Concentrations Of Hydrolysis Products Research Articles

In vitro application of Eruca vesicaria subsp. sativa leaf extracts and associated metabolites reduces the growth of Oomycota species involved in Kiwifruit Vine Decline Syndrome.

This study aimed to determine whether leaf extracts from seven Eruca vesicaria subsp. sativa cultivars and their biochemically active compounds (glucosinolates and downstream-derived products) inhibit mycelia growth of three well-known pathogenic oomycetes, Phytopythium chamaehyphon, Phytopythium vexans and Phytophthora citrophthora; being the most significant in the development of Kiwifruit Vine Decline Syndrome (KVDS). Leaf extract quantity of 10, 20 and 30 mg were inoculated in Petri dish (90 mm Ø, each 22 mL of liquid medium - Potato Dextrose Agar), for in vitro bioassays. A pathogen plug was placed in the centre of each plate and the Oomycota colony perimeter was marked 5 days after inoculation. Radial colony growth was measured from 4 marks per plate 5, 10, and 15 days after inoculation, further elaborated with Image J software image analysis. Growth rates for all strains were inhibited by around 67% after 15 days. This was most pronounced when applying the highest concentration of leaf extract. By using Liquid Chromatography Mass Spectrometry (LC-MS) and Gas Chromatography Mass Spectrometry (GC-MS), fifteen glucosinolate compounds, of which glucosativin was found in the highest quantity, were identified. Concentrations of hydrolysis products produced by leaves (erucin and sativin) were also investigated, and were significantly associated with colony radial growth, especially towards Pp. chamaehyphon and Pp. vexans. Three downstream products of glucosinolates (two pure isothiocyanates, AITC and PEITC; and one indole I3C; all commonly present in Brassicaceae) were also tested, and a statistically significant inhibition of growth was observed at the highest concentration (0.6 µL).

Kinetic characterization of digestive proteinases extracted from the processing waste of South Atlantic fish

In Argentina, the fishing industry is highly developed, producing large quantities of waste products from fish processing which contain digestive enzymes with potential biotechnological uses. Among these enzymes are proteinases that hydrolyze proteins and can convert them into functional ingredients. To use these value-added compounds in industrial processes, kinetic characterization of these proteinases under specific conditions, such as contact time, pH, and temperature, must first be carried out. The objective of the present work was to perform a kinetic characterization of crude extracts of alkaline proteinases from intestine-ceca of Merluccius hubbsi, Percophis brasiliensis, Urophycis brasiliensis, and Cynoscion guatucupa. Results showed that the reached maximum values of A440 depended on both the species analyzed and the initial concentration of azocasein. The maximum concentration of hydrolysis products obtained at long times was proportional to the initial concentration of the target protein. P. brasiliensis was the most suitable tested species to obtain high hydrolysis rates at high azocasein concentrations, and exhibited the highest proteinase activities, so it can be considered a candidate species for future biotechnological applications.

Effect of fluid hydrodynamic situations on enzymatic hydrolysis of mixed microalgae: Experimental study and simulation

The enzymatic hydrolysis of microalgae has already been studied for bioethanol production. However, the enzymatic hydrolysis of microalgae under various fluid hydrodynamic conditions has not been simulated yet. Accordingly, the present study investigated the effect of various stirrer speed values, impeller types, and the presence of baffles on glucose extraction from the mixed microalgae using cellulase. The Michaelis-Menten kinetic constants of enzymatic hydrolysis were calculated in the AQUASIM and were used for simulation in COMSOL. The simulated values agreed with the experimental results and revealed that the low stirring speed reduced glucose extraction through undesirable enzyme distribution and the formation of regions with a high concentration of hydrolysis products to inhibit the enzyme's operation. The higher uniformity of glucose concentration at higher stirring speeds compared to lower stirring speeds ensured the higher efficiency of the mixing process in this reactor. The study findings suggested that a sufficient understanding of the mixing effects in the enzymatic hydrolysis could improve the economic feasibility of the process. Furthermore, compared to the unbaffled reactor, the proper mixing in the baffled reactor increased the enzymatic hydrolysis rate. It was found that altering the impeller type leads to a negligible change in the enzymatic hydrolysis rate.

