Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis

Enzyme and Microbial Technology 130 (2019) 109363

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Increase in the thermostability of GH11 xylanase XynJ from Bacillus sp. strain 41M-1 using site saturation mutagenesis

T

Teisuke Takitaa, Kota Nakatania, Yuta Katanoa, Manami Suzukia, Kenji Kojimaa, Naoki Sakab, ⁎ Bunzo Mikamib, Rie Yatsunamic, Satoshi Nakamurac, Kiyoshi Yasukawaa, a

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan c Department of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama, 226-8501, Japan b

ARTICLE INFO

ABSTRACT

Keywords: Crystallographic analysis GH11 Site saturation mutagenesis library Thermostability Xylanase

GH11 xylanase XynJ from Bacillus sp. strain 41M-1 has a β-jellyroll fold composed of eight β strands with a deep active-site cleft. We hypothesized that the thermostability of XynJ will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity. To verify this hypothesis, we introduced random mutations into Tyr13–Arg104 and Gly169–Tyr194, both of which are located in the β-jellyroll fold of XynJ, to construct a site saturation mutagenesis library. By screening 576 clones followed by site saturation mutation analysis of Thr82, T82A was selected as the most thermostable variant. In the hydrolysis of beechwood xylan at pH 7.8, the temperatures required to reduce initial activity by 50% in 15 min were 61 °C for the wild-type XynJ (WT) and 65 °C for T82A. The optimum hydrolysis temperatures were 60 °C for WT and 65 °C for T82A. There was little difference in the kcat and Km values and the pH dependence of activity between WT and T82A. Crystallographic analysis of WT and T82A revealed that thermostabilization by the T82A mutation might result from the removal of unfavorable van der Waals interactions. Thus, a highly thermostable XynJ variant was generated without impairing activity using this mutation strategy.

1. Introduction Xylanase [EC 3.2.1.8] is a glycoside hydrolase (GH) that catalyzes the hydrolysis of internal β-1,4 linkages in xylan, the second most abundant woody polysaccharide. Xylanases from fungi such as Aspergillus spp. and those from bacteria such as Bacillus spp. are extensively used in the paper and pulp, food, and biofuel industries [1–3]. Most xylanases have been classified as GH10 or GH11 xylanases [4]. GH10 xylanase has a (β/α)8 TIM barrel fold with a shallow active-site cleft, while GH11 xylanase has a β-jellyroll fold composed of eight β strands with a deep active-site cleft [5]. Both xylanases have two catalytic Glu residues in the active site, but they do not have sequence similarity. Industrial reactions with xylanase are carried out under high temperature and pH conditions, necessitating the development of xylanases with high stability under such conditions [6]. Various protein engineering methods have been used to increase the thermostabilities of GH10 [7–14] and GH11 [15–20] xylanases, including the mutation of active-site aromatic residues [7,8], engineering of the N- or C-terminal

flexible loop [9,10,14], replacement of amino acid residues in the external α-helix with proline or glutamate [11], replacement of proline and glutamate [13], replacement of amino acid residues on the molecular surface with arginine [16], introduction of disulfide bridges [17–19], random mutations [12], and computational library design [20]. The alkaliphilicity of a GH11 xylanase has also been improved by the engineering of salt bridges in the catalytic cleft [16]. Site saturation mutagenesis library is a random mutation methods. Unlike error-prone PCR, this method can randomize a set of codons at any one of the amino acid residues in a target region and produce a library of variants in which one amino acid residue is substituted by one of the other 19 amino acid residues [21–23]. Using this method, we previously generated the thermostable variant D200C of Moloney murine leukemia virus (MMLV) reverse transcriptase [24]. We also generated the thermostable variant S92E of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 [25]. In the thermostabilization of MMLV RT, protein regions playing an important role in stability cannot be specified. Thus, 400 amino acid residues (Ala70−Arg469) corresponding to 62% of the entire 648 residues were set as a target for mutation. In the

Abbreviations: DNS, 3,5-dinitrosalicylic acid; RBB-xylan, Remazol Brilliant Blue-xylan ⁎ Corresponding author. E-mail address: [email protected] (K. Yasukawa). https://doi.org/10.1016/j.enzmictec.2019.109363 Received 14 March 2019; Received in revised form 29 May 2019; Accepted 17 June 2019 Available online 18 June 2019 0141-0229/ © 2019 Elsevier Inc. All rights reserved.

