Production of Minor Ginsenosides C-K and C-Y from Naturally Occurring Major Ginsenosides Using Crude ß-Glucosidase Preparation from Submerged Culture of Fomitella fraxinea.

Minor ginsenosides, such as compounds (C)-K and C-Y, possess relatively better bioactivity than those of naturally occurring major ginsenosides. Therefore, this study focused on the biotransformation of major ginsenosides into minor ginsenosides using crude ß-glucosidase preparation isolated from submerged liquid culture of Fomitella fraxinea (FFEP). FFEP was prepared by ammonium sulfate (30-80%) precipitation from submerged culture of F. fraxinea. FFEP was used to prepare minor ginsenosides from protopanaxadiol (PPD)-type ginsenoside (PPDG-F) or total ginsenoside fraction (TG-F). In addition, biotransformation of major ginsenosides into minor ginsenosides as affected by reaction time and pH were investigated by TLC and HPLC analyses, and the metabolites were also identified by UPLC/negative-ESI-Q-TOF-MS analysis. FFEP biotransformed ginsenosides Rb1 and Rc into C-K via the following pathways: Rd → F2 → C-K for Rb1 and both Rd → F2→ C-K and C-Mc1 → C-Mc → C-K for Rc, respectively, while C-Y is formed from Rb2 via C-O. FFEP can be applied to produce minor ginsenosides C-K and C-Y from PPDG-F or TG-F. To the best of our knowledge, this study is the first to report the production of C-K and C-Y from major ginsenosides by basidiomycete F. fraxinea.

The efficacy of lyticase and ß-glucosidase enzymes on biofilm degradation of Pseudomonas aeruginosa strains with different gene profiles.

BACKGROUND: Pseudomonas aeruginosa is a nosocomial pathogen that causes severe infections in immunocompromised patients. Biofilm plays a significant role in the resistance of this bacterium and complicates the treatment of its infections. In this study, the effect of lyticase and ß-glucosidase enzymes on the degradation of biofilms of P. aeruginosa strains isolated from cystic fibrosis and burn wound infections were assessed. Moreover, the decrease of ceftazidime minimum biofilm eliminating concentrations (MBEC) after enzymatic treatment was evaluated. RESULTS: This study demonstrated the effectiveness of both enzymes in degrading the biofilms of P. aeruginosa. In contrast to the lyticase enzyme, ß-glucosidase reduced the ceftazidime MBECs significantly (P < 0.05). Both enzymes had no cytotoxic effect on the A-549 human lung carcinoma epithelial cell lines and A-431 human epidermoid carcinoma cell lines. CONCLUSION: Considering the characteristics of the ß-glucosidase enzyme, which includes the notable degradation of P. aeruginosa biofilms and a significant decrease in the ceftazidime MBECs and non-toxicity for eukaryotic cells, this enzyme can be a promising therapeutic candidate for degradation of biofilms in burn wound patients, but further studies are needed.

Role of intestinal microbiota-mediated genipin dialdehyde intermediate formation in geniposide-induced hepatotoxicity in rats.

Geniposide is a natural hepatotoxic iridoid glycoside. Its hydrolysate of intestinal microbiota, genipin, is thought to be responsible for the hepatotoxicity. However, the underlying mechanism that genipin contributes to the hepatotoxicity of geniposide is not well understood. In this study, we found that genipin spontaneously converted into a reactive dialdehyde intermediate and covalently bond to the primary amine group of free amino acids in both of the phosphate buffers and geniposide-treated rats. Furthermore, genipin dialdehyde can form the covalent linkage to the primary amino group (ε) of lysine side chains of the hepatic proteins in geniposide-treated rats. Pretreatment with ß-glucosidase or antibiotics significantly modulated the genipin dialdehyde formation and protein modification, revealing the essential role of microbial glycosidases. The levels of protein adduct were that mapped onto the hepatotoxicity of geniposide. In summary, this study demonstrates that the intestinal microbiota mediated covalent modification of the hepatic protein by genipin dialdehyde may play a crucial role in the liver injury of geniposide. The study is also helpful for understanding the contribution of intestinal microbiota to the metabolic activation of xenobiotics.

