Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and solid-state cultures and recovery of hydrophobin proteins

The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-state (SSC) cultures. The maximum CO₂ production rate in SmC (V_max = 11.7 mg CO₂ L_g⁻¹ day⁻¹) was three times lower than in SSC (V_max = 40.4 mg CO₂ L_g⁻¹ day⁻¹). The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein⁻¹, but in SmC, this protein was not detected. The PLHYD showed a critical micelle concentration of 0.45 mg mL⁻¹. In addition, the PLHYD modified the hydrophobicity of Teflon from 130.1 ± 2° to 47 ± 2°, forming porous structures with some filaments <1 μm and globular aggregates <0.25 μm diameter. The interfacial studies of this PLHYD could be the basis for the use of the protein to modify surfaces and to stabilize compounds in emulsions.     

Diel activities of antioxidant enzymes,photosynthetic pigments and malondialdehyde content in stationary-phase cells of Tetraselmis gracilis (Prasinophyceae)

The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as photosynthetic pigment contents and free malondialdehyde (MDA), were determined in senescent batch cultures of Tetraselmis gracilis (Kylin) Butcher, under a cyclic light regime. A 2.6-fold increase in SOD activity (from 53 to 137 U mg⁻¹ protein) was observed in the light phase, contrasting with a 9-fold increase in CAT (from 1 to 9 μmol H₂O₂ min⁻¹ mg⁻¹ protein) and a 1.7-fold increase in APX (from 3 to 5 μmol ascorbate min⁻¹ mg⁻¹ protein) activities, both enzymes peaking in the dark phase. The β-carotene and lycopene content did not vary significantly with the light–dark cycle. The Chl a, Chl b, lutein, zeaxanthin, violaxanthin and neoxanthin pigments exhibited the highest values in the first half (3–6 h) of the light phase, followed by a declining trend and a plateau or a slight increase 3 h from the beginning of the dark phase onwards. The highest values for prasinoxanthin were observed in the second half of the dark phase and the first half of the light phase. None of the pigments displayed any discernible cyclic trend. The possibility of the xanthophyll cycle occurring during senescence is discussed in light of the high value (∼0.9) obtained for the zeaxanthin/(zeaxanthin + violaxanthin) ratio. The free MDA content was enhanced during the experimental period, which may be an indicator of oxidative stress in aging cell cultures. Our results indicated the occurrence of an imbalance between the production of reactive oxygen species and the antioxidant defense in stationary T. gracilis cells.     

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters K_M and V_max for free and immobilized cutinase were found to be 0.55 mM and 1410 μmol min⁻¹ mg⁻¹ protein, 2.99 mM and 996 μmol min⁻¹ mg⁻¹ protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.     

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.     

Activity of hydroperoxide lyase under aqueous and micro-aqueous conditions

Hydroperoxide lyases (HPL E.C. 4.1.2.) are part of the lipoxygenase pathway in plants and catalyze the conversion of fatty acid hydroperoxides into oxo acids and short chain aldehydes. These aldehydes have desirable properties for the food and agricultural industry. HPL activity can be modulated by salts and surfactants, but the mechanisms governing the modulation are not fully understood. Recombinant HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. To investigate solubility issues of the two different substrates, an aqueous and a two-phase micro-aqueous reaction medium was used. The highest HPL activity (8.7 μmol min⁻¹ mg⁻¹) was achieved under aqueous conditions with high salt (1.5 M) and low surfactant (0%, v/v) concentrations and LA-OOH as a substrate. Maximal activity (2.4 μmol min⁻¹ mg⁻¹) under micro-aqueous conditions was achieved with high salt (1.5 M) and high surfactant (0.01%, v/v) concentrations and LS-OOH as a substrate. A significant interaction between salt and surfactant as well as salt and substrate could be identified and a hypothesis for the interaction phenomena is presented.     

