Partial biochemical characterization of α- and β-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae)

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of α- and β-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of α- and β-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Activity of α- and β-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of α- and β-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 μmol⁻¹ min⁻¹ mg protein⁻¹, respectively. Results showed that the optimal pH for α- and β-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for α- and β-glucosidase activity in the midgut was obtained as 45 °C. The addition of CaCl₂ (40 mM) decreased midgut β-glucosidase activity whereas α-glucosidase activity was significantly increased at this concentration. The α-glucosidase activity, in contrast to β-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive β-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

Digestive glucosidases in the midgut of Apodiphus amygdali Germar (Hemiptera: Pentatomidae)

Apodiphus amygdali or stink bug of fruit trees is one of the polyphagous species from pentatomid bugs that attack many of fruit trees and ornamental trees. In the current study, activities of α- and β-glucosidases were measured in the midgut of A. amygdali adults. It was found the higher activity of β-glucosidase than α-glucosidase in addition to different enzymatic properties of the enzymes. Optimal pHs for enzymatic activities were found to be 5 and 7 for α- and β-glucosidases, respectively. Values regarding optimal temperatures were obtained at 30?°C for both α- and β-glucosidases. Among ions used on α-glucosidase activity, K⁺ and Ca²⁺ significantly increased enzymatic activity, Na⁺ had no effect, and Cu²⁺, Fe²⁺ and Mg²⁺ had the significant negative effects on the enzyme activity. Ca²⁺ and Fe²⁺ increased β-glucosidase activity in the midgut of A. amygdali, Na⁺ had no effect, and other ions significantly decreased the enzyme activity. Ethylene glycol-bis (β-aminoethylether) N,N,N?,N-tetraacetic acid (EGTA), citric acid, ethylenediamide tetraacetic acid (EDTA) and sodium dodecylsulfate (SDS) significantly decreased α-glucosidase activity but EGTA, triethylenetetramine hexaacetic acid (TTHA), EDTA and SDS decreased β-glucosidase activity in the midgut of A. amygdali. Characterisation of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful for better understanding of enzyme roles in nutritional physiology of insects in addition to reach safe and useful controls of insect pests.     

Construction and characterization of different fusion proteins between cellulases and β-glucosidase to improve glucose production and thermostability

A β-glucosidase from Clostridium cellulovorans (CcBG) was fused with one of three different types of cellulases from Clostridium thermocellum, including a cellulosomal endoglucanase CelD (CtCD), a cellulosomal exoglucanase CBHA (CtCA) and a non-cellulosomal endoglucanase Cel9I (CtC9I). Six bifunctional enzymes were constructed with either β-glucosidase or cellulase in the upstream. CtCD-CcBG showed the favorable specific activities on phosphoric acid swollen cellulose (PASC), an amorphous cellulose, with more glucose production (2 folds) and less cellobiose accumulation (3 folds) when compared with mixture of the single enzymes. Moreover, CtCD-CcBG had significantly improved thermal stability with a melting temperature (T_m) of 10.9 °C higher than that of CcBG (54.5 °C) based on the CD unfolding experiments. This bifunctional enzyme is thus useful in industrial application to convert cellulose to glucose.     

Ectohydrolytic enzyme activities of bacteria associated with Orbicella annularis coral

Ectohydrolytic enzyme activity (EEA) potential of 37 bacterial isolates derived from Orbicella annularis coral and 2 coral pathogens (Vibrio shilonii and V. coralliilyticus) was measured as model to infer the role of bacteria in organic matter processing within coral reef ecosystems. Bacterial cell-specific activities of eight enzyme types were measured after incubation in organic matter enriched and unenriched filtered seawater. Max value of activities of alkaline phosphatase, oleate-lipase, stearate-lipase and proteinase were 769.3, 327.6, 82.9 and 36.7 amol cell⁻¹ h⁻¹, respectively. Chitinase, α-mannosidase, α-glucosidase and β-glucosidase were generally lower by comparison (max 4.7–20.7 amol cell⁻¹ h⁻¹). No “super” isolates (bacteria expressing high levels of all ectohydrolases) were found suggesting a “specialization” among individual bacterial strains. Cumulatively, the 39 isolates tested displayed a broad range of cell-specific enzyme activities in both organic matter conditions. Culture-independent measurement of coral mucus layer EEA in O. annularis off a Panama reef showed comparable EEA patterns and diversity as the isolates. Volume-specific EEAs of all enzymes except alkaline phosphatase were 8–48 times higher in mucus than in surrounding seawater (SSW) samples. However, cell-specific EEAs in mucus were generally lower than in the SSW partly due to more abundant cells in the mucus than in SSW. For field samples, ≥ 85% of proteinase was cell-bound, while lipase was preferentially dissolved (40–96%). In general, the production of dissolved EEAs varied among measurements depending on sample source and enzyme types, suggesting a potential role of ectoenzyme size distribution in linking the whole reef ecosystem. Our findings support that the cumulative ectoenzyme expression (“ectoenzymome”) of the coral microbiome has the potential to maintain the functional resilience of the coral holobiont and response to stress through its contribution to organic matter processing within coral reef ecosystems.

