Structural Insights into the Thermophilic Adaption Mechanism of Endo

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Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-#-Xylanase from Caldicellulosiruptor owensensis Xin Liu, Tengfei Liu, Yuebin Zhang, Fengjiao Xin, Shuofu Mi, Boting Wen, Tianyi Gu, Xinyuan Shi, Fengzhong Wang, and Lichao Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03607 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 17, 2017

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Journal of Agricultural and Food Chemistry

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Structural Insights into the Thermophilic Adaption Mechanism of

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Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis

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Xin Liu1*, Tengfei Liu2*, Yuebin Zhang3*, Fengjiao Xin1, Shuofu Mi4, Boting Wen1, Tianyi Gu1,

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Xinyuan Shi2, Fengzhong Wang1*, Lichao Sun1*

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Xin Liu, Tengfei Liu and Yuebin Zhang contributed equally to this work.

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1. Laboratory of Food Enzyme Engineering, Institute of Food Science and Technology, Chinese

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Academy of Agricultural Sciences, Beijing 100193, P.R. China 2. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102,

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P.R. China 3. State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics,

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Chinese Academy of Sciences, 457 Zhongshan Rd, Dalian 116023, P.R. China 4. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

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Chinese Academy of Sciences, Beijing 100190, P.R. China.

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*

Corresponding author: Feng Z. Wang. Telephone:+86 10 62817417. Email: [email protected]. Li C. Sun. Telephone:+86 10 62815969. Email: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT

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Xylanases (EC 3.2.1.8) are a kind of enzymes degrading xylan to xylooligosaccharides

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(XOS) and have been widely used in a variety of industrial applications. Among them, xylanases

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from thermophilic microorganisms have distinct advantages in industries that require high

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temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10

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xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was

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overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90 °C with

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a half-life of about 1 h at 80 °C and exhibited highest activity at pH 7.0. CoXynA activity was

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affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood

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xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular

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dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA

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was proposed to be due to the increased overall protein rigidity resulting from the reduced length

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and fluctuation of Loop 7.

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KEYWORDS: GH10 xylanase, Caldicellulosiruptor owensensis, Thermostability, Crystal

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structure, Loop 7

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INTRODUCTION

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Heteroxylans are major components of plant hemicelluloses.1 The internal β-1,4-xylosidic

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bonds of heteroxylans were mainly cleaved by endo-β-1,4-xylanases (EC 3.2.1.8), leading to the

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release of xylooligosaccharides (XOS).

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hydrolases (GH), were widely used in pulp bleaching,3–4 food manufacturing,5-6 animal feed

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preparation,7 brewing industry,8 and biofuel production.9 In particular, xylanases from

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thermophilic microorganisms possess extraordinary properties for adapting to high temperature

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conditions in industrial applications, such as higher acticity and lower microbial contamination.10

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According to substrate specificity, sequence similarity, and three-dimensional structural

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features, xylanases are mainly classified as glycoside hydrolases 10 (or F) and 11 (or G).

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Glycoside hydrolase 10 (GH10) xylanases are among the most extensively studied families and

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can operate over broad pH and temperature ranges, suggesting potential roles in commercial

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applications.11 The overall structures of GH10 xylanases exhibit a classic (β/α)8 TIM barrel fold

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and are strictly conserved. The substrate is commonly suited at the groove of catalytic module.12

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Usually, xylanases from different prokaryotic and eukaryotic organisms have distinct catalytic

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mechanisms and substrate specificities.11

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Thus, endo-β-1,4-xylanases, a kind of glycoside

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Structural differences among the different GH10 xylanases produce their diverse features.

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An in-depth knowledge of the structures of thermophilic xylanases enables understanding of

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their thermostable properties and may provide useful evolutionary strategies for protein

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engineering. Generally, thermozymes are more rigid than mesoenzymes. For example, Zhang et

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al. determined the structures of Caldicellulosiruptor bescii Xyn10B (CbXyn10B) and the

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complex with XOS.13 They found that the thermostable CbXyn10B forms extensive hydrogen

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bond interactions between Loop 7 and Loop 8 in the catalytic module, and revealed that an

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aromatic cluster connecting the N- and C-termini may enhance the protein rigidity of

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CbXyn10B.

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Several factors reported to evaluate the protein rigidity were widely used in the

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measurement of enzyme thermostability. Crystallographic B factor is one of the most widely

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used factors to measure the protein flexibility and thermal fluctuations. According to the

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conserved sequences of thermophilic GH10 xylanases, Chen et al. increased the melting

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temperature (Tm) of Streptomyces sp. XynAS9 by 6.9 °C through replacing Val81 with proline

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and replacing Gly82 with glutamic acid, resulting in lower crystallographic B factor and

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increased protein rigidity.14 The root mean square displacement (RMSD), represents the Cα-

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atomic range from the initial status to that at high temperature. RMSD is determined using

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molecular dynamics (MD) simulations and is negatively correlated with protein rigidity and

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thermostability. According to the sequences of Thermoascus aurantiacus (TaXyn10), Wang et al.

