Biotechnol Lett (2014) 36:1495–1501 DOI 10.1007/s10529-014-1512-7
ORIGINAL RESEARCH PAPER
Three-dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions Wenqin Bai • Cheng Zhou • Yanfen Xue Chun-Hsiang Huang • Rey-Ting Guo • Yanhe Ma
•
Received: 22 January 2014 / Accepted: 4 March 2014 / Published online: 30 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The alkaline xylanase Xyn11A-LC from the alkalophilic Bacillus sp. SN5 was expressed in E. coli, purified and crystallized. The crystal structure ˚ . Xyn11A-LC was determined at a resolution of 1.49 A has the b-jelly roll structure typical of family 11 xylanases. To improve its thermostability and thermophilicity, a mutant SB3 was constructed by introducing three arginines on the different sides of the protein surface. SB3 increased the optimum temperature by 5 °C. The wild type and SB3 had the halflives of 22 and 68 min at 65 °C at pH 8.0 (Tris/HCl buffer), respectively. CD spectroscopy revealed that the melting temperature (Tm) of the wild type and SB3 were 55.3 and 66.9 °C, respectively. These results
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1512-7) contains supplementary material, which is available to authorized users. W. Bai C. Zhou Y. Xue Y. Ma (&) National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected] W. Bai e-mail: [email protected] C. Zhou e-mail: [email protected] Y. Xue e-mail: [email protected]
showed that the introduction of arginines enhance the thermophilicity and thermostability of Xyn11A-LC. Keywords Alkaline xylanase Arginines Thermophilicity Thermostability ThreeDimensional Structure X-ray crystallography Xylanase
Introduction Xylanase (EC 3.2.1.8) catalyzes the hydrolysis of b1,4-xylosidic linkages of xylan, the major hemicellulose component in the plant cell wall. Xylanases have attracted increasing attention because of potential industrial applications in the pulp bleaching process, baking industry, animal feed preparation, biofuel
W. Bai School of Life Science, Shanxi Normal University, Linfen 041004, China C.-H. Huang R.-T. Guo National Engineering Lab for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China e-mail: [email protected] R.-T. Guo e-mail: [email protected]
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development and so on (Collins et al. 2005). Thermophilic or thermostable xylanases have obvious advantages in industrial applications where the cooling process is tedious and uneconomical or in which a high temperature is needed to improve the bioavailability and/or solubility of substrates, lowering the viscosity and/or reducing the risk of contamination (Collins et al. 2005). Although a large variety of xylanases have been isolated and studied, most of them are mesophilic. In order to withstand the high temperature environment in many applications, it is required to improve the thermostability and thermophilicity of the existing mesophilic xylanase. To date, a lot of crystal structures of family 11 xylanases have been determined (http://www. cazy.org/GH11_characterized.html). They all display the b-jelly roll structure which is composed of two large b-sheets and one or two helices. The structures of thermophilic xylanases are similar to those of mesophilic xylanases, and the stability of thermophilic xylanases can be achieved by an array of minor modifications throughout the molecule without significant change in the backbone conformation (Collins et al. 2005; Hakulinen et al. 2003; Harris et al. 1997; Turunen et al. 2001, 2002; Umemoto et al. 2009; Wang et al. 2012; Zhang et al. 2010). These modifications could enhance the intramolecular or intermolecular interactions, which lead to a more rigid and stable enzyme. Several studies have shown that surface arginines are beneficial to protein stability and substitution of arginines for other residues can increase the thermostability of many proteins because of hydrogen bonds and/or salt bridges formed by arginines and nearby groups (Chan et al. 2011; Lam et al. 2011; Matsutani et al. 2011; Mrabet et al. 1992; Strub et al. 2004; Turunen et al. 2002; Vogt et al. 1997). Xyn11A-LC is an alkaline mesophilic xylanase from alkalophilic Bacillus sp. SN5 with the highest catalytic activity reported to date. It is a potential candidate for industrial applications, especially in the paper and pulp industry (unpublished data). In this paper, the three-dimensional structure of Xyn11A-LC was determined by X-ray crystallography at a resolu˚ . Based on the structural analysis and tion of 1.49 A comparison with other xylanases, three arginines were introduced into Xyn11A-LC by site-directed mutagenesis. The mutant can improve the thermophilicity and thermostability of the enzyme and is of more potential interest for industrial applications.
