Modares Journal of Biotechnology

Abstract In this article, the interaction between lysozyme and CdTe nanoparticles was investigated by UV-Vis spectroscopy, fluorescence, thermal stability, kinetics, and circular dichroism (CD) spectroscopic methods at pH 7.25. It was proved that the fluorescence quenching of lysozyme by CdTe NPs was mainly a result of the formation of the CdTe–lysozyme complex. By the fluorescence quenching results, the Stern–Volmer quenching constant (K_SV), binding constant (Ka), and binding sites (n) were calculated. Under pH 7.25 conditions, the level of binding constant is determined to be 2.33×10³ from fluorescence data. The hydrogen bond or van der Waals force is involved in the binding process. The blue shift of the fluorescence spectral peak of protein after the addition of CdTe nanoparticles reveals that the microenvironments around tryptophan residues are disturbed by CdTe nanoparticles. The effect of CdTe NPs on the conformation of lysozyme has been analyzed by means of UV-Vis spectra and CD spectra, which provided evidence that the secondary structure of lysozyme has been changed by the interaction of CdTe NPs with lysozyme.

Abstract Glucoamylase, is an important economic enzyme due to its ability to hydrolyze starch and β-D-glucose polymers. Understanding of factors affecting the thermal stability of the glucoamylase enzyme is critical in the production of isoenzymes with high heat or cold stability. In this study, the effect of temperature on the structure and properties of each of the isoenzymes of the mesophilic, thermophilic and psychrophilic glucoamylase were studied. For this purpose, molecular dynamics simulation was used to assess these factors and structural differences. 240 nanosecond of MD simulation was done for three isoenzymes of glucoamylase in four temperatures at 300, 350, 400 and 450 K. The variations of each of these parameters were compared for three isoenzymes, and it was found that among the computable factors in molecular dynamics simulation, electrostatic energy of protein with water, van der Waals energy between proteins and water, free energy solubility (∆G_solvation), instability parameter, nonpolar solvent accessible surface, and total solvent accessible surface can be used to predict thermal stability of a protein during increase of temperature.

Abstract Some diseases such as gout, the formation of kidney stones, Lesch-Nyhan syndrome, Heart disease, diabetes type II and metabolic syndrome are caused due to the high concentration of uric acid. Within drugs, uricase significantly decreases the level of uric acid in plasma. The production, formulation and preservation proteins need special conditions so that there was no alteration in their structure and highest activity and response, at the same time the lowest immunogenicity can be achieved.In this study, uricase from Aspergillus flavus was cloned and expressed in Escherichia coli BL21. The protein was then purified using affinity chromatography. The enzyme activity and stability were compared with the common industrial Rasburicase. Results showed higher activity and stability at different temperatures (50, 37, 25, 4, and-20°C). Since uricase has an important role in the prevention and cure of mentioned diseases, therefore, the stable form of this enzyme could be a potential candidate for drug development.

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 K_m and increase of V_max in sucrose substrate and 5-fold increase in K_cat/K_m 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.

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.

Abstract Aims: Studies based on thermal stability are considered as one of the methods for investigating the physicochemical properties of proteins in biotechnology. The aim of this study was to evaluate the effect of replacement of Arginine 39 amino acid with lysine on the heat denaturation of mnemiopsin photoprotein 1.
Materials and Methods: In the current experimental study, R39K mutated mnemiopsin was compared with wild protein (in which arginine 39 amino acid was converted to the lysine amino acid). In order to investigate the effect of mutation on the content of the secondary structure, a rotation interpolation method was used. To investigate the possible changes in the rate of thermal stability of mutated and wild proteins, heat denaturation measurements were performed by differential scanning calorimeter. Bioinformatics software were used to compare the structure of two types of proteins.
Findings: The mutated R39K compression decreased in comparison with wild protein. No significant change was observed in the values of thermodynamic parameters, especially T_m. The upward movement of arginine 187 amino acid in the mutated protein decreased the thermal stability of this protein. Increasing the accessible surface of lysine 188 in the mutated protein increased its stability.
Conclusion: In thermal stability of the R39K mutated protein, various factors are effective, including the molecular movements of amino acids, their accessible surface, and the content of the secondary structure of protein stabilizing. This mutation reduces the mutated R39K compression rather than the wild protein; increasing ASA related to Lys188 amino acid in the mutated R39K compared with wild protein increases protein stability, but reducing the amount of secondary structure in this mutated, accompanied by an increase in the molecular upward movement in the Arg187 amino acid serves to reduce the stability of this mutated.

Abstract Aspartic proteases (APs) (EC 3.4.23.X) catalyze the hydrolysis of peptide bonds, a reaction that is fundamental to many biological processes. All of the vertebrate and most of the fungal APs are synthesized as zymogens. Porcine pepsin (EC 3.4.23.1) belongs to the aspartic protease family. Pepsin is a gastric aspartic protease and one of the three principal protein degrading enzymes in the digestive system. Pepsin is an industrial enzyme in the food industry. In this study, thermal stability of pepsin investigated in the different concentrations of aluminium in presence and absence of organic solvents ) butanol, ethanol, 1,4-Butanediol and glycerol). Thermal stability of pepsin increased in the presence of aluminium and decreased in presence of organic solvents ) butanol, ethanol, 1,4-butanediol ) and unchanged in presence of glycerol .Thermal stability of pepsin increased in presence organic solvents with adding of aluminium to its absence. possibly aluminum ions through electrostatic and dative interactions with carboxylate groups of Aspartic acid and Glutamic acid residues are bonded to pepsin structure, and causing to condense enzyme structure which leading to increasing thermal stability of pepsin. Mechanism of increasing thermal stability of pepsin is unknown in presence of aluminium. Therefore, we can reduce the instability of pepsin in presence of organic solvents by
Aluminium.

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.