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One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant... Background: One-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombi- nant Escherichia coli through one-step fermentation was investigated in this study. Results: An endo-β-1,4-xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60 °C and pH 8.0–8.5. Its activity retained more than 60% after incubation at 70 °C for 4 h, showing a good stability. The recombinant E. coli BL21(DE3) could secreted xylanases that directly hydrolyzed de-starched wheat bran to XOS in fermentation medium. The XOS generated from hydrolysis consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The XOS concentration reached 5.3 mg/mL at 10% loading of wheat bran, which is higher than those of previous researches. Nitrogen source type could also affect production of XOS by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied using response surface optimization. The maximal concentration emerged at 44.3 °C, pH 7.98, which is affected by characteristics of the xylanase as well as growth conditions of E. coli. Conclusions: This work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and final concentration for producing XOS. Results can also provide theoretical basis for large-scale production and contribute to the wide adoption of XOS. Keywords: Prebiotics, Bacillus agaradhaerens, Single-step fermentation, Xylanase, Response surface optimization Background Prebiotics, namely some kinds of oligosaccharides, can specifically promote the activity of beneficial bacteria in gastrointestinal tract [1, 2]. With insight into the effect of gut microbiota on human overall health, prebiotics have *Correspondence: leonliu2013@126.com been the hotspot in consumption and research currently Jiawen Liu and Cong Liu contributed equally to this work [3]. Xylo-oligosaccharides (XOS) are emerging probiot- Jiangsu Key Laboratory of Phylogenomics and Comparative Genomics, School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, ics which consist of several β-1,4 linked xylose units [4]. Tongshan District, Xuzhou 221116, Jiangsu Province, China They have been attached importance to recent years due © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. BMC Biotechnology (2022) 22:6 Page 2 of 11 to remarkable prospect of application in food, medicine, fermentation so far. These microorganisms, however, can poultry and other fields [5–7]. Furthermore, XOS are all utilize XOS as carbon source, which prejudices the more efficient than other prebiotics in enhancing growth accumulation of XOS in medium. In addition, critical of certain bifidobacteria and in protecting lactobacilli restriction limiting XOS production remains unknown under stress environments [8–10]. XOS also present for such integrated fermentation, which leads to difficulty good heat and pH stability, which is beneficial to retain - in substantial improvement in yield. ing more nutritional properties in digestive tract [11]. Escherichia coli has been used to produce food addi- Because of these advantages, market demand for XOS is tives and drugs for decades, which has been proved to be rising quickly and expected to reach 130 million U.S. dol- safe and reliable [23, 24]. E. coli is probably suited to fer- lars in 2023 at an annual growth of 5.3% [12]. mentation for producing XOS because it cannot consume Enzymatic hydrolysis is one of the major methods to this kind of oligosaccharides [25]. However, feasibility of produce XOS, which is more environmentally friendly one-step fermentation employing E. coli lacks sufficient and generates less undesired by-products than chemi- study. Wheat bran is a xylan-rich by-product of white cal hydrolysis [13]. Xylanases are the critical factor for flour milling and has been used as cheap raw materials enzymatic production of XOS, which act on backbone for XOS production previously [26, 27]. Bacillus agarad- of xylan and convert it into XOS as well as xylose. Xyla- haerens C9 is an alkaliphilic strain with lignocellulose- nases belonging to glycoside hydrolase (GH) family 11 degrading ability. Secretion of alkali-tolerant xylanases attack unsubstituted sites of xylan, whose hydrolysate by B. agaradhaerens C9 was verified in our previous work mainly consists of xylobiose and xylotriose; GH10 xyla- [28]. Bioinformatics analysis of its genome revealed an nases can accommodate a decorated xylopyranosyl resi- GH11 xylanase that was named Baxyl11. In this study, due at − 1 subsite, resulting in production of both linear Baxyl11 was expressed using E. coli BL21(DE3) and enzy- and substituted XOS with low degree of polymerization matic characteristics of recombinant Baxyl11 (rBaxyl11) (DP); GH30 xylanases prefer branched xylan than the lin- were then investigated. Moreover, producing XOS from ear one so substituted XOS are their principal products wheat bran by the recombinant E. coli BL21(DE3) con- [14, 15]. Hydrolyzing extracted xylan or raw lignocellu- taining rBaxyl11 was carried out. Effects of wheat bran losic biomass using these xylanases to produce XOS has concentration, nitrogen source type and fermentation been widely reported, and XOS yields are very attractive conditions (pH and temperature) on XOS production in some works [16, 17]. However, preparation of these were finally investigated. These results would contribute purified enzymes is unwieldy and costly. In addition, high to overcoming yield and cost challenge in the production temperature is commonly needed for an efficient enzy - of XOS, and promote its wide adoption. matic hydrolysis, which also prejudices the cost of pro- duction process [18, 19]. To cope with these issues, some Results researches devoted to integrating production of xylanases Enzymatic characteristics of rBaxyl11 and XOS into a single process. In such process, microor- Baxyl11 gene was cloned from genomic DNA of B. aga- ganisms extracellularly secrete xylanases and meanwhile, radhaerens C9 and ligated with plasmid pET22b(+). these enzymes directly convert xylan or lignocellulosic rBaxyl11 was then expressed using E. coli BL21(DE3). biomass into XOS in medium. For example, a wild-type The purified rBa xyl11 showed electrophoretic homoge- Bacillus subtilis was reported to produce XOS by direct neity and its molecular weight corresponded to the cal- fermentation utilizing brewers’ spent grain, and XOS culated value of 28.9 kD (Fig.  1a). rBaxyl11 presented could yield further increase when B. subtilis was geneti- hydrolytic activity to glucuronoxylan and arabinoxylan cally modified [20]. Some fungi, such as Trichoderma but not to cellulose, mannan, starch and 4-nitrophe- reesei and Aspergillus nidulans, exhibited potential of nyl-beta-D-xylopyranoside (pNPX), which demonstrated producing XOS in one-step fermentation as well [21, 22]. that rBaxyl11 is an endoxylanase. These integrated production of XOS left out separate To evaluate its catalytic activities, kinetic parameters process for preparing xylanases, and generally adopted of rBaxyl11 against arabinoxylan and glucuronoxylan mild fermentation conditions, which contributes to over- were measured (Table  1). V and K against arabi- max cat coming cost challenge [12]. Nevertheless, XOS yields noxylan were approximately two times as high as those of one-step fermentation are commonly disadvantaged against glucuronoxylan, showing higher activity against comparing with those of enzymatic hydrolysis. Indeed, arabinoxylan. However, lower K against glucuron- yields can be improved by optimizing types of medium, oxylan indicated the preference for such polysaccharide substrates, fermentation microorganisms and conditions, than arabinoxylan. As a result, the K /K of rBaxyl11 cat m but researches about these issues are scarce. For example, against glucuronoxylan was higher than that against only a few bacillus and fungi are employed for one-step arabinoxylan. Liu  et al. BMC Biotechnology (2022) 22:6 Page 3 of 11 Fig. 1 Electrophoresis and sequence analysis of rBaxyl11. a Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of rBaxyl11. Line 1: soluble cell extract containing rBaxyl11; Line 2: rBaxyl11 after purification; 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 To investigate the optimal conditions for catalysis, Table 1 Kinetic parameters of rBaxyl11 for xylans activities of rBaxyl11 were measured at different tem - Substrate V (μΜ/s) K (/s) K (g/L) K /K (L/g/s) max cat m cat m peratures and pH values (Fig.  2a, b). rBaxyl11 showed Arabinoxylan 44.2 ± 3.7 599.0 ± 49.7 10.9 ± 0.9 55.0 ± 0.3 highest activity at 60 °C and its optimal pH ranged from Glucuronoxylan 24. 