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Materials and methods Strains, plasmids, culture conditions, and chemicals Escherichia coli BL21 (DE3) (Novagen) was used as host for gene expression. Plasmid pET28a (Novagen) was employed as gene expression vector. E. coli strains carrying the xylanase expression plasmids were grown at 37 °C in LB medium with 50 lg kanamycin/ ml. Restriction endonucleases, T4 DNA ligase, Pyrobest DNA polymerase were purchased from Takara, Japan. Beechwood xylan was from Sigma. Cloning, expression and purification The expression plasmid pET28a-Xyn11A-LC (pET28a carrying Xyn11A-LC gene) was constructed as follows: using the genomic DNA of Bacillus sp. SN5 as template, the gene Xyn11A-LC was amplified using the primer F (50 -GCATGGATCCCAAATCACTGGA AATGAAATCG-30 ) and R2 (50 -GCTACTCGAGTA TCGTTAAGTTATTTCGATAAAC-30 ). The underlined sequences corresponded to the BamHI and XhoI restriction sites, respectively. The PCR products were purified, digested with BamHI and XhoI and inserted into the pET28a treated with the same restriction endonucleases. The recombinant plasmid pET28a-Xyn11A-LC was transformed into E. coli BL21 (DE3) competent cells. The transformants were grown in LB medium containing 50 lg kanamycin/ml at 37 °C until the OD600 reached 0.6–0.8, and then the culture was induced by 0.5 mM IPTG for 4 h. Cells were harvested by centrifugation (5,0009g, 10 min) and disrupted by ultrasonication. The crude enzyme was purified by using the HisBind kits (Novagen). The purity of the recombinant protein was analyzed by SDS-PAGE. The concentration of purified recombinant protein was measured using a protein assay kit (Bio-Rad, USA). Crystallization The purified recombinant xylanase Xyn11A-LC was crystallized using the sitting-drop vapour diffusion method at 20 °C. Crystal screening of the enzyme was performed using Crystal Screens kit (Hampton Research, California, USA). Drops containing 1 ll 0.5 mg protein
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solution/ml and 1 ll precipitant were equilibrated against 300 ll reservoir solution. Data collection, structure determination and refinement The X-ray diffraction datasets were collected at beam lines BL13B1 of the National Synchrotron Radiation Research Center (NSRRC, Hsinhu, Taiwan) and processed by using the program of HKL2000 (Otwinowski and Minor 1997). Prior to structural refinement, 5 % randomly selected reflections were set aside for calculating Rfree as a monitor. The crystal structure of Xyn11A-LC was determined by the molecular replacement method with the program PHASER (Mccoy et al. 2007), using the structure of the xylanase XynJ from Bacillus sp. 41 M-1 as a search model (PDB code: 2DCK), which have 81.6 % sequence identity with Xyn11A-LC. The model and map were further improved by computational refinement using CNS (Brunger et al. 1998) and Coot (Emsley and Cowtan 2004) programs. Details of the data collection statistics and refinements statistics are given in Supplementary Table 1.
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activity was defined as the amount of enzyme releasing 1 lmol reducing sugar from xylan per min. The optimal temperature for the enzyme activity was determined from 50 to 65 °C in McIlvaine buffer (citric acid/Na2HPO4; pH 7.5) containing 1 % (w/v) beechwood xylan. To measure the half-lives of thermal inactivation, the wild type and mutant were incubated in 50 mM Tris/HCl (pH 8.0) without the substrate at 65 °C and taken out at various time intervals. The residual activity was determined as described earlier (Bai et al. 2012). All the assays were performed in triplicate. Measurement of the melting temperature The melting temperature (Tm) was determined by circular dichroism (CD) spectroscopy (Zhou et al. 2010). A Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, UK) was used to measure the Tm of the wild and mutant from 200 to 260 nm and from 36 to 82 °C. The tests were carried out in 20 mM Tris/HCl (pH 8.0) containing 8 lM protein sample. The analysis of experimental data was done using the Global 3 Analysis Software (http://www.photophysics.com/ software/global-3-analysis-software).
Mutant construction Swiss-PdbViewer (Guex and Peitsch 1997) was used a tool to examine the structure of Xyn11A-LC. The mutation was generated by using PCR primers containing the mutated codons as described elsewhere (Turunen et al. 2001). The plasmid pET28a-Xyn11ALC was used as the template. The PCR cycling conditions consisted of an initial step of 2 min at 95 °C, a second step of 18 cycles including 35 s at 95 °C, 35 s at 58 °C and 6.5 min at 68 °C, and a final extension step of 20 min at 68 °C. The amplification products were digested with DpnI at 37 °C overnight and transformed into E. coli BL21 (DE3) competent cells. The mutant xylanase gene from the transformants was confirmed by sequencing. The purification procedure of the mutant was the same to the above. The purity and concentration of the mutant protein were assayed as described above. Enzyme assay Xylanase activities were determined by the DNS method (see Bai et al. 2012). One unit (U) of xylanase
