Melda Altikatoglu

Decolorization of Naphthol Blue Black using the Horseradish Peroxidase

This study evaluates the potential of the enzyme horseradish peroxidase in the decolorization of one common industrial azo dye, naphthol blue black. Studies are carried out to understand the process parameters such as pH, temperature and reaction time. The enzymatic decolorization of the dye was examined by UV-Vis spectrophotometer and LC-MS measurements. Temperature and pH conditions were optimized for obtaining high azo-dye decolorization. Azo-dye removal at a pH range 4-6 was found to be the highest for all temperatures. After 5 minutes of treatment, the color removal of dye was ca. 80-90%. The LC-MS and spectrophotometric analyses indicated that the decolorization of the azo dye with enzyme was due to the reduction of the azo bonds. This study verifies the viability of the use of the horseradish peroxidase in the decolorization of naphthol blue black.

Stabilization of horseradish peroxidase by covalent conjugation with dextran aldehyde against temperature and pH changes

Stabilization of Horseradish Peroxidase (HRP; EC 1.11.1.7) against temperature and pH via the formation of the conjugates obtained by multipoint covalent bonding of dextran aldehyde (DA) to the enzyme were studied. Hence, three different molar weighted dextrans (17.5 kD, 75 kD, 188 kD) were covalently bonded to purified enzyme with different molar ratios (nHRP/nDA 20/1, 10/1, 1/1, 1/5, 1/10, 1/15, 1/20). The thermal stabilities of the obtained conjugates were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time. Conjugates formed were characterized by gel-permeation chromatography (GPC) and fluorescence techniques. The conjugate synthesized using dextran 75 kDa with nHRP/nDA 1/10 molar ratio showed better thermal stability than other conjugates and purified enzyme at pH 7. This conjugate also has wider activity pH range than purified enzyme. In addition, mentioned conjugate at pH 7 had very long storage lifetime compared to purified enzyme at +4°C and room temperature; which is considered a favorable feature for usage in practice.

Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime

Improvement of enzyme stability via non-covalent complex formation with dextran against temperature and storage lifetime The optimal methodology to prepare the novel modified enzyme, polymer-enzyme complex, was developed to give a high catalytic activity in aqueous solution. The non-covalent complexes of two different enzymes (horseradish peroxidase and glucose oxidase) were prepared with various molar ratios (nD/nE 0,05; 0,1; 1; 5; 10; 15; 20) by using 75kDa dextran. The thermal stabilities of the obtained complexes were evaluated with the activities determined at different temperatures (25, 30, 35, 40, 50, 60, 70, 80°C) applying 60 minutes incubation time for pH 7. The complexes with the molar ratio nD/nHRP: 10 and nD/nGOD: 5 showed the highest thermal stability. Its activity was very high (ca. 1,5-fold higher activity than pure enzyme for HRP-dextran complexes) and almost the same between applying one hour incubation time and without incubation, and could also be measured at high temperatures (70, 80 °C). We finally succeeded in preparing dextran-enzyme complexes which showed higher activity than pure enzyme in aqueos solution at all temperatures for pH 7. In addition, the mentioned complexes at pH 7 had very long storage lifetime compared to purified enzyme at +4 °C; which is considered as a good feature for the usage in practice.

Enhanced Stability and Decolorization of Coomassie Brilliant Blue R-250 by Dextran Aldehyde-modified Horseradish Peroxidase

Abstract: Horseradish peroxidase (EC 1.11.1.7) was chemically modified by periodate-activated dextran. The activities of free and modified enzyme against organic-aqueous interface and some chemicals were determined. Modified HRP remained fully active in the presence of organic solvent for 4 h. However, the unmodified enzyme lost 50% of its activity within the first 2 h. The effects of possible inhibitors on enzyme activity were investigated. In addition, Coomassie Brilliant Blue R-250 was efficiently decolorized using the free and modified HRP. After 5 minutes of treatment, the color removal of dye was 80-90%. Modified HRP showed effective performance compared to free HRP.

Measuring calcium, potassium, and nitrate in plant nutrient solutions using ion-selective electrodes in hydroponic greenhouse of some vegetables.

Generally, the life cycle of plants depends on the uptake of essential nutrients in a balanced manner and on toxic elements being under a certain concentration. Lack of control of nutrient levels in nutrient solution can result in reduced plant growth and undesired conditions such as blossom‐end rot. In this study, sensitivity and selectivity tests for various polyvinylchloride (PVC)‐based ion‐selective membranes were conducted to identify those suitable for measuring typical concentration ranges of macronutrients, that is, NO3−, K+, and Ca2+, in hydroponic solutions. The sensitivity and selectivity of PVC‐membrane‐based ion‐selective sensors prepared with tetradodecylammoniumnitrate for NO3−, valinomycin for K+, and Ca ionophore IV for Ca2+ were found to be satisfactory for measuring NO3−, K+, and Ca2+ ions in nutrient solutions over typical ranges of hydroponic concentrations. Potassium, calcium, and nitrate levels that were utilized by cucumber and tomato seedlings in the greenhouse were different. The findings show that tomato plants consumed less amounts of nitrate than cucumber plants over the first 2 months of their growth. We also found that the potassium intake was higher than other nutritional elements tested for all plants.

