Effects of high pressure homogenization on the activity, stability, kinetics and three-dimensional conformation of a glucose oxidase produced by Aspergillus niger.

Alline Artigiani Lima Tribst,Beata G Vertessy,Marcelo Cristianini,Júnio Cota,Mario Tyago Murakami

doi:10.1371/journal.pone.0103410

Abstract

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5–6.0 and a remarkable activity increase (30–300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.

Highlights

Glucose oxidase (GO) (b-D-glucose:oxygen 1-oxidoreductase) (EC 1.1.3.4) is a dimeric glycoprotein, consisting of two polypeptide chains covalently linked by disulfide bounds [1]
Effects of High pressure homogenization (HPH) treatment on GO activity The enzyme activity was measured at temperatures ranging from 15 to 85uC and at pH values ranging from 4.5 to 6.5
The maximum activity (3,224,591 U.g21), defined as 100%, was determined at pH 5.0 and 50uC. These results were subsequently compared with the HPH-treated enzyme to evaluate if the process promoted changes in the enzyme activity under optimum and non-optimal conditions

Summary

Introduction

Glucose oxidase (GO) (b-D-glucose:oxygen 1-oxidoreductase) (EC 1.1.3.4) is a dimeric glycoprotein, consisting of two polypeptide chains covalently linked by disulfide bounds [1]. Previous results obtained for a commercial GO indicated that HPH improved the enzyme activity at high temperatures (75uC) and enhanced enzyme stability after one day of storage under specific conditions [19].

Results

Conclusion