Bacterial peroxide forming enzymes

Dissertação de mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2015

Lignin, after cellulose, is the most abundant organic polymer on Earth and has vital functions as a constituent of plant cell walls including structural resistance and protection against pathogens and hydrolysis. Notwithstanding lignin degradation by microbes represents a key-step in the completion of the carbon cycle in land ecosystems. The most well-known microorganisms involved in lignin degradation are fungi using oxidoreductases such as laccases, high redox peroxidases and peroxide forming enzymes. Bacterial lignin degradation is much less characterized and might allow the development of new cost-affordable bioprocesses for lignin depolymerisation, considering the relative easiness of bacterial enzyme production and engineering systems. This work focuses on in silico screening, cloning, expression and characterisation of bacterial lignin-degrading auxiliary enzymes, more specifically, H2O2 forming enzymes such as aryl-alcohol oxidase, pyranose-2-oxidases, glyoxal oxidases or galactose oxidases with the intent to advance the scope of our understanding of lignin degradation in bacteria. Here we report the successful cloning of the first bacterial pyranose-2-oxidase and also the heterologous cloning, production and preliminary biochemical characterization of a bacterial galactose oxidase, both from Arthrobacter siccitolerans. The recombinant pyranose oxidase revealed activity towards D-glucose and the typical ultra-violet/visible spectrum of a flavoenzyme. The recombinant galactose oxidase showed an optimal temperature between 25-35°C and an optimal pH of 8.0. The A. siccitolerans galactose oxidase is able to oxidize the typical substrates such as D-galactose, D-raffinose, lactose and glycerol, but also exhibited activity towards L-arabinose which has not been reported for other galactose oxidases. It is expected that this work will open new perspectives for the understanding of the structure-function relationships of bacterial hydrogen peroxide producing enzymes and optimization of multienzymatic systems, along with bacterial dye-decolorizing peroxidases and bacterial laccases, recently identified in the laboratory, for the set-up of lignin depolymerisation and valorisation.