نشریه زیست فناوری دانشگاه تربیت مدرس
سال چهارم شماره 1 (پیاپی 5، بهار و تابستان 1392)

Most patients with liver diseases are in the waiting list of liver implantation for a long period of time because of the lack of enough donors. Liver differentiation potential of Mesenchymal Stem Cells (MSCs) is a new perspective in curing these patients. Tissue engineering improves hepatocyte differentiation by coating the culture surfaces with Glycosaminoglycans (GAGs) such as Heparan Sulfate (HS). Cell detachment and death during hepatogenic differentiation hamper the efficiency of cell therapy. This study aims to establish a matrix. mimicking the liver extracellular matrix، which supports the attachment and proliferation potential of MSCs، as well. Collagen was physically coated on polystyrene plates. Collagen-GAG matrix was constructed by covalently immoblizing the HS molecules on collagen by EDC. Cell attachment and proliferation were evaluated by direct cell-counting and MTT methods. GAG presence on collagen was verified by Safranin O staining. Comparisons showed that the highest attachment belonged to collagen، collagen-HS and polystyrene، respectively. Collagen matrix showed also the highest cell proliferation. Collagen-GAG provided more suitable matrix for cell proliferation compared to polystyrene. The results further showed that biomimicked collagen-GAG matrix supports superior attachment and viability for MSCs compared to polystyrene.

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#).

DNA molecules contain nitrogenous bases that look like fluorophores; however they are weakly or non-fluorescent. Hence، it is important to identify DNA binding-ligands that do not show fluorescence emission in the Free State، though their fluorescence intensity increases upon binding to DNA. Here،we report metal ions (either K and Na) induced guanine quadruplex formation with PS2. M، d (GTG3TAG3CG3T2G2)، which shows peroxidase function when complexed with hemin. Ultravioletvisible absorption spectroscopy revealed activity of the PS2. M oligomer as DNAzyme، and Circular Dichroism spectroscopy showed the formation of G-quadruplex structure of PS2. M. We also studied the intrinsic fluorescence of G-quadruplex forming peroxidase-like DNAzyme. The fluorescence spectra showed increment in the intrinsic fluorescence of folded DNA in comparison with its unfolded structure of the same sequence. Moreover، unsymmetrical cyanine dye (SYBR gold) was utilized as a probe for the study of the extrinsic fluorescence of G-quadruplex DNA، where it could discriminate between the single and four-stranded structures of DNA. Also the G-quadruplex dye interaction was also investigated using Circular Dichroism and Fluorescence spectroscopies.

The production of bioethanol from lignocellulosic biomass could be considered as an appropriate and economic option to remove environmental disasters and improve energy security. In fact، lignocellulosic material is mainly composed of cellulose، hemicellulose and lignin. Lignin works as the adhering prevents the bioconversion of cellulose into sugars and ultimately to ethanol. To address the problem، various chemical، physical، physicochemical and biological methods have been suggested. Enjoying convenient operating conditions، production of non-hazardous wastes، and having no harmful side effects، make the biological methods a potentially proper option. Unfortunately، the biological methods are slower and less efficient in comparison with the other processes. In the present study، an attempt is made to resolve this problem in an enzymatic degradation of lignin of a rice straw sample. Several peroxidase enzymes were produced by a white rot fungus، and their effects on lignin removal from the biomass samples were investigated in shaking flasks. Lignin concentration and enzymes'' activity were measured by the acetyl bromide-soluble lignin spectrophotometric method and optical density method using special reagents، respectively. The results revealed that the enzymatic treatment could remove at least 30% of the lignin content of the lignocellulosic biomass. To achieve the maximum activity of the enzymes، The chemical composition of the culturing medium was optimized for the concentration of important metal ions including Cu2+، Mn2+ and Zn2+ through Box Behnken response surface methodology. The enzymes'' activity at the obtained optimal conditions increased four times for Manganese peroxidase، and lignin peroxidase.

