Hydrolysis Of Glucosides Research Articles

Compositional and Tissue Modifications Induced by the Natural Fermentation Process in Table Olives

Olive fruits contain high concentrations of phenols that include phenolic acids, phenolic alcohols, flavonoids, and secoiridoids. The final concentration of phenols is strongly affected by brine conditions. The factors involved in modification by brine are still partially unknown and can include hydrolysis of secoiridoid glucosides and the release of hydrolyzed products. In this study olives from various Italian cultivars were processed by natural fermentation (e.g., without a preliminary treatment of olives with NaOH) using a selected Lactobacillus strain. Processed olives are characterized by a low phenolic concentration of phenols, consisting mainly of phenyl alcohols, verbascoside, and the dialdehydic form of decarboxymethylelenolic acid linked to (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA), whereas a high level of phenols occurs in olive brine from all the cultivars studied. Olives of the Coratina cultivar, control and with fermentation by Lactobacillus pentosus 1MO, were analyzed in a frozen hydrated state by cryo scanning electron microscopy and energy-dispersive X-ray microanalysis, on both surface and transversal freeze-fracture planes. Structural modifications, found in olives after fermentation, may explain the phenol release in brine.

Enrichment of isoflavone aglycones in soymilk by fermentation with single and mixed cultures of Streptococcus infantarius 12 and Weissella sp. 4

Soymilk was fermented with either Streptococcus infantarius 12 (Si 12), Weissella sp. 4 (Ws 4), or their mixed cultures with different mixing ratios (Si 12:Ws 4=1:1, 1:3, 1:5, and 1:10, v/v) for 12h at 37°C. All cultures in soymilk readily proliferated and reached about 108–9CFU/mL. After 12h, pH and titratable acidity of soymilk ranged 4.19–4.47 and 0.57%–0.64%, respectively. The pH of soymilk fermented with Si 12 was the lowest while that obtained with Ws 4 the highest. A sharp increase in β-glucosidase (β-glu) activity corresponded well with a rapid decrease in isoflavone glucosides and an increase in aglycone contents. The rate of hydrolysis of isoflavone glucosides was the least with Si 12 while the highest with Ws 4, resulting in about 23%–33% and 98%–99% hydrolysis of the glucosides with Si 12 and Ws 4, respectively, after 12h. Mixed cultures with 1:3, 1:5, and 1:10 ratios seem to be more effective starters for bioactive fermented soymilk with more aglycones and appropriate acidity in a short time than single cultures.

Cloning, Expression, and Characterization of Two β-Glucosidases from Isoflavone Glycoside-Hydrolyzing Bacillus subtilis natto

On the basis of the genomic sequence of Bacillus subtilis 168, two beta-glucosidase genes (bglH and yckE) from B. subtilis natto, which has been reported to have high isoflavone glucoside-hydrolyzing activity, were cloned and overexpressed in E. coli M15. The temperature for the optimal p-nitrophenyl-beta-D-glucoside hydrolyzing activity of both enzymes was between 37 and 45 degrees C, but BglH had a higher thermal stability than YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than BglH. Recombinant BglH was inhibited 73%, 63%, and 43% by 1.0 mM Cd(2+), Fe(2+), or Cu(2+), respectively, while other divalent metal ions resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BglH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-beta-D-fructopyranoside. Both BglH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BglH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher kcat). Our results suggest that recombinant BglH may be applicable in the process of isoflavones deglycosylation.

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl − salts) the effectiveness follows the series: Cu +2, Fe +2 > Zn +2 > Li + > Ca +2 > Mg +2 > Cs + > NH 4 + > Rb + > K + > Na + and for anions (Na + salts) the series is: I − > ClO 4 − > −SCN > Br − ≈ NO 3 − > Cl − ≈ −OAc > F − ≈ SO 4 − 2 . The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k cat/ K m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic p K as and not to an effect on the pH-independent second-order rate constant, ( k cat/ K m) lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent p K a1 increases (from 3.7 to 4.9) and the apparent p K a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent ( k cat/ K m) lim (= 9 × 10 4 M − 1 s − 1 ) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the p K as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced p K a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a p K a of 3.7 and dissociated from the group with a p K a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two p K as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, ( k cat/ K m) lim, of the monoprotonated species. For other enzymes which may show such salt-induced p K a shifts, this provides a convenient test for the role of reverse protonation.

