Modares Journal of Biotechnology

Abstract Ethanol as a renewable biofule is an appropriate and viable alternative to the challenging fossil fuels. Bacillus subtilis, a gram positive bacterium, seems to be a promising choice since it has many useful features. For example B.subtilis ferments broad range of sugars derived from lignocellulosic hydrolysis. Transformation of this cellulytic bacterium to an ethanologenic one was accomplished via metabolic engineering techniques and Ethanol production operon of Z.mobilis was introduced to the B.subtilis. SR1 and SR21 strains expressed plasmid-borne ethanologenic genes of Z.mobilis but the genes had been integrated into the SR22 genomic DNA. Also lactate dehydrogenase gene had been knocked-out in SR21 and SR22 strains. Defect of cell growth in SR21 and SR22, suggests that NAD+ oxidation by lactate dehydrogenase is important for anaerobic growth. Considering the impact of Fe2+ ion on alcohol dehydrogenase II activity, in further experiments Fe2+ was added to the culture media and improvement in growth rates was seen. Final yield of ethanol production of SR1, SR21, and SR22 strains were 53.8%, 86.7%, and 83.9% respectively.

Abstract The increasing rate of depositing new protein sequences and structures to biological databases such as Protein Data Bank (PDB) indicates the importance of structural comparison to explore the evolutionary relationship among different protein families, prediction of function in annotated proteins and classification of protein structure and folds. Due to the high computational cost, protein multiple structural alignment programs in comparison with the conventional multiple sequence alignment programs are slower and represent approximate answers. Therefore, designing new algorithms is an open problem. In this paper, a novel algorithm named PSNetAl for multiple structural alignments of proteins based on graph matching is introduced. PSNetAl inputs are protein structural files in PDB format. Undirected, distance-based graphs are constructed from pdb input files and multiple alignments of graphs are performed by a progressive algorithm. The largest common subgraph as the output of multiple alignment includes the common nodes among all networks. If there is any structural or evolutionary similarity among networks, it will be expected after multiple alignments structural motifs to be present in the largest common subgraph. To evaluate the functionality of PSNetAl, a dataset containing 76 protein families with 50-90% sequence identity and at least 3 members were extracted from HOMSTRAD database. The obtained results show in 67 out of 76 families more than 90% of structural motifs are observed in the largest common subgraph of the multiple alignment.

Abstract One of the newest methods in plant breeding programs is mapping quantitative trait loci (QTL) with molecular markers. In order to identify QTL associated with some chemical traits such as chlorine, nicotine, sugar concentrations and ash in oriental tobacco, a population of 55 recombinant inbred lines coming from the cross Basma seres 31 × SPT406 were evaluated for above mentioned traits. QTL mapping was performed using linkage map developed on 103 recombinant inbred lines by 64 molecular markers including 14 SSR, 24 ISSR and 26 retrotransposone. The linkage map is composed of 7 linkage groups (LGs). Composite interval mapping revealed 5 QTLs associated with studied traits. Phenotypic variation explained by identified QTLs varied between 0.34 and 0.70. Any QTL was not detected for sugar concentration in tobacco leaves. Common markers between some of studied traits can be due to linkage or pleiotropic effects. The common markers lead to increase the efficiency of marker-assisted selection in plant breeding programs via simultaneously selection for several traits.

Abstract Production and characterization of bacterial thermophilic avicelase Fatemeh Azadian, Arastoo Badoei-dalfard*, Abdolhamid Namaki-Shoushtari, Mehdi Hassanshahian Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran Nowadays, developing processes for effectively converting agricultural wastes for production of high value chemicals has gained considerable interest. Avicelases are important industrial enzymes for the most bioconversion processes. In this study, samples were picked up and inoculated in AVI broth for 7 days at 50 ºC. The bacterial strains with the clear halo (represent extracellular avicelase) have been purified. AV8 isolate which showed the highest clear halo was selected for further studies. This strain was identified as Bacillus genus based on biochemical tests and 16S rRNA analysis. Avicelase production was considered under varying environmental parameters. The best carbon and nitrogen sources for maximum avicelase production were 0.5% sucrose and 0.25% yeast extract, respectively. Avicelase from this strain has been partially purified using ammonium sulphate fractionation followed by dialysis and ion exchange Q-Sepharose chromatography. Results showed that enzyme was active and stable between 30-70 ºC and itʼs maximum temperature activity was observed in 70 ºC. The optimum avicelase activity and stability was observed at pH 6.0. These are characteristics indicating that this enzyme could be an acidophilic and thermophilic avicelase. Furthermore, the avicelase activity improved by methanol (138 %) and chloroform (107 %). These results indicated that AV8 avicelase has potential applications in various industries.

Abstract The Cu dependent restriction deoxyribozyme is the unique example of known deoxyribozymes. The uniqueness of this deoxyribozyme is originated from specific cleaving of single stranded DNA and formation of triple helix DNA structure which is necessary for substrate recognition and binding. The most established method for measuring the kinetic parameters of deoxyribozyme is based on use of radiolabeled substrates which have several difficulties. In this study we present accurate, fast and inexpensive methods for kinetic study of the deoxyribozyme which is based on extrinsic fluorescence and UV-visible spectroscopy techniques. As mentioned above, DNA triple helix formation is necessary for substrate identification and also enzyme activity. Circular dichroism spetropolarimetery is used for structural study of enzyme. Analysis of spectrum results from this technique indicates structural changes which is a direct evidence for the triple helix formation in enzyme-substrate complex. Extrinsic fluorescence experiment is based on high affinity of SYBR gold to double stranded DNA compared to single stranded DNA. Enzyme activity can be measured by SYBR gold fluorescence emission upon addition of cofactor to the enzyme-substrate complex. Continuous hyperchromicity assay method which is based on UV-visible spectroscopy was used for measuring of enzyme activity by hyperchromicity of the enzyme-substrate complex after addition of cofactor. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method, because of this method is based on intrinsic physicochemical properties of DNA without interference of external factors.

