Hydrolysis of Arabinoxylo-oligosaccharides by α-L-Arabinofuranosidases and β-DXylosidase from Bifidobacterium dentium

Two α-L-arabinofuranosidases (BfdABF1 and BfdABF3) and a β-D-xylosidase (BfdXYL2) genes were cloned from Bifidobacterium dentium ATCC 27679, and functionally expressed in E. coli BL21(DE3). BfdABF1 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 25°C. This exo-enzyme could hydrolyze p-nitrophenyl arabinofuranoside, arabino-oligosaccharides (AOS), arabinoxylo-oligosaccharides (AXOS) such as 32-α-L-arabinofuranosyl-xylobiose (A3X), and 23-α-Larabinofuranosyl-xylotriose (A2XX), whereas hardly hydrolyzed polymeric substrates such as debranched arabinan and arabinoxylans. BfdABF1 is a typical exo-ABF with the higher specific activity on the oligomeric substrates than the polymers. It prefers to α-(1,2)-L-arabinofuranosidic linkages compared to α-(1,3)-linkages. Especially, BfdABF1 could slowly hydrolyze 23,33-di-α-L-arabinofuranosyl-xylotriose (A2+3XX). Meanwhile, BfdABF3 showed the highest activity in sodium acetate at pH 6.0 and 50°C, and it has the exclusively high activities on AXOS such as A3X and A2XX. BfdABF3 mainly catalyzes the removal of L-arabinose side chains from various AXOS. BfdXYL2 exhibited the highest activity in sodium citrate at pH 5.0 and 55°C, and it specifically hydrolyzed p-nitrophenyl xylopyranoside and xylo-oligosaccharides (XOS). Also, BfdXYL2 could slowly hydrolyze AOS and AXOS such as A3X. Based on the detailed hydrolytic modes of action of three exo-hydrolases (BfdABF1, BfdABF3, and BfdXYL2) from Bf. dentium, their probable roles in the hemiceulloseutilization system of Bf. dentium are proposed in the present study. These intracellular exo-hydrolases can synergistically produce L-arabinose and D-xylose from various AOS, XOS, and AXOS.

J. Microbiol. Biotechnol. and endo-hydrolase genes, and their expression can be properly regulated [11,12]. Recent genomic, transcriptomic, and proteomic approaches revealed that Bifidobacterium spp. sensitive to the prebiotic AXOS can timely express any combination of core arabinoxylan-degrading enzyme genes including ABF and XYL [13,14]. To date, several ABFs have been studied from Bf. adolescentis [15,16], Bf. longum [17][18][19], and Bf. breve [20]. Also, a few XYLs has been reported from Bf. adolescentis [21], Bf. longum, and Bf. breve [22]. Although exo-hydrolases were previously cloned from some Bifidobacterium species, their enzymatic characterizations were mainly carried out with a few simple-structured substrates such as p-nitrophenyl sugars. Therefore, the detailed hydrolytic modes of action and preferences towards various natural complex substrates have not been well-understood to date. Unlike most common Bifidobactrium species being frequently isolated from the gastrointestinal tract, Bf. dentium spp. are identified from the oral cavity, dental caries, or feces of human and animals [23]. Although Bf. dentium is recently considered as one of major Bifidobacterium species, the information about its enzymatic hemicellulose-utilization system and related genes are not focused yet. Based on the microbial genome information, meanwhile, five open reading frames probably encoding exo-acting hemicellulose-hydrolases were found Bf. dentium ATCC 27679 which was isolated from a human vagina.
In this study, the genes encoding two ABFs (hereafter, BfdABF1 and BfdABF3) and a XYL (BfdXYL2) were cloned from Bf. dentium, and their enzymatic properties, substrate specificities, and hydrolytic modes of action towards various natural substrates such as AXOS were comparatively characterized in detail. Finally, the roles of these exo-hydrolases were proposed for the degradation and utilization of AXOS in Bf. dentium.

Enzyme Activity Assays
The enzyme activity on 1 mM p-nitrophenyl sugars (p-NPAf and p-NPXp) was determined at 405 nm by measuring the p-nitrophenol being released. L-Arabinose/D-Galactose assay kit (Megazyme) was utilized to measure L-arabinose produced from 0.5% polymeric or oligomeric substrates at 340 nm. One unit of ABF or XYL activity was defined as the amount of enzyme liberating 1 μmol of L-arabinose, D-xylose, or p-nitrophenol from each substrate for 1 min, respectively.