3 ± 0.6 330.1 ± 7.7 4.1 ± 0.1 79.7 ± 1.2 8.0 to 8.5, indicating it is an alkaline xylanase. Stabil- ity of rBaxyl11 was then studied (Fig.  2c, d). Activity of Concentration of rBaxyl11 was 220 nΜ for measurement. All data are presented as mean ± standard deviation (n = 3) Fig. 2 Eec ff t of temperature and pH on activity and stability of rBaxyl11. a Eec ff t of temperature on activity of rBaxyl11. b Eec ff t of pH on activity of rBaxyl11. c Eec ff t of temperature on stability of rBaxyl11. d Eec ff t of pH on stability of rBaxyl11. In (a) and (b), the maximal activity was designated as 100%. In (c) and (d), activity of enzyme without incubation was designated as 100%. Measurement at pH 5.0–8.0 and 8.0–10.5 was carried out in Na HPO -NaH PO buffer and Na CO -NaHCO buffer, respectively. All data are presented as means ± standard deviations (n = 3) 2 4 2 4 2 3 3 Liu et al. BMC Biotechnology (2022) 22:6 Page 4 of 11 rBaxyl11 retained more than 80% after incubation at 70  °C for 30  min, and even after 4  h, 60% of its activity could be maintained. Moreover, rBaxyl11 showed good stability when incubated at the pH ranging from 5.0 to 9.0, which is commonly the appropriate pH range for fermentation. One‑step fermentation for XOS production To save cost and simplify process, direct fermentation by rBaxyl11-transformed E. coli BL21(DE3) to produce XOS from wheat bran was carried out. Starch in wheat bran was removed in advance for a better XOS yield. Employ of the recombinant E. coli BL21(DE3) in the presence of isopropyl-1-thio-β-D-galactopyranoside (IPTG) and wheat bran for fermentation resulted in a reducing sugar yield of 1.41  mg/mL at the 24th hour (Fig.  3a). By con- trast, fermentation without wheat bran or using E. coli BL21(DE3) containing raw plasmid only produced negli- gible reducing sugars, demonstrating that rBaxyl11 from E. coli could produce XOS by acting on wheat bran in such one-step fermentation. In the presence of rBaxyl11 and wheat bran, xylanase activity increased rapidly in the first 6 h and slowly then (Fig.  3b). Activity in the medium without IPTG showed Fig. 3 The time course of a XOS concentration and b extracellular similar trend but at lower level. It is noteworthy that xylanase activity during one-step fermentation. “With IPTG”: employ use of IPTG raised xylanase activity by 40% while only of inducer (IPTG), wheat bran and recombinant E. coli containing increased XOS concentration by 18%, suggesting that rBaxyl11; “Without IPTG”: employ of wheat bran and recombinant xylanase activity is not the most important factor to yield E. coli containing rBaxyl11 without inducer; “Empty vector”: (see “Effect of nitrogen source type on one-step fermen - employ of inducer, wheat bran and recombinant E. coli containing unmodified pET22b(+); “Without wheat bran”: employ of inducer and tation” section for details). recombinant E. coli containing rBaxyl11 without wheat bran. Wheat bran concentration: 2%. All data are presented as means ± standard Product composition of rBaxyl11 acting on wheat bran deviations (n = 3) To study the product composition of rBaxyl11 acting on wheat bran, its hydrolysate was analyzed using high pres- sure ion chromatography (HPIC). Results demonstrated that xylose, xylobiose and xylotriose are the primary To study how wheat bran concentration affects pro - product (Fig. 4a). Further quantitative analysis basing on duction of XOS, xylanase activity and growth of E. chromatogram showed that xylose, xylobiose and xylo- coli BL21(DE3) were also measured. Xylanase activ- triose respectively accounted for 23.1%, 37.3% and 39.6% ity increased with wheat bran concentration while the (Fig. 4b). In other words, about 77% of its product is low- biomass of E. coli BL21(DE3) showed opposite trend, DP XOS when rBaxyl11 hydrolyzed wheat bran. indicating that high-concentration wheat bran stimu- lated the synthesis and secretion of rBaxyl11 and inhib- Eec ff t of wheat bran concentration on one‑step ited the growth of E. coli BL21(DE3) (Fig.  5). A high fermentation xylanase activity would contribute to XOS production, Effect of wheat bran concentration on XOS yield was but the huge increase of final concentration was not investigated here. As showed in Fig.  5a, XOS concentra- exclusively due to this reason. Specifically, XOS con - tion increased with wheat bran concentration in 0–10% centration increased nearly 16-fold when wheat bran of loading range, and further raise in substrate load- concentration raised tenfold from 1 to 10%, and mean- ing would lead to an excessive viscosity of medium. At while, xylanase activity only increased by 103%. It is 10% of wheat bran concentration, 6.9  mg/mL of reduc- obvious that augmentation of XOS yield resulted from ing sugar was obtained. That means the XOS concentra - combined effect of the increase in both wheat bran con - tion reached 5.3 mg/mL (excluding xylose), which is very centration and xylanase activity, where the former con- considerable. tributed more. In other words, substrate concentration Liu  et al. BMC Biotechnology (2022) 22:6 Page 5 of 11 Fig. 4 Product composition of rBaxyl11 acting on wheat bran analyzed by HPIC. a Product composition of the hydrolysis. “Standards”: mixture of xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose with respective concentration of 2 mg/mL; “Hydrolysate”: XOS produced by rBaxyl11 from wheat bran; “Control”: sample of hydrolysate without employ of rBaxyl11. b Quantitative analysis of xylose and XOS produced by rBaxyl11 Fig. 5 Eec ff t of wheat bran concentration on one-step fermentation. a Eec ff t of wheat bran concentration on XOS production, xylanase activity and biomass of recombinant E. coli. b Correlation between XOS concentration and xylanase activity as well as wheat bran concentration. Fermentation time: 12 h. Temperature: 37 °C. Initial pH: 7.0. In (a), data are presented as means ± standard deviations (n = 3) is the decisive factor to XOS production in such fer- assay, medium after dialysis was also employed for meas- mentation process instead of enzymatic activity. urement (Fig. 6b). Results demonstrated a positive linear correlation between XOS concentration and xylanase Eec ff t of nitrogen source type on one‑step fermentation activity regardless of whether medium was treated with Effect of nitrogen source type on one-step fermentation dialysis. By comparison, no credible correlation between was investigated here (Fig.  6a). Measurement of reduc- XOS concentration and biomass was observed. There - ing sugar indicated that maximal XOS concentration fore, types of nitrogen source affected XOS production was obtained when using glycine as nitrogen source, mainly by changing xylanase activity. which is slightly higher than that using yeast extract (p value = 0.065). The lowest three yields showed when Optimizing XOS production by response surface NH NO, NaNO and NH SO were employed, indicat- methodology 4 3 3 4 4 ing such inorganic salts are not suited to production of To study effect of fermentation conditions, tempera - XOS. ture, pH and glycine concentration were chosen as vari- The correlation analysis was then conducted to evaluate ables for optimization using Box-Behnken design. After the effect of nitrogen sources on XOS. To avoid the effect 12-h fermentation, concentrations of reducing sugars of difference in nitrogen sources on enzymatic activity varied in the range of 1.629–1.895 mg/mL (Table 2 and Liu et al. BMC Biotechnology (2022) 22:6 Page 6 of 11 Fig. 6 Eec ff t of nitrogen source type on one-step fermentation. a Eec ff t of nitrogen source type on XOS production, xylanase activity and biomass of recombinant E. coli. “Activity”: xylanase activity measured using supernatant of medium; “Activity after dialysis”: xylanase activity measured using dialysis-treated supernatant of medium. b Correlation between XOS concentration and xylanase activity as well as wheat bran concentration. Wheat bran concentration: 2%. Fermentation time: 12 h. Temperature: 37 °C. Initial pH: 7.0. In (a), data are presented as means ± standard deviations (n = 3) (60 °C) and higher than the best growth temperature of Table 2 Experimental design to study the effect of pH, fermentation temperature and glycine concentration on XOS E. coli (37 °C). production Discussion Run pH Temperature Glycine (%) Concentration (mg/mL) (°C) High cost is a challenge limiting the enzymatic produc- tion of XOS. One-step fermentation is a cost-efficient 1 8.1 40 0.2 1.629 ± 0.008 way to produce XOS, but its yield was commonly modest 2 8.1 44 2.6 1.895 ± 0.009 comparing with that of enzymatic hydrolysis (Table  3). 