Characterisation of recombinant peroxidases in respect to efficiency and reaction characteristics for selected substrates

Abstract

Peroxidases are oxidoreductases produced by a number of microorganisms and plants. They are heme proteins containing iron (III) protoporphyrin IX as the prosthetic group. Peroxidases have a molecular weight ranging from 30,000 to 150,000 Da and catalyze the reduction of peroxides, such as hydrogen peroxide but also oxidations of various organic and inorganic compounds. Peroxidase activity has been identified in plants, microorganisms and animals, where they play important roles. The potential of peroxidases is substantial and therefore they are the focus of interest of industries. Eucodis Bioscience is interested in an industrial use of heme-peroxidases and therefore produces various types of that enzyme. To understand the effect of different peroxidases specific enzyme-substrate reactions were studied in this thesis using an unsaturated fatty acid and its methyl ester as substrate. An analytical strategy was developed to analyze reaction samples by means of thin layer chromatography (TLC) and an efficient staining protocol but also with GC coupled to mass spectrometry (GCMS). This method combination allowed to determine the conversion rates of the enzymes and subsequently by studying the conversion rates the enzymatic reaction conditions were systematically optimized by qualitative and semi-quantitative evaluation of reaction products. In this thesis several reaction products could be identified by GCMS.