Results and discussion Crystallization, data collection, structure determination and refinement Crystals of Xyn11A-LC could be formed under several crystallization screening conditions in about a week. The high quality crystals were obtained from the precipitant which contained 0.1 M LiSO4H2O, 0.1 M ADA (pH 6.5), 12 % (w/v) PEG4000 and 2 % (v/v) 2-propanol. The crystals of Xyn11A-LC belong to the tetragonal space group P43, with unit-cell parameters a = b = ˚ , c = 153.7 A ˚ . The crystal structure was deter59.4 A mined by molecular replacement using the structure (PDB code: 2DCK) (Lu et al. 2000) of the xylanase XynJ from alkaliphilic Bacillus sp. 41 M-1 as a search model. The model was refined to a resolution of ˚ . Ramachandran plots show that all the residues 1.49 A are in the allowed regions with acceptable values of bond angle geometry. Details of data collection and refinement statistics of the crystals are summarized in
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Fig. 2 The mutation sites of the SB3. The figure was obtained using PyMOL software Fig. 1 The three-dimensional structure of Xyn11A-LC and the location of three mutations in 3D structure of Xyn11A-LC. The two b-sheets are shown in blue (sheet A) and yellow (sheet B), and the helices are shown in red. The structural features of right hand are labelled as in Torronen et al. (1994)
Supplementary Table 1. The atomic coordinates and structure factors have been submitted to the Protein Data Bank under accession code 4IXL. Overall structure of Xyn11A-LC Xyn11A-LC displays the b-jelly roll structure typical of family 11 xylanases, as shown in Fig. 1. The structure is composed of two b-sheets (A and B) and two helices. Two b-sheets pack against one another. The sheet A consists of five antiparallel b-strands (A2–A6), and the sheet B consists of nine mostly antiparallel b-strands (B1–B9). The hydrophilic face of sheet A is rich in serine and threonine residues, so it is called ‘Ser/Thr surface’. Sheet B is highly twisted and forms the catalytic cleft. Two helices are embedded on the external side of sheet B. The overall structure is also likened to the shape of a ‘right hand’ (Torronen et al. 1994), in which two b-sheets forms ‘fingers’ and the twist part of b-sheet B and the ahelices form a ‘palm’. The loop between the b-strands B7 and B8 forms a ‘thumb’ that partly encloses the catalytic cleft. The long loop between the b-strands B6 and B9 makes a cord that also encloses the cleft from the side (Fig. 1).
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The pH optimum of family 11 xylanases is influenced by the residues adjacent to the acid/base catalyst. This residue is aspartic acid in xylanases that function optimally under acidic condition; while it is asparagines in those that function optimally under more alkaline conditions (Joshi et al. 2000). By sequence and structure comparison of Xyn11A-LC with other family 11 xylanases (Torronen and Rouvinen 1997), the catalytic residues Glu98 and Glu183 locate on the strands B6 and B4, respectively, and the residue close to the acid/base catalyst (Glu183) is Asn44, which is consistent with the alkaline pH optimum (pH 7.5) of Xyn11A-LC. Mutagenesis site selection To improve the thermostability and thermophilicity of Xyn11A-LC, three Arg residues were introduced into the enzyme. Mutational sites were chosen by sequence and structure comparison of Xyn11A-LC with other family 11 xylanases. The sites replaced by arginines were selected on the basis that these sites were occupied by charged residues forming salt bridges with nearby oppositely charged residues in some other family 11 xylanases. In view of the slight effect of single mutation, the combined mutation SB3 was constructed by introducing three arginines (Q51R, T55R and K111R). Three arginines substitution might introduce three putative salt bridges (D14R51, R55-D89 and R111-E125) into the enzyme (Fig. 2).
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1499
120
1.0
Concentration values
Relative activity (%)
WT SB3
100 80 60 40
WT SB3
0.8 0.6 0.4 0.2 0.0
20 50
55
60
40
65
50
60
70
80
Temperature ( oC)
o
Temperature ( C) Fig. 3 Temperature-dependent activity profile of the wild type and the mutant. Their respective maximum value was set as 100 %. The absolute value of 100 % of the wild type and the mutant SB3 were 4,512 and 4,462 U/mg, respectively
Characterization of the mutant and structural analysis The optimal temperature of the mutant SB3 was 60 °C, which was 5 °C higher than that of the wild type (Fig. 3). The Vmax value of the mutant SB3 at the optimal temperature was similar to that of the wild type (Table 1). Xyn11A-LC had a half-time of 22 min at 65 °C at pH 8.0 (Tris/HCl buffer). Under the same condition, the half-time of the mutant SB3 was 68 min (Table 1). CD spectroscopy revealed that the Tm of the wild type and SB3 were 55.3 and 66.9 °C, respectively (Fig. 4). Compared with the wild type, the Tm of SB3 was elevated by 11.6 °C. These results showed that the introduction of arginines could both enhance the thermophilicity and thermostability of Xyn11A-LC. Several studies indicate that there is a correlation between the stability of protein and the number of arginines on the molecular surface of protein (Argos et al. 1979; Strub et al. 2004; Vogt et al. 1997). Compared with the mesophilic counterparts, thermophilic proteins generally increase arginine content, decrease the lysine content and increase the Arg/Lys ratio on their surface (Argos et al. 1979; Vogt et al. 1997).
