Biochemical Properties of an Alkaline Xylanase From Bacillus Agaradhaerens C9 and Its Application in Producing Xylo-oligosaccharides With High Degree of Polymerization From Wheat Bran

A xylanase of Bacillus agaradhaerens C9 was heterologously expressed and was then investigated. The recombinant xylanase (rBaxyl11) showed maximal activity at 60°C and pH 8.0-9.0. Under optimal conditions, K cat of rBaxyl11 for arabinoxylan and glucuronoxylan were 599 s -1 and 330 s -1 , respectively. rBaxyl11 showed a good stability at pH ranging from 5.0 to 9.0, and retained 50% of activity after 6-hour incubation at 70°C. However, it was markedly inactivated by transition elements including Fe 3+ , Ni 2+ , Mn 2+ , Co 2+ , Zn 2+ , Cu 2+ and Fe 2+ . rBaxyl11 generated xylo-oligosaccharides (XOS) whose degree of polymerization (DP) is greater than 3 when hydrolyzing arabinoxylan, while the DP of XOS ranged from 2 to 6 when acting on glucuronoxylan. Simultaneously producing xylanase and XOS by recombinant E. coli containing rBaxyl11 were then carried out. Results showed that the engineering E. coli generated xylanase and high-DP XOS extracellularly using wheat bran as substrate, and concentration of XOS reached 73 mg/g substrate after 12-hour fermentation. This study indicates the feasibility of producing XOS by a single-step fermentation approach with low cost using rBaxyl11.


Introduction
Xylo-oligosaccharide (XOS), which is composed of several β-1,4 linked xylose units, presents increasing commercial value because of great potential for application in many elds. The role of XOS as prebiotic food is the most known, which supports growth of bene cial intestinal microorganisms and stimulates the production of metabolites with helpful effect on health (1). Moreover, XOS improves growth performance of livestock or poultry in breeding industry (2). Additionally, XOS can also be made into microcapsules to load drugs (3). With various functions, market demand for XOS has increased in recent years. For example, the annual growth rate of global prebiotics market is predicted to exceed 10%, and it is expected to reach 7.37 billion USD by 2023 (4).
Enzymatic hydrolysis is one of the major methods to produce XOS, which is more environmentally friendly and generates less undesired by-products than chemical hydrolysis (5). Xylanases are essential to enzymatic production of XOS and application of those from glycoside hydrolase (GH) family 10, 11 and 30 has been extensively reported (6). However, most xylanases generate XOS with wide range of degree of polymerization (DP) as well as xylose, resulting in the low content of target product and the di culty to puri cation.
Utilization of xylanases with higher speci cities would improve yield or proportion of desired XOS, and bene t to the downstream process. Moreover, XOS with low DP such as xylobiose and xylotriose are principal product of most reported xylanases (7)(8)(9). Medium-chain and long-chain XOS, however, are of speci c functions that short-chain XOS do not possess. For example, XOS mixture with high DP is regarded as distally fermentable substrates of caecal and colonic microbiota (10). Therefore, xylanases producing high-DP XOS deserve concerns.
High cost is a severe challenge to enzymatic production of XOS. To cope with this issue, many researchers seek for utilization of cheap raw materials, or employ of e cient enzymes to save fermentation time (11,12).
Another remarkable method is process simpli cation. For instance, a wild-type Bacillus subtilis was employed to produce XOS by direct fermentation of brewers' spent grain, and yield of XOS further increased when B. subtilis was genetically modi ed (13). Trichoderma reesei also presented XOS production potential in one-step process (14). A recombinant Escherichia coli containing bifunctional xylanase/feruloyl esterase was reported to produce XOS and ferulic acid simultaneously through direct fermentation of wheat bran (15). Although a few efforts have been devoted to develop single-step fermentation, reports are still limited. In addition, e ciency of XOS production in such integrated process need to be further improved.
Bacillus agaradhaerens C9 is alkaliphilic strain with lignocellulose-degrading ability. Secretion of alkalitolerant xylanase by B. agaradhaerens C9 was veri ed in our previous work (16). Bioinformatics analysis of its genome identi ed two xylanases, one of which belongs to GH 11 and was named Baxyl11. Although the catalytic mechanism for 4-nitrophenyl-beta-D-xylopyranoside (pNPX) of Baxyl11 have been previously reported, its enzymatic characteristics of hydrolyzing xylan are still unknown (17). In this study, Baxyl11 was heterogeneously expressed in E. coli and enzymatic characteristics of recombinant Baxyl11 (rBaxyl11) were investigated. Also, employ of the recombinant E. coli to produce XOS from wheat bran though single-step fermentation was carried out to assess the potential of saving cost and process.