3 7.6 48 2.6 1.780 ± 0.009 For example, hydrolyzing mahogany employing a xyla- 4 7.6 44 0.2 1.673 ± 0.014 nase of Clostridium resulted in a XOS concentration of 5 8.1 48 5.0 1.721 ± 0.014 4.5  mg/mL [29]. The concentrations could even exceed 6 8.6 44 5.0 1.784 ± 0.039 8 mg/mL when using extracted xylan as substrate [16]. By 7 8.1 40 5.0 1.721 ± 0.013 comparison, only 0.8–1.1  mg/mL of XOS were obtained 8 8.1 44 2.6 1.895 ± 0.009 employing B. subtilis or Trichoderma species in one-step 9 8.1 48 0.2 1.709 ± 0.018 fermentation despite optimization [20, 21]. A higher con- 10 8.1 44 2.6 1.895 ± 0.009 centration of 3.2 mg/mL was obtained when using wheat 11 7.6 44 5.0 1.862 ± 0.044 middlings and B. subtilis, but the fermentation time, 48 h, 12 8.6 44 0.2 1.743 ± 0.003 was less competitive [30]. This study described a consid - 13 7.6 40 2.6 1.703 ± 0.011 erable XOS concentration of 5.3  mg/mL with only 12-h 14 8.1 44 2.6 1.895 ± 0.009 fermentation, which is much higher than those of previ- 15 8.6 48 2.6 1.714 ± 0.008 ous works. Moreover, substrate concentration was found 16 8.1 44 2.6 1.895 ± 0.009 to be the most influential factor to XOS production here. 17 8.6 40 2.6 1.685 ± 0.055 This is probably the cause of modest XOS yields in previ - All data are presented as mean ± standard deviation (n = 3) ous reports because XOS yield were prejudiced by a large loading of substrates using B. subtilis and fungi [20, 21]. Therefore, employing E. coli BL21(DE3) is promising to eliminate the disadvantage in XOS production by one- Fig.  7). Glycine concentration is the most influential step fermentation. variable with p-value = 0.0006, followed by temperature Considerable reducing sugar (1.19 mg/mL at the 24th with p value = 0.0132. It was predicted that the opti- hour) were produced even without IPTG, which could mal concentration of 1.904  mg/mL would be obtained be attributed to induction of certain saccharides from at 44.3  °C, pH 7.98 with 3.36% of glycine, which cor- wheat bran. Further study indicated that XOS concen- responds approximately to the central-point condition tration induced by wheat bran alone reached 78% of the of the design. The optimal pH for fermentation corre - maximum with 1 mM of IPTG, and adding only a small sponded to that of catalysis by rBaxyl11 (Fig. 2b), while amount of IPTG (0.02  mM) could lead to the maximal the optimal temperature is lower than that for catalysis Liu  et al. BMC Biotechnology (2022) 22:6 Page 7 of 11 Fig. 7 Response surface showing effect of temperature, pH and glycine concentration on XOS production. a Eec ff t of temperature and pH on XOS production. b Eec ff t of glycine concentration and pH on XOS production. c Eec ff t of glycine concentration and temperature on XOS production. Wheat bran concentration: 2%. Fermentation time: 12 h. All experiments were conducted in triplicate Table 3 XOS production by enzymatic hydrolysis and one-step fermentation Substrate Enzyme or strain Reaction XOS yields XOS production method References time (h) c d mg/mLmg/g substrate e e Wheat bran Engineering E. coli BL21(DE3) 12 0.8–5.3 53-80 One-step fermentation (37 °C) This study e e Wheat middlings Bacillus subtilis 48 3.2 64 One-step fermentation (37 °C) [30] Brewers’ spent grain Engineering Bacillus subtilis 12 1.1 34 One-step fermentation (45 °C) [20] Brewers’ spent grain Trichoderma reesei 72 0.8 40 One-step fermentation (30 °C) [21] Rice husk Engineering Aspergillus nidulans 48 – 24 One-step fermentation (37 °C) [22] Pistachio shell Commercial xylanase 10 2.7 – Enzymatic hydrolysis (45 °C) [43] e e Mahogany Xylanase from Clostridium strain 24 4.5 90 Enzymatic hydrolysis (50 °C) [29] BOH3 Sugarcane bagasse Xylanase from Bacillus subtilis 15 3.6 119 Enzymatic hydrolysis (50 °C) [44] e e Rice straw Commercial xylanase 24 0.1 2 Enzymatic hydrolysis (50 °C) [45] e e Rice straw Xylosidase from Weissella cibaria 10 2.6 70 Enzymatic hydrolysis (37 °C) [46] e e Beechwood xylan Xylanase from Mycothermus thermo- 12 8.0–8.8 800–880 Enzymatic hydrolysis (65 °C) [16] philus Xylan from corn cobs Xylanase from Thermomyces lanugi- 8 6.9 345 Enzymatic hydrolysis (45 °C) [47] nosus e e Xylan from data seed Xylanase from Aspergillus niger 4 4.1 411 Enzymatic hydrolysis (38 °C) [48] Xylan from vetiver grass Xylanase from Aureobasidium melano- 92 4.7 194 Enzymatic hydrolysis (28 °C) [49] genum Reaction time indicates the hydrolysis or fermentation time when XOS concentration reaches the presented value Xylose is not included Yields are presented as final concentration (mg/mL) of XOS in fermentation medium Yields are presented as mass (mg) of XOS obtained from a gram of substrate These data are measured using liquid chromatogram and others are measured using DNS method XOS concentration as well as xylanase activity in the XOS production, but also played an important role in fermentation medium (Additional file  1: Fig. S1). Also, stimulating the secretion of rBaxyl11 and served as E. coli BL21(DE3) could still grow and secrete xylanases nutrient, which is conducive to economical use of extra without additional nitrogen source, suggesting certain inducer and to saving cost. Fermentation without wheat components like crude protein of wheat bran could be bran or using E. coli BL21(DE3) containing raw plasmid utilized as nitrogen source (Fig.  6). These results sug - also produced tiny amounts of reducing sugar, which gested that wheat bran not only acted as substrate for probably resulted from reducing metabolites secreted by E. coli BL21(DE3) (Fig. 3). Liu et al. BMC Biotechnology (2022) 22:6 Page 8 of 11 Xylanase activity is another factor influencing XOS at industrial level. A test using lab-scale fermentation production. For example, type of nitrogen source actually tank is constructive research as well as the first step to affected XOS production mainly by changing xylanase promote it from laboratory to factory. activity (Fig.  4), and the increase of activity also contrib- uted to the production in the experiment of optimiz- Conclusions ing wheat bran concentration (Fig.  5). However, a huge This work demonstrates that E. coli is appropriate for raise in xylanase activity commonly leading to a limited producing XOS with a competitive concentration thereby increase in XOS concentration (Fig.  3 and Fig.  6a), sug- overcoming the current weakness of one-step fermenta- gesting that mere pursuit of high activity or large loading tion. The critical factor leading to the breakthrough in of xylanase could be less effective than expected in large- yield is efficient production of XOS by E. coli at high sub - scale production of XOS. Interestingly, the biomass of E. strate concentration. The optimal conditions, especially coli BL21(DE3) was very low when the optimal nitrogen pH, for fermentation are highly affected by enzymatic source or high wheat bran concentration was employed characteristics of the xylanase used. This work provides (Figs.  5a, 6a). It seems that ideal condition for fermenta- theoretical basis for overcoming yield and cost challenge, tion prejudices bacteria growth but stimulates the accu- and contributes to the wide adoption of XOS. mulation of heterologous proteins. The GH11 xylanase of B. agaradhaerens AC13, BadX, Methods was previously reported to hydrolyze pNPX [31]. How- Strains, plasmid, and substrates ever, rBaxyl11 of B. agaradhaerens C9 was actually an B. agaradhaerens C9 was isolated from saline-alkali soil, endo-β-1,4-xylanase, and was not able to act on pNPX and has been maintained in our laboratory since then according to our results. This difference could be attrib - [36]. E. coli DH5α was used for gene cloning and plas- uted to the change from lysine to glutamic acid of amino mid maintenance. E. coli BL21(DE3) was used for gene acid sequence (Fig. 1b). Xylobiose and xylotriose were the expression as well as fermentation. pET22b(+), which main products by rBaxyl11 acting on wheat bran. Such was previously used for extracellular production of low-DP XOS commonly present better prebiotic efficacy recombinases in many researches [37, 38], was employed [32, 33]. Moreover, alkaline environment is optimal for for constructing recombinant plasmid. fermentation by E. coli containing rBaxyl11, which could Arabinoxylan, glucuronoxylan and XOS with DP rang- prevent hemicellulose from autohydrolysis during heat ing from 2 to 6 were all purchased from Megazyme (Ire- sterilization thereby avoiding