Showing 6 results for Site-Directed Mutagenesis
Roseata Zonouzi, Khosro Khajeh, Majid Monajemi, Naser Ghaemi,
Volume 3, Issue 2 (11-2012)
Abstract
In the Bacillusamyloliquefaciens α-amylase (BAA), the loop (residues from 177-185; region І) is the constructive part of the cage responsible for attachment to calcium. It has two more amino acid residues than the α-amylase from Bacillus licheniformis (BLA). Arg176 in this region makes an ionic interaction with Glu126 from region ІІ (residues 118-131) but this interaction is lost in BLA due to substitution of R176Q and E126V. It is the common feature of α-amylases that calcium ion is required for their thermal stability. The present work quantitatively estimates the effect of ionic interaction on the overall stability of the enzyme. To clarify the functional and structural significance of corresponding salt bridge, first an automated homology model of the mutant enzyme (∆E126) was built by the Swiss-Model Protein Modeling Server. Bacillus amyloliquefaciens α-amylase (3BH4.pdb) was used as the template and examined by GETAREA and WHAT IF programs, then Glu126 was deleted (∆E126) by site-directed mutagenesis and the thermostability was examined for the wild-type and mutant enzymes. Modeling results showed that deletion of salt bridge affected on the hydrophobic and hydrophilic residues orientation of two discussed regions (Ι, ΙΙ). The mutant enzyme also exhibited lower thermostability relative to the wild-type enzyme. Thus, it may be suggested that salt bridge could affect on accessible surface area of the discussed regions, decrease water diffusion, prevent diffusion of cations and improve the thermostability of the whole protein.
, , Reza Hassan Sajedi, , , ,
Volume 5, Issue 1 (11-2014)
Abstract
Mnemiopsin, a Ca2+-regulated photoprotein from ctenophore Mnemiopsis leidyi, as coelentrate photoproteins emits flash blue light upon reacting with coelenterazine. In contrast to coelenterate photoproteins, there is a little information about the structure of chromophore binding site and bioluminescence mechanism in ctenophore photoproteins. In this study, three important amino acid residues in coelenterazine binding cavity of mnemiopsin were substituted by corresponding residues in the well-known coelentrate photoproteins. W59K, N105W and L127W mutants were constructed and characterized for investigation of hydrogen bond network around the important rings of coelenterazine. All three mutants are completely inactivated. In addition, the results of structural studies including CD, intrinsic and extrinsic fluorescence together with theoretical studies showed that these mutants, especially for N105W and L127W, have found different structural features. These results suggest the presence of the residues in binding cavity and/or a mechanistic role for these residues. It seems that arrangement of amino acid residues in the binding cavity of coelenterate and ctenophore photoproteins are different, so that the replacement of these residues with their corresponding residues in other group (such as mutations in this study) perturbs the structural integrity needed for bioluminescence activity.
, , , , , ,
Volume 5, Issue 1 (11-2014)
Abstract
Laccase enzymes are polyphenol oxidase that catalyze the oxidation of wide range of phenolic components including phenols, polyphenols, aromatic amines and non-phenolic substitution with molecular oxygen as electron acceptor. So these enzymes have biotechnological application such as wastewater treatment system, bioremediation of soil pollution and etc. Result from previous studies showed an increase in thermal stability of bacterial laccase from Bacillus sp. HR03 using site directed mutagenesis and the effect of E188 residue on the surface regions at the interface between domain 1 and 2 in stability was confirmed. The aim of the present work was to investigate the effect of this amino acid substitution on enzyme activity in the presence of dimethyl sulfoxide and dimethylformamide as organic solvents. Compression of kinetic parameters including Kcat / Km ، ∆∆G‡, C50 showed significant increases in the mutant enzyme than wild type enzyme, that industrial application of the enzyme will be easy.
S. Arjmand, L. Ghobadi , S.o. Ranaei-Siadat , Y. Sefidbakht, F. Farzaneh,
Volume 9, Issue 3 (9-2018)
Abstract
Aims: Invertase is an enzyme that is widely used in industries. The main source of industrial production of invertase is yeast Saccharomyces cerevisiae (S. cerevisiae). Increasing thermal stability makes an important contribution to improving productivity in related production. The aim of this study was increasing thermal stability of Saccharomyces cerevisiae recombinant protein invertase by site-directed mutagenesis.
Materials and Methods: In the present experimental study, using invertase enzyme from thermophilic bacteria, Thermotoga maritima as template, it was decided to replace the threonine 345 and asparagine 349 amino acid with alanine, using site-directed mutagenesis and in Pichia pastoris, cloning was performed with the SOEing polymerase chain reaction. The activity of natural and mutant recombinant invertase enzymes at different temperatures, different pHs, stability duration, and thermal-performance stability, and Michaelis–Menten kinetics were drawn.
Findings: The thermal-structural stability of the natural and mutant invertease enzymes at 55°C showed that the mutant enzyme had a higher thermal stability at 55°C compared with the natural enzyme. Both natural and mutant enzymes exhibited a similar trend in functional stability. Reduction of Km and increase of Vmax in sucrose substrate and 5-fold increase in Kcat/Km ratio of mutant enzyme was observed.
Conclusion: Site-directed mutagenesis has no negative effect on the amount of production as well as the secretion of recombinant protein invertase and increases enzyme activity. The mutant enzyme has a higher structural stability than the natural enzyme without altering its functional stability.
S. Jarchi , Farangis Ataei, S. Hosseinkhani,
Volume 10, Issue 2 (7-2019)
Abstract
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.
Vahab Jafarian, Elahe Karimipour,
Volume 14, Issue 1 (3-2023)
Abstract
Nowadays, the peptides and proteins possessing anti-cancer, anti-allergic and anti-inflammatory properties are used for disease treatment. Brazzein is a sweet protein containing 54 amino acids and according to reports, it has anti-cancer properties based on sequence and structurehas sequence. In this study, the role of position 40 aspartate in the structure and function of wild brazzein protein and mutants as well as the anti-cancer properties of the peptides obtained on the TLR5 receptor were investigated. For this, several models of mutated forms were designed and constructed using Modeller.v.9.20 software. Then, the accuracy of the models and the physico-chemical properties of wild type (WT) and mutants of D40N, D40R and D40Deletion were evaluated using various bioinformatics servers and softwares including ProtParam, ProtScale, SAVES, PIC, ModEval, and PredyFlexy. For predicting anticancer properties, the sequence of WT protein and mutants was examined and compared using ACPred and iACP servers. The quality and analysis of WT protein and mutants binding as a ligand with TLR5 receptor, triggering an anti-cancer signaling pathway, were investigated through molecular docking using HADDOCK software.The results of bioinformatics parameters analysis indicated the possibility of improving the stability of brazzein structure and function, and the probability of increasing the available surface to bind to the receptor. Moreover, based on the results of molecular docking analyses, the ability binding TLR5 receptor was higher in D40R than the other proteins indicating an increased probability in anti-cancer properties of the mutant.
