Recombinant spore-displayed enzymes for cellulose hydrolysis: dual-enzyme spores versus mixed single-enzyme spores
- 1Indian Institute of Technology Bombay
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.
Related
- Computational Prediction of Recombinant Expression Efficiency for Industrial Enzymes: An Integrated Co-Translational Sequence Analysis Platform
- Development and diagnostic validation of a recombinant Hexon L1-based indirect enzyme-linked immunosorbent assay for detecting antibodies to fowl adenovirus serotype 8b in chickens.
- Displayed judgment certainty reorganizes source selection in self-formed networks
- Formin Is a Processive Motor that Requires Profilin to Accelerate Actin Assembly and Associated ATP Hydrolysis
- Deep Learning Based Cell Segmentation of 3D Confocal Images of Wood for Enzymatic Hydrolysis Applications
BibTeX
@misc{james2026recombinant,
title = {{Recombinant spore-displayed enzymes for cellulose hydrolysis: dual-enzyme spores versus mixed single-enzyme spores}},
author = {R. P. James and S. B. Noronha},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.21.760755},
url = {https://www.biorxiv.org/content/10.64898/2026.10.21.760755v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Recombinant spore-displayed enzymes for cellulose hydrolysis: dual-enzyme spores versus mixed single-enzyme spores
AU - R. P. James
AU - S. B. Noronha
PY - 2026
DA - 2026/10/21
PB - bioRxiv
DO - 10.64898/2026.10.21.760755
UR - https://www.biorxiv.org/content/10.64898/2026.10.21.760755v1
AB - 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.
ER -