Isothiocyanates, Nitriles, and Epithionitriles from Glucosinolates Are Affected by Genotype and Developmental Stage in Brassica oleracea Varieties.

Vegetables of the Brassica oleracea group, such as broccoli, cauliflower, and cabbage, play an important role for glucosinolate consumption in the human diet. Upon maceration of the vegetable tissue, glucosinolates are degraded enzymatically to form volatile isothiocyanates, nitriles, and epithionitriles. However, only the uptake of isothiocyanates is linked to the cancer-preventive effects. Thus, it is of great interest to evaluate especially the isothiocyanate formation. Here, we studied the formation of glucosinolates and their respective hydrolysis products in sprouts and fully developed vegetable heads of different genotypes of the five B. oleracea varieties: broccoli, cauliflower as well as white, red, and savoy cabbages. Further, the effect of ontogeny (developmental stages) during the head development on the formation of glucosinolates and their respective hydrolysis products was evaluated at three different developmental stages (mini, fully developed, and over-mature head). Broccoli and red cabbage were mainly rich in 4-(methylsulfinyl)butyl glucosinolate (glucoraphanin), whereas cauliflower, savoy cabbage and white cabbage contained mainly 2-propenyl (sinigrin) and 3-(methylsulfinyl)propyl glucosinolate (glucoiberin). Upon hydrolysis, epithionitriles or nitriles were often observed to be the main hydrolysis products, with 1-cyano-2,3-epithiopropane being most abundant with up to 5.7 μmol/g fresh weight in white cabbage sprouts. Notably, sprouts often contained more than 10 times more glucosinolates or their hydrolysis products compared to fully developed vegetables. Moreover, during head development, both glucosinolate concentrations as well as hydrolysis product concentrations changed and mini heads contained the highest isothiocyanate concentrations. Thus, from a cancer-preventive point of view, consumption of mini heads of the B. oleracea varieties is recommended.

Proposed Method for Estimating Health-Promoting Glucosinolates and Hydrolysis Products in Broccoli (Brassica oleracea var. italica) Using Relative Transcript Abundance.

Due to the importance of glucosinolates and their hydrolysis products in human nutrition and plant defense, optimizing the content of these compounds is a frequent breeding objective for Brassica crops. Toward this goal, we investigated the feasibility of using models built from relative transcript abundance data for the prediction of glucosinolate and hydrolysis product concentrations in broccoli. We report that predictive models explaining at least 50% of the variation for a number of glucosinolates and their hydrolysis products can be built for prediction within the same season, but prediction accuracy decreased when using models built from one season's data for prediction of an opposing season. This method of phytochemical profile prediction could potentially allow for lower phytochemical phenotyping costs and larger breeding populations. This, in turn, could improve selection efficiency for phase II induction potential, a type of chemopreventive bioactivity, by allowing for the quick and relatively cheap content estimation of phytochemicals known to influence the trait.

Profiles of Glucosinolates, Their Hydrolysis Products, and Quinone Reductase Inducing Activity from 39 Arugula (Eruca sativa Mill.) Accessions.