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Fig. 1. Structure of XynJ. (A) Primary structure of whole protein (GenBank accession no. AB029319.1). The dashed box indicates the catalytic domain. The β strands are underlined. The three targeting regions (Tyr13–Thr58, His59–Arg104, and Gly169–Tyr194) are colored (in orange, green, and blue, respectively). Two catalytic Glu residues (Glu93 and Glu183) are colored in red, while Thr82 are colored in magenta. (B) Tertiary structure of the catalytic region Ala1–Gly201 (PDB entry 2DCJ). The colors of the three targeting regions, the two catalytic Glu residues, and Thr82 are the same in (A) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

thermostabilization of XynR, we hypothesized that the substrate binding region of XynR is flexible and that the thermostability of XynR will increase if the flexibility of the substrate binding region is decreased without impairing the substrate binding ability. Thus, 99 amino

acid residues (Tyr43–Lys115 and Ala300–Asn325) corresponding to 28% of the entire 351 residues were set as the target. XynJ is a thermophilic and alkaline GH11 xylanase, identified in the culture broth of Bacillus sp. strain 41M-1 isolated from soil sample from 2

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Chiba, Japan [26]. The molecular mass of XynJ is 36 kDa [26]. The XynJ gene was cloned from strain 41M-1 [27]. Sequence analysis reveals that XynJ is synthesized as a preprotein consisting of a leader peptide (27 residues) and a mature sequence (327 residues) (Fig. 1A, B). The mature sequence consists of the catalytic domain (Ala1−Pro222) and xylan-binding domain (Tyr223−Arg327). Crystallographic analysis of Escherichia coli-produced recombinant protein revealed that XynJ has a β-jellyroll fold composed of eight β strands with a deep active-site cleft (unpublished; PDB entry 2DCJ). Site-specific mutagenesis study has revealed several residues thought to be involved in the thermostability and pH dependence of its activity [27,28]. Considering that high stability at high temperature and pH conditions is important for the industrial use of xylanase, thermostabilization is an attractive strategy. Accordingly, in this study, we selected XynJ as a target for thermostabilization. As can be seen from Fig. 1, ca. 30% of the amino acid residues of XynJ are involved in β-sheets, and the β-jellyroll fold occupies a large part of the catalytic domain. In the thermostabilization of enzymes by mutations, β-sheets are not typically regarded as promising target regions since they are, in general, fairly rigid in well-folded proteins. However, it should be noted that the mechanism by which β-sheets are stabilized is very complex. The stability of β-sheets is governed by multiple factors such as the position of side chains, van der Waals interactions, salt bridges, and hydrogen bonds bonding [29]. The optimal residue at a specific residue position varies depending on the surrounding structure [30,31]. This raises the possibility that the β-strands in the β-jellyroll fold of XynJ have the potential to be improved, leading to the hypothesis that the thermostability of XynJ may be increased if the flexibility of the β strands in the jellyroll structure is decreased without impairing the substrate binding and catalytic ability. This could be achieved by increasing (or decreasing) interactions that are favorable (or unfavorable) for stability. Based on this hypothesis, we attempted to increase the thermostability of XynJ using a site saturation mutagenesis library.

oligonucleotides listed in Table S1 as primers. Transformation and expression were carried out as described above. 2.3. Hydrolysis of RBB-xylan Hydrolysis of Remazol Brilliant Blue (RBB)-xylan was measured as described previously [25,32]. Briefly, the reaction (50 μl) was carried out in 225 mM Tris−HCl buffer (pH 7.8), 0.45% w/v RBB-xylan (Sigma, St. Louis, MO), 10% v/v E. coli extracts at 37 °C. After 15 min, 50 μl of 99% ethanol v/v was added to the reaction mixture. The reaction mixture was incubated at 4 °C for 15–30 min and centrifuged (3200 × g, 5 min, 4 °C). An aliquot (100 μl) of the supernatants was collected and added to 100 μl of water, and the absorbance at 540 nm (A540) was measured with an EnSight Multimode Plate Reader (PerkinElmer, Waltham, MA). Based on the results, relative activity of each clone was calculated by the following terms.