Aroma composition and sensory quality of fruit juices processed from cultivars of elderberry (Sambucus nigra L.)

The experiment included identification and quantification of 25 aroma compounds and determination of the sensory quality of fruit juices processed from 32 cultivars of black elder using enzymation. It was concluded that disappearance of 15 esters and the increases of four alcohols, six terpenoids, six carbonyls, six aldehydes, three ketones, eight alcohols, trans-rose oxide, nerol oxide, and dihydroedulan primarily might be due to enzymatic conversion of esters of fatty acids and enzymatic hydrolysis of glucosides as found by processing of juices from other fruits. Juices processed from the elderberry cultivars significantly differed with respect to aroma compound composition and sensory characteristics. Factor analysis of the combined data from measurement of aroma composition and sensory evaluation resulted in extraction of four factors with factor scores that could be used for selection of cultivars with different promising sensory properties and varying concentrations of phenolic acids and flavonoids that may be useful for processing of functional elderberry juices with health promoting properties.

Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific

Bacterioplankton nutrient metabolism in the Eastern Tropical North Pacific (ETNP) was assessed using specific activities of intracellular nitrogen (N) assimilation enzymes and hydrolytic ectoenzymes during amendment experiments, mesocosms, and diel studies of in situ rates. Glutamine synthetase (GS) and assimilatory nitrate reductase (ANR) were used to investigate N bioavailability, alkaline phosphatase (AP) to assess phosphorous (P) bioavailability and β-glucosidase (β-Glu) to detect shifts in the use of labile dissolved organic carbon (DOC). Conditions regulating activity of each enzyme were tested using incubations of < 0.6 mm size-fractionated seawater amended with different combinations of N, P, and DOC as glucose. Overall, N-deficiency was indicated by pronounced growth stimulation and repression of GS and ANR activity in incubations amended with dissolved free amino acid and ammonium. Phosphate and glucose amendments produced little or no growth stimulation, but did influence activity of all enzymes measured. Enzyme activities of bacterioplankton in mesocosms of whole plankton indicated enhanced N-deficiency and glucoside hydrolysis when the plankton community was released from any P-deficiency. Spatially, enzyme activity of bacterioplankton during two diel studies (at one slope and one open-ocean station) suggested greater N-deficiency at surface depths than within the chlorophyll maximum where activity of AP and b-Glu was often greatest. There was also greater GS and ANR activity at the open-ocean station, which had lower concentrations of dissolved inorganic N (DIN) relative to soluble reactive P (SRP), than along the continental slope of Mexico. These data suggest that bacterioplankton in surface waters of the ETNP require a large flux of DOC to drive N-deficiency; whereas, bacterioplankton deeper in the chlorophyll maximum depend on hydrolysis of complex DOC and DOP to meet their carbon demand in the presence of elevated nutrients with a low DIN:SRP ratio.

Conversion of Isoflavone Glucosides to Aglycones in Soymilk by Fermentation with Lactic Acid Bacteria

Four lactic acid bacteria (LAB), Lactobacillus paraplantarum KM, Enterococcus durans KH, Streptococcus salivarius HM and Weissella confusa JY, were isolated from humans and tested for their capabilities of converting isoflavone glucosides to aglycones in soymilk. Changes in growth, pH, and titratable acidity (TA) were investigated during fermentation at 37 degrees C for 12 h. After 6 to 9 h of fermentation, each population of 4 LAB reached 10(8) to 10(9) CFU/mL. The initial pH of 6.3 +/- 0.1 decreased while the TA of 0.13%+/- 0.01% increased as fermentation proceeded, resulting in the final range between 4.1 +/- 0.2 and 4.6 +/- 0.1 for pH and between 0.51%+/- 0.02% and 0.67%+/- 0.06% for TA after the 12 h of fermentation. The glucoside concentrations were significantly decreased in soymilks fermented with either L. paraplantarum KM, S. salivarius HM, or W. confusa JY with fermentation time (P < 0.05). L. paraplantarum KM was the best in percent conversion of glucosides to corresponding aglycones, resulting in 100%, 90%, and 61% hydrolysis of genistin, daidzin, and glycitin, respectively, in 6 h. Consequently, the aglycone concentrations in soymilk fermented with L. paraplantarum KM were 6 and 7-fold higher than the initial levels of daidzein and genistein, respectively, after 6 h of fermentation. Changes in the daidzin and genistin levels were not significant in soymilk fermented with E. durans KH. The rates of hydrolysis of glucosides varied depending on the species of LAB. Especially, L. paraplantarum KM seems to be a promising starter for bioactive-fermented soymilk based on its growth, acid production, and isoflavone conversion within a short time.

