Rekombinante Herstellung und Charakterisierung phenoloxidierender Enzyme aus Geobacillus stearothermophilus zur Evaluierung einer biosensorischen Anwendung

In the current thesis the genetic structure of the phenol hydroxylase of Geobacillus stearothermophilus has been clarified. The single components Phe A1 (oxygenase component), Phe A2 (Flavin reductase component) and a tandem construct consisting of both components were successfully produced with an E. coli host strain. Due to hexahistidin residue, with which all components were provided, all enzymes could be expressed and purified to homogeneity for the first time. The highest specific activity for the Phe A2 component achieved after optimisation was 0,6 U mg-1. The phenol hydroxylase complex was purified to homogeneity. For the oxidation of substrates no soluble FAD was needed due to direct transfer of electrons between the components. A specific activity of 0,62 U mg-1 was achieved for the phenol hydroxylase. This is the highest described value for thermostable phenol hydroxylases. The characterisation of the enzyme complex showed the following results, the temperature optimum was 56,5°C, the activation energy was 44,6 kJ mol-1 and the inactivation energy was 260 kJ mol-1. The optimal pH Value was about 7,8. The kinetic data was measured and calculated for phenol and FAD as electron acceptors. The Km values were 138 µM for phenol and 34,6 µM for FAD. For the first time the reaction mechanism of the phenol hydroxylase was well investigated. Testing showed that phenol hydroxylase from Geobacillus stearothermophilus in contrast to the current opinion in science consists of two subunits. On is the Phe A1 component and the second is a dimmer of Phe A2 subunits. At the end it was successfully proven that the recombinant phenol hydroxylase complex is suitable for biosensor applications. The enzyme complex was immobilised into an acrylamide matrix and coupled to an optical oxygen detector. This setup formed a stable biosensor for phenolic compounds with a linear measurement range from 0,6 to 56,5 µM.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Jäntges, Uwe Konrad

Contributors dc:contributor

• Hartmeier, Winfried

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Enzym-Biosensor
• Phenol
• Heterologe Genexpression
• Phenolhydroxlase
• Tandemexpression
• Immobilisierung
• phenol hydroxylase
• tandem expression
• immobilisation

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123038%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61367