Enzymatic Hydrolysis Of Glycosides Research Articles

Study of 400MHz microwave conduction loss effect for a hydrolysis reaction by thermostable β-Glucosidase HT1.

Microwave irradiation at different frequencies gave molecular selective effects, namely higher frequency microwave effects for waters while lower frequency effects for ions. We already reported that 2.45GHz and 5.80GHz microwave irradiation gave different results for a hydrolysis reaction by thermostable β-Glucosidase HT1. Here, we designed and made a reactor, employed 400MHz microwave irradiation, and studied the effectiveness of 400MHz microwave for HT1 reaction, then 400MHz and 2.45GHz had the ability to accelerate HT1 reaction. In consideration of the general mechanism of enzymatic glycoside hydrolysis, our results would be reasonable if ions are key reaction species because 400MHz microwave activated ions selectively. In addition, the phenomenon that 400MHz microwave would not affect water molecules by dielectric heating might contribute the enzyme stability. This report should support that microwave is not only a tool to heat reactions efficiently but also can bring unique effects for reactions.

The precursors of C8 alcohols from soybean: Purification, distribution and hydrolysis properties of glycosidically bound volatiles.

C8 alcohols, such as 1-octen-3-ol and 3-octanol, have been variously described as mushroom, musty or earthy and are common volatile compounds of soymilk. C8 alcohols were the major volatiles formed during soybean soaking, and about 95% of the total C8 alcohols were originated from the enzymatic hydrolysis of glycosides of alcohols in soybean hypocotyls. In addition to 1-octen-3-yl β-primeveroside, a new glycoside was purified from the methanol extracts of soybean hypocotyls and were confirmed as 3-octanyl β-primeverosides by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The crude glycosidase extracted from soybean hypocotyls showed much higher hydrolysis activities toward C8 alcohol β-primeverosides than that of almond β-glycosidase. Besides, increasing nonenzymatic hydrolysis was observed when the purified β-primeverosides were heated at pH values lower than 6. The nonenzymatic hydrolysis activation energies (Ea ) were 30.41kJ/mol and 38.08kJ/mol for 1-octen-3-yl β-primeveroside and 3-octanyl β-primeveroside, respectively. In addition to C8 alcohols, nonenzymatic hydrolysis also resulted in the formation of 2,3-octanedione, 3-hydroxy-2-octanone and 3-octanone. The nonenzymatic hydrolysis of β-primeverosides in the soy milk was also studied under the same conditions as those in the model systems. The degree of hydrolysis was similar but the molar yields of the volatile components were lower than those obtained in the model system. PRACTICAL APPLICATION: The results indicated that a different strategy or technology is required to suppress the formation of C8 alcohols than that applied to off-flavor compounds formed de novo during processing of soybean seeds.

Circular Permutation of Bacillus circulans Xylanase: A Kinetic and Structural Study

The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a beta-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within beta-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.

Chemical changes in the volatile fractions of Brazilian honeys during storage under tropical conditions

The chemical transformations of the volatile fractions of two different Brazilian honeys (cashew and marmeleiro) were monitored during storage under tropical conditions. Five systems, namely: 1 – fresh samples; 2 and 3 – samples heated for 3 and 6 months at 35 – 40 °C; 4 and 5 – samples kept under similar conditions to systems 2 and 3, but containing sodium metabisulphite, were tested. The transformations noted in the volatile fractions of these honeys could be mainly associated to acid or enzymatic hydrolysis of glycosides, volatilization, oxidation or esterification processes. The formation of furan derivatives, linalool derivatives and esters appears to be quite affected by the sodium metabisulphite. The concentrations of most powerful odorants increased during the storage (e.g.: benzenemethanol and isovaleric acid) or remained unchangeable (e.g.: vanillin, furfuryl mercaptan, 2-methoxyphenol).

Free and bound volatiles of rocket (Eruca sativa Mill.)