A comparison of changes in the transformation of isoflavones in soymilk using varying concentrations of exogenous and probiotic-derived endogenous ?-glucosidases

To compare endogenous and exogenous beta-glucosidases for the hydrolysis of the predominant isoflavone glucosides in soymilk in order to improve the biological activity. beta-glucosidase activity of probiotic organisms, including Bifidobacterium animalis ssp. lactis Bb12, Lactobacillus acidophilus ATCC 4461 and Lactobacillus casei 2607 in soymilk, was evaluated and was related to the increase in the concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model HPLC with an Amperometric electrochemical detector. The aglycone composition, also known as aglycone equivalent ratio, has been considered to be important for the delivery of health benefits of isoflavones, and was monitored during the fermentation of soymilk. Comparison of the hydrolytic effectiveness of both exogenous and endogenous enzyme during 4-h incubation in soymilk was conducted using the Otieno-Shah (O-S) index. Results showed that exogenous enzyme exhibited faster rate of isoflavone glucoside hydrolysis than that by endogenous enzyme. Highest O-S indices were obtained after 4, 3 and 2 h of incubation with enzyme solution having beta-glucosidase activity of 0.288 U ml(-1), 0.359 U ml(-1) and 0.575 U ml(-1), resulting into aglycone concentration increments of 5.87-, 6.07- and 5.94-fold, respectively. Conversely, aglycone concentration in the soymilk with B. animalis ssp. lactis Bb12, L. casei 2607 and L. acidophilus 4461 increased by 3.43-, 2.72- and 3.03-fold, respectively, after 4 h of fermentation at 37 degrees C. In addition, the O-S index of endogenous enzyme was much lower than that of the exogenous enzyme over the same 4-h incubation period. Optimum aglycone equivalent ratios coincided with highest O-S indices and highest aglycone concentrations in soymilk hydrolysed with exogenous enzyme. The same correlation of O-S indices and highest aglycone concentrations occurred for endogenous enzyme during the 24 h of fermentation. Obtaining highest aglycone concentration and optimum aglycone equivalent ratio could provide a critical beginning point in clinical trials for the realization of unique health benefits of soy isoflavones. Screening for beta-glucosidase activities of probiotics in soymilk and comparing their hydrolytic potentials with that of exogenous beta-glucosidase could find wide applications in the development of different aglycone-rich functional soy beverages.

Glucosylated aroma precursors and glucosidase(s) in vanilla bean (Vanilla planifoliaG. Jackson)

Introduction. Les traitements thermiques appliques aux gousses du vanillier (Vanilla planifolia G. Jackson) pendant la transformation ont differents objectifs, dont celui de favoriser l'hydrolyse des precurseurs glucosyles de composes d'arome par une (ou plusieurs) glucosidase(s) endogene(s). Les glucosides de la gousse du vanillier. Une quinzaine de glucosides ont pu etre identifies dans le fruit vert, parmi lesquels la glucovanilline est le plus important quantitativement. Trente semaines apres la pollinisation, sa concentration peut atteindre 15% de la matiere seche du fruit. Ce glucoside represente la principale forme d'accumulation de la vanilline. -D-glucosidase(s) de la gousse du vanillier. Une -D-glucosidase a ete purifiee et caracterisee a partir de la gousse du vanillier, mais il est possible que plusieurs glucosidases existent qui resteraient a etudier. L'activite glucosidasique semble tres instable au cours des traitements thermiques, ce qui pose des questions quant au processus d'hydrolyse des glucosides pendant la transformation de la vanille. Compartimentation entre activite glucosidasique et glucosides dans la gousse du vanillier. Apres differentes controverses, il semble desormais acquis que la glucovanilline et l'activite glucosidasique sont majoritairement localisees dans la partie placentaire de la gousse. Leur localisation cellulaire respective reste a preciser, mais de nombreux elements confortent l'hypothese que l'hydrolyse enzymatique est regulee par une compartimentation cellulaire differente entre enzyme et substrat. Conclusion. De nombreux travaux de recherche restent a entreprendre sur les glucosides et glucosidase(s) de la gousse du vanillier et il serait notamment interessant de mieux connaitre le deroulement du processus d'hydrolyse enzymatique des precurseurs d'aromes pendant la transformation. (Resume d'auteur)

