Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904

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Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904 Qin Li a,c , Baoguo Sun a,b , Xiuting Li a,c,∗ , Ke Xiong c , Youqiang Xu a,c , Ran Yang c , Jie Hou c , Chao Teng b a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, China Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU), Beijing 100048, China c School of Food and Chemical Engineering, Beijing Technology and Business University, No.33, Fucheng Road, Beijing 100048, China b

a r t i c l e

i n f o

Article history: Received 27 August 2017 Received in revised form 30 September 2017 Accepted 3 October 2017 Available online xxx Keywords: GH10 xylanase Salt-tolerant Xylobiose Catalytic characteristics

a b s t r a c t A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37 ◦ C for 1 h in 3.0 M and 5.0 M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties. © 2017 Published by Elsevier B.V.

1. Introduction Endo-1,4-␤-d-xylanase (EC 3.2.1.8), isolated from various microorganisms, is an important member of the xylanase class of enzymes that catalyzes the hydrolysis of the ␤-1,4-xylosidic linkages of xylan. Most xylanases are classified into glycoside hydrolase (GH; http://www.cazy.org) families 10 and 11, while others are categorized as families 5, 7, 8, 16, 26, 43, 52, and 62, based on the amino acid sequence similarities of their catalytic domains [1–3]. GH10 xylanases typically have high molecular mass and display an (␣/␤)8 -barrel fold, which has been likened to a “salad bowl” [4–6], when compared to a “true xylanase”, GH11. However, they exhibit higher catalytic versatility than GH11 xylanases. Additionally, GH11 xylanase products can be further hydrolyzed by GH10 [7]. In recent years, xylanases have been widely applied in the feed, pulp, paper, food, drinks, and xylooligosaccharide industries [8–10]. In order to meet the increasing demands of these industries, xylanases, including various GH10 xylanases, have been extensively studied. For example, the hyperthermostable GH10 xylanase, Tlxyn10A, from Talaromyces leycettanus JCM12802

∗ Corresponding author at: School of Food and Chemical Engineering, Beijing Technology and Business University, No.33, Fucheng Road, Beijing 100048, China. E-mail addresses: [email protected], li [email protected] (X. Li).

retains 81.7%, 74.8%, and 58.2% initial activity after incubation for 1 h at 85 ◦ C, 90 ◦ C, and 95 ◦ C, respectively [11]; the thermostable GH10 xylanase, XynE15, from Microcella alkaliphila is stable during incubation at 75 ◦ C for 30 min, while exhibiting a half-life of 48 h at 50 ◦ C [12]; a thermo-alkali-stable xylanase from Bacillus halodurans TSEV1 is optimally active at 80 ◦ C with T1/2 of 35 min [13]; the alkalophilic xylanase, XynT, from Bacillus alcalophilus exhibits a high binding affinity and optimal hydrolytic activity toward the insoluble xylan between pH 7–9 [14]; an alkalinetolerant and thermostable xylanase from Streptomyces chartreusis L1105 retains more than 80% activity after a 30 min incubation at 50 ◦ C between pH 6–10 [15]. Moreover, xylanases from halophilic/halotolerant microorganisms have also been investigated as potential resources in applications such as wastewater treatment, degradation of marine products, and production of bioethanol from seaweeds, owing to their notable salt-tolerant characteristics [16–19]. In our previous work, a xylanase gene (Srxyn10) from Streptomyce rochei L10904 [20] was cloned and expressed in Escherichia coli BL21(DE3). The recombinant enzyme exhibited effective salt-tolerant properties and mainly produced xylobiose. Protein engineering was conducted on Srxyn10 by removing the C-terminal carbohydrate-binding modules (CBM) in order to improve its catalytic characteristics.

https://doi.org/10.1016/j.ijbiomac.2017.10.013 0141-8130/© 2017 Published by Elsevier B.V.

