Engineering of Enzymes to Improve Lignin Breakdown for Use in Fuel Ethanol Production

Abstract

Lignin is the second most abundant biopolymer on Earth, behind cellulose. Lignin contains aromatic rings similar to those in fossil fuels, and breaking down lignin into smaller polymers is the first step in using it as a source of fuel. Once lignin is broken down, additional enzymes can then hydrolyze the cellulose and hemicelluloses, so that sugar fermentation and production of ethanol can occur. Lignin is difficult to break down, although some fungi can slowly degrade it using lignin peroxidase and/or laccase enzymes. Laccases break down the complex structure of lignin, while lignin peroxidases oxidize the aromatic rings. These are slow processes and are not practical for mass‐producing fuels from lignin. We are engineering these enzymes with the goal of improving the efficiency of lignin degradation. We have begun by studying BglB, an enzyme involved in the hydrolysis of cellulose, as a proof of concept. We have successfully expressed, purified, and assayed the activity of the BglB protein. We have also used FoldIt software to identify mutations for modifying the activity of the BglB protein. One specific series of mutants characterized had the amino acid substitutions W402S, W412F, E409Q, and W410H. We have characterized the activity of these mutants, and found W412F had similar activity as the wild type, while the activities of W402S, E409Q, and W410H were lower than wild type. We are now ready to expand these engineering methods to lignin peroxidase and laccase enzymes, to generate more efficient enzymes for the production of fuel ethanol from lignin.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.