New thermostable endoglucanase from Spirochaeta thermophila and its mutants with altered substrate preferences.

Endoglucanases are key elements in several industrial applications, such as cellulosic biomass hydrolysis, cellulose fiber modification for the production paper and composite materials, and in nanocellulose production. In all of these applications, the desired function of the endoglucanase is to create nicks in the amorphous regions of the cellulose. However, endoglucanase can be diverted from its activity on the fibers by other substrates-soluble oligosaccharides. This issue was addressed in the current study using enzyme engineering and an enzyme evolution approach. To this end, a hypothetical endoglucanase from a thermostable bacterium Spirochaeta thermophila was for the first time cloned and characterized. The wild-type enzyme was used as a starting point for mutagenesis and molecular evolution toward a preference for the higher molecular weight substrates. The best of the evolved enzymes was more active than the wild-type enzyme toward high molecular weight substrate at temperatures below 45 °C (3-fold more active at 30 °C) and showed little or no activity with low molecular weight substrates. These findings can be instrumental in bioeconomy sectors, such as second-generation biofuels and biomaterials from lignocellulosic biomass. KEY POINTS: • A new thermostable endoglucanase was characterized. • The substrate specificity of this endoglucanase was changed by means of genetic engineering. • A mutant with a preference for long molecular weight substrate was obtained and proposed to be beneficial for cellulose fiber modification.