جستجوی مقالات مرتبط با کلیدواژه « luciferase » در نشریات گروه « زیست شناسی »

The conversion of chemical energy to light by a living organism, bioluminescence, is an alluring process that catalyzes by enzymes generically called luciferases. This enzyme has wide applications as a reporter gene in in vivo imaging. In spite of wide range of luciferase applications, some inherent properties limit further application and development of this technology, including the low stability of the enzyme, a low turnover number, and a high Km for the substrate ATP.Due to the low stability of luciferase enzyme, the present study was performed to evaluate the effect of single amino acid mutations on the stability of luciferase enzyme of Iranian species Lamphyris Turkestanius using bioinformatics web servers and molecular dynamics simulation.For this purpose, to determine the stability, the mutations were tested using I-Mutant-2, Pop-music, Cupsat, istable, MUpro and Protparam and foldx web servers under Yasara software. Web servers and foldx predicted that two mutations, Q35L and H9M, would stabilize the structure. The web server Polyphen-2 predicted that these two mutations would not have a damaging effect on enzyme function. To further confirm these two mutations, molecular dynamics simulations were performed. The results of RMSD and RMSF and the radius of gyrus and hydrophobicity also showed that these mutations may cause increase the stability of this enzyme.

The bioluminescence process is a widespread phenomenon in Nature. These enzymes are identified in some domains of life, but the luciferases from the lampyridea genus are considered for biological applications. The molecular cloning of a new type of Iranian firefly luciferase from Lampyroidea maculata was reported, previously. In this study, we analyzed the rare codons of the Iranian insect luciferase gene using the computational databases as ATGme, RACC, LaTcOm, and Sherlocc. Also, the structural modeling process of this enzyme was performed. Next, the status of these rare codons in this structural model was studied using SPDBV and PyMOL software. In the following, the substrate binding site was studied using the AutoDock Vina. By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. AutoDock Vina was used in the molecular docking that recognizes Asp531 that yield closely related to luciferin and AMP binding site.. This bioinformatics analyzes play an important role in the design of new drugs.

Luciferase from firefly Photinus pyralis (P .py) is a peroxisomal enzyme that converts a heterocyclic substrate luciferin to an excited state oxyluciferin in the presence of Mg+2-ATP and O2. Excited oxyluciferin with the emission of visible light is changed to its ground state. The combination of rapidity, sensitivity, and convenience has led to the development of a broad range of luminescence applications. In spite of wide ranges applications, firefly luciferase is unstable against changes in chemical and physical conditions, thereby reduce its precision and sensitivity. The most undesirable instability of the luciferase is low thermostability and high susceptibility to proteolytic degradation. According to previous studies, limited proteolysis by trypsin of P .py luciferase indicated six cleavage sites on two accessible regions: 206-220 (Including K206, R213, and R218) and 329-341 (Including K329, R330, and R337) on N-terminal domain. In this study, we used site-directed mutagenesis to introduce one point mutation on the 329-341 accessible regions of P. py luciferase, in order to investigate the role of R330 on the enzyme structure and function which R330 changed to Q. Based on limited proteolysis data, R330Q mutant didn’t significantly change compared to wild type, but this mutation caused several alterations in enzymatic properties including shifting the pH optimum from 7.5 to 8 and increasing the thermal inactivation. Based on the results, it can be concluded that whilst Arg330 is a conserved residue but not effects on trypsinolysis stability.

The role of Hsp70 chaperone from Rutilus frisii kutum in the thermal inactivation of luciferase in E. coli cell carrying Hsp70 and firefly luciferase was investigated. Material and Methods: Co-transformation of E. coli cell was carried out with two expression vectors containing Hsp70 and firefly luciferase. The co-transformed cells carrying Hsp70 and luciferase were expressed under optimum conditions. After adding tetracycline, the cells were then incubated for 60 min at 40, 42, 44, 46, 48 and 50°C treatments. Finally, the luminescence activity of samples was calculated. After 30 min at 40 and 42°C temperatures, the luciferase activity in control samples reached almost zero, while in the co-transformed samples, about 72% and 60% of the luminance activity maintained compared with control samples (before heat treatment), and even after 60 min, up to 36% and 22% of the activity remained. Also, at 44 and 46°C temperatures in the initial times after stress, a significant difference was observed between the luciferase activity of the co-transformed and the control samples. In contrast, at 48 and 50°C temperatures, the changes of the luciferase activity of co-transformed samples were small compared with control samples even in the early stages of stress. The thermal aggregation of luciferase as a significant reporter protein is inhibited at high temperatures via the activity of Hsp70 in the bacterial cell, which this process can be widely used in the food and pharmaceutical industries and the providing cancer diagnostic kits.

The probability of establishing electrostatic interactions due to the abundance of charged hydrophilic residues and especially arginine is considered the most important thermal stabilizing factor of thermophilic enzymes. The current study was conducted with the aim of comparing thermodynamic stability and kinetic refolding of Lampyris turkestanicus and some of its mutants. In the present experimental thermal stability and the way of refolding Lampyris turkestanicus and 3 mutations, including ERR, ERR/I232R, ERR/Q35R/I182R/I232R were investigated by various spectroscopic techniques. In order to high expression of proteins, a single clone of each sample was selected and inoculated into 10ml of LB culture medium, containing Kanamycin at a concentration of 50μg/mg and incubated at 37°C with an ideal aeration for 12-15 hours. The culture medium was centrifuged for 5 minutes at 5000g at 4°C to provide the cellular contents of the bacteria. The results were obtained through spectroscopic methods of remote and near circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and kinetics experiments, using fluorescence-stopped flow technique. Along with the increase in the number of arginine residues at the protein level, the stability and structural compression of the mutated enzymes in comparison with the wild enzyme were increased and the thermograms obtained from differential scanning calorimetry showed a slight increase in Tm and calorimetric enthalpy of mutated proteins in comparison with wild protein. The rate constant of refolding mutated enzymes has increased compared with the wild type. The improvement of thermodynamic and kinetic parameters results from the improvement of electrostatic interactions, which results in a higher degree of compression and structural density.

Deep eutectic solvents (DES) are significantly important in enzymatic reactions. These solvents increase the efficient contact between substrate and enzyme and therefore, may increase enzyme stability and activity. Bioluminescence is the production and emission of light by a living organism. The principal chemical reaction in bioluminescence involves the light-emitting pigment luciferin and the enzyme luciferase. The enzyme catalyzes the oxidation of luciferin, which requires the energy-carrying molecule adenosine triphosphate (ATP). Because of its easily detectable product, luciferase has found a wide range of application in biotechnology and molecular biology. However, its low thermostability, low turnover number and high Km for ATP restrict further use of luciferase based systems in commercial application. Newly developed method which can be used to increase the stability of proteins and biocatalysts is to take advantage of Deep eutectic solvents (DESs). One group of Deep eutectic solvents are generally composed of organic salts with hydrogen bond donors, as a result of which hydrogen bonds are formed. In this study, we investigated the effects of these solvents on kinetic properties of wild type and E354R/Arg356 mutant Lampyris turkestanicus luciferases in the presence of choline chloride: glycerol with molar ratio of 2:1 as DES. For this, both wild type and mutant, expressed in Escherichia coli BL21, the protein of interest purified through affinity chromatography and used for kinetic studies. Based on the obtained results, DES has positive effect on the thermostability of wild type and mutant luciferases.

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.