Fig. 4 The melting temperature measurement using CD spectroscopy. Measurements were from 36 to 82 °C
Mutagenesis studies also indicated that substitution of arginines for other residues could stabilize the protein (Matsutani et al. 2011; Mrabet et al. 1992; Strub et al. 2004). It is proposed that one important reason that arginine can stabilize the protein is the stronger hydrogen bonding between the large guanidinium group of arginine and neighbouring polar group (Borders et al. 1994; Mrabet et al. 1992; Strub et al. 2004). For example, 14 solvent-exposed hydrophobic residues of acetylcholinesterase were replaced by arginine and half of the mutants showed an increased stability because of addition of new hydrogen bonds with the solvent (Strub et al. 2004). Another conceivable reason for the increased stability is the stronger salt bridges formed by arginines and nearby negatively charged group (Matsutani et al. 2011; Mrabet et al. 1992). Many studies have shown that the salt bridges play an important role in protein stabilization (Chan et al. 2011; Kumar et al. 2000; Lam et al. 2011; Vogt et al. 1997). In family 11 xylanases, there is a correlation between alkalophilicity and salt bridges. However, thermophilic xylanases have, on average, no more salt bridges than mesophilic xylanases (Hakulinen et al. 2003). In this paper, three putative salt bridges (D14-R51, R55-D89
Table 1 Comparison of the enzymatic properties of the wild type (WT) and the mutant SB3 Amino acid changes
Optimum temperature (°C)
Tm (°C)
t1/2 at 65 °C (min)
Vmax (lmol/min/mg)
kcat (s-1)
Km (mg/ml)
WT
–
55
55.3
22
7,178
2,690
3.3
SB3
Q51R, T55R, K111R
60
66.9
68
7,166
2,700
3.2
Xylanase
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1500
and R111-E125) were introduced into the molecular surface of Xyn11A-LC by substituting arginines for other residues. The optimal temperature and thermostability of the mutant SB3 were both increased. Furthermore, the mutant SB3 shifted the pH optimum of the wild-type enzyme from 7.5 to 8.0 (data not shown). The putative salt bridge (D14-R51) links the B2 strand with the loop between B3 strand and 310-helix (Fig 1d). Another putative salt bridge (R55-D89) links the loop between the strands B5 and B6 with the loop between the strand B3 and 310-helix. The third putative salt bridge (R111-E125) links the strand B8 with the ‘cord’ between the strands B6 and B9. These interactions may enhance the stability of the local region, which results in the whole conformation stability. In conclusion, the crystal structure of the alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. ˚ . It is the SN5 was determined at a resolution of 1.49 A b-jelly roll structure typical of family 11 xylanases. The structure is composed of two b-sheets packing against each other and two helices. In order to improve the thermophilicity and/or thermostability of Xyn11A-LC, arginines residues were introduced into the enzyme. The results showed that three putative salt bridges formed by introducing arginines could improve the thermostability and the activity at elevated temperature. The mutant is of more potential interest for industrial applications. Acknowledgments 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).
References Argos P, Rossman MG, Grau UM, Zuber H, Frank G, Tratschin JD (1979) Thermal stability and protein structure. Biochemistry 18:5698–5703 Bai WQ, Xue YF, Zhou C, Ma YH (2012) Cloning, expression and characterization of a novel salt-tolerant xylanase from Bacillus sp SN5. Biotechnol Lett 34:2093–2099 Borders CL Jr, Broadwater JA, Bekeny PA, Salmon JE, Lee AS, Eldridge AM, Pett VB (1994) A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens. Protein Sci 3:541–548 Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M,
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Biotechnol Lett (2014) 36:1495–1501 Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921 Chan CH, Yu TH, Wong KB (2011) Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding. PLoS ONE 6:e21624 Collins T, Gerday C, Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29:3–23 Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132 Guex N, Peitsch MC (1997) SWISS-MODEL and the SwissPdb-Viewer: an environment for comparative protein modelling. Electrophoresis 18:2714–2723 Hakulinen N, Turunen O, Janis J, Leisola M, Rouvinen J (2003) Three-dimensional structures of thermophilic beta-1,4xylanases from Chaetomium thermophilum and Nonomuraea flexuosa. Comparison of twelve xylanases in relation to their thermal stability. Eur J Biochem 270:1399–1412 Harris GW, Pickersgill RW, Connerton I, Debeire P, Touzel JP, Breton C, Perez S (1997) Structural basis of the properties of an industrially relevant thermophilic xylanase. Proteins 29:77–86 Joshi MD, Sidhu G, Pot I, Brayer GD, Withers SG, McIntosh LP (2000) Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J Mol Biol 299:255–279 Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191 Lam S, Yeung R, Yu T, Sze K, Wong K (2011) A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity. PLoS Biol 9:e1001027 Lu N, Moriyama H, Nakamura S, Sato T, Tanaka N (2000) Crystallization and initial X-ray analysis of