Modares Journal of Biotechnology

Abstract Botulinum neurotoxins are the most known toxic biological compounds, which cause neuroparalysis. The enzymatic activity of these enzymes causes the inhibition of acetylcholine release. The aim of this study is the recombinant production and high purification of BoNT/A light chain and evaluation of its enzymatic activity. The sequence of this target gene was obtained from NCBI. After codon usage optimization for E. coli, the final gene sequence was ordered for the synthesis on pET28a (+). The recombinant expression vector was transformed into host cell E.coli BL21 (DE3). The expression process was performed under standard conditions. In order to the protein production in a soluble form, optimization of host cell culturing and protein expression was carried out. The expressed protein was purified by Ni-NTA affinity chromatography, and confirmed by specific antibody.In this study, the high yield expression in soluble form was obtained at OD = 0.5, in 0.5 mM IPTG at 18°C in 18 hours. Western blot and ELISA analyses confirmed the BoNT/A light chain.The results indicated that the light chain of BoNT/A was produced in soluble form, and the purification process was performed with high quality so that the final protein was acquired with 98% purity index.

Abstract RNAs play a fundamental role in many biological and medical processes and the activity of RNA is directly dependent to itsstructure. Designing RNA structures is a basic problem in biology that is important in the treatment and nanotechnology. In this regard, some algorithms have been formed to predict RNA secondary structure. In this paper, we present an algorithm to accurately predict RNA secondary structure based on minimum free energy and maximum number of adjacent base pairs. This algorithm stands on a heuristic approach, which employs a dot matrix representation of all possible base pairs in RNA. Afterward, stems are extracted from the dot matrix and decreasingly sorted based on their length. Then the stems with equal length are increasingly sorted according to the free energy. Finally, the stems are orderly selected to form RNA secondary structure. The proposed algorithm is performed on some datasets containing CopA, CopT, R1inv, R2inv, Tar, Tar*, DIS, IncRNA54, and RepZ in the bacteria. Experimental results showed high accuracy of 95.71% of the proposed algorithm. This algorithm is run in lower computational time in comparison to the other similar approaches.

Abstract The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizer. Apart from increasing the stability and catalytic activity, these osmolytes do not change the structure of enzyme. There are a few general schemes about the stabilization mechanism of these osmolytes but the details of their mechanism have not been found so far. In this study, we investigated the simultaneous effects of sorbitol and trehalose on the activity and structural stability of Pseudomonas cepacia lipase (PCL) using UV–visible, fluorescence and circular dichroism (CD) spectroscopy. In order to trace the refractive index and dielectric constant alterations upon the addition of osmolytes, microenvironment of the enzyme (PCL) was studied by means of SPR technique.The results revealed that osmolytes increased catalytic activity and intrinsic fluorescence intensity of PCL. In the presence of both osmolytes the activity of enzyme is greater than when each of the osmolytes is used individally. Far-UV CD spectra indicated that the secondary structural content of protein has been some what increased upon interacting with these osmolytes.The results of SPR technique indicated none of the above osmolytes could change the dielectric constant of medium considerably. This study revealed the synergy of two osmolytes toward increasing the activity and stability of enzyme.

Abstract In biological methods, microorganisms such as bacteria, fungi, actinomycets and yeasts are used to produce metal nanoparticles. Fungi are extremely good candidates in the synthesis of silver nanoparticles because of their ability to secrete large amounts of enzymes. The aim of this study was the biosynthesis of silver nanoparticles by Penicillium spp. isolated from the soil of plump and zinc mine in Zanjan city (Iran). After culturing, growth of colonies and isolation of Penicillium spp., 15 g of the fungal biomass was mixed into 1 mM silver solution for 72 h incubation. The production of silver nanoparticles was characterized by UV-vis spectroscopy, X-ray diffraction(XRD) and transmission electron microscopy. Among the sixteen kinds of isolated fungi, six species were recognized as Penicillium of which just the fungus Penicilliumbrevicompactum was found to be able to produce silver nanoparticles. The production of silver nanoparticles was preliminarily approved by observing the color change of the reaction solution from colorless to yellowish brown. The synthesis of silver nanoparticles was confirmed by observing the characteristic peak at 406-425 nm. The presence of crystalline silver nanoparticles was confirmed by observing peaks in (111), (200), (220), (311) in the XRD analysis. Transmission electron microscopy images showed that silver nanoparticles were produced in the size range of 50 -100 nm in spherical shape mainly extracellular at the surface of mycelium. The fungus was recognized to be Penicilliumbrevicompactumusing slide culture method, growth on Czapek yeast agar and Keratin-sucrose agar.