Glucosinolates, their hydrolysis product concentrations, and the quinone reductase (QR) inducing activity of extracts of leaf tissue were assayed from 39 arugula (Eruca sativa Mill.) accessions. Arugula accessions from Mediterranean countries (n = 16; Egypt, Greece, Italy, Libya, Spain, and Turkey) and Northern Europe (n = 2; Poland and United Kingdom) were higher in glucosinolates and their hydrolysis products, especially glucoraphanin and sulforaphane, compared to those from Asia (n = 13; China, India, and Pakistan) and Middle East Asia (n = 8; Afghanistan, Iran, and Israel). The QR inducing activity was also the highest in Mediterranean and Northern European arugula accessions, possibly due to a significant positive correlation between sulforaphane and QR inducing activity (r = 0.54). No nitrile hydrolysis products were found, suggesting very low or no epithiospecifier protein activity from these arugula accessions. Broad sense heritability (H(2)) was estimated to be 0.91-0.98 for glucoinolates, 0.55-0.83 for their hydrolysis products, and 0.90 for QR inducing activity.

Transcriptome and Metabolome Analyses of Glucosinolates in Two Broccoli Cultivars Following Jasmonate Treatment for the Induction of Glucosinolate Defense to Trichoplusia ni (Hübner)

Lepidopteran larvae growth is influenced by host plant glucosinolate (GS) concentrations, which are, in turn, influenced by the phytohormone jasmonate (JA). In order to elucidate insect resistance biomarkers to lepidopteran pests, transcriptome and metabolome analyses following JA treatments were conducted with two broccoli cultivars, Green Magic and VI-158, which have differentially induced indole GSs, neoglucobrassicin and glucobrassicin, respectively. To test these two inducible GSs on growth of cabbage looper (Trichoplusia ni), eight neonate cabbage looper larvae were placed onto each of three plants per JA treatments (0, 100, 200, 400 µM) three days after treatment. After five days of feeding, weight of larvae and their survival rate was found to decrease with increasing JA concentrations in both broccoli cultivars. JA-inducible GSs were measured by high performance liquid chromatography. Neoglucobrassicin in Green Magic and glucobrassicin in VI-158 leaves were increased in a dose-dependent manner. One or both of these glucosinolates and/or their hydrolysis products showed significant inverse correlations with larval weight and survival (five days after treatment) while being positively correlated with the number of days to pupation. This implies that these two JA-inducible glucosinolates can influence the growth and survival of cabbage looper larvae. Transcriptome profiling supported the observed changes in glucosinolate and their hydrolysis product concentrations following JA treatments. Several genes related to GS metabolism differentiate the two broccoli cultivars in their pattern of transcriptional response to JA treatments. Indicative of the corresponding change in indole GS concentrations, transcripts of the transcription factor MYB122, core structure biosynthesis genes (CYP79B2, UGT74B1, SUR1, SOT16, SOT17, and SOT18), an indole glucosinolate side chain modification gene (IGMT1), and several glucosinolate hydrolysis genes (TGG1, TGG2, and ESM1) were significantly increased in Green Magic (statistically significant in most cases at 400 µM) while UGT74B1 and MYB122 were significantly increased in VI-158. Therefore, these metabolite and transcript biomarker results indicate that transcriptome profiling can identify genes associated with the formation of two different indole GS and their hydrolysis products. Therefore, these metabolite and transcript biomarkers could be useful in an effective marker-assisted breeding strategy for resistance to generalist lepidopteran pests in broccoli and potentially other Brassica vegetables.

The applicability of Impranil®DLN for gauging the biodegradation of polyurethanes

Polyurethane-based polymers and their eventual degradation products pervade modern society. One common method for determining whether a microorganism or protein can degrade this class of polymer is to qualitatively assess its ability to “clear” a polyester-polyurethane colloid branded Impranil®DLN (Impranil), whose formulation is proprietary. However, its colloidal state has ultimately made Impranil a precarious choice for determining if an organism or enzyme can degrade polyurethanes. In this work, the chemical hydrolysis products from Impranil using 0.1 M HCl or 0.1 M NaOH were identified and compared to the concentration of hydrolysis products formed using three commercial enzymes by proton nuclear magnetic spectroscopy (1H NMR) and Fourier-transform infrared spectroscopy (FT-IR). The differences in the integrated signal intensities from key 1H NMR signals were used to calculate the amount of Impranil that was hydrolyzed. These data were then correlated with the change in optical density of colloid containing reaction mixtures (termed as “clearing”). The enzymes (a Pseudomonas fluorescens recombinant esterase, Pseudomonas sp. lipase, and Bacillus sp. protease) showed significant esterase activities and partially-cleared, completely-cleared, or aggregated Impranil, respectively. However, only the Pseudomonas sp. lipase significantly degraded Impranil based on NMR and IR data. This study illustrates how Impranil can be used to quantitatively assess biodegradation rather than just be a qualitative “clearing” indicator of biodegradation.