English

Cassava roots and leaves constitute energy-rich and protein-rich foods, respectively, for the populations in Central Africa, where they are consumed as staple foods. But cassava roots and leaves contain some cyanide in the form of cyanogenic glucosides, notably the linamarine, which can constitute a poison for the consumers when roots or leaves are processed improperly. Cassava roots and leaves processing in Congo, as in most central African countries, involve fermentation. The fermentation of the cassava roots is a lactic fermentation (pH 3.8) with&nbsp;Lactobacillus&nbsp;as dominant microflora whereas that of the cassava leaves is an alkaline fermentation (pH 8.5) where&nbsp;Bacillus&nbsp;constitute the main microflora. The hydrolysis of cyanogenic glucosides takes place as well in acid medium during the cassava tubers fermentation as in basic medium with the cassava leaves fermentation. The cyanide content decreases during the fermentation of cassava roots and leaves by more than 70% through the activities of the bacterial produced linamarase, allowing the hydrolysis of cyanogenic glucosides. Certain lactic bacteria present in the environment of fermentation are resistant to the strong cyanide concentrations of between 200 and 800 ppm. &nbsp; Key words:&nbsp;Fermentation, cassava roots, cassava leaves, lactic acid bacteria, bacillus, cyanogenic glucosides.

Isoflavonoid production by adventitious-root cultures of Iris germanica (Iridaceae)

We established the adventitious-root cultures of Iris germanica, a monocotyledonous plant known for isoflavonoid production. Irigenin (5,7,3′-trihydroxy-6,4′,5′-trimethoxyisoflavone) and iristectorigenin A (5,7,3′-trihydroxy-6,4′-dimethoxyisoflavone) along with their 7-O-β-D-glucosides, iridin and iristectorin A, respectively, were found as the major components in adventitious roots in the liquid medium, and the total isoflavone content was about 3.6 µmol per g fresh weight in 3-week-old cultures, which was much higher than the 0.7 µmol per g dry weight in the rhizome previously reported (Ali et al. 1983 Phytochemistry 22: 2061). Abiotic stress was applied by addition of 3 mM cupric chloride (CuCl2) to the liquid medium. The isoflavone glucoside content was increased during the initial 6 h of CuCl2 treatment, which was followed by a decrease; the aglycone level continued to increase throughout the 48 h of treatment. The decrease in glucoside content was negatively correlated with the increase in aglycone content between 6 and 48 h. The total isoflavone content (glucosides+aglycones) at 6 h after the start of CuCl2 treatment was 1.4-fold the initial value, and nearly the same content was maintained for 48 h. Thus, the main effect of CuCl2 treatment appeared to be the induction of hydrolysis of isoflavone glucosides. A cDNA of chalcone synthase was cloned, and the mRNA was expressed in the culture producing isoflavones.

Probing the transition states of four glucoside hydrolyses with 13C kinetic isotope effects measured at natural abundance by NMR spectroscopy.