[(A540 for the reaction w ith each clone)-(A540 for the reaction with buffer)] [(A540 for the reaction with WT)-(A540 for the reaction with buffer)] 2.4. Preparation of purified XynJ Preparation of purified WT and T82A was carried out as described previously [25]. Their concentrations were determined using Protein Assay CBB Solution (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin (Nacalai Tesque) as a standard. 2.5. SDS-PAGE

2. Materials and methods

Samples were mixed with five volumes of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (0.25 M Tris−HCl buffer (pH 6.8), 50% v/v glycerol, 10% w/v SDS, 5% v/v 2-mercaptoethanol, 0.05% w/v bromophenol blue) and were boiled for 10 min. The solution (10 μl) was applied to a 12.5% w/v polyacrylamide gel. After electrophoresis, gels were stained with 0.25% Coomassie Brilliant Blue R-250, 50% methanol, and 7% acetic acid.

2.1. Screening of thermostable XynJ variants

2.6. Hydrolysis of beechwood xylan

Site saturation mutagenesis library was constructed using QuikChange HT Protein Engineering System (Agilent Technologies, Santa Clara, CA), as reported previously [24,25]. Briefly, three oligonucleotide sets each targeting Tyr13–Thr58, His59–Arg104, and Gly169–Tyr194 were designed, synthesized by Agilent Technology with a microarray technology, and amplified by PCR. Each of amplified products was used as primers for the thermal cycling reaction with the wild-type XynJ expression plasmid pET-21b(+)-XynJ (Fig. S1) as a template. Amplified products were transformed into E. coli strain BL21(DE3). The overnight culture of each transformant (15 μl) in a 96-well deep plate was added to 500 μl of LB broth containing 50 μg/ml ampicillin in another 96-well deep plate and incubated at 37 °C with shaking. When OD660 reached 0.3, 12.5 μl of 5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added. After the cultivation at 37 °C for 2 h, the cultural material was centrifuged (3200 × g, 10 min, 4 °C). The precipitates were collected and suspended in 100 μl of BugBuster Protein Extraction Reagent (Merck Bioscience, Tokyo, Japan) containing 0.025 units/ml Benzonase (Merck Bioscience). After the incubation at room temperature for 20 min, the suspension was centrifuged (3200 × g, 20 min, 4 °C). The supernatants were collected and stored at 4 °C before use as E. coli extracts.

Hydrolysis of beechwood xylan was measured as described previously [15,25,26]. Briefly, the reaction (200 μl) was carried out in 225 mM Tris−HCl buffer (pH 7.8), 0.23–2.43% w/v beechwood xylan (Megazyme, Bray, Ireland), and 0.1 μM of WT or T82A at 37 °C. An aliquot (40 μl) was taken from the reaction solution at predetermined times and immediately added to 40 μl of DNS solution (0.5% w/v 3,5dinitrosalicylic acid (Nacalai Tesque), 1.6% w/v NaOH, 30% w/v potassium sodium tartrate). The solution was incubated at 100 °C for 10 min and at 4 °C for 15 min. Then, 80 μl of the solution and 120 μl of water were mixed, and A540 was measured with an EnSight appratus. The initial reaction rate was estimated from the time-course of the production of reducing sugars with xylose as a standard. 2.7. Irreversible thermal inactivation of XynJ XynJ (0.1 μM) was incubated at predetermined temperature in 20 mM Tris−HCl buffer (pH 7.4) for specified durations followed by the incubation on ice for 5 min. The remaining beechwood xylan-hydrolyzing activity was determined at 37 °C as described above. Assuming that the thermal inactivation reaction of xylanase is irreversible and consists of only one step, thermodynamic analysis was made. 2.8. Crystallographic and X-ray diffraction of XynJ

2.2. Preparation of Thr82 variants

One μl of protein solution (26.3 mg ml–1 of WT or 28.0 mg ml–1 of T82A in 20 mM Tris−HCl buffer (pH 7.4)) was mixed with 1 ul of reservoir solution and was equilibrated against 100 μl of reservoir

For the expression of Thr82 variants, site-directed mutations were introduced into pET-21b(+)-XynJ using QuikChange method with the 3

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solution at 20 °C using the sitting drop vapour-diffusion method in a 96well plate. The reservoir solution for WT contained 0.1 M HEPES-NaOH buffer (pH 7.8), 0.35 M sodium citrate, and 10% 2-methyl-2,4-pentanediol, while that for T82A contained 0.1 M HEPES-NaOH buffer (pH 7.5), 0.2 M sodium citrate tribasic dehydrate, and (+/-) 2,4-pentanediol. Crystals, obtained after a few weeks, were briefly transferred into a cryoprotectant solution consisting of 10–30% v/v ethylene glycol in mother solution, and then flash-cooled. Diffraction data were collected using synchrotron radiation on beamlines BL26B1 and BL44XU at Spring-8, Hyogo, Japan, after an in-house diffraction check using Cu Kα radiation generated by a MAC Science M18XHF rotating–anode generator and a Bruker HI-STAR multiwire area detector. The collected diffraction data were processed with HKL-2000 [33].

