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1.
Electron. j. biotechnol ; 31: 10-16, Jan. 2018. graf, tab, ilust
Article in English | LILACS | ID: biblio-1022030

ABSTRACT

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.


Subject(s)
Paracoccus/enzymology , Chlorpyrifos/metabolism , Esterases/metabolism , Organophosphates/metabolism , Biodegradation, Environmental , Catalysis , Mutagenesis , Cloning, Molecular , Sequence Analysis , Esterases/isolation & purification , Esterases/genetics , Hydrolysis , Metals/metabolism
2.
Braz. j. microbiol ; 47(1): 143-149, Jan.-Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-775118

ABSTRACT

Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.


Subject(s)
Aspergillus/enzymology , Lipase/metabolism , Cations, Divalent/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Enzyme Activators/analysis , Enzyme Inhibitors/analysis , Hydrogen-Ion Concentration , Lipase/chemistry , Lipase/isolation & purification , Molecular Weight , Mercaptoethanol/metabolism , Metals/metabolism , Temperature
3.
Electron. j. biotechnol ; 18(5): 333-337, Sept. 2015. ilus, graf, tab
Article in English | LILACS | ID: lil-764017

ABSTRACT

Background Ethylene is capable of promoting seed germination in some plant species. Mobilization of metals such as Fe, Cu, Mn, and Zn in mature seeds takes place when seeds are germinating. However, whether ethylene is involved in the regulation of soybean seed germination and metal element mobilization during early seed germination stage remains unknown. In the present study, seeds were treated with ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) and ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and double distilled H2O (ddH(2)0) treatment was used as control. Ethylene emission, ACC synthase (ACS) expression, ACS enzyme activity and Ca, Zn, Mn, Cu and Fe content in hypocotyls were qualified to analyze the relationship between ethylene and mobilization of these elements. Results The results showed that ACS expression, ACS enzyme activity and ethylene emission peaked at 1 and 7 d after sowing. AVG inhibited ethylene production, promoted the hypocotyls length, ACS expression and its activity, concentrations of total and HCl-extractable Zn, and HCl-extractable Fe in hypocotyls, while ACC caused opposite effects. AVG and ACC treatment had no significantly effects on total and HCl-extractable Ca, Cu and HCl-extractable Mn. Total Mn concentration was promoted by AVG at 1, 3, and 5 d significantly, while ACC treatment tended to have no significantly effects on Mn concentration. Conclusion These findings suggested that ethylene is at least partly involved in the regulation of soybean seed germination. Remobilization of Zn and Fe may be negatively regulated by ethylene.


Subject(s)
Seeds , Soybeans , Germination , Ethylenes , Lyases/metabolism , Metals/metabolism , Zinc/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Iron/metabolism
5.
Biol. Res ; 48: 1-8, 2015. ilus, graf, tab
Article in English | LILACS | ID: biblio-950788

ABSTRACT

BACKGROUND: The development of clean or novel alternative energy has become a global trend that will shape the future of energy. In the present study, 3 microbial strains with different oxygen requirements, including Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, were used to construct a hydrogen production system that was composed of a mixed aerobic-facultative anaerobic-anaerobic consortium. The effects of metal ions, organic acids and carbohydrate substrates on this system were analyzed and compared using electrochemical and kinetic assays. It was then tested using small-scale experiments to evaluate its ability to convert starch in 5 L of organic wastewater into hydrogen. For the one-step biohydrogen production experiment, H1 medium (nutrient broth and potato dextrose broth) was mixed directly with GAM broth to generate H2 medium (H1 medium and GAM broth). Finally, Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D of three species microbial co-culture to produce hydrogen under anaerobic conditions. For the two-step biohydrogen production experiment, the H1 medium, after cultured the microbial strains Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, was centrifuged to remove the microbial cells and then mixed with GAM broth (H2 medium). Afterward, the bacterial strain Clostridium acetobutylicum ATCC 824 was inoculated into the H2 medium to produce hydrogen by anaerobic fermentation. RESULTS: The experimental results demonstrated that the optimum conditions for the small-scale fermentative hydrogen production system were at pH 7.0, 35°C, a mixed medium, including H1 medium and H2 medium with 0.50 mol/L ferrous chloride, 0.50 mol/L magnesium sulfate, 0.50 mol/L potassium chloride, 1% w/v citric acid, 5% w/v fructose and 5% w/v glucose. The overall hydrogen production efficiency in the shake flask fermentation group was 33.7 mL/h-1.L-1, and those the two-step and the one-step processes of the small-scale fermentative hydrogen production system were 41.2 mLVh-1.L-1 and 35.1 mL/h-1.L-1, respectively. CONCLUSION: Therefore, the results indicate that the hydrogen production efficiency of the two-step process is higher than that of the one-step process.


