Protein Min Research Articles

Characteristics and activity of SpmoB domain of particulate methane monooxygenase expressed in Escherichia coli BL21 (DE3)

Particulate methane monooxygenase is one of the methane monooxygenases of methanotrophic bacteria. The enzyme can convert methane to methanol in mild conditions. Cupredoxin domain recombinant protein (SpmoB) of particulate methane monooxygenase can oxidize methane to methanol. This protein was expressed in Escherichia coli BL21 (DE3) with Lac operon-based induction. SpmoB protein was isolated and refolded from the E. coli recombinant inclusion body. The pH and temperature dependence of SpmoB activity was also investigated to increase its activity. the SpmoB protein expressed by E. coli BL21 (DE3) is about 39 kDa. The SpmoB inclusion body was solubilized in 8 M urea followed by stepwise dialysis to get the active form SpmoB protein. The specific activity of the refolded protein was 0.46 methanol mg protein-1 min-1, which was higher than that of SpmoB from the previous study. The SpmoB was an acidic protein with the highest methanol production at pH six and a temperature of 30°C, which are higher than full-length pMMO. The SpmoB activity was stable at pH 6 to 8, and the Vmax and Km were 0.380 μM methanol s-1 and 44.27 μM, respectively.

Effect of Seed Priming with Plant Growth Regulators on Growth Parameters, Biochemical Changes of Rice (Oryza sativa L.)

The investigation entitled “Effect of seed priming with plant growth regulators on growth parameters, biochemical changes of rice (Oryza sativa L.)” was conducted during kharif season, 2018 at Student Instructional Farm (SIF) of Acharya Narendra Deva University of Agriculture & Technology, Kumarganj, Ayodhya-224 229 (U.P.) in randomized block design with eight treatments and three replications on rice variety Sarju-52.The treatments were comprised of priming with plant growth regulators of different concentrations viz salicylic acid (50 ppm, 100 ppm and 150 ppm), KNO3 (1%, 1.5% and 2%) along with unprimed control and hydro-priming. Seed priming was done for 12 hours in different treatments and nursery was raised. Twenty one days old seedlings were transplanted. Observations were recorded on various growth parameters at 30, 60, 90 DAT such as leaf area index (LAI), relative water content (RWC), biochemical parameters like chlorophyll content (SPAD Value), catalase activity (EU mg-1 protein min-1), total soluble sugar (mg g-1 dry weight), at harvest stages. All the plant growth regulators viz salicylic acid (50 ppm, 100 ppm and 150 ppm) and KNO3 (1%, 1.5% and 2%) influenced positively on the growth characters, chlorophyll content, & yield content in rice crop. Seed priming of KNO3 @ 2% was found superior followed by salicylic acid 150 ppm over rest of the treatments.

Biochemical Evaluation of Cotton Genotypes using Soluble Protein, Esterase (EST), Peroxidase (POX) And Polyphenol Oxidase (PPO) and their Role in Plant Disease Resistance

Isozyme analysis is a powerful biochemical technique that has numerous applications in plant biology. It has long been used by geneticists to study the population genetics. The isozyme esterase, peroxidase and polyphenol oxidase were standardized for upland cotton (Gossypium hirsutum L.) germplasm lines collected from all over the country. The knowledge of nature and magnitude of genetic diversity present in the germplasm is most important pre-requisite for the success of any breeding program. The thirty-four cotton germplasm lines were screened for prime three isozymes based on quantification assay and qualitative PAGE profiling. Among the material, the genotype AKH – 24 (190.60 mg ml-1), AKH – 053 (189.42 mg ml-1) and VIKAS (184.53 mg ml-1) recorded high protein content, whereas the enzymatic activities of esterase, peroxidase and polyphenol oxidase exhibited remarkable differences along with the protein content. The genotype LRA–5166 exhibited high esterase (462.68 mM mg protein-1 min-1) and peroxidase activity (250.97mM mg protein-1 min-1), while AKH – 24 recorded the maximum polyphenol oxidase activity (131.45 mM mg protein-1min-1). The banding pattern of biochemical markers revealed that the maximum number of bands were recorded in esterase analysis (fifteen) followed by protein (twelve) whereas, only five bands each were detected in peroxidase and polyphenol oxidase analysis indicating limited polymorphism. The Relative Mobility (Rm) values were ranged from 0.083 to 0.883 (protein), 0.100 to 0.971 (esterase), 0.033 to 0.283 (peroxidase) and 0.048 to 0.206 (polyphenol oxidase). The present study demonstrated that cotton genotypes could be differentiated by their quantity and quality through electrophoretic banding profiles. These results could be of practical value for cultivar identification, purity testing along with associated prediction of pest and disease resistance. However, the major constraint is that these biochemical markers do not able to reproduce the similar kind of variation pattern, but can provide strong distinguishing polymorphism each time.

