(675f) Biological and Enzymatic Demethylation of Kraft Lignin and Lignin-Like Model Compounds by Boreal Forest Fungi Using a Novel Technique: Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS)

Selected Ion Flow Tube-Mass Spectrometry (SIFT-MS) is a rapid and highly sensitive analytical method for detecting volatile organic compounds by chemical ionization using selected ions. The objective of this work was to detect methanol released from Kraft lignin (KL) and lignin-like model compounds (LMCs) by the action of enzymes produced by ligninolytic fungi that specifically cleave-off O-methyl groups. Lignin demethylation is considered a new enzyme activity, and such enzymes can be described specifically as lignin demethylases. The culture collection at Lakehead University harboring some 450 wood-decay fungi collected from the Boreal Forest in northern Canada were grown on minimum salts (Vogel) nutrient medium containing Kraft lignin and different low MW LMCs under stationary conditions at room temperature for 4 weeks. Growth on these substrates induced the formation of enzymes that specifically liberated methanol as determined by SIFT-MS. The most promising fungi examined producing methanol from KL were isolates LU-86 (84 ppb), LU-122 (125 ppb), BRI-269 (112 ppb) and BRI-270 (180 ppb), and with LMCs as substrates: BRI-122 (30 ppb), BRI-269 (140 ppb) and BRI-270 (171 ppb). The low levels of methanol produced are attributable to these isolates also producing an enzyme (methanol oxidase) that metabolizes the methanol released. Confirmation of demethylation of lignin was supported by an increase in phenolic (vicinal hydroxyl) content in the treated lignin. In another series of experiments, the same fungal isolates (6) were cultivated on media containing KL for 4 weeks. Mycelia were removed by centrifugation, and the supernatants recovered (ECF) and used as the source of enzyme. Enzymatic demethylation of KL and different LMCs was determined by adding ECF and measuring the amount of methanol liberated by SIFT-MS. Different lignin substrates, such as lignosulfonate (Sigma), periodate-oxidized lignin, Kraft lignin, and lignins extracted from softwood and hardwood black liquors, were also examined for the release of methanol by enzymatic demethylation. The release of methanol from these ligninolytic substrates as measured by SIFT-MS demonstrated unequivocally that O-methyl groups were cleaved from these compounds by the fungal and enzyme systems studied. The results obtained will be discussed in detail.

Supported by funds from NSERC-CRD (CRDPJ 38079-09: Dekker) Canada