In Silico Insights into Carbohydrate-Active Enzymes (CAZymes) of Bacillus subtilis T7: Lignin and Polysaccharide Degradation Mechanisms
TL;DR
Overall structural integrity for both proteins was confirmed and dynamic dynamics were more variable—xylotetraose remained bound within the Abhydrolase_1 active site for approximately 75 ns before partial displacement, whereas cellotetraose exhibited dynamic association along the GH9 catalytic channel, consistent with processive substrate translocation in endoglucanases.
Abstract
In silico structural characterization of carbohydrate-active enzymes (CAZymes) in Bacillus subtilis T7 reveals mechanistic insight into the strain’s capacity for consolidated bioprocessing of untreated lignocellulosic biomass. Homology models for 13 CAZymes were constructed using SWISS-MODEL, with Cu2+ and FAD cofactors incorporated into the AA10 lytic polysaccharide monooxygenase and AA3 oxidoreductase models, respectively. Blind molecular docking across the full protein surface identified energetically favorable binding pockets on GH9 endoglucanase and Abhydrolase_1. Among 12 enzyme–ligand pairs screened, Abhydrolase_1 exhibited the highest affinity for xylotetraose (−7.7 kcal/mol) and GH9 showed the strongest preference for cellotetraose (−7.0 kcal/mol). Site-specific docking confirmed six hydrogen bonds with Gly32, Phe33, Thr34, Ser36, Arg179, and His256, supplemented by two carbon–hydrogen bonds with Ile180 and Ser39, anchoring xylotetraose within the Abhydrolase_1 binding cavity, and seven hydrogen bonds stabilizing cellotetraose in the GH9 catalytic groove, with key contacts at Tyr141, Trp145, Asp194, Trp193, Arg254, Tyr255, and Tyr354. One-hundred nanosecond all-atom molecular dynamics simulations (GROMACS 2023.2, CHARMM36 force field, triplicate runs) confirmed overall structural integrity for both proteins: Abhydrolase_1 maintained a compact conformation (Rg = 18.11 ± 0.09 Å; backbone RMSD 2–3 Å), while GH9 was similarly stable (Rg = 30.36 ± 0.33 Å; RMSD 2–5 Å). Ligand dynamics were more variable—xylotetraose remained bound within the Abhydrolase_1 active site for approximately 75 ns before partial displacement, whereas cellotetraose exhibited dynamic association along the GH9 catalytic channel, consistent with processive substrate translocation in endoglucanases. These computational findings line up with the strain’s experimentally observed hydrolytic clearance zones (cellulase 24.5 mm; xylanase 11.6 mm), 63.4% alkali lignin decolorization, transient accumulation of ferulic acid and vanillin, and a hydrogen yield of 1.41 mol H2/mol substrate from untreated food waste. Together they give a molecular-level picture of substrate-specific CAZyme recognition in B. subtilis T7 and support its potential as a pretreatment-free platform for lignocellulosic biohydrogen production.