Reversible denaturation behavior of immobilized glucose oxidase.
Glucose oxidase (GOD) was immobilized by using glutaraldehyde crosslinking and various stabilizing agents such as BSA, gelatin, lysozyme, and polyethylenimine (PEI). Studies on the denaturation of the soluble as well as immobilized GOD were carried out for 1 h at various concentrations of guanidine hydrochloride (GdmCl) in 50 mM phosphate buffer, pH 6.0 at 25 +/- 1 degrees C. The soluble enzyme required a GdmCl concentration of 5 M for total activity loss, whereas for GOD immobilized with BSA, gelatin, lysozyme, and heat-inactivated lysozyme, the corresponding GdmCl concentration required was 8 M. GOD immobilized with PEI, however, was more stable and retained 25% activity when denatured for 1 h using 8 M GdmCl. However, after undergoing denaturation for 1 h, GOD immobilized with lysozyme regained 72% original activity within 20 min of renaturation, while GOD immobilized with BSA, PEI, gelatin, and heat-inactivated lysozyme regained only 39, 21, 20, and 25% of activity, respectively. After five cycles of repeated denaturation and renaturation with 8 M GdmCl, GOD immobilized with lysozyme retained 70% of the original activity. Refolding ability of lysozyme, glutaraldehyde crosslinkages between lysozyme and GOD, together with ionic interactions between them, appear to play an important role in the denaturation-renaturation behavior of the immobilized enzyme.
Gouda
M D
MD
Fermentation Technology and Bioengineering Department, Central Food Technological Research Institute, Mysore, India.
Thakur
M S
MS
Karanth
N G
NG
D002021:Buffers; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004566:Electrodes; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010100:Oxygen; D010710:Phosphates; D011489:Protein Denaturation; D013997:Time Factors
D002021:Buffers; D004800:Enzymes, Immobilized; D010710:Phosphates; D005949:Glucose Oxidase; D019791:Guanidine; D010100:Oxygen; D005976:Glutaral