Improving the thermostability and catalytic efficiency of GH11 xylanase PjxA by adding disulfide bridges

International Journal of Biological Macromolecules 128 (2019) 354–362

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Improving the thermostability and catalytic efficiency of GH11 xylanase PjxA by adding disulfide bridges Chao Teng a,b,c,1, Yuefeng Jiang b,c,1, Youqiang Xu c, Qin Li e, Xiuting Li a,b,c,⁎, Guangsen Fan c, Ke Xiong b,c, Ran Yang b,c, Chengnan Zhang c, Rong Ma d, Yunping Zhu c, Jinlong Li c, Changtao Wang a,f,⁎ a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100048, China Beijing Engineering and Technology Research Center of Food Additives, Beijing 100048, China School of Food and Chemical Engineering, Beijing Technology and Business University, No. 11, Fucheng Road, Beijing 100048, China d School of Foreign Language, Beijing Technology and Business University, No. 33, Fucheng Road, Beijing 100048, China e School of Food and Biological Engineering, Hubei University of Technology, No. 28, Nanli Road, Wuhan 430068, China f School of Science, Beijing Technology and Business University, No. 11, Fucheng Road, Beijing 100048, China b c

a r t i c l e

i n f o

Article history: Received 29 October 2018 Received in revised form 17 January 2019 Accepted 19 January 2019 Available online 22 January 2019 Keywords: Xylanase Disulfide bridge Catalytic characteristics

a b s t r a c t In order to increase the thermostability and catalytic efficiency of acidophilic GH11 xylanase, two disulfide bonds were introduced into crucial region of the enzyme. The xylanase PjxA, from Penicillium janthinellum MA21601, has attracted considerable attention due to its favorable acid-resistance; however, its poor thermostability and low enzymatic hydrolysis efficiency limit its application. In this study, two disulfide bonds were introduced into crucial regions of three recombined xylanases (DB-s1s3, DB-s1s4, and DB-s3s4). All three xylanases remained acid-resistant while gaining improved hydrolytic and thermostability properties, of which DB-s1s3 was the most noteworthy. The optimal temperature of recombined xylanase DB-s1s3 increased from 50 °C to 70 °C. The specific activity of DB-s1s3 was 4.76-fold higher than that of wild-type xylanase PjxA. Moreover, DB-s1s3 showed a 2.14-fold increase in kcat/Km. In addition, DB-s1 s3 showed improved hydrolytic characteristics, of which the most noteworthy was its enhanced ability to produce xylose and xylobiose from polymeric substrates. Compared with PjxA, combining DB-s1 s3 with cellulase improved the hydrolytic yield of corncob powder, procuring concentrations of xylose (X1) and xylobiose (X2) of 204.4% and 24.4%, respectively. Thus, these mutants offer great potential for application in the agricultural residue degradation industry. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Agricultural wastes are the most abundant and lowest-cost raw materials available for lignocellulose biotransformation. These renewable biomasses have been used as fuel, pulp and feed for centuries but not with great economy or efficiency [1]. Corncobs are one of the most abundant renewable agricultural wastes in China, with about 46,000,000 tons produced annually) [2]. Corncobs, which mainly contain lignin, cellulose and the hemicellulose, are important by-products of the corn industry and are used either as animal feed or returned to harvested fields as fertilizer [3]. Xylanases (endo-β-1,4-xylanase, EC 3.2.1.8) act on the main chain of xylan by hydrolyzing β-1,4 linkages between xylose molecules, leading to a mixture of xylooligosaccharides of different sizes [4]. Xylanases have important applications in industry because of their enormous potential to transform lignocellulosic materials. They are widely used as ⁎ Corresponding authors at: Beijing Technology and Business University, No 11 Fucheng Street, Haidian District, Beijing 100084, China. E-mail addresses: [email protected] (X. Li), [email protected] (C. Wang). 1 Chao Teng and Yuefeng Jiang contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.01.087 0141-8130/© 2019 Elsevier B.V. All rights reserved.

raw materials in a large number of industrial processes, such as in the food, animal feed, paper, pulp, textile and biofuel industries [5]. A reasonable biomass hydrolysis yield usually requires either a large amount of enzymes or a long hydrolysis time, which considerably limits the economic feasibility of this bio-refinery concept [6,7]. In order to further improve hydrolysis efficacy, higher-temperature hydrolysis with correspondingly thermostable enzymes has attracted much interest in the field of bio-refinery processes [8]. For example, the results of Katewadee et al. indicate that enzymatic hydrolysis at higher temperatures with modified xylanase produces 2–3 fold more bagasse hydrolysis compared to using the wild-type enzyme [9]. A recent study by Peng et al. showed that the synergistic interactions between thermophilic enzymes from C. owensensis and the commercial enzyme cocktail CTec2 (Novozymes) result in the efficient deconstruction of native lignocellulosic biomass, even without pretreatment [10]. Acidic xylanases have garnered increasing attention in recent years due to their unique properties. They have been widely used in different fields, such as for the bioconversion of lignocellulosic materials into fermentative products, improvement of the digestibility of animal feedstock [11–13], and clarification of juices [13,14]. Acidic xylanases have been reported from Aspergillus usamii E001 (pH 4.6, 50 °C) [15],