Screening exogenous fibrolytic enzyme preparations for improved in vitro digestibility of bermudagrass haylage.

Our objectives were to evaluate the effects of 12 exogenous fibrolytic enzyme products (EFE) on ruminal in vitro neutral detergent fiber digestibility (NDFD) and preingestive hydrolysis of a 4-wk regrowth of bermudagrass haylage (BH), to examine the accuracy of predicting NDFD with EFE activity measures, and to examine the protein composition of the most and least effective EFE at increasing NDFD. In experiment 1, effects of 12 EFE on NDFD of BH were tested. Enzymes were applied in quadruplicate to culture tubes containing ground BH. The suspension was incubated for 24 h at 25 °C before addition of rumen fluid media and further incubation for 24 h at 39 °C. The experiment was repeated twice. In addition, regression relationships between EFE activity measures and NDFD were examined. Compared with the values for the control, 9 EFE-treated substrates had greater NDFD (37.8 to 40.4 vs. 35.6%), 6 had greater total VFA concentration (59.1 to 61.2 vs. 55.4 mM), and 4 had lower acetate-to-propionate ratios (3.03 to 3.16 vs. 3.24). In experiment 2, EFE effects on preingestive fiber hydrolysis were evaluated by incubating enzyme-treated and untreated bermudagrass suspensions in quadruplicate for 24 h at 25 °C and examining fiber hydrolysis measures. Compared with values for the control, 3 EFE reduced neutral detergent fiber concentration (62.8 to 63.7 vs. 67.3%), 10 increased release of water-soluble carbohydrates (26.8 to 58.5 vs. 22.8 mg/g), and 8 increased release of ferulic acid (210 to 391 vs. 198 µg/g). Regression analyses revealed that enzyme activities accurately [coefficient of determination (R(2)) = 0.98] predicted preingestive hydrolysis measures (water-soluble carbohydrates, ferulic acid), moderately (R(2) = 0.47) predicted neutral detergent fiber hydrolysis, but poorly (R(2) ≤ 0.1) predicted dry matter and NDFD. In experiment 3, proteomic tools were used to examine the protein composition of the most and least effective EFE at improving NDFD. Relative to the least effective, the most effective EFE at increasing NDFD contained 10 times more endoglucanase III, 17 times more acetylxylan esterase with a cellulose-binding domain 1, 33 times more xylanase III, 25 times more ß-xylosidase, and 7.7 times more polysaccharide monooxygenase with cellulose-binding domain 1 and 3 times more swollenin. The most effective EFE had a much greater quantity of fibrolytic enzymes and key proteins necessary for hemicellulose and lignocellulase deconstruction. This study identified several EFE that increased the NDFD and in vitro fermentation of 4-wk BH and revealed why some EFE are more effective than others.

Changes in plant cell-wall structure of corn stover due to hot compressed water pretreatment and enhanced enzymatic hydrolysis.

Corn stover is a potential feedstock for biofuel production. This work investigated physical and chemical changes in plant cell-wall structure of corn stover due to hot compressed water (HCW) pretreatment at 170-190 °C in a tube reactor. Chemical composition analysis showed the soluble hemicellulose content increased with pretreatment temperature, whereas the hemicellulose content decreased from 29 to 7 % in pretreated solids. Scanning electron microscopy revealed the parenchyma-type second cell-wall structure of the plant was almost completely removed at 185 °C, and the sclerenchyma-type second cell wall was greatly damaged upon addition of 5 mmol/L ammonium sulfate during HCW pretreatment. These changes favored accessibility for enzymatic action. Enzyme saccharification of solids by optimized pretreatment with HCW at 185 °C resulted in an enzymatic hydrolysis yield of 87 %, an enhancement of 77 % compared to the yield from untreated corn stover.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures.