Strains, plasmid, and substrates
B. agaradhaerens C9 was isolated from saline-alkali soil, and has been maintained in our laboratory since then. E. coli DH5α was used for gene cloning and plasmid maintenance. E. coli Rosetta(DE3) was used for gene expression. pET22b(+) was used for construction of recombinant plasmid. Arabinoxylan, glucuronoxylan, linear xylan and XOS with DP ranging from 2 to 6 were used as substrates of xylanase, and were all purchased from Megazyme (Ireland).
Wheat bran was purchased from a our mill in Huainan city, China. Starch presenting in wheat bran was removed before hydrolysis as follows: milled wheat bran was treated with amylase and papain successively.
These enzymes were then denatured by boiling for 25 min. After that, wheat bran was washed several times to remove enzyme and starch. The de-starched wheat bran was nally dried and screened through 80 meshes sieve for hydrolysis. The xylan content of wheat bran increased from 28.3-59.4% after de-starched treatment, which were determined based on the method offered by National Renewable Energy Laboratory (18).
Gene expression was induced in LB-ampicillin medium using 0.6 mM of isopropyl-1-thio-β-Dgalactopyranoside at 37°C, 200 rpm for 3 hours. Bacterial cells were then harvested by centrifuging to remove medium, and were resuspended using Tris-HCl buffer (20 mM, pH8.0) for ultrasonication. After that, soluble cell extracts containing rBaxyl11 were collected by centrifuging at 4°C and were ltered with 0.45-µm lters. rBaxyl11 was puri ed by a nity chromatography as follows: 5 mL of soluble cell extracts were loaded into a Ni 2+ His-tag column that was previously equilibrated with binding buffer (20 mM Tri-HCl, 500 mM NaCl, pH 8.0). 12 mL of washing buffer (20 mM imidazole, 20 mM Tri-HCl, 500 mM NaCl, pH 8.0) and 6 mL of elution buffer (250 mM imidazole, 20 mM Tri-HCl, 500 mM NaCl, pH 8.0) were then loaded to remove unobjective proteins and to elute rBaxyl11, respectively. Saline ions in eluent was removed through dialysis and rBaxyl11 was nally freeze-dried for reserve.

Enzyme assay
The freeze-dried rBaxyl11 was dissolved with deionized water, and protein concentration was determined according to the absorbancy at 280 nm and the extinction coe cients of rBaxyl11. To measure enzymatic activity, 50 µL of diluted enzyme solution and 100 µL of substrate solution were mixed and incubated at 60°C, pH 8.0 for 5 min except speci cally indicated, and the reducing sugars were then determined by dinitrosalicylic acid assay. Kinetic parameters were worked out using Lineweaver-Burk plot based on enzyme activities measured with xylan solution whose concentration ranged from 1 to 20 mg/mL. The optimal reaction conditions were investigated by determining enzymatic activities at different temperatures or pH values. To study stability, activities of rBaxyl11 was measured after incubated at 70℃ for different time or incubated in buffer with different pH values for one hour. Fe 3+ , K + , Ni 2+ , Mn 2+ , Ca 2+ , Mg 2+ , Co 2+ , Zn 2+ , Cu 2+ , Fe 2+ , SDS and EDTA were respectively added to substrate solution in advance to determine the effect of metal ions and chemical reagents on enzymatic activity.

