The potential of using thermostable xylan-binding domain as a

Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 12520−12526

pubs.acs.org/journal/ascecg

The Potential of Using Thermostable Xylan-Binding Domain as a Molecular Probe to Better Understand the Xylan Distribution of Cellulosic Fibers Lingfeng Long,† Jinguang Hu,*,‡ Xun Li,† Yu Zhang,† and Fei Wang*,†

Downloaded via GUILFORD COLG on August 1, 2019 at 05:42:06 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Jiangsu Key Lab for the Chemistry and Utilization of Agro-forest Biomass, College of Chemical Engineering, Nanjing Forestry University, No 159, Long Pan Road, Nanjing, 210037, PR China ‡ Department of Chemical and Petroleum Engineering, University of Calgary, 2500 University Drive NW, Calgary, T2N 1N4, Canada S Supporting Information *

ABSTRACT: Understanding the roles and functions of xylan is crucial for lignocellulose-based fuel and material production; however, the tracking of xylan in cellulosic substrates has been challenging. In this study, a xylan specific binding domain from the thermostable Thermotoga thermarum DSM 5069 xylanase (Xyn10A) N-terminal domain (N1−N2) was cloned and characterized as a potential molecular probe to monitor the interfacial xylan of cellulosic fibers. The results showed that the N1−N2 could selectively interact with both insoluble and soluble xylanolytic substrates (no affinity with either crystalline or amorphous cellulose). The N1−N2 probe was thermostable at 80 °C and still maintained the high xylanbinding affinity even after 2 h of incubation at 90 °C. Visualization of fluorescently labeled N1−N2 by confocal microscopy showed distinguishable distribution of surface xylan from bleached hardwood (BHK, 17.2% xylan) and softwood (BSK, 8.4% xylan) Kraft pulps, respectively. These differences were similar to the images measured by confocal Raman microscopy. Our results showed that the patterns of interfacial xylan of cellulosic fiber varied among different plant species, and also demonstrated the potential of using thermostable xylan-binding domain as a molecular probe to map the location of surface xylan. KEYWORDS: Thermostable xylan-binding module (XBD), Xylan distribution, Specific mapping, Kraft pulps

■

INTRODUCTION Climate change, energy insecurity, and environmental issues have all encouraged governments around the world to develop the so-called bioeconomy. Lignocellulosic biomass is the most abundant and renewable biomass resource on the earth, which can be used for the sustainable production of next generation fuels, chemicals, and materials.1−3 Although lignocellulose has been utilized for thousands of years along with human history (e.g., pulps and papers and construction materials), our knowledge of its detailed architecture (the network of different carbohydrate polymers and lignin) is still limited. This has considerably hindered the current application of lignocellulose in value-added bioproducts such as fuels and nanomaterials.4−6 Therefore, the development of specific techniques to localize and visualize the spatial distribution of the major polymer compositions of lignocellulose is crucial if we want to better understand and control the fine processing of lignocellulosic biomass. Although the tracking of the cellulose accessible area and surface morphology has been extensively studied in the past few years,7−10 our knowledge about hemicellulose location is © 2019 American Chemical Society

limited. Among hemicelluloses, xylan-type polysaccharides are often the most abundant and complex components in plant cell wall, especially for hardwood and annual plants.11 Xylan plays a vital role in plant cells (e.g., conducting water, maintaining cell wall integrity, providing mechanical support in strengthening secondary wall, etc.), and it has been acknowledged as a physical barrier that limits both lignin extraction and cellulose accessibility in pulp and paper and bioconversion industries accordingly.11−14 For example, even high purity cellulosic pulp product dissolving pulp still contains around 4% of xylan,15−17 and the selective removal/cleavage of xylan could considerably open up the lignocellulose structure during the enzymatic deconstruction process. Therefore, the tracking of xylan that remains on the cellulosic fibers helps us to not only better understand plant cell wall architecture in detail and its enzymatic decomposition mechanism but also improve the Received: April 23, 2019 Revised: May 20, 2019 Published: June 13, 2019 12520

DOI: 10.1021/acssuschemeng.9b02261 ACS Sustainable Chem. Eng. 2019, 7, 12520−12526

Research Article

ACS Sustainable Chemistry & Engineering

Figure 1. (A) Primary structure of xylanase from Thermotoga thermarum DSM 5069 (T. thermarum), Thermotoga maritima (T. maritima), Thermotoga neapolitana (T. neapolitana), Caldicellulosiruptor sp. Rt69B.1 (C. Rt69B.1), and Thermoanaerobacterium xylanolyticum (T. xylanolyticum). XBD, xylan-binding domain; CBD, cellulose binding domain; GH10, family 10 catalytic domain. (B) Sequence alignment of the N-terminal domains from T. thermarum, T. maritima, T. neapolitana, C. Rt69B.1, and T. xylanolyticum. Conserved aromatic amino acids were shaded. Sequencing alignment was performed by Clustal X.

