Computer-Assisted Enzyme-Cocktail Approach Highly Improves Bioethanol Yield

Environmental concerns caused by fossil fuels and exponential energy demands have motivated the growth of the bioethanol industry, a promising renewable and green fuel. Enzymatic hydrolysis holds the promise to facilitate the most critical and rate-limiting hydrolysis steps in the cellulosic biomass conversion to ethanol process. However, a remaining challenge is how to rationally design the enzyme mixture without being labor-intensive and time-consuming. Herein, we reported a computer-assisted enzyme-cocktail (ComEC) design approach to improve feedstock utilization and ethanol yield. After screening of 17 structural and dynamics observables, two determinants, the hydration shell and water molecules in the active site, were identified from 20 molecular dynamics (MD) simulations of cellulase in 0–16% (v/v) ethanol and then used to direct the overall hydration-guided strategy and active-site inhibition-guided strategy for mixing the CBHI-CBHII-EG-BG cocktail. Notably, both strategies combining with fed addition technology achieved up to 5.8% improvement on ethanol yield compared to non-cellulase in situ corn fiber conversion. These results proved that the ComEC approach, based on a comprehensive molecular enzyme–solvent interaction landscape, allows intelligent design of enzyme-cocktail at the time–dosage resolution level and opens the gate of gaining higher production of bioethanol and even other renewable liquid fuels.