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Botryotinia fuckeliana B05–10 (pH 4.5–5.0, 38–42 °C) [16], Pichia stipitis (pH 4.5–5.0, 45 °C) [17], Penicillium purpurogenum (pH 3.5, 50 °C) [18], and Penicillium sp. CGMCC 1669 (pH 4.5, 40 °C) [19]. However, most of these acidic xylanases have poor thermostability [13]. A number of molecular techniques, such as genome-walking PCR, recombination methods, site-directed mutagenesis, etc., are powerful tools for improving the characteristics of enzymes for greater commercial application in industry [20]. For example, the thermostability of xylanase from Thermomyces lanuginosus VAPS24 has been enhanced by the addition of polyols or agar-agar entrapment. Its production has been maximized by using low-cost agro-industrial residues and hybrid optimization tools that facilitate various industrial applications [21,22]. Some factors that are known to increase protein thermostability are: improved hydrogen bonding networks [23–25], stabilization of α– helices by helix capping residues [26,27], shortening of loops between secondary structural elements [28], optimization of electrostatic surfaces [29–31], and increased solvation in specific regions of the protein [32]. The packing density of residues in the hydrophobic core can also influence protein thermostability [33]. Efforts to improve enzyme thermostability by structure-guided protein engineering have, therefore, concentrated on the improvement of intra- and inter-molecular interactions by introduction of disulfide bridges [34,35], and stacked aromatic residues [36,37]. Paës and O'Donohue used the introduction of disulfide bridges to prolong enzyme half-life (at a high temperature of 70 °C) N10-fold that of the wild enzyme [38]. Fenel et al. engineered a disulfide bridge at the N-terminal region of Trichoderma reesei endo-1,4-βxylanase [39]. They found that mutational changes increased the thermal inactivation half-life of this mesophilic enzyme from 40 s to 20 min at 65 °C, and from b10 s to 6 min at 70 °C [39]. In previous work, the acidic xylanase gene (named PjxA) from Penicillium janthinellum MA21601 was expressed in E. coli BL21 (DE3) [40]. Its poor thermostability was one of the crucial limiting factors for industrial applications, although its acid resistance is a valuable property. To increase the hydrolytic characteristics, techniques like structure-based protein engineering and site-directed mutagenesis have been used [41]. In this study, three mutants (DB-s1s3, DB-s1s4, DB-s3s4) were constructed by introducing two disulfide bonds. After site-directed mutation, all the genes were expressed in E. coli and characterized. Moreover, the production of xylobiose (X2) and xylose (X1) from corncob powder by PjxA-DB and mutants with the co-application of cellulase was also evaluated. 2. Materials and methods 2.1. Bacterial strains, plasmids, and substrates Acidic xylanase gene PjxA and mutants were obtained from the Beijing Advanced Innovation Center for Food Nutrition and Human Health. Plasmid pMD18-T, E. coli DH5α for cloning, plasmid PET-28a, E. coli BL21 (DE3) for expression, and Taq polymerase were obtained from Takara (Japan). Restriction endonucleases, T4 DNA ligase and Q5® HighFidelity DNA polymerase were obtained from NEB Inc. (USA). Bovine serum albumin (BSA) was purchased from Roche (738328), and beechwood, birchwood, and oat spelt xylans were purchased from Sigma (X4252, X0502, and X0627, respectively). Xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose (X5) were obtained from Megazyme (6860-47-5, 47592-59-6, 22416-58-6, and 49694-20-4 respectively). Cellulase was obtained from Biotopped. The dried corncob powder was sifted through a 65–40 mesh sieve. 2.2. Construction of mutant xylanases In order to both improve the thermostability and hydrolysis characteristics of the xylanases, DB-s1s3, DB-s1s4 and DB-s3s4 were created by generating two disulfide bonds. This strategy further improved the thermostability [42–44] and special hydrolysis characteristics [42,45–47] of the xylanases. All genes of the mutants were cloned by

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overlap extension PCR. For details of the primer and the formation of mutants, see Table S1 in the Supplementary Data. 2.3. Expression and protein purification Mutated genes from PCR were digested with NcoI and NotI and cloned into PET-28a, then transformed into E. coli BL21 (DE3) for expression. The positive transformants were obtained based on kanamycin resistance tests. After successful sequencing, they were grown overnight in LB medium containing 0.1% kanamycin (37 °C at 200 rpm), until reaching a cell density of OD600 = 0.8. Expression was initiated with the addition of 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). The crude enzyme solution was collected through the following steps: cells were collected by centrifugation (6000 g, 5 min, 4 °C) and suspended in 50 mM sodium acetate buffer (pH 5.5). The xylanases were collected after cell wall-breaking by ultrasonication and centrifugation (18,000 g, 10 min, 4 °C). (Histidine)6tagged protein was purified using HisTrap HP columns (1 mL, GE Healthcare, 29-0510-21) with 20 mM sodium phosphate buffer (pH 7.4), including 500 mM NaCl and different concentrations of imidazole, working on an ÄKTA FPLC purification system (GE Healthcare, Uppsala, Sweden). The purity of the xylanase was assayed by Coomassie (R250)-stained SDS-PAGE. All protein concentrations were determined by the BCA method (BSA as the protein standard) [48]. The formation of disulfide bonds was determined by the method described by Wakarchuk et al. [49]. In brief, the xylanases were pretreated with different concentrations of dithiothreitol (DTT), 100 mM iodoacetamide and 1% SDS at 70 °C for 5 min. Reducing agent DTT was added to the control group to break the disulfide bonds but was not added to the treatment group. Iodoacetamide was used to bind covalently with the thiol group of cysteine, so that the free sulfhydryl would be protected and disulfide bonds would not form during heat treatment. In the meantime, protein could not reform disulfide bonds in the control group [50–52]. 2.4. Xylanase activity and protein concentration assay Xylanase activity was assayed according to the method reported by Bailey [53]. The reaction mixture contained 0.9 mL of 1% (w/v) beechwood xylan and 0.1 mL of a suitably diluted enzyme solution (50 mM acetate buffer, pH 5.5) and was mixed at 55 °C for 5 min. The amount of reducing sugar liberated was determined by the 3,5dinitrosalicylicacid (DNS) method [54], using xylose (X1) as a standard. One unit (U) of xylanase activity was defined as the amount of enzyme that catalyzed the release of 1 μmol of xylose equivalent per min under the assay conditions. All experiments were performed in triplicate. 2.5. Biochemical characterization of the purified mutants The effect of pH on the activity of xylanase was studied at 55 °C in two different buffers with a pH range of 2.0–6.5 (50 mM). These were glycine hydrochloride buffer (pH 2.0–4.0) and citrate buffer (pH 3.5–6.5). To determine the pH stability of the enzyme, the xylanase was incubated at different pH levels (3.0, 4.0, and 5.0) at 40 °C for 30 min followed by cooling on ice water for 30 min. The residual xylanase activities were measured by the standard assay procedure. The optimum temperature for xylanase activity was determined at a range of temperatures (40–85 °C) in 50 mM citrate buffer (pH 4.0). To determine the temperature stability of the enzyme, xylanase was incubated at different temperatures (40, 50, and 60 °C) at pH 4.0 for 30 min followed by cooling on ice water for 30 min. Then, the residual xylanase activities were measured following the standard assay procedure. Relative activity (calculated as the percentage of residual activity to initial activity) was used to evaluate the pH and temperature stability. To confirm whether the introduction of disulfide bridges improved thermal stability, variants PjxA, PjxA-DB, DB-s1s3, DB-s1s4, and DB-s3s4 were

