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. 2023 Apr 25;16(7):1536–1547. doi: 10.1111/1751-7915.14267

Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase‐d3 and endo‐xylanase activities and its application in the hydrolysis of cereal arabinoxylans

Ruonan Wang 1,2, Yu Zhang 1, Liang Liu 1, Jinshui Yang 1, Hongli Yuan 1,✉
PMCID: PMC10281360  PMID: 37096984

Abstract

Xylanolytic enzymes, with both endo‐xylanase and arabinoxylan arabinofuranohydrolase (AXH) activities, are attractive for the economically feasible conversion of recalcitrant arabinoxylan. However, their characterization and utilization of these enzymes in biotechnological applications have been limited. Here, we characterize a novel bifunctional enzyme, rAbf43A, cloned from a bacterial consortium that exhibits AXH and endo‐xylanase activities. Hydrolytic pattern analyses revealed that the AXH activity belongs to AXHd3 because it attacked only the C(O)‐3‐linked arabinofuranosyl residues of double‐substituted xylopyranosyl units of arabinoxylan and arabinoxylan‐derived oligosaccharides, which are usually resistant to hydrolysis. The enzyme rAbf43A also liberated a series of xylo‐oligosaccharides (XOSs) from beechwood xylan, xylohexaose and xylopentaose, indicating that rAbf43A exhibited endo‐xylanase activity. Homology modelling based on AlphaFold2 and site‐directed mutagenesis identified three non‐catalytic residues (H161, A270 and L505) located in the substrate‐binding pocket essential for its dual‐functionality, while the mutation of A117 located in the −1 subsite to the proline residue only affected its endo‐xylanase activity. Additionally, rAbf43A showed significant synergistic action with the bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A from the same bacterial consortium on insoluble wheat arabinoxylan and de‐starched wheat bran degradation. When rXyn10A/Fae1A was added to the rAbf43A pre‐hydrolyzed reactions, the amount of released reducing sugars, xylose and ferulic acid increased by 9.43% and 25.16%, 189.37% and 93.54%, 31.39% and 32.30%, respectively, in comparison with the sum of hydrolysis products released by each enzyme alone. The unique characteristics of rAbf43A position it as a promising candidate not only for designing high‐performance enzyme cocktails but also for investigating the structure–function relationship of GH43 multifunctional enzymes.

1. A novel bifunctional xylanolytic enzyme (rAbf43A) was obtained.2. rAbf43A exhibits arabinoxylan arabinofuranohydrolase‐d3 and endo‐xylanase activities.3. rAbf43A shows obvious synergy with cognate bifunctional xylanase/feruloyl esterase.

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INTRODUCTION

Xylans, which account for about 30% of total plant cell wall's dry weight (Garrido et al., 2022; Smith et al., 2017), are the predominant hemicelluloses in hardwoods and grasses (Li et al., 2022; Naidu et al., 2018). Using xylans as cheap materials for the production of diverse value‐added products, such as biofuels (Boonchuay et al., 2018; Surmeli & Sanli‐Mohamed, 2023), prebiotic arabinosylated xylo‐oligosaccharides (AXOSs) and linear xylo‐oligosaccharides (XOSs) (Lian et al., 2020), low‐calorie sweetener L‐arabinose (Saleh et al., 2017), anti‐oxidant ferulic acid (FA) (Xue et al., 2017), etc. is of great interest and value. However, different types and degrees of side‐chain substitutions (L‐arabinofuranosyl, O‐acetyl, FA, D‐glucopyranosyl uronic groups and so on) on the β‐1,4 linked D‐xylose chain backbone renders xylans recalcitrant to enzymatic degradation and thus limits its large‐scale bio‐refinery (Biely et al., 2016; Kaur et al., 2015; Limsakul et al., 2021).

Arabinofuranose (Araf) is the major side‐chain decoration of grass xylan, which is linked to the backbone mainly through α‐1,3 or 1,2 bonds (Geng et al., 2019; Lin et al., 2023). In the milling byproduct wheat bran, the major component is arabinoxylan (AX), with an average of 35%–40% Araf substitution (Xue et al., 2020). Among them, mono‐substituted L‐arabinofuranosyls at the C(O)‐2 or C(O)‐3 position account for 10%–25% and double‐substituted L‐arabinofuranosyls at both C(O)‐2 and C(O)‐3 positions on the same xylopyranosyl units account for 10%–20% (Xue et al., 2020). In addition, Araf substitution is also the base point for the covalent cross‐link of xylan‐xylan and xylan‐lignin (Freeman et al., 2017). The reason is that its C(O)‐5 position can be esterified with FA and these feruloyl substituents will form diferulates between xylans through dehydrodimerization or be bonded to lignin directly (Lin et al., 2023; Liu et al., 2021; Sumiyoshi et al., 2013). Therefore, efficient removal of Araf decoration is the key to decrease xylans recalcitrance, increase enzymatic accessibility and improve utilization rate of lignocellulose.

