Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5
Wenqin Bai1,2,3 Yanfen Xue1 Cheng Zhou1 ∗ Yanhe Ma1
1 National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of China 2 National Engineering Lab for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, People’s Republic of China 3 College of Life Science, Shanxi Normal University, Linfen, People’s Republic of China
Abstract A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 ◦ C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5–8.5) with optimal activity at pH 5.5 and 7.5–8.0. They had high alkali tolerance, retaining over 80% residual activity after
preincubation at pH 8.5–11.0 at 37 ◦ C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper C 2014 International Union of Biochemistry and Molecular industry. Biology, Inc. Volume 62, Number 2, Pages 208–217, 2015
Keywords: alkali tolerance, carbohydrate-binding module, catalytic domain, xylanase
1. Introduction Xylan, a major component of hemicelluloses in plant cell walls, is the most abundant renewable polysaccharide in nature [1]. Endo-1, 4-beta-xylanase (EC 3.2.1.8) is the key enzyme-degrading xylan and catalyzes the hydrolysis of β-1,4-xylosidic linkages of xylan into short xylooligosaccha-
Abbreviations: CBMs, carbohydrate-binding module; CTAB, hexadecyltrimethylammonium bromide; DNS, 3,5-dinitrosalicylic acid; GH, glycoside hydrolase; LB, Luria Bertani; MC, microcrystalline cellulose; NA, not available; ORF, open reading frame; PAGE, polyacrylamide gelelectrophoresis.. ∗ Address
for correspondence: Dr. Ma Yanhe, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, People’s Republic of China. Tel.: +86 10 64807618; Fax: +86 10 64807616; e-mail: [email protected]. Received 6 March 2014; accepted 18 June 2014 DOI: 10.1002/bab.1265 Published online 22 September 2014 in Wiley Online Library (wileyonlinelibrary.com)
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rides [2]. Xylanases have great potential applications in the food, animal feed, textile, paper, and biofuel industries, as well as in the production of xylooligosacharides [3]. In the past decades, xylanases have been increasingly used in the paper industry for bleaching purposes. During the bleaching process, the hydrolysis of xylan by xylanases could facilitate the removal of lignin, which would reduce the amount of toxic chlorinated chemicals (the conventional bleaching agent). Because pulp is processed at high temperature and alkaline pH condition in pulping and bleaching processes, thermostable and alkali-tolerant xylanases have obvious advantages to the minimization of temperature and pH adjustment [4]. Therefore, it is of great interest to discover xylanases that are stable and active under high temperature (about 60 ◦ C) and alkaline (pH 8–10) conditions. Numerous xylanases contain a catalytic domain, one or more carbohydrate-binding modules (CBMs), and/or other functional domains joined by a linker region [5]. On the basis of primary structure alignment and hydrophobic cluster analysis of the catalytic domain, xylanases are mainly
classified into families 10 and 11 of glycoside hydrolase (GH) [3]. Compared with family 10 counterparts, family 11 xylanases have no cellulase activity and lower molecular mass (about 20 kDa) [6]; therefore, they can easily penetrate cellulose fiber network without damaging the fiber, thus more suitable in pulp bleaching process. CBMs are divided into 64 families based on their amino acid sequence similarity (http://www.cazy.org/fam/acc CBM.html). In general, CBMs can increase the binding ability or catalytic activity of enzyme to insoluble substrates [7, 8]. It is also reported that CBMs affect the enzymatic properties such as enzyme stability of catalytic domains [5, 9–11]. In this study, a xylanase gene (xyn11A) was cloned from alkaliphilic Bacillus sp. SN5. The mature polypeptide consisted of a family 11 catalytic domain, a linker region, and a family 36 CBM. The intact Xyn11A and the catalytic domain (Xyn11ALC) were expressed in Escherichia coli and characterized. The results showed that the CBM of Xyn11A could decrease the thermal stability of the enzyme, change the pH–activity profile, and improve the binding ability and hydrolytic activity to insoluble xylan. Xyn11A-LC was a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which made it a promising candidate in the pulp and paper industry.
2. Materials and Methods 2.1. Strains, plasmids, culture conditions, and chemicals Alkaliphilic Bacillus sp. SN5, which exhibited the highest xylanase activity, was isolated from a soda lake in Inner Mongolia, People’s Republic of China [12]. The culture was deposited in the China General Microbiological Culture Collection Center under the accession number CGMCC 1.12485. E. coli DH5α (Invitrogen) and E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) were used as host cells for gene cloning and expression. The E. coli strains were grown at 37 ◦ C in Luria-Bertani (LB) medium with 100 µg/mL ampicillin or 50 µg/mL kanamycin to select plasmid transformants. The pUC18 (TaKaRa, Dalian, People’s Republic of China) and pET28a (Novagen) plasmids were employed as gene cloning and expression vectors, respectively. Oatspelt xylan, birchwood xylan, beechwood xylan, and other polysaccharides were all purchased from Sigma, St. Louis, MO, USA. The standard xylooligosaccharides were purchased from Megazyme (Bray, Ireland). All other reagents used were of the highest purity commercially available.
2.2. Cloning and sequence analysis of the xylanase gene xyn11A Genomic DNA of Bacillus sp. SN5 was prepared from 100 mL of culture. The cell pellet was treated with 200 µg/mL lysozyme for 30 Min at 37 ◦ C, followed by 2% SDS and 100 µg/mL proteinase K for 1 H at 55 ◦ C. After addition of CTAB/NaCl for 10 Min at 65 ◦ C, the DNA was extracted by phenol and chloroform and precipitated with absolute ethanol. The genomic library was
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constructed by standard protocols [13]. Genomic DNA was partially digested with Sau3AI. The DNA fragments between 2 and 8 kb in size were recovered using the gel extraction kit (Omega). The recovered DNA fragments were ligated into the dephosphorylated BamHI site of pUC18 and transformed into the competent E. coli DH5α. Transformants were grown on LAX medium (LB medium containing 100 µg/mL ampicillin and 0.2% [w/v] Remazol Brilliant Blue xylan) [12]. The xylanasepositive clones were identified by showing clear halos around colonies on LAX medium. The DNA insert fragment of positive clone was sequenced by SinoGenoMax Co. (Beijing, People’s Republic of China). The gene sequence was analyzed and compared with sequence databases by using available online tools (http://www.ncbi.nlm.nih.gov/ and http://www.expasy.ch/). The signal peptide was predicted with the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP).
2.3. Construction of expression plasmids To further compare the enzymatic properties of the intact Xyn11A and the truncated Xyn11A-LC, the expression plasmids pET28a-xyn11A (pET28a carrying the mature open reading frame [ORF] region of xyn11A) and pET28a-xyn11A-LC (pET28a carrying the catalytic region of xyn11A) were constructed. The construction procedure was as follows: xyn11A was amplified using the primers F1 (5 -GCATGGATCCCAAATCACTGGAAATGAAATCG-3 ) and R1 (5 -GCTACTCGAGGTGAATTTCTAAGTAGTCGATATAAG3 ). The underlined sequences corresponded to the BamHI and XhoI restriction sites, respectively. Xyn11A-LC was amplified using the primer F1 and the primer R2 (5 GCTACTCGAGTATCGTTAAGTTATTTCGATAAAC-3 ) containing the XhoI restriction site (underlined). The PCR cycling conditions consisted of an initial step of 5 Min at 94 ◦ C, a second step of 30 cycles including 30 Sec at 94 ◦ C, 30 Sec at 55 ◦ C, and 90 Sec at 72 ◦ C and a final extension step of 10 Min at 72 ◦ C. The PCR products were purified using the gel extraction kit (Omega), digested with BamHI and XhoI, and ligated into the pET28a treated with the same restriction endonucleases.
