Enzymatic Degradation of Monocrotophos by Extracellular Fungal OP Hydrolases

The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO₄ precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml^?1) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K _deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day^?1 and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.     

Extraction and Detection of Pesticides by Cholinesterase Inhibition in a Two‐Phase System: a Strategy to Avoid Heavy Metal Interference

Abstract

A novel method of extraction has been developed to avoid the presence of heavy metals during the measurement of pesticides based on acetylcholinesterase (AchE) inhibition. Heavy metals have been in fact demonstrated in this article to interfere when the assay is performed by using the classic spectrophotometric Ellman's method. We present the results obtained with an assay system using two different phases, one organic and the other aqueous, in which the pesticide and the enzyme are, respectively, solubilized. In a first step, the concentration of the substrate acetylthiocholine (1 mM), of the enzyme (7 mU mL^?1), and the reaction time (20 min) for measurement of enzyme activity were optimized in aqueous solution. Next, the effect of an organic phase on the enzyme activity was studied by the addition of various solvents with the activity being evaluated after 10 min of mixing. It was found that by using hexane, the enzyme retained almost 100% of its activity, and this solvent was chosen for further development of the pesticide assay. Hexane was spiked with different concentrations of pesticides and then added to the enzyme aqueous phase. The pesticides were shown to be able to inhibit the enzyme by interaction at the interface between the two solutions. The degree of inhibition obtained with increasing amounts of pesticide was evaluated. A 50% inhibition was observed for a paraoxon solution of 9×10^?7 M.     

Genome Annotation and Validation of Keratin-Hydrolyzing Proteolytic Enzymes from <Emphasis Type="Italic">Serratia marcescens</Emphasis> EGD-HP20

Stabilization and utilization of poultry waste demand efficient biodegradation either by mixture of enzymes or by microbial system that can produce different types of protein-hydrolyzing enzymes. For utilization of this keratinous biomass, in the present study, genome was sequenced and annotated for a bacterium having multiple enzymatic options for hydrolysis of different soluble and insoluble protein fractions of poultry waste. Among the soluble protein substrates, optimum production of enzyme and soluble protein was observed in case of casein, whereas among the insoluble protein substrates, maximum production of enzyme was achieved when broken nails were used. Conditions for enhanced enzyme activity with concurrent degradation of keratin-rich poultry feather waste to protein-rich hydrolysate were optimized for different growth parameters. The bacterium grew well and highest protease production occurred in 144 h at mesophilic temperature (30 °C) and alkaline condition (pH 8–10) with enzyme activities of 134 and 168 U/mL, respectively.     

Poly(allylamine hydrochloride) Functionalized Multiwalled Carbon Nanotube Modified Carbon Paste Electrode as Acetylcholinesterase Biosensor Transducer

In this work, a sensitive, practical and reliable acetylthiocholine (ATCh) biosensor based on poly(allylamine hydrochloride) functionalized multiwalled carbon nanotube (PAH/MWCNT) was fabricated and used for pesticide detection. As far as we know, this is the first work that constitutes the usage of PAH and MWCNT for ATCh biosensor. The developed system was characterized by using scanning electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The influence of parameters such as enzyme amount and pH were examined and a linearity between 5×10^?5 M?2.0×10^?3 M for ATCh was obtained. The proposed biosensor was applied for a model pesticide, monocrotophos, detection. The analytical curve showed an excellent linearity in the monocrotophos concentration range of 1–25 pg/mL with an incubation time of 5 min. Limit of detection and limit of quantification values were calculated as 0.88 and 2.9 pg/mL, respectively. The system was also applied for detection of monocrotophos in grape, tomatoe, tap and mineralized water samples and promising recovery values were obtained.     

A New and Highly Sensitive Spectrophotometric Determination of Monocrotophos in Environmental,Agricultural and Biological Samples

A new and highly sensitive spectophotometric method is developed for the determination of parts per million levels of widely used organophosphorus pesticide monocrotophos. The method is based on alkaline hydrolysis of monocrotophos to N‐methylacetoacetamide followed by coupling with diazotized p‐amino acetophenone in alkaline medium. The absorption maxima of the reddish‐violet coloured compound formed is measured at 560 nm. Beer's law is obeyed over the concentration range of 1.2 to 6.8 μg in a final solution volume of 25 mL. The molar absorptivity and Sandell's sensitivity were found to be 7.1 × 10⁵ (±100) L mole^?1 cm^?1 and 0.008 μg cm^?2, respectively. The standard deviation and relative standard deviation were found to be ± 0.005 and 2.05%, respectively. The method is simple, sensitive and free from interferences of other pesticides and diverse ions. The method has been satisfactorily applied to the determination of monocrotophos in environmental, agricultural and biological samples.     