Chromate reduction by resting cells of Achromobacter sp. Ch-1 under aerobic conditions

Chromate reduction was carried out by resting cells of Achromobacter sp. Ch-1 with lactate as electron donor under aerobic conditions. The reduction activity of the samples supplemented with lactate was two times as those without lactate. The reduction rate was influenced by initial pH and lactate concentration. Under the optimal conditions, pH 9.0 and 4000 mg l⁻¹ lactate supplement, reduction rate was 5.45 mg l⁻¹ min⁻¹. The reduction rate decreased with increasing of Cr(VI) concentrations and increased with cell densities proportionally. The maximum reduction limit of Ch-1 cells was obtained at 2107 mg l⁻¹ of Cr(VI).     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL⁻¹) and ultrafiltration, followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 °C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba²⁺ increased the enzyme activity by 96% and its thermal stability by 30%, besides increasing its stability at acid pH. The apparent K_m with apple pectin as substrate was 1.82 mg mL⁻¹ and the V_max was 81 μmol min⁻¹ mg⁻¹.     

GLP-1(32–36)amide,a novel pentapeptide cleavage product of GLP-1, modulates whole body glucose metabolism in dogs

We have previously demonstrated in human subjects who under euglycemic clamp conditions GLP-1(9–36)amide infusions inhibit endogenous glucose production without substantial insulinotropic effects. An earlier report indicates that GLP-1(9–36)amide is cleaved to a nonapeptide, GLP-1(28–36)amide and a pentapeptide GLP-1(32–36)amide (LVKGR amide). Here we study the effects of the pentapeptide on whole body glucose disposal during hyperglycemic clamp studies. Five dogs underwent indwelling catheterizations. Following recovery, the dogs underwent a 180 min hyperglycemic clamp (basal glucose +98 mg/dl) in a cross-over design. Saline or pentapeptide (30 pmol kg⁻¹ min⁻¹) was infused during the last 120 min after commencement of the hyperglycemic clamp in a primed continuous manner. During the last 30 min of the pentapeptide infusion, glucose utilization (M) significantly increased to 21.4 ± 2.9 mg kg⁻¹ min⁻¹compared to M of 14.3 ± 1.1 mg kg⁻¹ min⁻¹ during the saline infusion (P = 0.026, paired t-test; P = 0.062, Mann–Whitney U test). During this interval, no significant differences in insulin (26.6 ± 3.2 vs. 23.7 ± 2.5 μU/ml, P = NS) or glucagon secretion (34.0 ± 2.1 vs. 31.7 ± 1.8 pg/ml, P = NS) were observed. These findings demonstrate that under hyperglycemic clamp studies the pentapeptide modulates glucose metabolism by a stimulation of whole-body glucose disposal. Further, the findings suggest that the metabolic benefits previously observed during GLP-1(9–36)amide infusions in humans might be due, at least in part, to the metabolic effects of the pentapeptide that is cleaved from the pro-peptide, GLP-1(9–36)amide in the circulation.     

Chemical compositions and characteristics of organic compounds in propolis from Yemen

Propolis is a gummy material made by honeybees for protecting their hives from bacteria and fungi. The main objective of this study is to determine the chemical compositions and concentrations of organic compounds in the extractable organic matter (EOM) of propolis samples collected from four different regions in Yemen. The propolis samples were extracted with a mixture of dichloromethane and methanol and analyzed by gas chromatography–mass spectrometry (GC–MS). The results showed that the total extract yields ranged from 34% to 67% (mean = 55.5 ± 12.4%). The major compounds were triterpenoids (254 ± 188 mg g⁻¹, mainly α-, β-amyryl and dammaradienyl acetates), n-alkenes (145 ± 89 mg g⁻¹), n-alkanes (65 ± 29 mg g⁻¹), n-alkanoic acids (40 ± 26 mg g⁻¹), long chain wax esters (38 ± 25 mg g⁻¹), n-alkanols (8 ± 3 mg g⁻¹) and methyl n-alkanoates (6 ± 4 mg g⁻¹). The variation in the propolis chemical compositions is apparently related to the different plant sources. The compounds of these propolis samples indicate that they are potential sources of natural bio-active compounds for biological and pharmacological applications.     

