Search results for: enzyme kinetics

Authors: Sergei Zhevnenko

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Сontinuous layer of tantalum boride is formed on the surface as a result of reactive wetting of oxidized tantalum by copper melt with boron at a temperatures above 1150 °C. An increase in the wetting temperature above 1400 °C leads to a change in the formation mechanism of tantalum borides, they are formed in the nanosized flakes. In the presented work, we studied the process of copper-based in-situ composite formation, strengthened by the particles of tantalum borides. We investigated the structure of the formed particles, the conditions, and the kinetics of their formation. Dissolving boride particles do not have time to mix uniformly in the melt upon sufficiently rapid cooling and form a macrostructure, partly repeating the shape of the metallic tantalum. This allows to set different gradient structures in the copper alloy. Such macrostructures have been obtained. Boride particles and microstructures were studied by scanning and transmission electron microscopy, and regions with particles were investigated by nanoindentation. In this work, we also measured the kinetics of impregnation of porous tantalum with copper-boron melt and studied the structures of the composite, in which the melt filling the interpore space is saturated with boride particles.

Keywords: copper, tantalum borides, in-situ composites, wetting, imbibition

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Authors: Jihane Saad, Thomas Lendormi, Caroline Le Marechal, Anne-marie Pourcher, Céline Druilhe, Jean-louis Lanoiselle

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Agricultural anaerobic digestion consists in the conversion of animal slurry and manure into biogas and digestate. They need, however, to be treated at 70 ºC during 60 min before anaerobic digestion according to the European regulation (EC n°1069/2009 & EU n°142/2011). The impact of such heat treatment on the outcome of bacteria has been poorly studied up to now. Moreover, a recent study¹ has shown that enterococci and clostridia are still detected despite the application of such thermal treatment, questioning the relevance of this approach for the hygienisation of digestate. The aim of this study is to establish the heat inactivation kinetics of two species of enterococci (Enterococcus faecalis and Enterococcus faecium) and two species of clostridia (Clostridioides difficile and Clostridium novyi as a non-toxic model for Clostridium botulinum of group III). A pure culture of each strain was prepared in a specific sterile medium at concentration of 10⁴ – 10⁷ MPN / mL (Most Probable number), depending on the bacterial species. Bacterial suspensions were then filled in sterilized capillary tubes and placed in a water or oil bath at desired temperature for a specific period of time. Each bacterial suspension was enumerated using a MPN approach, and tests were repeated three times for each temperature/time couple. The inactivation kinetics of the four indicator bacteria is described using the Weibull model and the classical Bigelow model of first-order kinetics. The Weibull model takes biological variation, with respect to thermal inactivation, into account and is basically a statistical model of distribution of inactivation times as the classical first-order approach is a special case of the Weibull model. The heat treatment at 70 ºC / 60 min contributes to a reduction greater than 5 log10 for E. faecium and E. faecalis. However, it results only in a reduction of about 0.7 log10 for C. difficile and an increase of 0.5 log10 for C. novyi. Application of treatments at higher temperatures is required to reach a reduction greater or equal to 3 log10 for C. novyi (such as 30 min / 100 ºC, 13 min / 105 ºC, 3 min / 110 ºC, and 1 min / 115 ºC), raising the question of the relevance of the application of heat treatment at 70 ºC / 60 min for these spore-forming bacteria. To conclude, the heat treatment (70 ºC / 60 min) defined by the European regulation is sufficient to inactivate non-sporulating bacteria. Higher temperatures (> 100 ºC) are required as far as spore-forming bacteria concerns to reach a 3 log10 reduction (sporicidal activity).

Keywords: heat treatment, enterococci, clostridia, inactivation kinetics

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Authors: C. Mekala, Indumathi M. Nambi

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Nitrogen pollution in groundwater arising from wastewater and fertilizer application through vadose zone is a major problem and it causes a prime risk to groundwater based drinking water supplies. Nitrogenous compounds namely ammonium, nitrate and nitrite fate and transport in soil subsurface were studied experimentally. The major process like sorption, leaching, biotransformation involving microbial growth kinetics, and biological clogging due to biomass growth were assessed and modeled with advection-dispersion reaction equations for ammonium, nitrate and acetate in a saturated, heterogeneous soil medium. The transport process was coupled with freundlich sorption and monod inhibition kinetics for immobile bacteria and permeability reduction due to biomass growth will be verified and validated with the numerical model. This proposed mathematical model will be very helpful in the development of a management model for a sustainable and safe wastewater reuse strategies such as irrigation and groundwater recharge.

Keywords: nitrogen species transport, transformation, biological clogging, biokinetic parameters, contaminant transport model, saturated soil

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Authors: T. Urushadze, R. Khvedelidze, L. Kutateladze, M. Jobava, T. Burduli, T. Alexidze

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There is an increasing interest in reliable, wasteless, ecologically friendly technologies. About 40% of enzymes produced all over the world are used for production of syrups with high concentration of glucose-fructose. One of such technologies complies obtaining fermentable sugar glucose from raw materials containing starch by means of amylases. In modern alcohol-producing factories this process is running in two steps, involving two enzymes of different origin: bacterial α-amylase and fungal glucoamylase, as generally fungal amylases are less thermostable as compared to bacterial amylases. Selection of stable and operable at 700С and higher temperatures enzyme preparation with both α- and glucoamylase activities will allow conducting this process in one step. S. Durmishidze Institute of Biochemistry and Biotechnology owns unique collection of mycelial fungi, isolated from different ecological niches of Caucasus. As a result of screening our collection 39 strains poducing amylases were revealed. Most of them belong to the genus Aspergillus. Optimum temperatures of action of selected amylases from three producers were estableshed to be within the range 67-80°C. A. niger B-6 showed higher α-amylase activity at 67°C, and glucoamylase activity at 62°C, A. niger 6-12 showed higher α-amylase activity at 72°C, and glucoamylase activity at 65°C, Aspergillus niger p8-3 showed higher activities at 82°C and 70°C, for α-amylase and glucoamylase activities, respectively. Exhaustive hydrolysis process of starch solutions of different concentrations (3, 5, 15, and 30 %) with cultural liquid and technical preparation of Aspergillus niger p8-3 enzyme was studied. In case of low concentrations exhaustive hydrolysis of starch lasts 40–60 minutes, in case of high concentrations hydrolysis takes longer time. 98, 6% yield of glucose can be reached at incubation during 12 hours with enzyme cultural liquid and 8 hours incubation with technical preparation of the enzyme at gradual increase of temperature from 50°C to 82°C during the first 20 minutes and further decrease of temperature to 70°C. Temperature setting for high yield of glucose and high hydrolysis (pasteurizing), optimal for activity of these strains is the prerequisite to be able to carry out hydrolysis of starch to glucose in one step, and consequently, using one strain, what will be economically justified.

