Engineering a xylanase from Streptomyce rochei L10904 by mutation to improve its catalytic characteristics

International Journal of Biological Macromolecules 101 (2017) 366–372

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Engineering a xylanase from Streptomyce rochei L10904 by mutation to improve its catalytic characteristics Qin Li a,b , Baoguo Sun b,c , Huiyong Jia d , Jie Hou b , Ran Yang b , Ke Xiong b , Youqiang Xu c , Xiuting Li a,b,∗ a

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing 100048, China School of Food and Chemical Engineering, Beijing Technology and Business University, No.33, Fucheng Road, Beijing 100048, China c Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University (BTBU), Beijing 100048, China d Department of Biology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA b

a r t i c l e

i n f o

Article history: Received 8 February 2017 Received in revised form 21 March 2017 Accepted 24 March 2017 Keywords: Xylanase N-terminal Cord Catalytic characteristics

a b s t r a c t Protein engineering was performed by N-terminal region replacement and site-directed mutagenesis in the cord of a xylanase (Srxyn) from Streptomyce rochei L10904 to improve its catalytic characteristics. Three mutants SrxynF, SrxynM and SrxynFM displayed 2.1-fold, 3.2-fold and 5.3-fold higher specific activities than that of Srxyn, respectively. Moreover, all of the mutants showed greater substrate affinity and kcat /Km than the native Srxyn. In addition, the enzymes showed improved hydrolysis characteristics, of which the most noteworthy is the enhanced ability of producing xylobiose (X2) and xylotriose (X3) from polymeric substrates. The engineered xylanases have greater potential for applications in oligosaccharide preparation industry. © 2017 Published by Elsevier B.V.

1. Introduction Xylanases are used in several different biotechnological applications, alone or in combination with other enzymes: processing aid of bakery products [1], starch separation, clarification of juices, animal feed biotechnology and production of functional food ingredients, especially for those have special properties [2,3]. Currently, an application of xylanases is the production of emerging prebiotics xylooligosaccharides due to the high specificity and little side product generation of the enzymatic preparation [4–8]. During the production of xylooligosaccharides, xylose (X1) is a undesirable component that requires additional costs to be removed [9]. Therefore, it’s necessary to explore appropriate xylanase by either traditional screening from special environment or the application of protein engineering. To date, many xylanases were reported to possess special hydrolysis characteristics in producing xylooligosaccharides. Xylanases from Bacillus Methylotrophicus CSB40, Streptomyces thermovulgaris TISTR1948, Streptomyces rameus L2001, Paenibacillus campinasensis BL11, Streptomyces matensis and xylanase from Aspergillus niveus

∗ Corresponding author at: Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University (BTBU), Beijing, 100048, China. E-mail addresses: [email protected], li [email protected] (X. Li). http://dx.doi.org/10.1016/j.ijbiomac.2017.03.135 0141-8130/© 2017 Published by Elsevier B.V.

expressed in Aspergillus nidulans, can produce xylooligosaccharides when using birchwood xylan, oat-spelt xylan, beechwood xylan or others as substrates [10–15]. Xylanase (␤-1,4-endoxylanase, EC3.2.1.8) is a category enzyme which cleave the ␤-d-xylopyranose bond between two Dxylopyranosyl residues linked by ␤-(1,4) bond, and the ones form the glycoside hydrolase family 11 (GH11) of the CAZy database are considered true xylanase [16–18]. GH11 xylanase is the overall conservation of the ␤-jelly-roll domain [19]. It resembles the shape of a partially closed right hand and consists of two twisted antiparallel ␤-sheets and a single ␣-helix. The ␤-sheet A is composed of a maximum of six ␤-sheets named A1-A6, whereas ␤-sheet B is composed of nine ␤-sheets denoted B1-B9 [20]. Both ␤-sheet A and B constitute the “fingers”, while the loop between ␤-sheets B7 and B8 makes the “thumb”. The twisted part of ␤-sheet B and the ␣-helix form the “palm” of the hand. As more than 60% of the residues are embedded in ␤-strands and ␣-helix of xylanase structure, the loops that collect all these elements are consequently short, about 5 residues on average. However, there are two exceptions: the “cord”, part of a long irregular loop (10 residue-long) joining the ␤-strands B6-B9, which connects the fingers with the base of the thumb and partially closes the active site on the aglycon side; the loop joining the ␤-strands B8-B7, about 12 residue-long [19]. Xylanases possessing special hydrolysis characteristics in producing xylooligosaccharides with a possible lowest X1 units have

