Microbial syringol utilization
Disclosed herein are compositions of non-naturally occurring enzymes to enable microbial syringol utilization with GcoAB.
1. A non-naturally occurring GcoAB enzyme comprising the amino acid sequence of SEQ ID NO: 1 capable of the O-aryl-demethylation of syringol and the O-aryl-demethylation of guaiacol and wherein a phenylalanine residue at the active site has been changed to an amino acid selected from the group consisting of alanine, serine, histidine, valine, isoleucine, and leucine.
2. The enzyme of claim 1 wherein a phenylalanine at the active site has been changed to alanine.
3. The enzyme of claim 1 comprising a k cat for guaiacol of at least 11 sec −1 and a k cat for syringol of at least 5.9 sec −1 .
4. The enzyme of claim 1 comprising a K M for guaiacol of less than about 40 mM and a K M for syringol of less than about 10 mM.
5. The enzyme of claim 1 comprising a kcat/K M for guaiacol of greater than about 290 mM −1 sec −1 and a kcat/K M for syringol of greater than about 600 mM −1 sec −1 .
6. The enzyme of claim 1 comprising increased specific activity for guiaicol when compared to the native GcoAB enzyme.
7. The enzyme of claim 1 wherein the specific activity for syringol is greater than 0.54 μmol sec −1 μmol −1 .
8. The enzyme of claim 1 wherein the specific activity for syringol is greater than 3.9 μmol sec −1 μmol −1 .
9. The enzyme of claim 1 wherein the specific activity for syringol is greater than 5.1 μmol sec −1 μmol −1 .