Enzyme kinetics and identification of the rate-limiting step of enzymatic arabinoxylan degradation

This study investigated the kinetics of multi-enzymatic degradation of soluble wheat arabinoxylan by monitoring the release of xylose and arabinose during designed treatments with mono-component enzymes at different substrate concentrations. The results of different combinations of α-l-arabinofuranosidases (EC 3.2.1.55), one derived from Aspergillus niger (AFAn) and one from Bifidobacterium adolescentis (AFBa), respectively, a β-xylosidase (EC 3.2.1.37) from Trichoderma reesei, and an engineered D11F/R122D variant of Bacillus subtilis XynA endo-1,4-β-xylanase (EC 3.2.1.8) were examined. The two selected α-l-arabinofuranosidases catalyze liberation of arabinose residues linked 1→3 to singly (AF_An) or doubly (AF_Ba) substituted xyloses in arabinoxylan, respectively. When added to arabinoxylan at equimolar levels, the AFBa enzyme catalyzed the release of more arabinose, i.e. had a higher rate constant than AF_An, but with respect to the xylose release, AF_An – as expected – exhibited a better synergistic effect than AF_Ba with β-xylosidase. This synergistic effect with AF_An was estimated to increase the number of β-xylosidase catalyzed cuts from ∼3 (with β-xylosidase alone) to ∼7 in each arabinoxylan substrate molecule. However, the synergistic effects between β-xylosidase and the α-l-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ∼25 times in 30min, to a yield equivalent to ∼104 β-xylosidase catalyzed cuts in each arabinoxylan substrate molecule. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate-limiting for the β-xylosidase catalyzed depolymerization to release xylose from arabinoxylan. The work provides clues to design efficient enzymatic degradation of arabinoxylan into fermentable monosaccharides.