Ahmet Colak

Karadeniz Technical University, Atrabazandah, Trabzon, Turkey

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Publications (26)25.46 Total impact

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    ABSTRACT: The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The K m and V max values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 μmol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.
    Applied Biochemistry and Microbiology 01/2014; 50(1). · 0.69 Impact Factor
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    ABSTRACT: The fatty acid and amino acid compositions of 11 mushroom species commonly consumed were collected from the East Black Sea region of Turkey and analyzed. All species were characterized by a high content of linoleic acid (C18:2n-6) and glutamic acid. The highest content of linoleic acid (78.0%) and glutamic acid (29.4 μg/mg dry weight [d.w.]) was found in Agaricus arvensis and the lowest in Cantharellus tubaeformis, 19.8% and 10.9 μg/mg d.w., respectively. The average content of amino acids for all species was 148 μg/mg d.w. Overall, these results demonstrate that the 11 different kinds of wild edible mushrooms gathered from the region represent substantial sources of fatty acids and amino acids that are essential in the diet of humans. Quality of the mushroom protein compares favorably with the FAO/WHO Standard. The present study demonstrates that macrofungi from the East Black Sea region (Turkey) are a good source of many nutrients essential to human well-being.
    International Journal of Food Sciences and Nutrition 12/2010; 62(4):328-35. · 1.26 Impact Factor
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    ABSTRACT: Some homo- and heteronuclear Cu(II) and Ni(II) complexes of new oxime-type ligands were tested against several pathogenic microorganisms to assess their antimicrobial potentials. The antimicrobial activities of complexes were evaluated in terms of minimum inhibitory concentration (MIC; μg/μL) and it was observed that the complexes possess moderate antimicrobial properties. The binding of the complexes with DNA were also investigated by using UV-Vis spectroscopy. It was seen that three of the complexes could bind to DNA through an intercalative mode while the other complexes could have other mechanisms. Furthermore, the antioxidant efficiencies of the metal complexes were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radical scavening activities. Due to the observed IC(50) values, they are potential drugs to eliminate the radicals.
    European journal of medicinal chemistry 11/2010; 45(11):5169-75. · 3.27 Impact Factor
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    ABSTRACT: Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The Km and Vmax values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.
    Food Chemistry. 01/2010; 119(3):1044-1049.
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    ABSTRACT: A crude extract was prepared from the fruiting body of Lepista flaccida, an edible mushroom and endoglucanase activity of the extract was increased 14-fold with ammonium sulphate precipitation. Maximum enzyme activity was seen at pH 4.0 and 50°C when carboxymethylcellulose was used as a substrate. K0.5 and Vmax values of the partially purified endoglucanase were 7.7mg/ml and 25±0.9U/mg protein, respectively. The enzyme was quite stable over a broad range of pH (2.0–9.0) at 4°C. When it was incubated at temperatures between 20°C and 60°C for 12h, it conserved much of its original activity (over 40%). The activity of the enzyme increased by 234±3.6% in the presence of 1mM Mn2+. The endoglucanase was inhibited by EDTA, PMSF, β-ME and DDT. In conclusion, pH and thermal stability of the L. flaccida endoglucanase could make it useful for industrial purposes.
    Food Chemistry - FOOD CHEM. 01/2010; 123(2):291-295.
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    ABSTRACT: Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes, belong to glycoside hydrolase family 13. A gene corresponding to MA in Geobacillus caldoxylosilyticus TK4 (GcaTK4MA) was cloned into pET28a(+) vector and expressed in Escherichia coli with 6xHis-tag at the N-terminus. Herein, we report on the biochemical properties of a new thermo- and pH-stable MA. GcaTK4MA has similar properties those of other MAases in terms of the primary structure, preference for CD over starch and having an extra domain at its N- and C-terminals. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. The purified enzyme exhibited optimal activity for β-CD hydrolysis at 50 °C and pH 7.0. When the enzyme was separately incubated at 4 °C and 50 °C in the buffer solutions (pH 3.0–9.0) up to 7 days, it was seen that the enzyme had the higher stability at 50 °C than 4 °C. The enzyme retained about 80% of its original activity when it was incubated at 50 °C for 7 days. The enzyme activity was significantly inhibited by SDS and EDTA at the final concentration of 1%. These results suggest that this is the first reported MA having an extremely pH- and thermal stabilities.