or decreased the activity. We selected 328 clones that exhibited 70% or more relative activity. We next investigated the thermostabilities of WT and the 328 clones based on Scheme 1:

N

3. Results and discussion 3.1. Screening of thermostable XynJ variants XynJ consists of 14 β-sheets, designated as β2–β15 in Bai et al. [39] and A2–A6 and B1–B9 in Watanabe et al. [13]. In this paper, the nomenclature for β-sheets is based on that by Bai et al. [39]. The targeting regions Tyr13–Thr58 and His59–Arg104 contain β3–β9, and that Gly169–Tyr194 contain β14 and β15. WT and each of the 576 clones (192 for Tyr13–Thr58, His59–Arg104, and Gly169–Tyr194) were expressed in E. coli using a 96-well deep plate. Then, the RBB-xylan-hydrolyzing activities of the E. coli extracts were measured. Table 1 shows the distribution for the number of clones with the same relative activity (activity compared to that of WT) for the 576 clones. The distribution profile was the valley-shaped for ≧90% of 265 clones (46%) and 0–5% of 129 clones (22%), indicating that a number of mutations eliminated

Residual activity after treatment at 65 °C (%)c

Number of clones out of the 328 clones

0≦x < 5 5 ≦ x < 10 10 ≦ x < 15 15 ≦ x < 20 20 ≦ x < 25 25 ≦ x < 30 30 ≦ x < 35 35 ≦ x < 40 40 ≦ x < 45 45 ≦ x < 50 50 ≦ x < 55 55 ≦ x < 60 60 ≦ x < 65 65 ≦ x < 70 70 ≦ x < 75 75 ≦ x < 80 80 ≦ x < 85 85 ≦ x < 90 90 ≦ x

129 (22)b 20 (5.5) 17 (3.0) 11 (1.9) 4 (0.7) 8 (1.4) 7 (1.2) 7 (1.2) 6 (1.0) 9 (1.6) 5 (0.9) 7 (1.2) 8 (1.4) 10 (1.7) 9 (1.6) 13 (2.3) 20 (3.5) 21 (3.6) 265 (46)

0≦x < 5 5 ≦ x < 10e 10 ≦ x < 15 15 ≦ x < 20 20 ≦ x < 25 25 ≦ x < 30 30 ≦ x < 35 35 ≦ x < 40 40 ≦ x < 45 45 ≦ x < 50 50 ≦ x < 55 55 ≦ x < 60 60 ≦ x < 65 65 ≦ x < 70 70 ≦ x < 75 75 ≦ x < 80 80 ≦ x < 85 85 ≦ x < 90 90 ≦ x

231 (70)d 40 (12) 18 (5.5) 11 (3.4) 6 (1.8) 5 (1.5) 5 (1.5) 1 (0.3) 3 (0.9) 2 (0.6) 0 (0) 1 (0.3) 1 (0.3) 0 (0) 1 (0.3) 2 (0.6) 0 (0) 0 (0) 1 (0.3)

a b c d e

(Scheme 1)

3.2. Analysis of thermostability of WT and T82A WT and T82A were expressed in the E. coli cells using a flask and purified from the cells by the anion exchange column chromatography. Upon SDS-PAGE of the purified preparations, WT and T82A yielded a single band with a molecular mass of 36 kDa (Fig. S2). These preparations were used for subsequent analysis. We investigated the residual beechwood xylan-hydrolyzing activities at pH 7.8 at 37 °C of WT and T82A that had been heat-treated for 15 min. The results for WT treated at 60–65 °C and T82A treated at 60–71 °C are shown in Fig. 3A. Both plots show the similar sigmoid curves. The residual activities of WT and T82A are nearly zero at 64 and 67 °C, respectively. The temperatures required to reduce initial activity by 50% (T50) were 61.8 °C for WT and 65.5 °C for T82A, indicating that ΔT50 for the mutation of Thr82 into Ala was +3.7 °C. We investigated the time-course of the thermal inactivation of WT at 61–65 °C and those of T82A at 64–68 °C. After heat treatment, the residual beechwood xylan-hydrolyzing activity at pH 7.8 at 37 °C was assessed. The results for WT treated at 61, 63, or 65 °C and for T82A treated at 65, 66, or 67 °C are shown in Fig. 3B, and others not shown. The natural logarithm of the residual activity plotted against the incubation time gave linear relationships at all temperatures examined, indicating that the inactivation followed pseudo-first-order kinetics. When incubated at 65 °C, the residual activity of WT decreased rapidly and fell below 10% by 5 min, while that of T82A decreased slowly and reached 45% at 45 min, indicating that T82A is more thermostable than WT. Fig. 3C shows an Arrhenius plot of kobs of the thermal inactivation of WT at 61–65 °C or T82A at 64–68 °C. The natural logarithm of kobs against 1/T showed a linear relationship. The activation energies (Ea) of

