Carboxyl group modification of Gymnoascella citrina glucoamylase: Cross-linking with hydrophobic nucleophile enhanced thermostability and thermophilicity

Abstracts

E Poster – [A-10-1034-1] Isolation and characterization of an acidophilic amylase from hot mineral spring of Dig Rostam Ahmad Asoodeh, Ashraf Alemi Ferdoosi Mashhad University, Mashhad, Iran E-mail addresses: [email protected] (A. Asoodeh), [email protected] (A. Alemi) Introduction: Amylases have potential application in a wide number of industrial processes such as food, fermentation and pharmaceutical industries. In this study, we purified and characterized an acidophilic amylolytic enzyme from a mesophilic bacterium isolated from Dig Rostam hot mineral spring in Kerman-Iran. Methods: The extracellular extract was concentrated using ammonium sulfate and ultrafiltration. The active enzyme was purified by using a Q-Sepharose ion exchange column. The characterization of the amylase was investigated and the reducing sugar released from starch was measured by Bernfeld method at 540 nm. Results: Biochemical studies showed that the bacterium has properties such as: positive in gram, capsule and indole tests, also negative in mobility, and gelatinase tests. Our findings showed that this amylase has a molecular weight of 69.63 kDa. The optimum pH and optimum temperature of this enzyme were observed to be 4.0 and 45 °C respectively. Conclusion: The isolated enzyme could hydrolyse polymers like starch at pH 4 which may be used in food industries. Furthermore, Iranian hot-springs, like the one at Dig Rostam, have a large amount of microbe storages and can be used as a source for different biological products such as enzymes. Keywords: Amylase, Dig Rostam, Purification, Mesophile bacterium doi:10.1016/j.clinbiochem.2011.08.209

Oral – [A-10-1088-11] Comparison effects of different doses of recombinant erythropoietin on serum paraoxonase and arylesterase activity, in male rats Amir Ghorbanihagjo, Nadereh Rashtchizadeh, Jaber Hashemzadeh Biochemistry Department, Tabriz University of medical sciences, Tabriz, Iran E-mail addresses: [email protected] (A. Ghorbanihagjo), [email protected] (N. Rashtchizadeh), [email protected] (J. Hashemzadeh) Introduction: Erythropoietin (EPO) is a glycoprotein hormone secreted by renal tissue in response to hypoxia and causes an increase in red blood cell (RBC) production. Recent studies demonstrate that EPO has multiple effects such as antiapoptotic, antioxidant, cardioprotective and neuroprotective on the body. Paraoxonase (PON), an antioxidant enzyme secreted by the liver hydrolyses the lipid peroxidation metabolites. This enzyme has a negative correlation with oxidative stress. The aim of this study is the evaluation of recombinant erythropoietin effects on serum PON and AE activity, in rats. Methods: 30 male adult wistar rats, were divided into 3 groups, randomly (n= 10). Groups A (high dose r-EPO) and B (low dose r-EPO) were injected as intraperitoneal (i.p.) for one and four weeks, respectively. Control Group (C) was injected in equal volume normal saline, intraperitoneally. After that, changes in PON and arylesterase(AE) activity, were analyzed. finally, the differences between three groups were analyzed by SPSS 16.0 softwar. Results: Data analyses showed that, in treated groups by high and low doses of r-EPO in contrast with control group, there is a significant increase (P< 0.05) in PON–AE activity, statistically. Also, we observed

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positive correlation between PON and AE activity, significantly in Groups A and B that received high and low doses of r-EPO. In spite of the expensive cost for use of high dose r-EPO in contrast with low dose, its effects are parallel with low dose r-EPO. Thus, the current study doesn't suggest the use of high dose r-EPO. Conclusion: Based on the results of the current study, recombinant erythropoietin(r-EPO) has the antioxidant properties and decrease of lipid peroxidation. Therefore, it can be use in oxidative stress conditions. Keywords: Recombinant erythropoietin (r-EPO), Paraoxonase (PON), Arylesterase (AE) doi:10.1016/j.clinbiochem.2011.08.210