Subject(s)
Fermentation/physiology , Microbial Consortia/physiology , Hydrogen/metabolism , Industrial Waste , Starch/metabolism , Time Factors , Kluyveromyces/metabolism , Carboxylic Acids/metabolism , Feasibility Studies , Enterobacter cloacae/metabolism , Coculture Techniques , Clostridium acetobutylicum/metabolism , Electric Conductivity , Microbial Interactions/physiology , Renewable Energy , Waste Water/analysis , Hydrogen/analysis , Ions/metabolism , Metals/metabolism
6.
Braz. j. microbiol ; 45(1): 127-134, 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-709465

ABSTRACT

Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. The enzyme was purified to homogeneity using (NH4)2SO4 precipitation, DEAE-Sepharose and Sephadex G-100 column chromatography. The final enzyme activity achieved 81UmL-1, specific activity 78 U mg-1 with purification fold of 130 and recovery 1.2% of the crude enzyme. SDS-PAGE indicated that the pure enzyme have a molecular mass of approximately 42 kDa. The optimum pH was between 4-5 and the optimum temperature was 25 ºC. The pure MnP activity was enhanced by Mn2+,Cu2+,Ca2+ and K+ and inhibited by Hg+2 and Cd+2.H2O2 at 5 mM enhanced MnP activity while at 10 mM inhibited it significantly. The MnP-cDNA encoding gene was sequenced and determined (GenBank accession no. AB698450.1). The MnP-cDNA was found to consist of 497 bp in an Open Reading Frame (ORF) encoding 165 amino acids. MnP from P. ostreatus could detoxify aflatoxin B1 (AFB1) depending on enzyme concentration and incubation period. The highest detoxification power (90%) was observed after 48 h incubation at 1.5 U mL-1 enzyme activities.


Subject(s)
Aflatoxins/metabolism , Peroxidases/isolation & purification , Peroxidases/metabolism , Pleurotus/enzymology , Biotransformation , Chemical Precipitation , Chromatography, Gel , Chromatography, Ion Exchange , DNA, Fungal/chemistry , DNA, Fungal/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Activators/metabolism , Enzyme Inhibitors/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Metals/metabolism , Open Reading Frames , Peroxidases/chemistry , Sequence Analysis, DNA , Temperature
7.
Braz. j. microbiol ; 44(4): 1305-1314, Oct.-Dec. 2013. ilus, tab
Article in English | LILACS | ID: lil-705271

ABSTRACT

An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45°C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca2+, Mg2+ and Na+ tend to increase the enzyme activity, whereas, Fe2+ and Mn2+ ions resulted in discreet decrease in the activity. Divalent cations Ca+2 and Mg+2 seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca+2 (5 mM) the optimum temperature shifted from 45°C to 55°C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of Km and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL-1 and 5000 μg.mL-1.min-1, respectively.