Exogenous methyl jasmonate treatment increases glucosinolate biosynthesis and quinone reductase activity in kale leaf tissue.

Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties ‘Dwarf Blue Curled Vates’ and ‘Red Winter’ in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar ‘Red Winter’ in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to combined increases in phenolics (quercetin and kaempferol) and GS hydrolysis product concentrations rather than by individual products alone.

Optimization of methyl jasmonate application to broccoli florets to enhance health-promoting phytochemical content

Spray treatment of methyl jasmonate (MeJA) has been shown to increase glucosinolate (GS) concentrations and health-promoting activity in Brassica vegetables. Since there is no reported standardized protocol, several MeJA treatment studies have been conducted to maximize human health bioactivity using the F1 broccoli cultivar 'Green Magic'. Foliar MeJA application 4 days prior to harvest of broccoli at commercial maturity resulted in enhanced total GS concentrations. Although a single application of 250 µmol L(-1) MeJA maximized GS concentrations in broccoli florets, two days of consecutive treatments (4 and 3 days prior to harvest) of 250 µmol L(-1) MeJA further enhanced neoglucobrassicin concentrations and floret extract quinone reductase (QR)-inducing activity. With increasing concentrations of MeJA in spray applications to broccoli florets, concentrations of the glucosinolates glucoraphanin, gluconasturtiin and neoglucobrassicin and the isothiocyanate sulforaphane as well as anticancer and anti-inflammatory bioactivities as measured by QR induction and inhibition of nitric oxide (NO) production respectively were significantly increased. Concentrations of these phytochemicals showed strong positive correlations with QR-inducing and NO-inhibitory activities. These application protocols were found to maximize GS and GS hydrolysis product concentrations and putatively enhance the health-promoting properties of broccoli heads for consumers.

Production of fructooligosaccharides by β‐fructofuranosidase from Aspergillus sp 27H

Abstractβ‐fructofuranosidase (EC 3.2.1.26) from Aspergillus sp 27H isolated from soil was investigated for production of fructooligosaccharides (FOS) using whole cells. It possesses hydrolytic and transfructosylating activities that can be altered by modifying the reaction conditions. The optimal conditions for the transfructosylating activity occur in the pH range 5.5–6.0 and at 60 °C, while hydrolytic activity was highest at pH 4.0 and 55 °C. At low sucrose concentration (10 g dm−3) there was rapid conversion of sucrose to glucose and fructose and very low concentrations of FOS were obtained. However, at sucrose concentrations higher than 216 g dm−3 the concentrations of hydrolysis products were reduced. Under the following conditions: pH 5.5, temperature 40 °C, sucrose concentration 615 g dm−3 and enzyme concentration 20β‐fructofuranosidase units g−1 of sucrose, the FOS concentration reached a maximum value of 376 g dm−3 (234 g dm−3 1‐kestose and 142 g dm−3 nystose) and the proportion of FOS in the solids in the reaction mixture was 600–620 g kg−1 at 6 h. These results suggest that β‐fructofuranosidase from Aspergillus sp 27H could be an appropriate enzyme for the commercial production of FOS. Copyright © 2004 Society of Chemical Industry

Membrane separation to improve degradation of road side grass by rumen enhanced solid incubation