The use of carbohydrate-binding modules (CBMs) to track specific carbohydrates within cellulosic fiber has garnered great attention in recent years.18,21,22 CBMs are special structural protein modules of carbohydrate active enzymes, with the main role of driving enzyme catalytic domains to their targeted polysaccharides in nature.23 Their high binding specificity and relatively simple operation conditions endow CBMs with the excellent potential to probe carbohydrate polymers within biomass. Along with others, we have developed various cellulose specific CBMs to assess cellulose accessibility and surface morphology of various cellulosic fibers in recent years.18,21,22,24,25 However, the utilization of xylan specific CBM probes to evaluate surface xylan has not been explored as much. During the last two decades, thermostable enzymes from thermophilic or hyperthermophilic microorganisms have become popular in many fields, since enzymatic reactions at

efficacy of biomass fragmentation and/or deconstruction as well as the corresponding bioproduct fabrication.18,19 Methods that have been explored to quantitively and qualitatively assess surface xylan of cellulosic fiber can be roughly divided into physical, chemical, and biological approaches. For example, the physical approach employs various spectrometric (e.g., Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD)) and microscopic techniques, while the chemical approach utilizes xylan-binding dyes. Biological approaches such as xylan specific antibodies have also been explored to map xylan present on the fiber surface.20,21 However, most of these methods are timeconsuming, often have limited specificity, and also require the access of specialized equipment and expertise. Therefore, the development of a diagnostic approach to track surface xylan in a rapid, easy, and specific manner would be beneficial. 12521


Research Article


xylan-binding domains (XBD) to bring xylanase to their targeted xylan substrates in nature.25,32,33 In most cases, the XBD domains of enzymes with A-domains are located on the N-terminal side of the catalytic domains (CD) while cellulose binding domains (CBD), if presented, are located on the Cterminal side.25,32 To gain deeper insight into the status of the N-terminal domains of Xyn10A, a multisequence alignment of the N-terminal domains of Xyn10A with some A-like domains was performed (Figure 1B). Results showed that the Nterminal domains of Xyn10A (N1 and N2) shared a high amino acid sequence similarity with that of the A-like domains of xylanase from Thermotoga maritima. The sequence alignment of A-like domains also exhibited the presence of conserved aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine, which could be responsible for carbohydrate recognition through hydrogen bonding and van der Waals interactions.34,35 Since previous works showed that xylanase with A-like domains has a high binding affinity toward xylan and also plays an important role in enzyme thermostability,30 these genes encoding N1, N2, and N1−N2 from Xyn10A were cloned and expressed, respectively. All three recombinant proteins (N1, N1 and N1−N2) exhibited a single band on SDS-PAGE after purification via Ni2+ affinity chromatography consistent with their theoretical molecular size (16, 20, and 34 kDa, respectively, Figure 2).

