Immobilization of horseradish peroxidase on chitosan for use in nonaqueous media

Abstract

Chitosan, a natural polysaccharide, was used for the covalent immobilization of horseradish peroxidase, an enzyme of high synthetic utility, with the carbodiimide method. Of the enzyme, 62% was immobilized on chitosan when 1-ethyl-3-(3-dimethylaminopropyl carbodiimide) was used as the peptide coupling agent. The influence of different parameters, such as the enzyme concentration, carbodiimide concentration, and incubation period, on the activity retention of the immobilized enzyme was investigated. Kinetic studies using horseradish peroxidase immobilized on chitosan revealed the effects of several parameters, such as the substrate hydrophilicity and hydrophobicity, the solubility of substrates in the medium, the solvent hydrophobicity, and the support aquaphilicity, on the catalytic activity of the immobilized enzyme in nonaqueous media. General rules for the optimization of solvents for nonaqueous enzymology based on the partitioning of the solvent were not applicable for the immobilized horseradish peroxidase. The catalytic efficiency was greatest when o-phenylene diamine was used as the substrate and least when guaiacol was used. The aquaphilicity of the support played an important role in the kinetics of the immobilized horseradish peroxidase in water-miscible solvents. The results were promising for the future development of chitosan-immobilized enzymes for use in organic media.

Stability Improvement of Immobilized Alkaline Phosphatase Using Chitosan Nanoparticles

“…The immobilization performed in this work was not optimized for ALP (Bindhu and Abraham, 2003). Some modifications were performed in steps to optimize the work.…”

“…In this study we synthesized chitosan nanoparticles using the Ionic Gelation method and immobilized alkaline phosphatase on them according to the method described by Bindhu and Abraham (2003). EDC (1-ethyl-3-(3-dimethylaminopropyl carbodiimide) was used for covalent immobilization of the enzyme, which is quite simple and efficient for enzyme immobilization.…”

Construção e aplicação de biossensores usando diferentes procedimentos de imobilização da peroxidase de vegetal em matriz de quitosana

“…Comprovaram que a enzima apresentou estabilidade superior quando comparada com a lacaselivre. A quitosana, quimicamente modificada, tem sido empregada tanto na forma de microesferas, quanto na de filmes, ou de pó, ou de membranas, como adsorvente de metais 10,[15][16][17] , na imobilização de diversas enzimas (creatinase deaminase 19 , galactose oxidase 20 , glutamato oxidase 20 , glicose oxidase 21 , peroxidase [22][23][24] , lactato oxidase 25 , sulfito oxidase 26,27 ) e na construção de biossensores [19][20][21][22][23][24][25][26][27] . A enzima peroxidase pode ser encontrada em vários vegetais e catalisa a oxidação de mono, di e polifenóis em presença de peróxido de hidrogênio 28,29 .…”

“…Comprovaram que a enzima apresentou estabilidade superior quando comparada com a lacaselivre. A quitosana, quimicamente modificada, tem sido empregada tanto na forma de microesferas, quanto na de filmes, ou de pó, ou de membranas, como adsorvente de metais 10,[15][16][17] 26,27 ) e na construção de biossensores [19][20][21][22][23][24][25][26][27] . …”

Electrochemistry of Hemoglobin in the Chitosan and TiO2Nanoparticles Composite Film Modified Carbon Ionic Liquid Electrode and Its Electrocatalysis

“…8 In this paper a new Hb composite film modified CILE was fabricated by using chitosan (CTS) and TiO 2 nanoparticles. CTS has been widely reported as an immobilization matrix for the preparation of biosensor and bioreactor, [9][10][11][12] which exhibit excellent film-forming ability as a biocompatible, biodegradable and nontoxic natural biopolymer. TiO 2 nanoparticles have been also revealed to afford good biocompatibility, large surface area, good dispersing properties and fast electron transfer ability, which had been applied to immobilize protein or enzyme for either mechanistic study or the fabrication of electrochemical biosensors.…”