Subject(s)
Lipase/metabolism , Pseudomonas aeruginosa/enzymology , Chemical Precipitation , Chromatography, Gel , Chromatography, Ion Exchange , Cations/metabolism , Enzyme Activators , Enzyme Stability , Enzyme Inhibitors/metabolism , Hydrogen-Ion Concentration , Kinetics , Lipase/chemistry , Lipase/isolation & purification , Metals/metabolism , Oxidants/metabolism , Pseudomonas aeruginosa/genetics , Solvents/metabolism , Temperature
8.
Braz. j. microbiol ; 44(2): 529-537, 2013. graf, tab
Article in English | LILACS | ID: lil-688598

ABSTRACT

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.


Subject(s)
Bacillus/enzymology , Bacillus/growth & development , Bacterial Proteins/metabolism , Carboxymethylcellulose Sodium/metabolism , Endopeptidases/metabolism , Ficus/microbiology , Industrial Waste , Bacterial Proteins/chemistry , Carboxymethylcellulose Sodium/chemistry , Enzyme Stability , Endopeptidases/chemistry , Enzyme Activators/metabolism , Hydrogen-Ion Concentration , Metals/metabolism , Temperature , Time Factors
9.
Indian J Biochem Biophys ; 2012 Oct; 49(5): 295-305
Article in English | IMSEAR | ID: sea-143551

ABSTRACT

Hydroxyl radicals (HO·) are derived in Fenton reaction with ferrous salt and H2O2 in acid medium, and at neutral pH, metal-oxyl radicals (M-O·) predominate. Evidence is accumulating that M-O· radicals are also active in oxidation reactions, in addition to metal-oxo (M=O) now shown in many publications. Reactivity of these radicals gives selective oxidized products useful in cellular activities, in contrast to purported indiscriminate cell damage by hydroxyl radicals. Reactions with vanadium compounds, such as diperoxovanadate, peroxo-bridged mixed valency divanadate, vanadium-oxyl radical, tetravalent vanadyl and decavanadate illustrates selective gain in oxidative capacity of oxo- and oxyl- species. Occurrence of ESR signals typical of hydroxyl radicals is demonstrated in cell homogenates and tissue perfusates treated with spin trap agents. It is known for a long time lipid peroxides are formed in tissue microsomal systems exclusively in presence of salts of iron, among many metals tested. Oxygen and a reducing agent, ascorbate (non-enzymic) or NADPH (enzymic) are required to produce 'ferryl', the chelated Fe=O active form (possibly Fe-O· and Fe-O-O-Fe ?) for the crucial step of H-atom abstraction. Yet literature is replete with unsupported affirmations that hydroxyl radicals initiate lipid peroxidation, an unexplained fixation of mindset. The best-known ·OH generator, a mixture of ferrous salt and H2O2, does not promote lipid peroxidation, nor do the many hydroxyl radical quenching agents stop it. The availability of oxo and oxyl-radical forms with transition metals, and also with non metals, P, S, N and V, calls for expansion of vision beyond superoxide and hydroxyl radicals and explore functions of multiple oxygen radicals for their biological relevance.


Subject(s)
Hydroxyl Radical , Lipid Peroxidation , Antioxidants/metabolism , Iron/metabolism , Metals/metabolism , Oxidants/metabolism , Vanadates
10.
J Environ Biol ; 2007 Apr; 28(2 Suppl): 367-76
Article in English | IMSEAR | ID: sea-113927

ABSTRACT

Phytoremediation is an emerging technology, which uses plants and their associated rhizospheric microorganisms to remove pollutants from contaminated sites. This plant based technology has gained acceptance in the past ten years as a cheap, efficient and environment friendly technology especially for removing toxic metals. Plant based technologies for metal decontamination are extraction, volatilization, stabilization and rhizofiltration. Various soil and plant factors such as soil's physical and chemical properties, plant and microbial exudates, metal bioavailability plant's ability to uptake, accumulate, translocate, sequester and detoxify metal amounts for phytoremediation efficiency. Use of transgenics to enhance phytoremediation potential seems promising. Despite several advantages, phytoremediation has not yet become a commercially available technology Progress in the field is hindered by lack of understanding of complex interactions in the rhizosphere and plant based mechanisms which allow metal translocation and accumulation in plants. The review concludes with suggestions for future phytoremediation research.