Effect of Plant Growth Regulators on Biochemical Studies of Mustard [Brassica juncea (L.) Czern & Coss.] under Sodic Soil

A field study was conducted to evaluate the “Effect of plant growth regulators on biochemical parameters of mustard [Brassica juncea (L.) Czern & Coss.] under sodic soil.” An experiment was laid out in randomized block design with seven treatments and three replications. The field experiment treatments consist of foliar spray with GA3 (50ppm, 100ppm, 150ppm) and salicylic acid (100ppm, 200ppm, 300ppm) were prepared on weight by volume basis as per desired concentration. Data confined to physiological changes like chlorophyll content, catalase activity were recorded at 40, 60 and 80 DAS. Oil content (%) in mature seeds was recorded at maturity. Significantly higher chlorophyll contents and catalase activity in leaves were estimated with the foliar spray of 100 ppm GA3 (2.51, 5.05, 2.24) chlorophyll content mg g-1 fresh weight and (69.17, 173.80, 171.30) catalase activity EU mg-1 protein min-1 and Significantly higher oil content (%) in mature seeds of mustard were estimated with the foliar application of 100 ppm GA3 (40.67 %) as compared to control. These biochemical parameters superior with foliar application of GA3 100ppm followed by Salicylic acid 200ppm.

Changes in Some Carbon and Nitrogen Metabolism Enzymes in Field-Grown Wheat

Objective: The activities of alanine and aspartate aminotransferases, NADmalate dehydrogenase in the ontogenesis have been determined in leaves of durum wheat genotypes (Barakatli 95, Garagylchyg 2) with contrasting drought tolerance, cultivated under natural soil drought conditions. Methods/ analysis: Enzymatic activities of aspartate and alanine transferases, NADmalate dehydrogenase were determined spectrophotometrically (Ultrospec 3300 pro, Amersham, USA). Measurements were taken at 340 nm for 1 min and the obtained results were expressed as mmol mg-1*protein min-1. The unequal variance two-tailed Student’s t-test was applied for the analysis of the significance of differences between plants cultivated under irrigated and natural drought conditions. P 0.05 was considered statistically significant. We used three samples for each treatment and performed the analysis twice. Findings : In flag leaves of the drought-tolerant Barakatli 95 genotype, the alanine aminotransferase activity increased ~2.7 and ~2.2 fold compared to the drought-sensitive Garagylchyg 2 genotype, under irrigated and natural drought conditions. According to the results, catabolism of amino acids is faster in the drought-sensitive Garagylchyg 2 genotype compared to the droughttolerant Barakatli 95 genotype under stress. Although the activity of all three enzymes studied varies in parallel in the ontogenesis of flag leaves, it is mostly dependent on leaf water content during the day. Novelty/improvement: The obtained data suggest that high enzyme activities in the Barakati 95 genotype play a role in achieving drought tolerance. Keywords: Wheat; flag leaf; natural drought conditions; aspartate aminotransferase; alanine aminotransferase; daytime

Different carbon sources influences the growth and digestive enzyme activity of grey mullet (Mugil cephalus) in biofloc based nursery rearing system