Keywords: amylase, glucose hydrolisis, stability, starch

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Authors: Niharika Keot, Manabendra Sarma

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A thorough investigation of Ln3+ complexes with more than one inner-sphere water molecule is crucial for designing high relaxivity contrast agents (CAs) used in magnetic resonance imaging (MRI). This study accomplished a comparative stability analysis of two hexadentate (H3cbda and H3dpaa) and two heptadentate (H4peada and H3tpaa) ligands with Ln3+ ions. The higher stability of the hexadentate H3cbda and heptadentate H4peada ligands has been confirmed by the binding affinity and Gibbs free energy analysis in aqueous solution. In addition, energy decomposition analysis (EDA) reveals the higher binding affinity of the peada4− ligand than the cbda3− ligand towards Ln3+ ions due to the higher charge density of the peada4− ligand. Moreover, a mechanistic overview of water exchange kinetics has been carried out based on the strength of the metal–water bond. The strength of the metal–water bond follows the trend Gd–O47 (w) > Gd–O39 (w) > Gd–O36 (w) in the case of the tris-aquated [Gd(cbda)(H2O)3] and Gd–O43 (w) > Gd–O40 (w) for the bis-aquated [Gd(peada)(H2O)2]− complex, which was confirmed by bond length, electron density (ρ), and electron localization function (ELF) at the corresponding bond critical points. Our analysis also predicts that the activation energy barrier decreases with the decrease in bond strength; hence kex increases. The 17O and 1H hyperfine coupling constant values of all the coordinated water molecules were different, calculated by using the second-order Douglas–Kroll–Hess (DKH2) approach. Furthermore, the ionic nature of the bonding in the metal–ligand (M–L) bond was confirmed by the Quantum Theory of Atoms-In-Molecules (QTAIM) and ELF along with energy decomposition analysis (EDA). We hope that the results can be used as a basis for the design of highly efficient Gd(III)-based high relaxivity MRI contrast agents for medical applications.

Keywords: MRI contrast agents, lanthanide chemistry, thermodynamic stability, water exchange kinetics

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Authors: Sameena Malik, Ghosh Prakash, Sandeep Mudliar, Vishal Waindeskar, Atul Vaidya

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In this study, the biodegradability enhancement along with COD color and toxicity removal of pharmaceutical effluent by O₃, O₃/Fe²⁺, O₃/nZVI processes has been evaluated. The nZVI particles were synthesized and characterized by XRD and SEM analysis. Kinetic model was reasonably developed to select the ozone doses to be applied based on the ozonation kinetic and mass transfer coefficient values. Nano catalytic ozonation process (O₃/nZVI) effectively enhanced the biodegradability (BI=BOD₅/COD) of pharmaceutical effluent up to 0.63 from 0.18 of control with a COD, color and toxicity removal of 62.3%, 93%, and 75% respectively compared to O₃, O₃/Fe²⁺ pretreatment processes. From the GC-MS analysis, 8 foremost organic compounds were predominantly detected in the pharmaceutical effluent. The disappearance of the corresponding GC-MS spectral peaks during catalyzed ozonation process indicated the degradation of the effluent. The changes in the FTIR spectra confirms the transformation/destruction of the organic compounds present in the effluent to new compounds. Subsequent aerobic biodegradation of pretreated effluent resulted in biodegradation rate enhancement by 5.31, 2.97, and 1.22 times for O₃, O₃/Fe²⁺ and O₃/nZVI processes respectively.

Keywords: iron nanoparticles, pharmaceutical effluent, ozonation, kinetics, mass transfer

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Authors: Melike Sultan Karasu Asnaz, Ayse Ozdogan Dolcek

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Crop drying, which aims to reduce the moisture content to a certain level, is a method used to extend the shelf life and prevent it from spoiling. One of the oldest food preservation techniques is open sunor shade drying. Even though this technique is the most affordable of all drying methods, there are some drawbacks such as contamination by insects, environmental pollution, windborne dust, and direct expose to weather conditions such as wind, rain, hail. However, solar dryers that provide a hygienic and controllable environment to preserve food and extend its shelf life have been developed and used to dry agricultural products. Thus, foods can be dried quickly without being affected by weather variables, and quality products can be obtained. This research is mainly devoted to investigating the modelling of drying kinetics of cantaloupe in a forced convection solar dryer. Mathematical models for the drying process should be defined to simulate the drying behavior of the foodstuff, which will greatly contribute to the development of solar dryer designs. Thus, drying experiments were conducted and replicated five times, and various data such as temperature, relative humidity, solar irradiation, drying air speed, and weight were instantly monitored and recorded. Moisture content of sliced and pretreated cantaloupe were converted into moisture ratio and then fitted against drying time for constructing drying curves. Then, 10 quasi-theoretical and empirical drying models were applied to find the best drying curve equation according to the Levenberg-Marquardt nonlinear optimization method. The best fitted mathematical drying model was selected according to the highest coefficient of determination (R²), and the mean square of the deviations (χ^²) and root mean square error (RMSE) criterial. The best fitted model was utilized to simulate a thin layer solar drying of cantaloupe, and the simulation results were compared with the experimental data for validation purposes.

Keywords: solar dryer, mathematical modelling, drying kinetics, cantaloupe drying

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Authors: Madhuresh Dwivedi, Navneet Singh Deora, Aastha Deswal, H. N. Mishra

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FT-NIR spectroscopy and electronic nose was used to study the kinetics of dough proofing. Spectroscopy was conducted with an optic probe in the diffuse reflectance mode. The dough leavening was carried out at different temperatures (25 and 35°C) and constant RH (80%). Spectra were collected in the range of wave numbers from 12,000 to 4,000 cm-1 directly on the samples, every 5 min during proofing, up to 2 hours. NIR spectra were corrected for scatter effect and second order derivatization was done to transform the spectra. Principal component analysis (PCA) was applied for the leavening process and process kinetics was calculated. PCA was performed on data set and loadings were calculated. For leavening, four absorption zones (8,950-8,850, 7,200-6,800, 5,250-5,150 and 4,700-4,250 cm-1) were involved in describing the process. Simultaneously electronic nose was also used for understanding the development of odour compounds during fermentation. The electronic nose was able to differential the sample on the basis of aroma generation at different time during fermentation. In order to rapidly differentiate samples based on odor, a Principal component analysis is performed and successfully demonstrated in this study. The result suggests that electronic nose and FT-NIR spectroscopy can be utilized for the online quality control of the fermentation process during leavening of bread dough.

