To date, the enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is mainly isolated from the horseradish root. Because of the cumbersome purification from the plant-source, many attempts to express the enzyme in different recombinant hosts have been progressed but several steps are required to obtain purified enzyme and yields are low. Yeasts, such as Saccharomyces cerevisiae and Pichia pastoris are valuable host organisms for large scale production of heterologous glycosylated proteins. However, P. pastoris hyperglycosylates secreted proteins which impairs a conventional chromatographic purification. Within the first part of this thesis, a fast and efficient 2-step protocol using hydrophobic charge induction chromatography (HCIC) and a monolithic colum for the purification of rHRP was developed, operating both systems in a negative mode. The final enzyme preparation was purified more than 12-fold from the crude fermentation broth, which makes the recombinant expression of rHRP in P. pastoris an interesting alternative to conventional HRP production. Moreover, the extensive glycosylation of rHRP produced in P. pastoris may cause severe immunogenic responses in humans when rHRP will be applied in medicine e.g. for targeted cancer treatment. For this reason, one of the N-glycosylation sites present in rHRP was removed in a molecular biology approach by substituting the Asparagine residue at site N57 to the three structural similar amino acids Glutamine, Serine and Arginine. The most stable and active enzyme variant was expressed in a bioreactor and characterized thoroughly and compared to the wild type enzyme from P. pastoris. The final enzyme variant N57S was >2-fold more active as the wild type enzyme and had a higher thermal stability, which was achieved by substituting a single amino acid only.