Arabinoxylan arabinofuranohydrolases (AXHs), an important kind of α‐L‐arabinofuranosidase (ABF), specifically remove the Araf substitutions from AXOSs, arabinoxylan and arabinan. However, the contents of AXHs are very low in many common commercial enzyme preparations, such as Celluclast 1.5 L, Accellerase 1500 and Cellic Ctec (Xin et al., 2019). Thus, exploring novel debranching AXHs, which can be used as key component to design high‐performance enzyme mixtures has attracted more and more attention. AXHs can be classified into three categories depending on its mode of action. The most common type of AXHs reported is AXHm2,3, which is only active on mono‐Araf substitutions at either position 2 or 3 (Leschonski et al., 2022; Long et al., 2020; Mroueh et al., 2019). But it is obvious that Araf decorations cannot be completely removed only by AXHm2,3 due to the existence of double‐Araf substitutions. Recently, very few AXHs with activity towards both mono and double Araf substitutions have been reported and defined as AXHm,d (Thakur et al., 2020). However, their activities against double‐Araf‐substituted substrates could only achieve 0.5%–7.2% of that against mono (Dos Santos et al., 2018; Geng et al., 2019; Wang et al., 2018). That is to say, double‐Araf‐substituted xylans are particularly recalcitrant to enzymatic attack (Liu et al., 2021).

The third type of AXHs is marked as AXHd3. They selectively hydrolyze only the α‐1,3‐linked Araf from doubly arabinosylated xylopyranosyl units, and thus, making arabinoxylan‐containing biomass accessible to AXHm2,3 and endo‐xylanase. Indeed, researchers found that the yield of arabinose or reducing sugars increased by 16.7%–111% when AXHd3 was added in hydrolysis mixtures containing AXHm2,3 or endo‐xylanase (Mroueh et al., 2019; Orita et al., 2017; Pouvreau et al., 2011; Sorensen et al., 2006). However, just a few AXHd3s have been reported to date (Liu et al., 2021; Mroueh et al., 2019; Orita et al., 2017; Pouvreau et al., 2011; Rogowski et al., 2015; Saito et al., 2020; Sorensen et al., 2006; van den Broek et al., 2005), which all belong to the GH43 family. In CAZy database, GH43 is one of the largest GH families and most reported multifunctional xylanolytic enzymes belong to this family (Limsakul et al., 2021; Mewis et al., 2016). The high cost of multiple enzyme production remains a major bottleneck of hydrolyzing arabinoxylan‐rich lignocellulosic biomasses. Supplementation of dual‐functional enzymes with endo‐xylanase (depolymerizing) and AXHd3 (debranching) activities is a way to address the problem. But such natural enzymes have rarely been reported (Limsakul et al., 2021).

In our previous studies, a stable lignocellulolytic consortium EMSD5 was isolated (Lv et al., 2008). A multi‐modular bifunctional xylanase/feruloyl esterase rXyn10A/Fae1A (GH10‐CBM13‐CE1‐CBM2) from it was obtained, which could alone liberate high‐value‐added products FA and XOSs from various agricultural residues (Wang et al., 2020). Interestingly, EMSD5 preferred to use highly Araf‐substituted wheat arabinoxylan (WAX) rather than low‐substituted beechwood xylan (BWX) as substrate for isopropanol production (Liu et al., 2019), which suggested that enzymes with AXH activity may be secreted by EMSD5. Indeed, its extracellular metaproteome showed that four GH43 proteins were co‐secreted with rXyn10A/Fae1A (Zhu et al., 2016). Among them, protein id_44723 (Abf43A) showed nearly 40% similarity to other reported AXHd3s. In the present study, protein id_44723 was heterogeneously expressed, and the enzymatic properties of recombinant enzyme (rAbf43A) were characterized. Then the essential residues responsible for its dual‐functionality of AXHd3 and endo‐xylanase were analysed by homology modelling and site‐directed mutagenesis. Furthermore, the synergism of rAbf43A with rXyn10A/Fae1A against complex substrates including insoluble wheat arabinoxylan (I‐WAX) and de‐starched wheat bran (DSWB) was investigated as well.