Keywords: FT-NIR, dough, e-nose, proofing, principal component analysis

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Authors: Fadilah Fadilah, Rochmadi Rochmadi, Siti Syamsiah, Djagal W. Marseno

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Glucomannan can be found in the tuber of porang together with starch and proteinaceous components which were regarded as impurities. An enzymatic process for obtaining higher glucomannan content from Porang flour have been conducted. Papain was used for hydrolysing proteinaceous components in Porang flour which was conducted after a simultaneous extraction of glucomannan and enzymatic starch hydrolysis. Three variables affecting the rate were studied, i.e. temperature, the amount of enzyme and the stirring speed. The ninhydrin method was used to determine degree of protein hydrolysis. Results showed that the rising of degree of hydrolysis were fast in the first ten minutes of the reaction and then proceeded slowly afterward. The optimum temperature for hydrolysis was 60 ^oC. Increasing the amount of enzyme showed a remarkable effect to degree of hydrolysis, but the stirring speed had no significant effect. This indicated that the reaction controlled the rate of hydrolysis.

Keywords: degree of hydrolysis, ninhydrin, papain, porang flour, proteinaceous components

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Authors: Akimitsu Kugimiya, Kouhei Iwato, Toru Saito, Jiro Kohda, Yasuhisa Nakano, Yu Takano

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The measurement of amino acid content is reported to be useful for the diagnosis of several types of diseases, including lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, and diabetes. The conventional detection methods for amino acid are high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS), but they have several drawbacks as the equipment is cumbersome and the techniques are costly in terms of time and costs. In contrast, biosensors and biosensing methods provide more rapid and facile detection strategies that use simple equipment. The authors have reported a novel approach for the detection of each amino acid that involved the use of aminoacyl-tRNA synthetase (aaRS) as a molecular recognition element because aaRS is expected to a selective binding ability for corresponding amino acid. The consecutive enzymatic reactions used in this study are as follows: aaRS binds to its cognate amino acid and releases inorganic pyrophosphate. Hydrogen peroxide (H₂O₂) was produced by the enzyme reactions of inorganic pyrophosphatase and pyruvate oxidase. The Trinder’s reagent was added into the reaction mixture, and the absorbance change at 556 nm was measured using a microplate reader. In this study, an amino acid-sensing method using histidyl-tRNA synthetase (HisRS; histidine-specific aaRS) as molecular recognition element in combination with the Trinder’s reagent spectrophotometric method was developed. The quantitative performance and selectivity of the method were evaluated, and the optimal enzyme reaction and detection conditions were determined. The authors developed a simple and rapid method for detecting histidine with a combination of enzymatic reaction and spectrophotometric detection. In this study, HisRS was used to detect histidine, and the reaction and detection conditions were optimized for quantitation of these amino acids in the ranges of 1–100 µM histidine. The detection limits are sufficient to analyze these amino acids in biological fluids. This work was partly supported by Hiroshima City University Grant for Special Academic Research (General Studies).

Keywords: amino acid, aminoacyl-tRNA synthetase, biosensing, enzyme reaction

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Authors: Christian Fercher, Jiaul Islam, Simon R. Corrie

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Fluorescence-based immunodiagnostics are an emerging field in biosensor development and exhibit several advantages over traditional detection methods. While various affinity biosensors have been developed to generate a fluorescence signal upon sensing varying concentrations of analytes, reagentless, reversible, and continuous monitoring of complex biological samples remains challenging. Here, we aimed to genetically engineer biosensors based on single-chain antibody fragments (scFv) that are site-specifically labeled with environmentally sensitive fluorescent unnatural amino acids (UAA). A rational design approach resulted in quantifiable analyte-dependent changes in peak fluorescence emission wavelength and enabled antigen detection in vitro. Incorporation of a polarity indicator within the topological neighborhood of the antigen-binding interface generated a titratable wavelength blueshift with nanomolar detection limits. In order to ensure continuous analyte monitoring, scFv candidates with fast binding and dissociation kinetics were selected from a genetic library employing a high-throughput phage display and affinity screening approach. Initial rankings were further refined towards rapid dissociation kinetics using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). The most promising candidates were expressed, purified to homogeneity, and tested for their potential to detect biomarkers in a continuous microfluidic-based assay. Variations of dissociation kinetics within an order of magnitude were achieved without compromising the specificity of the antibody fragments. This approach is generally applicable to numerous antibody/antigen combinations and currently awaits integration in a wide range of assay platforms for one-step protein quantification.

Keywords: antibody engineering, biosensor, phage display, unnatural amino acids

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Authors: Debajyoti Bose

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Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Chitosan is a positively charged natural biodegradable and biocompatible polymer. It is a linear polysaccharide consisting of β-1,4 linked monomers of glucosamine and N-acetylglucosamine. Chitosan can be mainly obtained from fungal sources during large fermentation process. In this study,three different fungal strains Aspergillus niger NCIM 1045, Aspergillus oryzae NCIM 645 and Mucor indicus MTCC 3318 were used for the production of chitosan. The growth mediums were optimized for maximum fungal production. The produced chitosan was characterized by determining degree of deacetylation. Chitosan possesses one reactive amino at the C-2 position of the glucosamine residue, and these amines confer important functional properties to chitosan which can be exploited for biofabrication to generate various chemically modified derivatives and explore their potential for pharmaceutical field. Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein (e.g. enzyme diastase) in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. It was noted that the chitosan loading and releasing efficiency as a nanocapsule, obtained from different fungal sources was almost near to initial enzyme activity(12026 U/ml) with a negligible loss. This signify, chitosan can be used as a polymeric drug as well as active component or protein carrier material in dosage by design due to its appealing properties such as biocompatibility, biodegradability, low toxicity and relatively low production cost from abundant natural sources. Based upon these initial experiments, studies were also carried out on modification of chitosan based nanocapsules incorporated with physiologically important enzymes and nutraceuticals for target delivery.

