Among the myriad biomass resources bestowed by nature, lignocellulosic biomass stands out as the most economical and abundant, predominantly found in agricultural and forestry wastes [1]. Xylan, a significant component (20–40%) of hemicellulose, is a polymer composed of xylose molecules. One of principal enzymes facilitating the degradation of xylan is endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the hydrolysis of glycosidic bonds, resulting in the production of xylose, xylooligosaccharides (XOS), and other derivatives [2], [3]. Xylanases are classified into several families according to their amino acid sequences and structural features, notably including GH5, GH8, GH10, GH11, GH43, and GH51. The majority of these enzymes are predominantly found within the GH10 and GH11 families. [4], [5], [6]. The effectiveness of xylanase is contingent upon various factors, including the nature of the substrate, the ionic strength of the milieu, reaction temperature, and the pH level [7]. Additionally, an important aspect impacting xylanase activity is the presence of xylanase inhibitory proteins (XIs) in raw materials [8], [9], [10].

As the main hydrolysis product of xylan, xylooligosaccharides (XOS) has demonstrated significant potential in the food, feed, and pharmaceutical sectors, exhibiting prebiotic properties and noted for their antioxidant activity [11], [12], [13], [14], [15], [16]. XOS has received approval from both the European Food Safety Authority (EFSA) and the U.S. Food and Drug Administration (FDA) for consumption. Batsalova et al. reported that a commercially available oligo-xylan could modulate TLR4 signaling and cellular redox status, exhibiting antitumor effects and underscoring XOS's potential in cancer research [17]. Acute oral toxicity tests on Wistar rats have shown non-toxicity of XOS derived from corncob, even at high doses of 5000 mg/kg body weight [18]. XOS obtained from peach palm waste via a commercial endo-xylanase demonstrated superior antioxidant capacity compared to XOS from commercial xylans [19]. Arthitaya Kawee-ai et al. found that a sonocatalytic-synergistic Fenton reaction, used as a pretreatment before enzymatic production of XOS from corn cobs, enhanced both the yield and antioxidant activity of the resultant XOS [20]. Antioxidants, crucial in counteracting oxidative stress-related pathologies, are especially appealing when derived from natural sources due to their biocompatibility, low toxicity, and minimal side effects, thus offering improved biological safety in chronic disease treatments [21], [22]. However, challenges in extraction and purification impede the widespread adoption of natural antioxidants, despite the abundant availability of lignocellulose as a raw material for XOS-based natural antioxidant development.

In this study, the family GH10 Aspergillus niger xylanase A (AnXylA10) was expressed in Pichia pastoris X33 and characterized. Then the antioxidant potential of XOS released from beechwood xylan by reAnXylA10 was investigated. Furthermore, we explored the intricate interactions between AnXylA10 and riceXIP, concentrating on competitive inhibition, which was achieved through Intrinsic fluorescence spectroscopy, molecular dynamics (MD) simulations, non-covalent interaction (NCI) analysis, and quantum chemical calculation.