Subject(s)
Biodegradation, Environmental , Chelating Agents/metabolism , Metals/metabolism , Plants/genetics , Plants, Genetically Modified/metabolism , Soil Microbiology , Soil Pollutants/metabolism
11.
Iranian Journal of Environmental Health Science and Engineering. 2006; 3 (2): 91-96
in English | IMEMR | ID: emr-164210

ABSTRACT

Investigation of the effectiveness of simultaneous adsorption and biodegradation [SAB] process over individual processes by using microbes Rhizopus oryzae and Stemphylium loti with granular activated carbon [GAC] as adsorbent was carried out. The maximum removal efficiency of cyanide had been achieved by biodegradation alone was 83% by R. oryzae, while it was 90% by S. loti at initial pH of 5.6 and 7.2 respectively and at initial CN[-] concentration of 150 mg/L. In the combined process efficiency of R. oryzae closer to S. loti [95.3% and 98.6% respectively]


Subject(s)
Water Pollutants, Chemical/metabolism , Adsorption , Biodegradation, Environmental , Metals/metabolism , Charcoal
12.
Biol. Res ; 39(1): 79-85, 2006. ilus, tab
Article in English | LILACS | ID: lil-430700

ABSTRACT

DMT1 _ Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 _ transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMT1 also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases about 50x over endogenous expression and about 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb Co Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.


Subject(s)
Animals , Humans , Rats , Cation Transport Proteins/metabolism , Metals/metabolism , RNA, Small Interfering , Biological Transport , Cation Transport Proteins/genetics
13.
J Environ Biol ; 2005 Jan; 26(1): 13-20
Article in English | IMSEAR | ID: sea-113770

ABSTRACT

Landfills are still the most widely used solid waste disposal method used across the world. Leachate generated from landfill areas exerts environmental risks mostly on surface and groundwater, with its high pollutant content, most notably metals, which cause an unbearable lower water quality. During dumping or after the capacity of the landfill has been reached, a decontamination and remediation program should be taken for the area. This study was conducted to assess the capacity and efficiency of Pennisetum clandestinum, a prostrate perennial plant, to accumulate chromium (Cr), copper (Cu), iron (Fe), nickel (Ni), zinc (Zn) and lead (Pb). Leachate, taken from the Sofulu Landfill Site, was given to Pennisetum clandestinum for 180 days, in 3 dilution sets as 1/1, 1/2 and 1/4, in batch configuration. An additional control set was also installed for comparison. Results showed that, even though the metal content of soil had risen, plants accumulated 2 to 8.5 times higher concentrations than the control set. It is important to see, the plant showed almost no stress symptoms even if the set was fed by pure leachate. Pennisetum clandestinum was observed to accumulate metals mostly in the upper bodies, excluding Fe and Cu. 76% of accumulated Cr, 85% of Ni, 66% of Zn and 100% of Pb was observed to accumulate in above-ground parts, where only 20% of Cu and 4% of Fe was accumulated. Due to the high pollution tolerance of Pennisetum clandestinum, makes this plant suitable for decontamination and remediation of landfill sites.


Subject(s)
Biodegradation, Environmental , Metals/metabolism , Plants/metabolism , Poaceae/metabolism , Refuse Disposal
14.
Asian Pac J Allergy Immunol ; 2003 Dec; 21(4): 259-67
Article in English | IMSEAR | ID: sea-37082

ABSTRACT

Gene fusion technique was successfully applied as a potential approach to create a metal-binding site to assist one-step purification of green fluorescent protein (GFP). The chimeric GFP carrying hexapolyhistidine (H6GFPuv) was purified to homogeneous protein via the Immobilized Metal Affinity Chromatography charged with zinc ions. Removal of metal tagger could readily be performed by using enterokinase enzyme. Engineering of the hexahistidine and enterokinase cleavage sites (DDDDK) onto the chimeric protein did not significantly affect the fluorescent property and the binding avidity to Burkholderia pseudomallei protease of a chimeric protease-binding GFP (H6PBGFPuv). This concludes that engineering of repetitive histidine regions onto interested target protein along with the enterokinase cleavage sites will ease the complication of protein purification.


