Emine Karakuş

Urea Biosensors Based on PVC Membrane Containing Palmitic Acid

A new urea biosensor was prepared by immobilizing urease with four different procedures on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid by using nonactine as an ammonium-ionophore. The analytical characteristics were investigated and were compared those of the biosensor prepared by using carboxylated PVC. The effect of pH, buffer concentration, temperature, urease concentration, stirring rate and enzyme immobilization procedures on the response to urea of the enzyme electrode were investigated. The linear working range and sensitivity of the biosensor were also determined. The urea biosensor prepared by using the PVC membranes containing palmitic acid showed more effective performance than those of the carboxylated PVC based biosensors. Additionally, urea assay in serum was successfully carried out by using the standard addition method.

Purification and Biochemical Characteristics of Pectinesterase from Malatya Apricot (Prunus armeniaca L.)

Abstract Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60°C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70°C. Km and Vmax values were 0.77 mM and 1.75 µmol min−1 mg−1 for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na2CO3, iodine, lauril sulphate, AgNO3, EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol−1 for the enzyme. Ascorbic acid, CaCl2, and KCl showed activatory effect on the apricot PE enzyme.

PRODUCTION AND OPTIMIZATION OF L-GLUTAMINASE ENZYME FROM Hypocrea jecorina PURE CULTURE

L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver–Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.

Determination of Creatine in Commercial Creatine Powder with New Potentiometric and Amperometric Biosensors

New potentiometric and amperometric biosensors were developed for the determination of creatine. The potentiometric creatine biosensor was prepared by immobilizing urease and creatinase on poly(vinylchloride) (PVC) ammonium membrane electrode containing palmitic acid prepared by using nonactine as an ammonium-ionophore. The linear working range of the biosensor was 1.0 × 10−5–1.0 × 10−3 M and the response time was about 60 s. The optimum pH, temperature, and buffer concentration were found to be 7.0, 20°C, and 5 mM, respectively. The slope of the electrode was 49.2 mV/p[creatine]. The storage stabilization of the biosensor was investigated and 40–45% decrease in the response was detected after 2 months. The amperometric creatine biosensor was prepared by immobilizing creatinase (CI) and sarcosine oxidase (SO) in a poly(vinylferrocenium) matrix onto the surface of a platinum working electrode by crosslinking with glutaraldehyde (GA) and bovine serum albumine (BSA). Determination of creatine was performed by the oxidation of enzymatically generated H2O2 at +0.7 V vs. Ag/AgCl. The linear working range of the biosensor was 2.0 × 10−5–3.2 × 10−4 M and the response time was about 50 s. The effects of pH, temperature, enzyme ratio and buffer concentration were investigated and optimum parameters were found to be 7.5, 37°C, 2.5:1 (CI:SO) and 0.05 M, respectively. The determination of creatine in commercial creatine powder was successfully carried out with these creatine biosensors by using the standard addition and calibration curve methods. The results were in good agreement with those obtained from Jaffé method at 95% confidence level.

Potentiometric Glucose Determination in Human Serum Samples with Glucose Oxidase Biosensor Based on Iodide Electrode

Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10−1–1.0 × 10−6 M and 1.0 × 10−2−1.0 × 10−4 M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.

Comparison of Covalent and Noncovalent Immobilization of Malatya Apricot Pectinesterase (Prunus armeniaca L.)

Abstract: Pectinesterase isolated from Malatya apricot pulp was noncovalently and covalently immobilized onto bentonite and glutaraldehyde-containing amino group functionalized porous glass beads surface at pH 8.0 and pH 9.0, respectively. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. The optimum temperature of covalently and noncovalently immobilized PE was 50°C. This value was 60°C for free PE. Although optimum pH of covalently-immobilized PE was 8.0, this parameter was 9.0 for free and covalently-immobilized PE. The noncovalently immobilized enzyme exhibited better thermostability than the free and covalently immobilized PE.

A New Potentiometric Ammonium Electrode for Biosensor Construction

New ammonium-selective membrane electrode based on poly(vinyl chloride) (PVC) membrane containing palmitic acid (a long-chain fatty acid) and nonactin as an ammonium ionophore for the determination of ammonim ions in the 10−7 − 10−1 mol/L concentration range was prepared and compared to those of the electrode prepared by using carboxylated PVC. Sebacate was used as a plasticiser for both of the ammonium sensor membranes. The analytical characteristics of the ammonium electrodes was investigated. The effect of pH, buffer concentration, temperature and stirring rate on the response to ammonium electrode was investigated. The linear working range and sensitivity of the electrodes were also determined. Ammonium electrodes give Nernstian response (52–58 mV/p[NH4+]) throughout the ammonium ion concentration range of 10−1 to 10−6M with detection limits of 10−6M ammonium ions. The ammonium-selective electrodes prepared by using the PVC membranes containing palmitic acid showed more effective performance than those of the carboxylated PVC. The ammonium ion sensor has potential application in the analysis of ammonium ions for biosensor construction.

A novel potentiometric biosensor for determination of L-lysine in commercial pharmaceutical L-lysine tablet and capsule

The construction of an L-lysine biosensor on ammonium-selective poly(vinylchloride) (PVC) membrane electrode is described in this study. The construction procedure occurs in two stages: (I) the preparation of ammonium-selective poly(vinylchloride) (PVC) membrane electrode and (II) the chemical immobilization of lysine oxidase on this ammonium-selective electrode by using glutaraldehyde. The ammonium ions produced after enzymatic reaction were determined potentiometrically. The sensitivity of the lysine biosensor against ammonium ions and lysine were studied. The response time, linear working range, reproducibility and life time of the biosensor were also determined. The interfering effect of other amino acids on the biosensor performance was also studied and potentiometric selectivity coefficients were calculated. Although the biosensor responded mainly against tyrosine, a lot of amino acids and ascorbic acid that can be present in some real samples did not show any important interference. Additionally, lysine assay in commercial pharmaceutical lysine tablets and capsules was also successfully carried out. The results were in good agreement with previously reported values.

Immobilization of glutaminase enzyme from Hypocria jecorina on polyacrylic acid: preparation and biochemical characterization

Abstract L-glutaminase enzyme produced from Hypocrea jecorina pure culture and polyacrylic acid (PAA) in the presence Cu+ ions were composed the ternary complex at pH 7. The properties of free and immobilized enzyme were defined. The effect of various factors such as pH, temperature, heat, and storage stability on immobilized enzyme were investigated. The properties of immobilized enzyme were investigated and compared to those of free enzyme. Optimum pH and temperature of both enzyme were determined to be 8.0 and 50°C, respectively. Kinetic parameters of the immobilized enzyme (Km and Vmax values) were also determined as 0.38 mM of the Km and 10.9 U/L of the Vmax. No drastic change was observed in the Km and Vmax values. Thermal and storage stability experiments were carried out. The thermal stability studies indicated that the immobilization process tends to stabilize the enzyme.

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.