Dilek ODACI, Suna TİMUR, Azmi TELEFONCU

Dilek ODACI, Suna TİMUR, Azmi TELEFONCU

Phenolic compounds are released into the environment by a large number of industrial wastes. Many of these phenolic compounds have toxic effects on animals and plants, resulting in an acute environmental problem. So the monitor and control of these pollutants are greatly important for protection of the environment. For phenol determination various spectrometric and chromatographic methods are in common use. Instead of those conventional methods, biosensor could be a cheap and easy alternative, getting increasing attention in the literature. Plant and animal tissues have been successfully employed as biocatalytic components in the construction of biosensors for about two decades. As compared to biosensors with immobilized isolated and pure enzymes, tissue based biosensors show potential advantages of low cost, high stability, longer life time and high level activity.

In this study, we described a biosensor for the determination of phenol based on Jerusalem artichoke (Helianthus tuberosus) plant tissue. The enzyme polyphenol oxidase (PPO) in the Jerusalem artichoke has been well characterized in previous works. Enzymatic reaction due to the action of polyphenol oxidases occurred in the plant tissue can be monitored amperometrically by using oxygenmeter. The tissue electrode response depends linearly on phenol concentration between 0.002 and 0.010 M in 10 min response time. Maximum electrode response was found in phosphate buffer at pH 8.0 and 35 ^oC. The reproducibility of the enzyme electrode was also tested by using standard phenol solutions (0.005 µM). The standard deviation (SD) and variation coefficient (cv) were calculated as 1.4x10^-4 M and 3.1 %, respectively.

P113

Raziye OZTURK UREK¹ , Nurdan KASIKARA PAZARLIOGLU²

Solid-state culture of white rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. The suitable medium and culture conditions have been chosen to favour the secretion of manganese peroxidase (MnP; EC 1.11.1.13). MnP is an extracellular heme-containing enzyme known to catalyze the oxidation of Mn⁺² to Mn⁺³ in a reaction requiring appropriate manganese chelator. The enzyme was isolated and purified from Phanerochaete chrysosporium and partially characterized.

Partial protein precipitation in crude enzyme was affected by using (NH₄)₂SO₄, polyethylene glycol, methanol and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification process attained 24,37 % activity yield with a purification factor of 7.96. According to data on gel filtration chromatography anda Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45000 1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30^oC respectively. This enzyme was stable in the pH range 4.5 to 6.0, 25^oC and also up to 35^oC at pH 4.5 for 1-h incubation period. MnP activity was inhibited by 2 mM NaN₃, Ascorbic acid, -mercaptoethanol and dithreitol. The Km values of MnP for Hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 M at pH 4.5 respectively.

P114

Funda KARTAL¹, Yusuf BARKUT¹,Ali KILINǹ,

Tansel ÖZTÜRK², İhsan YAŞA², Figen ZİHNİOĞLU<sup>1

1</sup>Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova/İZMİR-TÜRKİYE

²Ege University, Faculty of Science, Biology Department, 35100 Bornova/İZMİR-TÜRKİYE

funda_kartal@hotmail.com

Lipases[triacyl glycerol acylhydrolases (E.C.3.1.1.3)] are enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoacylglycerols and glycerol. Many applications of lipases include resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and chemical analyses. Lipases which play a key role in the biological turnover of lipids are distributed among higher animals, microorganisms, and plants, but only microbial lipases are commercially significant.

Microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze a wide variety of reactions in aqueous and non-aqueous media. Therefore, these enzymes are nowadays extensively studied for their potential industrial applications.

In this study, 7 different yeast induced with fish oil were checked for their lipase activity and protein content inorder to find the most appropriate microorganism for lipase production and Hansenula nonfermentans, was choosen as enzyme source. Extracellular lipase was purified 10 fold by using ammonium sulphate (25-75%) treatment, acetone precipitation(50%), ion exchange (DOWEX 1X4 –200: Strongly basic Anion Exchanger) chromatography, respectively.The purity and molecular weight of the enzyme was tested by 10% SDS polyacrylamide gel electrophoresis and the relative molecular weight was estimated approximately as 39 kDa, which seems closer to the other yeast lipases purified before.