E Poster – [A-10-1119-1] Mercaptopyruvate sulphurtransferase activity in different tissues of pigeons and chickens Hossein Tayefi-Nasrabadi, Reza Rahmani Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran E-mail addresses: [email protected], [email protected] (H. Tayefi-Nasrabadi) Introduction: Cyanide is a cytotoxic compound that rapidly combines with cytochrome oxidase and inhibits respiratory chain, thus preventing intracellular oxygen utilisation. Small quantities of cyanide are physiologically detoxified by detoxifying enzyme such as 3mercaptopyruvate sulphur transferase (3-MST) in the animal body to the less toxic thiocyanate (SCN). In this study, the activity of the 3-MST in the crude extracts of different tissues from pigeons and chickens have been compared. Methods: Tissue (liver, kidney, proventriculus and heart) homogenates were prepared in phosphate buffer (25 mM, pH 7.4) and centrifuged for 15 min at 4000 rpm. For activity assay, reaction mixture in a final volume of 1 mL contained 0.38 M of Tris HCl buffer, pH 7.8, 0.5 M potassium cyanide, 0.3 M mercaptopyruvate and 30 μl of enzyme solution (supernatant). The reaction was carried out for 10 min at 37 °C and stopped by adding 0.2 mL 15% formaldehyde and 1.5 mL of Sorbo reagent. Absorbance was measured at 460 nm. Results: The enzyme was present in all tissue studies, albeit in different concentrations. In pigeon, the highest specific activity of 3-MST was found for proventriculus, thereafter for kidney, liver, and heart. In chicken, 3-MST specific activity in the proventriculus and liver was considerably greater than the values of the kidney and heart. Discussion: This study showed widespread tissue distribution of 3MST activity in both pigeon and chicken. The results suggest that this enzyme may be functional in many physiological activities besides cyanide detoxification. Keywords: Mercaptopyruvate sulphurtransferase, Pigeon, Chicken doi:10.1016/j.clinbiochem.2011.08.211

E Poster – [A-10-1244-1] Carboxyl group modification of Gymnoascella citrina glucoamylase: Cross-linking with hydrophobic nucleophile enhanced thermostability and thermophilicity Mubashir Niaza, Tehreema Iftikhara, Mohammad Hamid Rashidb a Govt. College University, Faisalabad, Pakistan b National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan E-mail addresses: [email protected] (M. Niaz), [email protected] (T. Iftikhar), [email protected] (M.H. Rashid)

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Abstracts

Introduction: Surface carboxyl modification can be used to confer desired property in enzyme. Glucoamylase is an industrially improtant hydrolase. Materials and methods: Three different single modifications of glucoamylases with trimethylamine(TMAC-2, TMAC-6 and TMAC-18) as a whole showed increase in activity as compared to native enzyme. Results: Temperature optima were shifted from 35 °C to 65 °C. Likewise a shift in pH optima, and changed pKa1 and pKa2 of active site residues were observed. Michaelis constants, specificity constants, thermostability and thermophilicity presented activations trend after modification. The coupled enzyme also showed slight activation when exposed to proteolysis and resistance against denaturants like 4 M urea. The data suggests that techniques of surface carboxyl group modification through increased hydrophobic interactions can be employed to enhance thermostability and thermophilicity. Keywords: Gymnoascella citrina, Glucoamylase, Solid state fermentation, Starch hydrolysis, Carboxyl group modification, Enzyme kinetics doi:10.1016/j.clinbiochem.2011.08.212