    Process Biochemistry. 01/2010;
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    ABSTRACT: Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p-nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p-methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases.PRACTICAL APPLICATIONSEsterases catalyzing the cleavage and formation of ester bonds are known α/β-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications.
    Journal of Food Biochemistry 06/2009; 33(4):482 - 499. · 0.76 Impact Factor
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    ABSTRACT: Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase. Phosphotriesterase is a hydrolytic, bacterial enzyme with unusual substrate specificity for synthetic organophosphate triesters, common constituents of chemical warfare agents and agricultural pesticides. PHP may belong to the family of proteins from which phosphotriesterase evolved. The PHP gene from the thermophilic bacterium Geobacillus caldoxylosilyticus TK4 was cloned and overexpressed in Escherichia coli with 6×His tag in the N-terminal. The recombinant protein was purified with nickel affinity chromatography and characterized in detail. The enzyme did not have any activity against paraoxon. The highest activities were observed with p-nitrophenyl acetate (pNPA) and p-nitrophenyl butyrate. pH and temperature optima were determined as 8.0 and 50 °C, respectively, with pNPA. The enzyme activity was not largely affected by the incubation of the enzyme at 50 °C in the different buffer solutions (pHs between 3.0 and 9.0) for 7 days. After the incubation at 90 °C for 7 days, G. caldoxylosilyticus TK4 PHP retained 62% of its original activity. The enzyme was also resistant to some metal ions and organic solvents. These results suggest that this is the first reported PHP having an extremely pH- and thermo-stable esterase activity.
    Process Biochemistry. 01/2009;
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    ABSTRACT: Characterisation of esterase activities from the edible mushroom species, Amanita vaginata var. vaginata and Tricholoma terreum, were investigated. Native electrophoresis of the crude extracts prepared from both mushroom samples showed the presence of esterolytic activities. The extracts had the greatest activity in the presence of p-nitrophenyl butyrate (pNPB) as a substrate. pH and temperature optima were found to be 8.0 and 30°C for both enzymes, respectively. Vmax and Km values were determined as 14.2U/l and 71μM for A. vaginata var. vaginata and 34.6U/l and 9.6μM for T. terreum, respectively. The pH-stability profile showed a stationary line between 3.0 and 10.0 for both enzymes. The esterolytic activities from the extracts were maintained between 10 and 40°C for 4h and started to decrease at 50°C. The effects of EDTA, NaN3, DTT and PMSF on the enzyme activity were also investigated.
    Food Chemistry - FOOD CHEM. 01/2009; 115(4):1486-1490.
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    ABSTRACT: Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The Vmax and Km value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu2+, Hg2+ and Al3+, for stage 2, Cu2+ and Hg2+, and for stage 3, Cu2+, Hg2+, Al3+ and Ca2+ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.
    Food Chemistry. 01/2008;
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    ABSTRACT: A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and V(max) and K(m) values of its activity were found to be 800 U/L and 176.5 microM, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was 60-80 degrees C and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at 30-70 degrees C for 1 h, the enzyme activity was fully lost at 80 degrees C for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.
    Journal of biochemistry and molecular biology 08/2007; 40(4):588-94. · 2.02 Impact Factor
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    ABSTRACT: The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.
    Journal of Biochemistry 07/2007; 141(6):817-25. · 2.72 Impact Factor
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    ABSTRACT: Crude enzyme extracts were prepared from Armillaria mellea (A. mellea), Lepista nuda (L. nuda) and Hypholoma fasciculare (H. fasciculare), which were harvested from the Lişer High Plateau-Maçka (Trabzon, Turkey). The crude polyphenol oxidase (PPO) extracts from each mushroom were highly active against 4-methylcatechol. Native polyacrylamide gel electrophoresis, stained by L-3,4-dihydroxyphenylalanine, showed the polyphenol oxidase potentials. The optimum pH value, for each enzyme, was 7.0. When enzyme extracts were incubated at pH 7.0 for 24 h at 4 °C, it was observed that L. nuda and H. fasciculare enzyme activities decreased by about 26% and 18%, respectively, but, A. mellea enzyme activity increased by about 11%. The temperature optima of A. mellea, L. nuda and H. fasciculare were, respectively, 30, 30 and 20 °C. Cr3+ and Cu2+ ions inhibited each activity. Also, sodium metabisulphite and ascorbic acid were strong inhibitors of the enzyme activities.
