Abstract
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.
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Data availability
The datasets generated during and/or analyzed during the current study are available from supplementary information files and from the corresponding author on reasonable request.
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This study was funded by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation program, project SWEETWOODS, grant number 792061.
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VH planned and performed experiments, performed computer modeling, analyzed data; JDDBL planned and performed experiments, analyzed data; YB and RAR performed experiments, analyzed data; and KB supervised the project, planned and performed experiments, analyzed data, wrote the paper. All authors contributed to discussing and revising the manuscript.
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Hämäläinen, V., Barajas-López, J.D.D., Berlina, Y. et al. New thermostable endoglucanase from Spirochaeta thermophila and its mutants with altered substrate preferences. Appl Microbiol Biotechnol 105, 1133–1145 (2021). https://doi.org/10.1007/s00253-020-11077-x
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DOI: https://doi.org/10.1007/s00253-020-11077-x