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treated with 10 mM DTT at 4 °C for 12 h. All experiments were performed in triplicate. 2.6. Substrate specificity and kinetic analysis To determine the substrate specificity of the enzyme, the purified xylanase was incubated with 1% (w/v) of each substrate in 50 mM citrate buffer (pH 4.0) at 55 °C for 10 min. The amounts of reducing sugars produced were estimated by the DNS method as described above. To determine the kinetic parameters, different concentrations of beechwood xylan ranging from 1 to 40 mg mL−1, were dissolved in 50 mM citrate buffer (pH 4.0) and incubated with the purified xylanase at 40 °C for 5 min [55]. All experiments were performed in triplicate. The Km and Vmax values were calculated by a non-linear regression using Grafit 7 software. 2.7. Hydrolysis characteristics of the purified PjxA and mutants To evaluate the hydrolysis characteristics of xylanases, xylan (beechwood xylan, birchwood xylan, oat-spelt xylan) were used as substrates. Reactions (1 mL) containing 10 mg mL−1 substrate were incubated at 50 °C for 12 h in 50 mM citrate buffer (pH 4.0) with 10 U xylanase. The reaction mixtures were treated with boiling water for 5 min to stop the reaction and filtered through a 0.22 μm membrane filter. The hydrolysis products were analyzed qualitatively by thin-layer chromatography (TLC) and quantitatively by high-performance liquid chromatography (HPLC). The above-mentioned hydrolysate (20 μL) was injected into a COSMOSIL sugar-D packed column (4.6 ID × 500 mm, 05397–51) with a differential refractive index detector (RID). Elution (1.0 mL min−1) was performed with an isocratic flow of chromatographically-pure acetonitrile and ultrapure water (v/v, 70: 30) for 15 min. The temperatures of the chromatographic column and RID were both 40 °C. X1, X2, X3, and X4 were used as standards. All experiments were performed in triplicate. For TLC analysis, a silica gel plate (Merck) was developed twice with butanol, acetic acid, and water (v/v/ v, 2:1:1), then sprayed with a methanol and sulfuric acid mixture (v/v, 20:1) at 105 °C. X1, X2, X3, X4, and X5 were used as standards. 2.8. Degradation of corncob powder by the combination of xylanase and cellulase Enzymatic hydrolysis of corncob powder (100 mg mL−1) was performed in 50 mM citric acid buffer (pH 4.0) in 1 mL reaction systems. Hydrolysis was performed at 50 °C for 12 h with continuous stirring (170 rpm) in an incubator, then stopped with boiling water for 5 min,

followed by centrifugation (18,000 g, 5 min). The hydrolysis samples and calibration standards were assessed using an HPLC equipped with an RID and an Aminex HPX-87H column (300 × 7.8 mm, 9 μm, 125–0140). The analysis was carried out at 63 °C using 5 mM sulfuric acid as the mobile phase at a flow rate of 0.6 mL min−1. To analyze the synergistic effect of xylanase and cellulase on corncob powder, reactions were conducted as described above. Five groups of experiments were performed, including a control group (buffer only), 80 U xylanase-added group, 100 U xylanase-added group, 80 U xylanase:20 U cellulose-added group, and 20 U cellulose-added group. Assays were carried out in triplicate. 2.9. Statistical analysis All experiments were conducted in triplicate and data are presented as means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVAs and Duncan's multiple range tests (SPSS 17.0). Results were considered statistically significant at the 95% confidence level (p b 0.05). 3. Results and discussion 3.1. Production of mutants Through site-directed mutagenesis, three derivatives, DB-s1s3 (S39C–S186C), DB-s1s4 (S27C–S39C), and DB-s3s4 (S27C–S186C), were engineered with potentially better thermostability and hydrolysis characteristics than PjxA by introducing two disulfide bridges. These three mutants were constructed via two methods according to previous work [41]. First, a disulfide bridge (T2C–T29C) in the N-terminus region was studied with the aim of enhancing PjxA-DB's thermostability while maintaining its acid resistance and highly specific enzyme activity. Second, a disulfide bridge (S39C–S186C, S27C–S39C, or S27C–S186C) had an effect on hydrolysis characteristics and enhanced hydrolysis. In order to explore the structural location of the mutation site, a structural model was constructed based on PDB (3wp3.1.A) using the SwissModel web server (http://swissmodel.expasy.org). DB-s1s3, DB-s1s4, and DB-s3s4 had 72.63% amino acid sequence identity with the modeling template 3wp3.1.A: xylanase 11C from Talaromyces cellulolyticus [56]. As shown in Fig. 1A (3D), Ser(27)Cys, Ser(39)Cys, and Ser(186) Cys form two disulfide bridges with each other. The formation of each protein is as shown in Fig. 1B. SDS-PAGE analysis of the purified mutants is shown in Fig. 2A. Gray scanning analysis of SDS-PAGE by Band Scan 5.0 software indicates that the purities of the target proteins were 94.9%, 94.0%, and 94.5%,

Fig. 1. (A) The structure of the mutant DB-pjxA based on PDB: 3wp3.1A, the original disulfide bridge between Cys2 and Cys29 displayed in purple; an additional disulfide bridge between Cys39 and Cys186, Cys27 and Cys39, Cys27 and Cys186 for DB-s1s3, DB-s1s4, DB-s3s4 displayed in yellow; (B) Schematic overview of recombinantly produced derivatives of PjxA. PjxA: Recombinant xylanase of Penicillium janthinellum (MA21601); PjxA-DB: Mutation of N-terminus amino acid to introduce an additional disulfide bridge (T2C–T29C); DB-s1s3: (T2C–T29C, S39C–S186C); DB-s1s4: (T2C–T29C, S27C–S39C); DB-s3s4: (T2C–T29C, S27C–S186C); s1s3: (S39C–S186C); s1s4: (S27C–S39C); s3s4: (S27C–S186C).

