7-30: The addition of recombinant Lentinula edodes laccases can enhance the enzymatic hydrolysis of steam-pretreated softwoods

Laccases (E.C.1.10.3.2) are multi-Cu oxidases that catalyse the oxidation of phenolic compounds using molecular oxygen as an oxidant, concomitantly releasing water molecule as a by-product.  With the addition of mediators the activity of laccases can be enhanced such that the oxidation of substrates with a high redox potential becomes feasible.  Laccases have been assessed for a number of industrial applications including bioremediation, pulping and bleaching.  Two recombinant allelic laccases from Lentinula edodes were expressed in Pichia pastoris and they were subsequently purified and characterised.  The recombinant enzymes were shown to have similar substrate specificities and stabilities and they were both able to catalyse the biodegradation of synthetic dyes and polyaromatic hydrocarbons.  These initial results suggested their potential as “accessory” enzymes when added to a typical cellulase enzyme mixture, with the hope they would improve the hydrolysis of the cellulose within pretreated softwoods to glucose.  We assessed a range of “cocktail additions” when adding the laccases and mediators to cellulases during the hydrolysis of various pretreated lignocellulosic substrates.  The optimised laccase-mediator system (LMS) was shown to enhance the hydrolysis of pretreated softwoods but not agricultural residues or hardwoods. The enzyme/mediator mixtures were further modified to both improve the hydrolysis of different softwoods (to obtain greater than 70% hydrolysis in as short a time as possible) and to help us better elucidate the relative catalytic contributions of both the LMS and cellulases to the overall hydrolytic activity of the mixture.