Aim: To assess the impact of different carbon sources on growth performance of grey mullet, Mugil cephalus fry in biofloc based culture system during nursery rearing phase. Methodology: The experimental trial was carried out for 60 days using three carbon sources, cassava, jaggery and barley in triplicate following a Completely Randomized Design. The carbon nitrogen ratio (C:N) in the systems were maintained at 15:1. Grey mullet fry (0.91g) were stocked @ of 1 fry 10 l-1 and were fed with pelleted feed (35% crude protein) @ 4% of fish body weight thrice daily. Results: Significantly higher (P<0.05) growth performance, in terms of Specfic Growth Rate (3.78 % day-1) with lower Feed Conversion Ratio (1.35) were found in barley based biofloc group. Similarly, enhanced digestive enzyme activity in terms of amylase (8.78 Unit mg protein-1 min-1), lipase (2.77 Unit mg protein-1 min-1) and protease (4.22 Unit mg protein-1 min-1) were found in barley based biofloc group. Non- specific immune parameters such as lysozyme, respiratory burst activity and myeloperoxidase activity were significantly higher in barley based treatment group followed by jaggery and cassava based treatment groups. Likewise, stress indicators in serum and antioxidant status in liver were found to be lower in barley based treatment compared to cassava and jaggery based treatment groups. Interpretation: Rearing of Mugil cephalus in biofloc culture system is practicable; however, the carbon source required for biofloc production could be barley, compared to cassava and jaggery, in order to achieve maximum growth and to reduce the stress.

Essential oil yield estimation by Gas chromatography-mass spectrometry (GC-MS) after Methyl jasmonate (MeJA) elicitation in in vitro cultivated tissues of Coriandrum sativum L.

Coriandrum sativum is an important spice plant known for its unique fragrance. Coriander oil is also one of the major essential oils in world global market. The oil yield varies with different coriander varieties; and the content and quality of oil is governed by several factors. In recent times, a variety of technologies have been exploited to improve phyto-compounds including essential oils. In this present study, Methyl jasmonate (MeJA) was amended in medium and the yield of essential oil was measured and compared in different cultivating tissues. The cultured tissues were nonembryogenic callus and embryogenic tissues (induction, proliferation and maturation stages of embryos). MeJA acts as a signaling molecule in accumulating secondary metabolites. Four different MeJA treatments i.e. T1 = 50, T2 = 100, T3 = 150 and T4 = 200μM, along with a control (T0) were used and the yield of coriander essential oil was estimated in different in vitro cultivating tissues by using Gas chromatography-mass spectrometry (GC-MS). The addition of MEJA enriched essential oil yield, maximum oil being in maturation stage of embryos at T3 (150μM). Other added treatments also had varied stimulatory role. The addition of MeJA induced stress as the stress marker enzymes like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) content were high compared to non treated tissue (T0). In T4, the CAT activity was maximum i.e. 5.83 and 6.28mg-1 protein min-1 in Co-1 and RS respectively in matured somatic embryos. The SOD activity was also high at maturation stage of embryos at T4 (5.3mg-1 protein min-1 in RS). The APX activity on the other, was high (3.32mg-1 protein min-1) in induction stage of embryogenesis at T3. The comparative biochemical (sugar, protein and proline) analyses of tissues were performed and presented that had high and low essential oil. MeJA induced stress may help in accumulating essential oils in C. sativum.

Is divalent magnesium cation the best cofactor for bacterial β-galactosidase?

β-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of β-galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight ~95 kDa exhibited specific activity of 137.7 U mg-1 protein with a purification of 11.22-fold and yield 12.42%. The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg+2 as a cofactor of β-galactosidase. In this present investigation, we have checked the kinetic behavior of the purified β-galactosidase in presence of several bivalent metals. Lowest Km with highest substrate (orthonitrophenyl- β-galactoside or ONPG) affinity was measured in presence of Ca2+ (42.45 μM ONPG). However, our results demonstrated that Vmax was maximum in presence of Mn+2 (55.98 μM ONP produced mg-1 protein min-1), followed by Fe=2, Zn+2, Mg+2, Cu+2 and Ca+2. A large number of investigations reported Mg+2 as potential co factor for bgalacosidase. However, β-galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn+2 or Fe2+.