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5L (15-25FPU/g glucan) was in the following order: glycerol/HCl>HCl>H2SO4>NaOH, with the glycerol system achieving ≈ 100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6-6%) obtained with mixtures of endo-1,4-ß-glucanase (EG) and ß-glucosidase (ßG) for the NaOH substrate were 2-6 times that of acid substrates. However, glucan conversions (15-60%) obtained with mixtures of cellobiohydrolase (CBH I) and ßG on acidified glycerol substrate were 10-30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

Effect of ß-glucosidase on the meat quality and digestibility in broilers.

This study investigated the effects of supplementary ß-glucosidase on the carcass composition, meat quality, weight of digestive organ and apparent digestibility in male broilers. Two hundred and forty male, 1-day avine broiler chicks were randomly allocated into four treatment groups and fed with corn-soya bean meal supplemented with 0 (control), 0.6, 1.2 and 1.8 U/g of ß-glucosidase respectively. The results showed that there were no significant differences (p > 0.05) among groups in carcass composition (percentages of eviscerated yield, half-eviscerated yield, muscle yield of breast and leg). However, adding 0.6 U/g ß-glucosidase to the diet not only altered the meat quality by decreasing the drip loss ratio (p < 0.05) and relative lightness (L*) value (p < 0.01), increasing relative redness (a*) value (p < 0.01), but also significantly decreased the pancreas to body weight ratio (p < 0.05), however, with little effect on liver, proventriculus and gizzard to body weight ratio (p > 0.05). The length and width of duodenum villus were not affected by the addition of ß-glucosidase, but the coefficients of total tract apparent digestibility of protein and fat increased by 9.02% (p < 0.05) and 7.40% (p < 0.01) respectively; the parameters of ash were not affected by ß-glucosidase addition (p < 0.05). This study provided valuable information for evaluation of the effect of supplementary ß-glucosidase on the meat quality and digestibility of broilers.

Using FTIR spectroscopy to model alkaline pretreatment and enzymatic saccharification of six lignocellulosic biomasses.

Fourier transform infrared, attenuated total reflectance (FTIR-ATR) spectroscopy, combined with partial least squares (PLS) regression, accurately predicted solubilization of plant cell wall constituents and NaOH consumption through pretreatment, and overall sugar productions from combined pretreatment and enzymatic hydrolysis. PLS regression models were constructed by correlating FTIR spectra of six raw biomasses (two switchgrass cultivars, big bluestem grass, a low-impact, high-diversity mixture of prairie biomasses, mixed hardwood, and corn stover), plus alkali loading in pretreatment, to nine dependent variables: glucose, xylose, lignin, and total solids solubilized in pretreatment; NaOH consumed in pretreatment; and overall glucose and xylose conversions and yields from combined pretreatment and enzymatic hydrolysis. PLS models predicted the dependent variables with the following values of coefficient of determination for cross-validation (Q²): 0.86 for glucose, 0.90 for xylose, 0.79 for lignin, and 0.85 for total solids solubilized in pretreatment; 0.83 for alkali consumption; 0.93 for glucose conversion, 0.94 for xylose conversion, and 0.88 for glucose and xylose yields. The sugar yield models are noteworthy for their ability to predict overall saccharification through combined pretreatment and enzymatic hydrolysis per mass dry untreated solids without a priori knowledge of the composition of solids. All wavenumbers with significant variable-important-for-projection (VIP) scores have been attributed to chemical features of lignocellulose, demonstrating the models were based on real chemical information. These models suggest that PLS regression can be applied to FTIR-ATR spectra of raw biomasses to rapidly predict effects of pretreatment on solids and on subsequent enzymatic hydrolysis.

Enhancement of amygdalin activated with ß-D-glucosidase on HepG2 cells proliferation and apoptosis.

The growth inhibition and induction of apoptosis brought by amygdalin and activated with ß-D-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC50 of stand-alone amygdalin and activation with ß-D-glucosidase on the proliferation of HepG2 cells for 48 h were 458.10 mg/mL and 3.2 mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (p<0.01). Together, these findings indicate that amygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with ß-D-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity.

