The tomato leaf miner Tuta absoluta is one of the most devastating pests for tomato crops. Digestive proteases and β-glucosidase enzymes were investigated using general and specific substrates and inhibitors. Maximal β-glucosidase and proteolytic activities occurred at temperature and pH optima of 30 and 40°C, 5 and 10–11 unit of pH, respectively. Zymogram analysis showed the presence of distinguished β-glucosidase exhibiting a specific activity of about 183 ± 15 µmol min−1 mg−1. In vitro inhibition experiments suggested that serine proteases were the primary gut proteases. Gel based protease inhibition assays demonstrated that the 28 and 73 kDa proteases might be trypsin-like and chymotrypsin-like enzymes, respectively. Overall gut trypsin-like and chymotrypsin-like activities were evaluated to be about 27.2 ± 0.84 and 1.68 ± 0.03 µmol min−1 mg−1, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that T. absoluta gut serine proteases are responsible for Bacillus thuringiensis Cry insecticidal proteins proteolysis. Additionally, bioassays showed that T. absoluta larvae development was more affected by the β-glucosidases inhibitor (D-glucono-δ-lactone) than the serine proteases inhibitor (soybean trypsin inhibitor). These results are of basic interest since they present interesting data of β-glucosidases and gut serine proteases of T. absoluta larvae.

Introduction. The traditional curing of vanilla pods includes “killing” and sweating steps when pods are exposed to heat (35–65 ℃) for various lengths of time. Although it is known that liberation of vanillin and other phenolics from their non-aromatic glucosides is due to the action of an endogenous β-glucosidase, its in vivo kinetics remained unknown. Materials and methods. Mature green vanilla pods were pretreated for 2 h at 50 ℃, 55 ℃ and 60 ℃, then stored for 118 days at 27 ℃. Phenolic glucosides and their aglycons were extracted at regular intervals during the storage period and analyzed by HPLC. Results and discussion. All phenolic β-glucosides were slowly hydrolyzed during the storage period with production of vanillin, p-hydroxybenzaldehyde, vanillic acid, and other unknown aglycons. Most of the β-glucosidase was heat-denatured by the pretreatment, and analysis of its kinetic parameters showed that it adopts, in vivo, an allosteric mode of functioning with a lower affinity for glucovanillin than in vitro, where it behaves as a Michaelian enzyme. Conclusion. Extensive research is needed to confirm the allosteric mechanism of the vanilla β-glucosidase in vivo.

Introduction. Mature green vanilla pods accumulate 4-O-(3-methoxy-benzaldehyde)-β-D-glucoside (glucovanillin), which, upon hydrolysis by an endogenous b-glucosidase, liberates vanillin, the major aroma component of vanilla. Little is known on the spatial distribution of aroma-generating phenolics, and the enzymes responsible for their liberation (β-glucosidase) and oxidation (peroxidase). We report here quantitative data with respect to these three components in relation to the anatomy of the pod. Furthermore, the spatial progression of oxidation is shown. Materials and methods. Mature green vanilla pods were analyzed for their contents of phenolics (HPLC), and β-glucosidase and peroxidase activities by spectrophotometric techniques using p-nitrophenyl glucoside and vanillin as substrates, respectively. Lipids were examined under fluorescence microscopy after Nile red staining. Oxidation development was observed on transverse slices of pods. Results and discussion. Phenolics, and β-glucosidase and peroxidase activities showed gradients of increasing-decreasing concentrations from the stem to the blossom end of pods. The β-glucosidase activity is distributed in between the placentae, mesocarp, and trichomes in a [7 / 2 / 1] proportion while that of peroxidase shows a [38 / 1] ratio in the mesocarp and placentae, and was absent from trichomes. Oxidation begins from the blossom end in the placentae, progressively invading the mesocarp and moving towards the stem end. Conclusion. The green mature vanilla pod is spatially heterogeneous for its phenolics, and β-glucosidase and peroxidase activities, its placentae playing an important role in the liberation of vanillin and its subsequent oxidation.

Two β-glycosidases were purified from the termite Macrotermes subhyalinus (Rambur) workers by chromatography on gel filtration, ion exchange and hydrophobic interaction columns. The preparations were shown to be homogeneous on polyacrylamide gel. Both enzymes have a similar molecular mass (68 KDa) and optimum pH (5.4) but differ in optimal temperature and thermal stability. The β-glycosidases preferred β-fucosides to β-glucosides, β-galactosides and β-xylosides, and hydrolysed glucose-glucose-β-(1–4) linkages better than β-(1–3), β-(1–2) and β-(1–6) linkages. They did not hydrolyse saccharides such as melibiose, sucrose, lactose, xylobiose, melizitose, stachyose, lactose, raffinose, laminarin, arabinogalactan, carboxymethylcellulose, inulin, lichenan and starch. β-Glycosidase A and β-glycosidase B of M. subhyalinus workers are capable of catalysing transglucosylation reactions. The yields of glucosylation of hydroxyamino acid derivatives and phenylethanol, catalysed by the two enzymes in the presence of cellobiose as glucosyl donor, were lower than those reported previously with conventional sources of β-glycosidases. In addition, the optimum pH is different for the hydrolysis and transglucosylation reactions. On the basis of this work, it is proposed that the physiological role of β-glycosidase A and β-glycosidase B of M. subhyalinus workers is the digestion of di- and oligosaccharides derived from hemicelluloses and celluloses.

Fasciola hepatica secretes proteolytic enzymes and other molecules that are essential for host penetration and migration. This mixture may include enzymes required for the degradation of supramucosal gels, which defend epithelial surfaces against pathogen entry. These contain hydrated mucins that are heavily glycosylated. Excretory-secretory products (ES) from F. hepatica were examined for a range of glycosidase activities, using synthetic 4-methylumbelliferyl glycosides as substrates. The ES product contained at least 8 different glycosidase activities, the most abundant of which were β-N-acetylhexosaminidase, β-galactosidase and β-glucosidase. Alpha-fucosidase, β-glucuronidase, α-galactosidase, α-mannosidase and neuraminidase were also present. β-N-acetylhexosaminidase and β-galactosidase were present in multiple isoforms (at least 4), whereas β-glucosidase appeared to exist as one isoenzyme with a pI <3·8. All three enzymes had acidic pH optima (4·5–5·0). Ovine small intestinal mucin was degraded by ES at pH 4·5 or 7·0, with or without active cathepsin L, the major protease found in F. hepatica ES. The ability of F. hepatica ES to degrade mucin in the presence or absence of active cathepsin L suggests that cathepsin L is not essential for mucin degradation. The abundance of β-galactosidase and β-hexosaminidase in ES supports a role for these enzymes in mucin degradation.