2.4. Expression and purification of the recombinant proteins The recombinant plasmid pET28a-xyn11A was transformed into E. coli BL21 (DE3) competent cells. The positive clone was grown in LB medium containing kanamycin (50 µg/mL) at 37 ◦ C overnight. Then, the culture was inoculated in the abovementioned fresh medium and incubated at 37 ◦ C until OD600 reached 0.6–0.8. After supplemented with a final concentration of 0.5 mM isopropylthiogalactoside, the culture was induced for 4 H. Cells were harvested by centrifugation at 5,000g and 4 ◦ C for 10 Min and disrupted by ultrasonication. The crude R Kits (Novagen) enzyme was purified by using the His·Bind according to the manual. The expression and purification of Xyn11A-LC were the same as described above. The apparent molecular mass and purity of the recombinant Xyn11A and Xyn11A-LC were analyzed by SDS-PAGE. The concentration of
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2.5. Characterization of Xyn11A and Xyn11A-LC The enzyme activities of the purified recombinant Xyn11A and Xyn11A-LC were determined by measuring the amount of reducing sugar released from xylan by the 3,5-dinitrosalicylic acid (DNS) method [14]. The reaction mixtures containing 20 µL of 1 µg/mL enzyme solution and 480 µL of 1% (w/v) beechwood xylan in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 10 Min, followed by adding 0.5 mL of DNS reagent to stop reaction. The mixture was boiled for 5 Min and cooled to room temperature. Then, the absorbance at 540 nm was determined. One unit (U) of xylanase activity was defined as the amount of enzyme required to release 1 µmol reducing sugar from xylan per minute under the assay condition. The assays were all performed in triplicate. The optimal temperature for the xylanase activity was determined over a range of temperatures from 25 to 70 ◦ C at pH 7.5 (McIlvaine buffer). The optimal pH was determined at 55 ◦ C over the range of 4.5–10.0. The buffers used were McIlvaine buffer for pH 4.5–8.0, 0.05 M boric acid/borate for pH 8.0–9.0, and 0.05 M borate/NaOH for pH 9.5–10.0, respectively. For determining the temperature and pH profiles, the maximum value was set as 100%. For thermal stability, the purified recombinant proteins were preincubated in 50 mM Tris–HCl (pH 8.0) without substrate at 60 ◦ C and taken out at an interval of 5 Min. For pH stability, the proteins were diluted in different buffers with pH ranging from 3.0 to 12.0 and preincubated without substrate at 37 ◦ C for 1 H. Three buffer systems, 50 mM McIlvaine buffer (pH 3.0–7.6), 50 mM Tris–HCl (pH 7.6–9.0), and 50 mM glycine–NaOH (pH 9.0–12.0), were used. All the residual activities were measured at 55 ◦ C and pH 7.5. For the thermal and pH stability, the enzyme activity of the purified protein without preincubation was set as 100%. The effects of various reagents on the activities of Xyn11A and Xyn11A-LC were determined by adding a final concentration of 5 mM metal ions, 5 mM or 5% (v/v) surfactants, and 5 mM chelator into the reaction, respectively. The enzyme activity in the reaction without additive was set as 100%. To determine the kinetic parameters, reactions mixtures containing 1 µg/mL purified recombinant protein and varying concentrations of beechwood xylan (1–10 mg/mL) were incubated at 55 ◦ C and pH 7.5 for 5 Min. The Michaelis–Menten parameters were calculated by nonlinear regression using Graphpad Prism 5.0 software (http://www.graphpad.com/prism/). The substrate specificity of the recombinant Xyn11A and Xyn11A-LC were assayed by measuring the release of reducing sugars from the substrates including oatspelt xylan, birchwood xylan, beechwood xylan, methyl cellulose, crystalline cellulose, citrus fruits pectin, maize starch, locust bean gum, and maize amylopectin. For the analysis of the hydrolysis products degraded by Xyn11A and Xyn11A-LC, the reaction mixtures containing 3 µg of purified Xyn11A (or 2 µg of purified Xyn11A-LC) and
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500 µL of 0.5 mg/mL beechwood xylan or 1 mM xylo-oligose in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 12 H. After incubation, the enzymes were removed from the mixtures using the Nanosep Centrifugal 3 K Device (Pall). The products were detected using high-performance anionexchange chromatography (HPAEC), as described previously [15] with minor modification. After CarboPac PA100 column (Dionex Corp.) was previously equilibrated in 100 mM NaOH at a flow rate of 0.5 mL/Min, the analysis was performed by the wash step of 40 Min consisting of 100 mM NaOH and a gradient of 0–60 mM sodium acetate. Xylose, xylobiose, and xylotriose were used as standards.
2.6. Insoluble substrate degradation and binding assays Insoluble and soluble xylan were made from oat spelt xylan by using a modified method of Yazawa et al. [8]. Three grams of oat spelt xylan was suspended in 60 mL deionized water, and then the suspension was adjusted to pH 10.5 with 1 M NaOH and stirred gently at room temperature for 2 H. Then, the pH was readjusted to 10.5 with 1 M NaOH. The suspension was stirred again for 1 H, boiled, and centrifuged at 6,000g for 10 Min. The supernatant was readjusted pH to 7.5 with 1 M citric acid and dried in vacuum as soluble xylan. The precipitate was washed three times with deionized water and once with McIlvaine buffer (pH 7.5). The pellet was finally washed with ethanol, dried in vacuum, and ground as insoluble xylan. For measuring the enzyme activities of the purified Xyn11A and Xyn11A-LC toward insoluble xylan, the reactions mixtures containing 20 µL of 1 µg/mL enzyme solution and 480 µL of 1% (w/v) insoluble xylan in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 10 Min. The activity of the purified enzymes toward soluble xylan was set as 100%. The binding ability of the purified xylanases to insoluble xylan or microcrystalline cellulose (MC) was assayed as follows: the mixture containing 1 µg/mL purified recombinant protein, 1 mg/mL BSA, 5 mM CaCl2 , and 1% (w/v) insoluble xylan or MC in 50 mM Gly–NaOH (pH 9.5) was shaken gently at 4 ◦ C for 2 H, and then centrifuged at 10,000g for 5 Min. The residual xylanase activity in the supernatant was measured under standard conditions. The loss of enzyme activity implied that enzyme bound to the insoluble xylan or MC. The negative control sample (without substrate) was treated as described above, and the residual enzyme activity in the supernatant was set as 100%.
2.7. Nucleotide sequence accession number The nucleotide sequence of the xylanase gene xyn11A from alkaliphilic Bacillus sp. SN5 has been submitted to GenBank with accession number KC163132.
3. Results 3.1. Gene cloning and sequence analysis Among about 10,000 transformants, five xylanolytic-activitypositive clones showing clear zones on LAX medium were obtained from the genomic library of Bacillus sp. SN5. The
Xylanase from Alkaliphilic Bacillus sp. SN5
FIG. 1
Multiple sequence alignment of xylanases Xyn11A, XylX, and XynJ. Boxes show the two signature patterns belonging to family 11 xylanases. Pentagrams indicate two catalytic residues, Glu120 and Glu210.
sequence analysis revealed that all the insert DNA fragments contained a proposed entire 1,101 bp ORF, designated as xyn11A. It encoded a polypeptide of 366 amino acids, including a predicted N-terminal signal peptide of 27 residues. Blast analysis showed that the polypeptide Xyn11A shared the highest identity (67%) with xylanase XylX from Paenibacillus campinasensis BL11 (GenBank accession No. ABB77852) [16], followed by 66% identity with alkalophilic xylanase XynJ from Bacillus sp. 41M-1 (BAA82316.1) [17]. Multiple sequence alignment of xylanases Xyn11A, XylX, and XynJ is shown in Figure 1. On the basis of homology comparison in GenBank, Xyn11A was a modular xylanase containing a GH family 11 catalytic domain consisting of 200 amino acids (from Gln28 to Ile227) and a family 36 CBM consisting of 119 amino acids (from Ser248 to His366). They were connected by a Gly–Pro-rich linker region consisting of 20 amino acids (from Gly228 to Gly247). Two signature patterns of family 11 xylanases Signature 1: PLVEFYIVDS (from Pro117 to Ser126) and Signature 2: TVEGWQSSGS (from Thr208 to Ser217) were identified in Xyn11A (Fig. 1). Two putative catalytic residues (Glu120 and Glu210) were predicted in the conserved catalytic region by amino acid sequence alignment of Xyn11A with other GH family 11 catalytic domains (Fig. 1) [18].
and 27 kDa, consistent with the theoretical molecular weights of the recombinant Xyn11A and Xyn11A-LC, respectively (Fig. 2).
3.3. Characterization of xylanase The optimal temperature of both Xyn11A and Xyn11A-LC was 55 ◦ C, but Xyn11A had higher relative activity than Xyn11ALC from 25 to 50 ◦ C (Fig. 3A). Both Xyn11A and Xyn11A-LC exhibited high activity at a wide pH range (>71.6% activity at pH 5.5–8.5). The optimal pH of Xyn11A was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5– 8.5) with optimal activity at pH 5.5 and 7.5–8.0 (Fig. 3B). These results showed that the CBM of Xyn11A could influence the pH and temperature profiles of the catalytic domain. Xyn11A-LC had good thermal stability, retaining 63.8% residual activity after preincubation in the pH 8.0 buffer without substrate at 60 ◦ C for 30 Min, whereas Xyn11A retained 18.5% residual
3.2. Expression and purification of the recombinant proteins The plasmids pET28a-xyn11A and pET28a-xyn11A-LC were constructed and transformed into E. coli BL21 (DE3) competent cells. The positive clones were induced, cultured, and disrupted to obtain the crude extract, and the recombinant Xyn11A and Xyn11A-LC were purified from the crude extracts to electrophoretic homogeneity by Ni-affinity chromatography. SDS-PAGE analysis exhibited protein bands of approximately 43
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FIG. 2
SDS-PAGE analysis of the purified recombinant Xyn11A and Xyn11A-LC. Lane M, the standard protein molecular mass markers; lanes 1 and 3, the total protein of the induced E. coli BL21 (DE3) harboring plasmid pET28a-Xyn11A and pET28a-Xyn11A-LC, respectively; lanes 2 and 4, purified recombinant Xyn11A and Xyn11A-LC, respectively.
