assessment-of-dna-contamination-in-rna-samples-based-on-ribosomal-dna

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

National Institute of Genetics Engineering and Biotechnology (NIGEB)

Most of the studies in the field of enzyme immobilization have sought to develop a simple, efficient and cost-effective immobilization system. In this study, probiotic Bacillus spores were used as a matrix for enzyme immobilization, because of their inherent resistance to extreme temperatures, UV irradiation, solvents and drying. Above all, their preparation is cost-effective. The alpha-amylase enzyme was immobilized on the spore surface by the covalent and adsorption methods. For the covalent method, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxysulfosuccinimide (NHS) were used. The maximum concentration of the alpha-amylase immobilized by the two methods onto the spore surface was 360 μg/1.2×10(11) spore. However, maximum activity was achieved at an enzyme concentration of approximately 60 μg/.4×10(10), corresponding to an estimated activity of 8×10(3) IU mg(-1)/1.2×10(11) spore for covalent immobilization and 8.53×10(3) for the adsorption method. After washing the enzyme with 1M NaCl and 0.5% Triton X-100, the enzyme immobilization yield was estimated to be 77% and 20.07% for the covalent and adsorption methods, respectively. The alpha-amylase immobilized by both methods, displayed improved activity in the basic pH range. The optimum pH for the free enzyme was 5 while it shifted to 8 for the immobilized enzyme. The optimum temperatures for the free and immobilized enzymes were 60 °C and 80 °C, respectively. The covalently-immobilized alpha-amylase retained 65% of its initial activity, even after 10 times of recycling. The Km and Vmax values were determined by the GraphPad Prism software, which showed that the Vmax value decreased moderately after immobilization. This is the first study which reports the covalent immobilization of an enzyme on the spore surface.

New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization.

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment (74YGSDVT79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (Vmax, Km, kcat and, kcat/Km) and activation energy (Ea) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance.

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

Here, we have studied the role of a histidine residue with the lowest solvent accessibility among other histidine residues at the end of a short connecting structure (189AELH192) of the catalytic domain of the exo-inulinase through creation of H192A mutant. Site-directed mutagenesis method was applied to create the mutant enzyme. Molecular dynamics (MD) simulations, spectroscopic, calorimetric and kinetics analysis were used to study the structural and functional consequences of His192 substitution. Accordingly, the thermo-stabilities and catalytic performance were decreased upon H192A mutation. In silico and experimental approaches evidently confirm that His192 residue of exo-inulinase possesses structural and functional importance regardless of the lack of direct interaction with the substrate or involvement in the catalytic activity of exo-inulinase.

The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase.

Chitinases can inhibit the growth of many fungal diseases which are a great threat for global agricultural production. Biological control of pathogens like fungi, is believed to be one of the best ways to eliminate the adverse effects of plant pathogens. To this end, we expressed and displayed a chitinase from Bacillus pumilus (ChiS) on the surface of Bacillus subtilis spores, as a biocontrol agent.

Article	Year
New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization. Enzyme and microbial technology\| PubMed ID: 25152412	2014
Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase. International journal of biological macromolecules\| PubMed ID: 27527695	2016
The importance of the non-active site and non-periodical structure located histidine residue respect to the structure and function of exo-inulinase. International journal of biological macromolecules\| PubMed ID: 28163127	2017
Display of B. pumilus chitinase on the surface of B. subtilis spore as a potential biopesticide. Pesticide biochemistry and physiology\| PubMed ID: 28755689	2017

Gholamreza Ahmadian

Biography

JoVE Journal Publications

Other Publications