alkaline xylanase. Acta Crystallogr D Biol Crystallogr 56:464–465 Matsutani M, Hirakawa H, Nishikura M, Soemphol W, Ali IA, Yakushi T, Matsushita K (2011) Increased number of Arginine-based salt bridges contributes to the thermotolerance of thermotolerant acetic acid bacteria, Acetobacter tropicalis SKU1100. Biochem Biophys Res Commun 409:120–124 Mccoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674 Mrabet NT, Van den Broeck A, Van den brande I, Stanssens P, Laroche Y, Lambeir AM, Matthijssens G, Jenkins J, Chiadmi M, van Tilbeurgh H et al (1992) Arginine residues as stabilizing elements in proteins. Biochemistry 31:2239–2253 Otwinowski z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Meth Enzymol 276: 307–326 Strub C, Alies C, Lougarre A, Ladurantie C, Czaplicki J, Fournier D (2004) Mutation of exposed hydrophobic amino acids to arginine to increase protein stability. BMC Biochem 5:9 Torronen A, Rouvinen J (1997) Structural and functional properties of low molecular weight endo-1, 4-beta-xylanases. J Biotechnol 57:137–149
Biotechnol Lett (2014) 36:1495–1501 Torronen A, Harkki A, Rouvinen J (1994) Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site. EMBO J 13:2493–2501 Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu X, Rouvinen J, Leisola M (2001) A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1, 4-beta-xylanase II increases the thermal stability through synergism. J Biotechnol 88:37–46 Turunen O, Vuorio M, Fenel F et al (2002) Engineering of multiple arginines into the Ser/Thr surface of Trichoderma reesei endo-1, 4-beta-xylanase II increases the thermotolerance and shifts the pH optimum towards alkaline pH. Protein Eng 15:141–145 Umemoto H, Ihsanawati, Inami M, Yatsunami R, Fukui T, Kumasaka T, Tanaka N, Nakamura S (2009) Improvement of alkaliphily of Bacillus alkaline xylanase by introducing
1501 amino acid substitutions both on catalytic cleft and protein surface. Biosci Biotech Bioch 73:965–967 Vogt G, Woell S, Argos P (1997) Protein thermal stability, hydrogen bonds, and ion pairs. J Mol Biol 269:631–643 Wang Y, Fu Z, Huang H, Zhang H, Yao B, Xiong H, Turunen O (2012) Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge. Bioresour Technol 112:275–279 Zhang S, Zhang K, Chen X, Chu X, Sun F, Dong Z (2010) Five mutations in N-terminus confer thermostability on mesophilic xylanase. Biochem Biophys Res Commun 395: 200–206 Zhou C, Xue Y, Ma Y (2010) Enhancing the thermostability of alpha-glucosidase from Thermoanaerobacter tengcongensis MB4 by single proline substitution. J Biosci Bioeng 110:12–17
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ORIGINAL RESEARCH PAPER
Three-dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions Wenqin Bai • Cheng Zhou • Yanfen Xue Chun-Hsiang Huang • Rey-Ting Guo • Yanhe Ma
•
Received: 22 January 2014 / Accepted: 4 March 2014 / Published online: 30 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The alkaline xylanase Xyn11A-LC from the alkalophilic Bacillus sp. SN5 was expressed in E. coli, purified and crystallized. The crystal structure ˚ . Xyn11A-LC was determined at a resolution of 1.49 A has the b-jelly roll structure typical of family 11 xylanases. To improve its thermostability and thermophilicity, a mutant SB3 was constructed by introducing three arginines on the different sides of the protein surface. SB3 increased the optimum temperature by 5 °C. The wild type and SB3 had the halflives of 22 and 68 min at 65 °C at pH 8.0 (Tris/HCl buffer), respectively. CD spectroscopy revealed that the melting temperature (Tm) of the wild type and SB3 were 55.3 and 66.9 °C, respectively. These results
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1512-7) contains supplementary material, which is available to authorized users. W. Bai C. Zhou Y. Xue Y. Ma (&) National Engineering Lab for Industrial Enzymes, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected] W. Bai e-mail: [email protected] C. Zhou e-mail: [email protected] Y. Xue e-mail: [email protected]
showed that the introduction of arginines enhance the thermophilicity and thermostability of Xyn11A-LC. Keywords Alkaline xylanase Arginines Thermophilicity Thermostability ThreeDimensional Structure X-ray crystallography Xylanase
Introduction Xylanase (EC 3.2.1.8) catalyzes the hydrolysis of b1,4-xylosidic linkages of xylan, the major hemicellulose component in the plant cell wall. Xylanases have attracted increasing attention because of potential industrial applications in the pulp bleaching process, baking industry, animal feed preparation, biofuel
W. Bai School of Life Science, Shanxi Normal University, Linfen 041004, China C.-H. Huang R.-T. Guo National Engineering Lab for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China e-mail: [email protected] R.-T. Guo e-mail: [email protected]