MATERIALS AND METHODS

Strains, plasmid and substrates

Escherichia coli DH5α and BL21 (DE3) were used for gene cloning and protein expression. Plasmid pET‐30a was used as an expression vector. These strains were cultured at 37°C using Luria‐Bertani (LB) broth supplemented with 50 μg/mL Kanamycin. p‐nitrophenyl‐β‐D‐xylopyranoside (pNPX) and p‐nitrophenyl‐α‐L‐arabinofuranoside (pNPAf) were purchased from Yuanye (Shanghai, China) and Megazyme (Wicklow, Ireland). Polysaccharides including BWX, WAX, I‐WAX, rye arabinoxylan (RAX) and sugar beet arabinan (SBA) were purchased from Megazyme (Wicklow, Ireland). Oligosaccharides including xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), 23‐α‐L‐arabinofuranosyl‐xylotriose (A2XX), 23,33‐di‐α‐L‐arabinofuranosyl‐xylotriose (A2,3XX) and 33‐α‐L‐arabinofuranosyl‐xylotetraose (XA3XX) were also purchased from Megazyme (Wicklow, Ireland). DSWB was prepared as the method of Xu (Xu et al., 2019).

Cloning and expression of rAbf43A and its mutants

Metagenomic DNA of EMSD5 was extracted according to the method published earlier (Zhu et al., 2016). Specific primers were used to amplify the gene sequence encoding rAbf43A (Table S1), by PCR using metagenomic DNA of EMSD5 as template. The PCR products and the pET30a vector were restricted with BamHI and SalI (Takara, Japan). Then, the BamHI/SalI‐digested gene fragment and vector were ligated with T4 DNA ligase (Promega, USA) to construct recombinant plasmids, which were then transformed into E. coli DH5a competent cells. After verifying by DNA sequencing, the resulting plasmids were transformed into E. coli BL21(DE3) competent cells for further protein expression. Recombinant enzyme induction was performed with 0.5 mM isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG) at 37°C and 200 rpm for 3 h. The site‐directed mutants of rAbf43A were constructed using Q5 site‐directed mutagenesis kit (New England BioLabs, USA) according to its protocol.

Purification of rAbf43A and its mutants

After induction, the cells were harvested by centrifugation (4°C, 8000 × g, 5 min), resuspended in ice‐cold binding buffer (pH 8.0, 20 mM Tris–HCl, 500 mM NaCl) and lysed by ultrasound (10 min, 2 s off, 2 s on). Supernatants were prepared through centrifugation (4°C, 8000 × g, 15 min). The supernatants were purified using Ni2+ His‐tag column with non‐linear imidazole gradient from 20 to 300 mM (in resuspension buffer). The active fractions were pooled and dialyzed for 24 h in deionized water at 4°C. Then, the purified enzyme was lyophilized and stored at −20°C. Ultimately, the lyophilized enzyme was dissolved in a buffer containing 20 mM Tris–HCl (pH 8.0) as the protein solution for further experiments. The purity of targeted proteins was estimated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis. Protein concentrations were determined using Quick Start™ Bradford Kit (Bio‐Rad, USA).

Enzyme activity assays

Polysaccharides (including BWX, WAX and SBA), oligosaccharides (including A2XX, A2,3XX and XA3XX) and synthetic substrates (pNPX and pNPAf) were used for enzymatic activity assay. As for polysaccharides, the reaction was performed in 0.05 M citric‐Na2HPO4 (pH 5.0) containing 1% substrate, and the mixture was incubated at 50°C for 10 min. Reducing sugars released were determined by the DNS method. One unit of enzyme activity was defined as the amount of enzyme catalysing the release of 1 μmol of reducing sugars in 1 min. As for oligosaccharides, the reaction system consisted of 5 μL properly diluted enzyme and 5 μL of 1 mM substrate and 10 μL of 0.05 M citric‐Na2HPO4 (pH 5.0). After 10 min incubation at 50°C, the reaction was stopped by adding 20 μL of 0.05 M sulfuric acid. The concentration of arabinose in the mixture was determined by Essentia LC‐15C high‐performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) using a MARS MOA‐organic acid column (Phenomenex, Los Angeles, CA, USA) and a RID‐10A refractive index detector (Shimadzu, Kyoto, Japan) at 60°C. Separation was performed within 15 min using a mobile phase consisting of 2.5 mM sulfuric acid at a rate of 0.6 mL per min. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of arabinose per min. As for pNPX and pNPAf, the reaction system consisted of 10 μL properly diluted enzyme and 90 μL of 1 mM substrate in 0.05 M citric‐Na2HPO4 (pH 5.0). After 10 min incubation at 50°C, the reaction was stopped by adding 400 μL of 1 M sodium carbonate. The liberation of p‐nitrophenol was monitored at 405 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μmol of p‐nitrophenol per min. The pH optima for endo‐xylanase and arabinofuranosidase were determined in 0.05 M citric‐Na2HPO4 buffer (pH 4.0–8.0) at 50°C and 40°C for 10 min, respectively. The optimal temperature was measured at temperatures ranging from 20 to 60°C in 0.05 M citric‐Na2HPO4 buffer (pH 5.0) for 10 min.