Linum album is an herbaceous and medicinal plant that has important lignan such as podophyllotoxin (PTOX). PTOX has antiviral and anticancer properties. Since the chemical synthesis of PTOX is an expensive process، production of PTOX using cell and cultures of linum species is a cost-effective alternative approach. Various strategies havebeen employed to increase the production of secondary metabolites in cell cultures. In this study، we have verified the effect of chitosan on cell growth، PTOX production in 1، 2، 3 and 5 days after treatment. Cells elicited with chitosan for 5 days yielded the highest amount of PTOX. To study mechanism of chitosan action، expression of phenylalanine ammonio-lyase (PAL)، cinnamoyl-CoA reductase (CCR)، cinnamyl alcohol dehydrogenase (CAD) and pinoresinol lariciresinol reductase (PLR) genes were investigated. The expression of genes were increased، reaching a peak at 3 day after treatment. Chitosan up-regulate the production of PTOX، by effecting on gene expression of PTOX biosynthesis pathway.

The aim of this study is to investigate the relationship between the structure and function of an osteocalcin derived peptide on hydroxyapatite nanocrystal formation. For this purpose،، a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and synthesized in both acidic and amidic forms using solid phase method. Circular dichroism (CD) and electron microscopy were performed to examine the structure and function of synthesized peptides. Moreover، the effect of these peptides on the viability of osteoblast cells was evaluated. Electron microscopy analysis showed the formation of plate-like HA nanocrystals in the presence of amidic peptide. In contrast، amorphous calcium phosphate was formed in the presence of acidic peptide. CD spectra analysis confirmed the random coil structure with lower molar elipticity for amidic peptide. In addition، the amidic peptide significantly increased the proliferation of osteoblast cells. It is concluded that increased bioactivity، which only occurred in amidic peptide is attributable to C-terminal amidation. It is also proposed that peptides with the ability to promote HA formation have the potential to be utilized in hard tissue regeneration high bioactivity and biocompatibility.

The use of enzymes in organic solvents has biotechnological and industrial importance. Organic solvents can decrease the stability of enzymes that is a challenge for the use of enzymes in organic media. There are several approaches such as protein engineering، chemical modification، and use of additives for stabilization of enzymes in organic solvents. In this study، activity and stability of trypsin were investigated in the presence of different organic solvents. Then the effect of sucrose on the stability of the enzyme was investigated in the absence and prescence of solvents. The result showed that the activity and stability of trypsin were decreased in the presence of organic solvents. DMF had a lowest effect on the activity and stability of the enzyme. The use of sucrose increased the stability of trypsin in the presence of organic solvents. The stabilization effect of sucrose in the presence of DMF was more than other solvents. Consequently، a mixture of DMF and sucrose is proposed for the use of trypsin in industrial applications.

Thermolysin is a thermostable protease produced by Bacillus thermoproteolyticus. This enzyme is industrially applicable especially for peptide synthesis. Due to industrial applications، numerous investigations have been performed on thermolysin. In the present study، the role of calcium on thermal، acidic pH، denaturant (urea and SDS) and salt stabilities was studied. In the absence of calcium، t1/2 at 80، 85 and 90 ÛC was 7، 3 and 1 min، respectively. However، in the presence of 10 mM calcium، t1/2 at the same temperatures was >95، 45 and 16 min، respectively. On the other hand، calcium had marginal effect on the pH stability of enzyme. The analyses revealed that SDS and urea contrarily affect on the enzyme stability. The concentration of SDS by which the enzyme activity diminished by half was 0. 1 and 0. 9 (%W/V) in the presence and absence of calcium، respectively. Oppositely، urea did not reduced and even promoted the enzyme activity in both conditions. Stability of thermolysin in NaCl up to 3M was slightly increased in the presence of calcium while slightly decreased in the absence of calcium. At higher concentrations of NaCl، however، the stability was decreased in both conditions. These results reveal that thermal and SDS stability of thermolysin are strongly Ca-dependent، stability against NaCl and urea are moderately Ca-dependent، and its acidic pH stability is Ca-independent.