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development and so on (Collins et al. 2005). Thermophilic or thermostable xylanases have obvious advantages in industrial applications where the cooling process is tedious and uneconomical or in which a high temperature is needed to improve the bioavailability and/or solubility of substrates, lowering the viscosity and/or reducing the risk of contamination (Collins et al. 2005). Although a large variety of xylanases have been isolated and studied, most of them are mesophilic. In order to withstand the high temperature environment in many applications, it is required to improve the thermostability and thermophilicity of the existing mesophilic xylanase. To date, a lot of crystal structures of family 11 xylanases have been determined (http://www. cazy.org/GH11_characterized.html). They all display the b-jelly roll structure which is composed of two large b-sheets and one or two helices. The structures of thermophilic xylanases are similar to those of mesophilic xylanases, and the stability of thermophilic xylanases can be achieved by an array of minor modifications throughout the molecule without significant change in the backbone conformation (Collins et al. 2005; Hakulinen et al. 2003; Harris et al. 1997; Turunen et al. 2001, 2002; Umemoto et al. 2009; Wang et al. 2012; Zhang et al. 2010). These modifications could enhance the intramolecular or intermolecular interactions, which lead to a more rigid and stable enzyme. Several studies have shown that surface arginines are beneficial to protein stability and substitution of arginines for other residues can increase the thermostability of many proteins because of hydrogen bonds and/or salt bridges formed by arginines and nearby groups (Chan et al. 2011; Lam et al. 2011; Matsutani et al. 2011; Mrabet et al. 1992; Strub et al. 2004; Turunen et al. 2002; Vogt et al. 1997). Xyn11A-LC is an alkaline mesophilic xylanase from alkalophilic Bacillus sp. SN5 with the highest catalytic activity reported to date. It is a potential candidate for industrial applications, especially in the paper and pulp industry (unpublished data). In this paper, the three-dimensional structure of Xyn11A-LC was determined by X-ray crystallography at a resolu˚ . Based on the structural analysis and tion of 1.49 A comparison with other xylanases, three arginines were introduced into Xyn11A-LC by site-directed mutagenesis. The mutant can improve the thermophilicity and thermostability of the enzyme and is of more potential interest for industrial applications.
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Materials and methods Strains, plasmids, culture conditions, and chemicals Escherichia coli BL21 (DE3) (Novagen) was used as host for gene expression. Plasmid pET28a (Novagen) was employed as gene expression vector. E. coli strains carrying the xylanase expression plasmids were grown at 37 °C in LB medium with 50 lg kanamycin/ ml. Restriction endonucleases, T4 DNA ligase, Pyrobest DNA polymerase were purchased from Takara, Japan. Beechwood xylan was from Sigma. Cloning, expression and purification The expression plasmid pET28a-Xyn11A-LC (pET28a carrying Xyn11A-LC gene) was constructed as follows: using the genomic DNA of Bacillus sp. SN5 as template, the gene Xyn11A-LC was amplified using the primer F (50 -GCATGGATCCCAAATCACTGGA AATGAAATCG-30 ) and R2 (50 -GCTACTCGAGTA TCGTTAAGTTATTTCGATAAAC-30 ). The underlined sequences corresponded to the BamHI and XhoI restriction sites, respectively. The PCR products were purified, digested with BamHI and XhoI and inserted into the pET28a treated with the same restriction endonucleases. The recombinant plasmid pET28a-Xyn11A-LC was transformed into E. coli BL21 (DE3) competent cells. The transformants were grown in LB medium containing 50 lg kanamycin/ml at 37 °C until the OD600 reached 0.6–0.8, and then the culture was induced by 0.5 mM IPTG for 4 h. Cells were harvested by centrifugation (5,0009g, 10 min) and disrupted by ultrasonication. The crude enzyme was purified by using the HisBind kits (Novagen). The purity of the recombinant protein was analyzed by SDS-PAGE. The concentration of purified recombinant protein was measured using a protein assay kit (Bio-Rad, USA). Crystallization The purified recombinant xylanase Xyn11A-LC was crystallized using the sitting-drop vapour diffusion method at 20 °C. Crystal screening of the enzyme was performed using Crystal Screens kit (Hampton Research, California, USA). Drops containing 1 ll 0.5 mg protein
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solution/ml and 1 ll precipitant were equilibrated against 300 ll reservoir solution. Data collection, structure determination and refinement The X-ray diffraction datasets were collected at beam lines BL13B1 of the National Synchrotron Radiation Research Center (NSRRC, Hsinhu, Taiwan) and processed by using the program of HKL2000 (Otwinowski and Minor 1997). Prior to structural refinement, 5 % randomly selected reflections were set aside for calculating Rfree as a monitor. The crystal structure of Xyn11A-LC was determined by the molecular replacement method with the program PHASER (Mccoy et al. 2007), using the structure of the xylanase XynJ from Bacillus sp. 41 M-1 as a search model (PDB code: 2DCK), which have 81.6 % sequence identity with Xyn11A-LC. The model and map were further improved by computational refinement using CNS (Brunger et al. 1998) and Coot (Emsley and Cowtan 2004) programs. Details of the data collection statistics and refinements statistics are given in Supplementary Table 1.
