Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris

Yao Guo; Fengxia Lu; Haizhen Zhao; Yanchong Tang; Zhaoxin Lu

doi:10.1007/s12010-009-8778-6

Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris

Yao Guo, Fengxia Lu, Haizhen Zhao, Yanchong Tang, Zhaoxin Lu

Nanjing Agricultural University

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a Km value of 16.95 mM and a Kcat value of 484.26 s−1.
Keyword: Glucose oxidase,Overexpression,Pichia pastoris,Aspergillus niger,Properties

Original language	English
Journal	Applied Biochemistry and Biotechnology
Volume	162
Pages (from-to)	498-509
ISSN	0273-2289
DOIs	https://doi.org/10.1007/s12010-009-8778-6
Publication status	Published - 2010
Externally published	Yes

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title = "Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris",

abstract = "A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a Km value of 16.95 mM and a Kcat value of 484.26 s−1. Keyword: Glucose oxidase,Overexpression,Pichia pastoris,Aspergillus niger,Properties",

author = "Yao Guo and Fengxia Lu and Haizhen Zhao and Yanchong Tang and Zhaoxin Lu",

year = "2010",

doi = "10.1007/s12010-009-8778-6",

language = "English",

volume = "162",

pages = "498--509",

journal = "Applied Biochemistry and Biotechnology",

issn = "0273-2289",

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TY - JOUR

T1 - Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris

AU - Guo, Yao

AU - Lu, Fengxia

AU - Zhao, Haizhen

AU - Tang, Yanchong

AU - Lu, Zhaoxin

PY - 2010

Y1 - 2010

N2 - A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a Km value of 16.95 mM and a Kcat value of 484.26 s−1. Keyword: Glucose oxidase,Overexpression,Pichia pastoris,Aspergillus niger,Properties

AB - A gene of glucose oxidase (GOD) from Aspergillus niger Z-25 was cloned and sequenced. The entire open reading frame (ORF) consisted of 1,818 bp and encoded a putative peptide of 605 amino acids. The gene was fused to the pPICZαA plasmid and overexpressed in Pichia pastoris SMD1168. The recombinant GOD (rGOD) was secreted into the culture using MF-α factor signal peptide under the control of the AOX1 promoter. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that rGOD exhibited a single band at around 94 kDa. The maximal GOD activity of approximately 40 U/mL was achieved in shake flask by induction under optimal conditions after 7 days. rGOD was purified by ammonium sulfate precipitate leading to a final specific activity of 153.46 U/mg. The optimum temperature and pH of the purified enzyme were 40 °C and 6.0, respectively. Over 88% of maximum activity was maintained below 40 °C. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The Lineweaver–Burk plotting revealed that rGOD exhibited a Km value of 16.95 mM and a Kcat value of 484.26 s−1. Keyword: Glucose oxidase,Overexpression,Pichia pastoris,Aspergillus niger,Properties

U2 - 10.1007/s12010-009-8778-6

DO - 10.1007/s12010-009-8778-6

M3 - Journal article

C2 - 19784554

SN - 0273-2289

VL - 162

SP - 498

EP - 509

JO - Applied Biochemistry and Biotechnology

JF - Applied Biochemistry and Biotechnology

ER -

Cloning and Heterologous Expression of Glucose Oxidase Gene from Aspergillus niger Z-25 in Pichia pastoris

Abstract

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