Regulation of Glutamine Synthetase (GS) Activity and Root Morphological Plasticity in the Nitrogen Fixing Azolla- Anabaena Symbiont under Nitrogen Limiting Conditions

The present study was conducted to identify the role of the endosymbiont, Anabaena azollae in fixing atmospheric N2 in the Azolla-Anabaena symbiosis. The Azolla-Anabaena azollae (association) grown in nitrogen-free modified Hoagland’s medium supplemented with 10% (v/v) N2 using an artificial gas mix, resulted in the increase in root number and root length. The GS specific activity of the association was 75 ± 6 nmole γ-glutamate mg protein-1 min-1 and the total N content was lesser in cultures grown in 10% (v/v) N2. Similarly, the artificially produced Anabaena free Azolla (endophyte-free), developed increased root length, root number and reduced GS specific activity (30 ± 10 nmole γ-glutamate mg protein-1 min-1), which on supplementation with 2.5 mM NH4NO3 did not show any change in the root length, root number and GS activity. However, the association cultured in 10% (v/v) N2+2.5 mM NH4NO3, reversed the effect of 10% (v/v) N2 treatment exhibited a GS specific activity of 295 ± 120 nmole γ-glutamate mg protein-1 min-1 and the morphological features were similar to the control plants. The western blot analysis of the GS protein from the association showed two isoforms of GS (GS1 and GS2) of equal abundance under all growth conditions, whereas, one diffused band of intermediate size between GS1 and GS2 was observed in the endophyte-free fern.

Evolution of the digestive enzymes and bacterial changes of the gastrointestinal tract of the Artemia urmiana during growth period

As the digestive enzymes and gastro-intestinal (GI) bacterial community contribute to the health and nutrition of the organism, this study aims to evaluate the ontogeny of bacterial population and digestive enzymes activities in the GI tract of Artemia urmiana from nauplii to the adult stage. Artemia cysts were hatched under standard conditions and stocked at a density of 20 nauplii ml-1 for 15 days. Samplings for growth, bacterial and enzymatic analysis were collected on days 1, 5, 10 and 15 of the experiment. The results indicated that the GI tract was sterile at the time of hatching. Artemia GI tract was active after hatching by protease (0.282 ± 0.001 U mg-1 protein min-1), lipase (0.182 ± 0.001 U mg-1 protein min-1) and amylase (0.295 ± 0.001 U mg-1 protein min-1) secretion and increased during the experiment. In addition, a significant relation (p < 0.05) was observed between Artemia total length and the activity of digestive enzymes (lipase (r2 = 0.98) amylase (r2 = 0.97) and protease (r2 = 0.98). A significant relation (p < 0.05) was observed between total aerobic bacteria and digestive enzyme secretion (lipase, r2 = 0.83; amylase, r2 = 0.66 and protease, r2 = 0.84) too. Such a relation was observed between total Bacillus spp. count and digestive enzyme activities (lipase r2 = 0.82; amylase r2 = 0.79; and protease; r2 = 0.74). These results suggest that in addition to the chemical composition of food, total length, GI bacteria enzyme secretion and the interaction of these factors contribute in digestive enzymes ontogeny during the growth period.

Physio-biochemical evaluation of soybean (Glycine max L.) genotypes exhibiting variable seed coat colour

Eighteen promising soybean genotypes having different seed coat colour i.e. yellow, green, brown and black were examined for physio-biochemical differences. The highest mean crude protein content was recorded in brown (42.86%), followed by yellow coloured seeds. The yellow coloured seeds recorded the highest mean crude fat content (19.86%). The mean crude fibre content was highest in brown seeds (6.36%), followed by black coloured seeds. Mean seed coat lignin (16.60%), trypsin inhibitor (62.81 TIU g-1) in defatted flour and seed coat peroxidase activity (530 hmoles H2O2 decomposed mg-1 protein min-1) was highest in black coloured seeds. The lowest mean per cent mechanical damage was recorded in black coloured genotypes by ferric chloride test and in brown coloured genotypes by sodium hypochlorite test. The highest seed coat proportion, mean mechanical strength and the lowest mean EC was recorded in black coloured seeds. Mean per cent germination, seed vigour index-I as well as seed vigour index-II and hundred seed weight were recorded highest in yellow seed coat coloured soybean genotypes.