undesired saccharides [34]. land). Wheat bran was purchased from a flour mill in Therefore, rBa xyl11 is promising to one-step fermenta- Huainan city, China. Starch presenting in wheat bran was tion for XOS production with advantages in, for example, removed according to the reported method before fer- specificity and stability. The best fermentation tempera - mentation [39]. In brief, milled wheat bran was treated ture is 44.3 °C, which is affect by both of the optimal tem - with amylase and papain successively. These enzymes perature for catalysis by rBaxyl11 (60 °C) and for growth were then denatured by boiling for 25  min. After that, of E. coli (37  °C). Comparing with enzymatic hydrolysis wheat bran was washed three times to remove enzymes using purified xylanases at high temperature, one-step and starch. The de-starched wheat bran was finally dried fermentation employing recombinant E. coli adopts mild and screened through 80 meshes sieve for fermentation conditions, which contributes to save energy and cost. and hydrolysis. Xylan content of wheat bran increased Future research would be made with focus on two com- from 28.3 to 59.4% after de-starched treatment, which ponents. Firstly, the XOS concentration could be further was measured according to the method offered by improved. For example, many other xylanases, substrates, National Renewable Energy Laboratory [40]. pretreatments or mediums are alternative for fermenta- tion [35]. In particular, the plasmid is worth optimizing. Heterologous expression and purification pET22b(+) was used for constructing recombinant E. Sequence of Baxyl11 gene is accessible in raw data and coli in this study. Strictly speaking, it is not an ideal plas- it is predicted as xylanase basing on BLAST (https:// mid for secretory expression because only a small part, blast. ncbi. nlm. nih. gov/ Blast. cgi). The gene was cloned about 30% according to our measurement, of recombi- with forward primer containing BamHI restriction site nant proteins was transported into medium. Developing (5′ - C TA G G A T CC G C A A AT CG T C A C CG A C A A T T C more appropriate plasmids for production is very prom- CA-3′) and reverse primer containing XhoI restriction ising to improve the yield. Secondly, application prospect site (5′-CCG CTC GAG ATT GTT TTT GTC CAA AGT of such one-step fermentation needs to be evaluated at TAT -3′). Baxyl11 gene was ligated into pET22b(+) after larger scale. It is obvious that a good XOS yield derived digested by endonucleases, and then transferred into from triangular flasks is no guarantee of the same thing E. coli DH5α. The validated recombinant plasmid was Liu  et al. BMC Biotechnology (2022) 22:6 Page 9 of 11 finally transferred into E. coli BL21(DE3) for heterolo - column (250 by 4 mm) was used for sugar separation. 25 gous expression. μL of sample was eluted with 250  mM NaOH (1.0  mL/ Inducing Baxyl11 gene expression was carried out in min) at 30  °C and detected by ED 3000 pulsed ampero- LB-ampicillin medium containing 0.6  mM of IPTG at metric detector. 37 °C, 200 rpm for 6 h. Bacterial cells were then harvested by centrifugation, and were resuspended using Tris–HCl One‑step fermentation buffer (20  mM, pH8.0) for ultrasonication. After that, One-step fermentation was carried out as follow- soluble cell extract containing rBaxyl11 was collected ing method if not specifically indicated. Recombinant by centrifuging at 4 °C and was filtered through 0.45-μm E. coli BL21(DE3) was inoculated into LB-ampicillin filters. rBa xyl11 was purified by affinity chromatography medium and grown at 37  °C overnight. Then, appropri - as follows: 5 mL of soluble cell extract was loaded into a ate amounts of cells were collected by centrifugation and Ni–NTA column that was previously equilibrated with further diluted to an initial OD = 1.0 into fermentation binding buffer (20 mM Tri-HCl, 500 mM NaCl, pH 8.0). medium. After that, ampicillin and IPTG were respec- 12 mL of washing buffer (20 mM imidazole, 20 mM Tri- tively added to 50  μg/mL and 0.6  mM, and cells were HCl, 500  mM NaCl, pH 8.0) and 6  mL of elution buffer cultured at 37  °C and 200  rpm immediately. After 12  h, (250 mM imidazole, 20 mM Tri-HCl, 500 mM NaCl, pH supernatant of medium was diluted to measure extracel- 8.0) were then loaded to remove undesired proteins and lular xylanase activity and XOS concentration (equivalent to elute rBaxyl11, respectively. Saline ions in eluent were xylose) as the method introduced in “One-step fermenta- removed by dialysis and rBaxyl11 was finally freeze-dried tion” section. Medium before fermentation was used as for reserve. control group for measurement of reducing sugar. Each The purified rBa xyl11 was detected by sodium dodecyl liter of fermentation medium contains: 20 g wheat bran, sulfate polyacrylamide gel electrophoresis. In brief, 15 4.8 g Na HPO ·12H O, 2.65  g K H PO , 4  g (N H ) SO , 2 4 2 2 4 4 2 4 μL of rBaxyl11 solution was mixed with 5 μL of loading 0.3 g MgSO ·7H O, 0.01  g FeSO ·7H O, 0.01  g 4 2 4 2 buffer and then incubated in boiling water bath for 5 min. CaCl ·2H O and 1  mL trace element solution (0.3  g/L 2 2 After that, the protein solution was load into a poly- H BO , 0.2  g/L CoC l ·6H O, 30  mg/L Z nSO ·7H O, 3 3 2 2 4 2 acrylamide gel (12.5%) for electrophoresis. The gel was 30 mg/L MnCl ·4H O, 30 mg/L N aMoO ·2H O, 20 mg/L 2 2 4 2 dyed using coomassie blue solution to detect the band of NiCl ·6H O and 10  mg/L CuSO ·5H O). The pH value 2 2 4 2 protein. of fermentation medium was adjusted to 7.0 before sterilization. To investigate the optimal nitrogen source, Enzyme assay NH SO in initial medium was replaced with equal-mass 4 4 The freeze-dried rBa xyl11 was dissolved using deion- tryptone, casein acids hydrolysate, yeast extract, urea, ized water, and protein concentration was determined beef extract, NH NO, NaNO or glycine for fermenta- 4 3 3 according to the absorbancy at 280  nm and the extinc- tion and the medium without additional nitrogen source tion coefficient of rBa xyl11. To measure enzymatic was used as control. All experiments were performed in activity, 50 μL of diluted enzyme solution and 100 μL of triplicate. substrate solution were mixed and incubated at 60  °C, pH 8.0 for 20  min, and the reducing sugars were then Measurement of biomass measured with dinitrosalicylic acid (DNS) assay and Growth of E. coli was measured by dilute plate method. calibration curve using xylose as standard [41]. Kinetic 100 μL of medium after fermentation was sampled and 5 7 parameters were worked out using Lineweaver–Burk diluted 10 –10 times using NaCl solution (0.9%). 200 μL plot according to enzyme activities which were measured of diluted medium was spread onto a LB-ampicillin agar with xylan solution whose concentration ranged from 1 plate and cultured at 37  °C overnight. Colony forming to 20 mg/mL [42]. The optimal reaction conditions were unit (CFU) was finally counted to evaluate the biomass of investigated by determining enzymatic activities at dif- E. coli in medium after fermentation. ferent temperatures or pH values. To study stability, activities of rBaxyl11 were measured after incubated at Optimization of XOS production 70 °C (Na HPO -NaH PO buffer, pH 8.0) for different Response surface methodology was employed to opti- 2 4 2 4 time or incubated in buffers (Na HPO -NaH PO buffer mize XOS production by one-step fermentation using 2 4 2 4 and Na CO -NaHCO buffer) with different pH (5.0– Box-Behnken experimental design [20]. Nitrogen source 2 3 3 10.0) value for one hour. concentration, pH and fermentation temperature were XOS produced by rBaxyl11 from wheat bran were ana- selected for optimization. Experimental design was pro- lyzed using HPIC with a Dionex ICS3000 system. Ana- vided in Table 2, and the range of these factors was cho- lytical CarboPac PA10 pellicular anion-exchange resin sen basing on pretest. Liu et al. BMC Biotechnology (2022) 22:6 Page 10 of 11 2. Wu Y, Li SJ, Tao Y, Li DD, Han YB, Show PL, Wen GZ, Zhou JZ. 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One-step fermentation for producing xylo-oligosaccharides from wheat bran by recombinant Escherichia coli containing an alkaline xylanase

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Abstract