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

Proteases are some of the most important industrial enzymes, and one of their main applications is for the production of cheese in the dairy industry. Due to a shortage of animal rennet, microbial coagulant proteases are being sought. In this work, the production of microbial rennet from Thermomucor indicae-seudaticae N31 was studied in submerged fermentation. The best enzyme production was obtained in a fermentation medium containing 4 % wheat bran as the substrate in 0.3 % saline solution, incubated for 72 h at 45 °C and 150 rpm. The value of the milk clotting activity (MCA) was 60.5 U/mL, and the ratio to proteolytic activity (MCA/PA) was 510. The crude enzyme showed optimum pH at 5.5 and two peaks of optimum temperature (MCA at 65 °C and PA at 60 °C). The MCA was stable in the pH range 4.0–4.5 for 24 h and up to 55 °C for 1 h. It was stable during storage at different temperatures (?20 to 25 °C) for 10 weeks. Based on these results, we conclude that microbial rennet from T. indicae-seudaticae N31 produced by submerged fermentation showed good prospects of replacing traditional rennet.     

Effect of Lead Contamination on Soil Microbial Activity Measured by Microcalorimetry

Microcalorimetry was used to investigate the microbial activity in three types of soil (orchard soil, crop soil, forest soil) in Wuhan, China, and to evaluate the influence of different concentrations of lead (Pb²⁺) on soil microbial activity. The experimental results revealed that due to different physical and chemical characteristics of the soils, soil microbial activity in three soil samples were in a descending sequence: orchards soil, crops soil, forest soil. Six levels of Pb viz. 0, 10, 20, 40, 80, 160 μg·g⁻¹ were applied in these soils, and the results showed that an increase of the amount of Pb²⁺ is associated with a decrease in microbial activity in the soils due to the toxic effect of Pb²⁺. In order to gain further insight of the sequential change of microorganisms, determination of colony forming units (CFU) was performed to provide a negative linear correlation between the heat effect and the respective number of microorganisms in the system.     

Application of INAA for phyto-accumulation study of selenium by chickpea plant

The phyto-accumulation efficacy of selenium (Se) from soil by chickpea plant is reported. Chickpea plants were grown in soil having different concentrations (1–4 mg kg^?1) of Se. Samples of soil and different parts of chickpea plants in Se rich soil were analyzed for determination of Se concentrations by instrumental neutron activation analysis (INAA). Samples were irradiated in self-serve facility of CIRUS reactor, BARC, Mumbai at a neutron flux of the order of 10¹³ cm^?2 s^?1. The gamma activity at 264.7 keV of ⁷⁵Se (119.8 d) was measured using a 45% relative efficiency HPGe detector coupled to MCA. Dependence of Se distribution in soil and plants on its spiking concentration was evaluated in this work. The Se concentrations determined in plant parts grown in control soil and in soil spiked with Se (4 mg kg^?1) are in the range of 0.6–0.8 and 65–68 mg kg^?1 respectively.     

Levels and effects of natural radionuclides in soil samples of Garhwal Himalaya

Distribution of natural radionuclide gives significant parameter to assess the presence of gamma radioactivity and its radiological effect in our environment. Natural radionuclides are present in the form of ²²⁶Ra, ²³²Th and ⁴⁰K in soil, rocks, water, air, and building materials. Distribution of natural radionuclides depends on the type of minerals present in the soil and rocks. For this purpose gamma spectrometer is used as tool for finding the concentration of these radionuclides. The activity concentration of naturally occurring radionuclides ²²⁶Ra, ²³²Th and ⁴⁰K in these soil samples were found to vary from of 8 ± 1 Bq/kg to 50 ± 10 Bq/kg with an average 20 Bq/kg, 7 ± 1–88 ± 16 Bq/kg with an Average 26 Bq/kg and 115 ± 18–885 ± 132 Bq/kg with an average 329 Bq/kg, respectively. In this paper, we are presenting the radiological effect due to distribution of natural radionuclide present in soil of Garhwal Himalaya.     