Keywords: fungi, chitosan, enzyme, nanocapsule

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Authors: Sema Şenoğlu, Sevgi Karakuş

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Hydrazone scaffold is important to design new drug groups and is found to possess numerous uses in pharmaceutical chemistry. Besides, hydrazone derivatives are also known for biological activities such as anticancer, antimicrobial, antiviral, and antifungal. Hydrazone derivatives are promising anticancer agents because they inhibit cancer proliferation and induce apoptosis. Human carbonic anhydrase IX has a high potential to be an antiproliferative drug target, and targeting this protein is also important for obtaining potential anticancer inhibitors. The protein construct was retrieved as a PDB file from the RCSB protein database. This binding interaction of proteins and ligands was performed using Discovery Studio Visualizer. In vitro inhibitory activity of hydrazone derivatives was tested against enzyme carbonic anhydrase IX on the PyRx programme. Most of these molecules showed remarkable human carbonic anhydrase IX inhibitory activity compared to the acetazolamide. As a result, these compounds appear to be a potential target in drug design against human carbonic anhydrase IX.

Keywords: cancer, carbonic anhydrase IX enzyme, docking, hydrazone

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Authors: Mohamed H. Khalifa, Fikry I. El-Shahawi, Nabil A. Mansour

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The cotton leafworm, Spodoptera littoralis (Boisduval) is a major insect pest of vegetables and cotton crops in Egypt, and exhibits different levels of tolerance to certain insecticides. Chlorantraniliprole has been registered recently in Egypt for control this insect. The susceptibilities of three S. littoralis populations collected from El Behaira governorate, north Egypt to chlorantraniliprole were determined by leaf-dipping technique on 4^th instar larvae. Obvious variation of toxicity was observed among the laboratory susceptible, and three field populations with LC₅₀ values ranged between 1.53 µg/ml and 6.22 µg/ml. However, all the three field populations were less susceptible to chlorantraniliprole than a laboratory susceptible population. The most tolerant populations were sampled from El Delengat (ED) Province where S. littoralis had been frequently challenged by insecticides. Certain enzyme activity assays were carried out to be correlated with the mechanism of the observed field population tolerance. All field populations showed significantly enhanced activities of detoxification enzymes compared with the susceptible strain. The regression analysis between chlorantraniliprole toxicities and enzyme activities revealed that the highest correlation is between α-esterase or β-esterase (α-β-EST) activity and collected field strains susceptibility, otherwise this correlation is not significant (P > 0.05). Synergism assays showed the ED and susceptible strains could be synergized by known detoxification inhibitors such as piperonyl butoxide (PBO), triphenyl phosphate (TPP) and diethyl-maleate (DEM) at different levels (1.01-8.76-fold and 1.09-2.94 fold, respectively), TPP showed the maximum synergism in both strains. The results show that there is a correlation between the enzyme activity and tolerance, and carboxylic-esterase (Car-EST) is likely the main detoxification mechanism responsible for tolerance of S. littoralis to chlorantraniliprole.

Keywords: chlorantraniliprole, detoxification enzymes, Egypt, Spodoptera littoralis

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Authors: Caiyun Liu, A. Hernandez-Manas, N. Grimi, E. Vorobiev

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Drying is one of the most used methods for food preservation, which extend shelf life of food and makes their transportation, storage and packaging easier and more economic. The commonly dried method is hot air drying. However, its disadvantages are low energy efficiency and long drying times. Because of the high temperature during the hot air drying, the undesirable changes in pigments, vitamins and flavoring agents occur which result in degradation of the quality parameters of the product. Drying process can also cause shrinkage, case hardening, dark color, browning, loss of nutrients and others. Recently, new processes were developed in order to avoid these problems. For example, the application of pulsed electric field provokes cell membrane permeabilisation, which increases the drying kinetics and moisture diffusion coefficient. Microwave drying technology has also several advantages over conventional hot air drying, such as higher drying rates and thermal efficiency, shorter drying time, significantly improved product quality and nutritional value. Rehydration kinetics of dried product is a very important characteristic of dried products. Current research has indicated that the rehydration ratio and the coefficient of rehydration are dependent on the processing conditions of drying. The present study compares the efficiency of two processes (1: room temperature air drying, 2: microwave/air drying) in terms of drying rate, product quality and rehydration ratio. In this work, potato slices (≈2.2g) with a thickness of 2 mm and diameter of 33mm were placed in the microwave chamber and dried. Drying kinetics and drying rates of different methods were determined. The process parameters included inlet air velocity (1 m/s, 1.5 m/s, 2 m/s) and microwave power (50 W, 100 W, 200 W and 250 W) were studied. The evolution of temperature during microwave drying was measured. The drying power had a strong effect on drying rate, and the microwave-air drying resulted in 93% decrease in the drying time when the air velocity was 2 m/s and the power of microwave was 250 W. Based on Lewis model, drying rate constants (kDR) were determined. It was observed an increase from kDR=0.0002 s-1 to kDR=0.0032 s-1 of air velocity of 2 m/s and microwave/air (at 2m/s and 250W) respectively. The effective moisture diffusivity was calculated by using Fick's law. The results show an increase of effective moisture diffusivity from 7.52×10-11 to 2.64×10-9 m2.s-1 for air velocity of 2 m/s and microwave/air (at 2m/s and 250W) respectively. The temperature of the potato slices increased for higher microwaves power, but decreased for higher air velocity. The rehydration ratio, defined as the weight of the the sample after rehydration per the weight of dried sample, was determined at different water temperatures (25℃, 50℃, 75℃). The rehydration ratio increased with the water temperature and reached its maximum at the following conditions: 200 W for the microwave power, 2 m/s for the air velocity and 75°C for the water temperature. The present study shows the interest of microwave drying for the food preservation.