Purification and biochemical properties of a glucose-stimulated beta-D-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse.

The effect of several carbon sources on the production of mycelial-bound beta-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated beta-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The beta-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50 degrees C, respectively. The purified enzyme was thermostable up to 60 min in water at 55 degrees C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-xylopyranoside, o-nitrophenyl-beta-D-galactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-beta-D-fucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude beta-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea beta-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Transgenic alfalfa that accumulates piceid (trans-resveratrol-3-O-beta-D-glucopyranoside) requires the presence of beta-glucosidase to inhibit the formation of aberrant crypt foci in the colon of CF-1 mice.

Plants have been genetically enhanced to produce a number of products for agricultural, industrial and pharmaceutical purposes. This technology could potentially be applied to providing chemoprevention strategies to the general population. Resveratrol (3,5,4'-trihydroxystilbene) is a compound that has been shown to have protective activity against a number of cancers and could be an ideal candidate for such an application. Alfalfa that was genetically modified to express resveratrol-synthase was used as a model in applying biotechnological approaches to cancer prevention. The transgenic alfalfa, which accumulates resveratrol as a glucoside (piceid = trans-resveratrol-3-O-Beta-D-glucopyranoside) (152 +/- 17.5 microg piceid/g dry weight), was incorporated into a standard mouse diet at 20% of the diet by weight and fed for 5 wk to 6-wk-old, female CF-1 mice (N = 17-30) that were injected with a single dose of azoxymethane (5 mg/kg body weight). While the addition of resveratrol-aglycone (20 mg/kg diet) to the basal diet reduced the number of aberrant crypt foci/mouse, the transgenic alfalfa did not inhibit the number, size, or multiplicity of aberrant crypt foci in the colon of the CF-1 mice relative to control alfalfa which does not accumulate resveratrol-glucoside. However, diets containing transgenic alfalfa with an exogenous Beta-glucosidase (860 U/kg diet) did significantly inhibit the number of aberrant crypt foci in the distal 2 cm of the colon of the mice relative to mice fed diets containing the transgenic alfalfa without the enzyme (P < 0.05; Fisher's Combination of p-values). The Beta-glucosidase alone appeared to have no effect on the inhibition of aberrant crypt foci. These results suggest that piceid in transgenic piceid-accumulating alfalfa was not bioavailable.

Metabolism of pyrene by aquatic crustacean, Daphnia magna.

The aquatic crustacean Daphnia magna is an important species for ecotoxicological study, and is often used as a test organism for environmental risk assessment. However, the mechanism of xenobiotic metabolism by this species has not been studied in detail. In the present study, pyrene was used as model substance to investigate the mechanism of xenobiotic metabolism in D. magna. The results of 24-h exposure experiments showed that D. magna could metabolize pyrene and biotransform it into water-soluble metabolites. On the other hand, the metabolism of pyrene was significantly inhibited by SKF-525A as the cytochrome P450 (CYP) inhibitor. These observations indicated that oxidation by CYP participated in the biotransformation of pyrene by D. magna. We also identified the pyrene metabolites formed by D. magna by HPLC with an electrospray ionization triple quadrupole mass spectrometry detector (LC/ESIMS/MS) and de-conjugation by sulfatase, beta-glucuronidase, and beta-glucosidase. One of the metabolites was ionized in ESI negative mode and formed a dominant mass of m/z 297 (MS) with the product ion of m/z 217 (MS2). Furthermore, this metabolite formed 1-hydroxypyrene on treatment with sulfatase. This metabolite was considered to be a sulfate conjugate of oxidized pyrene (1-hydroxypyrenesulfate). Furthermore, we quantified the deconjugated 1-hydroxypyrene formed by the above enzyme treatment. It showed that 52% of the total metabolized pyrene was biotransformed into 1-hydroxypyrene-sulfate, and more than 73% was biotransformed into oxidized pyrene conjugate. These results indicated that CYP and several conjugation enzymes participate in its biotransformation, and sulfation is important in D. magna for metabolism and elimination of xenobiotics.