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Fig. 1. Formation and alignment sequences of Srxyn10 and Srxyn10M. (A) The sequence of 1XYF (PDB code) was obtained from Protein Data Bank and signal peptide was deleted. The predicted signal peptide and linker were boxed in blank. (B) Schematic overview of Srxyn10 and Srxyn10M. Srxyn10: recombinant xylanase from Streptomyce rochei L10904; Srxyn10M: deletion of the C-terminal carbohydrate-binding module (CBM).

2. Materials and methods 2.1. Bacterial strains, vectors, and substrates The genome of Streptomyce rochei L10904 [20] was used as a template for gene cloning. E. coli DH5␣ was used for propagation and manipulation of plasmids, and E. coli BL21(DE3) was used for protein expression. Plasmids pMD18-T and pET-28a(+) were used for gene cloning and expression in E. coli, respectively. Taq polymerases were obtained from Takara (Japan). Restriction ® endonucleases, T4 DNA ligase, and Q5 High-Fidelity DNA polymerase were obtained from NEB Inc. (USA). Bovine serum albumin was obtained from Roche (738328). Beechwood xylan was purchased from Megazyme (9014-63-5); birchwood xylan (X0502) and

oat-spelt xylan (X0627) were obtained from Sigma. Xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), xylohexaose (X6), and wheat arabinoxylans, including those of low viscosity (∼10 cSt), medium viscosity (∼31 cSt), and high viscosity (∼42 cSt) were obtained from Megazyme (6860-47-5, 47592-59-6, 2241658-6, 49694-20-4, 9040-27-1). Water-soluble and water-insoluble corncob xylans were obtained from the alkali extraction of corncobs as described by Li et al. [21]. 2.2. Gene cloning and expression of xylanase Streptomyce rochei L10904 was grown in Luria-Bertani (LB) medium. Genomic DNA was isolated the cells and subjected to polymerase chain reaction (PCR) amplification with degenerate primers

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Fig. 2. SDS-PAGE analysis of the purified Srxyn10 and Srxyn10M. Lane 1: the molecular mass standards; Lane 2: Srxyn10; Lane 3: purified Srxyn10 (48.1 kDa); Lane 4: Srxyn10M; Lane 5: purified Srxyn10M (34.7 kDa).

Strex10F and Strex10R. The full-length xylanase gene (Srxyn10) was amplified using a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) technique [22]. Srxyn10, including the predicted secretion signal, was cloned from the chromosomal DNA by PCR with the primers, Sig.L10904 × 10-NcoF and L10904 × 10-XhoR (Table S1 in Supplementary data). The amplified DNA fragment was subcloned into pMD18-T and transformed into E. coli DH5␣ cells. The gene was subcloned into pET-28a(+) at the NcoI and XhoI restriction sites and transformed into E. coli BL21(DE3) for expression. The expressed recombinant xylanase was denoted as Srxyn10. 2.3. Engineering and purification of xylanases The mutant Srxyn10M was created by removing the C-terminal carbohydrate-binding module (CBM) of Srxyn10. For primer details and details regarding the formation of Srxyn10 and Srxyn10M, see Table S1 in Supplementary data and Fig. 1B, respectively. Srxyn10M was cloned and expressed in E. coli BL21(DE3). The cells bearing the recombinant plasmids were cultured and expressed in LB medium at 37 ◦ C by shaking at 200 rpm. Isopropyl ␤-d-1thiogalactopyranoside (IPTG; 1 mM) was used to induce xylanase expression. The enzymes were collected via the following steps: centrifugation at 5000 rpm for 5 min and 4 ◦ C, resuspension of cells with 0.05 M Tris-HCl buffer (pH 7.0), cell lysis with ultrasonication, centrifugation at 10,000 rpm for 10 min at 4 ◦ C, and separation of the supernatant containing the enzymes. Srxyn10 and Srxyn10M were purified using Ni Sepharose HP column (1 × 10 cm) with 50 mM phosphate buffer (pH 7.8) containing 300 mM NaCl and different concentrations of imidazole, on an ÄKTA fast protein liquid chromatography (FPLC) purification system (GE Healthcare, Uppsala, Sweden). The purified enzymes were confirmed as homogeneous after examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were determined by Coomassie brilliant blue dyeing method using bovine serum albumin (BSA) as the standard. 2.4. Xylanase activity assay Xylanase activity was measured according to the method elucidated by Bailey et al. [24]. The reaction mixture contained 0.9 mL of 1% (w/v) beechwood xylan and 0.1 mL of a suitable diluted enzyme

