Glyco-variant library of the versatile enzyme horseradish peroxidase.
Bottom Line: 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.
Affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.Show MeSH
Related in: MedlinePlus
Mentions: We observed similar trends of Km and Vmax for the substrate H2O2, as the majority of HRP glyco-variants showed a reduced catalytic efficiency compared with the wt enzyme (Table IV). In fact, N57S was the only glyco-variant showing similar or even higher catalytic efficiency with both substrates compared with the wt. The Michaelis–Menten kinetics for the wt enzyme and for variant N57S for both substrates is exemplarily shown in Figure 2, whereas illustrations for the other enzyme variants are shown in the Supplementary data, Figure S3.Fig. 2.
Affiliation: Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna University of Technology, Vienna 1060, Austria.