Thin layer chromatography
rBaxyl11 and substrate were mixed and incubated. Subsequently, 8 µL of mixture was spotted onto a silica gel plate (Merk, Germany) and developed in a n-butanol-acetic acid-water (2:1:1, v/v/v) solvent system. The silica gel plate was then immersed in a solution containing methanol and sulfuric acid (4:1, v/v) for 20 s, followed by heating at 105°C for 10 min to detect XOS produced by rBaxyl11.

Integrated fermentation of XOS
250 µL of recombinant E. coli Rosetta(DE3) seeds was inoculated to 25 mL of LB-ampicillin medium containing 0.5 g of de-starched wheat bran, and was cultivated at 37°C for 2.5 hours in a shaker at 200 rpm.
Expression of rBaxyl11 was then induced by 0.6 mM of isopropyl-1-thio-β-D-galactopyranoside under the same condition for fermentation. 100 µL of medium was sampled and diluted to measure extracellular xylanase activity and XOS concentration during this process. E. coli Rosetta(DE3) containing empty vector was used as control group.
Sequence alignment was carried out using DNAMAN v6 software package.

Biochemical properties of rBaxyl11
The Baxyl11 gene was successfully expressed in E. coli. Puri ed rBaxyl11 showed electrophoretic homogeneity and the molecular weight is consistent with calculated value of 28.9 kD (Fig. 1a). rBaxyl11 displayed hydrolytic activities for both linear and branched xylans but not for cellulose, mannan, starch and pNPX, which demonstrates that rBaxyl11 is an endo-xylanase. To evaluate its catalytic activities, kinetic parameters of rBaxyl11 against arabinoxylan and glucuronoxylan were determined (Table 1). V max and K cat against arabinoxylan were approximately two times as high as those against glucuronoxylan, showing higher activity for arabinoxylan. However, lower K m against glucuronoxylan indicated the preference for such polysaccharide than arabinoxylan, suggesting that arabinofuranosyl side chains interfere with the interaction between rBaxyl11 and substrate. As a result, the K cat /K m of rBaxyl11 towards glucuronoxylan is higher than that towards arabinoxylan. Concentration of rBaxyl11 was 220 nΜ for determination. Data re ect the mean ± standard deviation (n = 3).
To investigate the optimal condition for catalysis, activities of rBaxyl11 were determined at different temperatures and pH values ( Fig. 2a and Fig. 2b). rBaxyl11 showed highest activity at 60°C and its optimal pH ranged from 8.0 to 9.0, indicating it is an alkaline xylanase. Stability of rBaxyl11 was then studied ( Fig. 2c and Fig. 2d). Activity of rBaxyl11 retained more than 80% when incubating at 70°C for 30 min, and more than 60% after incubation for 240 min. rBaxyl11 showed good stability when incubated at the pH range of 5.0 to 9.0, but was inactivated when pH value further increased.
The GH11 xylanase of B. agaradhaerens was reported to hydrolyze pNPX with optimal pH of 5.6 (17). rBaxyl11 of B. agaradhaerens C9, however, was not able to act on pNPX in this study, which can be attributed to subtle difference in amino acid sequence and catalytic sites (Fig. 1b). rBaxyl11 showed optimal activity in alkaline environment when hydrolyzing xylan. This characteristic gives rBaxyl11 unique advantages in treatment of biomass pretreated with alkali, because the substrate need not wash to neutral. The good stability is also bene cial to the application of rBaxyl11.

Effect of metal ions and chemical reagents on activity of rBaxyl11
Effects of common metal ions, ethylene diamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) on the catalytic activity for arabinoxylan and glucuronoxylan were investigated (Fig. 3). All the tested transition elements (Fe 3+ , Ni 2+ , Mn 2+ , Co 2+ , Zn 2+ , Cu 2+ and Fe 2+ ) markedly reduced activity of rBaxyl11. Inactivation of xylanases caused by metal ions was widely reported and the mechanism can be interpreted as occupying the binding or catalytic sites of enzymes (19)(20)(21)(22). By contrast, alkaline-earth metal ions, including K + , Ca 2+ and Mg 2+ , had weaker in uence on rBaxyl11, and their effects were related to substrate type. For instance, Mg 2+ stimulated the hydrolysis of arabinoxylan, but did not increase the activity of rBaxyl11 for glucuronoxylan. Such substrate-dependent effect suggested that these alkaline-earth metal ions may in uence the interaction between rBaxyl11 and substrates with speci c structure. Effects of tested chemical reagents were similar to that of alkaline-earth metal ions. Speci cally, both EDTA and SDS positively affected enzymatic activity for arabinoxylan while inhibited that for glucuronoxylan. Inactivation caused by EDTA was weak, indicating rBaxyl11 did not rely on metal ions to catalyze.