Kinetic parameters

The xylanase kinetic parameters were measured at 50°C in 0.05 M citric‐Na2HPO4 buffer (pH 5.0) for 10 min, using BWX and WAX at concentrations of 0–20.0 mg/mL. The arabinofuranosidase kinetic parameters were measured at 40°C in 0.05 M citric‐Na2HPO4 buffer (pH 5.0) for 10 min, using SBA at concentrations of 0–40.0 mg/mL. The AXHd3 kinetic parameters were measured at 40°C in 0.05 M citric‐Na2HPO4 buffer (pH 5.0) for 10 min, using A2,3XX at concentrations of 0–40.0 mM. The kinetic parameters were calculated by non‐linear regression of Michaelis–Menten using Graphpad prism 7 software.

Hydrolysate analysis

To explore the action mode of rAbf43A towards polysaccharides (including BWX, WAX, RAX and SBA) and oligosaccharides (including X2, X3, X4, X5, X6, A2XX, A2,3XX and XA3XX), hydrolysis experiments were performed. The purified rAbf43A (0.2 μg) was incubated with 1% polysaccharide or 0.2% oligosaccharide in 0.05 M citric‐Na2HPO4 buffer (pH 5.0) at 40°C for 24 h in a final volume of 10 μL. The reaction was terminated by boiling at 95°C for 10 min and centrifugated (8000 × g, 10 min). The degradation products were then analysed by HPLC and TLC. Arabinose, xylose and oligosaccharides (including X2, X3, X4, X5, X6, A2XX, A2,3XX and XA3XX) were used as standards for HPLC analysis as described above. For TLC analysis, 5 μL of the hydrolysates were spotted onto a silica gel 60 F254 plates (Merk, Germany). The plates were developed in a solvent system consisting of butanol: acetic acid: water at 5:4:1 (v/v/v). After spraying with a mixture of sulfuric acid: methanol at 1:4 (v/v), the plates were heated in an oven at 105°C for 10 min to detect sugar spots.

Synergistic action of rAbf43A with rXyn10A/Fae1A on the hydrolysis of I‐WAX and DSWB

To elucidate the synergistic collaboration between rAbf43A and rXyn10A/Fae1A, hydrolysis experiments of I‐WAX and DSWB were performed. All enzymatic synergy reactions were carried out in 0.05 M citric‐Na2HPO4 buffer (pH 6.0) with 2% (w/v) substrate. The enzymes were added independently or in combination, at a concentration of 0.05 mg/mL for each individual enzyme. The reactions were carried out at 40°C and 200 rpm for 12 h. Thereafter, the mixtures were boiled for 10 min and centrifugated (8000 × g, 10 min). The released reducing sugars and the concentration of xylose, arabinose and FA in the supernatant were estimated using the DNS method and HPLC, respectively. Except for the general simultaneous hydrolysis, sequential reactions were also performed. The reaction with the first enzyme was carried out for 12 h as described above. After 12 h incubation, the first reactions were terminated in boiling water for 10 min. When cooled down to 37°C, the second enzyme was added and the procedure was repeated.

All biochemical assays and hydrolysis experiments were performed in triplicate. An enzyme reaction without substrate and substrate without enzyme treatments were included under the same conditions as controls. Statistical significances of the data were determined using SPSS, version 23.

Bioinformatics analysis

Signal peptide was predicted at SignalP 5.0 server (https://services.healthtech.dtu.dk/service.php?SignalP‐5.0), and domain architecture was annotated using dbCAN (http://bcb.unl.edu/dbCAN2/). Multiple sequence alignment analysis was performed using DNAMAN software program. Structural sequence alignment images were created employing the ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi‐bin/ESPript.cgi) server. The residue frequencies were constructed with WebLogo (https://weblogo.berkeley.edu/logo.cgi). The three‐dimensional (3D) structural model was constructed using the opensource program AlphaFold2 (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb#scrollTo=_sztQyz29DIC). The predicted model was further visualized and analysed using PyMOL.

