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Expression of glucose oxidase by using recombinant yeast
1Institute for Molecular Biology and Genetics, Chonbuk National University, Dukjindong 664-14, Chonju, Chonbuk 561-756, South Korea.
Journal of Biotechnology
|August 11, 2000
Summary
This study optimized glucose oxidase (GO) production in yeast using various promoters and terminators. The ADH2-GPD hybrid promoter and UV mutagenesis significantly enhanced GO yield, with corn steep liquor offering a cost-effective alternative substrate.
Area of Science:
- Biotechnology
- Molecular Biology
- Enzyme Engineering
Background:
- Glucose oxidase (GO) is an important enzyme with applications in various industries.
- Efficient and cost-effective production of GO in microbial hosts is crucial for its commercial viability.
- Recombinant DNA technology offers a pathway to enhance enzyme production in yeast.
Purpose of the Study:
- To clone and express the glucose oxidase gene (GO) from Aspergillus niger in Saccharomyces cerevisiae.
- To evaluate the impact of different promoters and terminators on GO expression and secretion.
- To optimize GO production through promoter engineering, mutagenesis, and alternative substrates.
Main Methods:
- Cloning of the Aspergillus niger glucose oxidase gene into the yeast shuttle vector YEp352.
- Transformation of Saccharomyces cerevisiae with recombinant vectors.
- Testing of four different promoters (GAL1, GAL10, GPD, ADH2-GPD) and two terminators (intrinsic GO, GAL7).
- Optimization of culture conditions (glucose and ethanol concentrations) and application of UV mutagenesis.
- Evaluation of corn steep liquor as an alternative substrate.
Main Results:
- The ADH2-GPD hybrid promoter significantly outperformed other tested promoters in GO production.
- The presence of intrinsic or GAL7 terminators greatly increased GO yield compared to their deletion.
- Optimal GO production was achieved with 2% glucose and 1.5% ethanol, yielding 260 IU/ml.
- UV mutagenesis further enhanced GO yield to 460 IU/ml without affecting cell growth.
- Corn steep liquor proved to be an effective and cost-efficient substrate for GO production.
Conclusions:
- The ADH2-GPD hybrid promoter is highly effective for enhancing glucose oxidase production in recombinant Saccharomyces cerevisiae.
- Genetic engineering and mutagenesis strategies can substantially increase enzyme yields.
- The use of cost-effective substrates like corn steep liquor is feasible for industrial-scale GO production.