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Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris

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Abstract

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40 kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04 M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.

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Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors kindly acknowledge East Anatolia High Technology Application and Research Center (DAYTAM, Atatürk University, Erzurum, Turkey) for the equipment support. M.A. and E.D.T also thank The Council of Higher Education (CoHE, 100/2000) PhD Scholarship Program, Turkey.

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  1. Department of Molecular Biology and Genetics, Faculty of Science, Atatürk University, Erzurum, Turkey

    Melek Acar, Seyda Yildiz, Ezgi Dag Taskesenligil & Yagmur Unver

  2. Department of Chemistry, Faculty of Science, Atatürk University, Erzurum, Turkey

    Nurgul Abul, Serpil Gerni & Hasan Ozdemir

  3. Department of Basic Sciences, Faculty of Science, Erzurum Technical University, Erzurum, Turkey

    Ramazan Kalin

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Acar, M., Abul, N., Yildiz, S. et al. Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris. Bioprocess Biosyst Eng 46, 523–534 (2023). https://doi.org/10.1007/s00449-022-02837-2

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