RESULTS AND DISCUSSION

Sequence analysis and heterogeneous expression of Abf43A

In dbCAN database, Abf43A was annotated as a multi‐modular hydrolase composed of three domains (in order from the N‐terminus to C‐terminus): a domain belonging to the 10 subfamilies of glycoside hydrolase family 43 (GH43_10), a carbohydrate‐binding module from family 13 (CBM13) and a CBM2 (Figure 1). Blastp analysis of Abf43A showed that it displayed the highest homology of 82.33% with a GH43 hydrolase (Genebank: WP_177180698.1) from Clostridium polysaccharolyticum. To further ascertain the activity of Abf43A, a homology‐based search in Protein Data Bank (PDB) database was made. Abf43A had the highest identity (50.00%) to the GH43_10 domain of a bifunctional α‐arabinofuranosidase/feruloyl esterase (BeGH43/FAE, PDB: 6MLY) from human colonic Bacteroides eggerthii, which exhibited AXH activity (Pereira et al., 2021). Abf43A also had a 32.18% of identity with the first reported fungal AXHd3 (HiAXHd3, PDB:3ZXJ) from thermophilic fungus Humicola insolens, which was able to remove C(O)‐3 arabinose from doubly arabinosylated xylopyranosyl units (McKee et al., 2012; Sorensen et al., 2006). These results suggested that Abf43A may be an AXHd3. Until now, 195 members of GH43 have been characterized. However, only eight proteins were reported to exhibit AXHd3 activity (Figure 1). Multiple sequence alignment of the Abf43A's catalytic domain with other AXHd3s indicated that it had conservative Asp51, Asp162 and Glu216 for catalysis (Figure S1). But, compared with previously reported AXHd3s, Abf43A has a low‐sequence similarity (30.70–39.90%) and different domain structure (Figure 1). Therefore, Abf43A is expected to be a novel AXHd3 with special properties.

FIGURE 1.

FIGURE 1

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Domain organization of Abf43A and other reported arabinoxylan arabinofuranohydrolase‐d3s. Black rectangle: signal peptide; GH43_10/36: glycoside hydrolase family 43 from different subfamilies; Doc: dockerin module; CBM2/6/13: carbohydrate‐binding module from different families.

Recombinant Abf43A without its signal peptide (consisting of residues 1–29) was expressed in E. coli BL21(DE3) and termed as rAbf43A. The SDS‐PAGE analysis showed that purified rAbf43A displayed consistent molecular weight with its calculated value of 95.88 kDa (Figure S2).

Biochemical characteristics of rAbf43A

The GH43 members have diverse enzyme activities (Mewis et al., 2016). Therefore, the substrate specificity of rAbf43A was evaluated using different types of substrates including polysaccharides (BWX, WAX and SBA) and synthetic substrates [p‐nitrophenyl‐β‐D‐xylopyranoside (pNPX) and p‐nitrophenyl‐α‐L‐arabinofuranoside (pNPAf)]. As shown in Figure 2A, rAbf43A showed high activity towards BWX (6.40 U/mg) and WAX (6.54 U/mg), moderate activity on SBA (2.37 U/mg) and low activity on synthetic substrates. When rAbf43A was incubated with SBA, only arabinose but not arabino‐oligosaccharides could be detected by TLC (Figure S3), indicating that rAbf43A attacked the arabinofuranose side‐chain of SBA and exhibited an exo‐acting α‐L‐arabinofuranosidase (ABF) activity. Interestingly, rAbf43A had very low activity on pNPAf (0.01 U/mg), which is different from typical ABFs. Previous researches have shown that low or even lack of pNPAf activity is a common feature of AXHd3 (Table S2), such as HiAXHd3 (no activity) (Sorensen et al., 2006), ClAbn7 (0.0075 U/mg) from Chrysosporium lucknowense (Pouvreau et al., 2011) and BadAbf43A (0.095 U/mg) from Bifidobacterium adolescentis (van den Broek et al., 2005). But surprisingly, in contrast to those reported AXHd3s, rAbf43A also displayed endo‐xylanase activity against BWX (no arabinosyl‐substituted) and WAX (mono arabinosyl‐substituted). These results suggested that rAbf43A was a bifunctional enzyme with both exo‐acting α‐L‐arabinofuranosidase and endo‐xylanase activities.

FIGURE 2.

FIGURE 2

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Biochemical characterization of rAbf43A. (A) Substrate specificity of rAbf43A. (B) Effect of temperature on the activity of rAbf43A. (C) Effect pH of on the activity of rAbf43A. Data reflect the mean ± SD (n = 3).

Enzymatic properties of rAbf43A were investigated using SBA and BWX as substrates. As shown in Figure 2B,C, the optimal temperatures for its ABF and endo‐xylanase activities were 40°C and 50°C, respectively. And over 90% of the maximal ABF activities were kept at 30°C and 50°C. The optimal reaction pH of both activities was 5.0. These similar optimal conditions for both activities suggested that rAbf43A probably hydrolyzed both substrates with the same active site.