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improved the optimal temperature of AuXyn10A by 10 °C and obtained a 10.4-fold half-life at

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55 °C through Ser286Gly/His288Phe double mutation, leading to an enhanced hydrophobic

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interaction and smaller RMSD value.15 Flexible regions and unstable residues could be identified

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through calculating the protein flexibility values. These sites may induce protein unfolding and

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be applied in protein engineering. Joo et al. successfully increased the transition temperature (Tm)

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of Bacillus circulans xylanase by 4.2 °C and obtained a 30-fold half-life at 50 °C by introducing

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a Phe48Tyr/Thr50Val/Asn52Tyr/Thr147Leu quadruple mutation into the unstable regions,

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leading to increased hydrophobic interactions and aromatic stacking interaction.16

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Recently, a large quantity of xylanases were identified from anaerobic thermophilic

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bacterium Caldicellulosiruptor species,17–21 in which a GH10 xylanase from Caldicellulosiruptor

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owensensis, CoXynA, displayed outstanding thermal stability.22 Thus, a detailed characterization

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of CoXynA and understanding of the thermophilic mechanism are desirable. In this study, the

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recombinant CoXynA enzyme was purified and its enzymatic kinetics were examined in detail.

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Furthermore, structural studies and molecular docking simulation were performed to illustrate

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the thermostable mechanism of CoXynA.

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MATERIALS AND METHODS

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Materials and Chemicals. The substrate, beechwood xylan, was purchased from

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Megazyme (Megazyme. USA). Other chemicals were purchased from Aladdin Co., Ltd. or

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Sigma. Competent cells used for protein expression were Escherichia coli (E. coli) Transetta

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(DE3) (TransGen Biotech, China). Plasmid pET-28b (Novagen, USA) carrying the CoXynA

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gene was provided by Prof. Yejun Han. The xylanase gene, CoXynA, was cloned into the pET

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vector at the NdeI and XhoI restriction sites. The sequence of His6 tag was in-frame at the front

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of the gene. The recombinant xylanase gene, CoXynA8, was constructed with a longer Loop 7 on

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the basis of CoXynA through duplication of 24 bp nucleotides at the C terminal of Loop 7.

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Protein Expression and Purification. Plasmid pET-28b carrying the CoXynA gene or the

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CoXynA8 gene was transformed into E. coli Transetta (DE3). Both the recombinant CoXynA

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and CoXynA8 contained an N-terminal His6 tag. Transformed bacterial colonies were first

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transferred into 50 mg/mL kanamycin-containing LB media and cultured overnight with shaking

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(200 rpm/min) at 37 °C. The culture was then transferred into 1 liter of fresh LB media (1%, v/v)

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and grown with shaking (200 rpm/min) at 37 °C until OD600 reached 0.6. The expression of

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target protein was induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 6 h at

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37 °C. The total cells were collected by centrifugation at 4 °C (5,000 rpm/min), re-suspended in

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60 mL of Tris buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl), and then disrupted using a high-

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pressure homogenizer (ATS, Germany). Cell lysates were then centrifuged (12,000 rpm/min) for 5



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30 min at 4 °C. The supernatant was subjected to a Ni2+-NTA sepharose column and the elutions

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were collected with elution buffer (50 mM Tris-HCl, pH 8.0, 250 mM imidazole). The proteins

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were then purified through Q anion exchange chromatography (GE healthcare), which is pre-

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treated with equilibration buffer (50 mM Tris-HCl, pH 8.0). The bound proteins were eluted

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using a linear gradient (300 mL) of 0-1 M NaCl, which is dissolved in 50 mM Tris-HCl solution

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(pH 8.0). The elutions were concentrated by Amicon Ultra-15 (10 kDa, Millipore) and were

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further purified through superdex-75 gel filtration chromatography (1.6 cm × 20 cm, GE

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healthcare) using 50 mM Tris-HCl solution containing 150 mM NaCl (pH 8.0). All fractions

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were subjected to 12% SDS-PAGE. Recombinant proteins were stored at –80 °C for further use.

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Enzyme Assay. Protein concentration was assayed using a DC Protein Assay Kit (Bio-Rad,

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USA). The bovine serum albumin (BSA) was used as a standard. To determine the substrate

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specificity of CoXynA, 0.5% (w/v) beechwood xylan, birchwood xylan, pNPX (p-nitrophenyl-β-

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D-xylopyranoside), and carboxymethyl cellulose (CMC-Na) dissolved in Na–citrate buffer (500

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µL, 100 mM, pH 6.0) was pre-incubated for 5 min at 70 °C. The reaction was initiated with the

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addition of CoXynA (0.1 µL, 5.1 mg/mL) and reacted for 5 min. The reducing sugars released

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from the distinct substrates were measured using the previously reported 3,5-dinitrosalicylic acid

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(DNS) method.23 The reaction with pNPX substrate was stopped by addition of 1 M Na2CO3 (2

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mL) and the absorbance of the solution was determined at 405 nm.

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The optimal pH of CoXynA (0.51 µg) was tested at 70 °C for 5 min with 0.5% (w/v)

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beechwood xylan dissolved in different buffer solutions (50 mM): NaOH-glycine buffer (pH

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9.0–10.0), Tris-HCl buffer (pH 8.0), sodium phosphate buffer (pH 7.0) and citrate buffer (pH

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3.0–6.0). The optimal temperature for CoXynA (0.51 µg) and CoXynA8 (1.12 µg) was

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determined using 0.5% (w/v) beechwood xylan dissolved in 50 mM sodium phosphate buffer

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(pH 7.0) and the reaction was performed within 40-100°C at 5 °C intervals.

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The thermal stability of CoXynA was determined at 60, 65, 70, 75, 80, 85, and 90 °C

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through incubation in sodium phosphate buffer (pH 7.0) for 0, 60, 120, 180, or 240 min. The pH

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stability of the CoXynA was estimated at 70 °C by dilution in different citrate phosphate buffers

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(pH 3.0–10.0) for 1 h.

