Immobilization of horseradish peroxidase by entrapment in natural polysaccharide

Abstract

Horseradish peroxidase (HRP), which catalyzes oxidation reduction reactions of large number of substrates, was entrapped in K-carrageenan beads using polyethyleneimine as hardening agent. The heat and storage stability was found to be better for entrapped horseradish peroxidase than free enzyme. The entrapped enzyme showed 50% retention of its activity after 4 cycles. Effective diffusion coefficient for diffusion of hydroquinone into Kcarrageenan beads was found to be 0.27 ϫ 10 Ϫ10 m 2 /s during enzyme-catalyzed oxidation of hydroquinone. Kinetic parameters calculated from Lineweaver-Burke plots were observed to be K m ϭ 8 ϫ 10Ϫ5 and V max ϭ 1.53 for free enzyme, and K m ϭ 8.3 ϫ 10 Ϫ5 and V max ϭ 2.18 for entrapped enzyme when enzyme concentration was kept constant and K m ϭ 4 ϫ 10 Ϫ11 and V max ϭ 0.45 for free enzyme and K m ϭ 4.5 ϫ 10 Ϫ11 and V max ϭ 0.58 for entrapped enzyme when substrate concentration was kept constant. This indicates that there is no conformational change during entrapment.

Studies on the activity and stability of immobilized horseradish peroxidase on poly(ethylene terephthalate) grafted acrylamide fiber

“…This reduction is due to the inactivation of the enzyme caused by the denaturation of protein. The number of reuse represents a more satisfying performance when compared to previous research [3,6,38]. …”

“…These results indicate that the thermal stability of the immobilized HRP is much better than that of the free one owing to the covalent bond between the enzyme and support, which prevents the conformation transition of the enzyme at high temperature. It was reported that the thermal stability enhancement was one of the general advantages of the immobilized enzymes [6,35].…”

“…This allows the biocatalyst to survive longer during process conditions and allows one to operate the process at higher temperature [3]. There are many immobilization techniques available such as adsorption [4], covalent attachment [5] and entrapment [6]. Non-covalent binding processes are simple but have the disadvantage of weak binding of enzyme.…”

Novel thermally and mechanically stable hydrogel for enzyme immobilization of penicillin G acylase via covalent technique

“…For example, Boadi et al, 20019 used alginate and carrageenan to entrap tannase then crosslinked the gel beads with chitosan followed by glutaraldehyde. Whereas, Shukla et al, 200410 used κ‐carrageenan gel to entrap horseradish peroxidase and crosslinked the gel beads using KCl and PEI. However, these supports suffer from one or more of the following problems: (a) poor thermal stability, (b) poor mechanical stability, (c) poor stability towards microbial attacks, d) they use the entrapment technique, which limits their industrial use as supports for enzyme immobilization due to enzyme leakage.…”

Biopolymeric Formulations for Biocatalysis and Biomedical Applications

“…For example, Boadi and Neufeld, 2001 [9], used alginate and carrageenan to entrap tannase and then cross-linked the gel beads with chitosan followed by glutaraldehyde, whereas Shukla et al, 2004 [24], used κ -carrageenan gel to entrap horseradish peroxidase and cross-linked the gel beads using KCl and PEI.…”