نشریه زیست فناوری دانشگاه تربیت مدرس
سال پنجم شماره 1 (پیاپی 7، بهار و تابستان 1393)

Sugar beet molasses is a well-known، inexpensive and available carbon source for microbial cell growth. Its sugar components are used to produce energy for microbial growth and non-sugar components، especially nitrogen components، have important roles in improvement of cell growth. On the other hand، immobilization of whole cell is establishment and physical limitation of intact cells in specific space that keeps their catalytic activity and provides the possibility of reuse of the cells. This technique allows continuous and accelerated biological processes. It also improves production efficiency and quality and simplifies recycling of product. Immobilized living cells، as controlled catalysts، are able to perform one-step enzymatic reaction and continuous fermentative processes. In this research، E. coli cells were immobilized in calcium alginate hydrogels and using sugar beet molasses as carbon source، were applied for tryptophan production reaction in the presence of its precursors، serine and indole. In comparison between free biocatalysts and immobilized bacterial cells that entrapped in alginate gels، indicated that larger amounts of amino acids (about 42/9%) can produce in calcium alginate. Also the production reaction was followed up for 9 sequential cycles، and results showed that the cells could produce tryptophan amino acid under above conditions. Use of sugar beet molasses (by-product of agriculture industries) for growth of microbial cells and tryptophan production، causes decrease in production cost and more economical production of tryptophan by immobilized E. coli.

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.

The chloroplast gene matK، formerly known as ORF509، has been identified as one of the most rapidly evolving genes at the nucleotide and corresponding amino acid levels. This gene is located in the large single-copy region of the chloroplast genome، and placed between the 5’ and the 3’ exons of trnK (tRNA-lysine) within a group II intron. The matK RNA and protein levels are affected by light and developmental stages، suggesting functional roles for this putative maturase that affect in photosynthesis indirectly. The matK has been considered as one of the most useful genes for resolving phylogenetic and evolutionary relationships at a range of taxonomic levels، from closely related species to the generic، familial، and even supra-familial levels among land plants، especially Angiosperms. The matK as a DNA barcode for land plants showed high levels of discrimination among angiosperm species that can be used single or in combination with other genes.

Mnemiopsin، a Ca2+-regulated photoprotein from ctenophore Mnemiopsis leidyi، as coelentrate photoproteins emits flash blue light upon reacting with coelenterazine. In contrast to coelenterate photoproteins، there is a little information about the structure of chromophore binding site and bioluminescence mechanism in ctenophore photoproteins. In this study، three important amino acid residues in coelenterazine binding cavity of mnemiopsin were substituted by corresponding residues in the well-known coelentrate photoproteins. W59K، N105W and L127W mutants were constructed and characterized for investigation of hydrogen bond network around the important rings of coelenterazine. All three mutants are completely inactivated. In addition، the results of structural studies including CD، intrinsic and extrinsic fluorescence together with theoretical studies showed that these mutants، especially for N105W and L127W، have found different structural features. These results suggest the presence of the residues in binding cavity and/or a mechanistic role for these residues. It seems that arrangement of amino acid residues in the binding cavity of coelenterate and ctenophore photoproteins are different، so that the replacement of these residues with their corresponding residues in other group (such as mutations in this study) perturbs the structural integrity needed for bioluminescence activity.

One of the largest gene families in plants is MYB which contain a DNA-binding domain regulating gene transcription. These transcription factors (TF) have various functions in a wide range of plants. For example، biosynthesis pathway of anthocyanin as secondary metabolites and responsible for coloration in different plant tissues is regulated by the above-mentioned TFs. In the current study، Using homologous genes in the species close to mulberry (Morus Alba)، a fragment of a new MYB transcription factor belonging to MYB gene family was isolated from this plant. First protein sequence of 14 different genes regulating anthocyanin from the species close to mulberry family (moraceae) were selected and then aligned، subsequently the region with the highest similarity was nominated. Following this، the cDNA sequence of these genes was aligned and a location which had a high rate of similarity was considered. With regard to large polymorphism in this sequence، degererate primers were designed and used to isolate the target gene.

Sharks are relatively large sea creatures by an extensive cartilaginous skeleton. The shark cartilage is a rich source of bioactive molecules including collagen protein and glycosaminoglycan. In the present study، Cetyl Piridinium Chloride cationic salt was used for extracting of glycosaminoglycans from dryed cartilage of Carcharhinus dussumieri and their anticoagulant properties were examined. FTIR spectrum was also used to identifing and structurally compare with heparin. The total amount of the extracted glycosaminoglycan was 42. 8 mg/g of the dry cartilage. Also، FTIR spectrum results confirmed the presence of heparin- like compounds in the extract. Finally، the anticoagulant properties of extracted glycosaminoglycans was examined by the Activated Partial Thromboplastin Time anticoagulant test (APTT) method in 410، 763، and 1250 concentrations، and Prothrombin Time (PT) method in the 1250 concentration on the human plasma. The anticoagulant time was 43، 50، and 85s in 410، 763، and 1250 concentrations of extracts، respectively، which extended the coagulation time 1. 3، 1. 5، and 2. 5 folds.

Regarding the importance of inhibiting VEGF and unique features of VHHs as a new generation of antibody-based therapeutics، the present study aimed to generate VHHs against the receptor binding domain of VEGF، thereby blocking of VEGF binding to its receptor. After preparing the gene repertoire of VHH fragments from an immunized camel، a VHH phage display library was constructed. We adopted a stringent successive biopanning to isolate the phages displaying VHH with high affinity to VEGF-RBD. A significant enrichment of phages that specifically bound to the target protein was obtained after six rounds of panning. Of the specific clones with high binding affinity screened by monoclonal phage ELISA، 52% shared the same VHH sequence، showing its high enrichment. Using molecular simulation of antigen-antibody interaction based on the crystallographic information of VEGF/VEGFR2، molecular dynamics simulations and MM/PBSA free energy calculations، we provide a reliable picture of the binding site of antibody on antigen. The key residues in the VEvhh1-VEGF interface were dissected and the energetics was analyzed by MM/PBSA. The results of studies revealed that VEvhh1 binds to the receptor binding site of VEGF with high binding energy and showed the highest affinity to the residues of VEGF which are responsible for VEGF binding to VEGFR2. Also the antibody potently covers these key functional residues of VEGF، thereby inhibiting VEGF binding to its receptor and probably abrogating its biological activity. This study may represent VEvhh1 as an anti-VEGF and anti-angiogenic candidate.