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activity was defined as the amount of enzyme releasing 1 lmol reducing sugar from xylan per min. The optimal temperature for the enzyme activity was determined from 50 to 65 °C in McIlvaine buffer (citric acid/Na2HPO4; pH 7.5) containing 1 % (w/v) beechwood xylan. To measure the half-lives of thermal inactivation, the wild type and mutant were incubated in 50 mM Tris/HCl (pH 8.0) without the substrate at 65 °C and taken out at various time intervals. The residual activity was determined as described earlier (Bai et al. 2012). All the assays were performed in triplicate. Measurement of the melting temperature The melting temperature (Tm) was determined by circular dichroism (CD) spectroscopy (Zhou et al. 2010). A Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, UK) was used to measure the Tm of the wild and mutant from 200 to 260 nm and from 36 to 82 °C. The tests were carried out in 20 mM Tris/HCl (pH 8.0) containing 8 lM protein sample. The analysis of experimental data was done using the Global 3 Analysis Software (http://www.photophysics.com/ software/global-3-analysis-software).
Mutant construction Swiss-PdbViewer (Guex and Peitsch 1997) was used a tool to examine the structure of Xyn11A-LC. The mutation was generated by using PCR primers containing the mutated codons as described elsewhere (Turunen et al. 2001). The plasmid pET28a-Xyn11ALC was used as the template. The PCR cycling conditions consisted of an initial step of 2 min at 95 °C, a second step of 18 cycles including 35 s at 95 °C, 35 s at 58 °C and 6.5 min at 68 °C, and a final extension step of 20 min at 68 °C. The amplification products were digested with DpnI at 37 °C overnight and transformed into E. coli BL21 (DE3) competent cells. The mutant xylanase gene from the transformants was confirmed by sequencing. The purification procedure of the mutant was the same to the above. The purity and concentration of the mutant protein were assayed as described above. Enzyme assay Xylanase activities were determined by the DNS method (see Bai et al. 2012). One unit (U) of xylanase
Results and discussion Crystallization, data collection, structure determination and refinement Crystals of Xyn11A-LC could be formed under several crystallization screening conditions in about a week. The high quality crystals were obtained from the precipitant which contained 0.1 M LiSO4H2O, 0.1 M ADA (pH 6.5), 12 % (w/v) PEG4000 and 2 % (v/v) 2-propanol. The crystals of Xyn11A-LC belong to the tetragonal space group P43, with unit-cell parameters a = b = ˚ , c = 153.7 A ˚ . The crystal structure was deter59.4 A mined by molecular replacement using the structure (PDB code: 2DCK) (Lu et al. 2000) of the xylanase XynJ from alkaliphilic Bacillus sp. 41 M-1 as a search model. The model was refined to a resolution of ˚ . Ramachandran plots show that all the residues 1.49 A are in the allowed regions with acceptable values of bond angle geometry. Details of data collection and refinement statistics of the crystals are summarized in
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Fig. 2 The mutation sites of the SB3. The figure was obtained using PyMOL software Fig. 1 The three-dimensional structure of Xyn11A-LC and the location of three mutations in 3D structure of Xyn11A-LC. The two b-sheets are shown in blue (sheet A) and yellow (sheet B), and the helices are shown in red. The structural features of right hand are labelled as in Torronen et al. (1994)
Supplementary Table 1. The atomic coordinates and structure factors have been submitted to the Protein Data Bank under accession code 4IXL. Overall structure of Xyn11A-LC Xyn11A-LC displays the b-jelly roll structure typical of family 11 xylanases, as shown in Fig. 1. The structure is composed of two b-sheets (A and B) and two helices. Two b-sheets pack against one another. The sheet A consists of five antiparallel b-strands (A2–A6), and the sheet B consists of nine mostly antiparallel b-strands (B1–B9). The hydrophilic face of sheet A is rich in serine and threonine residues, so it is called ‘Ser/Thr surface’. Sheet B is highly twisted and forms the catalytic cleft. Two helices are embedded on the external side of sheet B. The overall structure is also likened to the shape of a ‘right hand’ (Torronen et al. 1994), in which two b-sheets forms ‘fingers’ and the twist part of b-sheet B and the ahelices form a ‘palm’. The loop between the b-strands B7 and B8 forms a ‘thumb’ that partly encloses the catalytic cleft. The long loop between the b-strands B6 and B9 makes a cord that also encloses the cleft from the side (Fig. 1).
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The pH optimum of family 11 xylanases is influenced by the residues adjacent to the acid/base catalyst. This residue is aspartic acid in xylanases that function optimally under acidic condition; while it is asparagines in those that function optimally under more alkaline conditions (Joshi et al. 2000). By sequence and structure comparison of Xyn11A-LC with other family 11 xylanases (Torronen and Rouvinen 1997), the catalytic residues Glu98 and Glu183 locate on the strands B6 and B4, respectively, and the residue close to the acid/base catalyst (Glu183) is Asn44, which is consistent with the alkaline pH optimum (pH 7.5) of Xyn11A-LC. Mutagenesis site selection To improve the thermostability and thermophilicity of Xyn11A-LC, three Arg residues were introduced into the enzyme. Mutational sites were chosen by sequence and structure comparison of Xyn11A-LC with other family 11 xylanases. The sites replaced by arginines were selected on the basis that these sites were occupied by charged residues forming salt bridges with nearby oppositely charged residues in some other family 11 xylanases. In view of the slight effect of single mutation, the combined mutation SB3 was constructed by introducing three arginines (Q51R, T55R and K111R). Three arginines substitution might introduce three putative salt bridges (D14R51, R55-D89 and R111-E125) into the enzyme (Fig. 2).
