Optimization of a recombinant multicomponent enzyme mixture for saccharification of ionic liquid pretreated biomass

Lignocellulolytic cocktails consist primarily of a mixture of several enzymes that synergistically solubilize the plant cell walls. The necessary loading of each enzyme in these mixtures may vary as a function of the biomass type and the pretreatment method, thus, it is important to optimize cellulolytic mixtures for the particular feedstock and pretreatment method used. Among the benefits of creating an optimal mixture is the resulting lowered cost of biomass saccharification. We optimized a cellulolytic enzyme mixture for the hydrolysis of ionic liquid 1-ethyl-3-methyl-imidazolium acetate ([C₂mim][OAc]) pretreated switchgrass (ILSG) and for use in the presence of the same ionic liquid (IL) at 70 ºC. Cellulolytic enzyme mixtures comprised of three recombinant enzymes previously shown to be tolerant of up to 20% [C₂mim][OAc] were optimized using an augmented simplex lattice a design-of-experiments (DOE). For comparison, we also optimized a mixture using a set of three commercially cellulases. The mixture of commercial cellulases exhibited 90% glucose release at pH 5 and 55ºC, but produced little glucose at 70 ºC and no glucose in the presence of [C₂mim][OAc] at any temperature. Mixtures optimized using our in-house set of [C₂mim][OAc] tolerant cellulases catalyzed conversion of up to 50% of the ILSG to glucose at 70°C and in the presence of 20% [C₂mim][OAc]. This DOE approach can be used for the formulation of more complex enzyme mixtures for the deconstruction of pretreated lignocellulosic substrates, particularly at the higher temperatures, pHs, and IL concentrations of relevance to industrial applications.