Mode of action of rAbf43A

To decipher the cleavage specificities of rAbf43A, defined AXOSs [23‐α‐L‐arabinofuranosyl‐xylotriose (A2XX), 23,33‐di‐α‐L‐arabinofuranosyl‐xylotriose (A2,3XX) and 33‐α‐L‐arabinofuranosyl‐xylotetraose (XA3XX)], XOSs [xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6)] and polysaccharides (BWX, WAX, RAX and SBA) were treated with rAbf43A and hydrolysis products were analysed by TLC or HPLC. As shown in Figure 3A, doubly arabinosylated A2,3XX was almost completely hydrolyzed to arabinose and monosubstituted A2XX after 24 h incubation, indicating that rAbf43A had AXH activity. And its Km, Kcat and Kcat/Km values for A2,3XX were 8.03 mM, 3474.00 min−1 and 432.63 min−1/mM−1, respectively (Table S2). Until now, only the kinetic parameters of XacAbf51 from Xanthomonas axonopodis pv. citri has been quantitatively measured using di‐substituted XA2,3XX (Dos Santos et al., 2018), while its Kcat/Km value (108 min−1/mM−1) was significantly lower than rAbf43A (fourfold). Although rAbf43A could also remove arabinose from monosubstituted XA3XX, the activity was very weak (0.37 U/mg) compared with A2,3XX (20.51 U/mg). Moreover, rAbf43A was not active towards A2XX, which could explain why A2XX accumulates in the hydrolysates of A2,3XX (Figure 3A). Additionally, besides AXOSs, rAbf43A is capable of releasing arabinose from long‐chain polysaccharides RAX and SBA, which contain disubstituted arabinosyl units (Figure S4). These results further demonstrated that rAbf43A should belong to the AXHd3 group as described previously.

FIGURE 3.

FIGURE 3

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Analysis of hydrolysis products of A2,3XX, xylan and XOSs by rAbf43A. (A) HPLC profiles of A2,3XX hydrolysate. (B) TLC patterns of BWX and WAX hydrolysates. (C) TLC patterns of XOSs (X2‐X6) hydrolysates.

When using BWX and WAX as the substrates, rAbf43A exhibited an endo‐mode of action, releasing a series of XOSs with a degree of polymerization higher than 3 (Figure 3B). And as shown in Figure 3C, rAbf43A preferred to hydrolyze long‐chain linear XOSs (xylopentaose and xylohexaose) rather than short‐chain XOSs (xylobiose, xylotriose and xylotetraose), thus providing a clear indication that it contained endo‐xylanase activity but no β‐xylosidase activity. Moreover, the truncated mutant of rAbf43A without CBM13 and CBM2 displayed the same specificity (Data not shown). Therefore, in contrast to other AXHd3 enzymes, rAbf43A showed unique properties of exo‐AXHd3 and endo‐xylanase activities in a single catalytic domain (GH43_10), which makes it an interesting enzyme.

Protein modelling and structural determinants of the dual‐functionality of rAbf43A

To investigate the possible residues responsible for the special dual‐functionality of rAbf43A, a structural model of its catalytic domain rAbf43A‐GH43 (consisting of residues 41–544) was generated through AlphaFold2. The quality check of this model was assessed by the tools on the SAVES server. The values in the Ramachandran plot showed that the number of residues in the favoured regions was 99.30% and the ERRAT test showed that the overall quality factor of this model was 90.43 (Data not shown). All these parameters indicated that the model was suitable for further studies. Inspection of the model reveals that rAbf43A‐GH43 harbours an N‐terminal five‐bladed β‐propeller domain and a C‐terminal β‐sandwich domain (Figure 4A). Notably, at the interface between the two domains, a solvent‐exposed shallow cleft is presented and the predicted active site pocket (including D51, D162 and E216) is located in a depression at the central region of the cleft (Figure 4B). Previous work on the first fungal AXHd3 (HiAXHd3, PDB:3ZXJ) has shown that the replacement of Tyr166 at the rim of the active site by a less bulky amino acid confers endo‐xylanase activity on HiAXHd3, while the resultant mutant (Y166A) retains its AXHd3 function (McKee et al., 2012). In addition, the distal subsites that line the interdomain cleft also participate in determining the specificity of HiAXHd3 (McKee et al., 2012). Compared with HiAXHd3, rAbf43A‐GH43 has a larger and wider pocket to accommodate long‐chain xylan (Figure 4B,C) and may explain why rAbf43A‐GH43 has the ability to hydrolysis BWX and WAX, while HiAXHd3 do not. To further ascertain the essential residues responsible for the conformational differences of the active site pocket of rAbf43A‐GH43 and HiAXHd3, some residues of rAbf43A‐GH43 that are different from HiAXHd3's residues located within 5 Å of the substrate XA2X (Figure 4D), were selected for mutagenesis and their effects on specificity were assessed.

FIGURE 4.

