Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.
Aims: The purpose of this study was to screen the bacteria producing cellulase enzymes and their bioinformatics studies. Background: Cellulose is a long-chain polymer of glucose that hydrolyzes by cellulases to glucose molecules. In order to design the new biotechnological applications, some strategies have been used as increasing the efficiency of enzyme production, generating cost-effective enzymes, producing stable enzymes and identification of new strains. Objective: On the other hand, some bacteria special features have made them suitable candidates for the identification of the new source of enzymes. In this regard, some native strains of bacteria were screened. Method: These bacteria were grown on a culture containing the liquid M9 media containing CMC to ensure the synthesis of cellulase. The formation of a clear area in the culture medium indicated decomposition of cellulose. In the following, the DNA of these bacteria were extracted and their 16S rDNA genes were amplified. Result: The results show that nine samples were able to synthesize cellulase. In following, these strains were identified using 16S rDNA. The results show that these screened bacteria belonged to the Bacillus sp., Alcaligenes sp., Alcaligenes sp., and Enterobacter sp.conclusionThe enzyme activity analysis shows that the Bacillus toyonensis, Bacillus sp. strain XA15-411 Bacillus cereus have produced the maximum yield of cellulases. However, these amounts of enzyme production in these samples are not proportional to their growth rate. As the bacterial growth chart within 4 consecutive days shows that the Alcaligenes sp. Bacillus cereus, Bacillus toyonensis, Bacillus sp. strain XA15-411 have a maximum growth rate. The study of the phylogenetic tree also shows that Bacillus species are more abundant in the production of cellulase enzyme. These bioinformatics analyses show that the Bacillus species have different evolutionary relationships and evolved in different evolutionary time. Other: However, for maximum cellulase production by this bacteria, some information as optimum temperature, optimum pH, carbon and nitrogen sources are needed for the ideal formulation of media composition. The cellulase production is closely controlled in microorganisms and the cellulase yields appear to depend on a variety of factors. However, the further studies are needed for cloning, purification and application of these new microbial cellulases in the different commercial fields as in food, detergent, and pharmaceutical, paper, textile industries and also various chemical industries. However, these novel enzymes can be further engineered through rational design or using random mutagenesis techniques.
Background: The 1,4-alpha-glucan branching protein (GlgB) plays an important role in the glycogen biosynthesis and the deficiency in this enzyme has resulted in Glycogen storage disease and accumulation of an amylopectin-like polysaccharide. Consequently, this enzyme was considered a special topic in clinical and biotechnological research. One of the newly introduced GlgB belongs to the Neisseria sp. HMSC071A01 (Ref.Seq. WP_049335546). For in silico analysis, the 3D molecular modeling of this enzyme was conducted in the I-TASSER web server. Methods: For a better evaluation, the important characteristics of this enzyme such as functional properties, metabolic pathway and activity were investigated in the TargetP software. Additionally, the phylogenetic tree and secondary structure of this enzyme were studied by Mafft and Prabi software, respectively. Finally, the binding site properties (the maltoheptaose as substrate) were studied using the AutoDock Vina. Results: By drawing the phylogenetic tree, the closest species were the taxonomic group of Betaproteobacteria. The results showed that the structure of this enzyme had 34.45% of the alpha helix and 45.45% of the random coil. Our analysis predicted that this enzyme has a potential signal peptide in the protein sequence. Conclusion: By these analyses, a new understanding was developed related to the sequence and structure of this enzyme. Our findings can further be used in some fields of clinical and industrial biotechnology.
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