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Fig. 2. (A) SDS-PAGE analysis of xylanases. Lane M: the molecular weight marker; lane 1, 3, 5: the crude enzyme (DB-s1s3, DB-s1s4, DB-s3s4, respectively); lane 2, 4, 6: the purified enzyme (DB-s1s3, DB-s1s4, DB-s3s4, respectively). (B) Validation of the disulfide bonds formation. Samples were treated in 1% SDS and the presence or absence of 10 mM DTT at 70 °C for 5 min. Lane M: the molecular weight marker; lanes 1, 3, 5, 7: PjxA, DB-s1s3, DB-s1s4, DB-s3s4 without DTT; lanes 2, 4, 6, 8: PjxA, DB-s1s3, DB-s1s4, DB-s3s4 with 10 mM DTT.

respectively. The molecular masses of DB-s1s3, DB-s1s4, and DB-s3s4 given by SDS-PAGE were all approximately 21.0 kDa, which is in agreement with the theoretical molecular weight of 20.6 kDa as a monomer (Fig. 2B). According to the analysis of SDS-PAGE, the mobility of the protein was accelerated by the presence of disulfide bonds, as shown in Fig. 2B. Disulfide bonds containing enzymes tended to bind less to SDS and thus migrate faster than enzymes without a disulfide bond [57]. Thus, mutants pretreated without the reducing agent DTT (Fig. 2B, lanes 3, 5, and 7) migrated faster than those with DTT (Fig. 2B, lanes 4, 6 and 8) in 12% SDS-PAGE. However, PjxA (no disulfide bonds) displayed no difference in mobility according to the presence or absence of DTT (Fig. 2B, lanes 1 and 2). In addition, previous research has shown that PjxA-DB without DTT forms a dimer and migrates as far as that with DTT.

3.2. Optimal pH and pH Stability The optimal pH of PjxA-DB and other mutants were similar to that of the wild-type enzyme PjxA; all favored acidic environments of pH 3.5–4.5 (Fig. 3A). As shown in Fig. 3C, all mutants were incubated at 40 °C for 30 min without substrate to measure the pH stability of the recombinant xylanases. After incubation between pH 3.0 and 5.0 for 30 min at 40 °C, all mutants were stable and showed over 50% residual activity. At pH 3.0, compared with the initial activities, DB-s1s4 and DB-s3s4 maintained the highest residual activities of 91.76% and 91.80%, respectively; whereas DB-s1s3 only maintained 56.33% activity. At pH 4.0, DB-s3s4 still had N90% activity, while DB-s1s3 and DB-s1s4 showed similar activities of 75.16% and 77.94%, respectively. At pH 5.0, DB-s1s3 showed the highest activity of 84.72%, but DB-s3s4 was slightly

Fig. 3. (A) Optimal pH and (C) pH stability of the three mutants. For pH stability, all proteins were incubated at 40 °C for 30 min before measuring for enzymatic activity. The sample controls without incubation were defined as 100%. (B) Optimal temperature and (D) thermostability of the three mutants. For thermal stability, all xylanases were incubated at 0.05 M pH 4.0 citrate buffer for 30 min before activity assay. The samples measure without incubation were defined as 100%.

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less active than at pH 3.0 and the activities of DB-s1s4 and DB-s3s4 were very similar between pH 3.0–5.0. In brief, the introduction of an additional disulfide bridge resulted in similar (DB-s1s3) or slightly higher (DB-s1s4 and DB-s3s4) pH stability. 3.3. Optimal temperature and temperature stability As shown in Fig. 3B, the optimal temperature of the mutants was 70 °C, which is about 20 °C higher than that of PjxA and 5 °C higher than that of PjxA-DB, indicating that the mutants acquired a thermophilic property after an extra disulfide bond was introduced. As shown in Fig. 3D, in terms of mutant thermal stability, all residual activities declined to about 50% after 30 min incubation at 60 °C. DB-s3s4 showed slightly higher residual activity (59.23%) than PjxA-DB (about 50%), while DB-s1s3 and DB-s1s4 displayed slightly lower residual activities at 60 °C (46.56% and 42.44%, respectively). For DB-s1s3 and DB-s3s4 incubated at 40 °C and 50 °C, there was almost no difference in xylanase activity compared with the untreated group. All activities remained above 93.00%; nonetheless, activity declined rapidly below 60% when the temperature increased to 60 °C. When incubated at 40 °C to 60 °C, DB-s1s4 showed smooth declines in activity of 86.36%, 71.32%, and 42.44%, respectively. To further determine the effect of disulfide bridges on thermal stability, after removing the disulfide bridges with DTT, all variants exhibited b6% residual activity after incubation at 50 °C for 30 min, while the control group (100%) and wild-type PjxA (N98%) displayed much higher residual activities (Table S2 in the Supplementary Data). In short, for thermostability, DB-s3s4 showed the highest residual activity after incubation, while DB-s1s4 displayed slightly lower residual activity than PjxA-DB. In addition, DB-s1s3 showed significant improvement in residual activity when incubated at 50 °C. Many studies have reported that disulfide bridges can significantly stabilize the native structures of proteins. In order to increase the thermostability of Tx-xyl, Paës introduced two disulfide bonds in strategic positions in the β-jelly roll structure of mesophilic GH-11 endoxylanases [38]. Furthermore, a slight difference in the positions of the disulfide bridges in the Nterminal region may affect xylanase thermostability, which may explain the difference between the three mutants [6]. Li et al. substituted both the N-terminal and cord regions of xylanase to increase the optimal temperature from 50 °C to 65 °C [58]. According to the comparison of pH and thermal stability, the three new mutants displayed stronger stability than PjxA and PjxA-DB, which is attributed to the additional disulfide bridge. A comparative three-dimensional structural analysis showed that an additional disulfide bridge was introduced in these substances, which would have led to a decrease in the configurational chain entropy of the unfolded polypeptide [26]. Masazumi et al. reported that a combination of disulfide bonds, each of which contributes to stability, can substantially improve the overall stability of a protein [26]. Similarly, Wang showed that both kinetics and thermostability are enhanced by the construction of disulfide bonds at N-terminals [6].

