Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger PCTC in Pichia pastoris

Abstract:

The aim of this study was to improve the production capacity of glucose oxidase.A gene of glucose oxidase(GOD) from Aspergillus niger TCPC was cloned and sequenced.The whole open reading frame(ORF) consisted of 1 772 bp bases and encoded a putative protein of 589 amino acids.The gene was fused to the pPIC9k plasmid and expressed in Pichia pastoris GS115.The recombinant GOD(rGOD) was secreted into the culture under the control of methanol and α factor signal peptide.After screening by G418 gradient resistance plate,color plate and shaker culture,one strain named GOD2-14 was obtained,which gave the higest enzyme activity(0.342 U/mL) after 4day induction by methanol.Shake flask experiments were conducted to optimize the culture conditions for the enhanced production of GOD.Highest enzyme activity of 25 U/mL was achieved in the shake flask by induction for 7 days under the optimal conditions(200 r/min,pH 5,22 ℃,inoculation amount of 50% and 1.5% methanol).The result showed that GOD gene has been successfully transferred and expressed in the Pichia pastoris GS115。