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

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

Heterologous expression of barley and wheat oxalate oxidase in an E-coli trxB gor double mutant

Oxalate oxidase catalyses the degradation of oxalic acid to carbon dioxide and hydrogen peroxide and is of commercial importance for clinical analyses of oxalate in biological samples. Novel potential applications for oxalate oxidase include the prevention of the formation of calcium oxalate incrusts in pulp and paper manufacture and rapid determination of oxalic acid in process waters. The potential in using oxalate-degrading enzymes in industrial processes increases the interest in finding systems for heterologous expression. Oxalate oxidase from barley is a secreted multimeric glycosylated manganese-containing enzyme with several disulfide bridges, which have been found to be essential for the catalytic activity. Attempts to achieve expression of active heterologous oxalate oxidase in bacteria have up to now met little success. In this study, one oxalate-oxidase-encoding cDNA from barley and two from wheat were cloned and tested with regard to expression in Escherichia coli. The results suggest that the selection of a novel commercially available E. coli host strain, which has the ability to form disulfide bridges in heterologous proteins expressed in its cytoplasm, was important for successful expression. Although a considerable part of the heterologous protein was produced in an insoluble and inactive form, this strain, E. coli Origami B(DE3), in addition yielded soluble and active barley and wheat oxalate oxidase. One of the wheat cDNAs, Ta(M)OXO1, gave three-fold higher activity than the barley cDNA, Hv(H)OXO1, while the other wheat cDNA, Ta(M)OXO2, gave no detectable activity. This indicates that the choice of cDNA was also critical despite the high identity between the cDNAs and the encoded polypeptides (88-89% on the nucleotide level and 88-92% on the amino-acid level). Gel filtration of cell extracts containing heterologous barley and wheat oxalate oxidase resulted in an increase in the activity. This indicates that low molecular weight inhibitory compounds were present in the E. coli lysates but could be removed by the introduction of a purification step

Treatment of pulp and paper industry process waters with oxalate oxidase: Compounds interfering with the activity. Applications of enzymes to lignocellulosics.

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Evaluation of Oxalate Decarboxylase and Oxalate Oxidase for Industrial Applications.

Increased recirculation of process water has given rise to problems with formation of calcium oxalate incrusts (scaling) in the pulp and paper industry and in forest biorefineries. The potential in using oxalate decarboxylase from Aspergillus niger for oxalic acid removal in industrial bleaching plant filtrates containing oxalic acid was examined and compared with barley oxalate oxidase. Ten different filtrates from chemical pulping were selected for the evaluation. Oxalate decarboxylase degraded oxalic acid faster than oxalate oxidase in eight of the filtrates, while oxalate oxidase performed better in one filtrate. One of the filtrates inhibited both enzymes. The potential inhibitory effect of selected compounds on the enzymatic activity was tested. Oxalate decarboxylase was more sensitive than oxalate oxidase to hydrogen peroxide. Oxalate decarboxylase was not as sensitive to chlorate and chlorite as oxalate oxidase. Up to 4 mM chlorate ions, the highest concentration tested, had no inhibitory effect on oxalate decarboxylase. Analysis of the filtrates suggests that high concentrations of chlorate present in some of the filtrates were responsible for the higher sensitivity of oxalate oxidase in these filtrates. Oxalate decarboxylase was thus a better choice than oxalate oxidase for treatment of filtrates from chlorine dioxide bleaching

Effects of ionic substances in bleaching filtrates and of lignosulfonates on the activity of oxalate oxidase from barley

The effects of ionic substances in seven industrial filtrates from kraft pulping, mechanical pulping, and sulfite pulping on the activity of oxalate oxidase from barley were investigated by pre-treatment of the filtrates with ion-exchange resins prior to enzymatic degradation of the oxalic acid in the filtrates. The pre-treatment resulted in increased oxalic acid degradation rates in all filtrates, except for one that was obtained from sulfite pulping. The possibility that lignosulfonates, which were present in the filtrate from sulfite pulping, could affect oxalate oxidase was investigated in a separate set of experiments involving four different preparations of lignosulfonates. At a lignosulfonate concentration of 50 mg/mL and a pH of 3.8, only 2-16% of the activity of oxalate oxidase remained. The results show the effects of anionic and cationic substances in bleaching filtrates on oxalate oxidase and indicate that there is an interaction between the enzyme, which has a positive net charge at pH 3.8, and the polymeric anionic lignosulfonates

Enzymatic degradation of oxalic acid for prevention of scaling

Formation of calcium oxalate incrusts, scaling, may cause severe problems in the pulp and paper industry. Enzymatic degradation of oxalic acid provides a novel approach to eliminate the problems with calcium oxalate precipitation. The performance of two oxalate-degrading enzymes, oxalate oxidase from barley and oxalate decarboxylase from Aspergillus, was tested in model experiments with respect to catalytic efficiency under different conditions, including pH, temperature and enzyme concentration. Oxalate decarboxylase was found to be more sensitive to temperature variations than oxalate oxidase, which was selected for further experiments. Authentic samples from pulp bleach plants were used to test the performance of oxalate oxidase. The results showed that oxalic acid could be degraded enzymatically also in the industrial bleaching filtrates, which were obtained from D-, E-, O-, OP-, PO-, Q-, QP-, and Z-stages. The bleaching filtrates contained compounds that inhibited the action of oxalate oxidase. The degree of inhibition was strongly dependent on the filtrate and could be alleviated by dilution

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose

Obtaining nanofibers from curauá and sugarcane bagasse fibers using enzymatic hydrolysis followed by sonication

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Nanofibers Produced from Agro-Industrial Plant Waste Using Entirely Enzymatic Pretreatments

Cellulose fibers can be freed from the cell-wall skeleton via high-shear homogenization, to produce cellulose nanofibers (CNF) that can be used, for example, as the reinforcing phase in composite materials. Nanofiber production from agro-industrial byproducts normally involves harsh chemical-pretreatments and high temperatures to remove noncellulosic polysaccharides (20–70% of dry weight). However, this is expensive for large-scale processing and environmentally damaging. An enzyme-only pretreatment to obtain CNF from agro-industrial byproducts (potato and sugar beet) was developed with targeted commercial enzyme mixtures. It is hypothesized that cellulose can be isolated from the biomass, using enzymes only, due to the low lignin content, facilitating greater liberation of CNF via high-shear homogenization. Comprehensive Microarray Polymer Profiling (CoMPP) measured remaining extractable polysaccharides, showing that the enzyme-pretreatment was more successful at removing noncellulosic polysaccharides than alkaline- or acid-hydrolysis alone. While effective alone, the effect of the enzyme-pretreatment was bolstered via combination with a mild high-pH pretreatment. Dynamic rheology was used to estimate the proportion of CNF in resultant suspensions. Enzyme-pretreated suspensions showed 4-fold and 10-fold increases in the storage modulus for potato and sugar beet, respectively, compared to untreated samples. A greener yet facile method for producing CNF from vegetable waste is presented here