Calcium, potassium and nitrate in plant nutrient solutions by measuring of with ion‐selective electrodes in hydroponic green house of some vegetables

Generally, the life cycle of plants depends on the uptake of essential nutrients in a balanced manner and on toxic elements being under a certain concentration. Lack of control of nutrient levels in nutrient solution can result in reduced plant growth and undesired conditions such as blossom‐end rot. In this study, sensitivity and selectivity tests for various polyvinylchloride (PVC)‐based ion‐selective membranes were conducted to identify those suitable for measuring typical concentration ranges of macronutrients, that is, NO3−, K+, and Ca2+, in hydroponic solutions. The sensitivity and selectivity of PVC‐membrane‐based ion‐selective sensors prepared with tetradodecylammoniumnitrate for NO3−, valinomycin for K+, and Ca ionophore IV for Ca2+ were found to be satisfactory for measuring NO3−, K+, and Ca2+ ions in nutrient solutions over typical ranges of hydroponic concentrations. Potassium, calcium, and nitrate levels that were utilized by cucumber and tomato seedlings in the greenhouse were different. The findings show that tomato plants consumed less amounts of nitrate than cucumber plants over the first 2 months of their growth. We also found that the potassium intake was higher than other nutritional elements tested for all plants.

Novel creatine biosensors based on all solid-state contact ammonium-selective membrane electrodes

Abstract Novel creatine bienzymatic potentiometric biosensors were prepared by immobilizing urease and creatinase on all solid-state contact PVC-containing palmitic acid and carboxylated PVC matrix membrane ammonium-selective electrodes without inner reference solution. Potentiometric characteristics of biosensors were examined in physiological model solutions at different creatine concentrations. The linear working range and long-term sensitivity of the biosensors were also determined. The creatine biosensors prepared by using the carboxylated PVC membrane electrodes showed more effective performance than those of the PVC containing palmitic acid membrane electrodes. Creatine assay in serum samples was successfully carried out by using the standard addition method.

Protective effect of dextrans on glucose oxidase denaturation and inactivation.

Abstract In the present study, the stabilizing effect of dextrans as additives on the denaturation and inactivation of glucose oxidase (GOD) was investigated. Three different molecular weighted dextrans (Mw 17.5, 75, 188 kD) were used with different concentrations. Dramatically increased enzyme activities were measured after one hour of incubation of enzyme with additives between 25–40°C in water bath. Highest activity value was measured with 75 kDa molecular weighted dextran (in concentration 30% w/v) at pH 5. Dextran as an additive supplied a long shelf-life to the enzyme at 4°C. In the presence of the 75 kDa dextran, the enzyme was more stable and its activity was increased 2.7-fold at 30°C. In addition, dextran protected GOD against inactivation by a n-heptane/aqueous buffer-stirred system.

Functional Stabilization of Cellulase from Aspergillus niger by Conjugation with Dextran-aldehyde

The covalent conjugates of cellulase from Aspergillus niger were prepared with various molar ratios by using dextran. The conjugate (nE/nD: 1/5) showed higher activity than purified enzyme at all temperatures after 1 h of incubation and its activity could also be measured at higher temperature. Also, this conjugate lost only 60% activity in 2 h at 70°C in comparison to the purified enzyme, which lost all its activity. In addition, conjugation protected cellulase against denaturation in the presence of sodium dodecylsulfate (residual activity of about 80%) and inactivation by air bubbles (residual activity of about 50% for 4 h).

Synthesis of glucose oxidase-PEG aldehyde conjugates and improvement of enzymatic stability

Abstract In this article, aldehyde derivative of poly(ethylene glycol) (PEG) was synthesized directly with sodium periodate agent. To obtain a conjugate which possesses better stability, PEG aldehyde was bonded to native enzyme with different molar ratios. The conjugation reaction turned out to be efficient and mild. Colorimetric method was applied to evaluate the enzymatic activity of native GOD and its derivatives by introducing another enzyme, horseradish peroxidase. The GOD–PEG aldehyde conjugate with polymeric chains exhibited reduced enzymatic activity towards the catalytical oxidation of glucose, but with significantly increased thermal stability and elongated lifetime. When GOD was modified with PEG aldehyde the enzymatic activity was decreased 40% at 30 °C. However, when incubated at 60 °C the GOD–PEG aldehyde conjugate still retained the enzyme bioactivity of 40% bioactivity left after 4 h, whereas the native GOD lost almost all the activity in 4 h. The polymer chain attached, the more reduction of the enzymatic activity resulted, however, the longer the lifetime and higher thermal stability of the enzyme obtained.