solution (50 mM acetate buffer, pH 5.5) at 55 ◦ C for 5 min. The amount of reducing sugar liberated was determined by the 3,5dinitrosalicylic acid (DNS) method, using xylose (X1) as a standard [25]. One unit (1 U) of xylanase activity was defined as the amount of enzyme that catalyzed the release equivalent to 1 ␮moL of X1 in 1 min under the assay condition. All experiments were performed in triplicate. 2.5. Enzyme characterization of xylanases The effect of pH on xylanase activity was investigated at 55 ◦ C using buffers (50 mM) with pH values ranging from 3.5–10.5: citrate buffer (pH 3.5–6.5); phosphate buffer (pH 6.0–8.0); and Tris-HCl buffer (pH 8.0–8.5); barbital sodium buffer (pH 8.5–9.5); and Gly-NaOH buffer (pH 9.5–10.5). The optimum temperature for xylanase activity was determined by incubating the enzyme at different temperatures between 40 and 75 ◦ C in 50 mM Tris-HCl buffer (pH 8.0). To determine the pH stability of the enzyme, xylanase was incubated in buffers with different pH: citrate buffer (pH 4.0 and 5.0); Tris-HCl buffer (pH 8.0); Gly-NaOH buffer (pH 10); and NaH2 PO4 -NaOH buffer (pH 11 and 12) at 37 ◦ C for 1 h. The residual xylanase activities were measured at 60 ◦ C in 50 mM Tris-HCl buffer (pH 8.0). To determine the temperature stability of the enzyme, xylanase was incubated at 50 ◦ C, 60 ◦ C, and 70 ◦ C for 1 h. After the treated enzyme samples were cooled on ice for 30 min, the residual xylanase activities were measured at 60 ◦ C in 50 mM Tris-HCl buffer (pH 8.0). All experiments were performed in triplicate. To determine the salt-tolerance of the enzyme, xylanase was incubated at 37 ◦ C for 1 h with different NaCl concentrations (0–5.0 M). To determine its resistibility to the external environment, xylanase was incubated at 37 ◦ C and 50 ◦ C in 50 mM Gly-NaOH buffer (pH 10.0) and 5.0 M NaCl for 2–24 h. The residual xylanase activities were measured at 60 ◦ C in 50 mM Tris-HCl buffer (pH 8.0). All experiments were performed in triplicate. 2.6. Substrate-specific activity and kinetic parameters To determine the substrate-specificity of the enzyme, purified xylanase was incubated with 1% (w/v) of each substrate in 50 mM Tris-HCl buffer (pH 8.0) at 60 ◦ C for 10 min. The amount of reducing sugars produced was estimated using the dinitrosalicylic acid method as described above. For the kinetic experiments, six dif-

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Fig. 3. (A) Optimal pH and (B) pH stability of Srxyn10 and Srxyn10M. For pH stability, all proteins were incubated at 37 ◦ C for 1 h before enzymatic assay. The samples measured without incubation were defined as 100%.