P115

IDENTIFICATION OF ESSENTIAL AMINO ACIDS FOR THE CATALYTIC ACTIVITY OF TWO ENDOGLUCANASES FROM A MUTANT STRAIN TRICHODERMA SP. M₇

Svetla PETROVA, Nadka BAKALOVA and Dimiter KOLEV

Endoglucanases (1,4--D-glucano-glucohydrolases, EC 3.2.1.4) attack randomly internal -1,4-glucosidic bonds of the amorphous regions of the cellulose, providing a sufficient number of chain ends for exoglucanases to act on. The mutant strain, Trichoderma sp. M₇, has proved to be one of the best in producing an optimal cellulase system when grown on lignocellulosic materials and has been used commercially for the conversion of delignified waste-cellulose fibers from the paper industry.

The aim of the present work was to investigate the properties endoglucanases from a mutant strain and to identify catalytically important amino acid residues by chemical modifications with specific reagents.The purification procedure of endoglucanases included consecutive chromatographic methods using Sephadex G-75, anion-exchangers DEAE Sephadex A-50 and Mono Q HR 5/5, followed by gel-filtration step on Superose 12 HR 10/30.The kinetics of the modification of the highly purified forms of the enzymes with group specific reagents was investigated to elucidate the mechanism and to determine the kinetic constants.

Two of the purified endoglucanases had Mr of 49.7 and 47.5 kDa, and estimated pI values of 3.7 and 6.35, respectively.The optimal pH and temperature values were determined to be pH 5.0 and 60 ^oC for the first cellulase, whereas pH 5.2 and 50 ^oC were optimal for the other. A water-soluble carbodiimide inactivated the one of the purified endoglucanases, while both were inhibited by jodoacetamide, indicating the involvement of carboxyl or thiol groups in the catalysis. N-Bromsuccinimide showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate (binding of the modified endoglucanases to Avicel is reduced by 40%).The modification studies are very important because the improvement of the role of the cellulases in biotechnological applications requires a fundamental understanding of their mode of action.

P116

ROLE OF LHC II PROTEINS IN THE THERMAL STABILITY OF PHOTOSYNTHETIC MEMBRANES

Anelia G. DOBRIKOVA ^a, Ivana B. PETKANCHIN ^b and Stefka G. TANEVA ^a

Electric light scattering method have been used to monitor the thermal sensitivity of thylakoid membranes from wild type and Chlorina f2 mutant of barley with a strongly reduced amount of the light-harvesting complex of photosystem II (LHC II). Our recent study has suggested that the major LHC II directly contribute to the surface electric properties of thylakoid membranes – transversal charge asymmetry distribution and electric polarizability. In this study, we compared the changes in the electric dipole moments after heat treatment of the thylakoid membranes from both barley genotypes. The permanent dipole moment and the dimension of the thylakoid membranes from the mutant which is deficient in the major LHC II exhibit higher thermal stability as compared to the wild type, while the electric polarizability per unit surface sharply decreases above 50 C. The thermal-induced changes in the dipole moments and the dimension of barley wild type thylakoids after heating in the range 20-75 C might be attributed to changes in the macroorganization of LHC II, which plays a decisive role in the stabilization of the thylakoid ultrastructure.

Acknowledgement: This work was supported by Contract MY-K-1003/00 from the Bulgarian National Science Fund.

P117

Tüge GÖKTUĞ, Mustafa Kemal SEZGİNTÜRK, Erhan DİNÇKAYA

Benzoic acid and sodium benzoate are used as food preservatives and are most suitable for foods, fruit juices, and soft drinks that are naturally in an acidic pH range. Their use as preservatives in food, beverages, toothpastes, mouthwashes, dentifrices, cosmetics, and pharmaceuticals is regulated. Benzoic acid occurs naturally in many plants and in animals. It is therefore a natural constituent of many foods, including milk products. Anthropogenic releases of benzoic acid and sodium benzoate into the environment are primarily emissions into water and soil from their uses as preservatives. Concentrations of naturally occurring benzoic acid in several foods did not exceed average values of 40 mg/kg of food. Maximum concentrations reported for benzoic acid or sodium benzoate added to food for preservation purposes were in the range of 2000 mg/kg of food.

Cases of urticaria, asthma, rhinitis, or anaphylactic shock have been reported following oral, dermal, or inhalation exposure to benzoic acid and sodium benzoate. The symptoms appear shortly after exposure and disappear within a few hours, even at low doses. The information concerning skin reactions caused by benzoic acid or sodium benzoate in the general population is limited.

