Recombinant spore-displayed enzymes for cellulose hydrolysis: dual-enzyme spores versus mixed single-enzyme spores

Abstract

Bacillus subtilis spore surface display has been widely used to immobilise single recombinant enzymes, yet many industrially relevant reactions are multi-enzyme cascades. This study examined whether two distinct, catalytically active enzymes can be co-displayed on a single spore, using the two-step hydrolysis of carboxymethyl cellulose (CMC) to glucose as a model cascade. An endoglucanase (EglS) and a beta-glucosidase (Unbgl1A) were expressed in E. coli, characterised, and surface-displayed on Bacillus subtilis WB800N by fusion to the coat anchors CotB, CotC and CotG. The best constructs were combined either by mixing two single-enzyme spore strains or by co-displaying both enzymes on one strain, and compared with the free-enzyme mixture on CMC. CotG was the most effective anchor for EglS and the only functional anchor for Unbgl1A, with improved thermal stability relative to the free enzymes and activity retained over repeated reuse: CotG-EglS retained approximately 46% activity after 12 h (versus approximately 27% for the free enzyme), and CotG-Unbgl1A retained approximately 30% activity after 8 h, whereas free Unbgl1A lost all activity within 6 h. The mixed single-enzyme spores gave the highest glucose yield (2.15 mM), exceeding both the single dual-enzyme strain (1.57 mM) and the free-enzyme mix (1.35 mM). Two functional enzymes were co-displayed on one spore, but mixing separate single-enzyme spores gave higher yield under the tested conditions and allowed the enzyme ratio to be tuned.