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great industrial application value [12,21]. In our previous work, a xylanase gene (Srxyn) from Streptomyce rochei L10904 [22] was cloned and expressed in Escherichia coli BL21(DE3). The recombinant enzyme showed attractive hydrolysis characteristic, with negligible X1 in hydrolysis products. However, the specific activity is too low to meet the requirement for broad industrial applications. In the present study, protein engineering was conducted by N-terminal region replacement and site-directed mutagenesis in the cord of the xylanase (Srxyn) from Streptomyce rochei L10904 to improve the catalytic characteristics. 2. Materials and method 2.1. Bacterial strains, vectors and substrates Streptomyce rochei L10904 was newly isolated from the soil in our previous study and identified using 16S rDNA PCR-RFLP [22]. 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 pET28a (+) were used for gene cloning and expression in E.coli, respectively. Taq polymerases were from Takara (Japan). ® Restricition endonucleases, T4 DNA ligase and Q5 High-Fidelity DNA polymerase were from NEB Inc. (USA). Bovine serum albumin was from Roche (738328). Beechwood, birchwood and oat spelt xylan were from Sigama(X4252, X0502 and X0627). Xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose (X5) were from Megazyme (6860-47-5, 47592-59-6, 22416-58-6, 49694-20-4).

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IPTG was used to induce xylanase expression. The enzymes were collected after centrifugation at 5000 rpm for 5 min at 4 ◦ C. The cells were re-suspended with 50 mM Tris-HCl buffer (pH 7.0), and subjected to cell wall-breaking with ultrasonic. The disrupted cells were centrifuged at 10000 rpm for 10 min at 4 ◦ C to obtain the supernatant with the enzymes ready for the following purification. Srxyn and the mutants were purified by Ni sepharose HP column (1 × 10 cm) with 50 mM phosphate buffer (pH 7.8) including 300 mM NaCl and different concentrations of imidazole, working on an ÄKTA FPLC purification system (GE Healthcare, Uppsala, Sweden). The purified enzymes were adjudged homogeneous after examination with SDS-PAGE. Protein concentrations were determined by Coomassie brilliant blue method with bovine serum albumin (BSA) as the standard. 2.5. Xylanase activity assay Xylanase activity was measured according to the procedure reported by Bailey [25]. The reaction mixture containing 0.9 mL of 1% (w/v) beechwood xylan and 0.1 mL of a suitable diluted enzyme solution was incubated (50 mM acetate buffer, pH 5.5) at 55 ◦ C for 5 min. The amount of reducing sugar liberated was determined by the 3,5-dinitrosalicylic acid (DNS) method, using X1 as the standard [26]. One unit (U) of xylanase activity was defined as the amount of enzyme releasing 1 ␮mol of X1 equivalent per min under the assay condition. 2.6. Characterization of pH and temperature properties

2.2. Gene cloning and expression of xylanase Streptomyce rochei L10904 was cultivated in LB medium. Srxyn (Genbank code: X81045) without the native secretion signal was cloned by polymerase chain reaction (PCR) from the chromosomal DNA with primers StreX11NcoF and StreX11XhoR (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 the pET28a(+) at NcoI and XhoI restriction sits and transformed into E. coli BL21(DE3) for expression. The expressed recombinant xylanase was denoted as Srxyn. 2.3. Engineering of xylanase Amino acid sequence homology analysis showed that Srxyn has a similarity of 91.36% (full length) to Streptomyces sp. JHA19 xylanase (Genbank: WP 055619964). Interestingly, six amino acids in Srxyn differed from Streptomyces sp. JHA19 xylanase (Fig. 1B). Four of the six are replaced by Thr in amino acid sequence of Streptomyces sp. JHA19 xylanase. Thus the four sites were selected according to the distinction of amino acid residue, excepting amino acids with similar characteristics, to be replaced with corresponding amino acids of Streptomyces sp. JHA19 xylanase (Fig. 1B). Furthermore, in an attempt to gain some insight into the functional role of the N-terminal of Srxyn (Fig. 1A), it has been replaced with a ␤-strand A1 “KFTVGNGQ” from Neocallimastix patriciarum xylanase [23,24], to create a hybrid enzyme named SrxynF. Excision method has usually been used to investigate the function of N-terminal, but we only obtained some inactivity mutants, whether long or short sequences have been deleted (data not shown). The primers containing mutated codons were used to introduce mutation (Table S1 in Supplementary data). 2.4. Production and purification of xylanases All the mutants were 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. 1.0 mM