Table 1 Distribution for the number of clones with the same activity or stability. Number of clones out of the 576 closes

D

where N, PD, and D represent the native, partially denatured, and denatured species, respectively. WT and the 328 clones were subjected to heat treatment at 65 °C for 15 min followed by reaction at 37 °C. Table 1 also shows the distribution for the number of clones with the same residual activity (activity compared to that before heat treatment) for the 328 clones. WT exhibited the residual activity of 5.3%. We selected six clones with relative activities of ≥55%. Sequence analysis showed that these six clones had the S26C/Q57R, M65I, T82A, M175 L, Y176A, and A191 V mutation(s), respectively. In a site saturation mutagenesis library, two amino acid substitution, like S26C/Q57R, is theoretically unlikely. However, in our previous study, the success rate of mutation was around 37%, and the failures included such two amino acid substitution [24,25]. We speculate that unexpected mutations may result from errors in oligonucleotides synthesis in the microarray. We investigated the thermostabilities of WT and these six clones (Fig. 2A). The residual activities (%) after the treatment at 65 °C for 15 min were 5.3 ± 1.9 for WT, 42.4 ± 7.9 for S26C/Q57R, 35.9 ± 11.5 for M65I, 91.5 ± 8.3 for T82A, 35.2 ± 5.5 for M175 L, 45.9 ± 5.4 for Y176A, and 27.8 ± 6.1 for A191 V, indicating that T82A was the most thermostable. To obtain more thermostable variants, we performed saturation mutation analysis of Thr82. WT and 19 single variants at amino acid position 82 were expressed in E. coli, and cellular soluble fractions were prepared. The residual activity of T82A after 15-min heat treatment at 65 °C was 82.0 ± 6.0%, which is almost equal to that described above (91.5 ± 8.3%), and that of T82 G was 78.0 ± 5.0%. Conversely, those of WT and the other 17 variants were below 25% (Fig. 2B), suggesting that amino acid position 82 is critical for thermostability. We thus selected T82A as the most thermostable variant.

2.8.1. Structural determination and refinement The crystal structures of WT and T82A were determined by the molecular replacement method using the atomic coordinates of WT (PDB entry 2DCJ) as a search model with MOLREP [34] from the CCP4 suite [35]. The structures were refined with phenix.refine from the PHENIX suite [36] after model rebuilding with Coot [37]. Figures in which protein structures are shown were prepared using PyMOL [38].

Relative activity (%)a

PD

The activity compared to that of WT. Relative value (%) compared to the number of total clones examined (576). The activity compared to that before heat treatment at 65 °C. Relative value (%) compared to the number of total clones examined (328). Corresponding to residual activity of WT (5.3%). 4

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Fig. 2. Screening of thermostable XynJ variants. RBB-xylan-hydrolyzing activities of E. coli-produced WT, S26C/Q57R, M65I, T82A, M175 L, Y176A, and A191 V (A) and T82X (B), which had received heat treatment at 65 °C for 15 min, are shown. Residual activity indicates the value compared to that before heat treatment. Error bars indicate SD values for three-times measurements.

thermal inactivation of WT and T82A, as calculated from the slope, showed a close similarity at 518 and 534 kJ mol−1, respectively. According to Eqs. (1)–(3), the Gibbs free energy changes of activation, ΔG‡, at 65 °C were calculated as 98 kJ mol−1 for WT and 106 kJ mol−1 for T82A, the enthalpy changes of activation, ΔH‡, were 515 kJ mol−1 for WT and 532 kJ mol−1 for T82A; and the entropy changes of activation, ΔS‡, were 1230 J mol−1 K−1 for WT and 1260 J mol−1 K−1 for T82A. There results indicated that in XynJ, large ΔH‡ values are compensated by large values of ΔS‡, resulting in small values of ΔG‡, which is so called enthalpy-entropy compensation.

the Plank constant ( = 6.626 × 10-34 J s), respectively. We investigated temperature dependence of the beechwood xylanhydrolyzing activities of WT and T82A. The results at 37–75 °C are shown in Fig. 3D. The relative activity against temperature showed a bell-shaped curve. The optimal temperature for WT was 60 °C, while that of T82A was 65 °C, indicating that T82A is more suitable for reaction at high temperature than WT.