Mitigating effects of paclobutrazol on flooding stress damage by shifting biochemical and antioxidant defense mechanisms in mungbean(Vigna radiata L.) at pre-flowering stage

Flooding stress is one of the major obstacles which hamper plant growth and development in various ways. Two screened mungbean genotypes, HUM-16 (flooding tolerant) and HUM-12 (flooding susceptible), were selected to evaluate the effect of paclobutrazol @10ppm and 25ppm along with its combinations through pre-seed soaking and foliar spray at pre-flowering stage. The flooding stress shock was imposed for seven days at pre-flowering stage. Paclobutrazol (PBZ) treatments significantly enhanced chlorophyll, soluble sugar, starch, MSI, APX, and SOD in both HUM-16 and HUM-12 genotypes under flooding stress. Among all the treatments, PBZ @10ppm seed treatment + 10ppm foliar spray revealed higher chlorophyll (2.47 and 1.97 mg g-1 FW), soluble sugar (24.57 and 20.53 mg g-1 FW), MSI (49.16% and 38.67%), APX (16.13 and 6.50 Units mg-1 protein min-1) and SOD (38.00 and 18.30 Units mg-1 protein min-1) in both tolerant and susceptible genotype, respectively, while it minimized the production of MDA (18.30% and 49.00%) and H2O2 (19.05% and 25.06%) in both tolerant and susceptible genotype, respectively, under flooding stress as compared to control. Paclobutrazol treatments, as evident, minimized the flooding stress damage in tolerant genotype as compared to susceptible genotype by enhancing chlorophyll, soluble sugar and antioxidant enzymes activity (APX and SOD) and by reducing the generation of H2O2 and MDA contents under flooding stress.

Changes in enzymatic and nonenzymatic antioxidant defense mechanisms of canola seedlings at different drought stress and nitrogen levels

To evaluate the responses of canola seedlings to different levels of drought stress and nitrogen at different growth stages, a factorial experiment was conducted in a completely randomized design with three replications at the experimental greenhouse of Mohaghegh Ardabili University in 2013. Treatments included three levels of drought stress (30%, 50%, and 70% of FC) and five levels of nitrogen (control, based on soil test results; 25% less than the recommended level; 50% less than the recommended level; 25% more than the recommended level; and 50% more than the recommended level). The recommended level of nitrogen, based on soil test results, was 0.09 g of nitrogen per kilogram of soil. Results showed that drought stress, nitrogen, and their interaction significantly affected the enzymatic activity of antioxidant catalase (CAT), polyphenol oxidase (PPO), and peroxidase (PO) at various stages of growth. Proline was also affected by drought stress and nitrogen. Maximum CAT activity (794.04 OD mg protein min-1) was observed at the 4-6 leaf stage under mild drought stress conditions (50% of FC) and a nitrogen application rate 50% less than the recommended level. Increased rates of nitrogen enhanced the PPO activity at the 4-6 and 6-8 leaf stages. PO had a negative response to increased rates of nitrogen application. The highest rate of increase in proline was at the 8-10 leaf stage: 205% and 207% higher under mild drought stress (50% of FC) and severe drought stress (30% of FC) conditions, respectively, compared to favorable moisture conditions (70% of FC). Increased nitrogen application led to an increase in proline production at all stages of sampling (4-6, 6-8, and 8-10 leaf stages).