AbstractVolatile fractions of rocket (Eruca sativa Mill.) flowers and leaves with green beans were isolated by hydrodistillation in a Clevenger apparatus from fresh plant material prior to and after autolysis and from dried plant material. All isolates of volatile compounds were analysed by gas chromatography/gas chromatography–mass spectrometry (GC/GC–MS). Sulphur and/or nitrogen compounds, fatty acids and esters, aliphatic alcohols and carbonyl compounds and some other compounds in smaller percentages were identified. The main sulphur and/or nitrogen components were glucoerucin degradation volatiles: 4‐methylthiobutyl isothiocyanate (9.0–81.7%) and 5‐methylthiopentanenitrile (2.4–17.7%). Also, fatty acids and esters were identified as major components, such as: decanoic acid (1.2–32.8%), hexadecanoic acid (2.7–22.3%), dodecanoic acid (0.6–8.7%), (E,E)‐9,12‐octadecadienoic acid (6.5%), tetradecanoic acid (1.5–6.4%), methyl linolenate (0.4–5.1%) and methyl hexadecanoate (0.3–1.2%). The O‐glycosides of volatile compounds were isolated from fresh plant material of rocket flowers and leaves with green beans. After the enzymatic hydrolysis of glycosides and analysis by GC and GC–MS, 27 volatile aglycones were identified for the first time. The main aglycones were: 2‐phenylethyl alcohol (23.4–62.8%), methyl vanillate (5.4–12.4%), benzyl alcohol (5.4–8.6%), eugenol (1.2–7.5%), p‐anisyl alcohol (0.4–4.6%), 3‐oxo‐α‐ionol (0.4–3.2%), 4‐oxo‐β‐ionol (3.2%), o‐methoxy phenol (1.1–2.8%), phenol (0.3–2.8%) and trans‐carveol (0.2–2.3%). When compared, only (Z)‐3‐hexen‐1‐ol, 2‐methylbutanoic acid and 2‐phenylacetaldehyde were present in volatiles obtained by hydrodistillation and the volatile O‐aglycone fractions. Copyright © 2008 John Wiley & Sons, Ltd.

Free and hydrolytically released volatile compounds of Vitis vinifera L. cv. Fiano grapes as odour-active constituents of Fiano wine

Grape-derived volatile compounds, including those released from odourless glycosidic precursor present in the grape, are strongly associated with the varietal aroma characteristic of non-aromatic wines. In this study, free and glycosidically bound volatile compounds of Fiano grapes have been identified and quantified by means of gas chromatography–mass spectrometry (GC–MS). The free volatile fraction of Fiano grapes was mainly characterised by the occurrence of several aliphatic alcohols, with minor amounts of the monoterpenes linalool and geraniol, and traces of the norisoprenoid β-damascenone. The volatile fraction obtained from either enzymatic or acid hydrolysis of juice glycosides was more complex, and contained compounds belonging to the chemical classes of terpenes, norisoprenoids, benzenoids, and aliphatic alcohols. Linalool, geraniol, teprinen-4-ol, 1,1,6-trimethyl-1,2-dihydronaphtalene (TDN), β-damascenone, ( E)-1-(2,3,6-trimenthylphenyl)buta-1,3-diene (TPB), ethyl cinnamate, and 4-vinyl guaiacol were detected by GC-olfactometry (GC-O) as odorants of young Fiano wine that were formed through hydrolysis of grape precursors. Pathways of formation of these compounds during winemaking were investigated. Yeast-driven enzymatic hydrolysis of glycosides was the major route for linalool and geraniol formation, while acid hydrolysis led to the formation of terpinen-4-ol, TDN, β-damascenone, TPB, and ethyl cinnamate.

Common Inhibition of Both β‐Glucosidases and β‐Mannosidases by Isofagomine Lactam Reflects Different Conformational Itineraries for Pyranoside Hydrolysis