Background: One-step fermentation is a cheap way to produce xylo-oligosaccharides (XOS), where production of xylanases and XOS is integrated into a single process. In spite of cost advantage, one-step fermentation is still short in yield so far due to the limited exploration. To cope with this issue, production of XOS from wheat bran by recombi- nant Escherichia coli through one-step fermentation was investigated in this study. Results: An endo-β-1,4-xylanase gene belonging to glycoside hydrolase family 11 of Bacillus agaradhaerens was employed to construct recombinant E. coli. This xylanase showed maximal activity at 60 °C and pH 8.0–8.5. Its activity retained more than 60% after incubation at 70 °C for 4 h, showing a good stability. The recombinant E. coli BL21(DE3) could secreted xylanases that directly hydrolyzed de-starched wheat bran to XOS in fermentation medium. The XOS generated from hydrolysis consisted of xylose, xylobiose and xylotriose accounting for 23.1%, 37.3% and 39.6%, respectively. Wheat bran concentration was found to be the most crucial factor affecting XOS production. The XOS concentration reached 5.3 mg/mL at 10% loading of wheat bran, which is higher than those of previous researches. Nitrogen source type could also affect production of XOS by changing extracellular xylanase activity, and glycine was found to be the best one for fermentation. Optimal fermentation conditions were finally studied using response surface optimization. The maximal concentration emerged at 44.3 °C, pH 7.98, which is affected by characteristics of the xylanase as well as growth conditions of E. coli. Conclusions: This work indicates that the integrated fermentation using recombinant E. coli is highly competitive in cost and final concentration for producing XOS. Results can also provide theoretical basis for large-scale production and contribute to the wide adoption of XOS. Keywords: Prebiotics, Bacillus agaradhaerens, Single-step fermentation, Xylanase, Response surface optimization Background Prebiotics, namely some kinds of oligosaccharides, can specifically promote the activity of beneficial bacteria in gastrointestinal tract [1, 2]. With insight into the effect of gut microbiota on human overall health, prebiotics have *Correspondence: leonliu2013@126.com been the hotspot in consumption and research currently Jiawen Liu and Cong Liu contributed equally to this work [3]. Xylo-oligosaccharides (XOS) are emerging probiot- Jiangsu Key Laboratory of Phylogenomics and Comparative Genomics, School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, ics which consist of several β-1,4 linked xylose units [4]. Tongshan District, Xuzhou 221116, Jiangsu Province, China They have been attached importance to recent years due © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. BMC Biotechnology (2022) 22:6 Page 2 of 11 to remarkable prospect of application in food, medicine, fermentation so far. These microorganisms, however, can poultry and other fields [5–7]. Furthermore, XOS are all utilize XOS as carbon source, which prejudices the more efficient than other prebiotics in enhancing growth accumulation of XOS in medium. In addition, critical of certain bifidobacteria and in protecting lactobacilli restriction limiting XOS production remains unknown under stress environments [8–10]. XOS also present for such integrated fermentation, which leads to difficulty good heat and pH stability, which is beneficial to retain - in substantial improvement in yield. ing more nutritional properties in digestive tract [11]. Escherichia coli has been used to produce food addi- Because of these advantages, market demand for XOS is tives and drugs for decades, which has been proved to be rising quickly and expected to reach 130 million U.S. dol- safe and reliable [23, 24]. E. coli is probably suited to fer- lars in 2023 at an annual growth of 5.3% [12]. mentation for producing XOS because it cannot consume Enzymatic hydrolysis is one of the major methods to this kind of oligosaccharides [25]. However, feasibility of produce XOS, which is more environmentally friendly one-step fermentation employing E. coli lacks sufficient and generates less undesired by-products than chemi- study. Wheat bran is a xylan-rich by-product of white cal hydrolysis [13]. Xylanases are the critical factor for flour milling and has been used as cheap raw materials enzymatic production of XOS, which act on backbone for XOS production previously [26, 27]. Bacillus agarad- of xylan and convert it into XOS as well as xylose. Xyla- haerens C9 is an alkaliphilic strain with lignocellulose- nases belonging to glycoside hydrolase (GH) family 11 degrading ability. Secretion of alkali-tolerant xylanases attack unsubstituted sites of xylan, whose hydrolysate by B. agaradhaerens C9 was verified in our previous work mainly consists of xylobiose and xylotriose; GH10 xyla- [28]. Bioinformatics analysis of its genome revealed an nases can accommodate a decorated xylopyranosyl resi- GH11 xylanase that was named Baxyl11. In this study, due at − 1 subsite, resulting in production of both linear Baxyl11 was expressed using E. coli BL21(DE3) and enzy- and substituted XOS with low degree of polymerization matic characteristics of recombinant Baxyl11 (rBaxyl11) (DP); GH30 xylanases prefer branched xylan than the lin- were then investigated. Moreover, producing XOS from ear one so substituted XOS are their principal products wheat bran by the recombinant E. coli BL21(DE3) con- [14, 15]. Hydrolyzing extracted xylan or raw lignocellu- taining rBaxyl11 was carried out. Effects of wheat bran losic biomass using these xylanases to produce XOS has concentration, nitrogen source type and fermentation been widely reported, and XOS yields are very attractive conditions (pH and temperature) on XOS production in some works [16, 17]. However, preparation of these were finally investigated. These results would contribute purified enzymes is unwieldy and costly. In addition, high to overcoming yield and cost challenge in the production temperature is commonly needed for an efficient enzy - of XOS, and promote its wide adoption. matic hydrolysis, which also prejudices the cost of pro- duction process [18, 19]. To cope with these issues, some Results researches devoted to integrating production of xylanases Enzymatic characteristics of rBaxyl11 and XOS into a single process. In such process, microor- Baxyl11 gene was cloned from genomic DNA of B. aga- ganisms extracellularly secrete xylanases and meanwhile, radhaerens C9 and ligated with plasmid pET22b(+). these enzymes directly convert xylan or lignocellulosic rBaxyl11 was then expressed using E. coli BL21(DE3). biomass into XOS in medium. For example, a wild-type The purified rBa xyl11 showed electrophoretic homoge- Bacillus subtilis was reported to produce XOS by direct neity and its molecular weight corresponded to the cal- fermentation utilizing brewers’ spent grain, and XOS culated value of 28.9 kD (Fig.  1a). rBaxyl11 presented could yield further increase when B. subtilis was geneti- hydrolytic activity to glucuronoxylan and arabinoxylan cally modified [20]. Some fungi, such as Trichoderma but not to cellulose, mannan, starch and 4-nitrophe- reesei and Aspergillus nidulans, exhibited potential of nyl-beta-D-xylopyranoside (pNPX), which demonstrated producing XOS in one-step fermentation as well [21, 22]. that rBaxyl11 is an endoxylanase. These integrated production of XOS left out separate To evaluate its catalytic activities, kinetic parameters process for preparing xylanases, and generally adopted of rBaxyl11 against arabinoxylan and glucuronoxylan mild fermentation conditions, which contributes to over- were measured (Table  1). V and K against arabi- max cat coming cost challenge [12]. Nevertheless, XOS yields noxylan were approximately two times as high as those of one-step fermentation are commonly disadvantaged against glucuronoxylan, showing higher activity against comparing with those of enzymatic hydrolysis. Indeed, arabinoxylan. However, lower K against glucuron- yields can be improved by optimizing types of medium, oxylan indicated the preference for such polysaccharide substrates, fermentation microorganisms and conditions, than arabinoxylan. As a result, the K /K of rBaxyl11 cat m but researches about these issues are scarce. For example, against glucuronoxylan was higher than that against only a few bacillus and fungi are employed for one-step arabinoxylan. Liu  et al. BMC Biotechnology (2022) 22:6 Page 3 of 11 Fig. 1 Electrophoresis and sequence analysis of rBaxyl11. a Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of rBaxyl11. Line 1: soluble cell extract containing rBaxyl11; Line 2: rBaxyl11 after purification; 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 To investigate the optimal conditions for catalysis, Table 1 Kinetic parameters of rBaxyl11 for xylans activities of rBaxyl11 were measured at different tem - Substrate V (μΜ/s) K (/s) K (g/L) K /K (L/g/s) max cat m cat m peratures and pH values (Fig.  2a, b). rBaxyl11 showed Arabinoxylan 44.2 ± 3.7 599.0 ± 49.7 10.9 ± 0.9 55.0 ± 0.3 highest activity at 60 °C and its optimal pH ranged from Glucuronoxylan 24. 