Biophysical aspects of lysozyme adduct with monocrotophos

The present study on in vitro formation and characterization of lysozyme adduct with monocrotophos (MP) evaluates the potential of lysozyme to be used as a sensitive biomarker to monitor exposure levels to the commonly used organophosphorus pesticide monocrotophos. Crystallization of lysozyme protein adduct with monocrotophos was also undertaken to understand the adduct formation mechanism at a molecular level. The binding of organophosphorus pesticides to lysozyme is one of the key steps in their mutagenicity. The formation and structural characterization of lysozyme adduct with monocrotophos was done using MALDI-TOFMS, fluorescence, UV/Vis spectroscopy, circular dichroism, and X-ray diffraction studies. We report the crystal structure of lysozyme adduct with monocrotophos at 1.9 Å. It crystallized in the P4₃ space group with two monomers in one asymmetric unit having one molecule of monocrotophos bound to each protein chain. The results proved that the fluorescence quenching of lysozyme by monocrotophos is due to binding of monocrotophos with a tryptophan residue of lysozyme. Monocrotophos interacts most strongly with the Trp-108 and Asp-52 of lysozyme. The interactions of the monocrotophos molecule with the lysozyme suggest the formation of a stable adduct. In addition, the alteration of lysozyme secondary structure in the presence of monocrotophos was confirmed by circular dichroism and fluorescence inhibition of lysozyme by increasing monocrotophos and UV/Vis spectrophotometry. The formation of lysozyme adduct with monocrotophos was confirmed by MALDI-TOFMS. Figure

Crystal Structure of lysozyme adduct with monocrotophos (MP) [ PDB ID 4TUN) and Ligplots shows the monocrotophos bonding distances and interactions with amino acid residues in lysozyme     

Transesterification of Waste Cooking Oil by an Organic Solvent-Tolerant Alkaline Lipase from Streptomyces sp. CS273

The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0?～?9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.     

Adsorption, desorption and activities of acid phosphatase on various colloidal particles from an Ultisol

Adsorption, desorption and activity of acid phosphatase on various soil colloidal particles and pure clay minerals were studied. Higher adsorption amounts and low percentage of desorption of acid phosphatase were found on fine soil clays (<0.2 μm). Electrostatic force and ligand exchange are the major driving forces that are involved in the adsorption of enzymes on soil clays. More enzyme molecules were adsorbed on soil clays in the presence of organic components. However, enzymes on organic clays were more easily released. One-third of the enzyme on goethite was adsorbed via ligand exchange process. Some other interactions, such as van der Waals force, hydrophobic force and hydrogen bonding may be more important in the adsorption of enzyme on kaolinite and the enzyme in this system cannot be easily removed. Coarse clays (0.2–2 μm) and inorganic soil clays had higher affinities for enzyme molecules than fine clays and organic clays, respectively. The activity of enzyme bound on soil clays was inhibited and the thermal stability was increased in the presence of organic matter. Data obtained in this study are helpful for a better understanding of the interactions of enzymes with inorganic and organic constituents in soil and associated environments.     

Concentration of arsenic in soil samples collected around the monazite processing facility, Thailand

Arsenic (As) in soil is a contaminant originated from human activities including pesticide use, mining and ore processing operations. In this work, As concentration in soil samples collected around the monazite processing facility, Pathum Thani, Thailand, was investigated. The collections of 24 soil samples were collected from the monazite processing area and 7 soil samples were collected from the control area without the processing activity of the same facility. Soils were digested with the mixture of HNO₃, HClO₄ and HF using a microwave digester. Inductively coupled plasma mass spectrometer (ICP-MS) equipped with an octopole reaction system (ORS) was used to determine the concentration of As in soils after the acid digestion. JB-3 (igneous rock) was the standard reference material used to check the accuracy of the method. It was found that the analytical results showed good agreement with the certified values. As concentration in soils collected from the monazite processing area ranged from 3.85 to 36.01 mg kg^?1 with the mean of 13.06 mg kg^?1. The concentration of As higher than the US EPA cancer soil screening level (22 mg kg^?1) was observed for only one sample. The control area showed As concentration varied from 9.59 to 14.19 mg kg^?1 with the mean of 11.97 mg kg^?1. The obtained results from this work were compared with the contaminated soil data of Amphoe Ron Phibun, Nakhon Si Thammarat, Thailand.     