higher temperatures could generally accelerate catalytic reaction, improve mass transfer, reduce slurry viscosity, and avoid microbial contamination.5,26 The utilization of thermostable enzymes in high-temperature bioprocesses has garnered significant attention due to the increased biocatalytic kinetic performance, the reduced risk of biocontamination, and the improved substrate rheology.5,27,28 Although some currently known carbohydrate binding modules (CBMs), such as CBM15, CBM27, CBM44, etc., have already shown potential as molecular probes to track the surface xylan of cellulosic fiber,18,21,22 these xylan-binding CBMs are not thermostable (have high xylan-binding affinity only at room temperature) which limits their potential applications in a relatively high temperature environment.5,29,30 Therefore, it would be beneficial to identify thermostable CBM probes which could broaden the potential application areas of the CBM mapping techniques. Recently, we identified a hyperthermostable xylanase (Xyn10A) from Thermotoga thermarum DSM 5069, which exhibited optimal kinetics at a high temperature (95 °C) with great thermostability (retaining almost 100% of its initial activity when incubated at 85 °C for 2 h with 5 mM Ca2+).31 Xyn10A contains three N-terminal signal peptides N1−N2− N3 (approximately 130 amino acid residues, respectively), followed by a ∼320 amino acid catalytic domain (CD) and two related ∼180 residue domains (C1−C2). Comparative amino acid sequence analyses revealed that the N-terminal domains of Xyn10A (N1−N2) shared a highly aligned sequence with the well-known A-like xylan-binding modules (A1 and A2) of xylanase from Thermotoga maritima.25 Thus, we hypothesized that the N1−N2 of Xyn10A might be the specific xylanbinding domains of thermostable Xyn10A. In the work reported here, the N-terminal noncatalytic domains (N1, N2, and the tandem N1−N2) of Xyn10A were cloned and expressed, and their binding affinity and specificity on various “model” carbohydrate polymers (e.g., beechwood xylan, carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC), etc.,) and “realistic” cellulosic pulps (bleached Kraft pulp from hardwood and softwood, respectively) were systematically assessed, with the goal of obtaining a thermostable xylan specific CBM probe to track interfacial xylan in cellulosic fiber. Our results indicated that such a probe selectively and effectively interacted with both soluble and insoluble xylan substrates, showed super stability at relatively high temperature, and could be used as a rapid and simple approach to help us better understand the mechanism of xylan distribution during/after lignocellulosic processing.

Figure 2. SDS-PAGE analysis of purified N1, N2, and N1−N2. Lane M, protein marker.

Determination of Xylan-Binding Specificity. Many carbohydrate enzymes in nature contain carbohydrate binding modules (CBMs), which facilitate the binding of enzymes to the targeted substrates.36 Although many of the cellulose binding CBMs have high specificity, previous studies showed that many xylan-binding domains also have an affinity for cellulosic polysaccharides, probably due to most of the xylan being tightly associated with cellulose in the plant cell wall structure.37,38 Since one of the major goals of this study was to obtain a specific probe to map xylan in cellulosic fibers, we first evaluated the binding affinity and specificity of the three recombinant proteins (N1, N1, and N1−N2) on microcrystalline cellulose (MCC) and insoluble beechwood xylan accordingly. SDS-PAGE analysis was carried out on proteins recovered from both supernatant and insoluble substrates after 2 h of adsorption (Figure 3). The protein bovine serum albumin (BSA), which has no binding affinity toward carbohydrates, was used as the control. It appeared that N1 and the tandem N1−N2 interacted specifically with xylan, while similar to the BSA control, no binding was detected

■

RESULTS AND DISCUSSION Sequence Analysis of N-terminal Domains of Xyn10A. The genus Thermotoga, which produces a number of thermostable enzymes, has been successfully isolated from terrestrial and marine hydrothermal areas.30,32 Structural analysis of thermostable xylanase from T. thermarum DSM 5069 (Xyn10A) showed that Xyn10A was composed of a glyosidic hydrolase family 10 (GH10) catalytic domain bordered by three N-terminal domains and two C-terminal domains (Figure 1A) (https://blast.ncbi.nlm.nih.gov). This modular structure was architecturally identical to the xylanase with A-like domains (e.g., xylanases from Thermotoga maritima, Thermotoga neapolitana, Caldicellulosiruptor sp. Rt69B.1, Thermoanaerobacterium xylanolyticumThermoanaerobacterium xylanolyticum, etc.), which have been designated as the specific 12522


