FOOD SCIENCE ›› 2013, Vol. 34 ›› Issue (9): 159-163.doi: 10.7506/spkx1002-6630-201309033

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Purification and Characterization of Recombinant Glucose Oxidase from Pichia pastoris

HAO Jie-qing,WANG Shuai-kun,SHI Hui,WANG Zhen-wei,MENG Yan-fa*   

  1. Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu 610064, China
  • Received:2012-10-21 Revised:2013-03-13 Online:2013-05-15 Published:2013-05-07
  • Contact: MENG Yan-fa E-mail:yfmeng0902@126.com

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.

Key words: Pichia pastoris, glucose oxidase, purification, characterization

CLC Number: