Structure and Functional Analysis of a Recombinant endo-β-1,4-xylanase (AcXyn30B_12) from Acetivibrio Clariflavus DSM 19732 and its Application in Deinking of Waste Papers

Abstract

A gene encoding an endo-β-1,4-xylanase of glycoside hydrolase family 30 (GH30) was identified from Acetivibrio clariflavus DSM 19732 genome and designated as AcXyn30B_12. AcXyn30B_12 was successfully cloned, expressed in E. coli BL21 (DE3) and purified by immobilized metal-ion affinity chromatography followed by SEC. The SDS-PAGE analysis of AcXyn30B_12 revealed a molecular mass, ~74 kDa. AcXyn30B_12 exhibited highest specific activity against partially acetylated birchwood xylan at optimal conditions, 70°C and pH 5.5. AcXyn30B_12 retained over 80% activity across a broad pH range (4.5-8.0) and was thermostable up to 60°C. Kinetic analysis revealed a Vmax of 133.3 U/mg and KM of 0.9 mg/mL. TLC, HPLC and MALDI-TOF MS analyses confirmed both endo- and exo-acting activities. Synergistic hydrolysis of different xylan substrates by AcXyn30B_12 and xylobiohydrolase, AcGH30A elevated the TRS production. When used to hydrolyze pretreated biomasses, AcXyn30B_12 produced both xylo-oligosaccharides and xylose. In-silico structure prediction of 3D model structure of AcXyn30B_12 using AlphaFold2 and validation by Ramachandran plot and ERRAT analyses demonstrated high model accuracy. Molecular docking of AcXyn30B_12 with xylotriose (ΔG=−11.2 kcal/mol) confirmed strong substrate affinity. Molecular dynamics simulations of AcXyn30B_12-xylotriose complex showed reduced RMSD, Rg and SASA values indicating enhanced stability upon ligand binding. SAXS and DLS analyses revealed a monodispersed, globular conformation with a hydrodynamic radius of 4.0 nm. The efficiency of AcXyn30B_12 was evaluated in enzymatic deinking of waste papers viz. newspaper and notebook paper. The findings establish AcXyn30B_12 as a robust, multifunctional biocatalyst with promising applications in biomass valorization and sustainable paper deinking industries.