosidase for industrial sector will be discussed. Moreover, Fermentation methods and various parameters affecting β-glucosidase production will be highlighted on the light of recent findings of different researchers. Finally, β-glucosidase applications in biofuel production, flavors enhancement, isoflavones hydrolysis, cassava detoxification and oligosaccharide synthesis will be described

1,6-Cyclophellitol Cyclosulfates : A New Class of Irreversible Glycosidase Inhibitor

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions

Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes

The development of small molecule activity-based probes (ABPs) is an evolving and powerful area of chemistry. There is a major need for synthetically accessible and specific ABPs to advance our understanding of enzymes in health and disease. α-Glucosidases are involved in diverse physiological processes including carbohydrate assimilation in the gastrointestinal tract, glycoprotein processing in the endoplasmic reticulum (ER), and intralysosomal glycogen catabolism. Inherited deficiency of the lysosomal acid α-glucosidase (GAA) causes the lysosomal glycogen storage disorder, Pompe disease. Here, we design a synthetic route for fluorescent and biotin-modified ABPs for in vitro and in situ monitoring of α-glucosidases. We show, through mass spectrometry, gel electrophoresis, and X-ray crystallography, that α-glucopyranose configured cyclophellitol aziridines label distinct retaining α-glucosidases including GAA and ER α-glucosidase II, and that this labeling can be tuned by pH. We illustrate a direct diagnostic application in Pompe disease patient cells, and discuss how the probes may be further exploited for diverse applications

1,6-Cyclophellitol Cyclosulfates : A New Class of Irreversible Glycosidase Inhibitor

The essential biological roles played by glycosidases, coupled to the diverse therapeutic benefits of pharmacologically targeting these enzymes, provide considerable motivation for the development of new inhibitor classes. Cyclophellitol epoxides and aziridines are recently established covalent glycosidase inactivators. Inspired by the application of cyclic sulfates as electrophilic equivalents of epoxides in organic synthesis, we sought to test whether cyclophellitol cyclosulfates would similarly act as irreversible glycosidase inhibitors. Here we present the synthesis, conformational analysis, and application of novel 1,6-cyclophellitol cyclosulfates. We show that 1,6-epi-cyclophellitol cyclosulfate (α-cyclosulfate) is a rapidly reacting α-glucosidase inhibitor whose 4C1 chair conformation matches that adopted by α-glucosidase Michaelis complexes. The 1,6-cyclophellitol cyclosulfate (β-cyclosulfate) reacts more slowly, likely reflecting its conformational restrictions. Selective glycosidase inhibitors are invaluable as mechanistic probes and therapeutic agents, and we propose cyclophellitol cyclosulfates as a valuable new class of carbohydrate mimetics for application in these directions

Novel pyrrolizidine alkaloid

The present invention involves a purified bioactive compound of the formula: ##STR1## wherein at least one of R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are H or an acyl having less than about five carbon atoms. More specifically the preferred purified bioactive compound is (1R, 1R, 3R, 7S, 7aR)-3-hydroxymethyl-1,2,7-trihydroxypyrrolizidine.Board of Regents, University of Texas Syste

INHIBITION OF ACTIVITIES OF INDIVIDUAL SUBUNITS OF INTESTINAL MALTASE-GLUCOAMYLASE AND SUCRASE-ISOMALTASE BY DIETARY PHENOLIC COMPOUNDS FOR MODULATING GLUCOSE RELEASE AND GENE RESPONSE

The occurrence of Type 2 diabetes is on the increase all over the world. Since free glucose is released through digestion of starch in the human diet, control of the starch digesting enzymes, particularly the intestinal mucosal alpha-glucosidases [Maltase-Glucoamylase (MGAM) and Sucrase-Isomaltase (SI)], has a potentially important role in both the etiology and treatment of this metabolic disorder. Inhibition of the activities of the intestinal alpha-glucosidases may be a promising way to moderate glucose delivery to the body. Although some commercial inhibitors, such as acarbose, have strong effect on these enzymes, and essentially block starch digestion, there is a need for new candidate inhibitors found in regular diets that still deliver glucose to the body in a slower way and have fewer side effects. Phenolics are known to have inhibitory effect on the intestinal alpha-glucosidases. For more precise control of glucose release in the small intestine, the concept of selective inhibition of the individual subunits (C terminal, Ct; N terminal, Nt) of MGAM and SI has been proposed. In this thesis work, we found that some phenolics selectively inhibit the individual mammalian recombinant subunits of MGAM and SI. For instance, chlorogenic acid and (-)-epigallocatechin gallate (EGCG) selectively inhibited the most active starch digesting subunit, Ct-MGAM (also called glucoamylase). We additionally used rat intestinal acetone powder and human intestinal tissue to investigate the inhibitory effects of selected phenolics on alpha-glucosidases. Chlorogenic acid and EGCG showed the high inhibitory potency for maltase, sucrase and isomaltase activities of rat intestinal acetone powder. Also, chlorogenic acid notably inhibited the sucrase activity of human immunoprecipitated SI, while EGCG the maltase activity of human immunoprecipitated MGAM. Also explored, and for the first time, were the effects of some phenolic compounds on gene expression levels of MGAM and SI. The presence of phenolic compounds in mouse explants caused the generation of different molecular size forms of MGAM, but with no effects on overall maltase activity of the intestinal epithelium. Overall results show that there is a potential to change the rate of digestion of starches, starch products, and other saccharides like sucrose by phenolics present in the diet. Each tested polyphenol displayed a distinctive pattern of inhibition in the MGAM and SI subunits, as well as variations in the relative potency of the enzymes derived from human, rat or mouse species. These results show that dietary phenolic compounds cause differential or selective inhibition of the different intestinal alpha-glucosidase activities. Of relevance is the finding that some phenolic compounds modify patterns of protein forms expression. We speculate that different alternative spliced forms of MGAM are present to digest starch efficiently in the presence of polyphenolic inhibitors