Deciphering the structural insights and concentration‐dependent dimerisation of endo‐β‐1,4‐xylanase ( <i>Ac</i> Xyn30B_12) from <i>Acetivibrio clariflavus</i> using <scp>SAXS</scp> and computational methods

Structure of full-length thermophilic multi-modular endo-β-1,4-xylanase, AcXyn30B_12 from Acetivibrio clariflavus DSM 19732 was determined by computational analysis and SAXS studies. AcXyn30B_12 comprised N-terminal glycoside hydrolase family 30 catalytic module, followed by family 6 carbohydrate-binding module and dockerin typeI. The experiments involved AcXyn30B_12 cloned into pET-28a(+) vector including an N-terminal His6-tag encoding 653 amino. The catalytic residues-Glu151 and Glu259-were conserved based on multiple sequence alignment and superposition with homologous protein sequence analysis. Circular dichroism analysis of AcXyn30B_12 showed 26.2% α-helices and 28.1% β-strands, corroborating with PSIPRED and SOPMA servers. Molecular docking revealed highest affinity of AcXyn30B_12 with xylotriose (-11.2 kcal mol^-1) followed by xylobiose (-9.6 kcal mol^-1). Molecular dynamics simulation showed that the docked AcXyn30B_12-xylotriose complex is more stable and compact than unbound AcXyn30B_12. SAXS analysis of AcXyn30B_12 confirmed the polydisperse and elongated structure of AcXyn30B_12 at 3 mg mL^-1 and 5 mg mL^-1. Dummy atom model analysis of AcXyn30B_12 revealed monomeric and dimeric form at 3 and 5 mg mL^-1, respectively. Dynamic light scattering analysis of AcXyn30B_12 confirmed the polydisperse nature at 1, 3 and 5 mg mL^-1. DLS study showed R_h, 4.2 nm, 4.5 nm and 5.3 nm at 1, 3 and 5 mg mL^-1, respectively, matching the R_g results from SAXS analysis. A lower zeta potential of -4.0 mV at 5 mg mL^-1 than -10.5 mV at 3 mg mL^-1 indicated protein aggregation and dimerisation at higher concentration. A stable-dimeric structure conserved the catalytic machinery of AcXyn30B_12, making it a promising biocatalyst for efficient hydrolysis of xylan into value-added products.