FIGURE 4

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Homology model of the catalytic domain of rAbf43A and specific activity of its site‐directed mutants. (A) Cartoon representation of the catalytic domain of rAbf43A. The catalytic residues are labelled and showed as sticks. (B) Surface representation of the catalytic domain of rAbf43A. The catalytic residues are coloured in blue. The dashed circle is the substrate‐binding pocket. (C) Surface representation of HiAXHd3. The catalytic residues are coloured in red. The dashed circle is the substrate‐binding pocket. (D) Substrate‐binding pocket of rAbf43A and HiAXHd3. Residues of rAbf43A and HiAXHd3, located in the substrate‐binding pocket are shown as palegreen and lightblue sticks, respectively; the conserved catalytic residues of rAbf43A and HiAXHd3 are shown as blue and red sticks, respectively. (E) WebLogo for GH43‐conserved WAP element in the sequences of characterized enzymes from glycoside hydrolase family 43. (F) Relative xylanase activity of rAbf43A and its mutants against BWX. (G) Relative AXHd3 activity of rAbf43A and its mutants against SBA. Data reflect the mean ± SD (n = 3). Statistical significance is indicated by stars on columns based on Student's t‐test. ***: p < 0.001, **: 0.001 < p < 0.01, *: 0.01 < p < 0.05.

As shown in Figure 4F,G, the catalytic residue mutants D162A and E216A displayed almost no endo‐xylanase or AXHd3 activity indicating that the dual‐functionality of rAbf43A utilize the same active site, which is consistent with other reported GH43 multifunctional enzymes (Basit et al., 2019; McKee et al., 2012). Three non‐conserved amino acids that appear to surround active site pockets, H161, A270 and L505, have been mutated to the corresponding residues found in HiAXHd3, Y166, H272 and W526. Surprisingly, unlike previous studies of HiAXHd3 (McKee et al., 2012), mutation of H161 to Tyr did not abolish the endo‐xylanase activity but slightly decreased it. However, when H161 was mutated to Gly, the activities were significantly reduced or even vanished, indicating the vital role of H161. The mutation of H161 might affect the spatial position or side chain of the adjacent conservative catalytic residue (Asp162), which is thought to play a role in pKa modulation and in maintaining the correct orientation of the general‐acid residue (Brux et al., 2006). The mutation of A270, which is located in the predicted shallow C(O)‐2 arabinose binding pocket that abuts the deep active site, to His resulted in the complete loss of both activities. One reason for this might be that the imidazole side chain of His makes the binding cleft too narrow to allow the substrate to enter the active site. Similarly, the replacement of L505 at the side edge of the interdomain cleft by a bulky Tyr resulted in dramatically decreased endo‐xylanase and AXHd3 activities (7.44% and 23.73% of the wild‐type, respectively). These data indicate that the occurrence of residues (A270 and L505) without bulky side chain in the surrounding area of the active site pocket of rAbf43A‐GH43 presumably makes room for the xylan chain to sit deeper in the cleft and to access the active site. In addition, it has been reported that GH43 members contain a non‐catalytic but conservative ‘WAP’ element (Figure 4E), which is part of the −1 subsite at the active site (Brux et al., 2006; Zanphorlin et al., 2019). But the corresponding element was ‘WAA’ (consisting of Trp115, Ala116 and Ala117) in rAbf43A. When A117 was mutated to Pro, the endo‐xylanase activity was almost completely lost, while little effect was observed on the AXHd3 activity (Figure 4F,G). That is to say, the endo‐xylanase activity of rAbf43A is conferred by the single residue changes in its −1 subsite. However, their exact roles in substrate recognition and catalysis need to be further elucidated by future structural crystallography studies. To our knowledge, there is no general principle to design novel multifunctional enzymes with a single domain through the mutation of several residues. These results as described above offer significant reference information to guide the rational design of other GH43 multifunctional enzymes.

Synergistic action of rAbf43A with rXyn10A/Fae1A on I‐WAX and DSWB hydrolysis

Enzymatic saccharification of arabinoxylan‐rich biomasses to produce value‐added bioproducts has attracted great attention. In our previous study, a bifunctional xylanase/feruloyl esterase Xyn10A/Fae1A that is co‐secreted with Abf43A by EMSD5 consortium was found (Zhu et al., 2016). It could alone liberate anti‐oxidant FA and prebiotic XOSs from recalcitrant I‐WAX and DSWB (Wang et al., 2020). It is worthy to note that both natural substrates contain the Araf substitutions to which FA is attached (Gao et al., 2020). Although synergism between AXHd3 and endo‐xylanase has been observed, the effect of the removal of Araf substitutions by AXHd3 on the production of FA remains unclear. Therefore, to elucidate the effect of AXHd3 on the yield of FA, rAbf43A and rXyn10A/Fae1A were incubated with cereal arabinoxylans (I‐WAX and DSWB), either separately, simultaneously or sequentially.