3 ± 0.6 330.1 ± 7.7 4.1 ± 0.1 79.7 ± 1.2 8.0 to 8.5, indicating it is an alkaline xylanase. Stabil- ity of rBaxyl11 was then studied (Fig.  2c, d). Activity of Concentration of rBaxyl11 was 220 nΜ for measurement. All data are presented as mean ± standard deviation (n = 3) Fig. 2 Eec ff t of temperature and pH on activity and stability of rBaxyl11. a Eec ff t of temperature on activity of rBaxyl11. b Eec ff t of pH on activity of rBaxyl11. c Eec ff t of temperature on stability of rBaxyl11. d Eec ff t of pH on stability of rBaxyl11. In (a) and (b), the maximal activity was designated as 100%. In (c) and (d), activity of enzyme without incubation was designated as 100%. Measurement at pH 5.0–8.0 and 8.0–10.5 was carried out in Na HPO -NaH PO buffer and Na CO -NaHCO buffer, respectively. All data are presented as means ± standard deviations (n = 3) 2 4 2 4 2 3 3 Liu et al. BMC Biotechnology (2022) 22:6 Page 4 of 11 rBaxyl11 retained more than 80% after incubation at 70  °C for 30  min, and even after 4  h, 60% of its activity could be maintained. Moreover, rBaxyl11 showed good stability when incubated at the pH ranging from 5.0 to 9.0, which is commonly the appropriate pH range for fermentation. One‑step fermentation for XOS production To save cost and simplify process, direct fermentation by rBaxyl11-transformed E. coli BL21(DE3) to produce XOS from wheat bran was carried out. Starch in wheat bran was removed in advance for a better XOS yield. Employ of the recombinant E. coli BL21(DE3) in the presence of isopropyl-1-thio-β-D-galactopyranoside (IPTG) and wheat bran for fermentation resulted in a reducing sugar yield of 1.41  mg/mL at the 24th hour (Fig.  3a). By con- trast, fermentation without wheat bran or using E. coli BL21(DE3) containing raw plasmid only produced negli- gible reducing sugars, demonstrating that rBaxyl11 from E. coli could produce XOS by acting on wheat bran in such one-step fermentation. In the presence of rBaxyl11 and wheat bran, xylanase activity increased rapidly in the first 6 h and slowly then (Fig.  3b). Activity in the medium without IPTG showed Fig. 3 The time course of a XOS concentration and b extracellular similar trend but at lower level. It is noteworthy that xylanase activity during one-step fermentation. “With IPTG”: employ use of IPTG raised xylanase activity by 40% while only of inducer (IPTG), wheat bran and recombinant E. coli containing increased XOS concentration by 18%, suggesting that rBaxyl11; “Without IPTG”: employ of wheat bran and recombinant xylanase activity is not the most important factor to yield E. coli containing rBaxyl11 without inducer; “Empty vector”: (see “Effect of nitrogen source type on one-step fermen - employ of inducer, wheat bran and recombinant E. coli containing unmodified pET22b(+); “Without wheat bran”: employ of inducer and tation” section for details). recombinant E. coli containing rBaxyl11 without wheat bran. Wheat bran concentration: 2%. All data are presented as means ± standard Product composition of rBaxyl11 acting on wheat bran deviations (n = 3) To study the product composition of rBaxyl11 acting on wheat bran, its hydrolysate was analyzed using high pres- sure ion chromatography (HPIC). Results demonstrated that xylose, xylobiose and xylotriose are the primary To study how wheat bran concentration affects pro - product (Fig. 4a). Further quantitative analysis basing on duction of XOS, xylanase activity and growth of E. chromatogram showed that xylose, xylobiose and xylo- coli BL21(DE3) were also measured. Xylanase activ- triose respectively accounted for 23.1%, 37.3% and 39.6% ity increased with wheat bran concentration while the (Fig. 4b). In other words, about 77% of its product is low- biomass of E. coli BL21(DE3) showed opposite trend, DP XOS when rBaxyl11 hydrolyzed wheat bran. indicating that high-concentration wheat bran stimu- lated the synthesis and secretion of rBaxyl11 and inhib- Eec ff t of wheat bran concentration on one‑step ited the growth of E. coli BL21(DE3) (Fig.  5). A high fermentation xylanase activity would contribute to XOS production, Effect of wheat bran concentration on XOS yield was but the huge increase of final concentration was not investigated here. As showed in Fig.  5a, XOS concentra- exclusively due to this reason. Specifically, XOS con - tion increased with wheat bran concentration in 0–10% centration increased nearly 16-fold when wheat bran of loading range, and further raise in substrate load- concentration raised tenfold from 1 to 10%, and mean- ing would lead to an excessive viscosity of medium. At while, xylanase activity only increased by 103%. It is 10% of wheat bran concentration, 6.9  mg/mL of reduc- obvious that augmentation of XOS yield resulted from ing sugar was obtained. That means the XOS concentra - combined effect of the increase in both wheat bran con - tion reached 5.3 mg/mL (excluding xylose), which is very centration and xylanase activity, where the former con- considerable. tributed more. In other words, substrate concentration Liu  et al. BMC Biotechnology (2022) 22:6 Page 5 of 11 Fig. 4 Product composition of rBaxyl11 acting on wheat bran analyzed by HPIC. a Product composition of the hydrolysis. “Standards”: mixture of xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose with respective concentration of 2 mg/mL; “Hydrolysate”: XOS produced by rBaxyl11 from wheat bran; “Control”: sample of hydrolysate without employ of rBaxyl11. b Quantitative analysis of xylose and XOS produced by rBaxyl11 Fig. 5 Eec ff t of wheat bran concentration on one-step fermentation. a Eec ff t of wheat bran concentration on XOS production, xylanase activity and biomass of recombinant E. coli. b Correlation between XOS concentration and xylanase activity as well as wheat bran concentration. Fermentation time: 12 h. Temperature: 37 °C. Initial pH: 7.0. In (a), data are presented as means ± standard deviations (n = 3) is the decisive factor to XOS production in such fer- assay, medium after dialysis was also employed for meas- mentation process instead of enzymatic activity. urement (Fig. 6b). Results demonstrated a positive linear correlation between XOS concentration and xylanase Eec ff t of nitrogen source type on one‑step fermentation activity regardless of whether medium was treated with Effect of nitrogen source type on one-step fermentation dialysis. By comparison, no credible correlation between was investigated here (Fig.  6a). Measurement of reduc- XOS concentration and biomass was observed. There - ing sugar indicated that maximal XOS concentration fore, types of nitrogen source affected XOS production was obtained when using glycine as nitrogen source, mainly by changing xylanase activity. which is slightly higher than that using yeast extract (p value = 0.065). The lowest three yields showed when Optimizing XOS production by response surface NH NO, NaNO and NH SO were employed, indicat- methodology 4 3 3 4 4 ing such inorganic salts are not suited to production of To study effect of fermentation conditions, tempera - XOS. ture, pH and glycine concentration were chosen as vari- The correlation analysis was then conducted to evaluate ables for optimization using Box-Behnken design. After the effect of nitrogen sources on XOS. To avoid the effect 12-h fermentation, concentrations of reducing sugars of difference in nitrogen sources on enzymatic activity varied in the range of 1.629–1.895 mg/mL (Table 2 and Liu et al. BMC Biotechnology (2022) 22:6 Page 6 of 11 Fig. 6 Eec ff t of nitrogen source type on one-step fermentation. a Eec ff t of nitrogen source type on XOS production, xylanase activity and biomass of recombinant E. coli. “Activity”: xylanase activity measured using supernatant of medium; “Activity after dialysis”: xylanase activity measured using dialysis-treated supernatant of medium. b Correlation between XOS concentration and xylanase activity as well as wheat bran concentration. Wheat bran concentration: 2%. Fermentation time: 12 h. Temperature: 37 °C. Initial pH: 7.0. In (a), data are presented as means ± standard deviations (n = 3) (60 °C) and higher than the best growth temperature of Table 2 Experimental design to study the effect of pH, fermentation temperature and glycine concentration on XOS E. coli (37 °C). production Discussion Run pH Temperature Glycine (%) Concentration (mg/mL) (°C) High cost is a challenge limiting the enzymatic produc- tion of XOS. One-step fermentation is a cost-efficient 1 8.1 40 0.2 1.629 ± 0.008 way to produce XOS, but its yield was commonly modest 2 8.1 44 2.6 1.895 ± 0.009 comparing with that of enzymatic hydrolysis (Table  3). 