ferent concentrations of each substrate were dissolved in 50 mM Tris-HCl buffer (pH 8.0), and incubated with purified xylanase at 60 ◦ C for 5 min. The Km and kcat values were calculated from the kinetic data using the GraphPad Prism software. 2.7. Hydrolysis characteristics of Srxyn10 and Srxyn10M To characterize the hydrolysis of xylanases, X6, beechwood xylan, birchwood xylan, oat-spelt xylan, and water-soluble and water-insoluble corncob xylans were used as substrates. X6: Reaction mixtures (500 ␮L), containing 0.5 mg mL−1 of substrate in 50 mM Tris-HCl buffer (pH 8.0), were incubated with 1 U of xylanase at 50 ◦ C for 12 h. Beechwood xylan, birchwood xylan and oat-spelt xylan: Reaction mixtures (1 mL), containing 10 mg mL−1 of substrate in 50 mM Tris-HCl buffer (pH 8.0), were incubated with 10 U of xylanase at 50 ◦ C for 12 h. Water-soluble and water-insoluble corncob xylans: Reaction mixtures (2 mL), containing 50 mg mL−1 of substrate in 50 mM Tris-HCl buffer (pH 8.0), were incubated with 100 U of xylanase at 50 ◦ C for 12 h. The reaction mixtures were treated with boiling water for 5 min, filtered with a 0.22 ␮m membrane filter, and injected (20 ␮L) into a COSMOSIL sugar-D packed column (4.6 ID × 500 mm, 05397-51) with a differential refractive index detector (RID). Isocratic elution (1.0 mL min−1 ) of chromatographically pure acetonitrile and ultrapure water (v/v, 70:30) was performed for 20 min, where the temperature of the chromatography column and RID was 40 ◦ C. X1, X2, X3, X4, and X5 were used as standards. All experiments were performed in triplicate. The hydrolysis products were observed using high-performance liquid

chromatography (HPLC; as described above) and thin-layer chromatography (TLC). For TLC analysis, silica gel plates (Merck) were developed with butanol, acetic acid, and water (2:1:1, v/v/v), followed by heating for a few minutes at 105 ◦ C. The plates were then sprayed with a methanol and sulfuric acid mixture (20:1, v/v). X1, X2, X3, X4, X5, and X6 were used as standards.

3. Results and discussion 3.1. Production of Srxyn10 and Srxyn10M For the Srxyn10 sequence, the first 41 amino acid residues were predicted to be a putative signal peptide. Srxyn10 is 82.41% identical to xylanase from Streptomyces olivaceoviridis E-86 (PDB code: 1XYF) [23]; an obvious difference between them was the linker (Fig. 1A). Srxyn10M was constructed based on the structure resolved from 1XYF. Analysis of the purified Srxyn10 and Srxyn10M by SDS-PAGE is shown in Fig. 2. When the Sxyn10 gene, excluding the predicted signal peptide, was cloned into pET28a(+) and transformed into E. coli BL21(DE3) for expression, the xylanase did not exhibit any activity (data not shown). Thus, the gene including the predicted signal peptide was used for further expression and deletion of the CBM. Each enzyme migrated as a single band. The molecular mass of Srxyn10 and Srxyn10M was approximately 48.1 kDa and 34.7 kDa, respectively. Analysis of the purified Srxyn10 and Srxyn10M by SDS-PAGE clearly indicated the decreases in molecular weight when compared to Srxyn10.

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xylanase from Thermotoga maritima, observed that the temperature for optimal xylanase activity and stability was strongly influenced by the presence of different modules, while also demonstrating the existence of inter- and/or intra-domain interactions [26]. Thermostability is not the result of a single domain alone, but a cooperative property of the entire molecule and is affected by the combination of numerous contributing factors including interaction between different domains [27]. 3.4. Salt-tolerance of Srxyn10 and Srxyn10M

Fig. 4. (A) Optimal temperature and (B) thermal stability of Srxyn10 and Srxyn10M. For thermal stability, all enzymes were incubated at pH 8.0 for 60 min before activity assay. The samples measured without incubation were defined as 100%.