Regulation of iron-siderophore uptake in a diazotrophic cyanobacterium Anabaena anabaena 7120

The regulation of 55Fe-siderophore uptake of was studied in a diazotrophic cyanobacterium Anabaena 7120. The uptake up to 20 min was found to be biphasic, a rapid first and steady second phase. A single kinetic system mediated the uptake of iron-siderophore with an apparent Km of 200 µM and a Vmax of 20 nmol iron taken up mg-1 protein min-1. 3-(3,4dichlorophenyl)-1, 1-dimethyl urea (DCMU), 2-n-heptyl-4-hydroxy quinoline (HOQNO), dicyclohexyl carbodiimide (DCCD), carbonyl cyanide p-fluoromethoxy phenyl hydrazone (FCCP) and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) inhibited the iron-siderophore uptake significantly, whereas KCN inhibited marginally. No iron-siderophore uptake was observed under dark-aerobic and dark-anaerobic conditions. The siderophore schizokinen supported the maximum iron-siderophore uptake. However, the siderophore aerobactin and desferal could support only 53 and 3.4% iron-siderophore uptake, respectively as compared to its schizokinen counterpart.

Protective effect of Codium tomentosum alga on SH-SY5Y model of neurotoxicity induced by 6-hydroxydopamine (6 – OHDA)

Event Abstract Back to Event Protective effect of Codium tomentosum alga on SH-SY5Y model of neurotoxicity induced by 6-hydroxydopamine (6 – OHDA) Joana Silva1*, Celso Alves1 and Rui Pedrosa1 1 School of Tourism and Maritime Tecnhology, Portugal Parkinson Disease (PD) is characterized by the loss of dopaminergic cell bodies in the substantia nigra and oxidative stress, mitochondrial dysfunction and apoptosis seem to be involved in the pathogenesis of the disease. The aim of this study was to evaluate the neuroprotective effect of different algae extracts on the cell death induced by by 6-OH-dopamine on a neuronal human cell model (SH- SY5Y). The neuroprotective effect of methanolic and dichloromethane algae extract (Padina pavonica, Sargassum muticum, Saccorhiza polyschides, Codium tomentosum, Ulva compressa) in the presence of 6-OH-DA was assessed using the MTT method. The activity of caspase-3 and the quantification of hydrogen peroxide (extracellular environment and real time production) production were studied in the presence and in the absence of the macroalgae extracts. The highest neuroprotective effect was exhibited by methanolic fractions (1000 µg/ml; 24 hours) of S. muticum (115.80 ± 8.53% of viable cells), S. polyschides (106.51 ± 4.26 of viable cells), P. pavonica (95.98 ± 3.95 of viable cells) and dichloromethane (1000 µg/ml; 24 hours) fraction of C. tomentosum alga (102.22 ± 4.24 of viable cells), when compared with 6-OH-DA condition (100μM; 24 hours) (67.40 ± 3.56 of viable cells). For the extracts that exhibited the highest neuroprotective effect was studied the possible alterations induced in the mechanisms of action previous selected. The dichloromethane fraction of Codium tomentosum alga (1000 µg/ml) highly reduced the 6-OH-DA induced increase of caspase-3 activity, the less quantity of H2O2 in the extracellular environment and less production of H2O2 on real time (2.58 ± 1.77 UAF mg of protein-1 min-1; 0.0 ± 0.005 µM of H2O2, 54.07 ± 6.66 UAF min-1, respectively) when compared with the 6-OH-DA condition at 100μM (4.5 ± 1:35 UAF mg of protein-1 min-1; ± 0.4 0.0019 µM of H2O2; 214.26 ± 8:46 UAF min-1, respectively). These results suggest that Codium tomentosum extracts has capacity to increase the SH-SY5Y viability in the presence of 6-OH-DA by the inhibition of caspase-3 activity and H2O2 production. In conclusion, Codium tomentosum could be a promising source of novel molecules with antioxidant activity. Keywords: algae, Parkinson Disease, Neuroprotection, 6-Hydroxidopamine, antioxidant activity Conference: IMMR | International Meeting on Marine Research 2014, Peniche, Portugal, 10 Jul - 11 Jul, 2014. Presentation Type: Poster Presentation Topic: BLUE BIOTECH Citation: Silva J, Alves C and Pedrosa R (2014). Protective effect of Codium tomentosum alga on SH-SY5Y model of neurotoxicity induced by 6-hydroxydopamine (6 – OHDA). Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2014. doi: 10.3389/conf.fmars.2014.02.00071 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 May 2014; Published Online: 18 Jul 2014. * Correspondence: Dr. Joana Silva, School of Tourism and Maritime Tecnhology, Peniche, Portugal, Joana.rita.ms@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joana Silva Celso Alves Rui Pedrosa Google Joana Silva Celso Alves Rui Pedrosa Google Scholar Joana Silva Celso Alves Rui Pedrosa PubMed Joana Silva Celso Alves Rui Pedrosa Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Pesticide detoxifying mechanism in field population of Spodoptera litura (Lepidoptera: noctuidae) from South India