ΔG‡ = – RT [ln(kobs) – ln(RT /Nh)]

(1)

ΔH‡ = Ea – RT

(2)

Fig. 4A shows the substrate-concentration dependence of the initial reaction rate in the hydrolysis of beechwood xylan at pH 7.8 at 37 °C. In the Lineweaver-Burk plot, linear relationships were observed between the reciprocal of v and the reciprocal of the substrate concentration. The kcat and Km values of WT and T82A were determined separately, which were similar (131 ± 21 and 113 ± 5 s−1, respectively, for kcat and

‡

‡

‡

ΔS = (ΔH – ΔG ) / T

3.3. Steady-state kinetic analysis of WT and T82A

(3) 23

Where N and h are the Avogadro number ( = 6.022 × 10

−1

mol

) and 5

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Fig. 3. Thermostability of WT and T82A. (A) Residual activity after heat treatment. WT or T82A at 0.1 μM was incubated at 60–65 °C or 60–71 °C, respectively, for 15 min. Then, the beechwood xylan-hydrolyzing reaction was carried out at 37 °C. The residual activity was defined as the ratio of the initial reaction rate with incubation for the indicated durations to that without incubation (4.72 μM s−1 for WT and 4.65 μM s−1 for T82A). (B) Time dependence of thermal inactivation. WT or T82A at 0.1 μM was incubated at 61–67 °C for the indicated durations. Then, the beechwood xylanhydrolyzing reaction was carried out at 37 °C. The first-order rate constant of the thermal inactivation (kobs) was estimated from the slope: WT, 0.53 × 10-3 s−1 at 61 °C (open circle), 2.31 × 10-3 s−1 at 63 °C (open triangle), and 4.78 × 10-3 s−1 at 65 °C (open square); T82A, 0.30 × 10-3 s−1 at 65 °C (closed circle), 0.41 × 10-3 s−1 at 66 °C (closed triangle), and 0.77 × 10-3 s−1 at 67 °C (closed square). (C) Arrhenius plots of kobs values. The activation energy (Ea) of thermal inactivation was calculated from the slope: WT (open circle), 518 kJ mol−1; T82A (closed circle), 534 kJ mol−1. (D) Dependence on temperature of the initial reaction rate. The reaction was carried out with WT or T82A at 0.1μM for 10 min at the indicated temperature. The relative activity is defined as the ratio of the initial reaction rate at the indicated temperature to that at the optimal temperature (12.2 μM s−1 for WT at 60 °C and 11.5 μM s−1 for T82A at 65 °C).

16.4 ± 1.4 and 13.2 ± 0.9 mg/ml, respectively, for Km), suggesting that the mutation of Thr82 to Ala did not affect the activity. Generally, there is a compromise between activity and stability in various enzymes [40]. Mutations that increase thermostability are, in most cases, accompanied with a decrease in enzyme activity. However, the results in this study do not appear to follow that logic. The kcat and Km values (Fig. 4A) and pH dependences of the activities (Fig. 4B) of WT and T82A are very similar. This suggests that the T82A and T82 G mutation did not affect the flexibility required for activity. Fig. 4B shows the pH dependence of the initial reaction rate in the hydrolysis of beechwood xylan at 37 °C. WT and T82A exhibited similar pH-dependences, with a catalytically optimum pH in the range of 5–9, indicating that the mutation of Thr82 to Ala did not affect the pH dependence of activity and that T82A retained high activity at alkaline conditions. Tailoring the pH dependence of catalytic activity is one of the aims of protein engineering, especially for enzymes in industrial use. In the case of xylanase, high activity and stability under high-pH conditions is desired. In the catalytic mechanism of XynJ, Glu93 acts as a nucleophile, and Glu183 as an acid-base catalyst [27,28,41,42]. In XynJ, the single mutations E16Q, D20 N, R48Q, K52Q, W144 F, and E177Q decreased the optimum pH by 2–3 [28,43], while the quintuple and sextuple mutations S26R/T34R/N74R/N76R/N192R and S26R/T34R/ K51R/N74R/N76R/N192R increased the optimum pH by 1 [16]. In Bacillus sp. GH11 xylanase Xyn11A-LC, single mutations of one of six amino acid residues (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187) adjacent to the acid-base catalyst decreased the optimum pH by 2, indicating that several active-site residues are responsible for the pH dependence of activity [39]. The next aim is to shift the optimum pH of T82A to the alkaline side without impairing thermostability. One strategy is the combination of T82A with the quintuple or sextuple mutations described above. Another strategy is the construction of a site-saturation mutagenesis library for T82A. In this case, regions containing amino acid residues close to the two catalytic Glu residues (Glu93 and Glu183) might be a target for mutation. As for further stabilization, one strategy is the combination of T82A with other stabilizing mutations observed in this study (S26C/Q57R, M65I, T82A,