3.2. Optimal pH and pH stability Both, Srxyn10 and Srxyn10M, showed the highest xylanase activity at pH 8.0, favoring a weak alkaline condition (Fig. 3A). For the pH stability experiments, Srxyn10 and Srxyn10M exhibited more than 90% activity at pH 8.0 and 10.0 compared to the initial activities. At pH 5.0, Srxyn10 possessed 80.12% activity, which was better than Srxyn10M (53.63%). At pH 11.0, Srxyn10 and Srxyn10M showed 58.21% and 45.27% original activities, respectively. At pH 4.0 and 12.0, Srxyn10 only exhibited 5.95% and 9.52% activity rates, respectively. Srxyn10M displayed a 21.61% activity at pH 12.0, which was more than that at pH 4.0 (7.26%; Fig. 3B). There were no differences in the optimal pH between Srxyn10 and Srxyn10M. However, some difference was observed between the pH stability of Srxyn10 and Srxyn10M. 3.3. Optimal temperature and thermal stability The optimal temperature of Srxyn10 and Srxyn10M was observed to be 60 ◦ C (Fig. 4A). With respect to thermal stability, both Srxyn10 and Srxyn10M exhibited more than 98% activity after 1 h incubation at 50 ◦ C. When incubated at 60 ◦ C, Srxyn10M maintained approximately 76.16% activity rate, which was better than that of Srxyn10 (28.63%). When incubated at 70 ◦ C, Srxyn10 and Srxyn10M displayed about 16.84% and 29.65% activity, respectively (Fig. 4B). Srxyn10M displayed more thermal stability than Srxyn10 at 60 ◦ C and 70 ◦ C. When the CBM of Srxyn10 was removed, the mutant Srxyn10M showed better thermal stability than Srxyn10. Verjans et al., by constructing truncated derivatives of a multidomain thermophilic

Incubating Srxyn10 and Srxyn10M at 37 ◦ C for 1 h with 3.0 M and 5.0 M NaCl, resulted in more than 95% and 91% activity rates, respectively (Fig. 5A). In order to explore the applied potential of Srxyn10 and Srxyn10M, they were incubated at 37 ◦ C in 5.0 M NaCl maintained at pH 10 to measure the residual xylanase activity. The results showed that the two enzymes still maintained more than 85% activity after a 10 h incubation. Following a longer incubation period of 24 h, Srxyn10 and Srxyn10M exhibited 76.59% and 85.05% activity rates, respectively (Fig. 5B). When the samples were treated at 50 ◦ C in 5.0 M NaCl maintained at pH 10 for 1–8 h, Srxyn10M displayed more activity than Srxyn10. After the 8 h incubation, Srxyn10M still possessed a 55.56% activity rate (Fig. 5C). Several studies have reported the salt-tolerance of GH10 to be extremely high, such as the salt-tolerant xylanase from Massilia sp. RBM26 that maintains an 86% activity rate even after being incubated at 37 ◦ C for 1 h in 5.0 M NaCl [28]; a halophilic xylanase from Zunongwangia profunda that retains nearly 100% activity when incubated at 30 ◦ C for 2 h in 5.0 M NaCl [29]; and the xylanase rXynAHJ3 from Lechevalieria sp. HJ3 that retains more than a 55% activity rate at concentrations ranging from 0.2–2.0 M NaCl and 26% for 4.0 M NaCl by incubating at 20 ◦ C for 72 h at pH 7.5 [30]. However, instances where xylanases were maintained in a notably extreme environment to explore their potential advantages rarely reported on the parameters of pH, temperature, and time simultaneously. In the present work, Srxyn10 and Srxyn10M demonstrated excellent potential for further application when the combination of a strong alkali environment (pH 10) along with temperature and time (37 ◦ C for 24 h and 50 ◦ C for 8 h) was used to evaluate their salt-tolerance. Moreover, the application possibilities are greatly enhanced with the improvement in the thermostability and stress tolerance of Srxyn10, by removing the C-terminal CBM to produce Srxyn10M. 3.5. Substrate specificity and kinetic parameters The substrate specificity of Srxyn10 and Srxyn10M was analyzed using beechwood xylan, birchwood xylan, and oat-spelt xylan. The specific activities of Srxyn10 and Srxyn10M with the beechwood xylan substrate were calculated to be 468.50 ± 6.56 and 1525.56 ± 18.61 U mg−1 , respectively, which was defined as 100%. Both enzymes showed a high specific activity with respect to the oat-spelt xylan substrate. Srxyn10M (99.56%) showed a higher specific activity than Srxyn10 (42.11%) for the birchwood xylan substrate (Table S2 in Supplementary data). In a previous study, two C-terminal modules of the xylanase rXTMA from Thermotoga maritima were deleted, and the resulting xylanase (rXTMAC) exhibited a 60% higher specific activity towards insoluble wheat arabinoxylan than rXTMA [26]. The kinetic parameter analysis of Srxyn10 and Srxyn10M with respect to polymeric substrates is shown in Table 1. Srxyn10M showed better substrate affinities and higher turnover rates than Srxyn10. In terms of catalytic efficiencies, Srxyn10M displayed about 18.65%, 41.77%, and 18.58% higher kcat /Km than Srxyn10, when using beechwood xylan, birchwood xylan, and oat-spelt xylan as the substrates, respectively. Both enzymes showed the highest catalytic efficiencies on the oatspelt xylan substrate than with any other substrate. Kleine and

