Abstract
Ionic liquid pretreatment of biomass has been shown to greatly reduce the recalcitrance of lignocellulosic biomass, resulting in improved sugar yields after enzymatic saccharification. However, even under these improved saccharification conditions the cost of enzymes still represents a significant proportion of the total cost of producing sugars and ultimately fuels from lignocellulosic biomass. Much of the high cost of enzymes is due to the low catalytic efficiency and stability of lignocellulolytic enzymes, especially cellulases, under conditions that include high temperatures and the presence of residual pretreatment chemicals, such as acids, organic solvents, bases, or ionic liquids. Improving the efficiency of the saccharification process on ionic liquid pretreated biomass will facilitate reduced enzyme loading and cost. Thermophilic cellulases have been shown to be stable and active in ionic liquids but their activity is typically at lower levels. Cel5A_Tma, a thermophilic endoglucanase from Thermotoga maritima, is highly active on cellulosic substrates and is stable in ionic liquid environments. Here, our motivation was to engineer mutants of Cel5A_Tma with higher activity on 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) pretreated biomass. We developed a robotic platform to screen a random mutagenesis library of Cel5A_Tma. Twelve mutants with 25–42% improvement in specific activity on carboxymethyl cellulose and up to 30% improvement on ionic-liquid pretreated switchgrass were successfully isolated and characterized from a library of twenty thousand variants. Interestingly, most of the mutations in the improved variants are located distally to the active site on the protein surface and are not directly involved with substrate binding.
Highlights
Uncertainties in petroleum supplies [1], concerns about climate change [2], and environmental pollution [3] have generated renewed interest in producing sustainable biofuels and bio-derived chemicals from lignocellulosic biomass [4,5]
Improving the efficiency of the saccharification process for the production of fuels from lignocellulosic biomass can be achieved by enhancing the catalytic efficiency and stability of lignocellulolytic enzymes
We developed an integrated and efficient high-throughput robotics platform for screening the activity of large libraries of thermophilic cellulases generated by random mutagenesis
Summary
Uncertainties in petroleum supplies [1], concerns about climate change [2], and environmental pollution [3] have generated renewed interest in producing sustainable biofuels and bio-derived chemicals from lignocellulosic biomass [4,5]. A major obstacle to the conversion of lignocellulosic biomass to fuels is that the highly complex lignocellulosic matrix consisting of cellulose, hemicellulose and lignin is highly recalcitrant to degradation into its individual components. Breaking this recalcitrance requires pretreatment using methods such as dilute acids [9], ammonia fiber expansion (AFEX) [10], hot water treatment [11] and steam explosion [12], before enzymes can efficiently access the cellulose and hemicelluloses and generate fermentable sugars.
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