Showing 3 results for Deep Eutectic Solvent
Fereshteh Rahmati, Amin Tashakor, , ,
Volume 6, Issue 2 (11-2015)
Abstract
Firefly luciferase is a light generating enzyme, which is used in different fields of biotechnology and molecular biology. Luciferase has found widespread applications in many areas of genetic analysis such as detecting gene expression, reporter gene assay and proteomics studies such as protein-protein interactions. Despite many advantages, there are some limitations in luciferase-based systems, the most important of which is its low stability. One of the newly developed methods to solve this problem is to take advantage of Deep Eutectic Solvents (DES). One group of DESs is those that composed of organic salts with hydrogen donor, due to which, intermolecular hydrogen bonds cause lower melting point in comparison with each of the component. In this study, we investigated the effects of DES on kinetic properties of wild type and I232R - E354R/Arg356 mutant Lampyris turkestanicus luciferases. For this, both enzymes, wild type and mutant, expressed in BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Here, we used choline chloride: glycerol as DES. According to the results, the wild type luciferase is much more thermostable in DES than I232R - E354R/Arg356 mutant. Furthermore, the remaining activity of both wild type and mutant luciferases are greater in the presence of DES than those in the absence of DES.
S. Mohseni , Kh. Khajeh, T. Tohidi Moghadam, B. Dabirmanesh, M. Haddadi,
Volume 9, Issue 3 (9-2018)
Abstract
Aims: Matrix Metalloproteinase 9 (MMP-9) plays an important role in the development of many diseases such as periodontitis, atherosclerosis, and cancer. One of the methods for stability of enzyme is using deep eutectic solvents (DESs). The aim of this study was to investigate the effect of deep eutectic solvent on stability and structure of Matrix Metalloproteinase 9 with therapeutic purpose.
Materials and Methods: Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) was expressed in Escherichia coli BL21, using the vector pET21a, and purification and refolding were conducted, using urea gradient method on Ni-NTA column, simultaneously. The effect of DES based on choline chloride and glycerol with a 1:1 mol ratio was investigated on activity, stability, and structure of MMP-9. The enzyme activity at different concentrations of gelatin in the presence of 15% and 30% volume/volume DESs at pH 7.8 was investigated for obtaining Vmax and km by Michaelis-Menten kinetics, using the Prism 5.0 software.
Findings: With an increase in the percentage of solvents up to 30%, the specific activity of enzyme increased, followed by a decreasing trend, and in the presence of a 30% volume/volume solvent at a temperature of 50°C and 60°C, compared with a 15% solvent and no solvent, contained more residue activity. The results showed more solubility of enzyme in 30% solvent.
Conclusion: MMp-9 has the highest activity in presence of 30% volume/volume DES based on choline chloride and glycerol. Increase in thermal stability of MMp-9 can be attributed to compactness of structure in the presence of DES.
Volume 20, Issue 139 (9-2023)
Abstract
Excessive use of antibiotics in the animal husbandry causes the accumulation of their residual amounts in food of animal origin such as dairy products. Since the consumption of these foods has a negative effect on human health, authorized organizations such as the European :union: have set maximum residue limits (MRLs) for antibiotics in food of animal origin. This research aimed to present a magnetic ionic solvent based extraction procedure for preparation and preconcentration of tetracycline, oxytetracycline and enrofloxacin residues in cheese samples and their determination by high-performance liquid chromatography equipped with a diode array detector. For this, the effect of various parameters on the extraction efficiency was investigated and optimized. The results showed that the residual amounts of oxytetracycline and tetracycline in 4 and 5 samples of the tested cheeses, respectively, were determined above the permissible limits. Enrofloxacin residue was not found in any of the samples. From the advantages of the proposed extraction method, we can point out the high separation power and the possibility of highly sensitive analyzing of mixed analytes, so that under optimized conditions, the recovery percentage ranges were 80-91. The limits of detection and quantification were respectively less than 1.8 and 6 ng/g, which is much lower than the MRLs set for target antibiotics in cheese (100 ng/g).
