Molecular Cloning, Expression, Purification, Biochemical Characterization and Structure Analysis of a Novel Obligate Xylobiohydrolase (AcGH30A) from Acetivibrio Clariflavus ATCC 19732

Abstract

The thesis includes a general introduction and literature review on xylan and xylan-degrading enzymes, followed by the cloning of the gene encoding the xylobiohydrolase (AcGH30A) from the Acetivibrio clariflavus genome. This study reports the biochemical and structural characterization of a novel GH30 xylobiohydrolase, AcGH30A, from Acetivibrio clariflavus. The gene was cloned in pET28a(+), expressed in E. coli BL21(DE3), and purified to homogeneity, revealing a ~58 kDa protein. AlphaFold2 modelling and SAXS analysis showed an α/β/α sandwich fold with Glu175 and Glu268 as catalytic residues. Molecular docking and molecular dynamics simulations confirmed high-affinity binding to xylobiose at subsites −1 and −2, supported by ITC (Ka = 7.83×10⁵ M⁻¹). AcGH30A displayed optimal activity at 80 °C, pH 7.0, stability from pH 4-7 and 30-70 °C, a melting temperature of 72 °C and a 21-day half-life at 4 °C. It hydrolysed various xylans exclusively to xylobiose, with highest activity against 4-O-methyl glucuronoxylan (Vmax = 139 U/mg, KM = 0.71 mg/ml). Enzymatic degumming of Boehmeria nivea (ramie) and Ananas comosus (pineapple leaf) fibres using AcGH30A alone or in combination with pectate lyase (CtPL1B) and mannanase (RfGH5_7) showed enhanced weight reduction and fibre smoothness. Dual-enzyme mixtures- AcGH30A + CtPL1B for ramie and CtPL1B + RfGH5_7 for PAL and the ternary mix showed superior performance. The enzymatic treatment yielded improved degumming efficiency and fibre mechanical properties. The thermostability, product specificity, and synergistic potential of AcGH30A highlight its potential for applications in the pulp, paper, and textile industries.