No abstract

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’.