Kinetic isotope effects (KIEs) were measured for methyl glucoside (4) hydrolysis on unlabeled material by NMR. Twenty-eight (13)C KIEs were measured on the acid-catalyzed hydrolysis of alpha-4 and beta-4, as well as enzymatic hydrolyses with yeast alpha-glucosidase and almond beta-glucosidase. The 1-(13)C KIEs on the acid-catalyzed reactions of alpha-4 and beta-4, 1.007(2) and 1.010(6), respectively, were in excellent agreement with the previously reported values (1.007(1), 1.011(2): Bennet and Sinnott, J. Am. Chem. Soc. 1986, 108, 7287). Transition state analysis of the acid-catalyzed reactions using the (13)C KIEs, along with the previously reported (2)H KIEs, confirmed that both reactions proceed with a stepwise D(N)A(N) mechanism and showed that the glucosyl oxocarbenium ion intermediate exists in an E(3) sofa or (4)H(3) half-chair conformation. (13)C KIEs showed that the alpha-glucosidase reaction also proceeded through a D(N)*A(N) mechanism, with a 1-(13)C KIE of 1.010(4). The secondary (13)C KIEs showed evidence of distortions in the glucosyl ring at the transition state. For the beta-glucosidase-catalyzed reaction, the 1-(13)C KIE of 1.032(1) demonstrated a concerted A(N)D(N) mechanism. The pattern of secondary (13)C KIEs was similar to the acid-catalyzed reaction, showing no signs of distortion. KIE measurement at natural abundance makes it possible to determine KIEs much more quickly than previously, both by increasing the speed of KIE measurement and by obviating the need for synthesis of isotopically labeled compounds.

Development of an isoflavone aglycone-enriched soymilk using soy germ, soy protein isolate and bifidobacteria

Soymilks comprising soy protein isolate (SPI) and soy germ (SG) at ratios of 9:1 (SPI/SG9:1), 6:4 (SPI/SG6:4) and 3:7 (SPI/SG3:7) were fermented with Bifidobacterium animalis Bb-12. Enumeration of microbial populations, pH measurements and quantification of isoflavones were performed at 12 and 24 h incubation and at 1, 7 and 14 day refrigerated storage. Isoflavone concentrations were 3, 7 and 12 times higher in SPI/SG9:1, SPI/SG6:4 and SPI/SG3:7 than soymilk made from SPI and microbial populations increased, respectively, by 1.2, 1.3 and 0.9 log 10 CFU/mL after 24 h. Hydrolysis of isoflavone glucosides increased the concentration of aglycones from 0.819 to 5.645 mg/100 mL (47% of total isoflavone) in SPI/SG9:1 after 24 h ( P<0.05), with isoflavone biotransformation less apparent in SPI/SG3:7. Fermented SPI/SG6:4 contained the highest concentration of isoflavone aglycone at 7.801 mg/100 mL (26% of total). Viability of B. animalis and composition of isoflavones were unchanged after 14 day refrigerated storage ( P>0.05).

Improvement on enzymatic hydrolysis of resveratrol glucosides in wine

An enzymatic hydrolysis method, useful for the analysis of total wine resveratrol, was optimised. The wine resveratrol O-glycosides were totally hydrolysed in ∼9 h after incubation with β-glucosidase at 50 °C; then the trans- and cis-aglycones were measured by high-performance liquid chromatography after solid phase extraction (SPE). Kinetic study of enzymatic hydrolysis at different temperatures showed that hydrolysis times were reduced with increasing temperature, up to 50 °C, because the enzyme retains its activity even at this temperature.

Absorption of quercetin-3-glucoside and quercetin-4′-glucoside in the rat small intestine: the role of lactase phlorizin hydrolase and the sodium-dependent glucose transporter

Two hypotheses on absorption mechanisms of flavonoid glucosides across the small intestine have been proposed: active uptake of the quercetin glucoside by the sodium-dependent glucose transporter (SGLT1) with subsequent deglycosylation within the enterocyte by cytosolic β-glucosidase, or luminal hydrolysis of the glucoside by lactase phlorizin hydrolase (LPH) and absorption by passive diffusion of the released aglycone. To test the above hypotheses we employed phlorizin (as an inhibitor of SGLT1) and N-( n-butyl)-deoxygalactonojirimycin (as an inhibitor of the lactase domain of LPH) in a rat everted-jejunal sac model. Quercetin-4′-glucoside mucosal hydrolysis was 10 times greater than quercetin-3-glucoside hydrolysis in the absence of inhibitors (449 and 47 nmol g −1 tissue, respectively), despite the similar amounts (13±4 and 9±1 nmol g −1, respectively) being transferred to the serosal compartment during the 15 min incubation. Apical hydrolysis of both quercetin glucosides was significantly reduced in the presence of NB-DGJ (80%), and transfer of quercetin (measured as quercetin metabolites) to the serosal solution was also significantly reduced (40–50%). In the presence of phlorizin, transfer of metabolites to the serosal solution was only reduced in the case of quercetin-4′-glucoside. Evidently the mechanism of absorption of quercetin-4′-glucoside involves both an interaction with SGLT1 and luminal hydrolysis by LPH, whereas quercetin-3-glucoside appears to be absorbed only following hydrolysis by LPH.