The effect of pH on the activity of xylanase was studied at 55 ◦ C and pH 4.0–8.5 (50 mM): citrate buffer for pH 4.0–6.5; Tris-HCl buffer for pH 7.0–8.5. To determine the pH stability of the enzyme, the xylanase was incubated in the above mentioned appropriate buffers of different pH at 50 ◦ C for 12 h, the residual xylanase activities were measured by the standard assay procedure. Relative activity calculated with percentage of residual activity to initial activity was used to evaluate the pH and temperature stability, respectively. The optimum temperature for xylanase activity was determined by incubating the enzyme at different temperature (40–85 ◦ C) in 50 mM citrate buffer (pH 6.0). To determine the temperature stability of the enzyme, the xylanase was incubated at different temperatures (50 ◦ C, 60 ◦ C and 70 ◦ C) at pH 6.0 for 60 min followed by cooling on ice for 30 min, and the residual xylanase activity was measured following the standard assay procedure. 2.7. Substrate specific activity and kinetic parameters 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 6.0) at 55 ◦ 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 different concentrations of each substrate were dissolved in 50 mM citrate buffer (pH 6.0), and incubated with the purified xylanase at 40 ◦ C for 5 min [27]. The Km and kcat values were calculated from the kinetic data using the GraphPad Prism software. 2.8. Hydrolysis characteristics for oligosaccharide To evaluate hydrolysis characteristics of xylanases, X3, X4 and X5 were used as substrates. Reaction systems (500 ␮l) containing 0.5 mg ml−1 substrate were incubated at 50 ◦ C for 8 h in 50 mM citrate buffer (pH 6.0) with 1 U xylanase. The mixtures were then heated in boiling water for 5 min. Reaction mixtures were filtered with 0.22 ␮m membrane filter and injected (20 ␮l) onto a COSMOSIL sugar-D packed column (4.6ID × 500 mm,

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Fig. 1. (A) Location of two mutations (N-terminal substituted sequence and mutation site in cord). (B) Full alignment (predicted signal peptides were deleted) of Srxyn and Streptomyces sp. JHA19 xylanase (named according to its NCBI database accession: WP 055619964). The sites of site-directed mutagenesis are indicated by red arrows. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

05397-51) with differential refractive index detector (RID). Elution (1.0 ml min−1 ) was isocratic of chromatographically pure acetonitrile and ultrapure water (70:30, v/v) for 15 min, the temperature of chromatographic column and RID were 40 ◦ C. X1, X2, X3, X4 were used as standards. 2.9. Hydrolysis of xylans Reaction systems (1 ml) containing 10 mg ml−1 substrate were incubated with 10 U xylanase in 50 mM citrate buffer (pH 6.0) at 50 ◦ C for 8 h. The reaction mixtures were treated with boiling water for 5 min and then filtered with 0.22 ␮m membrane filter. The hydrolysis products were observed using high-performance liquid chromatography (HPLC) above described and thin-layer chromatography (TLC). For TLC analysis, the silica gel plate (Merck) was developed with butanol, acetic acid, and water (2:1:1, v/v/v), followed by heating for few minutes at 105 ◦ C; the plate was then sprayed with a methanol and sulfuric acid mixture (20:1, v/v). X1, X2, X3 and X4 were used as standards. 3. Results and discussion 3.1. Production of Srxyn and mutants Through sequence alignment and site-directed mutagenesis, a derivative SrxynM (Ile(99)Thr) were obtained with a greater xylanase activity than Srxyn. SrxynF was composed of the Srxyn

Fig. 2. Schematic overview of recombinantly produced derivatives of Srxyn. Srxyn: recombinant xylanase from Streptomyce rochei L10904; SrxynF: substitution of N-terminal with an ␤-strand A1, sequence “ATTITT” has been replaced with “AFTVGNGQ”; SrxynM: Ile(99) has been replaced with Thr in cord; T-XynFM: combination of SrxynF and SrxynM.