Thearmy worm Spodoptera litura(fabricius) is one of the most damaging pests of ground nut and castor in India. Wedetermine the susceptibility of S. litura reared on a castor leaves, tothree insecticides (Temephos, Dichlorvos, and lambda-cyhalothrin) underlaboratory conditions, associated withthe enzyme activities of acetylcholinesterase (AChE), carboxylesterase (CarE),glutathione S-transferase (GST) and glutathione S- reductase (GSH) in thislarvae. Among treatments Dichlorvos showed high mortality then lambdacyhalothrin and Temephos in whole body of the insect. The AChE activities of Spodoptera litura treated with lambdacyhalothrin, Temephos at 10ppm were almost 1.5 fold high. The CarE activity at10ppm was almost 3 fold high. The GSH and GST activity were low then control(0.2 ± 0.4, 1.5 µM mg protein-1 min-1). The present studysuggests that esterase and acetylcholine esterase may play a role in detoxificationof synthetic pyrethroid and organophosphates in Spodoptera litura from South India.

Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4.

BackgroundFusarium wilt is an economically devastating disease that affects banana production. Although Cavendish banana cultivars are resistant to Fusarium oxysporum f.sp. cubense race 1 (FOC1) and maitain banana production after Gros Michel was destructed by race 1, a new race race 4 (FOC4) was found to infect Cavendish.ResultsAn exopolygalacturonase (PGC2) was isolated and purified from the supernatant of the plant pathogen Fusarium oxysporum f.sp. cubense race 4 (FOC4). PGC2 had an apparent Mr of 63 kDa by SDS-PAGE and 51.7 kDa by mass spectrometry. The enzyme was N-glycosylated. PGC2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. To obtain adequate amounts of protein for functional studies between the PGC2 proteins of two races of the pathogen, pgc2 genes encoding PGC2 from race 4 (FOC4) and race 1 (FOC1), both 1395 bp in length and encoding 465 amino acids with a predicted amino-terminal signal sequence of 18 residues, were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC2 products, r-FOC1-PGC2 and r-FOC4-PGC2, were expressed and purified as active extracellular proteins. Optimal PGC2 activity was observed at 50°C and pH 5. The Km and Vmax values of purified r-FOC1-PGC2 were 0.43 mg.mL-1 and 94.34 units mg protein-1 min-1, respectively. The Km and Vmax values of purified r-FOC4-PGC2 were 0.48 mg.mL-1 and 95.24 units mg protein-1 min-1, respectively. Both recombinant PGC2 proteins could induce tissue maceration and necrosis in banana plants.ConclusionsCollectively, these results suggest that PGC2 is the first exoPG reported from the pathogen FOC, and we have shown that fully functional PGC2 can be produced in the P. pastoris expression system.