Structure, Function and Metabolism of Biomolecules E Poster – [A-10-315-1] Non-amyloid fibrils of yeast hexokinase type B have a cross-β structure but weak tendency to thioflavin T and Congo red binding Hassan Ramshini Department of Biology, Payam Noor University, 19395-4697, Iran E-mail address: [email protected] Introduction: Amyloid fibril formation is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology and the biology of living organisms. Images acquired by transmission electron microscopy show that amyloid fibrils are long, rigid, unbranched and usually consist of a number (typically 2– 6) of protofilaments, each about 2–5 nm in diameter. The polypeptides undergoing aggregation are generally small in size. Materials and methods: The fibrils have the ability to bind specific dyes such as thioflavin T (ThT) and Congo red (CR) and are characterized by an extended cross-β structure, as revealed by X-ray fiber diffraction. Results: In the present study we have investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) as a large protein to form amyloid-like fibrils in vitro. The results showed that YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated morphology distinct from typical amyloid fibrils. Analysis carried out with X-ray diffraction confirmed the presence of cross-β structure. Such β-structured aggregates were also biologically harmful as their addition to the extracellular media of cultured human SH-SY5Y cells impaired cell viability to a significant extent. Conclusion: Taking together various findings mentioned above, it is suggested that aggregates must acquire all of the morphological, structural, and tinctorial properties explained above that were allowed to be classified as amyloid fibrils and thus in this case the aggregates were classified as non-amyloid aggregates. Keywords: Non-amyloid aggregation, Yeast hexokinase type B, X-ray diffraction

doi:10.1016/j.clinbiochem.2011.08.213

E Poster – [A-10-507-1] A calorimetric study on the concentration dependent of hemoglobin tetramer assembly Samaneh Zolghadri, Ali Akbar Saboury, Maliheh Sadat Atri Islamic Azad University, Iran E-mail addresses: [email protected] (S. Zolghadri), [email protected] (A.A. Saboury), [email protected] (M.S. Atri) In combination with structural information and spectroscopic results, isothermal titration calorimetry provides a thorough description of the interactions of biological macromolecules. As a practical application of the method, we describe the use of ITC to study the interaction between subunits of hemoglobin and determine the concentration of hemoglobin tetramerization and association. In the solution, subunit assembly and the ratio between dimer and tetramer of Hemoglobin depends on its concentration. We have measured the self-association kinetics over a range of concentration from about 0.1 to 10 mM where the sample is predominantly monomer to where it is predominantly tetramer. Very good agreement with literature values has been obtained. Keywords: Hemoglobin, Self-association, Isothermal titration calorimetry doi:10.1016/j.clinbiochem.2011.08.214

Oral – [A-10-704-2] Effect of G-quadruplex structure on the peroxidase-like DNAzyme activity Zahra Karamia, Bijan Ranjbara, Arastoo Badoei-Dalfardb a Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran b Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran E-mail addresses: [email protected] (Z. Karami), [email protected] (B. Ranjbar), [email protected] (A. Badoei-Dalfard) Introduction: In cells, DNA typically consists of two antiparallel strands arranged in a double-helical structure. In laboratories, DNA can be readily synthesized as a single-stranded polymer that can adopt many other types of structures including some that have been shown to catalyze chemical transformations. These catalytic DNA molecules are commonly referred to as DNAzymes. The complexes formed by hemin and some G-quadruplexes can be developed as a new class of DNAzyme with peroxidase activity. But to date, the actual G-quadruplex structure that can provide hemin with enhanced peroxidase activity is in doubt. Materials and methods: Colorimetric analyses of hemin–aptamer interaction characterized by UV–visible Spectroscopy Measurements of the reaction kinetics were performed by monitoring the appearance of the ABTS radical cation(ABTS) at 421 nm using a UV–Vis Spectrophotometer.The structure–function relationship of G-quadruplex– hemin complexes with peroxidase activity was studied by circular dichroism (CD) spectra of PS2.M, an 18-nucleotide DNA oligomer. Results: Compared with other reported CD results revealed that PS2.M may be a unimolecular parallel quadruplex rather than a unimolecular antiparallel quadruplex or a multistranded parallel quadruplex. Also catalytically active form of peroxidase-like DNAzyme with parallel structure exhibited increased catalytic activity compared to the other structures. Conclusion: According to the experimental results, the formation and strands orientation of G-quadruplexes are crucial factors in determining the peroxidase activity of G-quadruplex–hemin complexes. The additions of hemin to G-quadruplex solutions did not have a significant influence on the CD spectra of the parallel G-quadruplex