Keywords: drying, microwave, potato, rehydration

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Authors: Dharshika Rajalingam, Jeffery W. Peng

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Acinetobacter baumannii is a challenging threat to global health, recognized as a multidrug-resistant pathogen. -lactamase is one of the principal resistant mechanisms developed by A. baumannii to survive against -lactam antibiotics. OXA24/40 is one of the types of -lactamases, a well-documented carbapenem hydrolyzing class D -lactamases (CHDL). It was revealed that OXA24/40 showed resistivity against doripenem, one of the carbapenems, by two different mechanisms as hydrolysis and -lactonization. Furthermore, it undergoes genetic mutations to broaden the -lactamase activity to survive against antibiotic environments. One of the crucial characterizations of prokaryotes to develop adaptation is post-translational modification (PTM), mainly phosphorylation. However, the PTM of OXA24/40 is an unknown feature, and the impact of PTM on antibiotic resistivity is yet to be explored. We approached these hypotheses using NMR and MS techniques and found that the OXA24/40 could be phosphorylated in vitro. The Ser81 at the active STFK motif of OXA24/40 of catalytic pocket was identified as the site of phosphorylation using 1D 31P NMR experiment, whereas S81 is required to form an acyl-enzyme complex between enzyme and -lactam antibiotics. The activity of completely phosphorylated OXA24/40 wild type against doripenem revealed that the phosphorylation of active Ser inactivates the -lactamases activity of OXA24/40. The 1D 1H CPMG NMR-based activity assay of phosphorylated OXA24/40 against doripenem confirmed that both deactivating mechanisms are inhibited by phosphorylation. Carbamylated Lysine at the active STFK motif is one of the critical features of CHDL required for the acylation and deacylation reactions of the enzyme. The 1D 13C NMR experiment confirmed that the K84 of phosphorylated OXA24/40 is de-carbamylated. Phosphorylation of OXA24/40 affects both active S81 and carbamylated K84 of OXA24 that are required for the resistivity of -lactamase. So, phosphorylation could be one of the reasons for the genetic mutation of OXA24/40 for the development of antibiotic resistivity. Further research can lead to an understanding of the effect of phosphorylation on the clinical mutants of the OXA24-like -lactamase family on the broadening of -lactamase activity.

Keywords: OXA24/40, phosphorylation, clinical mutants, resistivity

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Authors: Heba Ahmed Nawafleh

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In this study, two commercial resins were evaluated to concentrate uranium from real solutions that were produced from analkaline leaching process of carbonate deposits. The adsorption was examined using a batch process. Different parameters were evaluated, including initial pH, contact time, temperature, adsorbent dose, and finally, uranium initial concentration. Both resins were effective and selective for uranium ions from the tested leaching solution. The adsorption isotherms data were well fitted for both resins using the Langmuir model. Thermodynamic functions (Gibbs free energy change ΔG, enthalpy change ΔH, and entropy change ΔS) were calculated for the adsorption of uranium. The result shows that the adsorption process is endothermic, spontaneous, and chemisorption processes took place for both resins. The kinetic studies showed that the equilibrium time for uranium ions is about two hours, where the maximum uptake levels were achieved. The kinetics studies were carried out for the adsorption of U ions, and the data was found to follow pseudo-second-order kinetics, which indicates that the adsorption of U ions was chemically controlled. In addition, the reusability (adsorption/ desorption) process was tested for both resins for five cycles, these adsorbents maintained removal efficiency close to first cycle efficiency of about 91% and 80%.

Keywords: uranium, adsorption, ion exchange, thermodynamic and kinetic studies

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Authors: S. Mat Radzi, N. J. Abd Rahman, H. Mohd Noor, N. Ariffin

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Ferulic acid has widespread industrial potential by virtue of its antioxidant properties. However, it is partially soluble in aqueous media, limiting their usefulness in oil-based processes in food, cosmetic, pharmaceutical, and material industry. Therefore, modification of ferulic acid should be made by producing of more lipophilic derivatives. In this study, a preliminary investigation of lipase-catalyzed trans-esterification reaction of ethyl ferulate and olive oil was investigated. The reaction was catalyzed by immobilized lipase from Candida antarctica (Novozym 435), to produce ferulate ester, a sunscreen agent. A statistical approach of Response surface methodology (RSM) was used to evaluate the interactive effects of reaction temperature (40-80°C), reaction time (4-12 hours), and amount of enzyme (0.1-0.5 g). The optimum conditions derived via RSM were reaction temperature 60°C, reaction time 2.34 hours, and amount of enzyme 0.3 g. The actual experimental yield was 59.6% ferulate ester under optimum condition, which compared well to the maximum predicted value of 58.0%.

Keywords: ferulic acid, enzymatic synthesis, esters, RSM

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Authors: Istvan Szilagyi, Marko Pavlovic, Paul Rouster

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Antioxidant enzymes are the most efficient defense systems against reactive oxygen species, which cause severe damage in living organisms and industrial products. However, their supplementation is problematic due to their high sensitivity to the environmental conditions. Immobilization on carrier nanoparticles is a promising research direction towards the improvement of their functional and colloidal stability. In that way, their applications in biomedical treatments and manufacturing processes in the food, textile and cosmetic industry can be extended. The main goal of the present research was to prepare and formulate antioxidant bionanocomposites composed of superoxide dismutase (SOD) enzyme, anionic clay (layered double hydroxide, LDH) nanoparticle and heparin (HEP) polyelectrolyte. To characterize the structure and the colloidal stability of the obtained compounds in suspension and solid state, electrophoresis, dynamic light scattering, transmission electron microscopy, spectrophotometry, thermogravimetry, X-ray diffraction, infrared and fluorescence spectroscopy were used as experimental techniques. LDH-SOD composite was synthesized by enzyme immobilization on the clay particles via electrostatic and hydrophobic interactions, which resulted in a strong adsorption of the SOD on the LDH surface, i.e., no enzyme leakage was observed once the material was suspended in aqueous solutions. However, the LDH-SOD showed only limited resistance against salt-induced aggregation and large irregularly shaped clusters formed during short term interval even at lower ionic strengths. Since sufficiently high colloidal stability is a key requirement in most of the applications mentioned above, the nanocomposite was coated with HEP polyelectrolyte to develop highly stable suspensions of primary LDH-SOD-HEP particles. HEP is a natural anticoagulant with one of the highest negative line charge density among the known macromolecules. The experimental results indicated that it strongly adsorbed on the oppositely charged LDH-SOD surface leading to charge inversion and to the formation of negatively charged LDH-SOD-HEP. The obtained hybrid materials formed stable suspension even under extreme conditions, where classical colloid chemistry theories predict rapid aggregation of the particles and unstable suspensions. Such a stabilization effect originated from electrostatic repulsion between the particles of the same sign of charge as well as from steric repulsion due to the osmotic pressure raised during the overlap of the polyelectrolyte chains adsorbed on the surface. In addition, the SOD enzyme kept its structural and functional integrity during the immobilization and coating processes and hence, the LDH-SOD-HEP bionanocomposite possessed excellent activity in decomposition of superoxide radical anions, as revealed in biochemical test reactions. In conclusion, due to the improved colloidal stability and the good efficiency in scavenging superoxide radical ions, the developed enzymatic system is a promising antioxidant candidate for biomedical or other manufacturing processes, wherever the aim is to decompose reactive oxygen species in suspensions.