Short-Time Effects of Pure and Formulated Herbicides on Soil Microbial Activity and Biomass

The short-time of six pure herbicides (atrazine, terbuthylazine, rimsulfuron, primisulfuron-methyl, glyphosate and gluphosinate-ammonium) with respect to the corresponding commercial formulations on microbial activity and biomass of sandy loam soil were investigated. Application rates were: agricultural rate, 20 and 200 µg a.i. g m 1 soil. Application at normal agricultural rates did not lead to significant effects on soil microbial activity, whereas soil microbial activity was markedly stimulated when pure and commercial formulations of the six herbicides were applied at 20 µg a.i. g m 1 soil. The addition of 200 µg a.i. g m 1 soil of four pure herbicides (atrazine, terbuthylazine, rimsulfuron, primisulfuron-methjyl) led to a significant decrease of soil microbial activity. Commercial formulations characterized by a higher relative a.i. concentration (atrazine and primisulfuron-methyl) approximately determined the same decreasing effect of the pure compound, whereas herbicide formulations with a lower relative a.i. concentration (terbuthylazine and rimsulfuron) produced a significant increase in soil microbial activity.     

The Effect of Iron and Copper as an Essential Nutrient on Mitochondrial Electron Transport System and Lipid Peroxidation in Trichoderma harzianum

Iron and copper are essential nutrients for all living organisms as cofactors of many enzymes and play important roles in electron transport system (ETS) enzymes which have heme and iron–sulfur centers. In the present study, ETS enzymes, namely, succinate dehydrogenase (SDH) and cytochrome c oxidase (COX), activities as well as adenine nucleotides and lipid peroxidation (LPO) levels of eukaryotic model Trichoderma harzianum grown in varied concentrations of iron (0–20 mg/l) and copper (0–25 mg/l) mediums have been examined. SDH and COX activities increased up to 10 mg/l of iron. COX and SDH activities increased significantly up to 15 and 1 mg/l of copper, respectively. ATP and ADP levels showed a positive correlation with SDH activity with respect to iron–copper concentrations. The trends of AMP were similar with those of ATP and ADP for iron concentrations, while AMP levels elevated until 5 mg/l of copper. As an indicative marker of membrane damage, LPO levels increased with iron and copper concentration. In conclusion, iron and copper concentrations are of critical importance on activities of the ETS enzymes besides adenine nucleotides and LPO levels by maintenance of this metal homeostasis.     

Estimation of enzymatic,microbial, and chemical properties in Brown soil by microcalorimetry

The study was conducted with the objective to assess soil enzymatic, microbial, and chemical properties by customary methods and results obtained by conventional methods, corroborated with microcalorimetry. The experiment was laid out in a randomized complete block design with ten treatments in triplicates. The RS and GM were used at three rates (0, 5, and 25 mg g^?1 soil, respectively). The soils were maintained at two water levels 25 % (W1) and 200 % (W2) of soil water-holding capacity. All soil enzymatic, microbial, and chemical properties were measured by standard methods. The incorporation of GM and RS, especially at high rates and water levels, 25 % (W1) and 200 % (W2) significantly (p < 0.05) affected the soil enzymatic, microbial, and chemical properties compared to controls. The microcalorimetric parameters P _max and k were positively correlated, whereas t _max negatively linked with the results of enzymatic, microbial, and chemical properties at p < 0.01. Conversely, Q elucidated non-significant correlation (p < 0.05) to urease (0.248), neutral phosphatase (0.281), dehydrogenase (0.291), MBC (0.283), MBP (0.277), DOC (0.269), DON (0.190), SOM (0.284), and pH (0.047). Our results suggested that calorimetric parameters P _max, t _max, and k are highly sensitive and could be used as indices of soil enzymatic, microbial, and chemical properties, while Q is an indigent indicator.     

Simultaneous Saccharification of Inulin and Starch Using Commercial Glucoamylase and the Subsequent Bioconversion to High Titer Sorbitol and Gluconic Acid

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.     