Characterization of Thermostable α-amylase from Thermophilic andAlkaliphilic Bacillus sp. Isolate DM-15

Bacillus sp. DM-15 was isolated from Ciftehan Thermal Spring, Turkey produced thermostable α-amylase at 55°C at pH 9. Analysis of the enzyme for molecular mass and amylolytic activity carried out by SDS-Starch-PAGE electrophoresis revealed a single band with high molecular mass 126 kDa. After extraction, the enzyme remained stable in a range of temperature and pH between 40-100°C and 4.5-10, respectively. The optimum enzyme activity was displayed at 60°C and pH 5.5-6.0. α-amylase production by thermophilic Bacillus sp. strain cultivated in liquid media reached a maximum at 12 h with levels of 27.5 μmol mg-1 protein min-1. The enzyme was stable for 15 min at 60°C while 24% of the original activity was lost at 100°C. Enzyme activity was increased in the presence of 5 mM CaCl2, Na2SO3 and KCl (105%) and inhibited in the presence of 5 mM EDTA, CuSO4, FeSO4, SDS (1%), Urea (8 M), Co up to 98, 79, 57, 36, 33 and 10%, respectively. The DM-15 α-amylase may be suitable for in liquefaction of starch in detergent and textile industries and in other industrial applications.

&lt;p&gt;&lt;strong&gt;Physiological Aspects of &lt;em&gt;Racomitrium crispipilum&lt;/em&gt; Taylor A. (Jaeger) during Dry Season in Páramo de Chingaza, Colombia&lt;/strong&gt;&lt;/p&gt;

In order to study the response to water deficit of the moss Racomitrium crispipilum (Taylor) A. Jaeger, a bryophyte typical of open sites and expose to water shortage, measurements of its soluble protein and proline contents, and enzymatic activity of catalase and peroxidase were made. Sampling was done in the Páramo de Chingaza, eastern mountain range of Colombia, in the zone of Lagunas de Buitrago during the dry season. We determined that R. crispipilum diminish its relative water content (RWC) from 50% to 4% of its dry mass (DM) (i.e. to &lt;10% of its capacity), without losing the ability to synthesize proteins in the rehydration. Declining RWC was associated with declining soluble protein content and increased activities of catalase and peroxidase. Enzyme activities peacked in the hours at which R. crispipilum exhibited its minimum RWC (21.02 U mg protein-1 min-1 for catalase and 172.66 for peroxidase). Proline was found to be high during the hours of maximum RWC and the earliest hours of diminishing RWC, between 60 and 87.07 mg g dry mass-1 and declines to 18.98 during the afternoon. The results reported in this study give the first evidence that the activities of catalase and peroxidase increase in this species in response to conditions of water deficit. This is likely to be an important component in the mechanism by which this moss tolerates drought periods.

Glucan, water dikinase phosphorylates crystalline maltodextrins and thereby initiates solubilization

Starch phosphorylation by glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step in the breakdown of native starch particles, but the underlying mechanisms have remained obscure. In this paper, the initial reactions of starch degradation were analyzed using crystallized maltodextrins as model carbohydrates. As revealed by X-ray diffraction analysis, the crystallized maltodextrins represent the B-type starch allomorph. Recombinant GWD phosphorylated crystalline maltodextrins with a high specific activity (55-60 nmol mg-1 protein min-1), but exhibited very little activity with the same maltodextrins that had been solubilized by heat treatment. Recombinant phosphoglucan, water dikinase (PWD; EC 2.7.9.5) utilized the crystalline maltodextrins only when pre-phosphorylated by GWD. Phosphorylation of crystalline maltodextrins, as catalyzed by GWD, initiated solubilization of neutral as well as phosphorylated glucans. In both the insoluble and the soluble state, mono-, di- and triphosphorylated alpha-glucans were observed, with wide and overlapping ranges of degree of polymerization. Thus, the substrate specificity of the GWD is defined by the physical arrangement of alpha-glucans rather than by structural parameters, such as the distribution of branching points or degree of polymerization. Unlike GWD and PWD, recombinant beta-amylase isozyme 3 (BAM3), which has been shown to be essential for plastidial starch degradation, preferentially degraded soluble maltodextrins rather than crystallized glucans. In summary, two conclusions were reached. Firstly, carbohydrate targets of GWD are primarily defined by the molecular order of glucan helices. Secondly, GWD-catalyzed phosphorylation mediates the phase transition of glucans from a highly ordered to a less ordered and hydrated state.