     

Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus

The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg + Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30 °C and it exhibited maximal activity at 25 °C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K_m and k_cat were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium.     

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K_m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.     

A method of purification, identification and characterization of β-glucosidase from Trichoderma koningii AS3.2774

In this study, we used native gradient-polyacrylamide gel electrophoresis and electroelution (NGGEE) to purify enzymatic proteins from Trichoderma koningii AS3.2774. With this method, we purified eight enzymatic proteins and classified them to the cellulase system by comparing secretions of T. koningii in inductive medium and in repressive medium. It resulted in 24-fold β-glucosidase (BG) purification with a recovery rate of 5.5%, and a specific activity of 994.6 IU mg^− 1 protein. The final yield of BG reached 8 μg under purifying procedure of NGGEE. We also identified BG using the enzyme assay with thin-layer chromatography and MALDI-TOFMS. This BG had one subunit with a molecular mass of 69.1 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The hydrolytic activity of the BG had an optimal pH of 5.0, an optimal temperature of 50 °C, an isoelectric point of 5.68 and a K_m for p-nitrophenyl-β-d-glucopyranoside of 2.67 mM. Taken together, we show that NGGEE is a reliable method through which μg grade of active proteins can be purified.     

Microbial activity profiles in Tagus estuary salt marsh sediments

This study provides some results about microbial activity in salt marsh sediments. Microbial activity was determined by profiling extracellular enzyme activities in three Tagus estuary marshes and in two sediments horizons: surface layer (0–2 cm) and depth (8–10 cm). Five enzymatic activities were examined (β-glucosidase, cellulase, alkaline phosphatase, potential nitrification and nitrate reductase). All extracellular enzymatic activities were highest in the surface layer and decreased with depth. β-glucosidase and alkaline phosphatase prevailed both in surface sediments (1150 and 1200 ηmol h⁻¹ g⁻¹, respectively) and in deeper sediments (150 and 200 ηmol h⁻¹ g⁻¹, respectively). Microbial activities differed significantly between salt marshes. The marsh location in the estuary seemed to contribute to these differences: marshes located in the proximity of urbanised and industrial areas had higher microbial activities.     

Secondary metabolite as therapeutic agent from endophytic fungi Alternaria longipes strain VITN14G of mangrove plant Avicennia officinalis

Endophytic fungi, especially from mangrove plants, are rich source of secondary metabolites, which plays a major role in various pharmacological actions preferably in cancer and bacterial infections. To perceive its role in antidiabetic activity we isolated and tested the metabolites derived from a novel strain Alternaria longipes strain VITN14G obtained from mangrove plant Avicennia officinalis. The crude extract was analyzed for antidiabetic activity and subjected to column chromatography. The isolated fractions were screened in vitro for α-glucosidase and α-amylase inhibitory activities. The cytotoxicity of the isolated fractions was studied on L929 cell lines. Following which, the screened fraction 2 was allowed for structure elucidation using gas chromatography-mass spectrometry, one-dimensional, two-dimensional nuclear magnetic resonance spectroscopy, ultraviolet, and Fourier-transform infrared analysis. The binding energies of the isolated fraction 2 with glycolytic enzymes were calculated by molecular docking studies using AutoDock Vina. The isolated fraction 2 identified as 2,4,6-triphenylaniline, showed no significant difference in α-amylase inhibition rates and a significant difference of 10% in α-glucosidase inhibition rates than that of the standard drug acarbose. Further, the cytotoxicity assay of the isolated fraction 2 resulted in a cell viability of 73.96%. Supportingly, in silico studies showed 2,4,6-triphenylaniline to produce a stronger binding affinity toward the glycolytic enzyme targets. The compound 2,4,6-triphenylaniline isolated from A. longipes strain VITN14G exhibited satisfactory antidiabetic activity for type 2 diabetes in vitro, which will further be confirmed by in vivo studies. Successful outcome of the study will result in a natural substitute for existing synthetic antidiabetic drugs.     