Table 1 Substrate Specificity of polymeric substrates of PjxA and the mutants. Xylanases

PjxA PjxA-DB DB-s1s3 DB-s1s4 DB-s3s4

Substrate Specificity (U mg−1) Beechwood xylan

Birchwood xylan

Oat spelt xylan

888.55 ± 25.51a 2871.57 ± 72.20a 4232.53 ± 48.99a 2109.19 ± 14.12a 3135.22 ± 52.97a

353.99 ± 13.70b 1495.94 ± 67.49b 3135.22 ± 52.97b 1742.18 ± 20.27b 2557.41 ± 46.09b

145.89 ± 8.30c 1279.20 ± 57.48c 2109.19 ± 140.12c 1094.46 ± 108.53c 1950.49 ± 69.73c

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

possessed higher specific activities than the single-disulfide-bond mutant, except that DB-s1s4 had a slight decrease when beechwood xylan and oat spelt xylan were used as substrates and had values 26.55% and 14.44% lower than that of PjxA-DB, respectively. The analyzed kinetic parameters of the mutants with beechwood xylan are shown in Table 2. The introduction of disulfide bridges in PjxA caused stronger affinities (Km). All the variants with single or double disulfide bonds exhibited 7.6–114.0% increases in catalytic efficiency (kcat/Km) compared to the wild-type enzyme PjxA under the same conditions, although not all mutants displayed higher Vmax and kcat values than wild-type PjxA. These phenomena are similar to the results that Boonyapakron et al. reported recently [9]. XynTTTE, which carries double disulfide bridges, displayed a 71% increase in kcat, and XynTT, which carries a disulfide bridge, showed a 58% increase compared with the wild type [9]. Evaluation the characteristics of variants with disulfide bridges in different enzyme locations indicates that the increased value of kcat may be due to a faster rate of product release, while the higher mobility may be associated with higher activity [59]. Together, these results suggest that the disulfide bridges in DB-s1 s3 are critical to enzyme thermostability and catalytic activity. Disulfide bonds rigidify the structure of xylanase, making it more resistant and thereby enhancing its thermostability [60]. However, a rigid structure may interfere with conformational changes, because flexibility is necessary for the catalysis of xylanase [61]. Similar to a previous study, the results show that the introduction of a disulfide bond has a negative effect on the catalytic activity of xylanase [57]. The presence of disulfide bonds of the same topography in different enzymes does not necessarily lead to the same improvement, and the addition of disulfide bonds does not always cumulate [13]. With the introduction of disulfide bonds in PjxA-DB, the specific activity of DB-s1 s4 slightly decreased as its active sites would interfere with the incorporation of an N-terminal disulfide bridge [49]. Meanwhile, DB-s1s3 and DB-s3s4 had positive effects on specific activity. The disulfide bond between T2C-T29C in the Nterminal stabilizes the irregular loop and the β-strand A2 interaction, which stabilizes the initial unfolding position. Meanwhile, the disulfide bonds between S39C–S186C, S27C–S39C, and S27C–S186C stabilize the critical regions on the β-strand A3 and A4, A2 and A3, and A2 and A4, respectively. A study by Boonyapakron et al. described the mutant XynAG, which carries a disulfide bond on the β-sheet of the interior β-layer of the enzyme, and which showed only 30% of the catalytic activity of

3.4. Substrate specificity and kinetic parameters of mutants Specific activities were measured with 10 mg mL−1 beechwood xylan at 55 °C in 50 mM citrate buffer (pH 4.0). PjxA possessed the lowest specific activity of 888.6 U mg−1, while the specific activities of PjxADB, DB-s1s3, DB-s1s4, and DB-s3s4 were improved to 2871.6 U mg−1, 4232.5 U mg−1, 2109.2 U mg−1, and 3135.2 U mg−1, respectively (Table 1). The substrate specificities of the mutants were assayed using polymeric substrates. In order to make the substrate specificity more intuitive, the specificity activity was defined as 100% for the beechwood xylan substrate. As shown in Table 1, all xylanases showed the highest specific activity to beechwood xylan and the lowest specific activity to oat spelt xylan. DB-s1s3 showed the highest specific activity to all polymeric substrates. The double-disulfide-bond mutants

Table 2 Kinetic parameters on beechwood xylan of PjxA and the mutants. Xylanases

PjxA PjxA-DB DB-s1 s3 DB-s1 s4 DB-s3 s4

Vmax

Km

kcat

kcat/Km

mg−1 min−1 μmol

mg mL−1

s−1

s−1 mg−1 mL

7.5 ± 0.6a 6.7 ± 0.4ab 6.2 ± 0.3b 6.6 ± 0.4ab 6.8 ± 0.6ab

1290 ± 45cd 1318 ± 27c 2250 ± 10a 1234 ± 21d 1647 ± 26b

172 ± 7.8d 197 ± 7.8c 362 ± 16.4a 185 ± 6.2cd 243 ± 10.8b

3757 ± 158c 3838 ± 82c 6541 ± 98a 3587 ± 55c 4788 ± 86b

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

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Fig. 4. Hydrolysates of xylan by PjxA and mutants. For TLC of hydrolysis products, control group with absence of xylanase: beechwood xylan (A); birchwood xylan (B); oat spelt xylan (C). Using beechwood xylan (A), birchwood xylan (B), oat spelt xylan (C) with different xylanases: PjxA (1), PjxA-DB (2), DB-s1s3 (3), DB-s1s4 (4), DB-s3s4 (5), s1s3 (6), s1s4 (7), s3s4 (8). Values are the mean of three replicates. Means within columns followed by the same letter were not significantly different (p N 0.05). ND, not detected.

