Laccase from a Litter Degrading Fungus: Evaluation for Environmental Applications and Modification of Lignin to a Bioadhesive

Sammanfattning: Popular Abstract in Swedish The phenol oxidase “laccase” was first described towards the end of the 19th century from the wound sap of the lacquer tree Rhus vernicifera. However, its use in one of the oldest biotechnologies, i.e. the making of lacquerware in East-Asia, dates back to 5000-6000 BC. The enzyme has the widest distribution in nature among the multicopper oxidases. It catalyzes one electron transfer oxidation in the presence of molecular oxygen producing water as by-product. Based on these highly desired oxidative properties and its wide range of substrates, immense efforts have marked the last decades regarding the search for new enzyme sources and potential laccases that are suitable for different applications. In the present thesis, we introduce a litter degrading fungus from Galerina sp. as a laccase producer. Besides laccase, the isolate is capable of producing lignin and manganese peroxidases as lignin modifying enzymes. The fungus has the ability to grow on different agricultural residues in submerged and solid state fermentation and to produce high amounts of laccase, especially after induction. The physical and biochemical characteristics of the major laccase, purified by chromatographic means from the crude culture supernatant, are treated in details. Sequences of internal peptides confirm the identity of the purified protein as a laccase. The enzyme is interestingly tolerant to elevated concentrations of hydrogen peroxide, and has high affinity towards common laccase substrates. The potential of the produced laccase is explored in a series of applications with important environmental implications. Indeed, the enzyme, in both free and immobilized forms, is able to decolorize an azo and triphenylmethane dyes. Moreover, laccase from Galerina Sp. is presented as a very good agent for the demethylation of hard wood Kraft lignin from eucalyptus as well as some lignin model compounds. In this context, a fast and sensitive spectrophotometric method for measuring the released methanol is adapted, presenting an alternative to gas chromatography. Laccase mediated lignin modification, including demethylation, is known to make it more reactive and prone to coupling. Therefore, this feature is further employed in the thesis to oxidize a range of lignin model compounds and catalyze their coupling to a substituted aromatic amine, highlighting the involvement of laccase in water and soil detoxification. Insights into the mechanism of coupling are given, and the kinetics of the enzymatic oxidations are studied using calorimetry. Based on the concept of lignin activation by laccase, another field of investigation is approached in this work covering the laccase-aided production of formaldehyde-free wood bioadhesives. A variety of polymeric materials (a synthetic polymer, a natural polysaccharide, and a protein) are reacted with Kraft lignin in the presence of laccase and a redox mediator in order to form composites with adhesive properties. The tensile and shearing strengths of the produced adhesives are evaluated. In addition, sorption isotherms are employed in order to characterize some of those composites in terms of their capacity to adsorb or release water vapour from their surface at different humidities. Results are useful for the evaluation of surface properties.