Abstract

Traditionally Horseradish peroxidase (HRP) the archetypal class III plant peroxidase has been investigated with respect to chemical modifications, organic solvent tolerance studies, reaction kinetics, kinetics in organic solvents, immunohistochemical and in biosensor fabrication. However HRP has many factors that prevent its use in industrial, and research areas. This is because of its inherent structure, and activity restrictions in regard to temperature, its pH range of activity, and solvents it is catalytically active in. There is another class III plant peroxidase, which has greater stability with regards to the above factors, this peroxidase is Soybean peroxidase (SBP). This is the peroxidase that is investigated in this work. Chemical modifications historically have targeted amino acid residues in the tertiary folded active form of an enzyme. HRP and many other enzymes have been chemically modified by modifying the E - N H 3 group of lysine (HRP has 3 lysines available for modification out of 5 lysines). SBP cannot be chemically modified in this fashion, because it has no lysines available for modification, out of 3 lysines). Therefore in order to attempt to successfully chemically modify SBP another approach needs to be studied. SBP is a glycosylated enzyme. By chemically modifying this catalytically inactive region of SBP, an increase in stability and activity can be achieved. Three methods of chemically modifying the carbohydrate shell, these were modifying by dextran dialdehyde, adipic acid dihydrazide (AADH), and ferrocene carboxcylic acid (FeCOOH). The dextran dialdehyde modification yielded a 2-fold modification. The AADH modification yielded a three-fold modification, but problems arose with the inhibition of the enzymes active site and the cross-linking agent. The FeCOOH modification was successful adding at least 2 extra irons to the enzyme. The modified was used in conjunction with a sensor. This modification also reduces the electron transfer distance between the enzyme and the transducer. This sensor was characterised and compared to a native SBP biosensor. Also investigated in this work was the non-heme peroxidase vanadium bromoperoxidase (VBrPO). VBrPO is a sourced enzyme, utilising vanadium at its active site. The native enzyme was characterised and the dextran dialdehyde modification was carried out on it. The modification was then characterised and compared to the native form of the enzyme.