hydrolyzed into xylooligosaccharides with a degree of polymerization (DP) no greater than five. However, when oat spelt xylan was used as a substrate, hydrolysis products with a degree of polymerization higher than X5 were observed, possibly due to arabinosyl, glucuronic acid, and other substituents of oat spelt xylan, which might contribute to steric hindrance and prevent xylanases from hydrolyzing, resulting in a higher degree of polymerization of hydrolysate [62]. It was observed that approximately 5 μmoL mL−1 × 1 and N10 μmoL mL−1 × 2 were hydrolyzed from beechwood xylan by the mutants, except s1s4 and s3s4. Additionally, DB-s1s3, DB-s1s4, and DBs3s4 increased by 17.40%, 14.63%, and 5.43% X1 compared with PjxADB, respectively. Regarding the increase of X2 in comparison with PjxA-DB, only DB-s1s3 had a higher increase (8.35%), while DB-s3s4 displayed a slight increase (1.62%) and DB-s1s4 exhibited a small decrease (−6.41%). When using birchwood xylan as a substrate, the double-disulfide-bridge enzymes DB-s1s3 (11.92%), DB-s1s4 (12.34%),

the wild type. Meanwhile, XynTT, which carries an exterior disulfide bond at the N-terminal, stabilized the β1-β4 interaction [9]. 3.5. Hydrolysis of commercial xylan using recombinant xylanase and mutants In order to determine the characteristics of hydrolysis, three commercial xylans were utilized as the substrates for PjxA and seven xylanase mutants [41] were engineered previously under the optimum conditions by analyzing the enzymatic hydrolysis products by TLC and HPLC. As indicated in Fig. 4 and Table 3 the most notable differences between the hydrolysis characteristics of PjxA and the mutants were the quantities of X1 and X2. It must also be mentioned that X2 was always the main hydrolysis product (N50%) and the hydrolysis of mutants introduced double disulfide bridges, which increased the maximum amount of X2. Beechwood and birchwood xylans were almost

Table 3 Partial data of primary hydrolysis products. Xylanases

Primary hydrolysis products (μmoL mL−1) Beechwood xylan

PjxA PjxA-DB DB-s1s3 DB-s1s4 DB-s3s4 s1s3 s1s4 s3s4

Birchwood xylan

Oat spelt xylan

X1

X2

X3

X1

X2

X3

X1

X2

X3

NDf 5.085 ± 0.021c 5.970 ± 0.042a 5.361 ± 0.038b 5.829 ± 0.023a 4.659 ± 0.046d 0.903 ± 0.009e 0.823 ± 0.013e

5.450 ± 0.029h 12.337 ± 0.098c 13.368 ± 0.114a 11.549 ± 0.097d 12.537 ± 0.084b 10.105 ± 0.076e 6.562 ± 0.048f 6.180 ± 0.530g

3.708 ± 0.014d 3.280 ± 0.010e 3.022 ± 0.009f 2.587 ± 0.017g 2.624 ± 0.014g 3.852 ± 0.013c 4.429 ± 0.037a 4.248 ± 0.039b

NDg 3.088 ± 0.027c 3.456 ± 0.034ab 3.469 ± 0.024a 3.421 ± 0.024b 2.675 ± 0.019d 0.572 ± 0.045f 0.695 ± 0.031e

4.355 ± 0.037h 6.786 ± 0.045e 8.769 ± 0.005a 7.751 ± 0.016c 8.117 ± 0.04b 7.153 ± 0.032d 4.828 ± 0.034g 5.208 ± 0.039f

3.227 ± 0.043c 2.600 ± 0.023e 2.684 ± 0.048d 2.169 ± 0.062h 2.361 ± 0.031g 2.501 ± 0.058f 3.454 ± 0.022b 3.654 ± 0.020a

NDf 1.027 ± 0.017d 1.471 ± 0.013b 1.392 ± 0.069c 1.709 ± 0.022a 1.390 ± 0.036c 0.632 ± 0.041e 0.591 ± 0.028e

1.687 ± 0.009g 2.453 ± 0.011e 3.251 ± 0.018b 3.015 ± 0.013d 4.066 ± 0.083a 3.091 ± 0.002c 2.368 ± 0.063f 1.589 ± 0.091h

1.073 ± 0.033c 1.098 ± 0.226c 1.171 ± 0.004b 1.078 ± 0.019c 1.292 ± 0.082a 1.071 ± 0.025c 1.191 ± 0.022b 0.868 ± 0.011d