Subject(s)
Binding Sites , Burkholderia pseudomallei/enzymology , Chromatography, Affinity/methods , Electrophoresis, Polyacrylamide Gel , Enteropeptidase/metabolism , Genetic Engineering , Green Fluorescent Proteins/chemistry , Histidine/metabolism , Metals/metabolism , Peptide Hydrolases/metabolism , Recombinant Fusion Proteins/chemistry , Zinc/metabolism
15.
J Environ Biol ; 2003 Oct; 24(4): 437-44
Article in English | IMSEAR | ID: sea-113270

ABSTRACT

In this study, the possible utilization of removed shoots and plant parts of banana as compost after fruit harvest were investigated. Three doses (15-30-45 kg plan(-1)) of the compost prepared from the clone of Dwarf Cavendish banana were compared with Farmyard manure (50 kg plant(-1), Mineral fertilizers (180 g N + 150 g P + 335 g K plant(-1)) and Farmyard manure + Mineral fertilizers (25 kg FM + 180 g N + 150 g P + 335 g K plant(-1)) which determined positive effects on the nutrient contents of banana leaves. The banana plants were grown under a heated glasshouse and in a soil with physical and chemical properties suitable for banana growing. The contents of N, P, K and Mg in compost and in farmyard manure were found to be similar. Nitrogen, phosphorus and potassium contents of leaves in all applications except control, and Ca, Mg, Fe, Zn, Mn, Cu contents in all applications were determined between optimum levels of reference values. There were positive correlations among some nutrient contents of leaves, growth, yield and fruit quality characteristics. Farmyard manure, Farmyard manure + Mineral fertilizers and 45 kg plant(-1) of compost increased the nutrient contents of banana leaves. According to obtained results, 45 kg plant(-1) of compost was determined more suitable in terms of economical production and organic farming than the other fertiliser types.


Subject(s)
Agriculture , Metals/metabolism , Musa/chemistry , Nitrogen/metabolism , Nutritive Value , Phosphorus/metabolism , Plant Structures/chemistry , Plant Leaves/metabolism , Potassium/metabolism , Soil
16.
Indian J Biochem Biophys ; 2001 Oct; 38(5): 335-41
Article in English | IMSEAR | ID: sea-27071

ABSTRACT

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.


Subject(s)
Binding Sites , Chromatography, DEAE-Cellulose , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Germination , Hot Temperature , Hydrogen-Ion Concentration , Isocitrate Dehydrogenase/chemistry , Metals/metabolism , Molecular Weight , Peas/enzymology , Seeds/enzymology
17.
Indian J Exp Biol ; 2000 Nov; 38(11): 1143-6
Article in English | IMSEAR | ID: sea-57153

ABSTRACT

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.


Subject(s)
Aeromonas/enzymology , Animals , Asparaginase/metabolism , Bivalvia/microbiology , Hot Temperature , Hydrogen-Ion Concentration , Metals/metabolism
18.
Indian J Biochem Biophys ; 1999 Dec; 36(6): 415-21
Article in English | IMSEAR | ID: sea-26552

ABSTRACT

From a study of the decay of the pH difference across vesicular membranes (delta pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M+ = K+, the rate of delta pH decay by H+/M+ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M+ = Na+) depending on whether the exchange ionophore chosen is monensin or nigericin. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anaesthetic (i) translocation rate constants increase, (ii) the concentrations of the M+ bound ionophores increase at the expense of H+ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigericin-H (Nig-H) for M+ = K+ whereas they are monensin-H (Mon-H) and nigericin-Na (Nig-Na) for M+ = Na+. Possible anaesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B), an associated increase in the membrane hydration (and membrane dielectric constant). An analysis of the consequent changes in the various transport step shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation of the observed changes in the delta pH decay rates. (b), Changes in the rates of fast equilibria at the interface contribute to changes in KH and KM. (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the delta pH decay data confirms the validity of the transport scheme used. In our experiments delta pH across the vesicular membrane was created by pH jump coming from a temperature jump.


