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Simona Capone

Simona Capone

Vienna University of Technology
Institute of Chemical Engineering
Austria

Title: Glyco-mutant library of the versatile enzyme horseradish peroxidase

Biography

Simona Capone finished her master degree studies in Industrial Biotechnology, in 2012, at the Bicocca University in Milan. She is currently attending her PhD program at the Technical University of Vienna, working on the research project “glycoengineered horseradish peroxidase for targeted cancer treatment”- Funded by FWF Projectnumber P24861-B19.

Abstract

When the 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 P. 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 in the bioreactor. 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 8 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 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. Here, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the horseradish peroxidase glyco-library.