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FIG. 3
Effects of temperature and pH on the activity and stability of the recombinant Xyn11A and Xyn11A-LC. (A) Effect of temperature on the activity of Xyn11A and Xyn11A-LC. The assay was performed at different temperature ranging from 25 to 70 ◦ C in pH 7.5 McIlvaine buffer for 10 Min. (B) Effect of pH on the activity of Xyn11A and Xyn11A-LC. The assay was performed in pH ranging from 4.5 to 10.0 at 55 ◦ C for 10 Min. (C) The thermostability of Xyn11A and Xyn11A-LC. The purified recombinant protein was incubated in 50 mM Tris/HCl (pH 9.0) without substrate at 60 ◦ C and taken out at an interval of 5 Min, and the residual xylanase activities were measured at 55 ◦ C and pH 7.5 (McIlvaine buffer). (D) The pH stability of Xyn11A and Xyn11A-LC. The recombinant protein was diluted in different buffers ranging from pH 3.0 to 12.0 and incubated without substrate at 37 ◦ C for 1 H. The residual xylanase activities were assayed at 55 ◦ C and pH 7.5 (McIlvaine buffer). The maximum value was set as 100%. The absolute values of 100% of Xyn11A and Xyn11A-LC were 3136.4 and 4511.9 U/mg, respectively.
activity under the same condition (Fig. 3C). The pH stability of Xyn11A-LC was similar to that of Xyn11A. They were both alkali tolerant and retained more than 80% residual activity after preincubation in alkaline pH range (pH 8.5–11.0) at 37 ◦ C for 1 H (Fig. 3D). These results indicated that the CBM of
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Xyn11A decreased the thermal stability and negligibly affected the pH stability of the catalytic domain. The effects of various reagents on the activities of Xyn11A and Xyn11A-LC are shown in Table 1. No metal ions used in the test could activate enzyme activities of Xyn11A and Xyn11A-LC. In contrast, most metal ions displayed inhibitory effects to different extents. For example, Hg2+ , Zn2+ , Mn2+ , Co2+ , and Cu2+ almost completely inhibited enzyme activities of Xyn11A and Xyn11A-LC, Ni2+ reduced the enzyme activities of them by about 80%, and Pb2+ reduced enzyme activities of Xyn11A and Xyn11A-LC by 95% and 80%, respectively. However, Ca2+ and Mg2+ showed little or no inhibition effects on the activity of Xyn11A and Xyn11A-LC. For the chemical agents assayed, 5 mM ethylenediaminetetraacetic acid (EDTA) slightly inhibited the activities of the Xyn11A and Xyn11A-LC. For anionic surfactants tested, 5 mM SDS strongly inhibited the enzyme activities of Xyn11A and Xyn11A-LC, retaining only 15.6% and 17.2% residual activities, respectively. The nonionic surfactant, Tween 20, increased the activities of Xyn11A and Xyn11A-LC by about 1.5- and 1.2-fold, respectively. Triton X-100 enhanced the activity of Xyn11A by about 1.4-fold, but it had no effect on Xyn11A-LC. The kinetic parameters of the recombinant Xyn11A and Xyn11A-LC toward beechwood xylan were assayed at 55 ◦ C and pH 7.5. The Km , Vmax , and kcat values of them are shown in Table 2. The Vmax and kcat values of Xyn11A-LC were
Xylanase from Alkaliphilic Bacillus sp. SN5
TABLE 1
Effects of metal ions and chemical reagents on the activities of Xyn11A and Xyn11A-LC
The substrate specificities of Xyn11A and Xyn11A-LC
TABLE 3
Metal ion or chemical agent
Concentration
Xyn11A
Xyn11A-LC
Substrate
No addition
0 mM
100.0 ± 4.2
100.0 ± 3.1
Ca2+ (CaCl2 )
5 mM
73.7 ± 6.6
72.5 ± 3.9
Co2+ (CoCl2 )
5 mM
1.4 ± 1.6
1.4 ± 1.5
(CuCl2 )
5 mM
12.1 ± 0.2
3.7 ± 0.4
Fe2+ (FeSO4 )
5 mM
23.2 ± 4.7
27.1 ± 3.6
5 mM
3.1 ± 1.5
7.1 ± 2.9
5 mM
93.4 ± 4.6
98.4 ± 5.0
5 mM
3.6 ± 6.6
10.8 ± 5.3
5 mM
17.2 ± 4.4
21.2 ± 4.6
(PbAc2 )
5 mM
5.4 ± 7.1
20.2 ± 5.8
Zn2+ (ZnSO4 )
5 mM
3.4 ± 0.8
3.1 ± 3.3
EDTA
5 mM
81.6 ± 5.8
81.8 ± 0.7
SDS
5 mM
15.6 ± 3.9
17.2 ± 2.0
Glycerol
5%
77.2 ± 6.0
64.5 ± 4.5
Tween20
5%
154.3 ± 4.7
125.5 ± 7.9
Triton X100
5%
138.5 ± 3.5
103.0 ± 5.1
Cu
2+
Hg
2+
Relative activity (%)
(HgCl2 )
Mg2+ (MgCl2 ) Mn
2+
(MnCl2 )
Ni2+ (NiSO4 ) Pb
2+
The kinetic parameters of Xyn11A and Xyn11A-LC
TABLE 2 Parameter Specific activity (U/mg) Km (mg/mL) Vmax (µmol/Min/mg) −1
kcat (S )
11A
11A-LC
3,136.4 ± 118.4
4,511.9 ± 79.6
3.6 ± 0.3
3.3 ± 0.2
4,767.0 ± 127.4
7,177.6 ± 186.2
2,971.4 ± 78.6
3,229.6 ± 83.8
higher than those of Xyn11A. The specific activities of Xyn11A and Xyn11A-LC for beechwood xylan were 3136.4 and 4511.9 U/mg, respectively.
3.4. Substrate specificity and hydrolysis products of xylan Xyn11A and Xyn11A-LC exhibited similar substrate specificities. They could degrade xylan from oat spelt, beechwood, and birchwood and both showed higher catalytic activity toward oat spelt xylan than beechwood and birchwood xylan (Table 3). No activity to methyl cellulose, crystalline cellulose,
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Specific activity (U/mg) Xyn11A
Xyn11A-LC
Beechwood xylan
3,136.4 ± 78.4
4,511.9 ± 54.1
Birchwood xylan
1,646.6 ± 31.3
3,979.5 ± 252.7
10,979.6 ± 272.9
6,348.2 ± 45.1
Oatspelt xylan
citrus fruits pectin, maize starch, locust bean gum, or maize amylopectin was observed, indicating that both of them were specific xylanases. The hydrolysis products of xylan from beechwood and xylooligosaccharides by Xyn11A and Xyn11A-LC were detected using HPAEC. Xyn11A was active on xylotriose, xylotetrose, xylopentaose, and xylohexaose but not active on xylobiose, and the hydrolysis products were xylose, xylobiose, and xylotriose, respectively (Figs. 4A–4D). However, Xyn11A was weak in hydrolyzing xylotriose, only a trace amount of xylose and xylobiose was detected after 12 H incubation (Fig. 4A). The major hydrolysis products of beechwood xylan by Xyn11A were xylose, xylobiose, and xylotriose (Fig 4E).The composition of the hydrolysis products of beechwood xylan by Xyn11ALC was similar to that by Xyn11A (data not shown). These results indicated that Xyn11A and Xyn11A-LC were strict endo-xylanase.
3.5. Insoluble substrate degradation and binding assays To investigate the role of the CBM of Xyn11A, the catalytic activity and binding ability of Xyn11A-LC were compared with those of Xyn11A. The activities of Xyn11A and Xyn11A-LC toward insoluble xylan were 57.7% and 37.8% of those toward soluble xylan, respectively. The result showed that the CBM of Xyn11A enhanced the catalytic activity of enzyme toward insoluble substrates. With respect to the binding assays, Xyn11A and Xyn11A-LC were both unable to bind to MC, whereas 65.5% of Xyn11A and 36.1% of Xyn11A-LC could bind to insoluble xylan. These results showed that the CBM of Xyn11A could increase binding ability to insoluble xylan but not to MC.
4. Discussion According to CAZY database (http://www.cazy.org/GH11 all.html), the great majority of GH family 11 xylanases have been isolated from various microorganisms including bacteria, actinomycetes, and fungi [5, 19, 20]. However, only a small number of alkaline- and alkali-tolerant xylanases have been found from alkaliphiles [21–28]. Because alkaline- and alkalitolerant family 11 xylanases have great potential applications
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FIG. 4
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HPAEC analyses of the hydrolysis products by Xyn11A. (A)–(E) The hydrolysis products of xylotriose, xylotetrose, xylopentaose, xylohexaose, and beechwood xylan by recombinant protein, respectively. X1, xylose; X2, xylobiose; X3, xylotriose.
Xylanase from Alkaliphilic Bacillus sp. SN5
in pulp bleaching process, it is very important to explore the cellulase-free xylanases possessing high stability and catalytic activity under alkaline condition. In this study, the catalytic domain Xyn11A-LC showed better thermal stability and higher specific activity to soluble xylan under alkaline conditions than Xyn11A. Xyn11A-LC, as a family 11 alkali-tolerant xylanase with high catalytic activity, represents a promising candidate to be used in paper bleaching process. Xyn11A shared high identity with xylanase XylX (67%) from P. campinasensis BL11 [16] and alkalophilic xylanase XynJ (66%) from Bacillus sp.41M-1 [17], but their enzymatic properties were different. The optimal temperature of Xyn11A, XylX, and XynJ were 55, 60, and 50 ◦ C, respectively; the optimal pH of them was 7.5, 7.0, and 9.0, respectively. Xyn11A retained 18.5% residual activity after preincubation at 60 ◦ C for 30 Min, XylX remained more than 88.6% residual activity at 60 ◦ C for 8 H [16], and XynJ was stable up to 55 ◦ C at pH 9.0 for 30 Min [17]. Xyn11A had good pH stability in alkaline pH range (from pH 8.5 to 11.5), retaining over 80% residual activity after preincubation at 37 ◦ C for 1 H, whereas XylX could keep stable between pH 5 and 9 and failed under extreme alkaline condition [16]. There are no reports about the pH stability of XynJ. XynJ from Bacillus sp.41M-1 was most active at pH 9.0, with Vmax value of 1,100 µmol/Min/mg when using larchwood xylan as the substrate at 37 ◦ C and pH 9.0 [22]. XBD was the catalytic domain of XynJ, and showed the highest specific activity (350 U/mg) toward birchwood xylan at pH 8.5 and 55 ◦ C [24]. Although Xyn11A-LC had the lower optimal pH (7.5–8.0) than XBD, its specific activity (3,310.9 U/mg) toward birchwood xylan at pH 8.5 was almost 10 times higher than that of XBD. Compared with other alkaline xylanases, Xyn11ALC exhibits good alkali tolerance and thermostability under alkaline condition and much higher catalytic activity, but it has lower optimal temperature or pH (Table 4). Most metal ions used in this study inhibited the xylanase activities of Xyn11A and Xyn11A-LC to different extents, consistent with the observation of xylanase XylX from P. campinasensis BL11 [16]. EDTA (5 mM) reduced the enzyme activity of XylX by 50% [16], but had no apparent effect on Xyn11A and Xyn11A-LC. The insensitivity to EDTA made Xyn11A and Xyn11A-LC highly valuable for application in the pulp bleaching process with chelating agent. Surfactants could enhance the enzyme activity because of increasing substrate availability to enzymes. However, surfactants might change enzyme conformation by electrostatic interaction, hydrogen bond, or hydrophobic interaction, resulting in a decrease of catalytic activity [29]. Although both anionic and nonionic surfactants were documented to inhibit the enzyme activity of XylX [16], our results showed that anionic surfactants strongly inhibited the enzyme activities of Xyn11A and Xyn11A-LC, whereas nonionic surfactant increased the enzyme activities to varying degrees.