    Food Chemistry. 01/2007;
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    ABSTRACT: In this study, Macrolepiota mastoidea, a wild edible mushroom, was evaluated for its polyphenol oxidase potential. Native electrophoresis, stained by l-dihydroxyphenylalanine, of the crude extracts from this species showed two bands having Rf values of 0.38 (minor) and 0.50 (major), supporting a polyphenol oxidase potential. The crude extracts showed monophenolase activity against 3-(4-hydroxyphenyl)-propionic acid and diphenolase activity against 4-methylcatechol as substrates. Monophenolase and diphenolase activities of enzyme extract prepared from M. mastoidea showed pH optimum values at pH 6.0 and pH 4.0, respectively. The extracts retained about 100% and 60% of their original monophenolase and diphenolase activities at their optimum pH values, respectively. It was estimated from thermodynamic data that M. mastoidea had a thermostable monophenolase activity. Thiourea and ascorbic acid were highly potential inhibitors for monophenolase, and ascorbic acid and sodium metabisulphite for diphenolase activity. It is clear from the present results that the enzyme extracts prepared from M. mastoidea possess polyphenol oxidase activities with interesting properties.
    Food Chemistry. 01/2007;
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    ABSTRACT: Diphenolases from Anoxybacillus kestanbolensis strains K1 and K4T, highly active against 4-methylcatechol were characterized in terms of pH- and temperature-optima, pH- and temperature-stability, kinetic parameters, and inhibition/activation behaviour towards some general polyphenol oxidase (PPO) inhibitors and metal ions. The temperature-activity optima, for Anoxybacillus kestanbolensis K1 and K4T catecholases in the presence of 4-methylcatechol, were 80 and 70C, respectively. Although catecholase from A. kestanbolensis K4T lost no activity after a period of 1 h incubation at its optimum temperature, the enzyme pH from K1 was stimulated by keeping at 80C. Both of the enzymes possessed pH optima at 9.5, and the pH-stability profiles showed that cathecholases from both preparations retained their activities at alkaline pH values. Both A. kestanbolensis K1 and K4T catecholase activities were totally inhibited by addition of 0.01mM sodium metabisulphite, ascorbic acid and l-cysteine. 1mM Mn2+ increased the activities of A. kestanbolensis K1 and K4T catecholases by 6.4- and 5.3-fold, respectively. These results indicate that both A. kestanbolensis K1 and K4T strains possess thermo- and alkalostable catecholases.
    World Journal of Microbiology and Biotechnology 05/2005; 21(4):501-507. · 1.26 Impact Factor
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    ABSTRACT: Poly-3-hydroxybutyrate (P3HB) degradation capabilities of a novel bacterium, Anoxybacillus gonensis G2, were investigated. Both changes on film surfaces of the solution-cast films monitored by scanning electron microscopy (SEM) and weight loss up to 24% after 72 h exposure to A. gonensis G2 cultures indicated secretion of an active esterase responsible for the degradation of P3HB films. Kinetic parameters, Vmax and Km for the esterase activity of crude enzyme from A. gonensis G2 in the presence of p-nitrophenylbutyrate as substrate were observed as 50 U/L and 0.125 mM, respectively, in 50 mM phosphate buffer, pH 7.5 at 60 degrees C. The stimulation of the activity by Ca2+ is an evidence for the requirement of Ca2+ as a cofactor for the enzyme activity which is a characteristic for lipases/esterases. Inhibition of the esterase activity by metal chelating agents such as ethylenediamine tetraacetate, azide and cyanide has also supported the requirement of a metal ion for the activity. The thermal and pH stability profiles for the enzyme showed that the thermophilic bacterium A. gonensis G2 secretes an extracellular thermoalkalophilic PHB depolymerase active at 60 degrees C, and stable at this temperature for 120 min at pH 7.5 and for 24 h at pH 7.5-9.5 range at 4 degrees C by retaining over 75% of its initial activities.
