Marine bifunctional processive xylanase with potent β-xylosidase activity for xylan based biomass conversion...

Enzymatic degradation of xylan, the most abundant hemicellulose in lignocellulosic biomass, is critical for efficient biomass conversion. This study reports a recombinant exo-β-1,4-xylanase, ZgGH43A of glycoside hydrolase family 43 from Zobellia galactanivorans was expressed using Escherichia coli BL21 (DE3) cells and purified by Immobilized Metal-ion Affinity Chromatography to homogeneity. Recombinant ZgGH43A exhibited high activity against various xylan polymeric substrates, with a marked preference for linear beechwood xylan (436.5 U/mg) at the optimum temperature of 40 °C and pH of 5.5. Consistent with its marine mesophilic origin, ZgGH43A displayed moderate thermal and pH stability retaining 90 % activity at 25 °C to 45 °C and 80 % activity at pH 5 to 6, after 90 min incubation. ZgGH43A also demonstrated substantial activity on partially substituted xylans, including 4-O-methyl-D-glucuronoxylan and acetylated birchwood xylan, highlighting its broad substrate specificity. Kinetic analysis showed catalytic efficiency (k_cat/K_M), 105.5 mL.mg^-1.s^-1 and turnover number (k_cat), 324.7 s^-1 with beechwood xylan. Mode of action analysis by TLC, HPLC and MALDI-TOF MS confirmed that ZgGH43A hydrolyses xylan directly to xylose, without the accumulation of intermediate xylo-oligosaccharides. TLC analysis of hydrolysed products of xylo-oligosaccharides further confirmed both exo-β-1,4-xylanase and β-xylosidase activities of ZgGH43A. Intrinsic fluorescence spectroscopic analysis revealed binding of ZgGH43A to xylose with ΔG, -10.0 kJ/mol, indicating single-site interaction. ZgGH43A exhibited moderate processivity against oat spelt xylan as processivity index was 6. Hydrolysis of pretreated rice straw by ZgGH43A and endo-β-1,4-xylanase, AcXyn30B_12 from Acetivibrio clariflavus released 27.4 mg/g TRS that was 10-fold higher than ZgGH43A (2.8 mg/g) alone displaying synergy between the two.