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Substrate specificity assays demonstrate that Xyn11A and Xyn11A-LC are able to hydrolyze xylan from different sources without activity to other polysaccharides, indicating that they are specific xylanases. The absence of cellulase activity makes Xyn11A and Xyn11A-LC excellent candidates used for pulp bleaching pretreatment. The activities of Xyn11A and Xyn11A-LC toward oat spelt xylan were much higher than those toward birchwood and beechwood xylan, suggesting that they were more suitable for straw pulp bleaching process. Analysis of xylan hydrolysis products indicated that both Xyn11A and Xyn11A-LC hydrolyzed xylan into short xylooligosaccharides and the CBM could not affect the mode of action. The major products of xylan hydrolysis catalyzed by Xyn11A and Xyn11A-LC are xylose, xylobiose, and xylotriose, but hydrolysates of xylan by other reported GH 11 xylanases were xylobiose, xylotriose, and higher oligosaccharides [16, 22]. The lack of activity toward xylobiose suggests that above-mentioned three xylosyl consecutive residues are required for Xyn11A and Xyn11A-LC, whereas at least four sites are required for the hydrolysis of XynJ from Bacillus sp. 41M-1 [22]. The structural basis determining product difference between Xyn11A and other family 11 xylanases is worthy of a further investigation. Although there are a lot of reports on the effect of the CBM on properties of catalytic domains, mainly in family 10, no consistent conclusion was drawn from them. The CBM could improve or decrease the thermal stability, pH stability, optimal pH, or optimal temperature of the catalytic domain, or had no effect on these properties [9–11]. To date, there are few reports about the effect of the CBM on the enzymatic properties of GH family 11 catalytic domains. In this study, the CBM of Xyn11A could affect the enzymatic properties of Xyn11A, especially the pH–activity profile and thermal stability. The CBM of Xyn11A has an unfavorable effect on the thermal stability, presumably because it changes the compact structure of the catalytic domain Xyn11A-LC. It has been documented that CBMs within a family and from different families have different binding specificities [30, 31]. For example, the family 9 CBM of xylanase 10A from Thermotoga maritima (CBM 9-2) can bind to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan [30], whereas the family 6 CBM from Clostridium stercorarium xylanase (XylA) can bind to insoluble oat spelt xylan and acid-swollen cellulose [31]. In this paper, the CBM of Xyn11A could bind to insoluble oat spelt xylan but not to MC. Several studies have shown that the CBM enhances the hydrolytic efficiency of the xylanase against insoluble xylan [10, 31]. Our data also showed that the CBM of Xyn11A enhanced the catalytic activity of the enzyme toward insoluble xylan, presumably because the CBM could increase the concentration of enzyme around the substrate by binding to insoluble xylan.
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216
Bacillus sp. SN5
Bacillus sp.41M-1
Bacillus pumilus BYG
Compost-soil metagenome
T. halotolerans YIM 90462(T)
Bacillus. sp JB 99
Xyn11A-LC
࢞XBD
XynBYG
Mxyl
Xylanase
Xylanase
70
70
80
50
55
55
8.0
8.0
9.0
8.0–9.0
8.5
7.5–8.0
Optimum pH
95% residual activity after 5 H-incubation at 60 ◦ C
85% residual activity at pH 5–9
Over 70% residual activity at pH 7.0 for 150 Min
Stable at pH 8.0–9.0
T1/2 was 2 H at 80 ◦ C and 15 Min at 90 ◦ C Over 90% residual activity after incubation at 70 ◦ C for 30 Min
75% residual activity at pH 9.0 for 120 Min
NA
Over 80% residual activity at pH 8.5–11.0 for 1 H
pH stability
Below 55% of its original activity for 30 Min at 55 ◦ C
NA
54% residual activity after incubation at 90 ◦ C and pH 9.5 for 30 Min
Thermostability
218.6
470.7
300
NA
NA
6,330.6
Vmax (µmol/ Min/mg)
NA, not available.
The values of Vmax and specific activity were determined toward birchwood xylan under respective optimal conditions of every enzyme.
Source
Optimum temperature (◦ C)
Comparison of characteristics of Xyn11A-LC and other alkaline xylanases
Enzyme abbreviation
TABLE 4
NA
NA
NA
NA
350
3,979.5
Specific Activity (U/mg)
4.8
3.5
8.0
NA
NA
3.3
Km (mg/mL)
[28]
[27]
[26]
[25]
[24]
NA
Reference
Biotechnology and Applied Biochemistry
Xylanase from Alkaliphilic Bacillus sp. SN5
5. Conclusions A xylanase gene (xyn11A) was cloned from alkaliphilic Bacillus sp. SN5. The mature xylanase Xyn11A contained a GH family 11 catalytic domain, a linker region, and a family 36 CBM. The catalytic domain Xyn11A-LC showed high activity at a wide pH range (>80% activity at pH 5.5–8.5), good alkali tolerance, and thermal stability and it also showed higher catalytic activity than other known family 11 alkaline xylanases. These favorable properties make Xyn11A-LC an excellent candidate for various industrial applications, especially in paper and pulp industry. A comparative study of enzymatic properties indicated that the CBM of Xyn11A could not only affect the pH profiles and thermal stability of the enzyme, but also increases the binding ability and hydrolytic efficiency to insoluble xylan. Future studies on the structure of Xyn11A and Xyn11A-LC will help us achieve a better understanding about the mechanism of action of the CBM.
6. Acknowledgements This study was supported by the National Basic Research Program of China (2011CBA00800 and 2009CB724700), Chinese National Programs for High Technology Research and Development (2011AA02A206 and 2012AA022100), and the Knowledge Innovative Program of Chinese Academy of Science (KSCX2-EW-G-8).
7. References [1] Berrin, J. G., and Juge, N. (2008) Biotechnol. Lett. 30, 1139–1150. [2] Biely, P. (1985) Trends Biotechnol. 3, 286–290. [3] Collins, T., Gerday, C., and Feller, G. (2005) FEMS Microbiol. Rev. 29, 3–23. [4] Subramaniyan, S., and Prema, P. (2000) FEMS Microbiol. Lett. 183, 1–7. [5] Li, N., Shi, P., Yang, P., Wang, Y., Luo, H., Bai, Y., Zhou, Z., and Yao, B. (2009) Appl. Microbiol. Biotechnol. 83, 99–107. [6] Biely, P., Vrsanska, M., Tenkanen, M., and Kluepfel, D. (1997) J. Biotechnol. 57, 151–166. [7] Boraston, A. B., Creagh, A. L., Alam, M. M., Kormos, J. M., Tomme, P., Haynes, C. A., Warren, R. A., and Kilburn, D. G. (2001) Biochemistry 40, 6240–6247.