amino acid sequence without N-terminal sequence “ATTITT”, which has been replaced with “AFTVGNGQ”, with replacement of the first Lys by Ala [23]. SrxynFM was constructed with the combination of SrxynF and SrxynM. In order to explore the structural location of the mutation site, structural model was modeled using the Swiss-Model webserver (http://swissmodel.expasy.org). Srxyn has 80.42% amino acid sequence identity with the modeling template, the xylanase B2 from Streptomyces lividans [28]. As shown in Fig. 1A, Ile(99)Thr located in the cord of Srxyn. The formation of each protein see Fig. 2. Analysis of the purified Srxyn and mutants on SDS-PAGE were shown in Fig. S1 (supplementary data). Each enzyme migrated as a single band. The molecular mass of Srxyn, SrxynF, SrxynM and SrxynFM displayed approximately equal molecules of 21.1 kDa, 21.2 kDa, 21.1 kDa, and 21.2 kDa, respectively.

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Fig. 3. (A) Optimal pH and (B) pH stability of Srxyn and the three mutants. For pH stability, all proteins were incubated at 50 ◦ C for 12 h before enzymatic assay. The samples measured without incubation were defined as 100%.

3.2. Optimal pH and pH stability Srxyn and the mutants all showed the highest xylanase activity at pH 6.0, favoring to a weak acid condition (Fig. 3). At pH 5.0, comparing with initial activities, Srxyn and SrxynM kept 98.30% and 98.54% activity, respectively; SrxynFM showed 64.33% activity; whereas SrxynF kept only 28.93% activity. At pH 6.0, both Srxyn and SrxynM still had more than 98% activity; SrxynFM possessed 85.46% activity which was better than that at pH 5.0; SrxynF still showed the lowest activity (31.90%). At pH 7.0, Srxyn and SrxynM still kept more than 98% activity; SrxynFM and SrxynF displayed 24.49% and 22.33% original activity, respectively (Fig. 3). Thus the pH stability of SrxynF and SrxynFM were weaker than those of Srxyn and SrxynM, indicating that the introduction of N-terminal has reduced the pH stability of the enzyme.

3.3. Optimal temperature and thermal stability The optimal temperature of SrxynF and SrxynFM was about 5 ◦ C lower than that of Srxyn and SrxynM (65 ◦ C). For thermal stability of Srxyn and the mutants, all residual activities declined below 25% after 60-min incubation at 70 ◦ C. When incubated at 60 ◦ C, Srxyn and SrxynM showed about 76.67% and 77.97% activity, respectively; SrxynF and SrxynFM possessed about 52.40% and 56.24% activity, respectively. When incubated at 50 ◦ C, there was almost no decreasing of xylanase activity compared with the untreated group (Fig. 4). According to the comparison of pH and thermal stability, SrxynF and SrxynFM displayed weaker stability than Srxyn and SrxynM attributing to the introduction of N-terminal substitution of “ATTITT” with “AFTVGNGQ”. A comparative amino acid analysis of them showed that an additional bulk benzene ring of Phe has been introduced, which would have led to a reduced N-terminus packing density. Xue et al. reported that the improvement of the

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Fig. 4. (A) Optimal temperature and (B) thermal stability of Srxyn and the three mutants. For thermal stability, all enzymes were incubated at pH 6.0 for 60 min before activity assay. The samples measured without incubation were defined as 100%.

