Glucosyl Ester Research Articles

Purification and some properties of a beta-glucosidase from Flavobacterium johnsonae.

Flavobacterium johnsonae was isolated as a microorganism that produced a beta-glucosidase with hydrolytic activity of beta-glucosyl ester linkages in steviol glycosides. The enzyme was purified to homogeneity from a cell-free extract by streptomycin treatment, ammonium sulfate fractionation, and column chromatographies on S-Sepharose and phenyl-Toyopearl. The molecular mass of the purified enzyme was about 72 kDa by SDS-PAGE. An isoelectric point of pI 8.8 was estimated by isoelectric focusing. The enzyme was most active at pH 7.0, and was stable between pH 3.0 and 9.0. The optimum temperature was 45 degrees C, and the enzyme was stable below 35 degrees C. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, and rubusoside, although it could not degrad beta-glucosidic linkages at site 13 of rebaudioside B or steviol bioside. The enzyme acted on aryl beta-glucosides such as p-nitrophenyl beta-glucoside, phenyl betaglucoside, and salicin, and glucobioses such as sophorose and laminaribiose. The enzyme activity on Rub was inactivated completely by Hg2+, and reduced by Fe3+, Cu2+, p-chloromercuric benzoate, and phenylmethylsulfonyl fluoride (residual activity; 67.9-84.8%). The pNPG hydrolysis was also inactivated to almost the same degrees. Kinetic behaviors in the mixed substrate reactions of rebaudioside A and steviol monoside, and of steviol monoglucosyl ester and phenyl beta-glucoside suggested the glucosidic and glucosyl ester linkages were hydrolyzed at a single active site of the enzyme.

On the Reversible Conjugation of

After administering [17-D(2)]GA(20) to Phaseolus coccineus L. cv. Preisgewinner seedlings, [17-D(2)]GA(20)-O-glucoside was identified by liquid chromatography (LC)/ESI-tandem mass spectrometry (MS). Likewise, by LC/ESI-tandem MS the metabolic formation of [17-D(2)]GA(20) glucosyl ester was established. The application of both [17-D(2)]-labeled GA(20) 13-O-glucoside and GA(20) glucosyl ester to Phaseolus coccineus L. seedlings resulted in free [17-D(2)]GA(20) by gas chromatography/MS. The results demonstrate that conjugation of GA(20) and the reconversion of the glucosyl conjugates are concomitant processes in plants.

Glycosylation of encapsulated crocetin by a Crocus sativus L. cell culture

Crocus sativus cell suspension culture converted crocetin into several glycosyl esters when the culture was fed with the encapsulated substrate. Glycosylated pigments were stored in vacuoles. Crocetin did not affect the cell growth at concentrations up to 30 mg 100 ml−1. The major pigment has been identified as the crocetin di-neapolitanosyl ester, a new pigment not yet reported in Crocus sativus plant. The other pigments were mixed forms of neapolitanosyl, gentiobiosyl, and glucosyl esters. Glucosyltransferase activity was measured during the culture cycle and indicated that the bioconversion capacity was higher at the end of the exponential phase. The formation of glycosyl esters was highest during the first 24 h of incubation and the maximal concentration of products was achieved after 4 days. The culture had the capacity to form up to 9 mg g−1 dry wt. glycosyl esters with 30 mg substrate per 100 ml culture. Glucose and 2,4-dichlorophenoxyacetic acid added to a 12-day-old culture enhanced the glucosyltransferase activity but did not affect the yield. The formation of glycosyl esters was higher when the substrate was added by steps each day rather than supplied all at once at the beginning of the incubation period, indicating the occurrence of impediment to crocetin uptake.