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Fig. 5. (A) salt tolerance of Srxyn10 and Srxyn10M and the resistibility for the notably extreme environment at (B) 37 ◦ C and (C) 50 ◦ C. For the salt tolerance, the residual activity was measured at 60 ◦ C at pH 8.0 after incubation of 37 ◦ C for 1 h with 0–5.0 M NaCl. The samples measured without incubation were defined as 100%. For the resistibility for the notably extreme environment, the residual activity was measured at 60 ◦ C at pH 8.0 after incubation of 37 ◦ C or 50 ◦ C at pH 10 for different time with 5.0 M NaCl. The samples measured without incubation were defined as 100%.

Table 1 Kinetic parameters of polymeric substrates of Srxyn10 and Srxyn10M. Substrate

Kinetic parametera

−1

Enzyme Srxyn10

Srxyn10M

Beechwood xylan

Km (mg mL ) kcat (s−1 ) kcat /Km (mg−1 s−1 mL)

8.79 ± 0.15 64.76 ± 1.62 7.37

8.07 ± 0.12 73.11 ± 1.3 9.06

Birchwood xylan

Km (mg mL−1 ) kcat (s−1 ) kcat /Km (mg−1 s−1 mL)

9.49 ± 0.15 49.33 ± 0.4 5.20

7.53 ± 0.07 67.25 ± 1.1 8.93

Oat-spelt xylan

Km (mg mL−1 ) kcat (s−1 ) kcat /Km (mg−1 s−1 mL)

6.63 ± 0.10 93.30 ± 1.5 14.07

6.33 ± 0.12 109.40 ± 1.8 17.28

a

Values are the mean of three replicates.

Liebl reported that the deletion enzyme XynAC of the xylanase XynA from Thermotoga maritima, which lacked the C domains, showed a higher affinity to the insoluble oat-spelt xylan and birchwood xylan substrates [31]. As reported, the xylanase from Streptomyces olivaceoviridis E-86 (PDB code: 1XYF), which shares the highest identity with Srxyn10 as per the Protein Data Bank (PDB; http://www.rcsb.org/pdb/home/home.do), has a Gly/Prorich linker region connecting the catalytic domain and CBD, which enables the two domains to move independently and possibly provides a 3-fold chance for substrate capture and catalysis [23]. The apparent difference between Srxyn10 and 1XYF is the linker, based on the analysis of the amino acid sequences. Srxyn10 consists of