M175 L, Y176A, and A191 V). 3.4. Crystallographic analysis of WT and T82A The thermostability of XynJ was enhanced by the replacement of Thr82 with the smallest amino acid Gly or the next smallest Ala (Fig. 2B). Thr82 locates in β8 (Tyr80–Thr87). All residues in β8 are well conserved except for Thr82. Interestingly, Chaetomium thermophilum xylanase (Xyn11A) has Ala at the amino acid position corresponding to Thr82 in XynJ (Fig. 5), and Xyn11A exhibited good residual activity of Xyn11A after heat treatment at 70 °C for 15 min [44]. This suggests that Ala is preferable for thermostability than Thr at amino acid position 82 in XynJ. To explore the effect of the mutation of Thr82 to Ala, we performed a crystallographic analysis of WT and T82A. Table 2 summarizes the data collection and structure statistics. The space group of the both WT and T82A crystals was P6122. Fig. 6A shows the backbone structures of WT obtained in this study and the wild-type XynJ reported previously (PDB entry 2DCJ). Slight backbone deviations were observed between the two structures. There was little difference in the backbone structure of WT and T82A (data not shown). For example, the RMSD values of the catalytic domain (A1-I200) are 0.139 Å for 2DCJ and WT, 0.140 Å for 2DCJ and T82A, and 0.095 Å for WT and T82A. In all cases, there were two molecules (molecule A and B) in the unit cell, and the electron density of the region from Ser204 to Glu207 was not clearly observed. Two Ca2+ ions, previously reported to be bound to the xylan-binding domain, were found in all four molecules. Fig. 6B shows the mutationsite structures of WT and T82A. The electron density map shows that Thr82 was replaced with Ala in T82A. Except for these residues, no appreciable difference in the conformation among WT, T82A, and 2DCJ was observed. Crystallographic analysis has revealed that the side-chain atoms of Thr82 in WT do not necessarily make favorable van der Waals interactions with the surrounding atoms (Table S2). The hydroxyl group of Thr82 is very close to the main chain of Ile96 (Fig. 6B). Ile96 is located in the next β-strand to that in which Thr82 is located. The distances of the OG1 of Thr82 from the C and O atoms of Ile96 are 3.2 Å and 3.0 Å, 6

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Table 2 Data collection and refinement statistics.

A. Diffraction data X-ray source Detector Wavelength (Å) Resolution range (Å) Space group Unit cell parameters a, c (Å) Unique reflections Multiplicity Completeness (%) Mean I/(I) Wilson B-factor (Å2) Rmerge (%) Rmeas (%) CC1/2 (%) B. Refinement statistics Resolution range used refinement Rwork (%) Rfree (%) Number of Ca2+/HEPES/citrate/c5diol water Protein residues R.m.s.d., bond lengths (Å) R.m.s.d., bond angles (?) Ramachandran favored (%) Rotamer outliers (%) Clashscore (%) Average B-factor ( Å2) PDB ID

WT

T82A

SPring-8/BL44XU DECTRIS EIGER X 16 M 0.9 50.00–2.45 (2.60–2.45) P6122

SPring-8/BL26B1 RAYONIX MX225HE 1.0 50.00–2.30 (2.40–2.36) P6122

125.94, 318.57 103196 (16,645) 3.2 (3.1) 99.6 (99.5) 13.3 (2.1) 50.275 6.4 (53.0) 7.7 (64.2) 99.7 (69.8)

125.40, 318.11 124,116 (20,005) 4.3 (3.8) 99.9 (99.5) 14.4 (2.1) 52.601 6.6 (62.0) 7.6 (72.4) 99.8 (65.7)