Optimization of in vitro conditions for stigma-like-structure production from half-ovary explants of Crocus sativus L

Studies were conducted to optimize the in vitro production of stigma-like-structures (SLS) that yielded the important biochemical constituents responsible for the color, taste, and aroma naturally found in the stigmas of autumn crocus. Immature half-ovary explants were evaluated for the frequency of proliferation of SLS by culture on five basal media supplemented with different combinations of plant growth regulators and vitamins. The optimum proliferation of SLS was observed on B5 basal medium containing α-naphthalene acetic acid (NAA) (5.4 µM), N6-benzyladenine (BA) (44.4 µM), MS organics, casein hydrolysate (0.05%), and l-alanine (11.2 µM) 50–60 d after inoculation. Some explants formed other structures (roots, corms, petals, leaves), the growth and development of which substantially reduced the development of SLS. Removal of brown tissues and other tissues during subculture (3-wk intervals) allowed continuous culture of halfovary explants for 9–10 mo. SLS that had deep red pigment and reached more than 0.5 cm length were removed from the explants and with concomitant reculturing of the ovaries, enabled SLS to be harvested three times. Activated charcoal (1%) added to B5 basal containing NAA (5.4 µM), BA (44.4 µM), and sucrose (3%) was found to be a helpful addendum to prevent browning of explants and accelerate the initiation, growth, and development of SLS. The amounts of crocin, crocetin glucosyl esters, picrocrocin, and safranal in SLS, as determined by high performance liquid chromatography analysis, were found similar to those in natural saffron.

Iridoid and eugenol glycosides from Nepeta cadmea

From the aerial parts of Nepeta cadmea, a new iridiod glucoside, 1,5,9-epideoxyloganic acid glucosyl ester, and a new eugenyl glucoside were isolated, together with the known compounds, 1,5,9-epideoxyloganic acid, eugenyl- O-β- d-glucoside, icarisides B 1 and B 2, (6 S,9 S)-roseoside, lariciresinol-4′- O-β- d-glucoside and rosmarinic acid. Sodium and potassium salts of 1,5,9-epideoxyloganic acid were also isolated. The structures of newly isolated compounds were elucidated by spectral and chemical analyses.

An Acyclic Monoterpene Glucosyl Ester fromLantana lilacia

A new acyclic monoterpene glucosyl ester was isolated from the leaves of Lantana lilacia together with the known compound, acteoside. The structure of the new compound was elucidated by spectroscopic and chemical analyses.

Detection and identification of gibberellins in needles of silver fir (Abies alba Mill.) by combined gas chromatography-mass spectrometry

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA9 was present in small amounts, while a major component was a cellulase-hydrolysable GA9 conjugate which was assumed to be GA9 glucosyl ester. It is proposed that GA9 glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles.

Purification and characterization of a novel β-glucosidase from Clavibacter michiganense that hydrolyzes glucosyl ester linkage in steviol glycosides

Clavibacter michiganense was identified as a microorganism that hydrolyzed the glucosyl ester linkages at site 19 of steviol glycosides. An enzyme that catalyzes the hydrolysis was purified from the cell-free extract using streptomycin treatment, ammonium sulfate fractionation, Q Sepharose anion exchange chromatography, Sephacryl S-100 gel filtration, and Ether Toyopearl hydrophobic chromatography. The purified enzyme migrated as a single protein band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, and isoelectric focusing. The molecular mass was estimated to be approximately 65 kDa, both by gel filtration and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. An isoelectric point, pI, of 4.6, was obtained using isoelectric focusing. The enzyme was most active at around pH 7.5 and at 45°C, and was stable between pH 6–10 and below 40°C. Both Hg 2+ and p-chloromercuric benzoate inhibited activity. The enzyme hydrolyzed glucosyl ester linkages at site 19 of rebaudioside A, stevioside, rubusoside, and steviol monoglucosyl ester, although it did not cleave 13- O-linked glucosyl residue of rubusoside and steviol monoside. A transglucosylation product having a cellobiosyl residue at site 19 was formed when rubusoside was used as a glucosyl donor and acceptor. The enzyme hydrolyzed glucosidic linkages in p-nitrophenyl β-glucoside faster than glucosyl ester linkages in the steviol glycosides. It also acted on phenyl β-glucoside and salicin, and faintly on sophorobiose and cellobiose. These results indicate that the enzyme is a novel β-glucosidase that hydrolyzes ester linkages.

