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Pichia yeast bacterium producing aspergillus-niger glucose oxidase and application thereof

A technology of glucose oxidase and Pichia pastoris, applied in the field of genetic engineering, can solve the problems of low yield and complicated purification process, and achieve the effect of high-efficiency expression

Inactive Publication Date: 2014-10-15
毕文祥 +2
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  • Abstract
  • Description
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Problems solved by technology

At present, GOD is mainly prepared and purified from Aspergillus niger and Penicillium, but the yield is low and the purification process is complicated. Therefore, the use of genetic engineering methods to construct better GOD production strains has been highly valued.

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  • Pichia yeast bacterium producing aspergillus-niger glucose oxidase and application thereof

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Embodiment Construction

[0007] 1 Materials and methods

[0008] 1.1 Materials

[0009] 1.1.1 Strains and plasmids

[0010] Aspergillus niger accc30161 was provided by Professor Dai Meixue, Department of Biotechnology, School of Life Sciences, Shandong Normal University; Pichia pastoris KM71 and plasmid pGAPZαA were purchased from Invitrogen; Escherichia coli DH5α was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0011] 1.1.2 Reagents

[0012] Restriction enzymes NotI, KpnI and EcoRI were products of TaKaRa; T4DNA ligase and restriction enzyme Avr II were products of Fermentas; 2×Easy Taq PCR SuperMix was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primers were synthesized And gene sequence determination was completed by Shenzhen Huada Gene Company; GOD standard was purchased from Sigma Company; rabbit anti-A. PVDF membrane and ECL luminescent liquid were purchased from Millipore Company of the United States; Omega SP Fungal DNA kit kit, plasmid small extraction kit and...

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Abstract

The invention discloses p pichia yeast bacterium producing aspergillus-niger glucose oxidase, a construction method and application thereof, and belongs to the technical field of gene engineering. A recombined pichia yeast KM71-GOD capable of producing glucose oxidase with relatively high activity is obtained by using a recombination DNA technology to clone and link glucose oxidase (GOD) gene of aspergillus niger accc30161 is cloned to pichia-yeast secretion-type expression vector pGAPZalphaA containing glyceraldehydes-3-phosphate dehydrogenase gene promoter, transforming pichia pastoris KM71 and performing screening and identification. The enzyme activity of glucose oxidase expressed by the bacterial strain under the regulation and control of glyceraldehydes-3-phosphate dehydrogenase gene promoter reaches 107 U / mL, and a good base is established for large-scale production and application of glucose oxidase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a new strain KM71-GOD of Pichia pastoris and the application of the strain. Background technique [0002] Glucose oxidase (Glucose oxidase, GOD) is an aerobic dehydrogenase, which is widely used in various fields such as protein deglycation, glucose quantitative analysis, medical detection, food industry and biosensor. At present, GOD is mainly prepared and purified from Aspergillus niger and Penicillium, but the yield is low and the purification process is complicated. Therefore, the use of genetic engineering methods to construct better GOD production strains has been highly valued. [0003] The expression system of Pichia pastoris is one of the most widely used eukaryotic expression systems for exogenous genes. Its protein expression is high, and there are few foreign proteins in the expressed protein. Pichia pastoris also has the advantages of being able to stric...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/04C12R1/84
Inventor 毕文祥邱占军孔峰
Owner 毕文祥
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