Keywords: clay, enzyme, polyelectrolyte, formulation

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Authors: Nagasamy Venkatesh Dhandapani, Amged Awad El-Gied

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Cefixime, a BCS class II drug, is insoluble in water but freely soluble in acetone and in alcohol. The aqueous solubility of cefixime in water is poor and exhibits exceptionally slow and intrinsic dissolution rate. In the present study, cefixime and β-Cyclodextrin (β-CD) solid dispersions were prepared with a view to study the effect and influence of β-CD on the solubility and dissolution rate of this poorly aqueous soluble drug. Phase solubility profile revealed that the solubility of cefixime was increased in the presence of β-CD and was classified as A_L-type. Effect of variable, such as drug:carrier ratio, was studied. Physical characterization of the solid dispersion was characterized by Fourier transform infrared spectroscopy (FT-IR) and Differential scanning calorimetry (DSC). These studies revealed that a distinct loss of drug crystallinity in the solid molecular dispersions is ostensibly accounting for enhancement of dissolution rate in distilled water. The drug release from the prepared solid dispersion exhibited a first order kinetics. Solid dispersions of cefixime showed a 6.77 times fold increase in dissolution rate over the pure drug.

Keywords: β-cyclodextrin, cefixime, dissolution, Kneading method, solid dispersions, release kinetics

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Authors: Boudjenah-Haroun Saliha, Isselnane Souad, Nouani Abdelwahab, Baaissa Babelhadj, Mati Abderrahmane

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Algeria is experiencing significant development of the dairy sector, where consumption of milk and milk products increased by 2.7 million tons in 2008 to 4,400,000 tons in 2013, and cheese production has reached 1640 tons in the year 2014 with average consumption of 0.7 kg/person/year. Although rennet is still the most used coagulating enzyme in cheese, its production has been growing worldwide shortage. This shortage is primarily due to a growing increase in the production and consumption of cheese, and the inability to increase in parallel the production of rennet. This shortage has caused very large fluctuations in its price). To overcome these obstacles, much research has been undertaken to find effective and competitive substitutes used industrially. For this, the selection of a local production of rennet substitute is desirable. It would allow a permanent supply with limited dependence on imports and price fluctuations. Investigations conducted by our research team showed that extracts coagulants from the stomachs of older camels are characterized by a coagulating power than those from younger camels. The objective of this work is to study the possibility of substituting commercial rennet coagulant by gastric enzymes from adult camels for coagulation bovine milk. Excerpts from the raw camel coagulants obtained are characterized through their teneures proteins and clotting and proteolytic activities. Milk clotting conditions by the action of these extracts were optimized. Milk clotting time all treated with enzyme preparations and under different conditions was calculated. Bovine rennet has been used for comparison. The results show that crude extracts from gastric adult camel can be good substituting bovine rennet.

Keywords: Algeria, camel, cheese, coagulation, gastric extracts, milk

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Authors: Heidarali Malmir

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The effects of acid rain (AR), smog’s cars (SC), and combined AR+SC on the antioxidants enzymes, lipid-soluble antioxidants, and water-soluble antioxidants were studied in the two cultivars of rice. The results showed that simulated AR significantly increased the total glutathione (TGSH), thiobarbituric acid (TBA), and α-tocopherol, accompanied by decreases in dry weight and leaves area in the two cultivars, and this change was more obvious in Shirudi cultivar than in Aus cultivar (p≤0.05). Under SC stress cultivar shirudi had higher H+-ATPase, glutathione peroxidase (GSH-px), and catalase (CAT) activities than cultivar Aus. The results of superoxide dismutase (SOD) activity, TGSH, and α-tocopherol levels affected by AR treatments were very different to those of SOD activity, TGSH, and α-tocopherol levels, as shown in SC treatment. It seems that SOD activity coupled with the water-soluble antioxidants and α-tocopherol levels correlated with the lipid-soluble antioxidants. It is suggested that α-tocopherol increases H+-ATPase activity.

Keywords: H+-ATPase, membrane permeability, lipid soluble antioxidants, water soluble antioxidants, associated enzyme

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Authors: Junie B. Billones, Maria Constancia O. Carrillo, Voltaire G. Organo, Stephani Joy Y. Macalino, Inno A. Emnacen, Jamie Bernadette A. Sy

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The drug discovery and development process is generally known to be a very lengthy and labor-intensive process. Therefore, in order to be able to deliver prompt and effective responses to cure certain diseases, there is an urgent need to reduce the time and resources needed to design, develop, and optimize potential drugs. Computer-aided drug design (CADD) is able to alleviate this issue by applying computational power in order to streamline the whole drug discovery process, starting from target identification to lead optimization. This drug design approach can be predominantly applied to diseases that cause major public health concerns, such as tuberculosis. Hitherto, there has been no concrete cure for this disease, especially with the continuing emergence of drug resistant strains. In this study, CADD is employed for tuberculosis by first identifying a key enzyme in the mycobacterium’s metabolic pathway that would make a good drug target. One such potential target is the lipoate protein ligase B enzyme (LipB), which is a key enzyme in the M. tuberculosis metabolic pathway involved in the biosynthesis of the lipoic acid cofactor. Its expression is considerably up-regulated in patients with multi-drug resistant tuberculosis (MDR-TB) and it has no known back-up mechanism that can take over its function when inhibited, making it an extremely attractive target. Using cutting-edge computational methods, compounds from AnalytiCon Discovery Natural Derivatives database were screened and docked against the LipB enzyme in order to rank them based on their binding affinities. Compounds which have better binding affinities than LipB’s known inhibitor, decanoic acid, were subjected to in silico toxicity evaluation using the ADMET and TOPKAT protocols. Out of the 31,692 compounds in the database, 112 of these showed better binding energies than decanoic acid. Furthermore, 12 out of the 112 compounds showed highly promising ADMET and TOPKAT properties. Future studies involving in vitro or in vivo bioassays may be done to further confirm the therapeutic efficacy of these 12 compounds, which eventually may then lead to a novel class of anti-tuberculosis drugs.

