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

Autor: Krainer FW; Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria., Pletzenauer R; Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria., Rossetti L; Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria., Herwig C; Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria., Glieder A; Austrian Centre of Industrial Biotechnology (ACIB, GmbH), Graz, Austria., Spadiut O; Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria. Electronic address: oliver.spadiut@tuwien.ac.at.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2014 Mar; Vol. 95, pp. 104-12. Date of Electronic Publication: 2013 Dec 14.
DOI: 10.1016/j.pep.2013.12.003
Abstrakt: 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.
(Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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