Thin Layer Chromatography (TLC) Analysis
The enzymatic hydrolysis patterns were comparatively analyzed by using thin layer chromatography (TLC). At the optimal condition, 0.5% of substrate was reacted with ABF or XYL for an appropriate time, and the resulting hydrolysates were separated on a 60F 254 silica gel glass TLC plate (Merck, Germany) with the solvent of chloroform/acetate/water (6:7:1). The product spots were visualized and identified by the developing solution (0.3% N-1-naphthyl-ethylenediamine and 5% H 2 SO 4 in methanol) at 110 o C for 10 min.

High performance Anion Exchange Chromatography (HPAEC) Analysis
Bio-LC system (ICS-3000; Thermo-Fisher, USA) equipped with a CarboPac PA1 column (4 × 250 mm) and an electrochemical detector (ED40) was utilized for the analysis of enzymatic hydrolysates. The samples were eluted with a linear gradient from 100% 150 mM NaOH (buffer A) to 15% buffer B (600 mM sodium acetate in buffer A) over 40 min. The flow rate of the mobile phase maintained at 1.0 ml/min through the analysis.

Gene Cloning and Expression of BfdABFs and BfdXYLs
Probable genes encoding three α-L-arabinofuranosidases (BfdABF1, 2, and 3) and two β-D-xylosidases (BfdXYL1 and 2) were found in the genome of Bf. dentium ATCC 27679, and cloned into an IPTG-inducible expression vector, pET-21a. Of these five genes, BfdABF2 and BfdXYL1 did not exhibit the detectable levels of gene expression and enzymatic activity on any of the substrates tested in this study. In contrast, the three genes encoding C-terminal 6-histidines-tagged BfdABF1, BfdABF3, and BfdXYL2 were successfully expressed in E. coli BL21(DE3), and purified by an Ni-NTA affinity chromatography (Fig. 1). The open reading frames of BfdABF1, BfdABF3, and BfdXYL2 encode 773 (85,395 Da), 572 (65,372 Da), and 543 (59,011 Da) amino acids, respectively. The SignalP 5.0 analysis predicted that all these exo-hydrolases without a detectable signal peptide sequence would likely be expressed as the intracellular enzymes. The apparent molecular mass of recombinant BfdABF1 (85 kDa), BfdABF3 (65 kDa), and BfdXYL2 (59 kDa) were similar to those being expected from the deduced amino acid sequences.

Optimal Reaction Conditions for BfdABFs and BfdXYL
BfdABF1 showed the highest activity on p-NPAf in 50 mM sodium acetate buffer at pH 5.0 and 25 o C (Fig. 2). Its enzymatic activity at pH 4.0 and 7.0 was less than 50% of optimal conditions. In contrast, BfdABF3 was highly active in 50 mM sodium citrate buffer at pH 5.0~6.0 and sodium acetate buffer at pH 5.5~6.0 at 50 o C. Most known  Relative activities of ABF and XYL on p-NPAf and p-NPXp were respectively measured at 405 nm. Sodium citrate (pH 3~6), sodium acetate (pH 4~6), sodium phosphate (pH 6~8), and Tris-HCl (pH 7~9) buffers were used for the activity assay. microbial ABFs have their own optimal reaction conditions at pH 5.0~7.0 and 40~60 o C [7]. Similarly, it was known that the ABFs from lactic acid bacteria show the highest activity at pH 5.5~6.0 and 30~50 o C. For example, AbfA and AbfB from Bf. adolescentis ATCC 15703 exhibited the highest activity at pH 6.0, and 30 o C and 50 o C, respectively [16]. The ABF from Levilactobacillus brevis DSM 20054 has an optimal temperature at 60~62 o C and pH 5.0~5.5 [24].
BfdXYL2 showed the highest activity in 50 mM sodium acetate buffer at pH 5.0 and 55 o C (Fig. 2). It was highly active in 50 mM sodium phosphate buffer at pH 6.0~6.5 as well. In the case of Bf. adolescentis LMG 10502, two βxylosidases (XylB and XylC) showed the highest activities at pH 5.5 and 60 o C, and pH 6.0 and 50 o C, respectively [21]. A β-xylosidase (BXA43) from Bf. animalis subsp. lactis BB-12 has the optimum of pH 5.5 and 50 o C [25], and XynB2 from Le. brevis DSM 20054 showed the highest activity at pH 6.0 and 50 o C [26].