Keywords: pharmacophore, molecular docking, lipoate protein ligase B (LipB), ADMET, TOPKAT

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Authors: Subrata Kar, Banani Kundu, Papiya Nandy, Ruma Basu, Sukhen Das

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Natural montmorilollite clay is a common ingredient in pharmaceutical products, both as excipients and active support; hence considered as suitable candidate for Drug Delivery System. In this work, cationic detergent CTAB is used to increase the interlayer spacing of Na+-Montmoriollite clay to intercalate curcumin and methotrexate. Methotrexate is a folic acid antagonist, anti-proliferative and immunosuppressive agent; while curcumin is a bioactive constituent of rhizomes of Curcuma longa, possessing remarkable chemo-preventive and anti-inflammatory properties. The resultant inorganic-organic hybrids are characterized by X-ray diffraction (XRD), Infrared spectroscopy (FTIR) and Thermo Gravimetric Analysis (TGA) to confirm successful intercalation of curcumin and Methotrexate within clay layers. Pharmaceutical investigation of the hybrids is explored by studying the drug loading (%), encapsulation efficiency and release kinetics. Finally in-vitro studies are performed using cancer cells to find the effect of released curcumin to improve the sensitivity of clay bound methotrexate to ameliorate cell death compared to their effectiveness when used without the inorganic aluminosilicate vehicle.

Keywords: montmorillonite, methotrexate, curcumin, loading efficiency, release kinetics, anticancer activity

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Authors: Somaye Eskandary

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Polycyclic aromatic hydrocarbons (PAHs) are present in crude oil and its derivatives contaminate soil and also increase carcinogen and mutagen contamination, which is a concern for researchers. Land farming is one of the methods that remove pollutants from the soil by native microorganisms. It seems that this technology is cost-effective, environmentally friendly and causes less debris problem to be disposed. This study aimed to refine the polycyclic aromatic hydrocarbons from oil-contaminated soil using the land farming method. In addition to examine the concentration of polycyclic aromatic hydrocarbons by GC-FID, some characteristics such as soil microbial respiration and dehydrogenase, peroxidase, urease, acid and alkaline phosphatase enzyme concentration were also measured. The results showed that after land farming process the concentrations of some polycyclic aromatic hydrocarbons dropped to 50 percent. The results showed that the enzyme concentration is reduced by reducing the concentration of hydrocarbons and microbial respiration. These results emphasize the process of land farming for removal of polycyclic aromatic hydrocarbons from soil by indigenous microorganisms.

Keywords: soil contamination, gas chromatography, native microorganisms, soil enzymes, microbial respiration, carcinogen

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Authors: Palwinder Singh, Baljit Kaur, Sukhmeet Kaur

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Amongst a library of rationally designed small peptides, (H)Gly-Gly-Phe-Leu(OMe) was identified, reducing prostaglandin production of COX-2 with IC50 60 nM vs. 6000 nM for COX-1. The 5 mg Kg-1 dose of this compound rescued albino mice by 80% from capsaicin-induced paw licking and recovered it by 60% from carrageenan-induced inflammation. The mode of action of the compound for targeting COX-2, iNOS, and VGSC was investigated by using substances P, L-arginine, and veratrine, respectively, as the biomarkers. The interactions of the potent compound with COX-2 were supported by the isothermal calorimetry experiments showing Ka 6.10±1.10x104 mol-1 and ΔG -100.3 k J mol-1 in comparison to Ka 0.41x103 ±0.09 mol-1 and ΔG -19.2±0.06 k J mol-1 for COX-1. This compound did not show toxicity up to 2000 mg Kg-1 dose. Furthermore, beyond the conventional mode of working with anti-inflammatory agents through enzyme inhibition, COX-2 was provided with a peptide-based alternate substrate. Proline-centered pentapeptide iso-conformational to arachidonic acid exhibited appreciable selectivity for COX-2 overcoming acetic acid and formalin-induced pain in rats to almost 80% and was treated as a substrate by the enzyme. Hence, we suggest small peptides as highly potent and promising candidates for their further development into an anti-inflammatory drug.

Keywords: small peptides, cyclooxygenase, inflammation, substrate

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Authors: Radka Obořilová, Hana Šimečková, Matěj Pastucha, Jan Přibyl, Petr Skládal, Ivana Mašlaňová, Zdeněk Farka

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Due to the spreading of antimicrobial resistance, alternative approaches to combat superinfections are being sought, both in the field of lysing agents and methods for studying bacterial lysis. A suitable alternative to antibiotics is phage therapy and enzybiotics, for which it is also necessary to study the mechanism of their action. Biosensor-based techniques allow rapid detection of pathogens in real time, verification of sensitivity to commonly used antimicrobial agents, and selection of suitable lysis agents. The detection of lysis takes place on the surface of the biosensor with immobilized bacteria, which has the potential to be used to study biofilms. An example of such a biosensor is surface plasmon resonance (SPR), which records the kinetics of bacterial lysis based on a change in the resonance angle. The bacteria are immobilized on the surface of the SPR chip, and the action of phage as the mass loss is monitored after a typical lytic cycle delay. Atomic force microscopy (AFM) is a technique for imaging of samples on the surface. In contrast to electron microscopy, it has the advantage of real-time imaging in the native conditions of the nutrient medium. In our case, Staphylococcus aureus was lysed using the enzyme lysostaphin and phage P68 from the familyPodoviridae at 37 ° C. In addition to visualization, AFM was used to study changes in mechanical properties during lysis, which resulted in a reduction of Young’s modulus (E) after disruption of the bacterial wall. Changes in E reflect the stiffness of the bacterium. These advanced methods provide deeper insight into bacterial lysis and can help to fight against bacterial diseases.