3 7.6 48 2.6 1.780 ± 0.009 For example, hydrolyzing mahogany employing a xyla- 4 7.6 44 0.2 1.673 ± 0.014 nase of Clostridium resulted in a XOS concentration of 5 8.1 48 5.0 1.721 ± 0.014 4.5  mg/mL [29]. The concentrations could even exceed 6 8.6 44 5.0 1.784 ± 0.039 8 mg/mL when using extracted xylan as substrate [16]. By 7 8.1 40 5.0 1.721 ± 0.013 comparison, only 0.8–1.1  mg/mL of XOS were obtained 8 8.1 44 2.6 1.895 ± 0.009 employing B. subtilis or Trichoderma species in one-step 9 8.1 48 0.2 1.709 ± 0.018 fermentation despite optimization [20, 21]. A higher con- 10 8.1 44 2.6 1.895 ± 0.009 centration of 3.2 mg/mL was obtained when using wheat 11 7.6 44 5.0 1.862 ± 0.044 middlings and B. subtilis, but the fermentation time, 48 h, 12 8.6 44 0.2 1.743 ± 0.003 was less competitive [30]. This study described a consid - 13 7.6 40 2.6 1.703 ± 0.011 erable XOS concentration of 5.3  mg/mL with only 12-h 14 8.1 44 2.6 1.895 ± 0.009 fermentation, which is much higher than those of previ- 15 8.6 48 2.6 1.714 ± 0.008 ous works. Moreover, substrate concentration was found 16 8.1 44 2.6 1.895 ± 0.009 to be the most influential factor to XOS production here. 17 8.6 40 2.6 1.685 ± 0.055 This is probably the cause of modest XOS yields in previ - All data are presented as mean ± standard deviation (n = 3) ous reports because XOS yield were prejudiced by a large loading of substrates using B. subtilis and fungi [20, 21]. Therefore, employing E. coli BL21(DE3) is promising to eliminate the disadvantage in XOS production by one- Fig.  7). Glycine concentration is the most influential step fermentation. variable with p-value = 0.0006, followed by temperature Considerable reducing sugar (1.19 mg/mL at the 24th with p value = 0.0132. It was predicted that the opti- hour) were produced even without IPTG, which could mal concentration of 1.904  mg/mL would be obtained be attributed to induction of certain saccharides from at 44.3  °C, pH 7.98 with 3.36% of glycine, which cor- wheat bran. Further study indicated that XOS concen- responds approximately to the central-point condition tration induced by wheat bran alone reached 78% of the of the design. The optimal pH for fermentation corre - maximum with 1 mM of IPTG, and adding only a small sponded to that of catalysis by rBaxyl11 (Fig. 2b), while amount of IPTG (0.02  mM) could lead to the maximal the optimal temperature is lower than that for catalysis Liu  et al. BMC Biotechnology (2022) 22:6 Page 7 of 11 Fig. 7 Response surface showing effect of temperature, pH and glycine concentration on XOS production. a Eec ff t of temperature and pH on XOS production. b Eec ff t of glycine concentration and pH on XOS production. c Eec ff t of glycine concentration and temperature on XOS production. Wheat bran concentration: 2%. Fermentation time: 12 h. All experiments were conducted in triplicate Table 3 XOS production by enzymatic hydrolysis and one-step fermentation Substrate Enzyme or strain Reaction XOS yields XOS production method References time (h) c d mg/mLmg/g substrate e e Wheat bran Engineering E. coli BL21(DE3) 12 0.8–5.3 53-80 One-step fermentation (37 °C) This study e e Wheat middlings Bacillus subtilis 48 3.2 64 One-step fermentation (37 °C) [30] Brewers’ spent grain Engineering Bacillus subtilis 12 1.1 34 One-step fermentation (45 °C) [20] Brewers’ spent grain Trichoderma reesei 72 0.8 40 One-step fermentation (30 °C) [21] Rice husk Engineering Aspergillus nidulans 48 – 24 One-step fermentation (37 °C) [22] Pistachio shell Commercial xylanase 10 2.7 – Enzymatic hydrolysis (45 °C) [43] e e Mahogany Xylanase from Clostridium strain 24 4.5 90 Enzymatic hydrolysis (50 °C) [29] BOH3 Sugarcane bagasse Xylanase from Bacillus subtilis 15 3.6 119 Enzymatic hydrolysis (50 °C) [44] e e Rice straw Commercial xylanase 24 0.1 2 Enzymatic hydrolysis (50 °C) [45] e e Rice straw Xylosidase from Weissella cibaria 10 2.6 70 Enzymatic hydrolysis (37 °C) [46] e e Beechwood xylan Xylanase from Mycothermus thermo- 12 8.0–8.8 800–880 Enzymatic hydrolysis (65 °C) [16] philus Xylan from corn cobs Xylanase from Thermomyces lanugi- 8 6.9 345 Enzymatic hydrolysis (45 °C) [47] nosus e e Xylan from data seed Xylanase from Aspergillus niger 4 4.1 411 Enzymatic hydrolysis (38 °C) [48] Xylan from vetiver grass Xylanase from Aureobasidium melano- 92 4.7 194 Enzymatic hydrolysis (28 °C) [49] genum Reaction time indicates the hydrolysis or fermentation time when XOS concentration reaches the presented value Xylose is not included Yields are presented as final concentration (mg/mL) of XOS in fermentation medium Yields are presented as mass (mg) of XOS obtained from a gram of substrate These data are measured using liquid chromatogram and others are measured using DNS method XOS concentration as well as xylanase activity in the XOS production, but also played an important role in fermentation medium (Additional file  1: Fig. S1). Also, stimulating the secretion of rBaxyl11 and served as E. coli BL21(DE3) could still grow and secrete xylanases nutrient, which is conducive to economical use of extra without additional nitrogen source, suggesting certain inducer and to saving cost. Fermentation without wheat components like crude protein of wheat bran could be bran or using E. coli BL21(DE3) containing raw plasmid utilized as nitrogen source (Fig.  6). These results sug - also produced tiny amounts of reducing sugar, which gested that wheat bran not only acted as substrate for probably resulted from reducing metabolites secreted by E. coli BL21(DE3) (Fig. 3). Liu et al. BMC Biotechnology (2022) 22:6 Page 8 of 11 Xylanase activity is another factor influencing XOS at industrial level. A test using lab-scale fermentation production. For example, type of nitrogen source actually tank is constructive research as well as the first step to affected XOS production mainly by changing xylanase promote it from laboratory to factory. activity (Fig.  4), and the increase of activity also contrib- uted to the production in the experiment of optimiz- Conclusions ing wheat bran concentration (Fig.  5). However, a huge This work demonstrates that E. coli is appropriate for raise in xylanase activity commonly leading to a limited producing XOS with a competitive concentration thereby increase in XOS concentration (Fig.  3 and Fig.  6a), sug- overcoming the current weakness of one-step fermenta- gesting that mere pursuit of high activity or large loading tion. The critical factor leading to the breakthrough in of xylanase could be less effective than expected in large- yield is efficient production of XOS by E. coli at high sub - scale production of XOS. Interestingly, the biomass of E. strate concentration. The optimal conditions, especially coli BL21(DE3) was very low when the optimal nitrogen pH, for fermentation are highly affected by enzymatic source or high wheat bran concentration was employed characteristics of the xylanase used. This work provides (Figs.  5a, 6a). It seems that ideal condition for fermenta- theoretical basis for overcoming yield and cost challenge, tion prejudices bacteria growth but stimulates the accu- and contributes to the wide adoption of XOS. mulation of heterologous proteins. The GH11 xylanase of B. agaradhaerens AC13, BadX, Methods was previously reported to hydrolyze pNPX [31]. How- Strains, plasmid, and substrates ever, rBaxyl11 of B. agaradhaerens C9 was actually an B. agaradhaerens C9 was isolated from saline-alkali soil, endo-β-1,4-xylanase, and was not able to act on pNPX and has been maintained in our laboratory since then according to our results. This difference could be attrib - [36]. E. coli DH5α was used for gene cloning and plas- uted to the change from lysine to glutamic acid of amino mid maintenance. E. coli BL21(DE3) was used for gene acid sequence (Fig. 1b). Xylobiose and xylotriose were the expression as well as fermentation. pET22b(+), which main products by rBaxyl11 acting on wheat bran. Such was previously used for extracellular production of low-DP XOS commonly present better prebiotic efficacy recombinases in many researches [37, 38], was employed [32, 33]. Moreover, alkaline environment is optimal for for constructing recombinant