Anesthetic effects on the structure and dynamics of the second transmembrane domains of nAChR α4β2

Channel functions of the neuronal α4β2 nicotinic acetylcholine receptor (nAChR), one of the most widely expressed subtypes in the brain, can be inhibited by volatile anesthetics. Our Na⁺ flux experiments confirmed that the second transmembrane domains (TM2) of α4 and β2 in 2:3 stoichiometry, (α4)₂(β2)₃, could form pentameric channels, whereas the α4 TM2 alone could not. The structure, topology, and dynamics of the α4 TM2 and (α4)₂(β2)₃ TM2 in magnetically aligned phospholipid bicelles were investigated using solid-state NMR spectroscopy in the absence and presence of halothane and isoflurane, two clinically used volatile anesthetics. ²H NMR demonstrated that anesthetics increased lipid conformational heterogeneity. Such anesthetic effects on lipids became more profound in the presence of transmembrane proteins. PISEMA experiments on the selectively ¹⁵N-labeled α4 TM2 showed that the TM2 formed transmembrane helices with tilt angles of 12° ± 1° and 16° ± 1° relative to the bicelle normal for the α4 and (α4)₂(β2)₃ samples, respectively. Anesthetics changed the tilt angle of the α4 TM2 from 12° ± 1° to 14° ± 1°, but had only a subtle effect on the tilt angle of the (α4)₂(β2)₃ TM2. A small degree of wobbling motion of the helix axis occurred in the (α4)₂(β2)₃ TM2. In addition, a subset of the (α4)₂(β2)₃ TM2 exhibited counterclockwise rotational motion around the helix axis on a time scale slower than 10^- 4 s in the presence of anesthetics. Both helical tilting and rotational motions have been identified computationally as critical elements for ion channel functions. This study suggested that anesthetics could alter these motions to modulate channel functions.     

Synthesis of α- and β-d-glucopyranosyl triazoles by CuAAC ‘click chemistry’: reactant tolerance, reaction rate, product structure and glucosidase inhibitory properties

Cu^I-catalysed azide alkyne 1,3-dipolar cycloaddition (CuAAC) ‘click chemistry’ was used to assemble a library of 21 α-d- and β-d-glucopyranosyl triazoles, which were assessed as potential glycosidase inhibitors. In the course of this work, different reactivities of isomeric α- and β-glucopyranosyl azides under CuAAC conditions were noted. This difference was further investigated using competition reactions and rationalised on the basis of X-ray crystallographic data, which revealed significant differences in bond lengths within the azido groups of the α- and β-anomers. Structural studies also revealed a preference for perpendicular orientation of the sugar and triazole rings in both the α- and β-glucosyl triazoles in the solid state. The triazole library was assayed for inhibition of sweet almond β-glucosidase (GH1) and yeast α-glucosidase (GH13), which led to the identification of a set of glucosidase inhibitors effective in the 100 μM range. The preference for inhibition of one enzyme over the other proved to be dependent on the anomeric configuration of the inhibitor, as expected.     

A glucose-tolerant β-glucosidase from Prunus domestica seeds: Purification and characterization

A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag⁺ and Hg²⁺. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a K_m of 3.09 mM and a V_max of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a K_m of 1.65 mM and a V_max of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with K_i values of 0.033 and 468 mM, respectively.     