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To investigate the influence of metal ions and chemicals on CoXynA activity, an array of

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metal ions (Na+, K+, Ag+, Mg2+, Li2+, Ca2+, Co2+, Fe2+, Zn2+, Ni2+, Mn2+, Pb2+, Sn2+ and Fe3+,)

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and EDTA, SDS, β-mercaptoethanol at final concentrations of 5 mM were preincubated in the

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reaction mixture at the optimal temperature for 5 min. The xylanase activity was then determined

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under optimal reaction conditions as described above. The xylanase activity without pre-

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incubation was defined as 100%.

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Protein Crystallization, X-Ray Data Collection, and Structure Determination. Rough

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screening for protein crystallization was performed in 24-well plates at 20 °C under 1152

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reservoir conditions including the Hampton Research kit, PACT, JSCG (QIGEN), and Wizard

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classic crystallization series (Rigaku, Washington, USA) using a hanging-drop diffusion method.

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Each drop contains 1 µL of reservoir buffer and 1 µL of protein (5.1 mg/mL). The optimized

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condition for CoXynA crystallization consisted of 5% isopropanol and 2.2 M (NH4)2SO4. Then,

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a suitable cryoloop was chosen to mount the selected crystal and the cryoprotectant buffer of

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50% ethylene glycol (v/v) was used to protect the crystal before frozen in liquid nitrogen. The

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best crystals were transferred to Shanghai Synchrotron Radiation Facility beamline BL19U

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(Shanghai, China) with an ADSC Quantum 315r CCD area detector for high-resolution

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diffraction data sets. The collected data was processed using the HKL2000 package.24 Then the

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crystal structure of CoXynA was solved using the structure of CbXyn10B from

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Caldicellulosiruptor bescii (PDB code: 4PMD) through molecular replacement method. Standard

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refinement was achieved using PHENIX25 and Coot.26 The structural graphics images were

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prepared with PyMOL (http://www.pymol.org). The processed data and refinement results are

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listed in Table 2..

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Simulation Procedures. The X-ray structures of apo CoXynA and mesophilic CmXyn10B

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from Cellvibrio mixtus (Topt: 40 °C, PDB code: 1UQY27) was subjected to molecular dynamics

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simulations. Both enzyme was embedded into a 10.5 × 10.5 × 10.5 Å box containing ~35,000

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TIP3P water molecules. Then, the system charges was neutralized by addition of 0.1 M NaCl.

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All simulations were performed at 367 K under atmospheric pressure using Gromacs 5.0.4

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package28 with CHARMM36 force field29. The relative algorithms and parameters were

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employed to control the simulation system following the previous work30, except that the cut-off

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value was set as 1.2 nm to compute electrostatic and van der Waals interactions. All structural

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images were generated with PyMOL (http://www.pymol.org).

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Protein Data Bank Accession Code. The structural factors and coordinates for the

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CoXynA structure were deposited in the Protein Data Bank (PDB code: 5Y3X).

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RESULTS

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Expression and Purification of CoXynA in E.coli. . The Calow_0124 gene encoding

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CoXynA in C. owensensis was overexpressed in E.coli Transetta (DE3). Recombinant CoXynA

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was obtained through purification procedure using Ni2+-NTA sepharose column, a Q anion

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exchange chromatography column, and a superdex-75 gel filtration column, successively. This

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afforded recombinant CoXynA with 98% purity, which was then analyzed using 12% SDS-

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PAGE (Figure 1A and 1B). The calculated molecular weight was about 43 kDa, in agreement

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with the theoretical molecular weight of 42.3 kDa as a monomer. The expression of CoXynA 8


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with high purity enabled detailed characterization of its enzymatic properties and protein

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crystallization.

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Biochemical Analysis of Thermophilic CoXynA. . Although majority of xylanases from

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fungi exhibit optimal temperatures between 45 and 75 °C,8,31,32 that of CoXynA was as high as

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90 °C (Figure 2A). To further study the thermostability of CoXynA, we tested the influence of

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higher temperatures on CoXynA activity. As shown in Figure 2B, CoXynA had a half-life of

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about 3 h at 75 °C and 1 h at 80 °C, indicating that CoXynA was a relatively thermostable

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xylanase.

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We also examined the pH profile of CoXynA. Similar to the previous study, CoXynA

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displayed optimal activity at pH 7.0 and was active in a wide range of pH. It retained more than

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70% of the maximal activity at pH 6.0–8.0 (Figure 2C). Moreover, CoXynA exhibited good pH

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stability feature at pH 5.0-10.0: more than 60% of the maximal activity was retained after

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reaction within this pH range for 1 h (Figure 2D).

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CoXynA showed different activities toward different substrates. Compared with its

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homolog, CbXyn10B, CoXynA exhibited much higher activity toward beechwood xylan and

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lower activity toward birchwood xylan (Table 1). The maximum activities of CoXynA on

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beechwood xylan and birchwood xylan were 594 U/mg (100%) and 283 U/mg (47.7%), while

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those of CbXyn10B were similar (497 U/mg and 448 U/mg, respectively). Moreover, CoXynA

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exhibited undetectable activity toward pNPX and carboxymethyl cellulose (CMC-Na),

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demonstrating its high substrate specificity toward xylan.

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Various metal ions and chemicals have been investigated extensively as either activator or

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inhibitor of xylanases.21,22 Thus, we detected their influence on the activity of CoXynA (Figure

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2E). As a result, Pb2+ had a positive effect, while Zn2+, Mg2+, Ca2+, and β-mercaptoethanol had

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no obvious effect. Fe3+, Ni2+, Fe2+, Na+, and Li2+ slightly inhibited the enzymatic activity, Mn2+

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and Sn2+ inhibited enzymatic activity by 50%, and Ag+ and SDS destroyed the enzymatic

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activity. EDTA addition also resulted in decreased enzyme activity, indicating that cations might

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play a role in the catalytic properties.