The role of nest surface temperatures and the brain in influencing ant metabolic rates

Thermal limits of insects can be influenced by recent thermal history: here we used thermolimit respirometry to determine metabolic rate responses and thermal limits of the dominant meat ant, Iridomyrmex purpureus. Firstly, we tested the hypothesis that nest surface temperatures have a pervasive influence on thermal limits. Metabolic rates and activity of freshly field collected individuals were measured continuously while ramping temperatures from 44 °C to 62 °C at 0.25 °C/minute. At all the stages of thermolimit respirometry, metabolic rates were independent of nest surface temperatures, and CT_max did not differ between ants collected from nest with different surface temperatures. Secondly, we tested the effect of brain control on upper thermal limits of meat ants via ant decapitation experiments (‘headedness’). Decapitated ants exhibited similar upper critical temperature (CT_max) results to living ants (Decapitated 50.3±1.2 °C: Living 50.1±1.8 °C). Throughout the temperature ramping process, ‘headedness’ had a significant effect on metabolic rate in total (Decapitated V̇CO₂ 140±30 µl CO₂ mg⁻¹ min⁻¹: Living V̇CO₂ 250±50 CO₂ mg⁻¹ min⁻¹), as well as at temperatures below and above CT_max. At high temperatures (>44 °C) pre- CT_max the relationships between I. purpureus CT_max values and mass specific metabolic rates for living ants exhibited a negative slope whilst decapitated ants exhibited a positive slope. The decapitated ants also had a significantly higher Q₁₀:25–35 °C when compared to living ants (1.91±0.43 vs. 1.29±0.35). Our findings suggest that physiological responses of ants may be able to cope with increasing surface temperatures, as shown by metabolic rates across the thermolimit continuum, making them physiologically resilient to a rapidly changing climate. We also demonstrate that the brain plays a role in respiration, but critical thermal limits are independent of respiration levels.     

Effects of intraspecific competition on growth and photosynthesis of Atriplex prostrata

We investigated the effect of intraspecific competition on growth parameters and photosynthesis of the salt marsh species Atriplex prostrata Boucher in order to distinguish the effects of density-dependent growth inhibition from salt stress. High plant density caused a reduction of 30% in height, 82% in stem dry mass, 80% in leaf dry mass, and 95% in root dry mass. High density also induced a pronounced 72% reduction in leaf area, 29% decrease in length of mature internodes and 50% decline in net photosynthetic rate. The alteration of net photosynthesis paralleled growth inhibition, decreasing from 7.6 ± 0.9 μmol CO₂ m⁻² s⁻¹ at low density to 3.5 ± 0.4 μmol CO₂ m⁻² s⁻¹ at high density, indicating growth inhibition caused by intraspecific competition is mainly due to a decline in net photosynthesis rate. Plants grown at high density also exhibited a reduction in stomatal conductance from 0.7 ± 0.1 mol H₂O m⁻² s⁻¹ at low density to 0.3 ± 0.1 mol H₂O m⁻² s⁻¹ at high density and a reduction in transpiration rate from 6.0 ± 0.3 mmol H₂O m⁻² s⁻¹ at low density to 4.3 ± 0.3 mmol H₂O m⁻² s⁻¹ at high density. Biomass production was inhibited by an increase in plant density, which reduced the rate of photosynthesis, stomatal conductance and leaf area of plants.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Repeated freezing induces oxidative stress and reduces survival in the freeze-tolerant goldenrod gall fly,Eurosta solidaginis