In this study, an amperometric biosensor based on mushroom tissue homogenate was developed for the determination of benzoic asit. Mushroom (Agaricus bisporus) tissue homogenate was used as the biological material. The principle of the measurements was based on the determination of the decrease in the differentiation of oxygen level which had been caused by the inhibition of polyphenol oxidases in the biological material by benzoic acid.

Characterization studies of the biosensor such as optimum substrate concentration, optimum pH, optimum temperature and thermal stability were carried out and a linearity in the benzoic acid concentration range 25-100 M was obtained when 200 M phenol was used as a substrate. The repeatability experiments were done and the average value ( x ), standard deviation (S.D.) and variation coefficient (C.V.) were calculated.

P118

A CLONING STRATEGY FOR DIRECTIONAL cDNA CLONING IN TOPO BASED VECTORS

İncilay SİNİCݹ, Michael B TROPAK², Don J MAHURAN², H. Asuman ÖZKARA¹

TOPO cloning is a quick, highly efficient, widely used, cloning strategy that facilitates cloning to other mammalian expression vectors. For this purpose, TOPO based pCR2.1-TOPO is designed as having appropriate sites for restriction endonucleases in the polycloning regions. It has advantage of direct insertion of Taq polymerase-amplified PCR products. Ligase and post-PCR procedures are not required. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine to 3’ ends of PCR products. pCR2.1-TOPO vector has single overhanging 3’-thymidine residues. This allows PCR inserts to ligate efficiently by annealing the A-T nucleotides. The insertion of cDNA can be in sense (5’ 3’) or antisense (3’ 5) orientations. It is important to check and capture the correct orientation, 5’ 3’ of inserted cDNA following the vector’s promoter, in clones for expression. In our study, while cloning PCR products into pCR2.1-TOPO vector has T7 promoter sequence. Forward (5’) primer, used for PCR, has T7 promoter sequence. Another forward (5’) primer, carrying no T7 promoter sequence, was designed for another two PCR. Both orientations were obtained in all 30 clones. A previous report is explaining that the repeats of DNA sequences acquire toxic functions and/or may direct cells into the apoptotic cycle (1). As a conclusion, if two repeats of T7 promoter sequence in the 5’ region are closer to each other, it would account for the death of cells carrying the other orientation by causing some toxic effects. If this is the situation, it is possible to propose a correct orientation cloning strategy of PCR products for TOPO cloning by designing 5’ primer having T7 promoter repeat sequence. Closer examination of the constructs led a possible potential novel technique for directional cDNA cloning using TOPO based vectors.

References:

1- Parniewski P, Staczek P. Molecular Mechanisms of TRS instability. Adv Exp Med Biol. 2002;516:1-25.

Serap EVRAN¹, İhsan YAŞA², Azmi TELEFONCU¹

The development of alternative preservatives for food, pharmaceuticals and cosmetics is necessary due to poor solubility, high toxigenic potential and narrow antimicrobial spectrum of preservative agents. Lysozyme, exhibiting a high water solubility as well as negligible toxigenic potential, is one of the most popular and safe bactericidal proteins, but it lacks an antimicrobial capability against Gram-negative bacteria. Lysozyme, a mucopolysaccharidase, acts against Gram-positive bacteria by transforming the insoluble polysaccharides of cell wall to soluble mucopeptides. The peptidoglycan layer in Gram-negative bacteria is protected from the lytic action of lysozyme, as the outer membrane of the bacteria functions as a permeability barrier. Hence, for lysozyme to broaden the antimicrobial specificity to Gram-negative bacteria, it must overcome the outer membrane barrier. Our study was based on the strategy in which the lysozyme molecule was equipped with a hydrophobic carrier by chemical modification to enable it to penetrate the bacterial membrane. Oleoyl chloride was covalently bound to the -amino group of lysyl residues of lysozyme. Lysozyme with various degrees of modification was obtained by changing the lysozyme/oleoyl chloride mass ratio. Lysozyme derivatives were removed from the reaction environment by precipitation at their isoelectric points taking into account the shift in the isoelectric point of lysozyme. A control sample was obtained by the same procedure without the addition of oleoyl chloride. The degree of modification was determined by measuring the free amino groups of both pure lysozyme and modified lysozyme using trinitrobenzenesulfonic acid (TNBS). The effect of the lipophilization on lytic activity of lysozyme derivatives against M. lysodeikticus cells was determined according to turbidimetric method based on the decrease in turbidity of the cell suspension following the addition of lysozyme derivatives. The antimicrobial activity of lysozyme derivatives was tested against both Gram-negative and Gram-positive bacteria by determining the viable cell numbers. Percentage survival was represented with respect to control mixture (no protein added). In conclusion, lysozyme derivatives are easily accessible by a simple chemical reaction and different strategies can be attempted to convert lysozyme to be active in killing Gram-negative bacteria.