    Bioresource Technology 01/2005; 96(5):625-631. · 4.75 Impact Factor
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    ABSTRACT: Diphenolases from two cherry laurel cultivars (Laurocerasus officinalis Roem. ‘Globigemmis’ and ‘Oxygemmis’) were highly active against 3-(3,4-dihydroxyphenyl)propionic acid (DHPPA) at acidic pH values with temperature optima of 50 and 40 °C, respectively. Although the pH-stability profiles showed that both enzymes were fully stable at pH 7.0, their stabilities decreased significantly at alkaline pH values. Thermal-stabilities of the cherry laurel diphenolases indicated that enzymes from the two cultivars share similar thermodynamic properties and heat-sensitivities as a result of heat-inactivation. In addition, ascorbate and metabisulfite, at 1 mM final concentrations, almost completely inhibited the oxidation of DHPPA by the enzymes, indicating the sensitivities of the cherry laurel diphenolases from the two cultivars towards general Polyphenol oxidases inhibitors. It can be concluded that the crude enzymes prepared from the cherry laurel fruits of the two cultivars, at an early stage of development, possess diphenolase activities sharing similar behaviours.
    Food Chemistry. 01/2005;
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    ABSTRACT: Nucleolytic activities of novel mononuclear Cu(II), homo- and heterodinuclear Cu(II)–Ni(II) complexes with two diester-type ligands were investigated on pCYTEXP by neutral agarose gel electrophoresis. The analyses of the cleavage products obtained electrophoretically indicate that the examined complexes induce very similar conformational changes on supercoiled DNA by converting supercoiled form to nicked form. At concentrations greater than 100M, the complexes possessed effective nucleolytic activities for 10min of incubation time. However, their nucleolytic activities did not increase significantly with longer periods of incubation. The pH-nucleolytic activity profiles of the complexes differed significantly. Metal complex induced DNA cleavage was also tested for inhibition by various radical scavengers. It could be proposed from the data that diffusible intermediate oxidants are not involved in these reactions or they are not necessary for DNA cleavage since none of antioxidants inhibited DNA cleaving activities of the complexes.
    Monatshefte fuer Chemie/Chemical Monthly 07/2004; 135(8):1023-1031. · 1.63 Impact Factor
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    ABSTRACT: A diphenolase from persimmon fruits (Diospyros kaki L.), active against catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine and 3-(3,4-dihydroxyphenyl)propionic acid, was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour toward some general PPO inhibitors. The substrate specificity of the persimmon PPO was very high for catechol and 4-methylcatechol. The enzyme was extremely stable at its optimum pH of 7.5 in the presence of 4-methylcatechol as the substrate and it retained over 90% of its original PPO activity after 24 h of incubation at 4 °C at that pH. Thermal properties of the persimmon enzyme, together with thermodynamic parameters, show that the persimmon enzyme is very heat-sensitive. Ascorbic acid, metabisulfite, azide and benzoic acid all inhibited the 4-methylcatechol oxidation by persimmon PPO, indicating its sensitivity toward the general PPO inhibitors, especially metabisulfite. All the data support the presence of a highly active diphenolase in persimmon fruits (Diospyros kaki L.) having similar properties to other plant polyphenoloxidases.
    Food Chemistry. 01/2004; 85(3):431-437.
  • Ahmet Colak, Saadettin Güner
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    ABSTRACT: Degradation of polyhydroxyalkanoate (PHA) by three Pseudomonas spp., isolated from fuel-oil contaminated soil and identified as Ps. fluorescens, Ps. aeruginosa and Ps. putida on the basis of 16S rRNA sequence analysis, was investigated. Degradation of PHAs was determined as molecular weight loss measured by gel permeation chromatography and morphological changes in poly-3-hydroxybutyrate films observed by scanning electron microscopy. Both, decrease in molecular weight (7–17%) and weight loss (up to 29%) of solution-cast films were evidence of the isolates secreting an active depolymerase responsible for degradation of PHA. The kinetic parameters, Vmax and Km, for depolymerase activity of Ps. aeruginosa in the presence of p-nitrophenylbutyrate as substrate were determined to be and , respectively, in phosphate buffer, pH 7.9, at 30°C. Stimulation of activity by Na+, K+, Ca2+ and Mg2+ at concentration indicated a requirement for metal ions as a cofactor for activity. Inhibition of the extracellular depolymerases of the three strains in the presence of p-methylphenyl sulfonylfluoride, cyanide, azide, deoxycholate or EDTA confirms the presence of an enzyme in the isolates similar to poly(3-hydroxybutyrate) degrading hydrolases reported earlier.
    International Biodeterioration & Biodegradation. 01/2004;