Enzymes that cleave ether bonds between lignin and non-glycosidic carbons of hemicellulose

Tethys Research has pioneered the discovery and development of enzymes that cleave ether bonds between lignin and non-glycosidic carbons of hemicellulose.  We have already identified, an enzyme that cleaves ether bonds between mannans and lignin phenyl groups, and cloned, sequenced and expressed its gene.  We have now discovered an enzyme that cleaves ether bonds between lignin and non-glycosidic carbons of xylan.  Birchwood xylan was derivatized on non-glycosidic carbons with 4-methylumbelliferone (4MU) to create 4MU-X, a fluorogenic substrate that barely fluoresces until the ether bond between xylan and 4-MU is broken, liberating highly fluorescent free 4MU.  Soil samples collected at sites of hardwood decay in Maine forests were incubated with microbiological media in which the sole carbon source was 4MU-X.  After several enrichments, the cultures were plated and individual colonies were tested for activity against 4MU-X.  A number of positive microbes were isolated, in a wide phylogenetic range of both eubacteria and fungi.  One microbe in particular, E518, produces a strongly inducible activity: the growth medium must contain either 4MU-X or benzylated xylan for E518 to express the activity.  The activity is abolished by proteases, indicating that the activity is due to an enzyme.  The enzyme is freely soluble in the medium and does not appear to require an energy cofactor.  In addition, Tethys is now isolating microorganisms that cleave ether bonds between non-glycosidic carbons of mannans and the α- and β-benzyl groups of lignin.  Characteristics of these novel enzymes and the microbes that express them will be presented.