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Overall Structure of Thermophilic CoXynA. To investigate the structural basis of the

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catalytic characteristics and thermostability of CoXynA, apo CoXynA was crystallized at 293 K.

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Then we solved the structure of CoXynA by molecular replacement. The final model was refined

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to 2.1 Å resolution with the space group P1 21 1 (Table 2). The crystal structure of CoXynA

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exhibited a classic GH10 (β/α)8-barrel fold (commonly known as a TIM barrel), similar to other

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GH10 homologs. The active site was located in the cleft between the loops linking the α-helices

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and β-strands. Among these loops, Loop 4 together with opposite Loops 7 and 8 formed a deep

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cleft on the surface (Figure 3A). Glu139 and Glu247, which are important for catalytic reaction,

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were conserved and situated in the end of the 4th and 7th strands, respectively. Structural

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comparison of apo CoXynA with xylobiose-binding CbXyn10B exhibited a similar active site,

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and yielded an RMSD value of 0.373 for 278 Cα atoms. The xylobiose fitted well in the apo

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CoXynA (Figure 3B), indicating that protein conformation was almost unchanged by binding of

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the ligand.

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Thermostable Mechanism of CoXynA. CoXynA showed outstanding hydrolytic activity

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towards beechwood xylan at high temperature. To explore the pivotal factors related to the

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thermostability of CoXynA, we compared the structure of CoXynA with CmXyn10B, which

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shows the optimum temperature of 40 °C ( PDB 1UQY)27, the most different region occurred in

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the Loop 7, with both the lengths and conformations displaying great differences (Figure 4A).

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The L7 loop of CoXynA was 16 residues long (residues 252–267), while thermally labile

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CmXyn10B had a longer L7 sequence of 29 residues (residues 268–296), and their

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conformations were also different. Therefore, the length and conformation of the L7 loop were

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potentially related to the thermal stability.

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To confirm our structural observations, we performed MD simulations of CoXynA and

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CmXyn10B in explicit solvent starting from their crystal structures at 367 K. CmXyn10B

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exhibited a larger RMSD fluctuation than CoXynA in the 100-ns simulations (Figure 4B),

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suggesting that the conformation of CmXyn10B was more flexible in solution at 367 K.

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Furthermore, the root mean square fluctuations (RMSF) showed the movements of each residue

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in CoXynA were reduced compared with those in CmXyn10B, and that the highest fluctuation

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region of CmXyn10B occurred in the L7 loop, which exhibited the most prominent change and

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high flexibility (Figure 4C). During our simulations, we observed that the longer Loop 7 of

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CmXyn10B deformed at 367 K and occupied the substrate binding site at the end of our

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simulations, which was not observed in the CoXynA simulations. For CoXynA, the shorter L7

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loop did not undergo a significant conformational change. Figure 4D compares the structural

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snapshots of CmXyn10B and CoXynA at the beginning and end of our MD simulations. These

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results indicated that the length of Loop 7 was negatively correlated with the thermal stability of

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the two GH10 xylanases and that a shortened Loop 7 in CoXynA exhibited higher thermal

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tolerance. Therefore, the substrate binding site of CoXynA would be less affected by large

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thermal perturbations.

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To provide more evidence for the importance of Loop 7 in thermal stability of CoXynA, we

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designed a new mutant by changing the length of Loop 7 of CoXynA. We extended the Loop 7

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of CoXynA through duplication of 8 amino acids at the C terminal of Loop 7 and constructed a

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new mutant, CoXynA8. As expected, the optimal temperature was 75 °C of CoXynA8, lower

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than that of CoXynA (Figure 4E). Therefore, the increase of length of Loop7 resulted in an

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obvious decrease of optimal temperature, indicating the importance of shortened Loop 7 in

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thermostability of CoXynA.

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DISCUSSION

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Thermozymes can efficiently work under high-temperature conditions, which are less

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susceptible to microbial contamination. Therefore, xylanases with high thermostability attract

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much industrial interest and are widely used in pulp bleaching and animal feed preparation, in

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which high-temperature treatments are usually companied with enzyme application. The several

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strategies generally used to obtain thermostable xylanases include screening from thermophilic

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microorganisms, evolution engineering of mesozymes, and protein immobilization on

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nanoparticles.3

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To gain structural insight into the thermophilic adaption mechanism of CoXynA, a GH10

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endo-β-1,4-xylanase identified from thermophilic bacterium Caldicellulosiruptor owensensis, we

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first expressed the recombinant CoXynA. CoXynA exhibited relative high thermostability. In the

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previous study, the optimal temperature of CoXynA was at 75 °C and the residual activity was

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totally abolished when heating at 80 °C for 2 h.22 However, the optimal temperature of CoXynA

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was elevated to 90 °C in our study. The half-life of CoXynA was approximately 1 h at 80 °C,

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which was also obviously longer than that previously reported (shorter than 0.5 h). Herein, we

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also purified CoXynA in the same condition with previous report22. As expected, the optimal

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temperature was reduced to 75 °C.