Assessment of 210Po deposition in moss species and soil around coal-fired power plant

In the present study, the depositions of ²¹⁰Po were assessed in the surface soil and some mosses species found in the area around coal fired power plant using radiochemical deposition and alpha spectrometry counting system. The purposes of the study were to determine activity concentrations of ²¹⁰Po in mosses and surface soil collected around coal-fired power plant in relation to trace the potential source of ²¹⁰Po and to identify most suitable moss species as a bio-indicator for ²¹⁰Po deposition. In this study, different species of mosses, Orthodontium imfractum, Campylopus serratus and Leucobryum aduncum were collected in May 2011 at the area around 15 km radius from Tanjung Bin coal-fired power plant located in Pontian, Johor. The ²¹⁰Po activity concentrations in mosses and soil varied in the range 102 ± 4 to 174 ± 8 Bq/kg dry wt. and 37 ± 2 to 184 ± 8 Bq/kg dry wt., respectively. Corresponding highest activity concentration of ²¹⁰Po observed in L. aduncum, therefore, this finding can be concluded this species was the most suitable as a bio-indicator for ²¹⁰Po deposition. On the other hand, it is clear the accumulation of ²¹⁰Po in mosses might be supplied from various sources of atmospheric deposition such as coal-fired power plant operation, industrial, plantation, agriculture and fertilizer activities, burned fuel fossil and forest; and other potential sources. Meanwhile, the main source of ²¹⁰Po in surface soil is supplied from the in situ deposition of radon decay and its daughters in the soil itself.     

Retrospective determination of 238Pu, 239,240Pu and 90Sr activities in the outer bark of Norway spruce (Picea abies (L.) Karst.) collected at various sites in the Czech Republic

The activity concentrations of ²³⁸Pu, ^239,240Pu and ⁹⁰Sr were determined in 25 archived spruce outer bark samples collected in coniferous forests across the Czech Republic in 1995. At three sampling sites the radionuclide activity concentrations were determined in forest soil. Data was provided on the cumulative deposition and vertical distribution of ²³⁸Pu, ^239,240Pu and ⁹⁰Sr in forest soil. The median activity concentration of ²³⁸Pu in the spruce bark samples was 0.009 Bq kg^?1. The median activity concentration of ^239,240Pu was 0.212 Bq kg^?1, and the median activity concentration of ⁹⁰Sr was 10.6 Bq kg^?1. The radionuclide activity concentration distribution was not significantly explained by the local long-term (1961–2000) mean annual precipitation totals, by site elevation, by bark acidity, by soil moisture and soil texture. The activity concentrations of the radionuclides in bark were found to be higher than or comparable with the published and measured figures for radionuclide activity concentrations in cultivated and uncultivated soils (0–20 cm layer) in the Czech Republic and abroad. The activity concentration ratio of ⁹⁰Sr/^239,240Pu in the investigated spruce bark samples was higher than in the relevant soil samples. We assume that the crucial radioactive contamination loads in bark occurred in the first half of the 1960s, when the concentration of the investigated radionuclides in the air was highest. Spruce trunk bark has preserved relatively high activity concentrations of these radionuclides. The mechanism governing the long-term radionuclide activity concentrations in outer bark proportional to the local radioactive fallout rates is not correctly known. Our results indicate the suitability of spruce bark for use as an effective monitor of radioactive plume fallout loads even several decades after the contamination episode.     

Determination of Organophosphorus and Carbamic Pesticides with a Choline and Acetylcholine Electrochemical Biosensor

Abstract

Pesticides as paraoxon and aldicarb have been determined with an amperometric hydrogen peroxide based choline sensor. These pesticides inhibit the enzyme acetylcholinesterase which in presence of its substrate, acetylcholine, produces choline. When these pesticides are in presence of acetylcholinesterase, the activity of this enzyme decreases; this causes a decrease of choline production which is monitored by a choline sensor and correlated to the concentration of pesticide in solution. Two different procedures were followed: one with both the enzymes acetylcholinesterase and choline oxidase immobilized, the second one with the acetylcholinesterase in solution and the choline oxidase immobilized. Parameters as pH, buffer, enzyme concentration, substrate concentration and reaction and incubation times were optimized. Results showed that these compounds can be detected in the range 10 – 100 ppb. The use of the enzyme in solution gave the best results with a detection limit of 2 ppb pesticide.