N-terminus packing density might enhance the thermostability of GH11 xylanase [29]. 3.4. Substrate specificity and kinetic parameters The substrate specificity of Srxyn and the mutants were assayed using beechwood xylan, birchwood xylan and oat-spelt xylan. Srxyn possessed the lowest specific activity of 58.16 ± 3.1 U mg−1 and the specific activity of SrxynF, SrxynM and SrxynFM were 122.94 ± 6.5, 183.69 ± 9.7 and 310.04 ± 11.5 U mg−1 , respectively, when beechwood xylan was used as a substrate and toward which the specificity activity was defined as 100%. All enzymes showed lower specific activity to birchwood. Srxyn (167.19%) and SrxynM (110.45%) showed higher specific activity than SrxynF (76.03%) and SrxynFM (65.28%) using oat-spelt xylan as a substrate (Table S2 in Supplementary data). Analyzing kinetic parameters of Srxyn and the mutants with beechwood xylan, the three mutants displayed higher affinity than Srxyn, but all the mutants excepting SrxynM displayed lower turnover rate than Srxyn (Table 1). All mutants had higher kcat /Km value than Srxyn. The specific activities of SrxynF, SrxynM and SrxynFM were 2.1-fold, 3.2-fold and 5.3-fold higher than that of Srxyn, respectively. The activity of constructed hybrid xylanase ATx was 3.6 and 5.4 times as high as those of parental xylanases by N-terminus replacement [30]. Interestingly, the activity of SrxynM with single mutation Ile99Thr was also improved. The activity increased mutant T91C-Y108C-insS92S93 of xylanase BsXynA from Bacillus subtilis was reconstructed by extending the cord and inserting a disulfide bridge [31]. In our study, in line with consensus in activity alteration, SrxynF, SrxynM and SrxynFM displayed higher substrate affinity and kcat /Km than those of Srxyn for beechwood xylan. Similarly, a xylanase named NTfus with a N-terminal region introduced displayed slightly lower stability while 17% increase in catalytic efficiency for the low viscosity wheat arabinoxylan compared to the

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Table 1 Kinetic parameters on beechwood xylan of Srxyn and the mutants. Kinetic parametera

Enzyme

Km (mg ml−1 ) kcat (s−1 ) kcat /Km (mg−1 s−1 ml) a

Srxyn

SrxynF

SrxynM

SrxynFM

12.16 ± 0.2 19.04 ± 0.3 1.57

8.69 ± 0.1 15.37 ± 0.2 1.77

7.72 ± 0.1 20.65 ± 0.3 2.67

4.32 ± 0.1 15.92 ± 0.2 3.69

Values are the mean of three replicates.

Table 4 Distribution of primary hydrolysis products from the degradation of X5. Enzyme

Srxyn SrxynF SrxynM SrxynFM

3.8. Hydrolysis pattern for xylans

Primary hydrolysis products (%) X1

X1 + X4

X2 + X3

ND ND ND ND

1.9 15.9 2.9 15.3

98.1 84.1 97.1 84.7

ND, not detected.

parental enzyme [24]. However, the BsXYnA mutant T91C-Y108CinsS92S93 displayed a reduction in apparent affinity for the tested substrate, which attributed to the steric hindrance of longer cord for substrate [31]. The residues of cord domain are almost exposed to the hydrophilic environment [28]. Substitute of Ile99Thr was located in the cord, a hydrophilic group, which may have provided a more suitable environment for the cord and the structure stability of xylanase. This might explain the higher activity and substrate affinity of SrxynM for the tested substrate.

3.5. Transglycosylation activity determination X3 was used as substrate to analyze the transglycosylation of Srxyn and the mutants. According to Abdul Manas et al., the ratio of the percentage of xylooligosaccharides with a degree of polymerization higher than X3 to the percentage of X1 and X2 was used as an indicator of enzyme transglycosylation activity [32]. From HPLC data (Table 2), SrxynFM possessed the highest transglycosylation activity (5.04) among the enzymes. The following were Srxyn (2.94), SrxynF (1.25) and SrxynM (0.32), respectively.

3.6. Hydrolysis favoritism for xylotetraose When using X4 as a substrate, Srxyn and the mutants displayed different distribution of primary hydrolysis products (Table 3). The ratio of the percentage of xylooligosaccharides with degrees of polymerization different from X4 to the percentage of X4 in the hydrolysed products was defined as hydrolysis favoritism on X4. The hydrolysis favoritism on X4 of Srxyn and SrxynM were greater than those of SrxynF and SrxynFM, indicating that X4 could be degraded more easily by Srxyn and SrxynM, compared with SrxynF and SrxynFM.

3.7. Hydrolysis pattern for xylopentaose X5 was used as a substrate for analyzing hydrolysis pattern of Srxyn and the mutants. During the course of X5 hydrolysis, the primary products of all enzymes were X2 and X3. Interestingly, when hydrolyzing X5, X1 + X4 levels of SrxynF and SrxynFM have risen to 15.9% and 15.3%, respectively (Table 4), showing a different hydrolysis pattern.

