Laccases and Oxalate-Degrading Enzymes Heterologous Expression and Novel Applications in Lignocellulose Processing

Sammanfattning: Lignocellulose constitutes a vast renewable resource for the production of, for example, paper and bioethanol. The potential of using laccase and oxalate-degrading enzymes in novel applications for the processing of lignocellulose was investigated in this work. Laccase cDNAs from the white-rot fungus Trametes versicolor were characterised and expressed in Saccharomyces cerevisiae and Pichia pastoris. The lcc2 cDNA was found to encode a laccase isoenzyme of 499 amino-acid residues preceded by a 21-residue signal peptide, and the sequence showed identity with Edman degradation data for T. versicolor laccase A. With S. cerevisiae, a 16-fold higher laccase activity was obtained by lowering the temperature of cultivation from 28ºC to 19ºC. P. pastoris transformants that were cultivated at 19ºC provided five times higher laccase activity than transformants kept at 28ºC, indicating the importance of low temperature for optimal production of heterologous laccase in yeast systems in general. The heterologous expression of laccase by S. cerevisiae was improved further by simultaneous overexpression of the homologous t-SNARE protein Sso2 and by optimising pH and aeration. Phenolic compounds inhibit the fermentation of sugars in lignocellulose hydrolysates to bioethanol by S. cerevisiae. Increased resistance to phenolic compounds and improved production of ethanol was obtained by using laccase-expressing S. cerevisiae for fermentation of a dilute acid hydrolysate of spruce. The formation of calcium oxalate in process water from the pulp and paper industry gives rise to the problem of calcium oxalate scaling. Oxalate-degrading enzymes could potentially be utilised for the prevention of calcium oxalate scaling in the pulp and paper industry, and for the analysis of the levels of oxalic acid in recirculated process water. Three cDNAs encoding oxalate oxidase from barley and wheat were expressed in Escherichia coli. The use of Origami B(DE3), a strain that allows disulphide formation in its cytoplasm, was found to be of critical importance in achieving successful expression in E.coli. Treatment of a series of six industrial bleaching filtrates with barley oxalate oxidase indicated that the efficiency of oxalic acid degradation was highly dependent on the chemical composition of the filtrate. Analysis of the filtrates showed the presence of compounds that had an inhibitory effect on oxalate oxidase. The inhibition could be alleviated by treatment of the filtrates with anion exchanger, cation exchanger and, to a lesser extent, uncharged resin. Chlorate, formic acid, sulphite and hydrogen peroxide, iron(II), iron(III) and copper(II) were identified as potential oxalate oxidase inhibitors in the bleaching filtrates. Comparison of oxalate decarboxylase and oxalate oxidase in eleven filtrates showed that oxalate decarboxylase performed better than oxalate oxidase in nine of the filtrates, whereas the opposite was observed in one filtrate. Analysis of the filtrates indicated that the difference in performance between the enzymes in D stage filtrates could be largely attributed to chlorate, which strongly inhibited oxalate oxidase but not oxalate decarboxylase.