The ninety sequence-based families of glycoside hydrolases (GHs) and the correspondingly large diversity of protein topologies are a rich framework for studying variations in the catalytic mechanism of enzymatic glycoside hydrolysis. Such hydrolysis features oxocarbenium-ion-like transition states in which the anomeric centre becomes sp hybridised and partial positive charge accumulates, primarily across the endocyclic O5 C1 bond. For pyranosides, such a species demands planarity of C5, O5, C1 and C2 at or near the transition state; a situation accommodated only by the H3 and H4 (half-chair) conformations (or their closely related envelope forms) and B and B2,5 boats. Initial assumptions that all glycosidases harness H3 conformations are incorrect; indeed, the utilisation of different transition states for the hydrolysis of glycosides is an emerging theme in glycobiology (Scheme 1) and one that suggests a route for specific enzyme inhibition. Isofagomine lactam (1) displays an “in-plane” carbonyl at C2 and is, not surprisingly, a reasonable b-glucosidase inhibitor, with family GH1 b-glucosidases from Thermotoga maritima (TmGH1) and sweet almond inhibited with Ki values of 130 nm (this work, Figure 1) and 29 mm, respectively. Compound 1 has previously been shown to be an equally potent b-mannosidase inhibitor, with the snail b-mannosidase inhibited with a Ki of 9 mm ; this is superficially extremely counter-intuitive. Here, such Ki values for mannosidases are rationalised through structural analysis of 1 in complex with both an exo b-mannanase/b-mannosidase and a b-glucosidase. This work strongly supports previous proposals that b-mannosidases utilise a novel conformational itinerary, featuring a B2,5 transition state. The ground-state axial O2 of mannose is thus pseudo-equatorial at the transition state in a way that should be harnessed in future generations of mannosidase inhibitors. Recently we described the conformational agenda of a retaining GH26 b-mannanase. Trapping of the S5 conformation for the “Michaelis” complex of unhydrolysed substrate, together with the S2 conformation for the covalent intermediate, suggested a novel conformational itinerary for these enzymes through a B2,5 transition state consistent with earlier proposals, notably by Sinnott and Horton. Glycoside hydrolases thus appear to be harnessing the full conformational itinerary in a way that is both enzyme and substrate dependent (this was recently reviewed in the context of inhibition by Vasella and colleagues). Conformational considerations suggest that retaining mannosidase transition-state mimics should thus feature the pseudo-equatorial O2 of the B2,5 conformation of mannose. Isofagomine lactam 1, synthesised by both Stick and Bols, contains an in-plane carbonyl at C2. In elegant work, Bols reports a Ki of 9 mm for the snail b-mannosidase; [4] this inspired us to study the three-dimensional structures of 1 bound to both TmGH1 and a Cellvibrio mixtus exo b-mannanase (CmMan5, which is totally specific for manno-configured substrates). “Dual” inhibition of b-glucosidases and b-mannosidases has also been reported for the hydropyridazone compounds, which show similarity to isofagomine lactam, as discussed below. The three-dimensional structures of TmGH1 and CmMan5 in complex with 1 were determined by X-ray [a] Dr. F. Vincent, T. M. Gloster, Prof. G. J. Davies Structural Biology Laboratory, Department of Chemistry The University of York, Heslington, York, YO10 5YW (UK). Fax: (+44)1904-328266 E-mail : davies@ysbl.york.ac.uk

Studies on aroma generation in lulo (Solanum quitoense): enzymatic hydrolysis of glycosides from leaves

Five C13-norisoprenoid glycosides, isolated from lulo (Solanum quitoense) leaves, were subjected to enzymatic hydrolysis with a commercial glucosidase (emulsin) and also with an enzymatic preparation having glycosidase activity, isolated from lulo fruit pulp. The volatile compounds generated after reaction were characterized by capillary GC and capillary GC–MS. Lulo fruit glycosidases were extracted by ammonium sulfate precipitation at pH 6.5 and subjected to fractionation by hydrophobic interaction chromatography (HIC) on phenyl Sepharose® gel, and some of their properties were determined. As the result of enzymatic hydrolysis with exogenous enzymes (emulsin), glycosides isolated from lulo leaves produced only the aglycone (glycosidically bound volatile); in contrast, enzymatic hydrolysis of glycosides, using endogenous enzymes (lulo fruit glycosidases), generated additional volatile compounds, some recognisable as components of fruit flavour. The role of lulo leaf glycosides as flavour precursors was confirmed.

Hydrolysis of Isoflavone Glycosides to Aglycones by β-Glycosidase Does Not Alter Plasma and Urine Isoflavone Pharmacokinetics in Postmenopausal Women

We investigated whether the bioavailability of isoflavones could be enhanced by enzymatic hydrolysis of glycosides to aglycones before consumption of a nonfermented soy food. Two drinks were formulated with an enriched isoflavone extract from soy germ (Fujiflavone P10), one of which was hydrolyzed enzymatically with beta-glucosidase to produce aglycones. In a randomized, double-blinded, cross-over study, six European, postmenopausal women consumed each soy drink at a 1-wk interval at a concentration of 1 mg total isoflavones/kg body. The plasma and urinary pharmacokinetics of daidzein, genistein and glycitein did not differ after consumption of the two beverages. Plasma total isoflavone concentrations reached 4-5 micro mol/L. The pharmacokinetics of glycitein were similar to those of daidzein. The isoflavone secondary metabolites detected were dihydrodaidzein in plasma and O-desmethylangolensin, equol, and dihydrogenistein in urine. The ratios of individual isoflavones to one another were not conserved from food to plasma to urine, indicating that the individual isoflavones do not have the same absorptions and body retentions. In conclusion, previous hydrolysis of glycosides to aglycones does not enhance the bioavailability of isoflavones in humans.