In vitro formation of crocetin glucosyl esters by Crocus sativus callus extract.

Callus extracts of Crocus sativus exhibited the ability to transform all-trans-crocetin into its related glycosides between pH 7.0 and 7.6 in the presence of uridine-diphospho-glucose. The reactions involved the step-wise addition of a glucose moiety to a free carboxyl function and the 1-->6 addition of a glucose moiety to a glucosyl ester function. The kinetics of synthesis for each glycoside seemed to indicate that two distinct glucosyl transferases were implicated in the synthesis of crocin, all-trans-crocetin di-(beta-D-gentiobiosyl) ester.

Glycosylation of Triterpene Alcohols and Acids of the Lupane and A-Secolupane Series

A series of 3- and 28-glucosides and glucosyl esters of betulinic acid (1a), 28-hydroxy-3,4-secolupa-4(23),20(29)-dien-3-oic acid (22a), dimethyl ester of 28-hydroxy-2,3-secolup-20(29)-en-2,3-dioic acid (43a), their 20(29)-dihydro derivatives (1b, 22b, 43b) and several other triterpenes of the lupane (12a, 12b) and 3,4-secolupane series (18a, 18b, 32a) has been prepared by reaction of tetra-O-acetyl-α-D-glucopyranosyl bromide in acetonitrile in the presence of mercury(II) cyanide and subsequent deacetylation of the obtained tetra-O-acetyl-β-D-glucopyranosyl derivatives. In several cases attempted glucosylation in the presence of silver silicate afforded predominantly the corresponding 1,2-orthoacetates of α-D-glucopyranose.

Hydrolysis of [17,17-2H2]gibberellin A20-glucoside and [17,17-2H2]gibberellin A20-glucosyl ester by Azospirillum lipoferum cultured in a nitrogen-free biotin-Based chemically-defined medium

Azospirillum lipoferum strain USA 5b, a gibberellin producing bacterium, was cultured in a nitrogen-free biotin-based chemically-defined medium in the presence of the glucosyl ester or the 13-O-glucoside of [17,17-2H2]-gibberellin A20. The [17,17-2H2]-gibberellin A20 conjugates were added at both the stationary phase of the cultures and at the beginning of the growth curve. Metabolism of the conjugates was examined after 72 h of incubation using capillary gas chromatography-mass spectrometry, with identification by full scan mass spectra. Metabolites identified were [17,17-2H2]-gibberellin A20, [17,17-2H2]-gibberellin A1 and [17,17-2H2]-gibberellin A3. Also, in the Azospirillum cultures fed at the beginning of the growth curve, gibberellin A5 and gibberellin A20 were characterized as endogenous by mass spectrometry/full spectrum. These results support the concept that the growth promotion in plants that is induced by Azospirillum infection may occur by a combination of both gibberellin production and gibberellin-glucoside/glucosyl ester deconjugation by the bacterium.

Liquid chromatography/electrospray ionization-tandem mass spectrometry: a tool for the identification and quantitication of abscisic acid glucosyl ester

LC/ESI-tandem MS techniques have been applied to the analysis of endogenous abscisic acid glucosyl ester (ABA-GE). By parent ion scanning (m/z 263) and constant neutral loss (162u) experiments, endogenous ABA-GE could be identified in extracts from wilted leaves ofPhaseolus vulgaris L. cv. Jutta. Using ABA-[6′-D2]GE as internal standard, the endogenous content of ABA-GE in wilted leaves ofPhaseolus vulgaris L. could be estimated by parent ion scanning ofm/z 263. From the signals of the [M-H]−-ions of ABA-GE (m/z 425) and ABA-[6′-D2]GE (m/z 427) obtained, the amount of the endogenous conjugate was calculated to be 280±9 ng ABA-GE/g plant material. This is the first accurate method for the direct quantification of ABA-GE as intact compound at endogenous levels.