Research Article


showed no affinity toward all three binding modules tested here (Figure 4D,E). A strong smear of the protein band was observed in Figure 4E. This was probably due to the presence of a charged group (carboxymethyl), which could result in the electrophoretic mobility of the polysaccharide.37 On the basis of these results, we also confirmed the previous prediction of A-like domains as the xylan specific binding module of thermostable xylanase.25 Influence of Temperature on Binding Properties of N1 and N1−N2. As mentioned earlier, in this study, we aimed to obtain thermostable xylan specific binding modules, which could potentially track/monitor the water-insoluble xylan of cellulosic fiber in an industrially relevant harsh environment (70−90 °C). Earlier studies have proved that the main functions of A-like domains are polysaccharide binding and thermostabilization.30 Therefore, the influence of temperature on xylan-binding performances of both N1 and the tandem N1−N2 was tested and comparisons were made by varying temperatures from 30 to 95 °C. Since our final target was the assessment of insoluble xylan on cellulosic fiber interface by using the thermostable probe from Xyn10A, an industrially relevant substrate (bleached commercial Kraft pulp from hardwood (BHK)) was selected as the “model” feedstock to evaluate the influence of temperature on xylan-binding affinity. It was apparent that both probes displayed high thermostability and retained approximately 40% of the original binding affinity after 2 h of incubation at 85 and 90 °C for N1 and the tandem N1−N2, respectively (Figure 5). However, a higher temperature (90 °C for N1 and 95 °C for N1−N2) led to precipitation and defunctionalization of the proteins. Although the two probes exhibited the same optimal temperature (80 °C), the maximum binding temperature of N1−N2 was 5 °C higher than that of N1, indicating that N2 played a pivotal role in contributing to the thermostability of Xyn10A. This was consistent with a previous result by Jun et al. (2009).30 Compared with the most currently known thermostable xylanbinding domains A1A2 from Thermotaga maritima xylanase A (denatured at 80 °C), the tandem N1−N2 in our study enabled an further increase in thermostability up to 95 °C.25 This improvement could be significant since many pulping conditions and biomass biorefinery processes often require such a high temperature.1,3,26 Unlike A1A2 that contained one binding site, the analysis of binding isotherms investigated by the sensitive ITC showed that N1−N2 had two binding sites (data not shown). This status (two binding sites) of N1−N2 was consistent with the binding results in Figure 4B, because both N1 and N2 had the ability to bind soluble xylan and barley β-glucan. As for affinity value, the tandem N1−N2

Figure 3. Binding affinity of N1, N2, and N1−N2 to insoluble beechwood xylan (A) and microcrystalline cellulose (B) accordingly, revealed by SDS-PAGE. Each recombinant protein was incubated with insoluble xylan or microcrystalline cellulose for 2 h. Lane M, protein marker; Lane 1, unbound protein fraction; Lane 2, the fifth washed-off supernatant; Lane 3, bound protein fraction.

between N2 and xylan (Figure 3AN2) or MCC (Figure 3BN2). Therefore, N1 and N1−N2 were selected as suitable molecular probes to track xylan within cellulosic fibers. To further assess the binding affinity of N1 and N1−N2, nondenaturing affinity polyacrylamide gel electrophoresis analysis was further carried out with various plant derived polysaccharides. Affinity electrophoresis is a simple and fast method to detect CBMs binding affinity to various soluble glycans, where the interaction between the studied proteins and the gel-embedded polysaccharides can be revealed by a reduced mobility due to protein−polysaccharide interaction.39 A significant retardation of N1 and N1−N2 was found in the gel where xylan and barley β-glucan were presented (Figure 4), which exhibited identical binding characteristics as the reported xylan specific binding modules from Thermotoga maritima xylanase 10A, Pseudomonas cellulose xylanase Xyn10C (CBM15), Cellulomonas fimi xylanase D, Paenibacillus barcinonensis xylanase 10C, etc.24,25,39,40 In addition, it appeared that N2 also showed a higher binding affinity to barley β-glucan (Figure 4C) in comparison to others. This information could be useful for those who are interested in the development of a β-glucan specific carbohydrate binding module. Carboxymethyl cellulose (CMC) and galactomannan

Figure 4. Nondenaturing affinity polyacrylamide gel electrophoresis analysis of purified N1, N2, and N1−N2. (A) Control (without adding polysaccharide), (B) 0.1% (w/v) beechwood xylan, (C) 0.1% (w/v) barley β-glucan, (D) 0.1% (w/v) galactomannan, (E) 0.1% (w/v) carboxymethyl cellulose (CMC). Lane M, maker; Lane 1, N1; Lane 2, N2; Lane 3, N1−N2. 12523


Research Article


Figure 5. Effect of temperature on the binding of N1 (A) and N1−N2 (B) to bleached hardwood Kraft pulp (BHK). After 2 h of incubation, binding was quantified by measuring the residual protein amount in the supernatant.

Figure 6. Bleached Kraft pulp fibers shown with (a) and without (b) the fluorescence distribution from fluorescently tagged N1−N2 probe. (c) Images of the fiber cross-section and the corresponding intensity profiles for the marked intersection. (A) Bleached Kraft pulp from hardwood (BHK), (B) bleached Kraft pulp from softwood (BSK).