The influence of methyl jasmonate (JA-Me) and B-glucosidase on induction of resistance mechanisms of strawberry against two-spotted spider mite (Tetranychus urticae Koch.).

The goal of the research was to study the influence of methyl jasmonate (JA-Me) and beta-glucosidase treatments on fecundity and preference to infestation and oviposition of two-spotted spider mite feeding on strawberry. The experiments were conducted in laboratory conditions on leaves of Aga and Kent cultivars. Leaves were treated with: a. solution of 0.1% JA-Me in 0.05% Triton X-100 (by spraying); b. beta-glucosidase dissolved in 0.1 M citrate buffer at pH 6 (by petiole); c. 0.05% solution of the Triton X-100 (by spraying); d. 0.1 M citrate buffer at pH 6 (by petiole). In the no-choice test, application of JA-Me on leaves of strawberry caused reducing of number of eggs laid during three days of the experiment. In the choice test, which was carried out for determination of non-preference mechanism of resistance, there was a statistically significant lower number of mites on leaves treated with JA-Me compared to leaves treated with other compounds as well as to non-treated leaves after 24 hours from solutions application. Moreover, at the same experiment, females of two-spotted spider mite laid the least number of eggs on leaves treated with JA-Me. Analysis conducted using liquid chromatography method, revealed increase of the level of phenolic compounds like chlorogenic acid and rutin on leaves treated with JA-Me. Thus, it appears that JA-Me may be involved in antybiosis or non-preference mechanisms of resistance of strawberry to two-spotted spider mite.

Enzyme activities and arylsulfatase protein content of dust and the soil source: biochemical fingerprints?

Little is known about the potential of enzyme activities, which are sensitive to soil properties and management, for the characterization of dust properties. Enzyme activities may be among the dust properties key to identifying the soil source of dust. We generated dust (27 and 7 microm) under controlled laboratory conditions from agricultural soils (0-5 cm) with history of continuous cotton (Gossypium hirsutum L.) or cotton rotated with peanut (Arachis hypogaea L.), sorghum [Sorghum bicolor (L.) Moench], rye (Secale cereale L.), or wheat (Triticum aestivum L.) under different water management (irrigated or dryland) and tillage (conservation or conventional) systems. The 27- and 7-microm dust samples showed activities of beta-glucosidase, alkaline phosphatase, and arylsulfatase, which are related to cellulose degradation and phosphorus and sulfur mineralization in soil, respectively. Dust samples generated from a loam and sandy clay loam showed higher enzyme activities compared with dust samples from a fine sandy loam. Enzyme activities of dust samples were significantly correlated to the activities of the soil source with r > 0.74 (P < 0.01). The arylsulfatase proteins contents of the soils (0.04-0.65 mg protein kg(-1) soil) were lower than values reported for soils from other regions, but still dust contained arylsulfatase protein. The three enzyme activities studied, as a group, separated the dust samples due to the crop rotation or tillage practice history of the soil source. The results indicated that the enzyme activities of dust will aid in providing better characterization of dust properties and expanding our understanding of soil and air quality impacts related to wind erosion.

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract.

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant beta-glucosidase from synthetic medium by Kluyveromyces marxianus.

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on beta-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and beta-glucosidase production rate. The highest product yield, specific product yield, and productivity of beta-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved beta-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L.h]) of beta-glucosidase occurred on cellobiose in the presence of CSL at 35 degrees C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of beta-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60 degrees C and may find application in some industrial processes.

Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1.

The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

In vitro degradation of willow salicylates.

