title: Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris.
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.
authors.affiliation: Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria.
authors.forename: Florian W
authors.initials: FW
authors.lastname: Krainer
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Pletzenauer
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria.
authors.forename: Laura
authors.initials: L
authors.lastname: Rossetti
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB, GmbH), Graz, Austria.
authors.forename: Anton
authors.initials: A
authors.lastname: Glieder
authors.affiliation: Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria. Electronic address: oliver.spadiut@tuwien.ac.at.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D002846:Chromatography, Affinity; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: Glycosylation; Horseradish peroxidase; Monolith; Negative chromatography; Pichia pastoris; Protein purification