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 Maltogenic Amylases (MAase) are a subfamily of Į-amylase family that can hydrolyze multiple substrates including starch, pullulan and cyclodextrins however, they prefer cyclodextrins to others, and unlike other Į-amylases, they are intracellular. This enzyme has the potential for use in many industrial processes such as food, fermentation and pharmacy. The effect of different concentrations of Ca2+ and K+ ions on irreversible thermoinactivation of the enzyme at 65 ÛC showed that Ca2+ and K+ decreased and increased its thermal stability. The CD spectra of the enzyme in the presence and absence of metal ions were measured to detect changes in the secondary structure contents. The spectra showed a decrease in the Į-helix content in the presence of 1 and 10 mM of Ca2+, but in the presence of 5 mM, a drastic increase in Į-helix content of the enzyme was witnessed. In the presence of 1 and 5 mM of Na+ the Į-helix content decreased, while it was increased in the presence of 10 mM. The results from intrinsic fluorescence of the protein (excitation at 280 nm) indicated that Ca2+ ion at 1 and 5 mM caused an increase in tertiary structure of the enzyme; however, at 10 mM, a decrease was observed in its tertiary structure. K+ ion at all concentrations increased the tertiary structure of the enzyme. These spectroscopic results are in a good agreement with the thermostability data. It was shown that destabilizing effect of calcium was enthalpic (decrease in ǻH#) whereas the stabilizing effect of potassium was entropic (decrease in ǻS#).

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β- Xylosidase from Selenomonas ruminantium (SXA) is one of the most important enzyme for the hydrolysis of cell wall hemicellulose. SXA has potential utility in industrial processes especially production of bioethanol from bagasse. However, this xylosidase lose activ‌ity drastically above 50 °C. Each monomer of this homotetramer has four free buried cysteine. It seems that cysteine 286 has no role in protein function. In this study, to investigate effects of free buried cysteine on protein thermal stability, Cys 286 was replaced with the same size amino acid, valine. The mutant and native protein have expressed in Pichia pastoris. Kinetic and thermostability parameters of mutant were compared with the wild type enzyme. While pH optimum, temperature profile and catalytic efficiency of recombinant mutant were be found similar to native enzyme, mutant showed about 65% increase in thermostability respect to the wild type at 55 ˚C. Our results showed that free thiol group of cysteine caused the destabilization. Moreover, hydrophobic side chain of valine could involve in a hydrophobic interaction to stabilize SXA. Elimination of a free cysteine enhanced thermal stability without changing the catalytic efficiency of the enzyme that could be very important for biotechnological applications.

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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.

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Enzymes play an essential role in catalyzing the reactions for multiple industrial applications. One of these critical industries with a worldwide application is paper and pulp, which is cost-effective in increasing attention. Xylanases are potential enzymes that proved their abilities in a broad range of applications, specifically in the paper and pulp industry as a biobleaching agent and dye removal biocatalyst. In these decades, the production of novel enzymes from natural sources is conceivable, especially with applying the culture-independent method of metagenome. This practical approach provides the opportunity to identify the novel enzymes from uncultivable microbial diversities. Concerning the importance of the thermostable enzymes for industrial applications and their better action in harsh conditions, this study aimed to identify novel thermostable xylanase from metagenomic data of sheep rumen by applying the in-silico screening. The thermostable xylanase was extracted from the ruminal DNA and after cloning and expression named PersiXyn5. The enzymeschr('39') kinetic parameters, including Km, Vmax, and its specific activity, were examined. The enzyme was optimally active at 80  and pH 8 and could retain 58% of its maximum activity after 2h of incubation at 90 . The thermostable, alkali PersiXyn5 was an efficient enzyme in the paper industry and poultry feed and fuel applications.

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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.

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Application of chemical pesticides has increased significantly worldwide and has raised serious concerns about environmental pollutions. One of the encouraging trends to minimize pesticide risk is production of resistant plants containing toxic proteins against insect pests. Considering the importance of purification and characterization of digestive enzymes in the production of resistant plants, in this study an α-glucosidase from the Naranga aenescens Moore's midgut was purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-sepharose, and concentrating through ultrafiltration. The apparent molecular mass of the enzyme was 48 kDa determined by SDS-PAGE. The optimum pH and temperature of the enzyme were 6.0 and 45°C, respectively. The irreversible thermoinactivation of the enzyme showed that it was highly stable at 35ºC but moderately stable at 40 and 45ºC. Zn²⁺, Hg²⁺, Co²⁺ at 10 and 20 mM, and Ba⁺²only in 20 mM strongly inhibited the α-glucosidase activity. Ba²⁺ and Ca²⁺ only at 10 mM, EDTA and Hg₂²⁺ only at 20 mM and Mg²⁺ at 10 and 20 mM significantly increased the enzyme activity. The K_m and K_cat values for the α-glucosidase were 0.54 mM and 3.62 min^-1, respectively, when p-Nitrophenyl-α-D-glucopyranoside (pNαG) was used as a substrate.