Abstract
Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a long-standing goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can “resurrect” efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30–90 °C), pH values (4–10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome.
Highlights
IntroductionAnother attempt to increase the activity of LFCA_EG is to incorporate it into scaffoldin, a non-catalytic scaffolding protein from a cellulosome, which is a macromolecular complex containing several lignocellulose-degrading enzymes anchored via dockerin protein domains
The carbohydratebinding module (CBM), a smaller subunit responsible for cellulose binding, does not align well, as some sequences had the CBM at the C terminus, while others have it at the N terminus, and there are numerous gaps
Introduction of the LFCA_EG in a cellulosome. Another attempt to increase the activity of LFCA_EG is to incorporate it into scaffoldin, a non-catalytic scaffolding protein from a cellulosome, which is a macromolecular complex containing several lignocellulose-degrading enzymes anchored via dockerin protein domains. Anaerobic cellulolytic bacteria such as C. thermocellum utilize the cellulosome to degrade cellulose very efficiently, and its use has been suggested for industrial applications, due to the increased cellulolytic activity observed when compared with the free enzymes[32]
Summary
Another attempt to increase the activity of LFCA_EG is to incorporate it into scaffoldin, a non-catalytic scaffolding protein from a cellulosome, which is a macromolecular complex containing several lignocellulose-degrading enzymes anchored via dockerin protein domains. Anaerobic cellulolytic bacteria such as C. thermocellum utilize the cellulosome to degrade cellulose very efficiently, and its use has been suggested for industrial applications, due to the increased cellulolytic activity observed when compared with the free enzymes[32]. LFCA-Dock incorporated into the cellulosome and LFCA_EGCBM is capable of binding microcrystalline cellulose Avicel (Supplementary Fig. 6), while the other proteins fail This indicates that, as expected, only when a CBM is present, specific microcrystalline cellulose binding can occur
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