The development of inexpensive and environmentally friendly enzyme immobilization techniques will facilitate the adoption of continuous flow biocatalysis (CFB) by industry and science. In this work, a relatively mild sulfite digestion process was used to remove lignin and hemicellulose from wood with minimal disruption of its native porous structure, resulting in aligned macroporous cellulose monoliths called cellulose backbones (CSs). By developing carbohydrate-binding modules (CBMs) at the termini of recombinant proteins, the CSs could be used as inexpensive, renewable and biodegradable materials for enzyme immobilization without further chemical functionalization. CBM-labeled fluorescent proteins were first used to demonstrate the proof of principle and to optimize the immobilization conditions; this resulted in initial protein loadings of 5.24% by weight and immobilization efficiencies of up to 97.1%. The process was then transformed into a CBM-labeled ω-transaminase (ωTA) Bacillus megateriumgiving enzyme loadings and immobilization efficiencies of up to 3.99 wt% and 82.4%, respectively. A demonstrative CFB reaction with the immobilized CBM-labeled ωTA is displayed approx. 95 ± 5% conversion efficiency relative to the free enzyme in solution under analogous conditions, suggesting that CBM-labeled recombinant enzymes immobilized on wood-derived CSs could potentially compete with other, more complex and costly enzyme immobilization technologies.

Graphic summary: Enzyme immobilization on wood-based cellulose frameworks via carbohydrate-binding module fusion constructs


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