Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris

Protein Expression and Purification
Florian W KrainerOliver Spadiut

Abstract

The plant enzyme horseradish peroxidase (HRP) is used in several important industrial and medical applications, of which especially biosensors and diagnostic kits describe an emerging field. Although there is an increasing demand for high amounts of pure enzyme preparations, HRP is still isolated from the plant as a mixture of different isoenzymes with different biochemical properties. Based on a recent next generation sequencing approach of the horseradish transcriptome, we produced 19 individual HRP isoenzymes recombinantly in the yeast Pichia pastoris. After optimizing a previously reported 2-step purification strategy for the recombinant isoenzyme HRP C1A by substituting an unfavorable size exclusion chromatography step with an anion exchange step using a monolithic column, we purified the 19 HRP isoenzymes with varying success. Subsequent basic biochemical characterization revealed differences in catalytic activity, substrate specificity and thermal stability of the purified HRP preparations. The preparations of the isoenzymes HRP A2A and HRP A2B were found to be highly interesting candidates for future applications in diagnostic kits with increased sensitivity.

References

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Citations

Sep 26, 2015·International Journal of Molecular Sciences·Simona CaponeOliver Spadiut
Feb 11, 2015·Acta Biochimica Et Biophysica Sinica·Haili ZhuWei Liu
Jan 13, 2015·Applied Microbiology and Biotechnology·Florian W Krainer, Anton Glieder
Nov 14, 2016·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·Angela Faustino JozalaAdalberto Pessoa
Jan 6, 2018·International Journal of Molecular Sciences·Suzanne M HuddyEdward P Rybicki
Oct 5, 2017·The Protein Journal·Yaser FattahianGholamreza Asadikaram
Mar 19, 2016·Cancer Medicine·Günther BonifertOliver Spadiut
Jan 17, 2021·International Journal of Biological Macromolecules·Soudabeh FarhadiMojtaba Mortazavi
Oct 15, 2020·Enzyme and Microbial Technology·Odwa D V BikoWillem H van Zyl
Sep 29, 2020·Protein and Peptide Letters·Veda P PandeyUpendra N Dwivedi
Aug 16, 2021·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Diana HumerOliver Spadiut

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Methods Mentioned

BETA
column chromatography
size exclusion chromatography
electrophoresis
Protein Assay
enzyme-linked immunosorbent assays
glycosylation

Software Mentioned

Sigma Plot
UVPC Optional Kinetics
NetNGlyc

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