Association of amphipathic lignin derivatives with cellobiohydrolase groups improves enzymatic saccharification of lignocellulosics

Amphipathic lignin derivatives (ALDs), prepared from hardwood acetic acid lignin and softwood soda lignin via coupling with a mono-epoxylated polyethylene glycol, have been reported to improve the enzymatic saccharification efficiency of lignocellulose while maintaining significant residual cellulase activity after saccharification. We previously demonstrated that the effect of ALDs was caused by a direct interaction between ALDs and Cel6A (or CBH II). In this study, a different ALD was prepared from softwood kraft lignin in addition to aforementioned ALDs. The interactions between all the ALDs and the enzymes other than Cel6A, such as Cel7A and Cel7B, in a cellulase cocktail were investigated using surface plasmon resonance. The kraft lignin-based ALD showed the highest residual cellulase activity among all ALDs and an improved cellulolytic enzyme efficiency similar to those of the other ALDs. All ALDs were found to directly associate with major enzymes in the cellulase cocktail, Cel6A and Cel7A (or CBH I), but not with Cel7B (or EG I). In addition, the ALDs showed a much higher affinity to amino groups than to hydroxy and carboxy groups. In contrast, polyethylene glycol (molecular mass 4000 Da), one part of the ALD and a previously reported enzymatic saccharification enhancer, did not adsorb onto any enzymes in the cellulase cocktail or the amino group. Size exclusion chromatography demonstrated that the ALDs formed self-aggregates in both water and chloroform; the formation process in the latter was especially unique. Therefore, we conclude that the high residual cellulase activity is attributed to the direct association of ALD aggregates with the CBH group.