Abstract In this study, nano drug delivery system based on graphene oxide and reduced graphene oxide- polyglycerol hybrids were constructed. Functionalization of nano graphene oxide and reduced graphene oxide was accomplished through noncovalent interaction between the π conjugated system of graphene materials and the aromatic segment in the focal point of polyglycerol polymer. Polyglycerol is a hydrophilic and biocompatible polymer that its conjugation with graphene materials was increased the colloidal stability and decreased the nonspecific interaction of graphene materials. Curcumin as an anticancer hydrophobic natural drug with low systemic bioavailability was simply loaded on these nanohybrids via π-π stacking force between the π conjugated systems of graphene materials and curcumin. Result showed that loading capacity of curcumin for reduced graphene oxide hybrid (49%) is higher than graphene oxide hybrid due to restored of π conjugated system in reduced graphene oxide. Anticancer efficiency of these drug hybrids was investigated by MTT assay. Results showed that these drug carriers have sufficient biocompatibility. Also these nano drug delivery systems showed a cytotoxic effect that was comparable to that of free curcumin. The reduced graphene oxide hybrid is preferred for delivery of curcumin due to its higher loading capacity that can provide efficient dose of drug in low level of carrier

Abstract Chitin is one of the most abundant renewable polysaccharides in nature that is widely found in the shell of the crustacean, insect cuticle and cell walls of fungi. Due to the unique properties such as biocompatibility, biodegradation and noun toxicity is widely used in various industries. In this study, we used of Banana shrimp, Penaeus merguinsis wastes (particle size 8-10 mm) to extract chitin using microbial fermentation method by Pseudomonas aeruginosa. Demineralization and deproteinization was carried out by incubating shrimp waste inoculated with bacteria at different concentration glucose (0%, 10%, 15% and 20% w/v) and inoculum (10%, 15% and 20% v/v) for 4 day in a shaking incubator (100 rpm) at 30°C. The results showed a direct correlation between the concentration of these parameters and deproteinization and demineralization rate. When studying the effect of these parameters, 20% glucose and 20% of the inoculum was determined as the optimum value, which leads to the production of chitin with a removal of minerals (76%) and protein (86%). Therefore, the microbial fermentation, as an ecofriendly and positive method, can be used to produce a high- quality chitin.

Abstract Immobilization of biosignal molecules including growth factors and cytokines is important for developing biologically active materials, because these materials will have important effects in targetted cell culture, photo- immobilization of visible-light induced crosslinkable biosealant in direct pulp capping material in the dental field, biosealants in tissue engineering and anti-adhesive agents for preventing tissue adhesions after surgery and design and fabrication of biological scaffold contributed to tissue engineering, photolithography. The photo-immobilization of biosignal molecules has more meanings than only immobilization of an enzyme in a bioreactor or ligand-receptor interactions, because the immobilized biosignal molecules work on cells which have very complex structures and functions. This review discusses so far progresses in immobilization of biosignal molecules including growth factors and cytokines with biological and medical applications. At first we will study on photolithography and cell patterning. Then biosignal molecules, photo-immobilization process and co-immobilization will be reviewed. Since material properties of surfaces directly affect the cellular functions and thereby affecting growth patterns, we will study on biological properties of surfaces such as cell adhesion, cell migration and cell growth. Finally different photo reactive biosystems including UV, visible and laser bio systems will be discussed.

Abstract Marine organisms are one of the valuable resource of pharmaceutical. In the last decade, the commercial value of these organisms and the use of compounds derived from them in biological research and drug development, have made them as an important new source for anti-cancer drugs. Microtubules are one of important drug targets in cancer cells therapy and their related inhibitors are being developed widely. In this study the structure of more than 3,000 compounds that contributed marine organism were constructed and optimized by ChemAxon. The affinity of compounds into colchicine/epothilone binding sites in αβ-tubulin structure, was examined using structure-based virtual screening (docking). Results of docking studies were shown that some compounds have high and better binding affinity than colchicine and epothilone inhibitors. MNP14107 and MNP0565 compounds have high affinity for the colchicine and epothilone binding sites respectively. We propose the MNP14107 and MNP0565 compounds as new and the best candidates for the inhibition of tubulin.

Abstract Chitin and chitosan are two very important biopolymer products that have so many usages in the high cost industries. Chitin Converts into chitosan via de-acetylation of chitin. It occurs by alkaline melting method in the absence of oxygen. Chemical structure change, severe environmental pollution and De-polymerization are of the major problems in producing high quality chitosan. In this study for conversion of chitin into chitosan fungus Aspergillus niger strains (ATHUM-10864), the generator of de-acetylases enzymes were used instead of chemicals. Chitosan quality was determined via elemental analysis infrared spectroscopy, X-ray tomography, molecular weight determination and estimation of crystallinity percent, color and molecular structure.The results showed 80±5% efficiency in the conversion of chitin into chitosan or de-acetylation degree of chitin. The gained chitosan contained of 44.4 % carbon, 8.9 % nitrogen, 2.7 % hydrogen and 39.5 % oxygen. The physical characteristics were as 94.5% Crystallinity and pale brown color. The chemical structure of per unit of chitosan was obtained as C6H12NO4. The results showed that replacing biological methods instead of chemicals was possible to access well quality products. It also eliminates the use of chemical materials such as concentrate sodium hydroxide that is damaging the environment.