Salicylates are defensive compounds against a great variety of generalist herbivores. Salicortin and its derivatives are labile compounds that degrade immediately when cell compartmentalization is ruptured, producing a 6-hydroxy-2-cyclohexenone (6-HCH) moiety that is a strong antifeeding cue. We studied the in vitro degradation of willow salicylates in the presence and absence of foliar enzymes at acidic, neutral, and alkaline pHs. Higher substituted salicylates were degraded in the absence of foliar enzymes at alkaline pH and in the presence of foliar enzymes at all three pHs. Salicin and its diglucoside, on the other hand, were degraded only in the presence of foliar enzymes at acidic pH, probably by beta-glucosidase activity. The main degradation products of higher substituted salicylates were salicin, 6-HCH, and catechol in both the absence and presence of enzymes, suggesting that the production of 6-HCH and catechol do not necessarily demand enzymatic activity. We propose that the degradation of salicylates begins with the cleavage of a 1-hydroxy-6-oxo-2-cyclohexen-1-carbonyl moiety by foliar esterases and/or alkaline condition. This moiety is decarboxylated in nonenzymatic reaction to an anion of 2-hydroxy-3-cyclohexenone, which is tautomerized to the enol form and further to the keto form, 6-HCH. Hydroxyketone can be also oxidized to catechol, a substrate of polyphenol oxidases.

The influence of methyl jasmonate and beta-glucosidase on induction of apple tree resistance mechanisms to two-spotted spider mite (Tetranychus urncae Koch.).

The objective of the research was to determine the influence of methyl jasmonate (JA-Me) and beta-glucosidase on the mechanisms of apple tree resistance to T. urticae like antibiosis and non-preference. The experiments were conducted on leaves of Close and Jester apple cultivars in laboratory conditions. Leaves were treated with: 1. solution of 0.1% JA-Me in 0.05% Triton X-100 (by spraying); 2. beta-glucosidase dissolved in 0.1 M citrate buffer at pH 6 (by petiole); 3. 0.05% solution of the Triton X-100 (by spraying); 4. 0.1 M citrate buffer at pH 6 (by petiole); 5. non-treated leaves. In the no-choice test, application of JA-Me on leaves of apple trees caused reducing of number of eggs laid up during three days of the experiment. In the choice test, which was carried out for determination of non-preference mechanism of resistance, there was not significant differences between number of mites on leaves treated with JA-Me compared to leaves treated with beta-glucosidase, and to non-treated leaves after 24 hours from solutions application. However, at the same experiment, females of T. urticae laid the least number of eggs on leaves treated with JA-Me. Analysis conducted using liquid chromatography method, revealed higher level of phenolic compounds on leaves treated with JA-Me compared to check and leaves treated with beta-glucosidase. Thus, it appears that JA-Me may be regarded as elicitor of induced resistance of apple tree to two-spotted spider mite.

The distribution characteristics of bacterial beta-glucosidase activity in Taiwan strait.

Twenty stations were established in the near-shore regions of South Fujian Shoal (116 degrees 10'-119 degrees 00' E, 21 degrees 20'-24 degrees 10' N) on summer and winter cruises during the period from August 1997 and February to March 1998. The distribution pattern of marine bacterial beta-glucosidase activity (beta-GlcA) has been investigated by using fluorogenic model substrate (FMS) technique in order to have better understanding of the beta-GlcA, as well as its relation to marine bacterial biomass, productivity and environmental factors in Taiwan strait. The results showed that: (1) In summer, the average of beta-GlcA at the Southern stations of Taiwan strait was 1.94 nmol/l h. While in winter, the average of beta-GlcA at the Northern stations was 0.86 nmol/l h and the range of variation (0.34-1.89 nmol/l h) was much more narrow than that in summer (0.31-8.1 nmol/l h). (2) According to the carbon conversion factor, the beta-GlcA was 0.14 and 0.062 ugc/l h in summer and winter respectively. These beta-GlcA values were higher than the bacterial production of the two seasons respectively. (3) The beta-GlcA gradually rises from offshore water to near-shore water. (4) The correlation between the beta-GlcA and the bacterial secondary production was not so obvious. (5) The correlation between the section distributions, daily varying of the beta-GlcA and the bacterial production was not obvious. (6) In the surface water, the distribution character of free-state beta-GlcA from bacteria was equal to that of the total beta-GlcA in the whole sea area.