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Background <p>Oxalate oxidase (OxOx) is an important enzyme commonly used in the determination of urine oxalate concentration. It is naturally expressed by a variety of plants and microorganisms and recombinant OxOx enzyme has been successfully expressed in yeast, but not in <i>Escherichia coli</i> (<i>E. coli</i>) due to its aggregation in insoluble inclusion bodies. This study aimed to (1) develop a method for purification of active OxOx following expression in <i>E. coli</i> and (2) demonstrate the feasibility of using filter paper coated with recombinant OxOx as a method to measure oxalate concentration in solution.</p> Results <p>A synthetic gene optimized for <i>E. coli</i> codon bias expression was derived from barley and cloned into the pRSET expression vector. Phenyl-Sepharose CL-4B and Q-Sepharose Fast Flow chromatography purified the active enzyme to over 90% purity. Using these methods, 21&#xa0;mg of purified active OxOx enzyme was obtained from a 1&#xa0;L culture. The purified OxOx exhibited an activity of 3.4 U/mg with the minimum oxalate concentration needed for visual detection on filter paper of 25 µM (range 25–500 µM).</p> Conclusion <p>Our study highlights the feasibility of using <i>E. coli</i> for the expression and purification of OxOx, providing a promising approach for future applications in the field of healthcare. Our study has also laid the foundation for further developing an oxalate test strip for urinary oxalate assays based on OxOx.</p>

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Efficient refolding, purification, and characterization of barley oxalate oxidase in Escherichia coli

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月6日

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  • William Donelan,
  • ShiWu Li,
  • Paul R. Dominguez-Gutierrez,
  • Dongqi Tang,
  • Li-Jun Yang,
  • Benjamin K. Canales

摘要

Background

Oxalate oxidase (OxOx) is an important enzyme commonly used in the determination of urine oxalate concentration. It is naturally expressed by a variety of plants and microorganisms and recombinant OxOx enzyme has been successfully expressed in yeast, but not in Escherichia coli (E. coli) due to its aggregation in insoluble inclusion bodies. This study aimed to (1) develop a method for purification of active OxOx following expression in E. coli and (2) demonstrate the feasibility of using filter paper coated with recombinant OxOx as a method to measure oxalate concentration in solution.

Results

A synthetic gene optimized for E. coli codon bias expression was derived from barley and cloned into the pRSET expression vector. Phenyl-Sepharose CL-4B and Q-Sepharose Fast Flow chromatography purified the active enzyme to over 90% purity. Using these methods, 21 mg of purified active OxOx enzyme was obtained from a 1 L culture. The purified OxOx exhibited an activity of 3.4 U/mg with the minimum oxalate concentration needed for visual detection on filter paper of 25 µM (range 25–500 µM).

Conclusion

Our study highlights the feasibility of using E. coli for the expression and purification of OxOx, providing a promising approach for future applications in the field of healthcare. Our study has also laid the foundation for further developing an oxalate test strip for urinary oxalate assays based on OxOx.