3.1 Biochemical properties of rBaxyl11
The Baxyl11 gene was successfully expressed in E. coli. Purified 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). Vmax and Kcat against arabinoxylan were approximately two times as high as those against glucuronoxylan, showing higher activity for arabinoxylan. However, lower Km 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 Kcat/Km of rBaxyl11 towards glucuronoxylan is higher than that towards arabinoxylan.
Table 1
Kinetic parameters of rBaxyl11 for xylans
Substrate
|
Vmax (µΜ/s)
|
Kcat (/s)
|
Km (g/L)
|
Kcat/Km (L/g/s)
|
Arabinoxylan
|
44.2 ± 3.7
|
599.0 ± 49.7
|
10.9 ± 0.9
|
55.0 ± 0.3
|
Glucuronoxylan
|
24. 3 ± 0.6
|
330.1 ± 7.7
|
4.1 ± 0.1
|
79.7 ± 1.2
|
Concentration of rBaxyl11 was 220 nΜ for determination. Data reflect 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 beneficial to the application of rBaxyl11.
3.2 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 (Fe3+, Ni2+, Mn2+, Co2+, Zn2+, Cu2+ and Fe2+) 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 (19–22). By contrast, alkaline-earth metal ions, including K+, Ca2+ and Mg2+, had weaker influence on rBaxyl11, and their effects were related to substrate type. For instance, Mg2+ 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 influence the interaction between rBaxyl11 and substrates with specific structure. Effects of tested chemical reagents were similar to that of alkaline-earth metal ions. Specifically, 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.
3.3 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 five, and could converted xylopentaose and xylohexaose into xylotriose and xylotetraose as primary products (Fig. 4a). This result indicated rBaxyl11 contains five 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 profiles produced by rBaxyl11 were different when hydrolyzing xylohexaose, glucuronoxylan and arabinoxylan, indicating structure and type of side chains would influence 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 specificity 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-CrO3-H3PO4 pretreated rice straw (24). Nevertheless, numerous monosaccharides were also produced in that system. By contrast, rBaxyl11 specifically 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.
Table 2
XOS production by reported xylanases using enzymatic hydrolysis and single-step fermentation
Substrate
|
Enzyme or strain
|
Reaction timea
|
XOS yieldb
|
MainDP
|
XOS production mode
|
Reference
|
De-starched wheat bran
|
Engineering E. coli
|
12 hours
|
7.3%
|
> 3
|
Single-step fermentation (37°C)
|
This study
|
De-starched wheat bran
|
Engineering E. coli
|
36 hours
|
/
|
/
|
Single-step fermentation (37°C)
|
(15)
|
Wheat middlings
|
Bacillus subtilis
|
48 hours
|
6.5%
|
3–4
|
Single-step fermentation (37°C)
|
(29)
|
Rice husk
|
Engineering Aspergillus nidulans
|
48 hours
|
2.4%
|
3–6
|
Single-step fermentation (37°C)
|
(28)
|
Brewers’ spent grain
|
Engineering Bacillus subtilis
|
12 hours
|
5.4%c
|
2–6
|
Single-step fermentation (45°C)
|
(13)
|
Brewers’ spent grain
|
Trichoderma reesei
|
72 hours
|
3.8%c
|
2–5
|
Single-step fermentation (30°C)
|
(14)
|
Beechwood xylan
|
Xylanase from Mycothermus thermophilus
|
12 hours
|
83%
|
2–3
|
Enzymatic hydrolysis (65°C)
|
(31)
|
Wheat bran
|
Xylanase from Aspergillus oryzae
|
24 hours
|
0.4%
|
2–4
|
Enzymatic hydrolysis (50°C)
|
(32)
|
Auto-hydrolysis pretreated corncobs
|
Mutant xylanase from Talaromyces thermophilus
|
2 hours
|
7.1%
|
2–3
|
Enzymatic hydrolysis (50°C)
|
(33)
|
Delignified sugarcane bagasse
|
Xylanase from Bacillus subtilis
|
15 hours
|
12.0%
|
2–11
|
Enzymatic hydrolysis (50°C)
|
(34)
|
Wheat husk
|
Crude xylanase from Aspergillus fumigatus R1
|
12 hours
|
/
|
≥ 2
|
Enzymatic hydrolysis (37°C)
|
(35)
|
Extracted xylan from corn cobs
|
Xylanase from Thermomyces lanuginosus
|
8 hours
|
34.5%
|
2–3
|
Enzymatic hydrolysis (45°C)
|
(36)
|
Birchwood xylan
|
Xylanase isolated from bovine rumen
|
0.5 hour
|
/
|
≥ 6
|
Enzymatic hydrolysis (35°C)
|
(23)
|
Extracted xylan from data seed
|
Commercial xylanase from Aspergillus niger
|
6 hours
|
36%
|
2–3
|
Enzymatic hydrolysis (38°C)
|
(37)
|
Alkali-microwave pretreated wheat bran
|
Xylanase from Bacillus halodurans
|
72 hours
|
26%
|
≥ 2
|
Enzymatic hydrolysis (70°C)
|
(38)
|
Microwave pretreated rice straw
|
Commercial xylanase
|
24 hours
|
0.4%
|
3–5
|
Enzymatic hydrolysis (50°C)
|
(24)
|
Extracted arabinoxylan from wheat bran
|
Xylanase from Geobacillus stearothermophilus
|
24 hours
|
53%
|
2–5
|
Enzymatic hydrolysis (50°C)
|
(39)
|
Extracted xylan from vetiver grass
|
Xylanase from Aureobasidium melanogenum
|
96 hours
|
23.6%
|
2–3
|
Enzymatic hydrolysis (28°C)
|
(40)
|
Steam-explosion corncobs
|
Xylanase from Paenibacillus barengoltzii
|
4 hours
|
23.4%
|
2–5
|
Enzymatic hydrolysis (50°C)
|
(12)
|
Extracted xylan from mahogany
|
Xylanase from Clostridium strain BOH3
|
24 hours
|
57.2%
|
2–5
|
Enzymatic hydrolysis (50°C)
|
(8)
|
Alkali pretreated mahogany
|
Xylanase from Clostridium strain BOH3
|
24 hours
|
9.0%
|
2–5
|
Enzymatic hydrolysis (50°C)
|
(8)
|
a Reaction time indicates the hydrolysis or fermentation time when XOS yield reaches the one represented here. |
b XOS yield is represented as amount of XOS per gram of substrates (mg/g). Xylose is not included. |
c These yields are expressed in xylose equivalents. |
3.4 Single-step fermentation for XOS production
Wheat bran is a cheap by-product from flour 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 purified 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 purification, and generally adopts mild conditions. Therefore, direct fermentation by microorganisms was believed to be a remarkable way to save cost and was beneficial 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 specifically 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 specificity of purified 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.