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[8] Yazawa, R., Takakura, J., Sakata, T., Ihsanawati, Yatsunami, R., Fukui, T., Kumasaka, T., Tanaka, N., and Nakamura, S. (2011) Biosci. Biotechnol. Biochem. 75, 379–381. [9] Ali, E., Araki, R., Zhao, G., Sakka, M., Karita, S., Kimura, T., and Sakka, K. (2005) Biosci. Biotechnol. Biochem. 69, 2389–2394. [10] Mamo, G., Hatti-Kaul, R., and Mattiasson, B. (2007) Extremophiles 11, 169–177. [11] Shin, E. S., Yang, M. J., Jung, K. H., Kwon, E. J., Jung, J. S., Park, S. K., Kim, J., Yun, H. D., and Kim, H. (2002) Appl. Environ. Microbiol. 68, 3496–3501. [12] Bai, W. Q., Xue, Y. F., Zhou, C., and Ma, Y. H. (2012) Biotechnol. Lett. 34, 2093–2099. [13] Sambrook, J., Fritsch, E. F., and Maniatis, T. (2001). Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [14] Miller, G. L. (1959) Anal. Chem. 31, 426–428. [15] Yang, P., Shi, P., Wang, Y., Bai, Y., Meng, K., Luo, H., Yuan, T., and Yao, B. (2007) J. Microbiol. Biotechnol. 17, 58–66. [16] Ko, C. H., Tsai, C. H., Tu, J., Lee, H. Y., Ku, L. T., Kuo, P. A., and Lai, Y. K. (2010) Process Biochem. 45, 1638–1644. [17] Nakai, R., Wakabayashi, K., Asano, T., Aono, R., and Horikoshi, K. N. S. (1994) Nucleic. Acids Symp. Ser. 31, 235–236. [18] Torronen, A., and Rouvinen, J. (1997) J. Biotechnol. 57, 137–149. [19] Yoon, K. H. (2009) J. Microbiol. Biotechnol. 19, 1514–1519. [20] Liu, W., Shi, P., Chen, Q., Yang, P., Wang, G., Wang, Y., Luo, H., and Yao, B. (2010) Appl. Biochem. Biotechnol. 162, 1–12. [21] Balakrishnan, H., Kamal Kumar, B., Dutta-Choudhury, M., and Rele, M. V. (2002) J. Biochem. Mol. Biol. Biophys. 6, 325–334. [22] Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R., and Horikoshi, K. (1993) Appl. Environ. Microb. 59, 2311–2316. [23] Poon, D. K. Y., Webster, P., Withers, S. G., and McIntosh, L. P. (2003) Carbohydrate. Res. 338, 415–421. [24] Umemoto, H., Ihsanawati, Inami, M., Yatsunami, R., Fukui, T., Kumasaka, T., Tanaka, N., and Nakamura, S. (2009) Biosci. Biotechnol. Biochem. 73, 965–967. [25] Wang, J., Zhang, W. W., Liu, J. N., Cao, Y. L., Bai, X. T., Gong, Y. S., Cen, P. L., and Yang, M. M. (2010) Mol. Biol. Rep. 37, 3297–3302. [26] Verma, D., Kawarabayasi, Y., Miyazaki, K., and Satyanarayana, T. (2013) PloS One 8, e52459. [27] Zhang, F., Chen, J. J., Ren, W. Z., Lin, L. B., Zhou, Y., Zhi, X. Y., Tang, S. K., and Li, W. J. (2012) J. Ind. Microbiol. Biotechnol. 39, 1109–1116. [28] Shrinivas, D., Savitha, G., Raviranjan, K., and Naik, G. R. (2010) Appl. Biochem. Biotechnol. 162, 2049–2057. [29] Liu, Y, Xu, J, and H, H. (2000) Acta Chim. Sin. 58. [30] Boraston, A. B., Bolam, D. N., Gilbert, H. J., and Davies, G. J. (2004) Biochem. J. 382, 769. [31] Mangala, S. L., Kittur, F. S., Nishimoto, M., Sakka, K., Ohmiya, K., Kitaoka, M., and Hayashi, K. (2003) J. Mol. Catal. B Enzym. 21, 221–230.
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Wenqin Bai1,2,3 Yanfen Xue1 Cheng Zhou1 ∗ Yanhe Ma1
1 National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People’s Republic of China 2 National Engineering Lab for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, People’s Republic of China 3 College of Life Science, Shanxi Normal University, Linfen, People’s Republic of China
Abstract A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 ◦ C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5–8.5) with optimal activity at pH 5.5 and 7.5–8.0. They had high alkali tolerance, retaining over 80% residual activity after
preincubation at pH 8.5–11.0 at 37 ◦ C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper C 2014 International Union of Biochemistry and Molecular industry. Biology, Inc. Volume 62, Number 2, Pages 208–217, 2015
Keywords: alkali tolerance, carbohydrate-binding module, catalytic domain, xylanase
1. Introduction Xylan, a major component of hemicelluloses in plant cell walls, is the most abundant renewable polysaccharide in nature [1]. Endo-1, 4-beta-xylanase (EC 3.2.1.8) is the key enzyme-degrading xylan and catalyzes the hydrolysis of β-1,4-xylosidic linkages of xylan into short xylooligosaccha-
Abbreviations: CBMs, carbohydrate-binding module; CTAB, hexadecyltrimethylammonium bromide; DNS, 3,5-dinitrosalicylic acid; GH, glycoside hydrolase; LB, Luria Bertani; MC, microcrystalline cellulose; NA, not available; ORF, open reading frame; PAGE, polyacrylamide gelelectrophoresis.. ∗ Address
for correspondence: Dr. Ma Yanhe, National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, People’s Republic of China. Tel.: +86 10 64807618; Fax: +86 10 64807616; e-mail: [email protected]. Received 6 March 2014; accepted 18 June 2014 DOI: 10.1002/bab.1265 Published online 22 September 2014 in Wiley Online Library (wileyonlinelibrary.com)
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rides [2]. Xylanases have great potential applications in the food, animal feed, textile, paper, and biofuel industries, as well as in the production of xylooligosacharides [3]. In the past decades, xylanases have been increasingly used in the paper industry for bleaching purposes. During the bleaching process, the hydrolysis of xylan by xylanases could facilitate the removal of lignin, which would reduce the amount of toxic chlorinated chemicals (the conventional bleaching agent). Because pulp is processed at high temperature and alkaline pH condition in pulping and bleaching processes, thermostable and alkali-tolerant xylanases have obvious advantages to the minimization of temperature and pH adjustment [4]. Therefore, it is of great interest to discover xylanases that are stable and active under high temperature (about 60 ◦ C) and alkaline (pH 8–10) conditions. Numerous xylanases contain a catalytic domain, one or more carbohydrate-binding modules (CBMs), and/or other functional domains joined by a linker region [5]. On the basis of primary structure alignment and hydrophobic cluster analysis of the catalytic domain, xylanases are mainly
classified into families 10 and 11 of glycoside hydrolase (GH) [3]. Compared with family 10 counterparts, family 11 xylanases have no cellulase activity and lower molecular mass (about 20 kDa) [6]; therefore, they can easily penetrate cellulose fiber network without damaging the fiber, thus more suitable in pulp bleaching process. CBMs are divided into 64 families based on their amino acid sequence similarity (http://www.cazy.org/fam/acc CBM.html). In general, CBMs can increase the binding ability or catalytic activity of enzyme to insoluble substrates [7, 8]. It is also reported that CBMs affect the enzymatic properties such as enzyme stability of catalytic domains [5, 9–11]. In this study, a xylanase gene (xyn11A) was cloned from alkaliphilic Bacillus sp. SN5. The mature polypeptide consisted of a family 11 catalytic domain, a linker region, and a family 36 CBM. The intact Xyn11A and the catalytic domain (Xyn11ALC) were expressed in Escherichia coli and characterized. The results showed that the CBM of Xyn11A could decrease the thermal stability of the enzyme, change the pH–activity profile, and improve the binding ability and hydrolytic activity to insoluble xylan. Xyn11A-LC was a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which made it a promising candidate in the pulp and paper industry.
2. Materials and Methods 2.1. Strains, plasmids, culture conditions, and chemicals Alkaliphilic Bacillus sp. SN5, which exhibited the highest xylanase activity, was isolated from a soda lake in Inner Mongolia, People’s Republic of China [12]. The culture was deposited in the China General Microbiological Culture Collection Center under the accession number CGMCC 1.12485. E. coli DH5α (Invitrogen) and E. coli BL21 (DE3) (Novagen, Darmstadt, Germany) were used as host cells for gene cloning and expression. The E. coli strains were grown at 37 ◦ C in Luria-Bertani (LB) medium with 100 µg/mL ampicillin or 50 µg/mL kanamycin to select plasmid transformants. The pUC18 (TaKaRa, Dalian, People’s Republic of China) and pET28a (Novagen) plasmids were employed as gene cloning and expression vectors, respectively. Oatspelt xylan, birchwood xylan, beechwood xylan, and other polysaccharides were all purchased from Sigma, St. Louis, MO, USA. The standard xylooligosaccharides were purchased from Megazyme (Bray, Ireland). All other reagents used were of the highest purity commercially available.
2.2. Cloning and sequence analysis of the xylanase gene xyn11A Genomic DNA of Bacillus sp. SN5 was prepared from 100 mL of culture. The cell pellet was treated with 200 µg/mL lysozyme for 30 Min at 37 ◦ C, followed by 2% SDS and 100 µg/mL proteinase K for 1 H at 55 ◦ C. After addition of CTAB/NaCl for 10 Min at 65 ◦ C, the DNA was extracted by phenol and chloroform and precipitated with absolute ethanol. The genomic library was
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constructed by standard protocols [13]. Genomic DNA was partially digested with Sau3AI. The DNA fragments between 2 and 8 kb in size were recovered using the gel extraction kit (Omega). The recovered DNA fragments were ligated into the dephosphorylated BamHI site of pUC18 and transformed into the competent E. coli DH5α. Transformants were grown on LAX medium (LB medium containing 100 µg/mL ampicillin and 0.2% [w/v] Remazol Brilliant Blue xylan) [12]. The xylanasepositive clones were identified by showing clear halos around colonies on LAX medium. The DNA insert fragment of positive clone was sequenced by SinoGenoMax Co. (Beijing, People’s Republic of China). The gene sequence was analyzed and compared with sequence databases by using available online tools (http://www.ncbi.nlm.nih.gov/ and http://www.expasy.ch/). The signal peptide was predicted with the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP).
2.3. Construction of expression plasmids To further compare the enzymatic properties of the intact Xyn11A and the truncated Xyn11A-LC, the expression plasmids pET28a-xyn11A (pET28a carrying the mature open reading frame [ORF] region of xyn11A) and pET28a-xyn11A-LC (pET28a carrying the catalytic region of xyn11A) were constructed. The construction procedure was as follows: xyn11A was amplified using the primers F1 (5 -GCATGGATCCCAAATCACTGGAAATGAAATCG-3 ) and R1 (5 -GCTACTCGAGGTGAATTTCTAAGTAGTCGATATAAG3 ). The underlined sequences corresponded to the BamHI and XhoI restriction sites, respectively. Xyn11A-LC was amplified using the primer F1 and the primer R2 (5 GCTACTCGAGTATCGTTAAGTTATTTCGATAAAC-3 ) containing the XhoI restriction site (underlined). The PCR cycling conditions consisted of an initial step of 5 Min at 94 ◦ C, a second step of 30 cycles including 30 Sec at 94 ◦ C, 30 Sec at 55 ◦ C, and 90 Sec at 72 ◦ C and a final extension step of 10 Min at 72 ◦ C. The PCR products were purified using the gel extraction kit (Omega), digested with BamHI and XhoI, and ligated into the pET28a treated with the same restriction endonucleases.