Liquid chromatography-electrospray ionization mass spectrometry for analysing plant hormone conjugates

A series of synthetic gibberellin glycosyl esters and abscisic acid glucosyl ester were investigated using LC-electrospray ionization (ESI) MS (negative- and positive-ion modes). From the spectra obtained, diagnostic ions were chosen to monitor HPLC separations on RP-18. The abundant [M + Na] + ion of the positive-ion ESI spectra enables sugar esters to be recognized from their molecular masses. Under negative-ion ESI conditions, the [M - sugar] − ion as the base peak allows one to search for a group of different sugar esters of a distinct aglycone. Under selected-ion monitoring conditions (positive-ion ESI), less as 3 · 10 −2 ng of abscisic acid glucosyl ester could be detected. The accessibility of ESI-MS results together with characterization by retention properties obtained by HPLC qualify LC-ESI-MS as a powerful method for the analysis of intact plant hormone conjugates, especially glucosyl ester, at endogenous levels.

Influence of phenolic compounds on Agrobacterium vir gene induction and onion gene transfer

Eight chalcones and benzalacetones were tested for their virulence induction on Agrobacterium tumefaciens. With one exception, they had a strong action, and in particular 4-(3,5-dimethoxy-4-hydroxy-phenyl)-but-3-en-2-one ( 10a) was very effective with a virulence induction about 1.5–2 times that of acetosyringone ( AS). When applied to Agrobacterium-mediated gene transfer of onion, both 10a and AS at 250 μM, led to increased gene transfer of between 25 and 35% when compared with the control. Many derivatives of AS were tested and the indispensable chemical functions required to induce vir genes were determined; for example, the presence of at least one methoxy group and a carbonyl group as in acetyl, aldehyde and acid functions. The most effective vir-inducing compound used was the original AS with two methoxy groups and an acetyl function. By testing the corresponding β-glucosides and glucosyl esters (from acids), we established that a phenolic function was also essential for virulence induction. These glucosides led to a decreased toxicity to the bacteria in relation to the original product. However, the presence of a β-glucosylated phenol function led to the total loss of vir induction while the corresponding esters, particularly the glucosyl sysingate ester, conserved a good vir induction.

Liquid chromatography/fast atom bombardment and electrospray ionization mass spectrometry of gibberellin A3 1,2‐trans‐glycosyl esters

AbstractThe glucosyl ester (2b), the galactosyl ester (3b), the xylosyl ester (4b) and the arabinosyl ester (5b) of gibberellin A3 (GA3) as well as the glucosyl ester (7b) and the xylosyl ester (8b) of isoGA3 were synthesized and their structures confirmed especially by 1H nuclear magnetic resonance. In order to identify such conjugates in nature, the glycosyl esters 2b, 3b, 4b, 5b, 7b and 8b were subjected to mass spectrometry analysis using fast atom bombardment and electrospray ionization techniques. The spectra obtained from both negative and positive ions were compared and interpreted on the basis of tandem mass spectrometry experiments. The differences between the fragmentation of GA3 glycosyl ester and that of isoGA3 glucosyl ester are discussed. Diagnostic ions were evaluated, which should be useful for the identification of these conjugates by liquid chromatography/mass spectrometry.

Analysis of endogenous gibberellins and gibberellin metabolites from Dalbergia dolichopetala by gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry

Qualitative gas chromatography-mass spectrometry (GC-MS) analysis of purified extracts from 4-d-germinated seeds of “jacaranda do cerrodo” (Dalbergia dolichopetala) detected gibberellin A1 GA1, epi-GA1 GA3, GA4, GA5, GA8, GA20 and G28. Quantitative analysis, using deuterated internal standards, showed that the germinated seed contained 29 ng GA1, 0.6 ng G4, 0.08 ng G5 and 1.7 ng GA20 · g−1 fresh weight. In metabolism experiments with germinated D. dolichopetala seeds, using 2H2, 3H-labelled GA1, GA4, GA5 and GA20, metabolic profiles were obtained by reversedphase high-performance liquid chromatography-radiocounting after which individual radiolabelled peaks were analysed by GC-MS and/or combined high-performance liquid chromatography-frit fast atom bombardmentmass spectrometry. The data obtained demonstrated the conversion of GA20 to GA20 glucosyl ester, and also to GA1 which was further metabolised, presumably via GA8, to an unidentified GA8 — O-glucoside with a mass spectrum different from those of the 2 — O — and 13 — O-glucosides of GA8. Gibberellin A1 was also conjugated to form GA1 — 3β-O-glucoside and a conjugate identified tentatively as GA1 — 3β-O-glucuronic methyl ester. Gibberellin A4 was converted to GA4 — 3β-O-glucoside and GA1. Gibberellin A5 was converted to GA5 glucosyl ester, GA3 and GA3 — 3β-O-glucoside.

Separation of picrocrocin, cis-trans-crocins and safranal of saffron using high-performance liquid chromatography with photodiode-array detection

High-performance liquid chromatography with photodiode-array detection was used to separate picrocrocin (bitter- tasting component, glucoside of safranal), cis/trans-crocins (carotenoids, glucosyl esters of crocetin) and safranal (flavour, monoterpene aldeyde) of saffron. All components of pure red Greek saffron were extracted from dried stigma with 50% methanol. These compounds were detected, separated collected and identified simultaneously using a Merck LiChroCART 125-4 Superspher 100 RP-18 (4 μm) column and as mobile phase a linear gradient from 20% to 100% acetonitrile in water in 20 min with a detection wavelength at 308 nm.

Synthesis of the β‐D‐glucosyl ester of [carbonyl‐13C]‐indole‐3‐acetic acid

AbstractAn efficient, operationally simple synthetic approach to 1‐O‐([carbonyl‐13C]‐indole‐3′‐ylacetyl)‐β‐D‐glucopyranose is described. The synthesis was carried out by fusing a fully benzylated 1‐O‐glucosylpseudourea intermediate with [carbonyl‐13C]‐indole‐3‐acetic acid, followed by hydrogenolytic removal of the protective groups.

Hydrolysis and reconjugation of gibberellin A20 glucosyl ester by seedlings of Zea mays L.

The [6-2H]glucosyl ester of [17-13C,3H]gibberellin A20 (GA20) was injected into light-grown 14-day-old seedlings of normal, dwarf-1, and dwarf-5 maize (Zea mays L.). The plant material was extracted 24 h later, and the extracts were purified by solvent partitioning, column chromatography, and HPLC. 13C-labeled metabolites were identified from the purified extracts by full-scan gas chromatography/mass spectrometry and selected ion current monitoring in conjunction with Kovats retention indices. The metabolites, [13C]GA20, [13C]GA29, [13C]GA20-13-O-glucoside, and [13C]GA29-2-O-glucoside, were identified from normal, dwarf-1, and dwarf-5 seedlings. [13C]GA8 and [13C]GA8-2-O-glucoside were also identified from normal and dwarf-5 seedlings but not from dwarf-1 seedlings. The data provide definitive evidence for the endogenous hydrolysis by the seedlings of the introduced conjugate and its reconjugation to three glucosides.

In vitro Glucosylation of Dihydrojasmonic Acid and Abscisic Acid

Summary Cell-free extracts from cell suspension cultures of Lycopersicon peruvianum were found to catalyze the glucosylation of [U- 3 H]DJA and [2- 14 C]ABA in the presence of UDP-glucose. The products of enzymatic reactions were identified as glucosyl esters of DJA and ABA by means of TLC, HPLC and hydrolysis with cellulase. The two activities were not separated by ammonium sulfate fractionation and gel filtration, but the transferase activity of the enzyme was enriched toward radioactive DJA about 60-fold and toward radioactive ABA nearly 2-fold. A DJA metabolyzing enzyme was described for the first time and can be classified as UDP-glucose: DJA glucosyltransferase. In addition to the glucosyl esters of DJA and ABA the formation of further labelled products with still unknown structure were observed during the incubation.