Substrate Specificity of BfdABFs and BfdXYL
The ABF activity against various substrates was measured using p-nitrophenyl sugar assay, DNS reducing sugar assay, and L-arabinose assay kit (Table 1). BfdABF1 has very weak, but detectable activity (0.23 U/mg) against branched SA polymer, whereas the hydrolytic activity against linear DA and various xylans was not significant. In contrast, BfdABF1 showed significantly high activity on oligomeric substrates, such as p-NPAf, AOS, and AXOS. These results revealed that BfdABF1 is an exo-type ABF specific to AOS and AXOS, not polymeric substrates. Among the various AOS, arabinobiose is the most preferred substrate for BfdABF1. Its hydrolytic activity against other AOS is less than 64% of that against arabinobiose. For AXOS, BfdABF1 could hydrolyze both A 3 X and A 2 XX. Its activity on A 2 XX was about 70% of that on A 3 X, while the activities on XA 3 XX and A 2+3 XX were very low. This means that BfdABF1 possesses high activity removing the single-substituted α-(1,2)-or α-(1,3)-arabinofuranosyl residues linked to D-xylose at the non-reducing terminus, not those linked to the internal D-xylose of XOS. Double-substituted AXOS, such as A 2+3 XX and XA 2+3 XX, were very slowly hydrolyzed when the excess amount of BfdABF1 was treated.
BfdABF3 has no detectable activity against most arabinan and xylan polymers, AOS, as well as p-NPAf (Table 1). In contrast, BfdABF3 has the highest activity on A 2 XX (71.4 U/mg), and considerable levels of activity on A 3 X (5.57 U/mg) and XA 3 XX (1.43 U/mg). On the contrary, the double-substituted AXOS including A 2+3 XX and XA 2+3 XX could not be hydrolyzed by BfdABF3. These results suggest that BfdABF3 is a single-substituted AXOSspecific exo-ABF. This enzyme can be easily distinguished from BfdABF1 due to the lack of detectable activity for AOS and p-NPAf, and a 16-fold higher and more specific debranching activity for A 2 XX.
BfdXYL2 showed very low activity (0.23 U/mg) only on beechwood xylan, but no activity on wheat and rye arabinoxylans, and arabinan polymers. The hydrolytic activity of BfdXYL2 on p-NPXp (12.14 U/mg) was much higher than that on p-NPAf (0.53 U/mg). As expected, BfdXYL2 was highly active against most linear XOS substrates. Therefore, BfdXYL2 is considered as an exo-type β-D-xylosidase highly specific for the β-Dxylopyranosidic linkages of linear XOS, not for AXOS and arabinoxylan polymers. Among XOS, BfdXYL2 showed the highest activity against the shortest xylobiose, and less than 41% of activity on xylotriose and the longer XOS. Although BfdXYL2 had much lower activity, it had the detectable activity against arabinobiose (0.13 U/mg) and A 3 X (0.17 U/mg), respectively, whereas its activities against A 2 XX, A 2+3 XX, and XA 2+3 XX were marginal.