Membrane separation proved to be an excellent means to maintain high residence time of microorganisms in an anaerobic hydrolysis reactor, and relatively low concentration of hydrolysis products. The microbial biocommunity typical for the rumen environment could be maintained, and the reactor efficiency of the reactor improved. Less than 4 days were reqired to reach almost complete hydrolysis of the grass fed into the reactor. To avoid blocking of the membrane unit, a backwash system is necessary. The membranes needed to be backwashed every 20 min with 4 bar gas-pressure for 10 s. After this treatment the initial permeability was regained. The plant was operated with a flux of 12 ml h(-1) cm(-2) on average. The transmembrane pressure was in the range of 0.8-0.9 bar. 90% of the dissolved fatty acids permeated through the membrane.

Dissolved organic carbon production and consumption in anoxic marine sediments: A pulsed‐tracer experiment

The degradation of phytoplankton and seagrass organic carbon (OC) in sulfate‐reducing (SR) and methaneproducing (MP) sediments was tracked by measuring concentrations of particulate OC (POC), hydrolysis products (HP), fermentation products (FP), and inorganic end products (EP). This experiment used the novel approach of amending sediment with organic substrates having δ13C values unique from that of the OC pool originally present in the sediment. As a result, we could monitor changes in the dynamic dissolved OC pool and gain insight into processes that control the fate of OC. Rates of hydrolysis, fermentation, and terminal metabolism were greater in the phytoplankton‐amended treatments than in the seagrass‐amended treatments during the period of active decomposition. At the end of the incubation, concentrations of HP and FP in amended treatments were not significantly different from those in the controls. An analysis of the δ13C values of HP from the amended treatments indicated that the addition of fresh organic matter stimulated the decomposition of carbon present in the sediment at the time of collection. Hydrolysis of this carbon accounted for &gt;=50% of the total carbon hydrolyzed in the sediment amended with seagrass.

Oxide Surface‐Catalyzed Hydrolysis of Carboxylate Esters and Phosphorothioate Esters

AbstractPathways and rates of organic pollutant transformation may be altered through interaction with the surfaces of oxides and other soil minerals. We examined oxide surface‐catalyzed hydrolysis of representative carboxylate esters and phosphorothioate esters. Ester and hydrolysis product concentrations were monitored in acetate‐buffered suspensions containing 10 g L−1 oxide. Carboxylate esters found susceptible to catalysis in this work possess an auxiliary ligand donor group that, along with the carbonyl O group, form a chelate with surface‐bound metal atoms. Esters possessing an O‐donor auxiliary group (e.g., phenyl salicylate) are susceptible to catalysis by Al2O3, TiO2, and FeOOH surfaces. Esters possessing a N‐donor auxiliary group (e.g., phenyl picolinate) are no longer susceptible to catalysis by Al2O3, reflecting the poor affinity of N‐donor groups for Al complexation. Methyl chlorpyrifos is the most susceptible of the three phosphorothioate esters to oxide‐catalyzed hydrolysis; it possesses an auxiliary ligand donor group analogous to the carboxylate esters examined. Methyl parathion and ronnel, which do not possess a suitable auxiliary ligand donor group, are also susceptible to oxide‐catalyzed hydrolysis, indicating that chelate formation is not required in order for surface catalysis to occur. The presence of methanol and acetonitrile cosolvents diminishes the catalytic effect. The widespread use of these cosolvents in fate studies may have caused rates of oxide and mineral surface‐catalyzed hydrolysis reactions to be underestimated.