As shown in Figure 5, when I‐WAX and DSWB were incubated with rAbf43A, only arabinose (371.46 μg/mL and 79.00 μg/mL, respectively) was detected (Figure 5D), while the reducing sugars yields were low (Figure 5A), suggesting that rAbf43A mainly exhibited AXHd3 activity in the degradation of natural cereal arabinoxylans but not endo‐xylanase activity. Similarly, trifunctional enzymes including PcAxy43A (Teeravivattanakit et al., 2016), PcAxy43B (Limsakul et al., 2021) and Ttxy43 (Basit et al., 2019) preferentially displayed their both endo‐xylanase and β‐xylosidase activities rather than AXH activity towards complex substrates. Therefore, we speculated that this phenomenon is quite common for multifunctional enzymes with a single catalytic domain. When I‐WAX and DSWB were incubated with rXyn10A/Fae1A, the yield of reducing sugars was much higher than rAbf43A (Figure 5A), FA and xylose were also detected (Figure 5B,C). In comparison with the sum of hydrolysis products by the individual enzymes, the amount of released reducing sugars, FA, and xylose increased by 9.43% and 25.16%, 31.39% and 32.30%, 189.37% and 93.54%, when rXyn10A/Fae1A was added followed by rAbf43A (Figure 5A,B,C). Especially, the highest yield of FA (3.86 and 2.86 mg/g substrate, respectively) was obtained from I‐WAX and DSWB by such a sequential addition strategy. These results revealed that high production of FA from arabinoxylan‐rich biomasses can be achieved by the first Araf removal with rAbf43A followed by main chain cleavage with rXyn10A/Fae1A. Therefore, rAbf43A is a potentially cost‐effective accessory enzyme for the production of value‐added products from cereal arabinoxylans.

FIGURE 5.

FIGURE 5

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Products of simultaneous or sequential reactions by rAbf43A and rXyn10A/Fae1A against I‐WAX or DSWB. (A) Reducing sugars; (B) Ferulic acid; (C) Xylose; (D) Arabinose. rXyn10A/Fae1A, hydrolysis with rXyn10A/Fae1A alone for 12 h; rAbf43A, hydrolysis with rAbf43A alone for 12 h; rXyn10A/Fae1A + rAbf43A, sum of rXyn10A/Fae1A and rAbf43A alone for 12 h; rXyn10A/Fae1A and rAbf43A, hydrolysis with rXyn10A/Fae1A and rAbf43A for 12 h; rXyn10A/Fae1A → rAbf43A, hydrolysis with rXyn10A/Fae1A for 12 h following supplementation with rAbf43A for another 12 h; rAbf43A → rXyn10A/Fae1A, hydrolysis with rAbf43A for 12 h following supplementation with rXyn10A/Fae1A for another 12 h. Data reflect the mean ± SD (n = 3). p values were determined using two‐tailed Student's t‐tests.

CONCLUSIONS

This study reported a novel xylanolytic enzyme rAbf43A with both exo‐acting arabinoxylan arabinofuranohydrolase‐d3 (debranching) and endo‐acting β‐xylanase (depolymerizing) activities. The AlphaFold2‐based structural modelling showed rAbf43A has a larger substrate‐binding pocket than common AXHd3 enzymes which makes it easier for long‐chain xylan substrate to enter. And structure‐guided mutagenesis within the substrate‐binding pocket identified four non‐catalytic residues (A117, H161, A270 and L505) responsible for its catalytic activities, thus providing possible hot spots for the rational design of other GH43 multifunctional enzymes. More importantly, rAbf43A significantly boosted liberation of FA and reducing sugars (XOSs/xylose) of bifunctional xylanase/feruloyl esterase on the I‐WAX and DSWB hydrolysis. Hence, rAbf43A is an important xylanolytic enzyme with high biotechnological potential.

AUTHOR CONTRIBUTIONS

Ruonan Wang: Data curation (lead); formal analysis (lead); funding acquisition (equal); investigation (lead); writing – original draft (lead); writing – review and editing (lead). Yu Zhang: Resources (supporting); writing – review and editing (supporting). Liang Liu: Resources (supporting). Jinshui Yang: Writing – review and editing (supporting). Hongli Yuan: Conceptualization (lead); formal analysis (lead); funding acquisition (equal); supervision (lead); writing – review and editing (lead).

CONFLICT OF INTEREST STATEMENT

None.

Supporting information

Appendix S1.

Click here for additional data file. (2MB, docx)

ACKNOWLEDGEMENTS

This research was supported by the National Key Research and Development Program of China (grant no. 2022YFA0912103) and the Henan Province Science and Technology Breakthrough Project (grant no. 222102320318).

Wang, R. , Zhang, Y. , Liu, L. , Yang, J. & Yuan, H. (2023) Discovery of a bifunctional xylanolytic enzyme with arabinoxylan arabinofuranohydrolase‐d3 and endo‐xylanase activities and its application in the hydrolysis of cereal arabinoxylans. Microbial Biotechnology, 16, 1536–1547. Available from: 10.1111/1751-7915.14267

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Appendix S1.

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