Expression and characterization of a novel halophilic GH10 β-1,4-xylanase from Trichoderma asperellum ND-1 and its synergism with a commercial α-L-arabinofuranosidase on arabinoxylan degradation

Abstract

Enzymatic hydrolysis of arabinoxylan is of cost-effective strategy to yield valuable macromolecules, e.g., xylooligosaccharides (XOS). A novel halophilic GH10 xylanase (TaXYL10) from Trichoderma asperellum ND-1 was over-expressed in Pichia pastoris and migrated as a single band (~36 kDa) in SDS-PAGE. TaXYL10 displayed >80 % activity in the presence of 4.28 M NaCl and 10 % ethanol. Moreover, TaXYL10 exhibited optimal activity at pH 6.0 and 55 °C, and remarkable pH stability (>80 % activity at pH 4.0-6.0). K⁺ and Al³⁺ could remarkably promote TaXYL10 activity, while the presence of 10 mM Fe²⁺, Zn²⁺, Cu²⁺ and Fe³⁺ decreased its activity. TaXYL10 possesses the highest catalytic activity towards beechwood xylan. TLC analysis revealed that it could rapidly degrade xylan and XOS with DP ≥ 3, yielding xylotriose and xylobiose. Site-directed mutagenesis indicated that Glu¹⁵⁴ and Glu²⁵⁹ are crucial active residues for TaXYL10, while Asp²⁹⁵ and Glu⁶⁹ played auxiliary roles in xylan hydrolysis. Additionally, TaXYL10 acted cooperatively with a commercial α-L-arabinofuranosidase (AnAra) towards arabinoxylan degradation (583.5 μg/mL), a greater synergy degree of 1.79 was obtained after optimizing enzymatic ratios. This work not only expands the diversity of Trichoderma GH10 xylanases, but also reveals the promising potential of TaXYL10 in various industrial applications.

Keywords: Catalytic sites; Cleavage pattern; Halophilic xylanase; Synergistic action; Trichoderma asperellum.