2.4. Expression and purification of the recombinant proteins The recombinant plasmid pET28a-xyn11A was transformed into E. coli BL21 (DE3) competent cells. The positive clone was grown in LB medium containing kanamycin (50 µg/mL) at 37 ◦ C overnight. Then, the culture was inoculated in the abovementioned fresh medium and incubated at 37 ◦ C until OD600 reached 0.6–0.8. After supplemented with a final concentration of 0.5 mM isopropylthiogalactoside, the culture was induced for 4 H. Cells were harvested by centrifugation at 5,000g and 4 ◦ C for 10 Min and disrupted by ultrasonication. The crude R Kits (Novagen) enzyme was purified by using the His·Bind according to the manual. The expression and purification of Xyn11A-LC were the same as described above. The apparent molecular mass and purity of the recombinant Xyn11A and Xyn11A-LC were analyzed by SDS-PAGE. The concentration of
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Biotechnology and Applied Biochemistry the purified recombinant protein was determined by protein assay kit (Bio-Rad).
2.5. Characterization of Xyn11A and Xyn11A-LC The enzyme activities of the purified recombinant Xyn11A and Xyn11A-LC were determined by measuring the amount of reducing sugar released from xylan by the 3,5-dinitrosalicylic acid (DNS) method [14]. The reaction mixtures containing 20 µL of 1 µg/mL enzyme solution and 480 µL of 1% (w/v) beechwood xylan in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 10 Min, followed by adding 0.5 mL of DNS reagent to stop reaction. The mixture was boiled for 5 Min and cooled to room temperature. Then, the absorbance at 540 nm was determined. One unit (U) of xylanase activity was defined as the amount of enzyme required to release 1 µmol reducing sugar from xylan per minute under the assay condition. The assays were all performed in triplicate. The optimal temperature for the xylanase activity was determined over a range of temperatures from 25 to 70 ◦ C at pH 7.5 (McIlvaine buffer). The optimal pH was determined at 55 ◦ C over the range of 4.5–10.0. The buffers used were McIlvaine buffer for pH 4.5–8.0, 0.05 M boric acid/borate for pH 8.0–9.0, and 0.05 M borate/NaOH for pH 9.5–10.0, respectively. For determining the temperature and pH profiles, the maximum value was set as 100%. For thermal stability, the purified recombinant proteins were preincubated in 50 mM Tris–HCl (pH 8.0) without substrate at 60 ◦ C and taken out at an interval of 5 Min. For pH stability, the proteins were diluted in different buffers with pH ranging from 3.0 to 12.0 and preincubated without substrate at 37 ◦ C for 1 H. Three buffer systems, 50 mM McIlvaine buffer (pH 3.0–7.6), 50 mM Tris–HCl (pH 7.6–9.0), and 50 mM glycine–NaOH (pH 9.0–12.0), were used. All the residual activities were measured at 55 ◦ C and pH 7.5. For the thermal and pH stability, the enzyme activity of the purified protein without preincubation was set as 100%. The effects of various reagents on the activities of Xyn11A and Xyn11A-LC were determined by adding a final concentration of 5 mM metal ions, 5 mM or 5% (v/v) surfactants, and 5 mM chelator into the reaction, respectively. The enzyme activity in the reaction without additive was set as 100%. To determine the kinetic parameters, reactions mixtures containing 1 µg/mL purified recombinant protein and varying concentrations of beechwood xylan (1–10 mg/mL) were incubated at 55 ◦ C and pH 7.5 for 5 Min. The Michaelis–Menten parameters were calculated by nonlinear regression using Graphpad Prism 5.0 software (http://www.graphpad.com/prism/). The substrate specificity of the recombinant Xyn11A and Xyn11A-LC were assayed by measuring the release of reducing sugars from the substrates including oatspelt xylan, birchwood xylan, beechwood xylan, methyl cellulose, crystalline cellulose, citrus fruits pectin, maize starch, locust bean gum, and maize amylopectin. For the analysis of the hydrolysis products degraded by Xyn11A and Xyn11A-LC, the reaction mixtures containing 3 µg of purified Xyn11A (or 2 µg of purified Xyn11A-LC) and
210
500 µL of 0.5 mg/mL beechwood xylan or 1 mM xylo-oligose in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 12 H. After incubation, the enzymes were removed from the mixtures using the Nanosep Centrifugal 3 K Device (Pall). The products were detected using high-performance anionexchange chromatography (HPAEC), as described previously [15] with minor modification. After CarboPac PA100 column (Dionex Corp.) was previously equilibrated in 100 mM NaOH at a flow rate of 0.5 mL/Min, the analysis was performed by the wash step of 40 Min consisting of 100 mM NaOH and a gradient of 0–60 mM sodium acetate. Xylose, xylobiose, and xylotriose were used as standards.
2.6. Insoluble substrate degradation and binding assays Insoluble and soluble xylan were made from oat spelt xylan by using a modified method of Yazawa et al. [8]. Three grams of oat spelt xylan was suspended in 60 mL deionized water, and then the suspension was adjusted to pH 10.5 with 1 M NaOH and stirred gently at room temperature for 2 H. Then, the pH was readjusted to 10.5 with 1 M NaOH. The suspension was stirred again for 1 H, boiled, and centrifuged at 6,000g for 10 Min. The supernatant was readjusted pH to 7.5 with 1 M citric acid and dried in vacuum as soluble xylan. The precipitate was washed three times with deionized water and once with McIlvaine buffer (pH 7.5). The pellet was finally washed with ethanol, dried in vacuum, and ground as insoluble xylan. For measuring the enzyme activities of the purified Xyn11A and Xyn11A-LC toward insoluble xylan, the reactions mixtures containing 20 µL of 1 µg/mL enzyme solution and 480 µL of 1% (w/v) insoluble xylan in McIlvaine buffer (pH 7.5) were incubated at 55 ◦ C for 10 Min. The activity of the purified enzymes toward soluble xylan was set as 100%. The binding ability of the purified xylanases to insoluble xylan or microcrystalline cellulose (MC) was assayed as follows: the mixture containing 1 µg/mL purified recombinant protein, 1 mg/mL BSA, 5 mM CaCl2 , and 1% (w/v) insoluble xylan or MC in 50 mM Gly–NaOH (pH 9.5) was shaken gently at 4 ◦ C for 2 H, and then centrifuged at 10,000g for 5 Min. The residual xylanase activity in the supernatant was measured under standard conditions. The loss of enzyme activity implied that enzyme bound to the insoluble xylan or MC. The negative control sample (without substrate) was treated as described above, and the residual enzyme activity in the supernatant was set as 100%.
2.7. Nucleotide sequence accession number The nucleotide sequence of the xylanase gene xyn11A from alkaliphilic Bacillus sp. SN5 has been submitted to GenBank with accession number KC163132.
3. Results 3.1. Gene cloning and sequence analysis Among about 10,000 transformants, five xylanolytic-activitypositive clones showing clear zones on LAX medium were obtained from the genomic library of Bacillus sp. SN5. The
Xylanase from Alkaliphilic Bacillus sp. SN5
FIG. 1
Multiple sequence alignment of xylanases Xyn11A, XylX, and XynJ. Boxes show the two signature patterns belonging to family 11 xylanases. Pentagrams indicate two catalytic residues, Glu120 and Glu210.
sequence analysis revealed that all the insert DNA fragments contained a proposed entire 1,101 bp ORF, designated as xyn11A. It encoded a polypeptide of 366 amino acids, including a predicted N-terminal signal peptide of 27 residues. Blast analysis showed that the polypeptide Xyn11A shared the highest identity (67%) with xylanase XylX from Paenibacillus campinasensis BL11 (GenBank accession No. ABB77852) [16], followed by 66% identity with alkalophilic xylanase XynJ from Bacillus sp. 41M-1 (BAA82316.1) [17]. Multiple sequence alignment of xylanases Xyn11A, XylX, and XynJ is shown in Figure 1. On the basis of homology comparison in GenBank, Xyn11A was a modular xylanase containing a GH family 11 catalytic domain consisting of 200 amino acids (from Gln28 to Ile227) and a family 36 CBM consisting of 119 amino acids (from Ser248 to His366). They were connected by a Gly–Pro-rich linker region consisting of 20 amino acids (from Gly228 to Gly247). Two signature patterns of family 11 xylanases Signature 1: PLVEFYIVDS (from Pro117 to Ser126) and Signature 2: TVEGWQSSGS (from Thr208 to Ser217) were identified in Xyn11A (Fig. 1). Two putative catalytic residues (Glu120 and Glu210) were predicted in the conserved catalytic region by amino acid sequence alignment of Xyn11A with other GH family 11 catalytic domains (Fig. 1) [18].
and 27 kDa, consistent with the theoretical molecular weights of the recombinant Xyn11A and Xyn11A-LC, respectively (Fig. 2).