Multiple sugar binding sites in α-glucosidase

Twenty-five analogs of d-glucose were examined as reversible inhibitors of yeast α-glucosidase (EC 3.2.1.20). The K i values range from 0.38 mM for 6-deoxy- d-glucose (quinovose) to 1.0 M for d-lyxose at pH=6.3 (0.1 M NaCl, 25°). All the monosaccharides and the three disaccharides (maltose, isomaltose and α,α-trehalose) were found to be linear competitive inhibitors with respect to α- p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [ K i=1.8(±0.1) mM], one by d-galactose [ K i=164(±11) mM] and one by d-mannose [ K i=120(±9) mM]. The pH dependence for glucose binding closely follows that of V/ K [p K a1=5.55(±0.15), p K a2=6.79(±0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme–product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.

Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

Hydrolysis of soybean isoflavone glucosides by lactic acid bacteria

Lactobacillus delbrueckii subsp. delbrueckii KCTC 1047, grown in de Man, Rogosa and Sharpe (MRS) or soymilk media, completely hydrolyzed the isoflavone glucosides, genistin and daidzin at 50 μg ml−1, into their respective aglycones, genistein and daidzein within 30 min. Other lactic acid bacteria did not produce β-glucosidase, the enzyme responsible for the hydrolysis of isoflavone glucosides, when cultured in MRS medium. Glucoside-hydrolyzing activity was induced in some lactic acid bacteria when cultured in soymilk medium. These strains hydrolyzed 70–80% of genistin into genistein and 25–40% of daidzin into daidzein.

Comparison of the Intestinal Absorption of Quercetin, Phloretin and Their Glucosides in Rats

Absorption and metabolism of quercetin and isoquercitrin (quercetin 3-O-glucose) were investigated in rats after in situ perfusion of jejunum plus ileum (15 nmol/min) for 30 min and compared with those of phloretin and phloridzin (phloretin 2'-O-glucose). After perfusion of the glucosides, the corresponding aglycone forms and conjugated derivatives appeared in the lumen. The conjugated metabolites were similar to those recovered after intestinal perfusion of the aglycone forms. Regardless of the aglycone or glucoside perfused, only conjugated forms were present in the mesenteric vein blood draining the perfused segment showing the importance of intestinal conjugation. The hydrolysis of glucosides was a prerequisite step before their conjugation by intestinal enzymes and their transport towards the mucosal and serosal sides. In contrast to phloridzin, lactase phloridzin hydrolase activity did not seem to be an essential pathway for isoquercitrin hydrolysis. The 3-O-glucosylation of quercetin improved the net absorption of the aglycone (P < 0.05), whereas phloretin absorption decreased when present as 2'-O-glucoside (P < 0.05). Whatever the perfused compound, the efficiency of the absorption seemed to be linked to the intestinal conjugation process and to the luminal secretion of metabolites.

Synthesis of polyhydroxylated cyclohexenyl sulfides and sulfoxides. Evaluation of their inhibitory activity on α- and β- d-glucosidases

Racemic polyhydroxylated sulfides and sulfoxides have been prepared as potential transition-state analogs of the glucoside hydrolysis reaction, through a reaction sequence involving transformations of a ketone group into thioacetal, followed by partial oxidation to sulfoxide then regioselective thermal elimination of the sulfoxide to vinyl sulfide. These sulfides with two, three or four hydroxyl groups have been oxidized to the corresponding diastereoisomeric sulfoxides. All compounds, summarily tested as inhibitors of α- and β-glucosidases, were found to be very weak inhibitors and thus their biological properties were not studied in depth. Curiously, however, their inhibitory properties, which are in the 10 mM range, do not really depend upon the number of hydroxyl groups or upon the presence of polar sulfoxide. In order to get more insights, 2-phenyl sulfoxide-3,4,5-trihydroxycyclohex-1-ene ( 9a) was studied in more detail using Brewers yeast α-glucosidase and was shown to give a mixed-type inhibition with a high K i of 45 mM.