Three kinds of xylans were used as substrates for determination of hydrolysis pattern. Srxyn and the mutants displayed different hydrolysis characteristics in the production of X1, X2 and X3 (Fig. 5). When using beechwood xylan as a substrate, few X1 was present in the hydrolysis products of all enzymes with the exception of SrxynFM. While the amount of X1 had a slight increase when either SrxynF or SrxynM was applied in hydrolysis of beechwood xylan, with 12.35% and 31.07% higher X1 than that of Srxyn, respectively, and the amounts of X2 and X3 were increased. SrxynFM displayed remarkable hydrolysis characteristic, with 41.32% more X2 and 64.29% more X3 were produced, and no X1 was detected. For hydrolysis of birchwood xylan, X1 was not detected in the hydrolysis products of SrxynF and SrxynFM. SrxynF produced fewer X2 and X3 than Srxyn in the hydrolysis products. SrxynFM still produced 24.53% X2 and 6.04% X3 higher than those of Srxyn, respectively. SrxynM displayed 47.83% X1, 17.91% X2 and 28.24% X3 higher than those of Srxyn, respectively. When using oat spelt xylan as a substrate, no X1 was detected in products of the three mutants. Furthermore, both SrxynM and SrxynFM are more efficient in producing X2 and X3 than Srxyn. In contrast, the products of SrxynF still had fewer X2 and X3 than that of Srxyn. According to the above experimental data, N-terminal replacement and site-directed mutagenesis in cord altered hydrolysis characteristics. SrxynF and SrxynM with a single mutation showed lower transglycosylation than Srxyn. Instead, SrxynFM, the combination of SrxynF and SrxynM, possessed a 1.7-fold increased capacity of transglycosylation. Many reports modulated the transglycosylation of enzyme by protein engineering previously [32–34]. The malto-oligosaccharide synthesis was improved using a structure-guided protein engineering method with two strategies. One was replacing the substrate-binding residues to weaken substrate binding and demolish the hydrolysis activity of maltogenic amylase; another was manipulating the hydrophobicity surrounding the active site to repel water molecules from entering the active site pocket [32]. Furthermore, we observed large amount of X3 still existing in the hydrolysis products of all the enzyme reactions when using X3 as substrate. However, Srxyn was distinct from the mutants when hydrolyzing X4. The data showed that Srxyn and SrxynM can hydrolyze X4 more easily. On the contrary, SrxynF and SrxynFM yielded products with the percentage of X4 up to 89.93% and 70.46%, respectively. Nevertheless, all enzymes displayed similar activity on X5, with hardly any X5 existed in degradation products. The hydrolysis predilection of SrxynF and SrxynFM was similar to a xylanase from Cellolosimicrobium sp. Strain HY-13 that the degradation of xylooligosaccharides with degrees of polymerization >4 appeared to be much faster than the degradation of X4 [35]. There was little drastic alteration in the hydrolysis pattern of Srxyn and the mutants, with only a slight shift to X1 + X4 displayed in the products of SrxynF and SrxynFM. Furthermore, Srxyn and the mutants efficiently degraded polymeric substrates. Gratifyingly, there was an increase in the amount of X2 and X3 when using SrxynM and SrxynFM for hydrolyzing beechwood xylan, birchwood xylan and oat spelt xylan. Especially important, X1 has not been detected in the hydrolysis products of SrxynFM.

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Table 2 Distribution of primary hydrolysis products from the degradation of X3. Transglycosylation activity was calculated as TR: the ratio of the percentage of X4 to the percentage of X1 and X2. Enzyme

Srxyn SrxynF SrxynM SrxynFM

Primary hydrolysis products (%)a

TR

X1

X2

X3

X4

ND ND ND ND

1.14 ± 0.01 5.69 ± 0.1 21.28 ± 0.4 1.09 ± 0.01

95.51 ± 1.5 87.17 ± 1.4 72.00 ± 1.4 93.42 ± 1.7

3.35 ± 0.02 7.14 ± 0.1 6.72 ± 0.1 5.49 ± 0.05

2.94 1.25 0.32 5.04

ND, not detected. a Values are the mean of three replicates.