Yeast alpha glucosidase inhibitory and antioxidant activities of six medicinal plants collected in Phalaborwa,South Africa

Recent decades have experienced a sharp increase in the incidence and prevalence of diabetes mellitus. One antidiabetic therapeutic approach is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate-digesting enzymes such as α-amylase and α-glucosidase and α-amylase. The aim of the current study was to screen six medicinal plant species, with alleged antidiabetic properties for α-glucosidase inhibitory activities. Powdered plant materials were extracted with acetone, and tested for ability to inhibit baker's yeast α-glucosidase and α-amylase activities. The largest mass (440 mg from 10 g) of the extract was obtained from Cassia abbreviata, while both Senna italica and Mormordica balsamina yielded the lowest mass of the extracts. Extracts of stem bark of C. abbreviata inhibited baker's yeast α-glucosidase activity with an IC₅₀ of 0.6 mg/ml. This plant species had activity at low concentrations, with 1.0 mg/ml and above resulting in inhibition of over 70%. The other five plant extracts investigated had IC₅₀ values of between 1.8 and 3.0 mg/ml. Senna italica only managed to inhibit the activity of enzyme-glucosidase at high concentrations with an IC₅₀ value of 1.8 mg/ml, while Tinospora fragosa extracts resulted in about 55% inhibition of the activity of the enzyme at a concentration of 3.5 mg/ml, with an estimated IC₅₀ value of 2.8 mg/ml. The bark extract of C. abbreviata was the most active inhibitor of the enzyme, based on the IC₅₀ values (0.6 mg/ml). The bark extract of C. abbreviata contains non-competitive inhibitor(s) of α-glucosidase, reducing V_max value of this enzyme from 5 mM·s^–1 to 1.67 mM·s^–1, while K_m remained unchanged at 1.43 mM for para-nitrophenyl glucopyranoside. Antioxidant activity of the extracts was also investigated. The C. abbreviata extract was more active as an antioxidant than the positive control, trolox. The extracts did not inhibit alphaamylase activity more than about 20% at the highest concentration tested.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Four glycosidases secreted by Collectotrichum lindemuthianum

The fungal plant pathogen, Collectotrichum lindemuthianum, was grown in culture with either galactose, arabinose or pectin as the carbon source resulting in the selective secretion of α-galactosidase, α-arabinofuranosidase and exopolygalacturonase, respectively. Each enzyme has been purified by ion exchange and gel permeation chromatography. In addition, a β-glucosidase was purified from the culture filtrate or arabinose grown fungus. The purified α-galactosidase and α-arabinosidase preparations were found to be essentially free of other carbohydrases while the β-glucosidase and exopolygalacturonase preparations contain contaminating activities.     

Effects of salinity, temperature and individual variability on the reproduction of Eurytemora affinis (Copepoda; Calanoida) from the Seine estuary: A laboratory study

The brackish water copepod Eurytemora affinis is the most abundant copepod species in the low salinity zone (2-15) of the Seine estuary. Despite its ecological importance, little is known about its population dynamics in the Seine. We studied the effects of temperature (10 °C and 15 °C) and salinity (5, 15 and 25) on reproduction under non-limiting food conditions. We used experiments to determine multiple reproductive parameters for E. affinis. In all experiments, we fed E. affinis a mixture of Rhodomonas marina and Isochrysis galbana. Couples of pre-adult females (C5) and adult males were mated until the female extruded a clutch of eggs and then individual females were observed every 6-12 hours until death to determine (a) embryonic development time, (b) inter clutch time and (c) clutch size throughout their adult lifespan. All reproductive parameters were negatively affected by low temperature (10 °C) and by high salinity (25). At 10 °C and a salinity of 25, mortality during the post-embryonic period was extremely high (85%). Differences in all reproductive parameters between salinities 5 and 15 were minimal. From 15 °C to 10 °C mean latency time (time between hatching of eggs and extrusion of new ones) increased from 0.8 to 2.25 days, the mean embryonic development time from 2.2 to 3.2 days and the mean clutch size decreased from 38 to 22 eggs female^- 1. The mean clutch size decreased when females reached a critical age. The hatching success was high (near 95%) under all conditions except at high salinity. Egg production rates showed no significant differences between salinities 5 and 15 and were significantly higher at 15 °C (13 eggs female^- 1 day^- 1 at salinity 5 and 15) than at 10 °C (4 eggs female^- 1 day^- 1). These values at 15 °C were higher compared to those from other populations of E. affinis in estuaries or lakes. The high reproductive potential of E. affinis from the Seine estuary at 15 °C and low salinities explain its high densities in the low salinity zone during spring and early summer.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.