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.

Abstract Stem cell therapy has been introduced as an innovative and promising treatment in Ischemic diseases. Mesenchymal stem cells are considered for cell therapy to some extent due to their immunemodulatory, differentiation potential, feasibility of isolation and proliferation properties. Stem cells, after transplantation, often encounter harsh and hypoxic environment in ischemic tissues, which leads to cell death and decreased therapeutic efficiency. On the other hand, the fate of stem cell viability and differentiation is still an ambiguous issue in cell therapy regenerative medicine. To overcome this problem, Hypoxic/Ischemic preconditioning has been reported as a powerful tool with beneficial effects on cell survival. The reported master regulator in this process is a transcription factor known as HIF-1α. This study aimed to over-express HIF-1α in mesenchymal stem cells along with eGFP by using lenti viral vectors. Bisistronic expression of eGFP and HIF-1α provides the possibilities of tracking the transplanted cells and mimicking the hypoxic conditions for genetically modified stem cells for future animal model studies.

Abstract Proper solubilization after precipitation and keeping the purified proteins in solution during the whole separation process are very critical to achieve accurate and high resolution patterns in two-dimensional gel electrophoresis (2-DE). Chaotropes and detergents are embedded in the sample and rehydration buffers in order to prevent hydrophobic interactions between the hydrophobic protein domains and avoid loss of proteins due to aggregation and precipitation. Unfortunately, detergents used for IEF must bear no net electrical charge and only week nonionic and zwitterionic detergents may be used in this process. Because of the low solubility of proteins at or very close to their isoelectric point, it seems that choice of chaotropes and detergents can dramatically affect on2D separations, especially in the case of very hydrophobic proteomes. Considering the physico-chemical heterogeneity of tear film protein content, it is deemed that solubilization can play an important role in 2D tear proteome analysis. So herein, we investigated the effect of some various detergents and chaotropes on the solubility of tear proteome during the sample preparation and IEF process. The results illustrated a very poor performance of non-ionic detergents (Triton­X-100 and Tween­80). Zwitterionic detergents (CHAPS and SB­3-10) had a better solubilization power and provided more reliable 2D maps. Last of all a great improvement in spot number and 2D resolution is achieved using a combination of urea/Thiourea in rehydration buffer and application of SDS in the sample buffer with a modified protocol, which ensures complete removal of anionic detergent during the first step of IEF and its replacement with the zwitterionic CHAPS.

Abstract Papain (EC2.22.4.3) is a thiol protease with high level of activity that has widespread industrial applications. The use of immobilized papain provides many advantages over its free form. In many applications, cysteine must be added as an activator. On the other hand, certain bivalent metal ions including Ca²⁺ behave as the inhibitors of papaein. In the present study, after preparation of Sepharose 6B with CNBr, a 5 mg/ml-protein solution was added to activate the gel for covalent attachment of enzyme and, subsequently, 2M glycine solution was added to block the remaining active groups on the gel. The immobilization process brought about significant enhancement of storage, thermal stability, stability at extreme pHs, and resistance against the inhibitory effect of bivalent metal ions with respect to papain. The optimum temperature of papain was increased by 20 °C (from 60 to 80 °C) and its optimum pH was shifted from 7 to 8.0 upon immobilization. Also k_m and k_cat of the enzyme altered due to the immobilization process.These results are important in particular if one considers that the major problem in enzyme immobilization is the loss of enzyme activity and catalytic efficiency.