Table 3 Distribution of primary hydrolysis products from the degradation of X4. Hydrolysis favoritism on X4 was calculated as HFR: the ratio of the percentage of X1, X2 and X3 to the percentage of X4. Enzyme

Srxyn SrxynF SrxynM SrxynFM

Primary hydrolysis products (%)a

HFR

X1

X2

X3

X4

ND ND ND ND

79.24 ± 1.5 ND 56.19 ± 1.1 11.76 ± 0.5

9.15 ± 0.4 10.07 ± 0.3 32.46 ± 0.6 17.77 ± 0.2

11.61 ± 0.2 89.93 ± 1.7 11.35 ± 0.3 70.46 ± 1.4

7.61 0.11 7.81 0.42

ND, not detected. a Values are the mean of three replicates.

Fig. 5. Hydrolysates of xylans by Srxyn and the mutants. For TLC of hydrolysis products, control group with absence of enzymes: 1 beechwood xylan; 2 birchwood xylan; 3 oat spelt xylan. Using beechwood xylan as substrate with different protein: A1, Srxyn; B1, SxynF; C1, SrxynM; D1, SrxynFM. Using birchwood xylan as substrate with different protein: A2, Srxyn; B2, SxynF; C2, SrxynM; D2, SrxynFM. Using oat spelt xylan as substrate with different protein: A3, Srxyn; B3, SxynF; C3, SrxynM; D3, SrxynFM. Values are the mean of three replicates. Means within columns followed by the same letter were not significantly different (P < 0.05). ND, not detected.

The N-terminal region of xylanases has been a hot research topic for a long time, especially focusing on the thermostability of xylanases. Shibuya et al. created two mutants Stx15 and Stx18 by incorporating N-terminal of the Thermomonospora fusca xylanase into the mesophilic xylanase from Streptomyces lividans, getting a recombinant enzyme with the optimal temperature of 75 ◦ C compared with 55 ◦ C of the native Streptomyces lividans xylanase [36]. Paës and O’Donohue introduced a disulphide bridge that could link the N- and C-terminal extremities, the mutant Tx-xyl-SS3 possessed 10-fold greater activity than the wild type enzyme [37]. Another team created a hybrid xylanase SlxB-M2 (T11Y, N12H, N13D, F15Y, 16F), which was 140-fold more stable at 70 ◦ C than wild the type SoxB [38]. The N-terminal region of Thermomyces lanuginosus GH11 xylanase was introduced into a disulfide bridge Q1C-Q24C, which increased the half-life of the enzyme by 20-fold at pH 8.0 and 70 ◦ C in the presence of substrate [39]. Yin et al. proved that the temperature optimum of mutant re-NhXyn1157 was 25 ◦ C higher than that of the parental xylanase re-AoXyn11 and it was stable at a temperature up to 65 ◦ C [40]. However, reports about whether the hydrolysis characteristic could be tuned by altering the N-terminal region of xylanase were limited [41,42]. Moreover, as a part of the structure of xylanase, cord flanks the aglycon side of the active site [31,43]. It observed that the positions of similar residues were different although the cord regions shared a similarity in amino-acid sequence according to the crystallographic

analysis of GH11 xylanases [43,44]. Pollet et al. investigated the hydrolysis behavior of GH11 BsXynA by engineering the aglycon subsites and the cord, considering that the aglycon subsites and the cord of BsXynA were important for substrate binding and hydrolysis [31]. The changing of the flexible region including cord between forms Aclose (closed active site cleft) and Bopen (open active site cleft) is presumed to induce and accelerate the enzyme reaction [45]. In this paper, three variants were constructed by short Nterminal region replacement and site-directed mutagenesis in the cord and their catalytic characteristics were successfully improved. To the best of our knowledge, improving hydrolysis characteristics of xylanase by engineering N-terminal region and cord have never been reported. 4. Conclusion Three mutants SrxynF, SrxynM and SrxynFM were constructed by engineering the N-terminal region and cord. The engineering was able to increase the catalytic characteristics in producing X2 and X3. It suggested that N-terminal region and cord play a crucial role in substrate hydrolysis, providing theoretical reference for engineering of GH11 xylanase to improve its characteristics, meanwhile, the successful improvement of Srxyn make the mutant enzymes valuable candidates for efficient bioconversion of polymeric substrates and production of functional oligosaccharide.

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