Insight into the role of α-arabinofuranosidase in biomass hydrolysis: cellulose digestibility and inhibition by xylooligomers

Abstract

Background: α-l-Arabinofuranosidase (ARA), a debranching enzyme that can remove arabinose substituents from arabinoxylan and arabinoxylooligomers (AXOS), promotes the hydrolysis of the arabinoxylan fraction of biomass; however, the impact of ARA on the overall digestibility of cellulose is controversial. In this study, we investigated the effects of the addition of ARA on cellulase hydrolytic action.

Results: We found that approximately 15% of the xylan was converted into AXOS during the hydrolysis of aqueous ammonia-pretreated corn stover and that this AXOS fraction was approximately 12% substituted with arabinose. The addition of ARA removes a portion of the arabinose decoration, but the resulting less-substituted AXOS inhibited cellulase action much more effectively; showing an increase of 45.7%. Kinetic experiments revealed that AXOS with a lower degree of arabinose substitution showed stronger affinity for the active site of cellobiohydrolase, which could be the mechanism of increased inhibition.

Conclusions: Our findings strongly suggest that the ratio of ARA and other xylanases should be carefully selected to avoid the strong inhibition caused by the less-substituted AXOS during the hydrolysis of arabinoxylan-containing biomass. This study advances our understanding of the inhibitory mechanism of xylooligomers and provides critical new insights into the relationship of ARA addition and cellulose digestibility.

Keywords: Biomass; Inhibition; Kinetics; Xylooligomers; α-Arabinofuranosidase.

Fig. 1

Effect of less-substituted AXOS on hydrolytic action of cellulases. Hydrolysis of 2% Avicel by CTec2 (10 FPU/g DM) and CEL (2 mg/g DM Cel5A, 8 mg/g DM Cel7A, and 0.2 mg/g DM Cel3A) with the addition of AXOS (5 mg/mL) and/or ARA (2 mg/g DM) at 50 °C for 48 h. The error bars represent the standard error of three independent experiments

Fig. 2

Schematic representation of the structure of arabinoxylan

Fig. 3

Role of ARA in the hydrolysis of pretreated corn stover. Hydrolysis of 10% corn stover after pretreatment with aqueous ammonia (AA-CS) by CTec2 (10 FPU/g DM) (a), or CEL (2 mg/g DM Cel5A, 8 mg/g DM Cel7A, and 0.2 mg/g DM Cel3A) (b), and XYL (2 mg/g DM), and/or ARA (2 mg/g DM) at 50 °C for 48 h. Hydrolysis of 10% AA-CS by CTec2 (10 FPU/g DM) and CEL (2 mg/g DM Cel5A, 8 mg/g DM Cel7A, and 0.2 mg/g DM Cel3A) with the addition of AXOS (4 mg/mL) and/or ARA (2 mg/g DM) at 50 °C for 48 h (c). The error bars represent the standard error of three independent experiments

Fig. 4

Hydrolytic action of individual cellulase in the presence of AXOS and/or ARA. Hydrolysis of pNPC (5 mM) by 8 mg/g DM Cel7A, HEC (10 mg/mL) by 2 mg/g DM Cel5A, and cellobiose (10 mg/mL) by 0.2 mg/g DM Cel3A, with the addition of AXOS (5 mg/mL) and/or ARA (2 mg/g DM) at 50 °C for 1 h. The error bars represent the standard error of three independent experiments

Fig. 5

Lineweaver–Burk plots of Cel7A activities at different XX (a), A³X (b), XXX (c), A²XX (d), and A²⁺³XX (e) (0.1, 0.5, and 1.0 mM) concentrations. The determination was carried out at 50 °C and pH 5.0 using pNPC as substrate

Fig. 6

The mechanism behind the increased inhibition. Arabinose substituents on the AXOS backbone block the access of AXOS to the active site of Cel7A