Kinetic Studies and Analytical Application of Cholesterol Oxidase and Peroxidase Immobilized to Synthetic Polymer

Abstract

A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and Km' compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise "Olinease"). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r = 0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.

Cholesterol Oxidation Using Hollow Fiber Dialyzer Immobilized with Cholesterol Oxidase: Preparation and Properties

“…In the past decade, several immobilization methods for cholesterol oxidase (COD) have been proposed. Yotova and Ivanov have reported a method for individual and simultaneous covalent immobilization of COD to the copolymer of acrylonitrile with acrylamide (8). Brahim et al coated COD onto polypyrrole-hydrogel membrane to determine the amount of cholesterol in serum (9).…”

Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized Trichosporon cutaneum cells

“…A copolymer of acrylonitrile with acrylamide, in which the acrylamide units amount to 15% of the total number of units, was obtained at the Department of Biotechnology in UCTM, Sofia. Granules were obtained by dropping the polymer under pressure into a water/methanol (7:3) mixture containing 0.8 g dm -3 NaCl according to reference [28]. Glucose, phenol, naphthalene, pyridine, toluene, salts for mineral medium and formaldehyde were obtained from Merck (Germany).…”

“…The immobilization procedure used is based on a method for covalent binding of enzymes to synthetic carriers containing active N hydroxymethyl groups, which bind to the amino acid residues of proteins [12,15,28]. The granulated carrier of copolymer of acrylonitrile with acrylamide was activated according to procedures described in reference [28]; 40 g of copolymer was activated with 12% (v/v) formaldehyde (dissolved in 0.1 M phosphate buffer, pH 7.8) and stirred for 4 h at 45°C in a closed vessel.…”

“…The granulated carrier of copolymer of acrylonitrile with acrylamide was activated according to procedures described in reference [28]; 40 g of copolymer was activated with 12% (v/v) formaldehyde (dissolved in 0.1 M phosphate buffer, pH 7.8) and stirred for 4 h at 45°C in a closed vessel. The activated carrier was then washed abundantly with distilled water until no more formaldehyde was observed in the rinsing waters.…”

Chemically Modified Sepharose as Support for the Immobilization of Cholesterol Oxidase

“…In the past decade, several immobilization methods for cholesterol oxidase had been proposed. Yotova and Ivanov [28] reported the method for individual and simultaneous covalent immobilization of cholesterol oxidase to the copolymer of acrylonitrile with acrylamide. Gilles et al [3] directly immobilized cholesterol oxidase onto Fe 3 O 4 magnetic nanoparticles.…”