Subject(s)
Anesthetics/pharmacology , Ion Transport , Membrane Lipids/metabolism , Metals/metabolism , Monensin/pharmacology , Nigericin/pharmacology , Phospholipids/metabolism , Protons
19.
Indian J Exp Biol ; 1999 Oct; 37(10): 941-51
Article in English | IMSEAR | ID: sea-63136

ABSTRACT

Attachment of Candida spp. to host tissues and plastic surfaces is the first and a crucial step that initiates colonization by yeast cells and subsequent development of disseminated fungal infection. These infections are associated with high degree of morbidity, mortality and extra cost. Modern trends have focused not only on how best to treat but also on how to prevent Candida infections. To achieve this goal, the factors that influence the adherence of Candida spp. to biological and non biological surfaces have been studied. C. albicans adheres at a degree higher than that of the other Candida spp. and C. tropicalis adheres to a lesser extent. This may reflect the higher pathogenicity of C. albicans compared to the other Candida spp. Germinated C. albicans cells adhere to host tissue more readily than do yeast-phase. Sugars play an important role in the adherence of Candida spp. Overall, galactose was found to promote the adherence of Candida spp. to host tissues and plastic surfaces more than any other mono or disaccharide. Amino sugars on the other hand inhibit the adherence of the yeast cells. Divalent ions such as Ca2+ and Mg2+ promote the adherence of Candida spp. more than monovalent ions. Candida spp. express on their surface receptors, which interact with a wide variety of host proteins including fibrinogen, fibronectin, lamanin, and type I and IV collagen thus binding Candida spp. To glycoproteinaceous conditioning film at the blood-polymer interface. Coaggregation of Candida spp. with other bacteria promotes colonization of yeast cells to oral biofilm, host tissues, and to surfaces of the indwelling vascular catheters. These factors form the basis for the interference with the adherence of Candida spp.


Subject(s)
Bacterial Adhesion/physiology , Candida/pathogenicity , Carbohydrate Metabolism , Humans , Hydrogen-Ion Concentration , Metals/metabolism , Microscopy, Electron, Scanning , Plastics , Protein Binding , Surface Properties , Temperature
20.
Rev. microbiol ; 30(3): 177-90, jul.-set. 1999. ilus, tab, graf
Article in Portuguese, English | LILACS | ID: lil-253771

ABSTRACT

Biocorrosion processes at metal surface are associated with microorganisms, or the products of their metabolic activities including enzymes, exopolymers, organic and inorganic acids, as well as volatile compounds such as ammonia or hydrogen sulfide. These can affect catholic and/or anodic reactions, thus altering electrochemistry at the biofilm/metal interface. Various mechanisms of biocorrosion, reflecting the variety of physiological activities carried out by different types of microorganisms, are identified and recent insights into these mechanisms reviewed. Many investigations have centered on the microbially-influenced corrosion of ferrous and copper alloys and particular microorganisms of interest have been the sulfate-reducing bacteria and metal (especially manganese)-depositing bacteria. The importance of microbial consortia and the role of extracellular polymeric substances in biocorrosion are emphasized. The contribution to the study of biocorrosion of modern analytical techniques, such as atomic force microscopy, Auger electron, X-ray photoelectron and Mössbauer spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy and microsensors, is discussed


Subject(s)
Metals/chemistry , Steel/chemistry , Copper/chemistry , Copper/metabolism , Corrosion , Metals/metabolism
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