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Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.

Essential oil samples of Cinnamosma fragrans from two regions in Madagascar, Tsaramandroso (38 samples) and Mariarano (30 samples), were analysed by GC/MS. Fifty-seven components were identified, accounting from 88.3% to 99.4% of the oils' composition. The major components were linalool (72.5 ± 23.3%) in Tsaramandroso and 1,8-cineole (47.3 ± 10.2%) in Mariarano. Samples B8 (95.8% linalool) from Tsaramandroso and B143 (71.6% 1,8-cineole) from Mariarano containing the highest proportions of the two main components identified, were selected to determine antimicrobial activities against 10 microbial strains. Bacillus subtilis and Staphylococcus aureus were the most sensitive strains to both oils. Minimum inhibitory concentration (MIC) values were lower for B143 against all tested Gram-negative strains than pure 1,8-cineole. B8 showed higher MIC values than pure linalool against Salmonella typhimurium and Vibrio alginolyticus, and similar MIC values to linalool towards the other Gram-negative strains. Both essential oils exhibited higher MIC values towards Fusarium oxysporum than their respective pure major component. These results suggested the occurrence of synergism or antagonism effects between the different oil constituents.

The morphology, anatomy and histology of mature green vanilla beans were examined by light and transmission electron microscopy. Beans have a triangular cross-section with a central cavity containing seeds. Each angle is lined with tubular cells, or papillae, while the cavity sides consist of placental laminae. The epicarp and endocarp are formed by one or two layers of very small cells, while the mesocarp contains large, highly vacuolarized cells, the cytoplasm being restricted to a thin layer along the cell walls. The radial distributions of glucovanillin and beta-glucosidase activity, measured on p-nitrophenyl-beta-glucopyranoside and glucovanillin, are superimposable and show how beta-glucosidase activity increases from the epicarp towards the placental zone, whereas glucovanillin is exclusively located in the placentae and papillae. Subcellular localization of beta-glucosidase activity was achieved by incubating sections of vanilla beans in a buffer containing 5-bromo-4-chloro-3-indolyl-beta-d-glucopyranoside as a substrate. Activity was observed in the cytoplasm (and/or the periplasm) of mesocarp and endocarp cells, with a more diffuse pattern observed in the papillae. A possible mechanism for the hydrolysis of glucovanillin and release of the aromatic aglycon vanillin involves the decompartmentation of cytoplasmic (and/or periplasmic) beta-glucosidase and vacuolar glucovanillin.

A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a ‘phenyloplast’.

Anatomy, histochemistry and biochemistry of glucovanillin, oleoresin and mucilage accumulation sites in green mature vanilla pod (Vanilla planifolia; Orchidaceae): a comprehensive and critical re-examination. Abstract-Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β-D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous β-glucosidase, liberates vanillin, the major aroma component of vanilla. Sites of storage of glucovanillin in the pod have been controversially reported for decades; we aim, using precise and widely accepted technical terminology, to clarify this controversy by providing an anatomical, histochemical and biochemical evidence-based picture of glucovanillin accumulation sites. The pod also synthesizes an oleoresin and a mucilage of unknown constitutions; we report here their localization and structures. Materials and methods. The pod anatomy was examined by light and epifluorescence microscopy. A protocol was established allowing fine hand-dissection of diverse anatomical parts of the pod (mesocarp, placentae, trichomes, intralocular interstitial cell-free region and seeds). Glucovanillin and γ-pyranones were extracted and analyzed by HPLC, while the structures of the mucilaginous polysaccharides were determined after permethylation. Results and discussion. Glucovanillin is essentially stored in the placentae (92%) and marginally in trichomes (7%); traces were measured in the mesocarp and intralocular interstitial cellfree medium. Trichomes store massive amounts of a fluorescing oleoresin (44%) rich in alkenylmethyldihydro-γ-pyranones and synthesize a mucilage made of a glucomannan and a pectic polysaccharide carrying monomeric arabinose and galactose side-chains. Conclusion. To date, the physiological roles of glucovanillin, long-chain pyranones, and mucilage remain unknown. France / Vanilla planifolia / Orchidaceae / vanilla / trichomes / vanillin / oleoresins / fluorescence / mucilages / polysaccharides Anatomie, histochimie, et biochimie des sites d'accumulation de la glucovanilline, d'une oléoresine, et d'un mucilage dans la gousse de vanille verte mature (Vanilla planifolia ; Orchidaceae) : ré-examen critique d'ensemble. Résumé-Introduction. Les gousses de vanille vertes matures accumulent du 4-O-(3méthoxy-benzaldehyde)-β-D-glucoside (glucovanilline), qui, par hydrolyse à l'aide une β-glucosidase endogène, libère de la vanilline, le composé d'arôme majoritaire de la vanille. Les sites de stockage de la glucovanilline dans la gousse ont été mentionnés depuis des décades de façon controversée; en utilisant une terminologie technique précise et largement acceptée, nous nous proposons de clarifier cette controverse en présentant un tableau, fondé sur des preuves anatomiques, histochimiques et biochimiques, des sites d'accumulation de la glucovanilline. La gousse synthétise aussi une oléorésine et un mucilage de compositions inconnues ; nous donnons ici leurs localisations et structures. Matériel et méthodes. L'anatomie de la gousse a été examinée en mi...

The aim of this research was to improve our understanding of the mechanism of glucovanillin hydrolysis by β‐d‐glucosidase activity in vanilla beans by studying their senescence, freezing and traditional curing. A batch of green pods from Madagascar was ripened at 30°C until fruits turned black; another batch was frozen for few days at −18°C and defrosted at 35°C for 24 h and a third batch was cured using traditional methods. During treatments, samples were analysed for the yield of glucovanillin hydrolysis, and β‐glucosidase activity was measured. Cellular structures were also examined by light and transmission electron microscopy. Green fruits had a low yield of glucovanillin hydrolysis (<5%), a high level of β‐glucosidase activity (∼1000 nkatal g−1 fresh weight) and a perfect cellular integrity. Senescent fruits had a high yield of glucovanillin hydrolysis (>95%), no measurable β‐glucosidase activity and complete cellular degradation. Similar results were observed in beans after defrosting. During curing, beans had a medium yield of glucovanillin hydrolysis (<50%), no measurable β‐glucosidase activity and partial cellular degradation compared with senescent or defrosted beans. Results show that the mechanism of glucovanillin hydrolysis in vanilla beans is regulated by cellular compartmentation and that the β‐glucosidase activity level is not the limiting factor for complete hydrolysis. If total decompartmentation is obtained, then complete glucovanillin hydrolysis is observed even if most of the β‐glucosidase activity is lost. The β‐glucosidase activity level only has an effect on glucovanillin hydrolysis kinetics.

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