3.3. Characterization of xylanase The optimal temperature of both Xyn11A and Xyn11A-LC was 55 ◦ C, but Xyn11A had higher relative activity than Xyn11ALC from 25 to 50 ◦ C (Fig. 3A). Both Xyn11A and Xyn11A-LC exhibited high activity at a wide pH range (>71.6% activity at pH 5.5–8.5). The optimal pH of Xyn11A was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5– 8.5) with optimal activity at pH 5.5 and 7.5–8.0 (Fig. 3B). These results showed that the CBM of Xyn11A could influence the pH and temperature profiles of the catalytic domain. Xyn11A-LC had good thermal stability, retaining 63.8% residual activity after preincubation in the pH 8.0 buffer without substrate at 60 ◦ C for 30 Min, whereas Xyn11A retained 18.5% residual
3.2. Expression and purification of the recombinant proteins The plasmids pET28a-xyn11A and pET28a-xyn11A-LC were constructed and transformed into E. coli BL21 (DE3) competent cells. The positive clones were induced, cultured, and disrupted to obtain the crude extract, and the recombinant Xyn11A and Xyn11A-LC were purified from the crude extracts to electrophoretic homogeneity by Ni-affinity chromatography. SDS-PAGE analysis exhibited protein bands of approximately 43
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FIG. 2
SDS-PAGE analysis of the purified recombinant Xyn11A and Xyn11A-LC. Lane M, the standard protein molecular mass markers; lanes 1 and 3, the total protein of the induced E. coli BL21 (DE3) harboring plasmid pET28a-Xyn11A and pET28a-Xyn11A-LC, respectively; lanes 2 and 4, purified recombinant Xyn11A and Xyn11A-LC, respectively.
211
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FIG. 3
Effects of temperature and pH on the activity and stability of the recombinant Xyn11A and Xyn11A-LC. (A) Effect of temperature on the activity of Xyn11A and Xyn11A-LC. The assay was performed at different temperature ranging from 25 to 70 ◦ C in pH 7.5 McIlvaine buffer for 10 Min. (B) Effect of pH on the activity of Xyn11A and Xyn11A-LC. The assay was performed in pH ranging from 4.5 to 10.0 at 55 ◦ C for 10 Min. (C) The thermostability of Xyn11A and Xyn11A-LC. The purified recombinant protein was incubated in 50 mM Tris/HCl (pH 9.0) without substrate at 60 ◦ C and taken out at an interval of 5 Min, and the residual xylanase activities were measured at 55 ◦ C and pH 7.5 (McIlvaine buffer). (D) The pH stability of Xyn11A and Xyn11A-LC. The recombinant protein was diluted in different buffers ranging from pH 3.0 to 12.0 and incubated without substrate at 37 ◦ C for 1 H. The residual xylanase activities were assayed at 55 ◦ C and pH 7.5 (McIlvaine buffer). The maximum value was set as 100%. The absolute values of 100% of Xyn11A and Xyn11A-LC were 3136.4 and 4511.9 U/mg, respectively.
activity under the same condition (Fig. 3C). The pH stability of Xyn11A-LC was similar to that of Xyn11A. They were both alkali tolerant and retained more than 80% residual activity after preincubation in alkaline pH range (pH 8.5–11.0) at 37 ◦ C for 1 H (Fig. 3D). These results indicated that the CBM of
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Xyn11A decreased the thermal stability and negligibly affected the pH stability of the catalytic domain. The effects of various reagents on the activities of Xyn11A and Xyn11A-LC are shown in Table 1. No metal ions used in the test could activate enzyme activities of Xyn11A and Xyn11A-LC. In contrast, most metal ions displayed inhibitory effects to different extents. For example, Hg2+ , Zn2+ , Mn2+ , Co2+ , and Cu2+ almost completely inhibited enzyme activities of Xyn11A and Xyn11A-LC, Ni2+ reduced the enzyme activities of them by about 80%, and Pb2+ reduced enzyme activities of Xyn11A and Xyn11A-LC by 95% and 80%, respectively. However, Ca2+ and Mg2+ showed little or no inhibition effects on the activity of Xyn11A and Xyn11A-LC. For the chemical agents assayed, 5 mM ethylenediaminetetraacetic acid (EDTA) slightly inhibited the activities of the Xyn11A and Xyn11A-LC. For anionic surfactants tested, 5 mM SDS strongly inhibited the enzyme activities of Xyn11A and Xyn11A-LC, retaining only 15.6% and 17.2% residual activities, respectively. The nonionic surfactant, Tween 20, increased the activities of Xyn11A and Xyn11A-LC by about 1.5- and 1.2-fold, respectively. Triton X-100 enhanced the activity of Xyn11A by about 1.4-fold, but it had no effect on Xyn11A-LC. The kinetic parameters of the recombinant Xyn11A and Xyn11A-LC toward beechwood xylan were assayed at 55 ◦ C and pH 7.5. The Km , Vmax , and kcat values of them are shown in Table 2. The Vmax and kcat values of Xyn11A-LC were
Xylanase from Alkaliphilic Bacillus sp. SN5
TABLE 1
Effects of metal ions and chemical reagents on the activities of Xyn11A and Xyn11A-LC
The substrate specificities of Xyn11A and Xyn11A-LC
TABLE 3
Metal ion or chemical agent
Concentration
Xyn11A
Xyn11A-LC
Substrate
No addition
0 mM
100.0 ± 4.2
100.0 ± 3.1
Ca2+ (CaCl2 )
5 mM
73.7 ± 6.6
72.5 ± 3.9
Co2+ (CoCl2 )
5 mM
1.4 ± 1.6
1.4 ± 1.5
(CuCl2 )
5 mM
12.1 ± 0.2
3.7 ± 0.4
Fe2+ (FeSO4 )
5 mM
23.2 ± 4.7
27.1 ± 3.6
5 mM
3.1 ± 1.5
7.1 ± 2.9
5 mM
93.4 ± 4.6
98.4 ± 5.0
5 mM
3.6 ± 6.6
10.8 ± 5.3
5 mM
17.2 ± 4.4
21.2 ± 4.6
(PbAc2 )
5 mM
5.4 ± 7.1
20.2 ± 5.8
Zn2+ (ZnSO4 )
5 mM
3.4 ± 0.8
3.1 ± 3.3
EDTA
5 mM
81.6 ± 5.8
81.8 ± 0.7
SDS
5 mM
15.6 ± 3.9
17.2 ± 2.0
Glycerol
5%
77.2 ± 6.0
64.5 ± 4.5
Tween20
5%
154.3 ± 4.7
125.5 ± 7.9
Triton X100
5%
138.5 ± 3.5
103.0 ± 5.1
Cu
2+
Hg
2+
Relative activity (%)
(HgCl2 )
Mg2+ (MgCl2 ) Mn
2+
(MnCl2 )
Ni2+ (NiSO4 ) Pb
2+
The kinetic parameters of Xyn11A and Xyn11A-LC
TABLE 2 Parameter Specific activity (U/mg) Km (mg/mL) Vmax (µmol/Min/mg) −1
kcat (S )
11A
11A-LC
3,136.4 ± 118.4
4,511.9 ± 79.6
3.6 ± 0.3
3.3 ± 0.2
4,767.0 ± 127.4
7,177.6 ± 186.2
2,971.4 ± 78.6
3,229.6 ± 83.8
higher than those of Xyn11A. The specific activities of Xyn11A and Xyn11A-LC for beechwood xylan were 3136.4 and 4511.9 U/mg, respectively.
3.4. Substrate specificity and hydrolysis products of xylan Xyn11A and Xyn11A-LC exhibited similar substrate specificities. They could degrade xylan from oat spelt, beechwood, and birchwood and both showed higher catalytic activity toward oat spelt xylan than beechwood and birchwood xylan (Table 3). No activity to methyl cellulose, crystalline cellulose,
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Specific activity (U/mg) Xyn11A
Xyn11A-LC
Beechwood xylan
3,136.4 ± 78.4
4,511.9 ± 54.1
Birchwood xylan
1,646.6 ± 31.3
3,979.5 ± 252.7
10,979.6 ± 272.9
6,348.2 ± 45.1
Oatspelt xylan
citrus fruits pectin, maize starch, locust bean gum, or maize amylopectin was observed, indicating that both of them were specific xylanases. The hydrolysis products of xylan from beechwood and xylooligosaccharides by Xyn11A and Xyn11A-LC were detected using HPAEC. Xyn11A was active on xylotriose, xylotetrose, xylopentaose, and xylohexaose but not active on xylobiose, and the hydrolysis products were xylose, xylobiose, and xylotriose, respectively (Figs. 4A–4D). However, Xyn11A was weak in hydrolyzing xylotriose, only a trace amount of xylose and xylobiose was detected after 12 H incubation (Fig. 4A). The major hydrolysis products of beechwood xylan by Xyn11A were xylose, xylobiose, and xylotriose (Fig 4E).The composition of the hydrolysis products of beechwood xylan by Xyn11ALC was similar to that by Xyn11A (data not shown). These results indicated that Xyn11A and Xyn11A-LC were strict endo-xylanase.
3.5. Insoluble substrate degradation and binding assays To investigate the role of the CBM of Xyn11A, the catalytic activity and binding ability of Xyn11A-LC were compared with those of Xyn11A. The activities of Xyn11A and Xyn11A-LC toward insoluble xylan were 57.7% and 37.8% of those toward soluble xylan, respectively. The result showed that the CBM of Xyn11A enhanced the catalytic activity of enzyme toward insoluble substrates. With respect to the binding assays, Xyn11A and Xyn11A-LC were both unable to bind to MC, whereas 65.5% of Xyn11A and 36.1% of Xyn11A-LC could bind to insoluble xylan. These results showed that the CBM of Xyn11A could increase binding ability to insoluble xylan but not to MC.