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Common thermostable features include increased interactions (such as hydrophobic

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interactions, hydrogen bonds, and disulfide bonds) and superior structure (such as increased

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rigidity, higher packing efficiency, lower tendency to unfold, larger protein stability and folding,

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conformational strain release, and α-helix stability).33 Therefore, structural insights into

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thermophilic xylanases are valuable for a better understanding of thermostable mechanisms. In

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this study, we solved the crystal structure of CoXynA with a resolution of 2.1 Å. The structural

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comparison of CoXynA with mesophilic counterpart CmXyn10B showed that deviations in Loop

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7 played an important role in their thermal properties. The shortened Loop 7 in CoXynA

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potentially decreased the structural flexibility and increased the thermal stability. Furthermore,

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the MD simulation study of CoXynA indicated a higher overall protein rigidity in contrast to

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CmXyn10B, which might endow CoXynA with high thermostability. The importance of Loop 7

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for thermal stability was further confirmed by the experimental data of CoXynA8, which has a

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longer Loop 7 and a reduced optimal temperature. Several studies had also reported the

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importance of shortened loops to structural stability in hyperthermostable enzymes34-36. Thus, the

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structural insights of the shortened Loop 7 in this study may provide useful evolutionary

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strategies for protein engineering of mesophilic xylanases. The thermostable mechanisms of

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thermophilic xylanases vary and depend on the specific enzyme. For example, thermostable

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CbXyn10B, a close homolog of CoXynA, displayed the highest activity at 70 °C.13 Zhang et al.

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showed that the extended Loop 7 and Loop 8 in CbXyn10B formed a relatively stable interaction

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at the catalytic groove through an extensive hydrogen bond interaction, restricting the protein

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flexibility and contributing to overall stability. Xylanase 10B from Thermotoga maritime MSB8

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(TmxB) is another thermostable enzyme, exhibiting highest activity at 90 °C.37 Ihsanawati et al.

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showed that the compact structure, contributing to the high thermostability of TmxB, was

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stabilized by the accumulation of aromatic clusters accompanied by a reduction of exposed

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hydrophobic sites and no cavity inside the protein. To better understand the thermostable

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mechanisms of thermozymes, further studies remain interesting and challenging undertaking.

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In this study, CoXynA exhibited relatively high catalytic activity for xylans. The catalytical

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activity of CoXynA on beechwood xylan was the highest compared with its homologues (Table

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2). The catalytical activity of CoXynA on birchwood xylan was 0.5-fold lower than that of

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CbXyn10B from C. bescii DSM 6725,19 similar to that of PbXyn10B from P. barcinonensis BP-

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23,38 and 4.5–5.6-fold higher than those of XynBE18 from Paenibacillus sp. E1839 and iXylC

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from C. laeviribosi HY-21, respectively.40 Unlike most homologues, CoXynA showed

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undetectable activity toward pNPX and CMC-Na, suggesting its high substrate specificity toward

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xylans.

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Xylanases have been widely used in paper and pulp bleaching to improve the pulp

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brightness. Consequently, the use of chlorine as a bleaching agent has been reduced.41 The most

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desirable characteristics for xylanases used in pulp bleaching are thermostability with an alkaline

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optimum pH.42 In this study, we characterized CoXynA as a thermostable xylanase with high

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stability at alkaline pH (Figure 2), which is promising in the pulp industry. In general, cofactor-

296

mediated activation is applied in pulp bleaching to enhance the catalytic efficiency of

297

xylanases.43 In this study, the catalytic activity of CoXynA was affected by a variety of cations,

298

suggesting that it is sensitive to metal ions. Therefore, in applications of CoXynA in the paper

299

industry, metal ion addition must be strictly controlled. Beyond the enzymatic analysis and the

300

thermophilic adaption mechanism, this study also provides a structural basis for the catalytic

301

improvement of CoXynA toward commercial purposes.

302 303 304 305

ABBREVIATIONS USED GH,

glycoside

thiogalactopyranoside;

hydrolase;

XOS,

xylo-oligosaccharides;

CMC-Na,

carboxymethyl

cellulose;

14


IPTG,

pNPX,

isopropyl-β-D-

p-nitrophenyl-β-D-

Page 15 of 29


306

xylopyranoside; DNS, 3,5-dinitrosalicylic acid; SDS-PAGE, Sodium dodecyl sulfate

307

polyacrylamide gel electrophoresis; EDTA, Ethylenediaminetetraacetic acid; SDS, sodium

308

dodecyl

309

Caldicellulosiruptor bescii Xyn10B; CoXynA, xylanase from Caldicellulosiruptor owensensis;

310

TaXyn10, xylanase from Thermoascus aurantiacus; CmXyn10B, Cellvibrio mixtus Xyn10B;

311

TmxB, xylanase 10B from Thermotoga maritime MSB8; MD simulation, molecular dynamics

312

simulation; RMSD, root mean square displacement; RMSF, root mean square fluctuations;

313

ACKNOWLEDGEMENT

sulfate;

Tm,

transition

temperature;

Tm,

melting

temperature;

CbXyn10B,

314

We thank Porf. Yejun Han for kindly offering the plasmid of CoXynA. We also thank the

315

Shanghai Synchrotron Radiation Facility (SSRF) for beam time allocation and data collection

316

assistance. This work was supported by the state key research and development plan “modern

317

food processing and food storage and transportation technology and equipment” (No.

318

2017YFD0400200) and National Natural Science Foundation of China (No. 31571963).

319

REFERENCES

320 321

(1)

Deutschmann, R.; Dekker, R. F. From plant biomass to bio-based chemicals: latest

developments in xylan research. Biotechnol. Adv. 2012, 30, 1627-1640.

322

(2) Motta, F. L.; Andrade, C. C. P.; Santana, M. H. A. A review of xylanase production by the

323

fermentation of Xylan: classification, characterization and applications. Sustainable Degradation

324

of Lignocellulosic Biomass - Techniques, Applications and Commercialization, 1st edition;

325

Chandel, A. K., Silva, S. S., Eds.; InTech: Rijeka, Croatia, 2013; 251-275.

