The need for large enzyme quantities due to the difficult hydrolysis of recalcitrant polysaccharides is still a major barrier to economical biomass conversion for biofuel production. To discover or develop new efficient wood degrading enzymes and add into enzyme cocktails are essential for optimizing enzymatic conversion of biomass.

Cellulases in glycoside hydrolase family 6 (GH6) play a key role and are obvious targets for enzyme discovery and engineering. However, convenient substrates for high throughput screening have not been available. In paper I, improved fluorogenic substrates for GH6 were rationally designed, synthesized and evaluated. Hydrolysis rates increased by 10–150 times with Hypocrea jecorina Cel6A. Enzyme-substrate structures showed that the modifications led to relief of the exo-anomeric effect and a better position of the glycosidic bond for protonation. In paper II, the first crystal structure of a bacterial GH6 cellobiohydrolase, Thermobifida fusca Cel6B, reveals that the enzymes has a much longer substrate binding tunnel than its fungal GH6 counterparts and that the tunnel exit is closed by a loop that needs to be displaced to allow cellobiose product release for processive action by the enzyme.

Recently, lytic polysaccharide monooxygenases (LPMOs) were discovered as a new class of enzyme for cleavage of recalcitrant polysaccharides with a novel oxidative mechanism. In paper III and IV, one fungal LPMO, Phanerochaete chrysosporium GH61D, was recombinantly expressed and shown to be metal-dependent, to cleave glycosidic bonds in wood lignocellulose and to oxidize preferentially at carbon C1. The PchGH61D crystal structure is the first structure of an LPMO from basidiomycetes. Its active center shows a type II copper binding configuration, which is common to other LPMOs. Structure comparison and molecular dynamic simulations indicate three loops and a series of aromatic and polar residues near the binding surface that may influence substrate recognition and binding.