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1499
120
1.0
Concentration values
Relative activity (%)
WT SB3
100 80 60 40
WT SB3
0.8 0.6 0.4 0.2 0.0
20 50
55
60
40
65
50
60
70
80
Temperature ( oC)
o
Temperature ( C) Fig. 3 Temperature-dependent activity profile of the wild type and the mutant. Their respective maximum value was set as 100 %. The absolute value of 100 % of the wild type and the mutant SB3 were 4,512 and 4,462 U/mg, respectively
Characterization of the mutant and structural analysis The optimal temperature of the mutant SB3 was 60 °C, which was 5 °C higher than that of the wild type (Fig. 3). The Vmax value of the mutant SB3 at the optimal temperature was similar to that of the wild type (Table 1). Xyn11A-LC had a half-time of 22 min at 65 °C at pH 8.0 (Tris/HCl buffer). Under the same condition, the half-time of the mutant SB3 was 68 min (Table 1). CD spectroscopy revealed that the Tm of the wild type and SB3 were 55.3 and 66.9 °C, respectively (Fig. 4). Compared with the wild type, the Tm of SB3 was elevated by 11.6 °C. These results showed that the introduction of arginines could both enhance the thermophilicity and thermostability of Xyn11A-LC. Several studies indicate that there is a correlation between the stability of protein and the number of arginines on the molecular surface of protein (Argos et al. 1979; Strub et al. 2004; Vogt et al. 1997). Compared with the mesophilic counterparts, thermophilic proteins generally increase arginine content, decrease the lysine content and increase the Arg/Lys ratio on their surface (Argos et al. 1979; Vogt et al. 1997).
Fig. 4 The melting temperature measurement using CD spectroscopy. Measurements were from 36 to 82 °C
Mutagenesis studies also indicated that substitution of arginines for other residues could stabilize the protein (Matsutani et al. 2011; Mrabet et al. 1992; Strub et al. 2004). It is proposed that one important reason that arginine can stabilize the protein is the stronger hydrogen bonding between the large guanidinium group of arginine and neighbouring polar group (Borders et al. 1994; Mrabet et al. 1992; Strub et al. 2004). For example, 14 solvent-exposed hydrophobic residues of acetylcholinesterase were replaced by arginine and half of the mutants showed an increased stability because of addition of new hydrogen bonds with the solvent (Strub et al. 2004). Another conceivable reason for the increased stability is the stronger salt bridges formed by arginines and nearby negatively charged group (Matsutani et al. 2011; Mrabet et al. 1992). Many studies have shown that the salt bridges play an important role in protein stabilization (Chan et al. 2011; Kumar et al. 2000; Lam et al. 2011; Vogt et al. 1997). In family 11 xylanases, there is a correlation between alkalophilicity and salt bridges. However, thermophilic xylanases have, on average, no more salt bridges than mesophilic xylanases (Hakulinen et al. 2003). In this paper, three putative salt bridges (D14-R51, R55-D89
Table 1 Comparison of the enzymatic properties of the wild type (WT) and the mutant SB3 Amino acid changes
Optimum temperature (°C)
Tm (°C)
t1/2 at 65 °C (min)
Vmax (lmol/min/mg)
kcat (s-1)
Km (mg/ml)
WT
–
55
55.3
22
7,178
2,690
3.3
SB3
Q51R, T55R, K111R
60
66.9
68
7,166
2,700
3.2
Xylanase
123
1500
and R111-E125) were introduced into the molecular surface of Xyn11A-LC by substituting arginines for other residues. The optimal temperature and thermostability of the mutant SB3 were both increased. Furthermore, the mutant SB3 shifted the pH optimum of the wild-type enzyme from 7.5 to 8.0 (data not shown). The putative salt bridge (D14-R51) links the B2 strand with the loop between B3 strand and 310-helix (Fig 1d). Another putative salt bridge (R55-D89) links the loop between the strands B5 and B6 with the loop between the strand B3 and 310-helix. The third putative salt bridge (R111-E125) links the strand B8 with the ‘cord’ between the strands B6 and B9. These interactions may enhance the stability of the local region, which results in the whole conformation stability. In conclusion, the crystal structure of the alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. ˚ . It is the SN5 was determined at a resolution of 1.49 A b-jelly roll structure typical of family 11 xylanases. The structure is composed of two b-sheets packing against each other and two helices. In order to improve the thermophilicity and/or thermostability of Xyn11A-LC, arginines residues were introduced into the enzyme. The results showed that three putative salt bridges formed by introducing arginines could improve the thermostability and the activity at elevated temperature. The mutant is of more potential interest for industrial applications. Acknowledgments 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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