Freeze tolerant insects must not only survive extracellular ice formation but also the generation of reactive oxygen species (ROS) during oxygen reperfusion upon thawing. Furthermore, diurnal fluctuations in temperature place temperate insects at risk of being exposed to multiple freeze–thaw cycles, yet few studies have examined metrics of survival and oxidative stress in freeze-tolerant insects subjected to successive freezing events. To address this, we assessed survival in larvae of the goldenrod gall fly Eurosta solidaginis, after being subjected to 0, 5, 10, 20, or 30 diurnally repeated cold exposures (RCE) to −18 °C or a single freeze to −18 °C for 20 days. In addition, we measured indicators of oxidative stress, levels of cryoprotectants, and total aqueous antioxidant capacity in animals exposed to the above treatments at 8, 32, or 80 h after their final thaw. Repeated freezing and thawing, rather than time spent frozen, reduced survival as only 30% of larvae subjected to 20 or 30 RCE successfully pupated, compared to those subjected to fewer RCE or a single 20 d freeze, of which 82% pupated. RCE had little effect on the concentration of the cryoprotectant glycerol (4.26 ± 0.66 μg glycerol·ng protein⁻¹ for all treatments and time points) or sorbitol (18.8 ± 2.9 μg sorbitol·mg protein⁻¹ for all treatments and time points); however, sorbitol concentrations were more than twofold higher than controls (16.3 ± 2.2 μg sorbitol·mg protein⁻¹) initially after a thaw in larvae subjected to a single extended freeze, but levels returned to values similar to controls at 80 h after thaw. Thawing likely produced ROS as total aqueous antioxidant capacities peaked at 1.8-fold higher than controls (14.7 ± 1.6 mmol trolox·ng protein⁻¹) in animals exposed to 5, 10, or 20 RCE. By contrast, aqueous antioxidant capacities were similar to controls in larvae subjected to 30 RCE or the single 20 d freeze regardless of time post final thaw, indicating these animals may have had an impaired ability to produce primary antioxidants. Larvae lacking an antioxidant response also had elevated levels of oxidized proteins, nearly twice that of controls (21.8 ± 3.2 mmol chloramine-T·mg protein⁻¹). Repeated freezing also lead to substantial oxidative damage to lipids that was independent of aqueous antioxidant capacity; peroxides were, on average, 5.6-fold higher in larvae subjected to 10, 20 or 30 RCE compared to controls (29.1 ± 7.3 mmol TMOP·μg protein⁻¹). These data suggest that oxidative stress due to repeated freeze–thaw cycles reduces the capacity of E. solidaginis larvae to survive freezing.     

Differential contributions of ankle plantarflexors during submaximal isometric muscle action: A PET and EMG study

The objective of this study was to investigate the relative contributions of superficial and deep ankle plantarflexors during repetitive submaximal isometric contractions using surface electromyography (SEMG) and positron emission tomography (PET). Myoelectric signals were obtained from twelve healthy volunteers (27.3 ± 4.2 yrs). A tracer ([¹⁸F]-FDG) was injected during the exercise and PET scanning was done immediately afterwards. The examined muscles included soleus (Sol), medial gastrocnemius (MG), lateral gastrocnemius (LG), and flexor hallucis longus (FHL). It was found that isometric maximal voluntary contraction (MVC) force, muscle glucose uptake (GU) rate, and SEMG of various plantarflexors were comparable bilaterally. In terms of %EMG MVC, FHL and MG displayed the highest activity (∼34%), while LG (∼21%) had the lowest activity. Cumulative SEMG from all parts of the triceps surae (TS) muscle accounted for ∼70% of the combined EMG signal of all four plantarflexors. As for GU, the highest quantity was observed in MG (2.4 ± 0.8 μmol * 100 g⁻¹ * min⁻¹), whereas FHL (1.8 ± 0.6 μmol * 100 g⁻¹ * min⁻¹) had the lowest uptake. Cumulative GU of TS constituted nearly 80% of the combined GU. The findings of this study provide valuable reference for studies where individual muscle contributions are estimated using models and simulations.     