exhibited a higher binding affinity (2×) for xylo-oligosaccharide (xylopentose, 8.8 × 105 M−1) than A1A2 (XBDs) from T. maritima (4.0 × 105 M−1).25 Furthermore, no affinity of N1− N2 was found toward cellopentaose by ITC analysis. It should be noted that both thermostable xylan-binding probes (N1− N2 and A1A2) revealed a very strong xylopentose affinity in comparison to that of mesothermal CBM15 (1.4 × 104 M−1), indicating that these thermostable probes hold great potential to specifically bind and thus monitor xylan during industrially relevant processing.24 Besides oligosaccharides, the binding affinities of N1−N2 toward insoluble xylan substrates were also compared by using beechwood, bagasse, and corn cob xylan. In general, xylan from bagasse had the highest degree of substitution, followed by corn cob xylan and beechwood xylan.41−43 It appeared that the N1−N2 probe was more capable to bind to xylan with a high degree of substitution, as it exhibited the highest binding affinity toward bagasse xylan

(∼86.2%), followed by corn cob xylan (∼63.6%) and beechwood xylan (∼39.0%). This was also in agreement with an earlier report that GH10 xylanases preferably cleave glycosidic linkages in the highly substituted xylan backbone within biomass.5 Confocal Imaging of N1−N2 Distribution on Bleached Kraft Pulps. Since the thermostable N1−N2 probe from Xyn10A showed great promise for xylan-binding at a molecular level, we next tested its potential to further visualize the binding profile of this probe on the commercial Kraft pulps derived from hardwood (BHK, 17.2% xylan) and softwood (BSK, 8.4% xylan), respectively. After being tagged with AMCA-X, the thermostable xylan-binding module (XBD) and BSA control were incubated with BHK and BSK accordingly. No notable green fluorescence could be observed under confocal images for tagged BSA (Figure S1). In contrast, obvious fluorescence was observed for labeled N1−N2 (Figure 12524



■

6). Briefly, the probe binding to the surface of BHK exhibited a very uniform and bright fluorescence distribution, whereas disordered localization of probe was observed on BSK fibers. This was most likely due to the different chemical compositions and structures of the hemicelluloses of hardwood and softwood derived pulp samples. It is known that hardwood hemicelluloses are mainly composed of xylan while softwood consists of mostly galactoglucomannan.44 Since hemicelluloses are thought to form a sheath on each cellulose microfibril, hardwood xylan might equally localize on the fiber surface, resulting in a uniform XBD distribution (Figure 6A).45 As for softwood, a scattered xylan distribution on the surface resulted in a disordered fluorescence distribution. The confocal z-stack measurement was also carried out to show cross-sectional images of pulp fibers stained with the fluorescence dye-linked xylan-binding proteins N1−N2 (Figure 6 A(c),B(c)). The results showed that the cross-sectional images of BHK (bleached hardwood kraft pulp) fibers were brighter and more evenly distributed than those of BSK (bleached softwood kraft pulp) fiber. The higher fluorescence intensity of BHK was expected since it contains twice the amount of xylan in BSK. While the distribution patterns of the xylan-binding probes were interesting, it seemed that the xylan in the BHK fiber was evenly distributed while the xylan in the BSK fiber was mainly located toward the primary cell wall and the cell lumen. The fluorescence intensity profiles across the fiber cross-sections also showed wider profile peaks with BSK fiber than with BHK fiber, indicating that the xylan-binding probes diffused deeper into BSK fiber. This was likely due to the higher accessibility of the BSK fiber (73 mg/g pulp, Simons’ stain values) than the BHK (45.5 mg/g pulp) as was demonstrated in our previous study.13 Unfortunately, we cannot detect xylan distribution at a nanometer scale at this moment because of the limited resolution of confocal laser scanning microscopy. Aside from the confocal laser scanning microscopy, confocal Raman microscopy was also further applied to in situ map xylan presented on the pulp fiber surfaces at molecular level (Figure S2). Results showed that similar to confocal microscopy images (Figure 6), a much brighter and evenly distributed xylan patterns were observed on BHK fiber surface than these on BSK fiber surface.

Research Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.9b02261.

■

Experimental section, fluorescence distribution from fluorescently tagged BSA, in situ tracking of xylan in hardwood and softwood Kraft pulps by confocal Raman microscopy (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jinguang Hu: 0000-0001-8033-7102 Xun Li: 0000-0002-1267-9263 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 31370572) and China Scholarship Council (CSC). This research was undertaken thanks in part to funding from the Canada First Research Excellence Fund.