Effects of the embryonic axis and phytohormones on proteolysis of the storage protein in buckwheat seed

The role of the embryonic axis in regulation of proteolysis of the main storage protein was studied in buckwheat (Fagopyrum esculentum) seed. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that removal of the embryonic axis had no effect on the first stage of hydrolysis, that is proteolytic modification, of 13S globulin. This modification took place in the growing seedlings also in the presence of cycloheximide, i.e. it was due to an enzyme present in dry seed. However, in isolated cotyledons the 13S globulin was not degraded completely. Incubation of isolated cotyledons with cytokinins, gibberellic acid and indoleacetic acid could not replace the excised embryonic axis. At the same time, proteolysis of the 13S globulin in the growing seedlings was strongly inhibited by casein hydrolyzate. It is suggested that a complete proteolysis of the modified storage protein is regulated by the concentration of hydrolysis products at the site of hydrolysis. The embryonic axis serves, most probably, as a site of efflux of the products of protein hydrolysis in the cotyledons during seedling growth and thus regulates the course of proteolysis.Abscisic acid (10–100 μM) was without effect on modification of the 13S globulin, but suppressed the complete proteolysis of the protein by inhibiting, apparently, the synthesis of the cysteine proteinase in the growing seedlings.

A comparative study of insecticide metabolism in photoperiod-entrained and unentrained bollworm larvae Heliothis zea (Boddie)

1. 1. The metabolism of P 32-labelled Di-syston R ( O, O-diethyl S-2(ethylthio)ethyl phosphorodithioate) in fifth instar bollworms, Heliothis zea (Boddie) from unentrained and light-synchronized populations was compared by the use of radiometric procedures. 2. 2. Larvae were treated by injection at consecutive 2-hr intervals for 24 hr and then analyzed to determine the concentrations of hydrolytic and oxidative metabolites of Di-syston R that were formed in 4 hr. 3. 3. Differences in metabolite concentrations in unentrained larvae were not significant; however, in entrained larvae variations were significantly different and followed a cyclic pattern. 4. 4. Maxima of hydrolysis product concentrations occurred at 24-hr intervals in internal extracts and at 12-hr intervals in excreta. 5. 5. Peak concentrations of oxidation products occurred at 12-hr intervals in internal extracts but did not follow a definite pattern in excreta.

The effect of ethyl methanesulfonate on the growth response, chromosome structure and mutation rate in barley

Effects of ethyl methanesulfonate (EMS) and X-rays were compared by several biological measures in barley. The criteria studied included rate of growth, survival and fertility of M 1 plants, frequencies of chromosomal aberrations in mitosis of seedlings and meiosis of M 1 spikes, and the frequencies of chlorophyll deficient mutations among M 2 seedling progeny. The doses of EMS and X-ray treatments chosen reduced the rate of seedling growth and survival to a comparable degree. The reduction of fertility was directly related to the frequency of chromosome aberrations and mutations after X-ray treatment. After EMS treatment, the decrease of fertility was pronounced; however, there was little effect on seedling growth and survival. Also, the frequency of chromosome aberrations observed in EMS treatments was negligible, whereas a high rate of chlorophyll deficient mutations was induced in both M 1 plants and M 2 progenies. Preliminary results indicated that the efficiency of EMS treatments can be further increased when the treatment solutions are buffered and the delayed action of the mutagen is prevented. Physiological damage, e.g., reduced seedling growth and survival, increased with concentration of hydrolysis products in the treatment solutions, except when buffers were added. The damage caused by drying seeds after EMS treatment was reduced considerably by soaking the treated seeds in distilled water before drying. This delayed effect may be due to active agent and/or by-products retained in the tissue after the defined treatment time.

A rapid method for the quantitative assay of proteolytic enzymes

1. 1. Methods for the preparation of stable dye-protein complexes for use in proteolytic enzymes are described. 2. 2. Indigo carmine-fibrin provides a suitable substrate for the quantitative assay of proteolytic enzymes of the pepsin type, whereas, Congo redhide powder provides a suitable substrate for the quantitative assay of proteolytic enzymes of the trypsin type. 3. 3. The dye concentration, as measured spectrophotometrically at 620 mμ, parallels the concentration of the enzymically obtained hydrolysis products as measured at 273 mμ. This suggests that the dye concentration is directly proportional to the concentration of hydrolysis products.