326

(3) Kumar, V.; Marin-Navarro, J.; Shukla, P. Thermostable microbial xylanases for pulp and

327

paper industries: trends, applications and further perspectives. World J.Microb Biot. 2016, 32, 34.

328

(4) Walia, A.; Guleria, S.; Mehta, P.; Chauhan, A.; Parkash, J. Microbial xylanases and their

15



329

industrial application in pulp and paper biobleaching: a review. Biotech. 2017, 7, 11.

330

(5) Oliveira, D. S.; Telis-Romero, J.; Da-Silva, R.; Franco, C. M. L. Effect of a thermoascus

331

aurantiacus thermostable enzyme cocktail on wheat bread qualitiy. Food Chem. 2014, 143, 139-

332

146.

333

(6) Falck, P.; Precha-Atsawanan, S.; Grey, C.; Immerzeel, P.; Stalbrand, H.; Adlercreutz, P.;

334

Karlsson, E. N. Xylooligosaccharides from hardwood and cereal xylans produced by a

335

thermostable xylanase as carbon sources for Lactobacillus brevis and Bifidobacterium

336

adolescentis. J. Agr. Food Chem. 2013, 61, 7333-7340.

337 338

(7) Beg, Q. K.; Kapoor, M.; Mahajan, L.; Hoondal, G. S. Microbial xylanases and their industrial applications: a review. Appl.Microbiol.Biot. 2001, 56, 326-338.

339

(8) Wang, X.; Luo, H.; Yu, W.; Ma, R.; You, S.; Liu, W.; Hou, L.; Zheng, F.; Xie, X.; Yao, B. A

340

thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry. Food

341

Chem. 2016, 199, 516-523.

342 343 344 345

(9) Dodd, D.; Cann, I. K. Enzymatic deconstruction of xylan for biofuel production. Glob.Change Biol. Bioenergy 2009, 1, 2-17. (10) Collins, T.; Gerday, C.; Feller, G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 2005, 29, 3-23.

346

(11) Pollet, A.; Delcour, J. A.; Courtin, C. M. Structural determinants of the substrate

347

specificities of xylanases from different glycoside hydrolase families. Crit.Rev.Biotechnol. 2010,

348

30, 176-191.

349 350 351

(12) Naumoff, D. G. GH10 Family of glycoside hydrolases: structure and evolutionary connections. Mol. Biol. 2016, 50, 151-160. (13) Zhang, Y.; An, J.; Yang, G. Y.; Zhang, X. F.; Xie, Y.; Chen, L. Q.; Feng, Y. Structure

16


Page 16 of 29

Page 17 of 29


352

features of GH10 xylanase from Caldicellulosiruptor bescii: implication for its thermophilic

353

adaption and substrate binding preference. Acta Bioch. Bioph. Sin. 2016, 48, 948-957.

354

(14) Chen, C. C.; Luo, H.; Han, X.; Lv, P.; Ko, T. P.; Peng, W.; Huang, C. H.; Wang, K.; Gao, J.;

355

Zheng, Y.; Yang, Y.; Zhang, J.; Yao, B.; Guo, R. T. Structural perspectives of an engineered beta-

356

1,4-xylanase with enhanced thermostability. J.Biotechnol. 2014, 189, 175-182.

357

(15) Wang, J. Q.; Tan, Z. B.; Wu, M. C.; Li, J. F.; Wu, J. Improving the thermostability of a

358

mesophilic family 10 xylanase, AuXyn10A, from Aspergillus usamii by in silico design. J. Ind.

359

Microbiol. Biot. 2014, 41, 1217-1225.

360

(16) Joo, J. C.; Pack, S. P.; Kim, Y. H.; Yoo, Y. J. Thermostabilization of Bacillus circulans

361

xylanase: computational optimization of unstable residues based on thermal fluctuation analysis.

362

J. Biotechnol. 2011, 151, 56-65.

363

(17) Su, X. Y.; Han, Y. J.; Dodd, D.; Moon, Y. H.; Yoshida, S.; Mackie, R. I.; Cann, I. K. O.

364

Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium

365

Caldicellulosiruptor bescii. Appl. Environ. Microb. 2013, 79, 1481-1490.

366

(18) Ying, Y.; Meng, D.; Chen, X.; Li, F. An extremely thermophilic anaerobic bacterium

367

Caldicellulosiruptor sp. F32 exhibits distinctive properties in growth and xylanases during xylan

368

hydrolysis. Enzyme Microb.Tech. 2013, 53, 194-199.

369

(19) An, J.; Xie, Y.; Zhang, Y.; Tian, D. S.; Wang, S. H.; Yang, G. Y.; Feng, Y. Characterization

370

of a thermostable, specific GH10 xylanase from Caldicellulosiruptor bescii with high catalytic

371

activity. J. Mol. Catal. B-Enzym. 2015, 117, 13-20.

372

(20) Jia, X. J.; Mi, S. F.; Wang, J. Z.; Qiao, W. B.; Peng, X. W.; Han, Y. J. Insight into

373

Glycoside Hydrolases for debranched xylan degradation from extremely thermophilic bacterium

374

Caldicellulosiruptor lactoaceticus. PloS One 2014, 9, e106482.