45.0–2.45 (2.49–2.45) 17.3 (29.0) 19.8 (32.2)

49.2–2.36 (2.40–2.36) 18.5 (24.0) 21.4 (24.8)

4/4/0/8 253 327 × 2 0.007 0.96 96.2 0.37 4.05 45.5

7/3/1/15 213 327 × 2 0.007 0.928 97.7 0.00 3.78 45.1

very close to four atoms (L81/C (3.1 Å), V97/CA (3.9 Å), L81/O (3.4 Å), and V83/N (3.2 Å)), and similar interactions are observed for the CB atom of Ala82 in T82A (Table S2). Thus, the thermostability enhancement of the jellyroll structure by the T82A and T82 G mutation may result from the removal of steric hindrance between the hydroxyl group of Thr82 with the main chain of Ile96. Such a narrow space will fully accommodate only Gly and Ala, but not Thr. In conclusion, based on the hypothesis that in GH11 xylanase XynJ from Bacillus sp. strain 41M-1, the thermostability will increase if the flexibility of the β strands in the jellyroll structure is decreased without impairing activity, we introduced random mutations using site saturation mutagenesis. By screening 576 clones, T82A was selected as the most thermostable variant. Crystallographic analysis of WT and T82A revealed that T82A was more thermostable than WT due to the removal of unfavorable van der Waals interactions by the mutation. Steady-state kinetic analysis revealed that T82A was indistinguishable from WT as assessed by hydrolysis of beechwood xylan. These results suggest that this strategy is effective for stabilization of GH11 xylanase.

Fig. 4. Steady-state kinetic analysis of WT and T82A. (A) Effects of substrate concentration. Hydrolyzing reaction of beechwood xylan was carried out at pH 7.5, at 37 °C with WT (open circle) or T82A (closed circle) each at 0.1 μM. Lineweaver-Burk plot is shown. Error bars indicate SD values for three-times measurements. (B) Effect of pH. Hydrolyzing reaction of beechwood xylan was carried out with WT (open symbols) or T82A (gray symbols) at with acetateNaOH buffer at pH 3.5–6.0 (circle), phosphate-NaOH buffer at pH 5.5–8.0 (triangle), Tris−HCl buffer at pH 8.0–9.0 (square), and carbonate-NaOH buffer at pH 9.0–11.0 (diamond). The relative activity is defined as the ratio of the initial reaction rate at the indicated pH to that at the optimal pH (7.10 μM s−1 for WT at pH 6.0 and 8.63 μM s−1 for T82A at pH 6.0).

respectively, both of which are shorter than the corresponding calculated values (the sum of the van der Waals radii of the pair of atoms) [45]. The OG1 atom of Thr82 forms a hydrogen bond with the O atom of Val83. However, the hydrogen bond appears to be weak, as judged from the O-H—O angle (107°). The CG2 atom of Thr82 is some distance from the surrounding atoms. Conversely, the CB atom of Thr82 in WT is

Fig. 5. Amino acid sequences of the β8 strand of xylanases. The ClustalW program was used for multiple sequence alignment of the amino acid sequences of xylanases from Bacillus sp. 41M-1 (XynJ) (Gene bank accession no. AB029319.1), Hypocrea jecorina (Xyn2) (X69573.1), Acrophialophora nainiana (Xyn6) (DQ641721.1), Streptomyces sp. S38 (Xyn1) (X98518.1), and Chaetomium thermophilum (Xyn11A) (AJ508931.1). The asterisk indicates the amino acid residues conserved. Thr82 of XynJ and corresponding amino acid residues are boxed with a broken line. Data of thermostability are derived from Paës et al. [46]. 7

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Fig. 6. Crystallographic analysis of WT and T82A. (A) Overall protein structure. The backbones structures of WT obtained in this study (cyan) and reported previously (PDB entry 2DCJ) (magenta) are shown. The low electron density region (Gly201-Ala209) is shown using a yellow dotted line. The spheres represent Ca2+ ions. (B) Mutation-site structure (Stereo view). Electron densities (blue cages) of Thr82 in WT (upper panel) and the introduced Ala in T82A (lower panel) are contoured at 1.5 σ in the 2Fo-Fc map. Hydrogen bonds are shown using blue lines. Unfavorable van der Waals interactions of the sidechain of Thr82 with the surrounding residues (within 4.0 Å from the OG1 or CG2 atom of Thr82) are shown using red lines (upper panel) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

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