Keywords: biosensors, atomic force microscopy, surface plasmon resonance, bacterial lysis, staphylococcus aureus, phage P68

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Authors: Agnieszka Krzemińska, Katarzyna Świderek, Vicente Molinier, Piotr Paneth

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In order to understand in details the interactions between ligands and the enzyme isotope effects were studied between clinically used drugs that bind in the active site of Human Immunodeficiency Virus Reverse Transcriptase, HIV-1 RT, as well as triazole-based inhibitor that binds in the allosteric pocket of this enzyme. The magnitudes and origins of the resulting binding isotope effects were analyzed. Subsequently, binding isotope effect of the same triazole-based inhibitor bound in the active site were analyzed and compared. Together, these results show differences in binding origins in two sites of the enzyme and allow to analyze binding mode and place of newly synthesized inhibitors. Typical protocol is described below on the example of triazole ligand in the allosteric pocket. Triazole was docked into allosteric cavity of HIV-1 RT with Glide using extra-precision mode as implemented in Schroedinger software. The structure of HIV-1 RT was obtained from Protein Data Bank as structure of PDB ID 2RKI. The pKa for titratable amino acids was calculated using PROPKA software, and in order to neutralize the system 15 Cl- were added using tLEaP package implemented in AMBERTools ver.1.5. Also N-terminals and C-terminals were build using tLEaP. The system was placed in 144x160x144Å3 orthorhombic box of water molecules using NAMD program. Missing parameters for triazole were obtained at the AM1 level using Antechamber software implemented in AMBERTools. The energy minimizations were carried out by means of a conjugate gradient algorithm using NAMD. Then system was heated from 0 to 300 K with temperature increment 0.001 K. Subsequently 2 ns Langevin−Verlet (NVT) MM MD simulation with AMBER force field implemented in NAMD was carried out. Periodic Boundary Conditions and cut-offs for the nonbonding interactions, range radius from 14.5 to 16 Å, are used. After 2 ns relaxation 200 ps of QM/MM MD at 300 K were simulated. The triazole was treated quantum mechanically at the AM1 level, protein was described using AMBER and water molecules were described using TIP3P, as implemented in fDynamo library. Molecules 20 Å apart from the triazole were kept frozen, with cut-offs established on range radius from 14.5 to 16 Å. In order to describe interactions between triazole and RT free energy of binding using Free Energy Perturbation method was done. The change in frequencies from ligand in solution to ligand bounded in enzyme was used to calculate binding isotope effects.

Keywords: binding isotope effects, molecular dynamics, HIV, reverse transcriptase

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Authors: Rahile Ozturk, Esra Maltas

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This study aimed to determine the effect of vinclozolin on some biochemical characteristics of Galleria mellonella (Lepidoptera: Pyralidae) which is an economically harmful species damaging the honeycomb in beekeeping. For experimental groups, the eggs obtained from stock were dropped into the mixed feed of vinclozolin at different doses (20, 40 and 60 ppm) and had the larvae fed with this feed. As result of the addition of vinclozolin at concentrations of 20, 40 and 60 ppm, glycogen contents of G. mellonella were determined and a significant reduction in the amount of glycogen was observed with increasing concentration of vinclozolin. In this study, activity of catalase enzyme, particularly effective in defense mechanism, activity of xanthine oxidase involved in nucleotide metabolism and activity of glucose oxidase in the metabolism of carbohydrates were measured. When compared with the results from control groups, the enzyme activities of the larvaes fed with the feed including 20, 40 and 60 ppm of vinclozolin were observed to vary or remain constant. Accordingly, glucose oxidase and catalase activities increased with the increase in amount of vinclozolin in the feed and the activity of xanthine oxidase remained stable.

Keywords: Catalase, Galleria mellonella, glucose oxidase, vinclozolin, xanthine oxidase.

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Authors: Aqeel Ahmed Shad, Tanveer Ahmad, Muhammad Farooq Iqbal, Muhammad Javaid Asad

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The supplemental effects of novel protease produced from Bacillus subtilis K-5 and beta-mannanase were evaluated on growth performance, carcass characteristics, nutrients digestibility, blood profile and intestinal morphometry of broilers fed guar meal (Cyamopsis tetragonoloba) based diets with reduced Crude Protein (CP), Essential Amino Acids (EAAs), and Metabolizable energy (ME) contents. One-day old Ross 308 broiler chicks (n=360) were randomly allotted to thirty six experimental units in a way that each of the nine dietary treatments received four replicates with ten birds per replicate. A control diet without guar meal (0GM) was formulated with standard nutrient specifications of Ross 308 for the starter and finisher phases. Two negative control diets, one with 5% (5GM) and second with 10% (10GM) guar meal, were formulated with reduction of 5% CP, 5% EAAs and 80 Kcal/kg ME. These three basal diets (no enzyme) were supplemented with novel protease enzyme (PROT) and commercial beta-mannanase (Beta-M) enzyme. The birds were reared up to 35d of age. The data on weekly body weight gain (BWG) and feed intake were recorded to compute feed:gain for the starter (0-21d) and finisher (22-35d) phases. At the end of 35d of experimental period, four birds per experimental unit were randomly selected for blood samples collection and later slaughtered for ileal digesta, intestinal tract and carcass trait sampling. The data on overall performance (1-35d) indicated improved (P<0.05) BWG and feed:gain in birds supplemented with PROT (1.41% and 1.67) and Beta-M (2.79% and 1.64) than non-supplemented groups. Improved (P<0.05) carcass yield, breast meat yield and thigh meat yield were noted with the supplementation of Beta-M. However, non-significant (P>0.05) effect on carcass traits was noted in broiler fed guar meal based PROT supplemented diets. Crude protein digestibility, nitrogen retention (Nret) and apparent digestibility coefficient for nitrogen (ADCN) were improved (P<0.05) only with PROT. The improvement in apparent metabolizable energy (AME) and apparent metabolizable energy corrected for nitrogen (AMEn) was noted (P<0.05) with both supplemented enzymes. However, no effect (P>0.05) of enzyme addition was noted on blood glucose, total protein and cholesterol. Improved villus height of duodenum, jejunum and ileum was noted (P<0.05) with the addition of both enzymes. The EAAs digestibility was improved (P<0.05) only with PROT. In conclusion, beta-mannanase and protease supplementation better improved the overall bird performance in low nutrient profile guar meal based diets than non-supplemented diets.

Keywords: novel protease, guar meal, broilers, low protein diets, low metabolizable energy diets, nutrients digestibility

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