Purification and Characterization of Recombinant Glucose Oxidase from Pichia pastoris

Abstract

Purpose: To explore the purification and properties of glucose oxidase(GOD) from recombinant Pichia pastoris.Methods: The crude glucose oxidase was purified by Q-Sepharose Fast Flow chromatography.Polyacrylamide gel electrophoresis(PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) were used to determine its molecular weight.Ultraviolet and fluorescence absorption spectroscopy were used to observe its spectral characteristics.Results: Purified GOD with a purification factor of 1.35 and a recovery of 92.7% was obtained.This enzyme exhibited a dimeric form with a molecular mass of 150 kD.The GOD showed the highest activity at pH 6.0 and 40 ℃ and was stable in a broad pH range of 5.0-8.0 and at 55 ℃ or below.The Km of the GOD enzyme was 21.06 mmol/L with glucose as the substrate.The enzyme was inhibited intensively by Hg2+,Fe2+,Ag+ and Cu2+ and showed a maximum ultraviolet and fluorescence absorption peak at 275 nm and 344 nm,respectively.The lyophilized enzyme was stable at 4 ℃ over a period of 6 months and could be stored for 3-5 d at normal temperature.Conclusion: The recombinant GOD has simple purification procedure,high recovery,good stability and promising application potential.