Hydrolytic Modes of Action of BfdABFs and BfdXYL
To investigate the detailed hydrolytic actions towards AXOS, XOS, and AOS, three exo-hydrolases were respectively reacted with various substrates, and the reaction products were comparatively analyzed with each other using TLC and HPAEC methods. After the excess amount of enzyme (1 U/ml) was reacted with AXOS for 15 h, BfdABF1 could cleave L-arabinose residues from A 3 X, A 2+3 XX, A 2 XX, A 3 XX, XA 2 XX, and XA 3 XX, while it had no activity on XA 2+3 XX (Figs. 3A and 3B). Time-course TLC analysis showed that BfdABF1 hydrolyze both α-(1,2)-and α-(1,3)-L-arabinofuranosyl linkages from the mixture of A 2 XX and A 3 XX to L-arabinose and xylotriose (Fig. 3C). It was observed that the hydrolytic rate of A 2 XX is relatively faster than that of A 3 XX. Even though BfdABF1 very slowly hydrolyzed A 2+3 XX, this enzyme preferentially attacked α-(1,3)-L-arabinofuranosidic linkages. At the initial reaction step, BfdABF1 slowly hydrolyzed α-(1,3)-linkage of A 2+3 XX to produce L-arabinose and A 2 XX, and then the resulting A 2 XX was rapidly degraded into arabinose and xylotriose as the final products. Accordingly, the intermediate A 2 XX or A 3 XX was not observed at all. Similarly, BfdABF1 could slowly hydrolyze the mixture of XA 2 XX and XA 3 XX to generate L-arabinose and xylotetraose. As XA 2 XX was rapidly disappeared, the amounts of L-arabinose and xylotetraose proportionally increased. Although XA 2 XX contains the internal α-(1,2)-L-arabinofuranosidic side chain, its hydrolysis was much faster than that of XA 3 XX. In contrast, the hydrolysis of XA 3 XX was too slow to be detected within 30 min, which coincides with the extremely low activity of BfdABF1 on XA 3 XX (Table 1).
BfdABF3 shares very similar AXOS hydrolysis patterns with BfdABF1, with the exception of A 2+3 XX (Fig. 4). BfdABF3 could not hydrolyze A 2+3 XX as well as XA 2+3 XX, whereas BfdABF1 hydrolyzed A 2+3 XX to liberate Larabinose residues. As expected from remarkably high specific activity of BfdABF3 against A 2 XX in Table 1, the time-course TLC analysis also showed that the hydrolysis of A 2 XX or XA 2 XX by BfdABF3 is much faster than that of A 3 XX or XA 3 XX (data not shown).
According to TLC analysis, BfdXYL2 could cleave mainly β-(1,4)-D-xylopyranosidic linkages of XOS substrates. As shown in Table 1 and Fig. 5A, BfdXYL2 is a typical exo-hydrolase exhibiting much higher activity against xylobiose, the shortest substrate, than the longer oligomeric and polymeric substrates. Even though the activity of BfdXYL2 against AOS was much lower than that against XOS, this enzyme could very slowly and partly remove the L-arabinose residues from the non-reducing end of AOS (Fig. 5B). A series of AOS intermediates was observed from the incomplete hydrolysis of AOS by BfdXYL2 because of the much lower activity against AOS. Especially, BfdXYL2 could hydrolyze A 3 X into L-arabinose and D-xylose (Fig. 5C). This result implies that BfdXYL2 slowly but distinctly cleaved α-(1,3)-L-arabinofuranosyl linkage of A 3 X, and the resulting xylobiose was rapidly hydrolyzed into D-xyloses.

Proposed Roles of exo-Hydrolases in Bf. dentium
The detailed hydrolytic modes of action of BfdABF1, ABF3, and XYL2 on various oligosaccharide substrates have been schematically summarized (Fig. 6). In conclusion, BfdABF1 is a typical exo-type α-L-arabinofuranosidase that removes only L-arabinose by acting on various AOS and branched AXOS. BfdABF3 is supposed to be a debranching enzyme which does not act on most common substrates for ABF including p-NPAf and AOS, but possesses the considerable activity only towards various AXOS substrates, including A 2 XX and A 3 X. On the other hand, BfdXYL2 is a typical β-D-xylosidase specific for β-(1,4)-D-xylopyranosidic linkages of XOS and beechwood xylan.
Recently, to help the understanding of the synbiotics (probiotics and prebiotics) system, the relationship between the synergistic action of microbial carbohydrate-activating enzymes and the target substrate was intensively studied [12,13]. In this study, two ABF and XYL genes were functionally expressed from Bf. dentium and their enzymatic properties were comparatively characterized with each other. Based on the hydrolysis pattern analysis, it was predicted that three exo-type hydrolases can synergistically hydrolyze AXOS to produce the fermentable monosaccharides, L-arabinose and D-xylose. By trimming the L-arabinosyl branches by BfdABF1 and ABF3, for instance, AXOS are converted to the linear XOS, which can be further decomposed into D-xylose by the successive actions of BfdXYL2. The resulting pentose sugars are expected to act as the prebiotics which can promote the growth of Bifidobacterium species [2,3]. For the more efficient arabinoxylan-utilization by Bf. dentium, however, the endo-type β-xylanases and the exo-type ABFs will be additionally necessary. β-Xylanases shorten arabinoxylan polymers to produce AXOS, which can be further degraded into L-arabinose and D-xylose by ABFs with the specific activities towards the double-substituted and/or α-(1,3)-L-arabinofuranosyl branches of AXOS.