Gülseren COŞKUN, Ayşe DİNÇER, Figen ZİHNİOĞLU

Glutathione-s-transferases(GSTs; EC: 2.5.1.18) are a family of multifunctional enzymes involved in the metabolism of xenobiotics and reactive compounds. These enzymes catalyze the nucleophilic attack of thiol group of glutathione (GSH) at an electrophilic site of a second substrate, yielding a GSH-conjugate, which is generally less toxic than the parent compound. The enzymes are found in mammals, insects, plants and microorganisms and are thought to play a major role in the protection of these organisms from the toxic effects of wide variety of electrophilic and hydrophobic compounds. In most organisms studied, GSTs have been found to exist in multiple forms. Plant GSTs were first identified with regard to herbicide detoxification and environmental safety. Additionally the exhibit a variety of further functions, such as auxin binding, cellular protection from oxidative stress and non-enzymatic binding and transport(ligandin function). A common feature of most of these functions in that, they are essential components of the plant’s defence system for environmental stress.

In this work, wheat(Triticum aestivum), which was treated with and without parathion-methyl(1.37 mM) were harvested 12 days after planting. The roots were homogenized and centrifuged at 10000xg for 30 minutes. The supernatants were subjected to 40-80% (NH₄)₂SO₄ precipitation and CDI(1-1’,carbonyldiimidazole) activated GSH-Sepharose 6B affinity column. GST from control and pesticide induced wheat was purified 145 and 159 fold, respectively. Optimum temperature, pH and some kinetic parameters were studied and compared. Relative molecular weights were estimated approximately as 25.2 kDa for control and 25.2 kDa and 23.7kDa for parathion-methyl induced wheat by the help of 11% SDS-gel electrophoresis. The results may imply that after induction of the plant, new isoform exists besides the control GST, which seems to be specific to the methyl-parathion. However, data obtained should be confirmed by structure analysis and substrate specificity experiments.

P121

THE EFFECTS OF KAINIC ACID AND MELATONIN ON THE RATIO OF Bcl-2 TO Bax IN THE RAT HIPPOCAMPUS

Ayfer YALÇIN¹, , Lütfiye KANIT² , Eser Y SOZMEN³

Kainic acid (KA) is an agonist of ionotropic glutamate receptors and excessive or persistent activation of glutamate-receptor gated ion channels (excitotoxicity) contributes to neuronal degeneration. Bcl-2 proto-oncogen is an common regulator of multiple apoptotic pathways. The active form of the Bcl-2 protein ,which promotes cell survival, is part of a heterodimer with Bax , which promotes cell death , and the ratio of Bcl-2 to Bax appears to determine the susceptibility to apoptotic stimuli. The pineal hormone melatonin is of particular interest as it can prevent neuronal degeneration induced by neurotoxins such as KA.

In this present study, we assessed the effect of kainic acid and melatonin on the ratio of Bcl-2 to Bax in excitotoxic hippocampal injury. Rats were received melatonin or kainic acid as i.p injection and divided into four experimental groups as saline, melatonin alone, kainic acid alone and kainic acid plus melatonin. In the latter group melatonin was administered to rats 30 min before kainic acid injection. Total RNA isolated from hippocampus tissues by isopropanol precipitation method followed phenol-chloroform extraction. Bcl-2 and Bax mRNA were quantified using real-time polymerase chain reaction followed reverse transcription. It is found that the ratio of Bcl-2 to Bax mRNA is significantly increased in kainic acid plus melatonin treated rats when compared to kainic acid alone treated group (p<0.01). This finding suggests that a possible neuroprotective effect of melatonin in relation to Bcl-2/Bax in excitotoxic hippocampal injury.

P122

IMMOBILIZATION OF -GLUCOSIDASE IN CHITOSAN-COATED POLY GALACTURONIC ACID(PGA) BEADS