plasmid. fermentation by E. coli containing rBaxyl11, which could Arabinoxylan, glucuronoxylan and XOS with DP rang- prevent hemicellulose from autohydrolysis during heat ing from 2 to 6 were all purchased from Megazyme (Ire- sterilization thereby avoiding undesired saccharides [34]. land). Wheat bran was purchased from a flour mill in Therefore, rBa xyl11 is promising to one-step fermenta- Huainan city, China. Starch presenting in wheat bran was tion for XOS production with advantages in, for example, removed according to the reported method before fer- specificity and stability. The best fermentation tempera - mentation [39]. In brief, milled wheat bran was treated ture is 44.3 °C, which is affect by both of the optimal tem - with amylase and papain successively. These enzymes perature for catalysis by rBaxyl11 (60 °C) and for growth were then denatured by boiling for 25  min. After that, of E. coli (37  °C). Comparing with enzymatic hydrolysis wheat bran was washed three times to remove enzymes using purified xylanases at high temperature, one-step and starch. The de-starched wheat bran was finally dried fermentation employing recombinant E. coli adopts mild and screened through 80 meshes sieve for fermentation conditions, which contributes to save energy and cost. and hydrolysis. Xylan content of wheat bran increased Future research would be made with focus on two com- from 28.3 to 59.4% after de-starched treatment, which ponents. Firstly, the XOS concentration could be further was measured according to the method offered by improved. For example, many other xylanases, substrates, National Renewable Energy Laboratory [40]. pretreatments or mediums are alternative for fermenta- tion [35]. In particular, the plasmid is worth optimizing. Heterologous expression and purification pET22b(+) was used for constructing recombinant E. Sequence of Baxyl11 gene is accessible in raw data and coli in this study. Strictly speaking, it is not an ideal plas- it is predicted as xylanase basing on BLAST (https:// mid for secretory expression because only a small part, blast. ncbi. nlm. nih. gov/ Blast. cgi). The gene was cloned about 30% according to our measurement, of recombi- with forward primer containing BamHI restriction site nant proteins was transported into medium. Developing (5′ - C TA G G A T CC G C A A AT CG T C A C CG A C A A T T C more appropriate plasmids for production is very prom- CA-3′) and reverse primer containing XhoI restriction ising to improve the yield. Secondly, application prospect site (5′-CCG CTC GAG ATT GTT TTT GTC CAA AGT of such one-step fermentation needs to be evaluated at TAT -3′). Baxyl11 gene was ligated into pET22b(+) after larger scale. It is obvious that a good XOS yield derived digested by endonucleases, and then transferred into from triangular flasks is no guarantee of the same thing E. coli DH5α. The validated recombinant plasmid was Liu  et al. BMC Biotechnology (2022) 22:6 Page 9 of 11 finally transferred into E. coli BL21(DE3) for heterolo - column (250 by 4 mm) was used for sugar separation. 25 gous expression. μL of sample was eluted with 250  mM NaOH (1.0  mL/ Inducing Baxyl11 gene expression was carried out in min) at 30  °C and detected by ED 3000 pulsed ampero- LB-ampicillin medium containing 0.6  mM of IPTG at metric detector. 37 °C, 200 rpm for 6 h. Bacterial cells were then harvested by centrifugation, and were resuspended using Tris–HCl One‑step fermentation buffer (20  mM, pH8.0) for ultrasonication. After that, One-step fermentation was carried out as follow- soluble cell extract containing rBaxyl11 was collected ing method if not specifically indicated. Recombinant by centrifuging at 4 °C and was filtered through 0.45-μm E. coli BL21(DE3) was inoculated into LB-ampicillin filters. rBa xyl11 was purified by affinity chromatography medium and grown at 37  °C overnight. Then, appropri - as follows: 5 mL of soluble cell extract was loaded into a ate amounts of cells were collected by centrifugation and Ni–NTA column that was previously equilibrated with further diluted to an initial OD = 1.0 into fermentation binding buffer (20 mM Tri-HCl, 500 mM NaCl, pH 8.0). medium. After that, ampicillin and IPTG were respec- 12 mL of washing buffer (20 mM imidazole, 20 mM Tri- tively added to 50  μg/mL and 0.6  mM, and cells were HCl, 500  mM NaCl, pH 8.0) and 6  mL of elution buffer cultured at 37  °C and 200  rpm immediately. After 12  h, (250 mM imidazole, 20 mM Tri-HCl, 500 mM NaCl, pH supernatant of medium was diluted to measure extracel- 8.0) were then loaded to remove undesired proteins and lular xylanase activity and XOS concentration (equivalent to elute rBaxyl11, respectively. Saline ions in eluent were xylose) as the method introduced in “One-step fermenta- removed by dialysis and rBaxyl11 was finally freeze-dried tion” section. Medium before fermentation was used as for reserve. control group for measurement of reducing sugar. Each The purified rBa xyl11 was detected by sodium dodecyl liter of fermentation medium contains: 20 g wheat bran, sulfate polyacrylamide gel electrophoresis. In brief, 15 4.8 g Na HPO ·12H O, 2.65  g K H PO , 4  g (N H ) SO , 2 4 2 2 4 4 2 4 μL of rBaxyl11 solution was mixed with 5 μL of loading 0.3 g MgSO ·7H O, 0.01  g FeSO ·7H O, 0.01  g 4 2 4 2 buffer and then incubated in boiling water bath for 5 min. CaCl ·2H O and 1  mL trace element solution (0.3  g/L 2 2 After that, the protein solution was load into a poly- H BO , 0.2  g/L CoC l ·6H O, 30  mg/L Z nSO ·7H O, 3 3 2 2 4 2 acrylamide gel (12.5%) for electrophoresis. The gel was 30 mg/L MnCl ·4H O, 30 mg/L N aMoO ·2H O, 20 mg/L 2 2 4 2 dyed using coomassie blue solution to detect the band of NiCl ·6H O and 10  mg/L CuSO ·5H O). The pH value 2 2 4 2 protein. of fermentation medium was adjusted to 7.0 before sterilization. To investigate the optimal nitrogen source, Enzyme assay NH SO in initial medium was replaced with equal-mass 4 4 The freeze-dried rBa xyl11 was dissolved using deion- tryptone, casein acids hydrolysate, yeast extract, urea, ized water, and protein concentration was determined beef extract, NH NO, NaNO or glycine for fermenta- 4 3 3 according to the absorbancy at 280  nm and the extinc- tion and the medium without additional nitrogen source tion coefficient of rBa xyl11. To measure enzymatic was used as control. All experiments were performed in activity, 50 μL of diluted enzyme solution and 100 μL of triplicate. substrate solution were mixed and incubated at 60  °C, pH 8.0 for 20  min, and the reducing sugars were then Measurement of biomass measured with dinitrosalicylic acid (DNS) assay and Growth of E. coli was measured by dilute plate method. calibration curve using xylose as standard [41]. Kinetic 100 μL of medium after fermentation was sampled and 5 7 parameters were worked out using Lineweaver–Burk diluted 10 –10 times using NaCl solution (0.9%). 200 μL plot according to enzyme activities which were measured of diluted medium was spread onto a LB-ampicillin agar with xylan solution whose concentration ranged from 1 plate and cultured at 37  °C overnight. Colony forming to 20 mg/mL [42]. The optimal reaction conditions were unit (CFU) was finally counted to evaluate the biomass of investigated by determining enzymatic activities at dif- E. coli in medium after fermentation. ferent temperatures or pH values. To study stability, activities of rBaxyl11 were measured after incubated at Optimization of XOS production 70 °C (Na HPO -NaH PO buffer, pH 8.0) for different Response surface methodology was employed to opti- 2 4 2 4 time or incubated in buffers (Na HPO -NaH PO buffer mize XOS production by one-step fermentation using 2 4 2 4 and Na CO -NaHCO buffer) with different pH (5.0– Box-Behnken experimental design [20]. Nitrogen source 2 3 3 10.0) value for one hour. concentration, pH and fermentation temperature were XOS produced by rBaxyl11 from wheat bran were ana- selected for optimization. Experimental design was pro- lyzed using HPIC with a Dionex ICS3000 system. Ana- vided in Table 2, and the range of these factors was cho- lytical CarboPac PA10 pellicular anion-exchange resin sen basing on pretest. Liu et al. BMC Biotechnology (2022) 22:6 Page 10 of 11 2. Wu Y, Li SJ, Tao Y, Li DD, Han YB, Show PL, Wen GZ, Zhou JZ. 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Journal

BMC Biotechnology – Springer Journals

Published: Feb 5, 2022

Keywords: Prebiotics; Bacillus agaradhaerens; Single-step fermentation; Xylanase; Response surface optimization

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