Hydrolytic modes of rBaxyl11
To investigate hydrolytic modes of rBaxyl11, its products were determined using thin layer chromatography.
rBaxyl11 did not hydrolyze xylo-oligosaccharides whose DP is less than ve, and could converted xylopentaose and xylohexaose into xylotriose and xylotetraose as primary products (Fig. 4a). This result indicated rBaxyl11 contains ve xylose-binding subsites for catalysis. When hydrolyzing glucuronoxylan, rBaxyl11 generated XOS with DP ≥ 3 at initial stage, and xylobiose was later generated after further incubation (Fig. 4b). When arabinoxylan was used as substrate, rBaxyl11 produced XOS with DP ≥ 4 in the whole stage (Fig. 4c). Moreover, migration rates of certain oligosaccharide products ranged between two XOS standards, which were probably arabinoxylan-oligosaccharides (AXOS), namely XOS containing arabinosyl side chains. Despite the same backbone of substrates, XOS pro les produced by rBaxyl11 were different when hydrolyzing xylohexaose, glucuronoxylan and arabinoxylan, indicating structure and type of side chains would in uence its hydrolytic mode.
Hydrolysis products of xylanases generally contained low-DP XOS such as xylobiose and xylotriose (Table 2). An exception is a xylanase isolated from bovine rumen, which converted birchwood xylan mainly into xylohexaose (23). This xylanase showed a good product speci city but was unsuitable for XOS production due to a low rate of xylan degradation. The other reported example is a commercial xylanase, which generated XOS with DP range of 3 to 5 from microwave-CrO 3 -H 3 PO 4 pretreated rice straw (24). Nevertheless, numerous monosaccharides were also produced in that system. By contrast, rBaxyl11 speci cally generated series of high-DP XOS and AXOS without xylose, xylobiose and xylotriose when hydrolyzing arabinoxylan. Being different from linear low-DP oligosaccharides, XOS with high molecular weight and AXOS with more complex structures were considered as slower fermenting prebiotics, thereby promoting health of distal intestinal tract (10,25,26). Therefore, rBaxyl11 showed promising potential for production of such prebiotics.

