A new method for immobilization of acetylcholinesterase

Abstract

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V (max)) and Michaelis-Menten constant (K (m)) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca-alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 degrees C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.

Biosensors based on cholinesterase inhibition for insecticides, nerve agents and aflatoxin B1 detection (review)

“…For the selection of the pH, it should be considered that certain enzymes have ionic groups on their active site and these groups must be in a suitable form such as the serine group in the catalytic site of ChE enzymes. As reported in literature, the optimum pH for the free enzyme is pH= 8 while the pH can be shifted when the enzyme is immobilised to pH=7 as in the case of AChE immobilised onto Ca-alginate gel beads [157]. However the acid pH should be avoided, in fact in the case of insecticide detection during wine fermentation [118] or in orange juice [158] it is necessary to adjust the pH towards neutral value.…”

Metalloporphyrin-based acetate-selective electrodes as detectors for enzymatic acetylcholine determination in flow-injection analysis system

“…Two methods of acetylcholinesteraze immobilization were used in this work: cross-linking on Amberlite XAD 7 and recently reported method [29] based on entrapment in calcium alginate beads. Preliminary experiments, conducted to optimize these methods, were carried out using peroxidase, due to its much lower price, as compared to acetylcholinesterase.…”

“…Amberlite beads with immobilized enzyme were washed with 300 mL of MES buffer solution (pH 7.4) and then kept refrigerated. [29,30] Peroxydase (3.48 mg) and sodium alginate (20 mg) were mixed with 2 mL of TRIS buffer solution, pH 7.0. Obtained suspension was placed into the syringe pump, mounted over the beaker containing 100 mL of CaCl 2 solution (0.3 M).…”

Reversible immobilization of uricase on conductive polyaniline brushes grafted on polyacrylonitrile film

“…The result readily indicates that the immobilized uricase exhibits an improved stability over that of the free enzyme. Of the immobilization methods, fixation of enzyme molecules on a surface often gives rise to the highest stabilization effect on enzyme activities because the active conformation of the immobilized enzyme is stabilized by multipoint non-covalent binding of enzyme molecules on the polyaniline-grafted film [37,38]. Thus, polyaniline-grafted film and the immobilization method provide higher shelf-life compared to that of its free enzyme.…”