17



375

(21) Jia, X. J.; Qiao, W. B.; Tian, W. L.; Peng, X. W.; Mi, S. F.; Su, H.; Han, Y. J. Biochemical

376

characterization of extra- and intracellular endoxylanse from thermophilic bacterium

377

Caldicellulosiruptor kronotskyensis. Sci. Rep. 2016, 6, 21672.

378

(22) Mi, S. F.; Jia, X. J.; Wang, J. Z.; Qiao, W. B.; Peng, X. W.; Han, Y. J. Biochemical

379

characterization of two thermostable xylanolytic enzymes encoded by a gene cluster of

380

Caldicellulosiruptor owensensis. PloS One 2014, 9, e105264.

381 382 383 384

(23) Miller, G. L. Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal. Chem. 1959, 31, 426-428. (24) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol. 1997, 276, 307-326.

385

(25) Adams, P. D.; Grosse-Kunstleve, R. W.; Hung, L. W.; Ioerger, T. R.; McCoy, A. J.;

386

Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.; Sauter, N. K.; Terwilliger, T. C. PHENIX:

387

building new software for automated crystallographic structure determination. Acta Crystallogr.

388

D. 2002, 58, 1948-1954.

389 390

(26) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. 2004, 60, 2126-2132.

391

(27) Pell, G.; Taylor, E. J.; Gloster, T. M.; Turkenburg, J. P.; Fontes, C. M. G. A.; Ferreira, L. M.

392

A.; Nagy, T.; Clark, S. J.; Davies, G. J.; Gilbert, H. J. The mechanisms by which family 10

393

glycoside hydrolases bind decorated substrates. J. Biol. Chem. 2004, 279, 9597-9605.

394

(28) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl, E.

395

GROMACS: High performance molecular simulations through multi-level parallelism from

396

laptops to supercomputers. SoftwareX 2015, s 1–2, 19-25.

397

(29) MacKerell, A. D.; Bellott, D. B.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer,

18


Page 18 of 29

Page 19 of 29


398

S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.;

399

Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.;

400

Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; Wirkiewicz-Kuczera, J.; Yin,

401

D.; and Karplus, M. All-atom empirical potential for molecular modeling and dynamics studies

402

of proteins. J. Phys. Chem. B. 1998, 102, 3586-3616.

403

(30) Zhang, Y. B.; Niu H. Y.; Li Y.; Chu H. Y.; Shen H. J.; Zhang D. L.; Li G. H. Mechanistic

404

insight into the functional transition of the enzyme guanylate kinase induced by a single

405

mutation. Sci. Rep. 2005, 5, 8405.

406

(31) Yin, Y. R.; Hu, Q. W.; Xian, W. D.; Zhang, F.; Zhou, E. M.; Ming, H.; Xiao, M.; Zhi, X. Y.;

407

Li, W. J. Characterization of a neutral recombinant xylanase from Thermoactinospora rubra

408

YIM 77501T. Antonie Van Leeuwenhoek 2017, 110, 429-436.

409

(32) Anbarasan, S.; Wahlstrom, R.; Hummel, M.; Ojamo, H.; Sixta, H.; Turunen, O. High

410

stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa GH10

411

xylanase in biomass-dissolving ionic liquids. Appl.Microbiol.Biot. 2017, 101, 1487-1498.

412 413

(33) Li, W. F.; Zhou, X. X.; Lu, P. Structural features of thermozymes. Biotechnol. Adv. 2005, 23, 271-281.

414

(34) Esposito, L.; Sica, F.; Raia, C. A.; Giordano, A.; Rossi, M.; Mazzarella, L.; Zagari, A.

415

Crystal structure of the alcohol dehydrogenase from the hyperthermophilic archaeon Sulfolobus

416

solfataricus at 1.85 Å resolution. J. Mol. Biol. 2002, 318, 463-477.

417 418

(35) Guy, J. E.; Isupov, M. N.; Littlechild, J. A. The structure of an alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix. J. Mol. Biol. 2003, 331, 1041-1051.

419

(36) Miyazono, K. I.; Sawano, Y.; Tanokura, M. Crystal structure and structural stability of

420

acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3. Proteins 2005,

19



421

61, 196-205.

422

(37) Ihsanawati; Kumasaka, T.; Kaneko, T.; Morokuma, C.; Yatsunami, R.; Sato, T.; Nakamura,

423

S.; Tanaka, N. Structural basis of the substrate subsite and the highly thermal stability of

424

xylanase 10B from Thermotoga maritima MSB8. Proteins 2005, 61, 999-1009.

425

(38) Gallardo, O.; Diaz, P.; Pastor, F. I. Characterization of a Paenibacillus cell-associated

426

xylanase with high activity on aryl-xylosides: a new subclass of family 10 xylanases. Appl.

427

Microbiol. and Biot. 2003, 61, 226-233.

428

(39) Shi, P.; Tian, J.; Yuan, T.; Liu, X.; Huang, H.; Bai, Y.; Yang, P.; Chen, X.; Wu, N.; Yao, B.

429

Paenibacillus sp. strain E18 bifunctional xylanase-glucanase with a single catalytic domain.

430

Appl. Environ. Microbiol. 2010, 76, 3620-3624.

431

(40) Kim, D. Y.; Han, M. K.; Oh, H. W.; Bae, K. S.; Jeong, T. S.; Kim, S. U.; Shin, D. H.; Kim,

432

I. H.; Rhee, Y. H.; Son, K. H.; Park, H. Y. Novel intracellular GH10 xylanase from Cohnella

433

laeviribosi HY-21: biocatalytic properties and alterations of substrate specificities by site-

434

directed mutagenesis of Trp residues. Bioresource Technol. 2010, 101, 8814-8821.