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

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and DB-s3s4 (10.78%) showed similar increases in X1. Meanwhile, DBs1s3, DB-s1s4, and DB-s3s4 displayed increases in X2 of 29.22%, 19.61%, and 14.22%, respectively. Compared with the parent enzyme PjxA-DB, mutants with double disulfide bonds displayed better ability to hydrolyze oat spelt xylan into X1 and X2: DB-s3s4, DB-s1s3, and DB-s1s4 had large increases of 66.41% and 65.76%, 43.23% and 32.53%, and 35.53% and 22.91%, respectively. As for the amounts of X3, these mostly decreased after modification. The experimental data demonstrate that the construction of disulfide bonds alters hydrolysis characteristics and thermostability. The TLC results showed that the xylanases from Penicillium citrinum and Paecilomyces thermophila had no detectable activity toward X2 but had noticeable hydrolytic activity toward X3 [13,63,64]. PjxA-DB, with a single disulfide bond, showed less X1 and X2 activity than the mutants with double disulfide bonds. Instead, the combinations of PjxA-DB with DB-s1s3, DB-s1s4, or DB-s3s4, exhibited varying degrees of increased X1 and X2 activity. This indicates that engineered disulfide bonds can increase the wild-type xylanase PjxA's affinity to X3. Many studies have modulated the thermostability of enzymes by introducing disulfide bridges; however, relatively few have investigated the hydrolytic capacity of disulfide bonds. Boonyapakron showed that introducing one or two disulfide bonds at the end of a β-sheet is important in improving the hydrolytic performance of lignocellulosic materials and their tolerances to higher temperature and pH compared with the wild type. Similar research has shown that double-disulfide-bonded xylanase has more efficient hydrolysis than the wild type; that is, Tx-xyl-SS3 released more X1 from wheat bran at 60 °C with nearly identical xylose/arabinose values [38]. Paës hypothesized that the superior performance of Tx-xyl-SS3 in wheat bran hydrolysis might result from some unidentified changes to its physicochemical properties. These may allow Tx-xyl-SS3 to better penetrate the wheat bran cell wall network and interact with various cell wall components. This improves the penetrative ability of Tx-xylSS3, allowing it greater access to suitable substrates [38]. According to the structure of SWISS-MODEL, disulfide-bonded mutants are located on the surfaces of subsites, which are crucial for substrate binding and correct orientation. Although engineering was not directly on the subsites, it still had a strong impact on the hydrolytic characteristics of the mutants. Similarly, the xylanase mutant SrxynFM from Streptomyce rochei L10904 showed enhanced ability to produce X2 and X3 [65]. Additionally, the xylanase mutant T-XynFM from Talaromyces thermophilus F1208 displayed formidable transglycosylation properties [66]. 3.6. Degradation of corncob by the combination of xylanase and cellulase PjxA-DB and the three new mutants possess special hydrolytic properties that provide high yields of X1 and X2. The potential of the engineered xylanases was explored with cellulase for application in the agricultural residue degradation industry. Dried corncob powder was used (65–40 mesh) as substrate. The optimal pH of cellulase is 4.0–5.0, which is similar in both wild and mutant enzymes. Reactions were performed at 50 °C, at which both xylanase and cellulase exhibited desirable thermostability. In the hydrolysis reactions performed with cellulase alone, X1 and XOS were not detected, and the main hydrolysis product was glucose. As shown in Tables S3 and Table 4, when an additional 20 U of cellulose [67] was added to the 80 U xylanase reaction, more X1 and X2 (44.0% and 5.36%, respectively) were released compared with adding 20 U xylanase (DB-s3s4) to the 80 U xylanase (DBs3s4) reaction. DB-s3s4 displayed the most prominent synergistic effect among the mutants, with increases of 35.21% X1 and 10.44% X2, while DB-s1 s3 released the most hydrolysis products. The X1, G, and X2 concentrations reached 12.09 μmoL mL−1, 12.43 μmoL mL−1, and 27.75 μmoL mL−1, with increases of 204.53%, 20.93%, and 24.44%, respectively, compared with PjxA. These phenomena are consistent with the biochemical properties of the mutants measured above. Song et al. used mixed enzyme groups (0.2 g/g:0.2 g/g) to hydrolyze natural substrates including corncob, corn stover, and rice straw, and the sugar yields

Table 4 Hydrolysis of corncob powder using various enzyme combinations. Xylanases

Variation of primary hydrolysis products (%) Δ1(20 U xylanase)

Δ2(20 U cellulase)

Δ3 = Δ2 − Δ1

X1

X1

X2

X1

X2

8.40 10.80 10.71 2.87 7.01 8.68 3.59 4.02

– 17.60 35.21 4.76 44.00 7.02 12.08 18.69

0.97 5.79 10.44 0.48 5.36 1.82 1.96 1.60

PjxA PjxA-DB DB-s1s3 DB-s1s4 DB-s3s4 s1s3 s1s4 s3s4

– 2.84 6.66 1.95 18.59 13.43 32.50 19.58

X2 7.44 5.01 0.27 2.39 1.65 6.85 1.63 2.42

– 20.44 41.87 6.71 62.58 20.44 44.58 38.26

Δ1 = (Y − W) / W × 100%. It means the percentage increase in X1 or X2, when xylanase use in hydrolysis reaction increased from 80 U to 100 U (equally added 20 U xylanase). Δ2 = (Z − W) / W × 100%. It means the percentage increase in X1 or X2, when xylanase use in hydrolysis reaction was equal but cellulase use increased from 0 U to 20 U (total 100 U enzyme, equally added 20 U cellulase). W: the concentration of X1 or X2 when use 80 U xylanase in the hydrolysis reaction. Y: the concentration of X1 or X2 when use 100 U xylanase in the hydrolysis reaction. Z: the concentration of X1 or X2 when use 80 U xylanase and 20 U cellulase in the hydrolysis reaction.

