Endo-β-1,4-xylanases (EC 3.2.1.8) are enzymes that catalyse the hydrolysis of β-1,4 xylosidic linkages in xylans [1]. Most xylanases are classified into glycoside hydrolase (GH; http://www.cazy.org) families 10 and 11, and other minorities belong to families 5, 7, 8, 16, 26, 43, 52, and 62 based on the amino acid sequence similarities of their catalytic domains [[2], [3], [4]]. The xylanases from the glycoside hydrolase family 11 (GH11) of the CAZy database are considered true xylanases [1,2,5]. Compared to other xylanases, GH11 xylanases exhibit several intriguing properties, such as remarkable substrate selectivity and catalytic efficiency, compact size (approximately 20 kDa), and a wide range of pH and temperature optima, rendering them suitable for diverse conditions in numerous applications [6]. The clarification of fruit juices for commercial production has recently been explored for the application of xylanase enzymes in the food industry [7]. The action of xylanases on freshly squeezed fruit juice facilitates the clarification process by removing excess polysaccharides, which contributes to its effective liquefaction and ultimately enhances its quality [8]. This process further enhances the yield of fruit juice and effectively prolongs the shelf life of packaged products. Simultaneously, it facilitates the recovery of juice aroma by mitigating the viscosity caused by excess sugars [9]. Fruits such as pineapple, which are rich in hemicellulose, were among the first to be processed using xylanase for juice clarification. The efficacy of xylanase isolated from Aspergillus niger DFR5, both individually and in conjunction with pectinases and cellulases, was assessed for its impact on the yield of pineapple juice [10]. The xylanase derived from Thermomyces lanuginosus exhibits remarkable efficacy in the clarification of apple juice, resulting in a rapid reduction in turbidity of approximately 32 % within a span of 90 min [11]. Furthermore, after pineapple juice was subjected to treatment at 50 °C and pH 4.5 for 120 min, turbidity was significantly decreased by 52.8 % [12]. The addition of xylanase Xyn11C from Fusarium sp.21 resulted in a significant increase in the percent transmittance of orange juice, with an improvement of approximately 48.3 % compared to the control group [13]. Peach juice was subjected to treatment with xylanase derived from Pediococcus acidilactici GC25 (0.15 U/mL), which resulted in a significant increase of 24.47 % in reducing sugar content and 21.22 % in the turbidity of the peach juice [14]. Compared with the control treatment, treatment with purified GvXyl derived from Geobacillus vulcani GS90 led to significant increases in the yield of apple and orange juices of 21.74 % and 12.12 %, respectively [15].

Given the acidic nature inherent in the environment of fruit juice clarification, xylanase must exhibit robust acid stability. The xylanases currently reported from Streptomyces thermovulgaris TISTR1948 [16], Fusarium sp. BVKT R2 [17], Trichoderma longibrachiatum KT693225 [18], A. niger [19], A. fumigatus Z5 [20], Tuber maculatum [21] and A. awamori AFE1 [22] all exhibit remarkable enzymatic activity under acidic conditions. Protein engineering technology has become an essential tool for studying the relationship between enzyme structure and function to facilitate the screening of xylanase resources suitable for industrial production and to effectively manage enzyme resource production demands. The acid resistance of xylanase has been the subject of successive studies [[23], [24], [25], [26], [27], [28]]. Despite some reports on the acid stability of xylanase, a paucity of studies have investigated the correlation between its molecular structure and resistance to acidic conditions, particularly compared to research focused on the thermal stability of xylanase. Among these studies, π-π stacking interactions have been reported to play a pivotal role in influencing the stability of GH11 xylanase under acidic conditions [23]. The pH stability and catalytic efficiency of xylanase can be enhanced through single or multipoint amino acid substitutions, as shown by other studies [[24], [25], [26], [27]]. However, limited reports exist regarding the significant relationship between the N-terminus of GH11 xylanase and the acid stability of xylanase [28]. The N-terminal region of xylanase is commonly acknowledged to be associated with thermal stability [[29], [30], [31], [32], [33], [34]]. If the N-terminal sequence of thermostable xylanase is replaced by the N-terminal sequence of acidic xylanase, would this substitution affect the corresponding enzymatic properties, such as thermal stability and acid stability? This molecular modification strategy will be implemented and verified in this paper. In the present study, we searched for xylanase resources in the National Center for Biotechnology Information (NCBI) database and identified two enzymes, namely, a thermally stable xylanase (xynA; GenBank code: KC422245.1) and an acidic xylanase [35] (pjxA; NCBI accession number: QDX01881.1) (the sequence information for xynA and pjxA is provided in the Supplementary data). Based on the predicted secondary structure (Supplementary Fig. S1 and Fig. S2), a strand formed by the N-terminus “FPTGNTTELEKRQTT” of xynA was replaced with a strand composed of the N-terminus “QTITTS” of pjxA, and the impact of this modification on its enzymatic properties and structural characteristics was investigated using a combination of enzyme property determination and computer molecular simulations. Subsequently, we evaluated the practical application of the modified enzyme in apple juice clarification, thereby comprehensively studying both its theoretical basis and practical implications.