435 436

(41) Viikari, L.; Kantelinen, A.; Sundquist, J.; Linko, M. Xylanases in bleaching: From an idea to the industry. FEMS Microbiol. Rev. 1994, 13, 335-350.

437

(42) Boonyapakron, K.; Jaruwat, A.; Liwnaree, B.; Nimchua, T.; Champreda, V.; Chitnumsub,

438

P. Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-

439

beta-1,4-xylanase for applications in pulp bleaching. J. Biotechnol. 2017, 259, 95-102.

440

(43) Wang, X.; Huang, H.; Xie, X.; Ma, R.; Bai, Y.; Zheng, F.; You, S.; Zhang, B.; Xie, H.; Yao,

441

B.; Luo, H. Improvement of the catalytic performance of a hyperthermostable GH10 xylanase

442

from Talaromyces leycettanus JCM12802. Bioresource Technol. 2016, 222, 277-284.

443

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444

FIGURE CAPTIONS

445

Figure 1. The purification and SDS-PAGE of CoXynA. (A) SDS-PAGE analysis of purified

446

CoXynA. M, marker; 1, the supernatant of cell lysates without IPTG; 2, the supernatant of cell

447

lysates with IPTG; 3, the elution purified by Ni-affinity column (50 mM Tris-HCl pH 8.0, 250

448

mM imidazole); 4, CoXynA purified by Q anion exchange chromatography; 5, CoXynA purified

449

by superdex-75 chromatography; (B) The gel filtration chromatography of CoXynA loaded on a

450

superdex-75 chromatography column.

451

Figure 2. The effect of temperature, pH and metals on CoXynA. (A) The effect of temperature

452

on the activity of CoXynA. The activity of CoXynA at optimal temperature was considered to be

453

100 %; (B) Thermostability of CoXynA at 70, 75, 80, 85°C. The activity of CoXynA without

454

heat treatment was considered to be 100 %. Error bars, SD (n = 5). (C) The effect of pH on the

455

activity of CoXynA. The activity of CoXynA at optimal pH was considered to be 100 %; (D) pH

456

stability of CoXynA determined at 70°C for 1 h. The activity of CoXynA without treatment was

457

considered to be 100 %. Error bars, SD (n = 5); (E) The effect of various additives (5 mM) on the

458

activity of CoXynA. The activity of CoXynA without additive was considered to be 100 %. Error

459

bars, SD (n = 5).

460

Figure 3. Overall structure of CoXynA. (A) Ribbon diagram of CoXynA with the α helices, β

461

strands and loops are drawn in cyan, magenta and red, respectively; (B) Superposition of the

462

CoXynA (blue) and the CbXyn10B (cyan). The catalytic residues of CoXynA are highlighted in

463

cyan sticks and the xylobiose molecule in CbXyn10B structure are shown in magenta sticks.

464

Figure 4. Molecular dynamics simulations and structural basis for thermostablility of CoXynA.

465

(A) Superposition of the CoXynA (blue) and the CmXyn10B (gray). The xylooligosaccharide

466

ligand in CmXyn10B structure are shown in green sticks; (B) Backbone RMSD values of the

21



467

CoXynA (red) and the CmXyn10B (blue) compared to corresponding crystal structure; (C)

468

Comparison of the RMSF values of the CoXynA (red) and the CmXyn10B (blue); (D)

469

Superposition of the 0-ns and the 100-ns snapshot of CmXyn10B (gray) and CoXynA (blue). The

470

largest deviation during the simulation occurs for the Loop 7, which is highlighted in red. (E)

471

The optimal temperature of recombinant CoXynA8. The Diagram showing the sequence

472

alignment of CoXynA and CoXynA8 is shown on the top. The Loop 7 sequences are highlighted

473

in box.

22


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Page 23 of 29


Figure 1

23



Figure 2

24


Page 24 of 29

Page 25 of 29


Figure 3

25



Figure 4

26


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Page 27 of 29


Table 1. Substrate specificity of CoXynA Enzyme

Source

CoXynA CbXyn10B PbXyn10B XynBE18 iXylC

C.owensis C.bescii DSM 6725 Paenibacillus barcinonensis BP-23 Paenibacillus sp. E18 C. laeviribosi HY-21

Specific activity (U/mg) Beech Birch CMC pNPX wood wood -Na 594 283 ND ND 497 448 2×10-3 0.05 249 233 286 NA NA 62 NA ND 44 52 58 NA

ND: Not detected. NA: Not available.

27


Identity

Reference

100% 81% 54% 49% 49%

This study Reference19 Reference38 Reference39 Reference40


Table 2. Data collection and refinement statistics CoXynA a

Data collection Space group Cell dimensions a, b, c (Å)

P1 21 1 126.6, 74.1, 137.7 90.0, 90.4, 90.0 30.00-2.10 (2.18-2.10) b 2542595 149034 11.3 (49.1) 15.3 (2.5) 99.9 (99.6) 6.5 (5.9)

α, β, γ (°)

Resolution (Å) No. of measured reflections No. of unique reflections Rmerge I / σI Completeness (%) Redundancy Refinement Resolution (Å) No. reflections Rwork / Rfree No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles (°)

29.11-2.10 (2.12-2.09) 148526 18.9/22.9 16634 0 1130 36.2 0 39.4 0.009 1.051

Ramachandran plot (%) Favored Allowed Disallowed

97.7 2.3 0.0

All data sets were collected from a single crystal. Values in the parentheses are for the highest-resolution shell.

28


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TOC graphic

Thermostable CoXynA

29


Structural Insights into the Thermophilic Adaption Mechanism of Endo

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