were improved by 133%, 164%, and 545%, respectively, which confirms that the cooperation between xylanase and cellulase was much higher than in either of the single enzyme treatments [68]. The X1 concentration of the corncob hydrolysis using xylanase alone was 1.6 mg mL−1, while that using both cellulase and xylanase was 2.3 mg mL−1. Agricultural residues like corncob are comprised of cellulose and hemicellulose (mainly xylan), which are tightly connected and intertwined. This phenomenon hampers hydrolysis by either cellulase or hemicellulase (such as xylanase) alone [69,70]. In order to expose cellulose or xylan to a corresponding enzyme, it would, theoretically, be helpful to conduct partial hydrolysis using a mixture of xylanase and cellulase [68,70]. In fact, scanning electron microscopy revealed that the mutual synergistic effect of xylanase and cellulase had a principal effect on the external substrates [71]. Xylanase not only causes fiber swelling at the cellulose surface, which causes eruptions and increases external accessibility but also promotes pore formation by hydrolyzing xylan, which improves internal accessibility so that substrates are more accessible to enzymes [7,67,71,72]. In this way, once the cellulose component of corncob is hydrolyzed using cellulase, exposure to xylan will increase and promote the activity of xylanase, which is beneficial to the exposure of cellulose. Some research has clarified that the hydrolysis time and content of xylan in the substrate increase the synergistic effect of xylanase and cellulase [73]. A higher proportion of xylanase was required when the substrate had a relatively high xylan content and at high substrate concentrations [74]. Through their synergistic effects, the way that modified xylanase and cellulase cooperate to degrade substrates has considerable potential for a broad variety of applications. These include converting agricultural residues to soluble saccharides for biofuels or to XOS for functional food. 4. Conclusion In this study, three mutants of PjxA acquired thermophilic properties, maintained similar acid resistance, and improved their hydrolytic activity for X1 and X2 after an additional disulfide bond was introduced by genetic engineering and protein engineering. It suggested that disulfide bonds play a crucial role in substrate hydrolysis, providing a theoretical reference for the engineering of GH11 xylanase to improve its characteristics. Meanwhile, the existence of cellulase renders it more accessible for xylanase to hydrolyze xylan, and the release of XOS and xylose may be enhanced. In this regard, the synergy of xylanase and

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cellulase has considerable potential for a broad variety of applications related to the degradation of agricultural residues. Funding This research was supported by The National Key Research and Development Program of China (2017YFD0400206), the National Natural Science Foundation of China (No. 31671798, No. 31371723, No. 31501416, No. 31671793, No. 31601408 and No. 31201449), the Foundation of Beijing Technology and Business University (No. PXM2017_014213_000036, No. PXM2018_014213_000033). Notes The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.01.087. References [1] V. Juturu, J.C. Wu, Microbial xylanases: engineering, production and industrial applications, Biotechnol. Adv. 30 (2012) 1219–1227. [2] S. Chang, J. Chu, Y. Guo, H. Li, B. Wu, B. He, An efficient production of high-pure xylooligosaccharides from corncob with affinity adsorption-enzymatic reaction integrated approach, Bioresour. Technol. 241 (2017) 1043–1049. [3] J.S. Brigham, W.S. Adney, M.E. Himmel, Hemicellulases: diversity and applications, Handb. Bioethanol, Routledge 2018, pp. 119–141. [4] M. Cayetano-Cruz, A.I.P.D.L. Santos, Y. García-Huante, A. Santiago-Hernández, P. Pavón-Orozco, V.E.L.Y. López, M.E. Hidalgo-Lara, High level expression of a recombinant xylanase by Pichia pastoris cultured in a bioreactor with methanol as the sole carbon source: purification and biochemical characterization of the enzyme, Biochem. Eng. J. 112 (2016) 161–169. [5] P. Boonchuay, C. Techapun, P. Seesuriyachan, T. Chaiyaso, Production of xylooligosaccharides from corncob using a crude thermostable endo-xylanase from Streptomyces thermovulgaris TISTR1948 and prebiotic properties, Food Sci. Biotechnol. 23 (2014) 1515–1523. [6] Y. Wang, Z. Fu, H. Huang, H. Zhang, B. Yao, H. Xiong, O. Turunen, Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an Nterminal disulfide bridge, Bioresour. Technol. 112 (2012) 275–279. [7] J. Hu, V. Arantes, A. Pribowo, J.N. Saddler, The synergistic action of accessory enzymes enhances the hydrolytic potential of a “cellulase mixture” but is highly substrate specific, Biotechnol. Biofuels. 6 (2013) 112–123. [8] L. Long, D. Tian, R. Zhai, X. Li, Y. Zhang, J. Hu, F. Wang, J. Saddler, Thermostable xylanase-aided two-stage hydrolysis approach enhances sugar release of pretreated lignocellulosic biomass, Bioresour. Technol. 257 (2018). [9] K. Boonyapakron, A. Jaruwat, B. Liwnaree, T. Nimchua, V. Champreda, P. Chitnumsub, Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-β-1,4-xylanase for applications in pulp bleaching, J. Biotechnol. 259 (2017) 95–102. [10] X. Peng, W. Qiao, S. Mi, X. Jia, H. Su, Y. Han, Characterization of hemicellulase and cellulase from the extremely thermophilic bacterium Caldicellulosiruptor owensensis and their potential application for bioconversion of lignocellulosic biomass without pretreatment, Biotechnol. Biofuels. 8 (2015) 131–145. [11] S. Vandeplas, R.D. Dauphin, P. Thonart, A. Théwis, Y. Beckers, Effect of the bacterial or fungal origin of exogenous xylanases supplemented to a wheat-based diet on performance of broiler chickens and nutrient digestibility of the diet, Can. J. Anim. Sci. 90 (2010) 221–228. [12] H.J. Yang, C.Y. Xie, Assessment of fibrolytic activities of 18 commercial enzyme products and their abilities to degrade the cell wall fraction of corn stalks in in vitro enzymatic and ruminal batch cultures, Anim. Feed Sci. Technol. 159 (2010) 110–121. [13] G. Paës, J.G. Berrin, J. Beaugrand, GH11 xylanases: structure/function/properties relationships and applications, Biotechnol. Adv. 30 (2012) 564–592. [14] E. Olfa, M. Mondher, S. Issam, L. Ferid, M.M. Nejib, Induction, properties and application of xylanase activity from Sclerotinia sclerotiorum S2 fungus, J. Food Biochem. 31 (2010) 96–107. [15] C. Zhou, H. Bai, S. Deng, J.J. Zhu, M. Wu, W. Wang, Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli, Bioresour. Technol. 99 (2008) 831–838. [16] A. Brutus, I.B. Reca, S. Herga, B. Mattei, A. Puigserver, J.C. Chaix, N. Juge, D. Bellincampi, T. Giardina, A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I, Biochem. Biophys. Res. Commun. 337 (2005) 160–166. [17] P. Basaran, Y.D. Hang, N. Basaran, R.W. Worobo, Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli, J. Appl. Microbiol. 90 (2010) 248–255.

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