a longer linker sequence (AGGGGNPDLDPEPGDGTA) when compared to 1XYF (GGSSTPPPSGGGQ). More importantly, the amino acids ‘Pro’ and ‘Gly’, located at the right of linker, were obstructed (AGGGGNPDLDPEPGDGTA), which could possibly result in negative flexibility of the linker. The amino acid composition of the linker against the coordination of the two domains working on the substrates may be a possible explanation as to the improvement in the catalytic properties of the truncated derivative, Srxyn10M. The presence of a linker with a specific length and degree of flexibility and hydrophilicity can play an essential role in maintaining cooperative inter-domain interactions [32,33]. In addition to the thermostability and salt tolerance, the specific activity, substrate affinity, and turnover rate of Srxyn10 could also be modified by altering its modular structure, thereby being efficiently applied in biorefineries specific for lignocellulostic materials. 3.6. Hydrolysis patterns of xylohexaose X6 was used as a substrate to analyze the hydrolysis patterns of Srxyn10 and Srxyn10M. During X6 hydrolysis, both X3 + X3 and X2 + X4 levels of Srxyn10 were calculated to be 50%. However, the X2 + X4 levels of Srxyn10M rose to 62% in response to the 38% of X3 + X3 levels (Fig. S1 in Supplementary data and Table 2), showing a different hydrolysis pattern. A drastic alteration in the hydrolysis patterns of Srxyn10 and Srxyn10M was observed, with a shift towards X2 + X4, as displayed by the products formed. A previous study regarding xylanase rXTMA from Thermotoga maritima and thermophilic xylanase A from Caldibacillus cellulovorans reported

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Fig. 6. Primary hydrolysis products of Srxyn10 (A and B) and Srxyn10M (C and D) with different substrates. BEX: Beechwood xylan; BIX: Birchwood xylan; OSX: Oat-spelt xylan; WAXL: Wheat arabinoxylan with low viscosity; WAXM: Wheat arabinoxylan with medium viscosity; WAXH: Wheat arabinoxylan with high viscosity. X1: Xylose; X2: Xylobiose; X3: Xylotriose; X4: Xylotetraose; X5: Xylopentaose; X6: Xylohexaose.

Table 2 Distribution of primary hydrolysis products from the degradation of X6. Enzyme

Srxyn10 Srxyn10M

Primary hydrolysis products (%) X3 + X3

X2 + X4

50 38

50 62

a variation in the xylooligomer cleavage pattern of xylanases with and without CBMs [26,34]. 3.7. Hydrolysis of xylan Six kinds of xylans were used as substrates to determine their hydrolysis patterns. Both, Srxyn10 and Srxyn10M, showed high production of X2 (at least 40%) in the degradation products. X2 was also the major product formed (more than 50%) when Srxyn10 and Srxyn10M acted on beechwood, birchwood and oat-spelt xylans (Fig. 6). The xylans were almost hydrolyzed into xylooligosaccharides with less than six degrees of polymerization when beechwood, birchwood, and oat-spelt xylans were used as substrates,. However, when wheat arabinoxylans with dif-

ferent viscosities were used as substrates, some products with polymerization higher than X6 were observed due to their rich ␣-l-arabinofuranose units attaching to the linear ␤-(1,4)-linked xylopyranose backbone [35]. One main difference is the varying sugar ratio (arabinose:xylose, w/w) between the different viscosities of the wheat arabinoxylans. The low, medium, and high viscosity wheat arabinoxylans showed sugar ratios in the range of 30:70, 22:78 and 26:74, respectively (as per the instructions provided by Megazyme). The production of X1 is consistent with the X1 levels in three kinds of arabinoxylan sugar ratios. Srxyn10 and Srxyn10M provided similar results using the six commercial xylans as substrates. 3.8. Synergetic hydrolysis of corncob xylans Srxyn10 and Srxyn10M possess special hydrolytic properties that provide a high yield of X2. However, X1 is more readily produced. Here, we tried to decrease the production of X1 and improve the quality of xylooligosaccharide, providing insight into their hydrolytic characteristics and cooperative effects. A previous study showed that a xylanase mutant, T-XynFM, from Talaromyces thermophilus F1208 possessed formidable transglycosylation prop-

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Table 3 Composition of hydrolysis products with water-insoluble and water-soluble corncob xylans. Enzyme

