An engineered cellobiohydrolase I for sustainable degradation of lignocellulosic biomass

Abstract

This study provides computational‐assisted engineering of the cellobiohydrolase I (CBH‐I) from Penicillium verruculosum with simultaneous enhanced thermostability and tolerance in ionic liquids, deep eutectic solvent, and concentrated seawater without affecting its wild‐type activity. Engineered triple variant CBH‐I R1 (A65R‐G415R‐S181F) showed 2.48‐fold higher thermostability in terms of relative activity at 65°C after 1\u2009h of incubation when compared with CBH‐I wild type. CBH‐I R1 exhibited 1.87‐fold, 1.36‐fold, and 1.57‐fold higher specific activities compared with CBH‐I wild type in [Bmim]Cl (50\u2009g/L), [Ch]Cl (50\u2009g/L), and two‐fold concentrated seawater, respectively. In the multicellulases mixture, CBH‐I R1 showed higher hydrolytic efficiency to hydrolyze aspen wood compared with CBH‐I wild type in the buffer, [Bmim]Cl (50\u2009g/L), and two‐fold concentrated seawater, respectively. Structural analysis revealed a molecular basis for the higher stability of the CBH‐I structure in which A65R and G415R substitutions form salt bridges (D64 … R65, E411 … R415) and S181F forms π–π interaction (Y155 … F181), leading to stabilize surface‐exposed flexible α‐helixes and loop in the multidomain β‐jelly roll fold structure, respectively. In conclusion, the variant CBH‐I R1 could enable efficient lignocellulosic biomass degradation as a cost‐effective alternative for the sustainable production of biofuels and value‐added chemicals.