Chemical modifications by ionic liquid-inspired cations improve the activity and the stability of formate dehydrogenase in [MMIm][Me2PO4]

The formate dehydrogenase (FDH, EC: 1.2. 1.2) from Candida boidinii was found to be inactivated and unstable in the presence of high concentration (>50%) of the water soluble dimethylimidazolium dimethyl phosphate ([MMIm][Me₂PO₄]) ionic liquid. In order to circumvent this problem, the enzyme was chemically modified by cations usually present in ionic liquids: cholinium (1), hydroxyethyl-methylimidazolium (2) and hydroxypropyl-methylimidazolium (3) cations were activated with carbonyldiimidazole before being reacted with the FDH leading to a heterogeneous population of 6–7 biocatalysts. FDH modified by (1) or (3) led to 3–9 modifications while FDH modified by (2) led to 6 proteins presenting 7–12 grafted cations. Specific activity of the modified enzymes was decreased by a 2.5–3-fold factor (0.10–0.15 μmol min⁻¹ mg⁻¹) compared to the non-modified FDH (0.33 μmol min⁻¹ mg⁻¹) when assayed in carbonate buffer (pH 9.7, 25 mM). After modification, the FDH still present 0.06 μmol min⁻¹ mg⁻¹ in 70% [MMIm][Me₂PO₄] (v:v) (30–45% of their activity in aqueous buffer) while the native enzyme is inactive at this ionic liquid concentration, proving the efficiency of this strategy. The half-life of the modified enzyme is also increased by a 5-fold factor after modification by (1) (t_1/2 of 9 days) and by a 3-fold factor after modification by (2) or (3) (t_1/2 of 6 and 5 days respectively) in aqueous solution. When stored in 37.5% [MMIm][Me₂PO₄] (v:v), both modified and unmodified FDH have an increased half-life (t_1/2 of 6–9 days). This grafting strategy is found to be good methods to mimic and study the stabilizing effect of ionic liquids on enzymes.     

Stress responses and specific metal exclusion on mine soils based on germination and growth studies by Australian golden wattle

We reported the Australian golden wattle as a copper stabilizer in abandoned copper mine soils earlier. Here we investigate to confirm this plant’s suitability to grow on metal contaminated mine soils based on stress indication. The seeds of Acacia pycnantha collected from mining area were germinated after heat and no heat treatment on two types of irrigation. The daily irrigated and heat treated seeds gave up to 85% germination on sandy soil. The A. pycnantha was grown under greenhouse condition in six different soils collected from abandoned copper mine at Kapunda in South Australia. Among the six soil samples, soil-1 with the highest copper concentration produced 2.05 mmol g⁻¹ tissue of proline. Proline expression was prominent in more saline soils (1, 5 and 6) having electrical conductivity (EC) 1184, 1364 and 1256 μS, respectively. Chlorophyll a, b and carotenoid levels in plants showed a gradually decreasing trend in all the soils as experiment progressed. The plants grown on soil sample-1, containing 4083 ± 103 mg kg⁻¹ of copper resulted in 18 ± 2 mg kg⁻¹ accumulation in its leaf. The calcium accumulation was significant up to 11648 ± 1209 mg kg⁻¹ in leaf. Although pore water samples showed higher Cu concentration in soils, an increased mobility of arsenic and lead was observed in all the soil samples. Our experiment points out the need for proper monitoring of revegetation processes to avoid revegetation and reclamation failure.     

Horseradish peroxidase production from Spodoptera frugiperda larvae: A simple and inexpensive method

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 10⁶ cells or 3.0 ± 0.1 mg l⁻¹ with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg⁻¹ larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The K_m and V_max were 12.1 ± 1.7 mM and 2673 ± 113 U mg⁻¹, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications.     

A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay

In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of alpha, mu or pi-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the pi-class with specific activities of 0.062, +/-0.0015 micromol min(-1) mg(-1) and 0.0019, +/-0.00064 micromol min(-1) mg(-1) for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, +/-0.003 micromol min(-1) mg(-1) and 0.002, +/-0.0005 micromol min(-1) mg(-1), respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the pi-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 micromol min(-1) mg(-1) and a negligible BSP activity (相似文献