4. Discussion According to CAZY database (http://www.cazy.org/GH11 all.html), the great majority of GH family 11 xylanases have been isolated from various microorganisms including bacteria, actinomycetes, and fungi [5, 19, 20]. However, only a small number of alkaline- and alkali-tolerant xylanases have been found from alkaliphiles [21–28]. Because alkaline- and alkalitolerant family 11 xylanases have great potential applications
213
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FIG. 4
214
HPAEC analyses of the hydrolysis products by Xyn11A. (A)–(E) The hydrolysis products of xylotriose, xylotetrose, xylopentaose, xylohexaose, and beechwood xylan by recombinant protein, respectively. X1, xylose; X2, xylobiose; X3, xylotriose.
Xylanase from Alkaliphilic Bacillus sp. SN5
in pulp bleaching process, it is very important to explore the cellulase-free xylanases possessing high stability and catalytic activity under alkaline condition. In this study, the catalytic domain Xyn11A-LC showed better thermal stability and higher specific activity to soluble xylan under alkaline conditions than Xyn11A. Xyn11A-LC, as a family 11 alkali-tolerant xylanase with high catalytic activity, represents a promising candidate to be used in paper bleaching process. Xyn11A shared high identity with xylanase XylX (67%) from P. campinasensis BL11 [16] and alkalophilic xylanase XynJ (66%) from Bacillus sp.41M-1 [17], but their enzymatic properties were different. The optimal temperature of Xyn11A, XylX, and XynJ were 55, 60, and 50 ◦ C, respectively; the optimal pH of them was 7.5, 7.0, and 9.0, respectively. Xyn11A retained 18.5% residual activity after preincubation at 60 ◦ C for 30 Min, XylX remained more than 88.6% residual activity at 60 ◦ C for 8 H [16], and XynJ was stable up to 55 ◦ C at pH 9.0 for 30 Min [17]. Xyn11A had good pH stability in alkaline pH range (from pH 8.5 to 11.5), retaining over 80% residual activity after preincubation at 37 ◦ C for 1 H, whereas XylX could keep stable between pH 5 and 9 and failed under extreme alkaline condition [16]. There are no reports about the pH stability of XynJ. XynJ from Bacillus sp.41M-1 was most active at pH 9.0, with Vmax value of 1,100 µmol/Min/mg when using larchwood xylan as the substrate at 37 ◦ C and pH 9.0 [22]. XBD was the catalytic domain of XynJ, and showed the highest specific activity (350 U/mg) toward birchwood xylan at pH 8.5 and 55 ◦ C [24]. Although Xyn11A-LC had the lower optimal pH (7.5–8.0) than XBD, its specific activity (3,310.9 U/mg) toward birchwood xylan at pH 8.5 was almost 10 times higher than that of XBD. Compared with other alkaline xylanases, Xyn11ALC exhibits good alkali tolerance and thermostability under alkaline condition and much higher catalytic activity, but it has lower optimal temperature or pH (Table 4). Most metal ions used in this study inhibited the xylanase activities of Xyn11A and Xyn11A-LC to different extents, consistent with the observation of xylanase XylX from P. campinasensis BL11 [16]. EDTA (5 mM) reduced the enzyme activity of XylX by 50% [16], but had no apparent effect on Xyn11A and Xyn11A-LC. The insensitivity to EDTA made Xyn11A and Xyn11A-LC highly valuable for application in the pulp bleaching process with chelating agent. Surfactants could enhance the enzyme activity because of increasing substrate availability to enzymes. However, surfactants might change enzyme conformation by electrostatic interaction, hydrogen bond, or hydrophobic interaction, resulting in a decrease of catalytic activity [29]. Although both anionic and nonionic surfactants were documented to inhibit the enzyme activity of XylX [16], our results showed that anionic surfactants strongly inhibited the enzyme activities of Xyn11A and Xyn11A-LC, whereas nonionic surfactant increased the enzyme activities to varying degrees.
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Substrate specificity assays demonstrate that Xyn11A and Xyn11A-LC are able to hydrolyze xylan from different sources without activity to other polysaccharides, indicating that they are specific xylanases. The absence of cellulase activity makes Xyn11A and Xyn11A-LC excellent candidates used for pulp bleaching pretreatment. The activities of Xyn11A and Xyn11A-LC toward oat spelt xylan were much higher than those toward birchwood and beechwood xylan, suggesting that they were more suitable for straw pulp bleaching process. Analysis of xylan hydrolysis products indicated that both Xyn11A and Xyn11A-LC hydrolyzed xylan into short xylooligosaccharides and the CBM could not affect the mode of action. The major products of xylan hydrolysis catalyzed by Xyn11A and Xyn11A-LC are xylose, xylobiose, and xylotriose, but hydrolysates of xylan by other reported GH 11 xylanases were xylobiose, xylotriose, and higher oligosaccharides [16, 22]. The lack of activity toward xylobiose suggests that above-mentioned three xylosyl consecutive residues are required for Xyn11A and Xyn11A-LC, whereas at least four sites are required for the hydrolysis of XynJ from Bacillus sp. 41M-1 [22]. The structural basis determining product difference between Xyn11A and other family 11 xylanases is worthy of a further investigation. Although there are a lot of reports on the effect of the CBM on properties of catalytic domains, mainly in family 10, no consistent conclusion was drawn from them. The CBM could improve or decrease the thermal stability, pH stability, optimal pH, or optimal temperature of the catalytic domain, or had no effect on these properties [9–11]. To date, there are few reports about the effect of the CBM on the enzymatic properties of GH family 11 catalytic domains. In this study, the CBM of Xyn11A could affect the enzymatic properties of Xyn11A, especially the pH–activity profile and thermal stability. The CBM of Xyn11A has an unfavorable effect on the thermal stability, presumably because it changes the compact structure of the catalytic domain Xyn11A-LC. It has been documented that CBMs within a family and from different families have different binding specificities [30, 31]. For example, the family 9 CBM of xylanase 10A from Thermotoga maritima (CBM 9-2) can bind to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan [30], whereas the family 6 CBM from Clostridium stercorarium xylanase (XylA) can bind to insoluble oat spelt xylan and acid-swollen cellulose [31]. In this paper, the CBM of Xyn11A could bind to insoluble oat spelt xylan but not to MC. Several studies have shown that the CBM enhances the hydrolytic efficiency of the xylanase against insoluble xylan [10, 31]. Our data also showed that the CBM of Xyn11A enhanced the catalytic activity of the enzyme toward insoluble xylan, presumably because the CBM could increase the concentration of enzyme around the substrate by binding to insoluble xylan.
215
216
Bacillus sp. SN5
Bacillus sp.41M-1
Bacillus pumilus BYG
Compost-soil metagenome
T. halotolerans YIM 90462(T)
Bacillus. sp JB 99
Xyn11A-LC
࢞XBD
XynBYG
Mxyl
Xylanase
Xylanase
70
70
80
50
55
55
8.0
8.0
9.0
8.0–9.0
8.5
7.5–8.0
Optimum pH
95% residual activity after 5 H-incubation at 60 ◦ C
85% residual activity at pH 5–9
Over 70% residual activity at pH 7.0 for 150 Min
Stable at pH 8.0–9.0
T1/2 was 2 H at 80 ◦ C and 15 Min at 90 ◦ C Over 90% residual activity after incubation at 70 ◦ C for 30 Min
75% residual activity at pH 9.0 for 120 Min
NA
Over 80% residual activity at pH 8.5–11.0 for 1 H
pH stability
Below 55% of its original activity for 30 Min at 55 ◦ C
NA
54% residual activity after incubation at 90 ◦ C and pH 9.5 for 30 Min
Thermostability
218.6
470.7
300
NA
NA
6,330.6
Vmax (µmol/ Min/mg)
NA, not available.
The values of Vmax and specific activity were determined toward birchwood xylan under respective optimal conditions of every enzyme.
Source
Optimum temperature (◦ C)
Comparison of characteristics of Xyn11A-LC and other alkaline xylanases
Enzyme abbreviation
TABLE 4
NA
NA
NA
NA
350
3,979.5
Specific Activity (U/mg)
4.8
3.5
8.0
NA
NA
3.3
Km (mg/mL)
[28]
[27]
[26]
[25]
[24]
NA
Reference
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Xylanase from Alkaliphilic Bacillus sp. SN5
5. Conclusions A xylanase gene (xyn11A) was cloned from alkaliphilic Bacillus sp. SN5. The mature xylanase Xyn11A contained a GH family 11 catalytic domain, a linker region, and a family 36 CBM. The catalytic domain Xyn11A-LC showed high activity at a wide pH range (>80% activity at pH 5.5–8.5), good alkali tolerance, and thermal stability and it also showed higher catalytic activity than other known family 11 alkaline xylanases. These favorable properties make Xyn11A-LC an excellent candidate for various industrial applications, especially in paper and pulp industry. A comparative study of enzymatic properties indicated that the CBM of Xyn11A could not only affect the pH profiles and thermal stability of the enzyme, but also increases the binding ability and hydrolytic efficiency to insoluble xylan. Future studies on the structure of Xyn11A and Xyn11A-LC will help us achieve a better understanding about the mechanism of action of the CBM.
6. Acknowledgements This study was supported by the National Basic Research Program of China (2011CBA00800 and 2009CB724700), Chinese National Programs for High Technology Research and Development (2011AA02A206 and 2012AA022100), and the Knowledge Innovative Program of Chinese Academy of Science (KSCX2-EW-G-8).
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