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Lignin is a highly methylated natural aromatic biopolymer found in the cell walls of plants. Biological demethylation bywood-
decay fungiremoves O-methyl groups from lignin increasing its phenolic content. The objective was to demonstrate a new
enzyme associated with lignin demethylation (lignin demethylase, LDM) that cleaves O-methyl groups from benzyl rings
comprising lignin making the latter more phenolic in structure toserve as phenol substitutes in formaldehyde-based polymers.
Assays for LDM activity were developedthat specifically measured (i) methanol liberated as a consequence of demethylation,
followed by (ii) an increase in pyrocatecholic structure content of modified lignin. Wood-decay fungi isolated from the Boreal
Forest (31 isolates growing solely on Kraft lignin, KL) were screened for enzymes that modify lignin.An assay for LDM consisted
of coupling an enzymatic oxidation step using alcohol oxidase to convert methanol released to formaldehyde and hydrogen
peroxide, and then employing specific reagents to yield colored complexes measured spectrophotometrically. Fivebiochemical
methods were developed to determine LDM using KL andlignin-like model compounds (LMC; guaiacol, veratryl, syringyl
alcohols). Formaldehyde was measured using (i) pentan-2,4-dione/ammonium acetate. (ii) Hydrogen peroxide was assayed
against ABTS in the presence of horseradish peroxidase. The increase in pyrocatecholic content of modified LMC?s and KLas
consequence of demethylation, was determined spectrophotometrically by either (iii)complexation with Ti(III)-nitrilotriacetic
acid, or (iv) reaction with4-aminoantipyrine. Biologically modified KL structures were determined by FT-IR. SIFT-MS(v), a
highly sensitive procedure that measuresvolatile organic compounds, was also used to measure methanol,and demonstrated
unequivocally that methanol was released from LMC?s and KL examined. The potential of ligninolytic enzymes to modify KL
and LMCs was performed on 6 ligninolytic fungal isolates, and the results obtained are discussed.Supported by NSERC-CRD.
Balaji Venkatesagowda completed his PhD at the University of Madras (India) and joined the Biorefining Research Institute in 2010. His specialty
is biodiesel production from seed oils using fungal lipases. Lately
, his research focuses on biological modification of lignin for polymer applications.
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