Single-step fermentation for XOS production
Wheat bran is a cheap by-product from our milling industry and consists of 29-42% of arabinoxylan (27). To save cost and simplify process, direct fermentation of de-starched wheat bran by engineering E. coli containing rBaxyl11 to produce XOS and AXOS was attempted (Fig. 5). Extracellular secretion of rBaxyl11 was detected after two-hour fermentation. The xylanase activity rapidly increased to 48% of the maximum in the next two hours, and the increment slowed down from the 8th hour. Concentration of XOS in medium showed similar trend comparing with xylanase activity, which increased rapidly from the 2nd hour to 8th hour.
It reached 1.46 mg/mL at the 12th hour and only further increased by 8.9% in the next 12 hours. Therefore, 12 hours were optimal fermentation time for XOS production considering the cost.
These results demonstrated the feasibility of single-step fermentation for XOS production by recombinant E coli containing rBaxyl11. Theoretically, rBaxyl11 was transported to the periplasm when using pET22b(+). According to our measurement, however, about 30% of rBaxyl11 was secreted to medium, which made the fermentation workable. Although some puri ed or commercial xylanases generated considerable XOS by hydrolysis, preparation of these enzyme prejudices economy of the production process. In addition, high temperature is commonly employed to maintain enzymatic activity and a large dose of xylanase is needed to cope inactivation, which are both adverse to cost ( Table 2). By contrast, an integrated production of XOS can leave out separate process for enzyme expression as well as puri cation, and generally adopts mild conditions. Therefore, direct fermentation by microorganisms was believed to be a remarkable way to save cost and was bene cial to industrial production of XOS (4).
There are a few cases of single-step fermentation so far (Table 2). Some fungi such as Aspergillus nidulans and Trichoderma reesei were showed to simultaneously produce xylanase and XOS using cheap biomass (14,28). Nevertheless, the yields of XOS were modest despite optimization, which could be attributed to lack of pretreatment to substrate. Bacillus subtilis was also reported to speci cally produce xylotriose, xylotetraose or xylopentaose from wheat middlings in a single-step fermentation process, but the fermentation required at least 48 hours for a high purity of XOS without xylose (29). By comparison, an recombinant B. subtilis could generate AXOS with DP ranging from 2 to 6 after 12-hour fermentation, despite slight reduction in yield (13). It seems that engineering bacteria is more promising in XOS production with low cost. As model bacteria, E. coli is widely utilized in bioengineering and, in addition, it neither uses XOS as carbon source nor produces other undesired saccharides, thereby contributing to enhance yield and purity of XOS. (30). A recombinant E. coli was recently reported to produce XOS and xylose by single-step fermentation, but the concentration and DP of XOS were not investigated (15). In this study, the engineering E coli. retained the speci city of puri ed rBaxyl11, which generated high-DP XOS and AXOS from wheat bran (Fig. 4d). Yield of XOS reached 73 mg/g substrate at 12th hour and exceed those of previous reports using single-step fermentation ( Fig. 5 and Table 2). Such system can also be further optimized to improve the overall yields or reduce cost to meet requirements of industrialization.

Conclusions
Enzymatic characteristics of rBaxyl11 from B. agaradhaerens C9 was studied. The alkaline xylanase of GH11 has good stability and activity to various xylans. DP of XOS produced by rBaxyl11 was in uenced by type of substrates. It was notable that rBaxyl11 converted arabinoxylan into high-DP XOS, which is different from most reported xylanases. Results of direct fermentation demonstrated the feasibility of simultaneously producing xylanase and XOS by recombinant E. coli containing rBaxyl11 in an integrated process. Thus, such single-step fermentation approach could be a promising strategy for XOS production at industrial scale.   Electrophoresis and sequence analysis of rBaxyl11 (a) Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of rBaxyl11. Line 1: soluble cell extracts containing rBaxyl11; Line 2: rBaxyl11 after puri cation; Line 3: marker. (b) Sequence alignment of Baxyl11 and BadX. Amino acid residues belonging to signal peptide are marked with yellow background. Different amino acid residues between Baxyl11 and BadX are marked with green background.

Figure 2
Effect of temperature and pH on activity and stability of rBaxyl11 (a) Effect of temperature on activity of rBaxyl11. (b) Effect of pH on activity of rBaxyl11. (c) Effect of temperature on stability of rBaxyl11. (d) Effect of pH on stability of rBaxyl11. For gure 1A and 1B, the maximal activity was designated as 100%. For gure 1C and 1D, activity of enzyme without incubation was designated as 100%. Determination at pH 5.0-8.0 and 8.0-10.5 was carried out in Na2HPO4-NaH2PO4 buffer and Na2CO3-NaHCO3 buffer, respectively. All data are presented as means ± standard deviations (n=3).

Figure 3
Effect of metal ions and chemical reagents on activity of rBaxyl11 Activity of rBaxyl11 was measured using 0.3% of xylan in the presence of metal ion or chemical reagent (5mM). Activity of control group without metal ion or chemical reagent was designated as 100%. Data between control and test groups differ statistically (p<0.05, t test) except the activity measured with K+ and glucuronoxylan (p=0.355, t test). All data are presented as means ± standard deviations (n=3).

Figure 5
Single-step fermentation by recombinant E. coli Circle and triangle represented xylanase activity and reducing sugar, respectively. Solid and dotted lines represented recombinant E. coli containing rBaxyl11 and empty vector, respectively. Fermentation started from the time of inducing. All data are presented as means ± standard deviations (n=3).