Free and Glycosidically Bound Volatiles of Mentha longifolia Growing in Croatia

The glycosides of volatile compounds and the essential oil were isolated from wild Mentha longifolia. After the enzymatic hydrolysis of glycosides, fourteen volatile aglycones were identified by GC/MS. The main aglycones were eugenol, 2-phenylethanol, benzyl alcohol, lavandulol, trans- and cis-carveol, 3-octanol, and 3-hexen-1-ol. The content of aglycones was 40.85 mg kg-1 of dried plant material. The main components of the essential oil (yield 0.93 w/w) were carvone, piperitenone oxide, limonene, and β-caryophyllene. Eugenol, carveol, 3-octanol, and α-terpineol were identified in the aglycones and in the essential oil.

ChemInform Abstract: “Heteroglycals” as New Potential Glycosidase Inhibitors. Synthetic Approaches from D-Arabinose.

Within the frame of an ongoing project on glycosidase inhibitors, we have been interested in the synthesis of “heteroglycals”, namely, glycal analogues with sulfur or nitrogen in the ring. Glycals2 are well known for their applications in sugar chemistry in particular for glycosyl transfer.3 They are also known as glycosidase inhibitors through a slow chemical reaction with the enzyme. Recently exo-glycals emerged as a new class of glycals4 which showed interesting features as glycosidase inhibitors but also as precursors of glycomimetics such as C-glycosides.5 We have undertaken investigations on related heteroglycals: such compounds are of interest because they combine a planar geometry at the anomeric center and a possible charge site - both elements known to be important to mimic the transition state of the enzymatic glycoside hydrolysis process.6

“Heteroglycals” As New Potential Glycosidase Inhibitors. Synthetic Approaches From D-Arabinose

Within the frame of an ongoing project on glycosidase inhibitors, we have been interested in the synthesis of “heteroglycals”, namely, glycal analogues with sulfur or nitrogen in the ring. Glycals2 are well known for their applications in sugar chemistry in particular for glycosyl transfer.3 They are also known as glycosidase inhibitors through a slow chemical reaction with the enzyme. Recently exo-glycals emerged as a new class of glycals4 which showed interesting features as glycosidase inhibitors but also as precursors of glycomimetics such as C-glycosides.5 We have undertaken investigations on related heteroglycals: such compounds are of interest because they combine a planar geometry at the anomeric center and a possible charge site - both elements known to be important to mimic the transition state of the enzymatic glycoside hydrolysis process.6

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5 B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase

The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.

Mutagenesis of glycosidases.

Enzymatic hydrolysis of glycosides can occur by one of two elementary mechanisms identified by the stereochemical outcome of the reaction, inversion or retention. The key active-site residues involved are a pair of carboxylic acids in each case, and strategies for their identification and for probing the details of their roles in catalysis have been developed through detailed kinetic analysis of mutants. Similarly the roles of other active-site residues have also been probed this way, and mutants have been developed that trap intermediates in catalysis, allowing the determination of the three-dimensional structures of several such key species. By manipulating the locations or even the presence of these carboxyl side chains in the active site, the mechanisms of several glycosidases have been completely changed, and this has allowed the development of "glycosynthases," mutant glycosidases that are capable of synthesizing oligosaccharides but unable to degrade them. Surprisingly little progress has been made on altering specificities through mutagenesis, although recent results suggest that gene shuffling coupled with effective screens will provide the most effective approach.

Mechanisms of enzymatic glycoside hydrolysis

The determination of a large number of three-dimensional structures of glycosidases, both free and in complex with ligands, has provided valuable new insights into glycosidase catalysis, especially when coupled with results from studies of specifically labelled glycosidases and kinetic analyses of point mutants.

Cyanogenesis in Canned Stone Fruits

ABSTRACTThe amount of cyanogenic glycosides in seeds and pulp of stone fruits, and the HCN content in heat processed unstoned fruit have shown that the decisive factors affecting HCN concentration in canned unstoned fruits are the glycoside content of raw fruits and the conditions of heat processing. Enzymatic hydrolysis of glycosides during canning is the main source of HCN in fruit products. HCN contents found in canned fruits (0‐4 mg‐kg−1) were not acutely toxic, but are not negligible.