Biochemical Studies on Enzymatic Treatment of Pulp and Paper Mill Biosludge and Polymeric Lignin

Anaerobic digestion is just becoming popular for the treatment of pulp and paper mill effluents. However, hydrolysis of complex organic matter represents the rate-limiting step of anaerobic digestion. Hence, this project is focused on improving the anaerobic digestibility of pulp and paper mill biosludge using enzymatic pretreatment. The abundance of proteins, carbohydrates, and lignin in pulp and paper mill biosludge prompted us to focus our research on the application of proteases, glycosidases, and lignin-oxidizing enzymes for enzymatic treatments of pulp and paper mill biosludge. Biosludge pretreatment with two proteases (from Bacillus licheniformis and Aspergillus oryzae) and two glycosidases (the SCO6604 glycosidase from Streptomyces coelicolor and lysozyme from chicken egg white) improved the anaerobic digestibility of pulp and paper mill biosludge, resulting in increased biogas yields (i.e., 6-26%).To identify novel lignin-oxidizing enzymes, we developed a high-throughput screen for oxidase activity of purified proteins against polymeric lignin, where we screened 45 purified bacterial multicopper oxidases (MCOs) and heme-peroxidases and identified oxidase activity against polymeric lignin in 22 proteins. Among them, the Bacillus subtilis laccase BSU0630 (CotA) demonstrated the highest activity towards both soluble substrates and polymeric lignin. Structure-based site-directed mutagenesis of BSU0630 revealed active site residues critical for catalytic activity, and three mutant variants with increased activity against polymeric lignin. Analyses of BSU0630 activity against kraft lignin using X-ray photoelectron spectroscopy (XPS), mass spectrometry (MS), liquid chromatography, and bright-field microscopy suggested oxidation and repolymerization of reaction products. We determined crystal structures of dye-decolorizing peroxidases (DyPs) FC2591 (from Frankia casuarinae), PF3257 (from Pseudomonas fluorescens), and PR9465 (from Pseudomonas rhizosphaerae). Using structure-based site-directed mutagenesis of FC2591, we identified active site residues essential for oxidase activity against polymeric lignin, as well as the S370A mutant showing enhanced oxidase activity against both soluble substrates (ABTS) and polymeric lignin. Similar to BSU0630, analysis of reaction products of the FC2591 activity towards lignin using liquid chromatography suggested polymerization of reaction products. Overall, these studies confirmed lignin transformation by oxidases, introduced a high throughput lignin screening assay, and indicated that enzymes have potential for improving biosludge digestibility, but further research is needed on protein engineering and reaction optimization.

Department dc:contributor.department

Chemical Engineering Applied Chemistry

Year dc:date.issued

2021

Author dc:creator

• Choolaei, Zahra

Advisors dc:contributor.advisor

• Yakunin, Alexander
• Edwards, Elizabeth

• Anaerobic digestion
• laccase
• Lignin
• peroxidase

Handle dc:identifier.uri

http://hdl.handle.net/1807/104889

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/104889