■

REFERENCES

(1) Chundawat, S. P. S.; Beckham, G. T.; Himmel, M. E.; Dale, B. E. Deconstruction of Lignocellulosic Biomass to Fuels and Chemicals. Annu. Rev. Chem. Biomol. Eng. 2011, 2, 121−45. (2) Adsul, M. G.; Singhvi, M. S.; Gaikaiwari, S. A.; Gokhale, D. V. Bioresource Technology Development of Biocatalysts for Production of Commodity Chemicals from Lignocellulosic Biomass. Bioresour. Technol. 2011, 102 (6), 4304−4312. (3) Zhao, X.; Zhang, L.; Liu, D.; Chemistry, A. Part II: Fundamentals of Different Pre-Treatments to Increase the Enzymatic Digestibility of Lignocellulose. Biofuel Bioprod Bior. 2012, 6, 561−579. (4) Hu, J.; Arantes, V.; Saddler, J. N. The Enhancement of Enzymatic Hydrolysis of Lignocellulosic Substrates by the Addition of Accessory Enzymes such as Xylanase: Is It an Additive or Synergistic Effect? Biotechnol. Biofuels 2011, 4 (1), 36−48. (5) Long, L.; Tian, D.; Zhai, R.; Li, X.; Zhang, Y.; Hu, J.; Wang, F.; Saddler, J. Thermostable Xylanase-Aided Two-Stage Hydrolysis Approach Enhances Sugar Release of Pretreated Lignocellulosic Biomass. Bioresour. Technol. 2018, 257, 334−338. (6) Peng, X.; Qiao, W.; Mi, S.; Jia, X.; Su, H.; Han, Y. Characterization of Hemicellulase and Cellulase from the Extremely Thermophilic Bacterium Caldicellulosiruptor Owensensis and Their Potential Application for Bioconversion of Lignocellulosic Biomass without Pretreatment. Biotechnol. Biofuels 2015, 8 (1), 131−144. (7) Chandra, R. P.; Au-Yeung, K.; Chanis, C.; Roos, A. A.; Mabee, W.; Chung, P. A.; Ghatora, S.; Saddler, J. N. The Influence of Pretreatment and Enzyme Loading on the Effectiveness of Batch and Fed-Batch Hydrolysis of Corn Stover. Biotechnol. Prog. 2011, 27 (1), 77−85. (8) Chandra, R.; Ewanick, S.; Hsieh, C.; Saddler, J. N. The Characterization of Pretreated Lignocellulosic Substrates prior to Enzymatic Hydrolysis. Biotechnol. Prog. 2008, 24, 1178−1185. (9) Hu, J.; Tian, D.; Renneckar, S.; Saddler, J. N. Enzyme Mediated Nanofibrillation of Cellulose by the Synergistic Actions of an Endoglucanase, Lytic Polysaccharide Monooxygenase (LPMO) and Xylanase. Sci. Rep. 2018, 8, 3195−3402.

■

CONCLUSION In this study, a thermostable molecular probe (N1−N2) from Thermotoga thermarum DSM 5069 xylanase (Xyn10A) was cloned and characterized. Similar to the reported binding substrate specificity of A-like XBDs, N1−N2 could interact with both soluble and insoluble xylan preparations and with mixed-linkage β-1,3/β-1,4-glucans (barleyβ-glucan) but showed no affinity for insoluble microcrystalline cellulose, carboxymethyl cellulose (CMC), and galactomannan. This probe exhibited high thermostability over a broad range of temperatures, 30−90 °C. The results of the confocal imaging of the probe distribution on industrially relevant Kraft pulps demonstrated that N1−N2 had great potential to bind and monitor xylan on the surface of bleached Kraft pulps. We believe that the CBM tracking strategy could be a useful tool to help us better understand the surface morphology and chemical composition of cellulosic fibers. This work could open the door to formulate more efficient CBM probes and further expand our knowledge of cellulosic materials. 12525


