title: Glyco-variant library of the versatile enzyme horseradish peroxidase.
abstract: When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.
authors.forename: Simona
authors.initials: S
authors.lastname: Capone
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.
authors.forename: Robert
authors.initials: R
authors.lastname: Pletzenauer
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna 1190, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Metzger
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna 1190, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Herwig
authors.affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria oliver.spadiut@tuwien.ac.at.
authors.forename: Oliver
authors.initials: O
authors.lastname: Spadiut
mesh_terms: D020816:Amino Acid Motifs; D001709:Biotechnology; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase
keywords: Pichia pastoris; bioprocess technology; glyco-engineering; glycosylation; horseradish peroxidase