Primary hydrolysis products (␮moL mL−1 )A Water-insoluble corncob xylan

Srxyn10 Srxyn10 + T-XynFM Srxyn10M Srxyn10M + T-XynFM

Water-soluble corncob xylan

X1

X2

X3

X4

X1

X2

X3

X4

8.58 ± 0.21a 6.32 ± 0.20b 5.30 ± 0.15c 2.73 ± 0.07d

24.35 ± 1.06a 24.87 ± 1.19a 13.52 ± 0.51b 14.42 ± 0.70b

1.50 ± 0.06a 0.82 ± 0.03b 0.98 ± 0.04c 0.89 ± 0.04bc

2.30 ± 0.10a 2.30 ± 0.11a 1.79 ± 0.07b 2.03 ± 0.09b

6.63 ± 0.28a 3.38 ± 0.13b 5.93 ± 0.26c 4.96 ± 0.20d

14.87 ± 0.60a 17.53 ± 0.71b 16.07 ± 0.75ab 16.14 ± 0.73ab

0.98 ± 0.03a 0.94 ± 0.04a 5.55 ± 0.26b 3.43 ± 0.12c

2.62 ± 0.11a 3.47 ± 0.13b 2.01 ± 0.08c 2.08 ± 0.08c

A Values are the mean of three replicates. Means within columns followed by the same letter were not significantly different (P < 0.05).

erties [36]. According to the optimal experimental procedure (Table S3 in Supplementary data), 70 U of Srxyn10 (Srxyn10M) and 30 U of T-XynFM were used to degrade water-insoluble corncob xylan and 90 U of Srxyn10 (Srxyn10M) and 10 U of T-XynFM were used to hydrolyze water-soluble corncob xylan during the hydrolytic processes. From Table 3, the production of X1 markedly decreased when compared to the control groups with 100 U of Srxyn10 (Srxyn10M). More X2 was observed in the Srxyn10 and Srxyn10M hydrolyzed products than any other compound using water-insoluble and water-soluble xylans extracted from the corncob keeping in line with commercial xylans (beechwood xylan, birchwood xylan and oat-spelt xylan). Srxyn10M showed a negative effect on the water-insoluble xylans, which corresponded with the xylanase from Streptomyces olivaceoviridis E-86, where the removal of the CBM resulted in a decrease in activity against insoluble xylan. However, the truncated CBM demonstrated minimal effect on the hydrolysis of soluble xylan. As revealed in the previous report, the enzymes use the CBM domain to capture the insoluble substrates and promote catalysis [23]. GH10 xylanases cleave glycosidic bonds via a double-displacement catalytic mechanism [37,38], where a covalent glycosyl-enzyme intermediate is formed [39] and a competitive process occurs between the acceptor and water molecule during hydrolysis to interact with the intermediate at the active site, in order to determine the type of reaction that will occur [40]. Water molecules enter the catalytic cleft, which are located on the surface of the C-terminal side of the central ␤-barrel [41], and attack the carbonium cation intermediate, cleaving the glycosidic linkage. Nevertheless, the reaction shifts toward transglycosylation in the presence of high concentrations of an acceptor sugar [42]. In this paper, the combination of Srxyn10 (Srxyn10M) and T-XynFM was produced to make the best use of the catalytic specialty of each enzyme and to allow for optimal performance of the enzymes in the biotechnological processes. The expected X2 is known to possess the highest prebiotic activity in xylooligosaccharides with respect to the proliferation of bifidobacteria, which plays an important role in maintaining healthy intestinal microflora [43]. To the best of our knowledge, this is the first report that aims to improve the catalytic property of a salt-tolerant GH10 xylanase, which forms X2 as the main product, by deleting the C-terminal CBM.

4. Conclusion A salt-tolerant GH10 xylanase, Srxyn10, and its C-terminal CBM truncated derivative, Srxyn10M, were successfully synthesized in this study. The catalytic characteristics of Srxyn10M were considered to be an improvement when compared to Srxyn10. Furthermore, both enzymes displayed special hydrolysis properties in order to produce X2. An effective salt-tolerant ability and the formation of X2 as a major hydrolysate makes these enzymes excellent candidates for further application.

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