Modares Journal of Biotechnology

Abstract Water pollution is one of the most important problems for human beings. BTEX (Benzene, Toluene, Ethylbenzene and Xylenes) have vast application in industry and their carcinogenic effect on human body has approved. Hence these part of water pollutants (water-soluble aromatic components) have more importance. Monitoring systems that can detect presence of BTEX in water supplies are much expensive such as gas chromatography so we need simple systems to reduce the number of samples that we are suspected to them for more analysis by much more expensive systems. Bioreporters are a subgroup of biosensors which are using for sensing and monitoring some signals or reagents. A bioreporter is an organism like a bacteria or a plant that is genetically manipulated to have a promoter which is sensitive to a chemical or physical signal. Activation of the promoter In presense of the signal leads to product of the reporter gene which can be sensed or calculated by our laboratory supplies. A green fluorescent protein gene has been used as a reporter gene downstream of PtbuA1 as a BTEX sensitive promoter in Escherichia coli and its response to BTEX has been investigated in this study. Our results show our bioreporter can sense Toluene.The optimum time and temperature for the bioreporter is also defined.

Abstract Lipases, as an important enzyme group, are able to catalyze hydrolysis or synthesis of esters.The lipase from pseudomonas fluorescens (E.C.3.1.1.3) is a thermophilic kind of lipases (MW around 33 Kd). In this study, the effect of different concentrations of sorbitol on the activity and conformational stability of Psedomonas fluorescence lipase was evaluated using UV/Vis and Circular Dichroism (CD), respectively. According to the results of thermodynamic studies the 0.6 M concentration of sorbitol was selected for refolding and unfolding kinetic measurements with stopped flow fluorescence apparatus. Kinetics data indicate that unfolding of lipase is performed via two different pathways; one of them is probably involves a synchronous unfolding and dissociation of subunits and the other one comprises a two step unfolding in which the subunits are first dissociated followed by complete unfolding of subunits. We found that more population of protein molecules unfolded with slow phase unfolding pathway when sorbitol is present in the unfolding buffer. Furthermore; refolding kinetics data suggest that in the presence of sorbitol the energy barrier of refolding is reduced.

Abstract Laccase enzymes are polyphenol oxidase that catalyze the oxidation of wide range of phenolic components including phenols, polyphenols, aromatic amines and non-phenolic substitution with molecular oxygen as electron acceptor. So these enzymes have biotechnological application such as wastewater treatment system, bioremediation of soil pollution and etc. Result from previous studies showed an increase in thermal stability of bacterial laccase from Bacillus sp. HR03 using site directed mutagenesis and the effect of E188 residue on the surface regions at the interface between domain 1 and 2 in stability was confirmed. The aim of the present work was to investigate the effect of this amino acid substitution on enzyme activity in the presence of dimethyl sulfoxide and dimethylformamide as organic solvents. Compression of kinetic parameters including Kcat / Km ، ∆∆G‡, C50 showed significant increases in the mutant enzyme than wild type enzyme, that industrial application of the enzyme will be easy.

Abstract Laccase (benzenediol: oxygen oxidoreductase), is a multicopper polyphenol oxidase enzyme which has glycoprotein structure. The Researches are indicated that laccase enzyme can play role in detoxification of aromatic pollutants (as petroleum derivatives) and conversion them to less toxic compounds. On the other hand, because of its extensive, fibrous root system; Festuca arundinacea, creates an appropriate environment that causes increased catalysis of petroleum contaminants. Considering the fact that increasing in catalysis of pollutants can be provided by presence and changes in activity of various plant enzymes, in this research; the changes in laccase activity of Festuca's vegetative organs under soil pollution with different concentration of diesel fuel has been investigated. For this purpose, at first, the seeds of Festuca were cultivated in pots containing diesel fuel polluted soils and also control pots in greenhouse conditions. Then in specified time treatments, plants were harvested and plant extract containing laccase were extracted from aerial parts and roots of the plant, separately. After doing centrifuge, changes in enzyme activity were calculated by spectrophotometer. The results show that creation of soil pollution treatments compared with control samples, leads to increase in laccase activity in many cases. In other words, by increasing in laccase activity, the plant will increase its potency of decomposition and assimilation of pollutant hydrocarbons.

Abstract 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.

Abstract 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#).

Abstract One of the most promising strategies in cancer therapy is to induce apoptotic pathway. For this purpose, several constructed agonists of Death Receptor 5 (DR5) are in clinical development. Extrinsic and intrinsic apoptosis pathways of various cancer cells are primarily induced through the activation of the proapoptotic DR5. The extracellular domain of DR5 is comprised of several functional domains, among them the cysteine-rich domains (CRDs) play a critical role in TRAIL-DR5-mediated apoptosis. It has recently been shown that the binding of agonistic monoclonal antibody to another N-terminal domain of DR5 could mediate its activation and apoptosis induction. Variable domains derived from heavy chain antibodies (hcAb) called VHHs or nanobodies are robust, efficient and smallest antigen binding fragments. These unique features of VHHs make them potential therapeutic and diagnostic candidates. In the present study, using phage display technology, a library containing VHH genes was generated of an immunized camel with hapten-peptide 1ITQQDLAPQQRA12 and used to isolate the binders of this peptide. Through screening the phage library, three binders with high binding ability to desired epitope in the NTR region were obtained. Considering to the key role of this epitope in apoptosis inducing, these selected binders could be potential candidates to trigger apoptosis in various cancer cells.

Abstract Biodegradable polymeric nanoparticles are highly regarded in drug delivery due to bioavailability, better encapsulation, controlled release and low toxicity. Drug encapsulation in polymeric nanoparticles may improve the therapeutic effects of these compounds. Polymers are divided in two types: natural and synthetic. Chitosan, as a natural polymer, can have many applications in drug delivery due to good properies. The purpose of this study is to optimization of the production of chitosan nanoparticles for drug delivery. Chitosan nanoparticles were prepared according to ionic gelation method and characterized. Prepared nanoparticle morphology investigated using SEM and particle size distribution, and surface charge and polydispersity index (PDI) were determined by Nanozeta Sizer. FTIR spectra of the lyophilized samples were recorded and proved the formation of nanoparticles. This study has shown that the particle size and zeta potential can be controlled by a change in the ratio of the weight and volume of chitosan and pH adjustment.

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 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.