Research Article


(30) Jun, H.; Bing, Y.; Keying, Z.; Xuemei, D.; Daiwen, C. Thermostable Carbohydrate Binding Module Increases the Thermostability and Substrate-Binding Capacity of Trichoderma Reesei Xylanase 2. New Biotechnol. 2009, 26, 2−8. (31) Shi, H.; Li, X.; Gu, H.; Zhang, Y.; Huang, Y.; Wang, L.; Wang, F. Biochemical Properties of a Novel Thermostable and Highly Xylose-Tolerant β-Xylosidase/α-Arabinosidase from Thermotoga Thermarum. Biotechnol. Biofuels 2013, 6 (1), 27−36. (32) Winterhalter, C.; Heinrich, P.; Candussio, A.; Wich, G.; Liebl, W. Identification of a Novel Cellulose-binding Domain the Multidomain 120 kDa Xylanase XynA of the Hyperthermophilic Bacterium Thermotoga maritima. Mol. Microbiol. 1995, 15 (3), 431−444. (33) Morris, D. D.; Gibbs, M. D.; Ford, M.; Thomas, J.; Bergquist, P. L. Family 10 and 11 Xylanase Genes from Caldicellulosiruptor Sp. Strain Rt69B.1. Extremophiles 1999, 3 (2), 103−111. (34) Tormo, J.; Lamed, R.; Chirino, A. J.; Morag, E.; Bayer, E. A.; Shoham, Y.; Steitz, T. A. Crystal Structure of a Bacterial Family-lIl Cellulose-Binding Domain: A General Mechanism for Attachment to Cellulose. EMBO J. 1996, 15 (21), 5739−5751. (35) Gilbert, H. J.; Knox, J. P.; Boraston, A. B. Advances in Understanding the Molecular Basis of Plant Cell Wall Polysaccharide Recognition by Carbohydrate-Binding Modules. Curr. Opin. Struct. Biol. 2013, 23 (5), 669−677. (36) Carrard, G.; Koivula, A.; Soderlund, H.; Beguin, P. CelluloseBinding Domains Promote Hydrolysis of Different Sites on Crystalline Cellulose. PNAS. 2000, 97 (19), 10342−10347. (37) Bolam, D. N.; Xie, H.; White, P.; Simpson, P. J.; Hancock, S. M.; Williamson, M. P.; Gilbert, H. J. Evidence for Synergy between Family 2b Carbohydrate Binding Modules in Cellulomonas Fimi Xylanase 11A. Biochemistry 2001, 40 (8), 2468−2477. (38) Filonova, L.; Gunnarsson, L. C.; Daniel, G.; Ohlin, M. Synthetic Xylan-Binding Modules for Mapping of Pulp Fibres and Wood Sections. BMC Plant Biol. 2007, 7, 1−10. (39) Tomme, P.; Boraston, A.; Kormos, J. M.; Warren, R. A. J.; Kilburn, D. G. Affinity Electrophoresis for the Identification and Characterization of Soluble Sugar Binding by Carbohydrate-Binding Modules. Enzyme Microb. Technol. 2000, 27, 453−458. (40) Simpson, P. J.; Bolam, D. N.; Cooper, A.; Ciruela, A.; Hazlewood, G. P.; Gilbert, H. J.; Williamson, M. P. A Family IIb Xylan-Binding Domain Has a Similar Secondary Structure to a Homologous Family IIa Cellulose-Binding Domain but Different Ligand Specificity. Structure 1999, 7 (7), 853−864. (41) Jayapal, N.; Samanta, A. K.; Kolte, A. P.; Senani, S.; Sridhar, M.; Suresh, K. P.; Sampath, K. T. Value Addition to Sugarcane Bagasse: Xylan Extraction and its Process Optimization for Xylooligosaccharides Production. Ind. Crop. Prod. 2013, 42, 14−24. (42) Puls, J. Chemistry and Biochemistry of Hemicelluloses: Relationship Between Hemicellulose Structure and Enzymes Required for Hydrolysis. Macromol. Symp. 1997, 120, 183−196. (43) Van Dongen, F. E. M.; Van Eylen, D.; Kabel, M. A. Characterization of Substitutents in Xylans from Corn Cobs and Stover. Carbohydr. Polym. 2011, 86, 722−731. (44) Sjoström, E. Wood chemistry fundamentals and applications; Academic Press: New York, 1993. (45) Grethlein, H. E. The Effect of Pore Size Distribution on the Rate of Enzymic Hydrolysis of Cellulosic Substrates. Bio/Technology 1985, 3 (2), 155−160.

