Binding profile of cellulolytic enzymes to steam-pretreated and alkali-pretreated sugarcane bagasse lignins

Plant cell wall is recalcitrant to enzymatic deconstruction. Lignin and hemicellulose act as barriers, limiting the access of enzymes to the glucan fibers. Physicochemical pretreatments are required to increase the cellulose accessibility, enhancing the enzymatic conversion to glucose. However, chemically increasing the accessibility comes at a price: chemical modifications in the residual lignin increase its protein-binding capacity. Because of that, during the hydrolysis, part of the enzymes may bind to lignin, reducing the amount of free enzymes to hydrolyze cellulose. We have shown previously that the residual lignin in sugarcane bagasse submitted to alkali and SO₂-catalyzed steam pretreatments unproductively bound Celluclast proteins. However, unproductive binding depends not only on lignin characteristics, but also on surface properties of proteins. We incubated Celluclast with lignins isolated from the substrates mentioned above (NaOH-lignin and SO₂-steam-lignin) and measured the activity in the supernatant before and after incubation. We also compared the SDS-PAGE band profile, measuring the bands intensity by densitometry. SO₂-steam-lignin bound more cellulases than NaOH-lignin, confirmed by activity assays and SDS-PAGE profiles. After the incubation with both lignins, β-glucosidase activity in the supernatant decreased dramatically, compared to cellulases. Similar experiment was conducted with commercial purified CBHI, EGII and β-glucosidase, incubating with SO₂-steam-lignin. The binding profiles and activities were different from Celluclast cellulases, and the amount of β-glucosidase bound to lignin was lower than CBHI and EGII. This experiment confirms that the source of enzymes reflects the lignin-binding properties, and also indicates why it is so important to supplement Celluclast with β-glucosidases.