(10) Falco, C.; Baccile, N.; Titirici, M.-M. Green Chemistry Morphological and Structural Differences between Glucose, Cellulose and Lignocellulosic Biomass Derived Hydrothermal Carbons. Green Chem. 2011, 13, 3273−3281. (11) Leschine, S. B. Cellulose Degradation in Anaerobic Environments. Annu. Rev. Microbiol. 1995, 49 (1), 399−426. (12) Hu, J.; Arantes, V.; Pribowo, A.; Saddler, J. N. The Synergistic Action of Accessory Enzymes Enhances the Hydrolytic Potential of A “cellulase Mixture” but Is Highly Substrate Specific. Biotechnol. Biofuels 2013, 6 (1), 1−112. (13) Long, L.; Tian, D.; Hu, J.; Wang, F.; Saddler, J. A XylanaseAided Enzymatic Pretreatment Facilitates Cellulose Nanofibrillation. Bioresour. Technol. 2017, 243, 898−904. (14) Scheller, H. V.; Ulvskov, P. Hemicelluloses. Annu. Rev. Plant Biol. 2010, 61, 263−89. (15) Andrade, M. F.; Colodette, J. L. Dissolving Pulp Production from Sugar Cane Bagasse. Ind. Crops Prod. 2014, 52, 58−64. (16) Christov, L. P.; Prior, B. A. Xylan Removal from Dissolving Pulp Using Enzymes of Aureobasidium Pullulans. Biotechnol. Lett. 1993, 15 (12), 1269−1274. (17) Gübitz, G. M.; Lischnig, T.; Stebbing, D.; Saddler, J. N. Enzymatic Removal of Hemicellulose from Dissolving Pulps. Biotechnol. Lett. 1997, 19 (5), 491−495. (18) Hébert-Ouellet, Y.; Meddeb-Mouelhi, F.; Khatri, V.; Cui, L.; Janse, B.; MacDonald, K.; Beauregard, M. Tracking and Predicting Wood Fibers Processing with Fluorescent Carbohydrate Binding Modules. Green Chem. 2017, 19 (11), 2603−2611. (19) Van Dyk, J. S.; Pletschke, B. I. A Review of Lignocellulose Bioconversion Using Enzymatic Hydrolysis and Synergistic Cooperation between Enzymes-Factors Affecting Enzymes, Conversion and Synergy. Biotechnol. Adv. 2012, 30 (6), 1458−1480. (20) Knox, J. P. In Situ Detection of Cellulose with CarbohydrateBinding Modules. Method. Enzymol. 2012, 510, 233−245. (21) Khatri, V.; Héb ert-Ouellet, Y.; Meddeb-Mouelhi, F.; Beauregard, M. Specific Tracking of Xylan Using Fluorescent-Tagged Carbohydrate-Binding Module 15 as Molecular Probe. Biotechnol. Biofuels 2016, 9 (1), 74−86. (22) Gourlay, K.; Hu, J.; Arantes, V.; Penttilä, M.; Saddler, J. N. The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- And Swollenin-Induced Deconstruction of Lignocellulosic Substrates. J. Biol. Chem. 2015, 290 (5), 2938−2945. (23) Boraston, A. B.; Bolam, D. N.; Gilbert, H. J.; Davies, G. J. Carbohydrate-Binding Modules: Fine-Tuning Polysaccharide Recognition. Biochem. J. 2004, 382 (3), 769−781. (24) Szabó, L.; Jamal, S.; Xie, H.; Charnock, S. J.; Bolam, D. N.; Gilbert, H. J.; Davies, G. J. Structure of a Family 15 CarbohydrateBinding Module in Complex with Xylopentaose: Evidence That Xylan Binds in an Approximate 3-Fold Helical Conformation. J. Biol. Chem. 2001, 276 (52), 49061−49065. (25) Meissner, K.; Wassenberg, D.; Liebl, W. The Thermostabilizing Domain of the Modular Xylanase XynA of Thermotoga Maritima Represents a Novel Type of Binding Domain with Affinity for Soluble Xylan and Mixed-Linkage β-1,3/β-1,4-Glucan. Mol. Microbiol. 2000, 36 (4), 898−912. (26) Turner, P.; Mamo, G.; Karlsson, E. N. Potential and Utilization of Thermophiles and Thermostable Enzymes in Biorefining. Microb. Cell Fact. 2007, 6, 1−23. (27) Harris, A. D.; Ramalingam, C. Xylanase and Its Application in Food Industry: A Review. Biotechnol. Biofuels 2010, 1, 01−11. (28) Wang, W.; Mozuch, M. D.; Sabo, R. C.; Kersten, P.; Zhu, J. Y.; Jin, Y. Production of Cellulose Nanofibrils from Bleached Eucalyptus Fibers by Hyperthermostable Endoglucanase Treatment and Subsequent Microfluidization. Cellulose 2015, 22, 351−361. (29) Zhai, R.; Hu, J.; Saddler, J. N. What Are the Major Components in Steam Pretreated Lignocellulosic Biomass That Inhibit the Efficacy of Cellulase Enzyme Mixtures? ACS Sustainable Chem. Eng. 2016, 4, 3429−3436. 12526


The potential of using thermostable xylan-binding domain as a

Recommend Documents