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A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81
New BIOTECHNOLOGY 53 (2019) 57–64
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A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81 Atilio Tomazinia,1, Paula Higasia,1, Livia R. Manzinea, Matthew Stottb, Richard Sparlingc, ⁎ David B. Levind, Igor Polikarpova, a
Sao Carlos Institute of Physics, University of Sao Paulo, São Carlos, São Paulo, Brazil School of Biological Sciences, University of Canterbury, New Zealand Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada d Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, Canada b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Thermogemmatispora sp. T81 Heterologous gene expression Thermostability Enzyme kinetics Enzyme structure
A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 °C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family β-D-xylosidase. Using 4-nitrophenyl β-D-xylopyranoside (pNPX) as a substrate, the KM, Vmax, kcat and kcat/KM kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s−1 and 156.8 mM−1 s−1, respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme’s low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family β-D-xylosidase.
Introduction Hemicellulose is the second most abundant heteropolysaccharide in nature and its main component is xylan [1]. Xylan has a backbone of covalently linked β-D-1-4-xylopyranose units, which can be decorated by L-arabinofuranose, 4-O-methyl-D-glucuronic acid, p-cumaric acid and ferrulic acid residues [1,2]. The ratio and degree of these decorations depend on xylan origin. Complete degradation of xylan into monosaccharide units requires a set of enzymes acting in synergy, known as a xylanolytic complex [3]. The core enzymes that depolymerizing the xylan backbone are β-D-1,4xylanases (EC 3.2.1.8) and β-D-1,4-xylosidases (EC 3.2.1.37). The xylanase hydrolyzes the β-1,4 linkages in an endo fashion, yielding xylooligosaccharides, which are degraded into xylose monomers by exoacting β-D-xylosidases [1,4]. To date, according to the CarbohydrateActive enZymes database (CAZy; www.cazy.org) [5], xylosidases are classified into glycoside hydrolase families (GHs) 1, 3, 5, 30, 39, 43, 51, 52, 54, 116, and 120, representing enzymes from all the kingdoms of life. All β-xylosidases hydrolyze their substrate with retention of the anomeric configuration, except for GH43 members, which are inverting
GHs. The GH3 and GH43 Families are the most prevalent, accounting for more than two thirds of the currently known β-xylosidases [6]. To date, nine β-xylosidase structures have been deposited in the PDB database (http://www.rcsb.org/), distributed between four different GH families (GH3 (1), GH39 (4), GH43 (3), GH52 (1)). Eight are of bacterial enzymes, of which Geobacillus stearothermophilus enzymes are the most extensively studied, with 4 structures determined (GH39 (2), GH43 (1) and GH52 (1)). No structural information is currently available for GH5 β-xylosidases. β-xylosidases have a number of biotechnological applications, including in the pulp and paper industry, the food industry and in bioethanol production [7]. However, enzymes intended for industrial application must optimally have specific characteristics such as thermostability, high activity and appropriate pH range, which translate into reduced amounts of enzyme required and a lower risk of microbial contamination [8]. Thermogemmatispora sp. T81 (Phylum Chloroflexi, Family Thermogemmatisporaceae) is an aerobic, thermophilic bacterium isolated from a geothermal hotspring [9], possessing a filamentous morphology with hyphaed mycelium and aerial spores capable of growth in
Abbreviations: CAZy, Carbohydrate-Active enZymes database; CMC, carboxymethyl-cellulose; GH, glycoside hydrolase family; SAXS, small-angle X-ray scattering; Trx, thioredoxin; DNS, 3,5-dinitrosalicylic acid; pNPX, 4-nitrophenyl β-D-xylopyranoside ⁎ Corresponding author. E-mail address: [email protected] (I. Polikarpov). 1 Equally contributing authors. https://doi.org/10.1016/j.nbt.2019.07.002 Received 7 April 2018; Received in revised form 9 June 2019; Accepted 6 July 2019 Available online 09 July 2019 1871-6784/ © 2019 Elsevier B.V. All rights reserved.
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gradient of 50–500 mM of imidazole. The eluted protein containing fractions were pooled, dialyzed (buffer A) to remove the imidazole and concentrated using Vivaspin 20 (GE Healthcare). The recombinant protein was incubated overnight with TEV protease (1 mg TEV protease per 500 mg of recombinant protein) and submitted to a second affinity chromatography step using Ni-NTA resin (1 mL of resin per 50 mg of recombinant protein) to separate the HisTrx tag from the target enzyme. The purified enzyme was concentrated and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using a 15% (v/v) gel [15], and the protein concentration determined by extinction coefficient (Ɛ = 101,885 M−1 cm−1) at 280 nm using a Nanodrop 1000 spectrophotometer (Thermo Scientific). SDS-PAGE gels were stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories) and destained with 10% (v/v) acetic acid.
mostly acidic and neutral pHs ranging from 3.1 to 7.5, and temperatures varying from 40 °C to 75 °C. The organism exhibits halotolerance and is able to use different substrates as a source of nutrients [9]. Genome analysis has identified two GH5 genes (A4R35_16780 and A4R35_07040) in the Thermmogematispora sp. T81 genome [10], of which the latter was annotated as a β-xylosidase. Given that most βxylosidases used in industrial applications are thermophilic enzymes [11], its biochemical characterization is a subject of interest. The β-xylosidase from Thermmogematispora sp. T81 belongs to subfamily 22 of GH5 [10]. The GH5 is one of the largest among 149 GH families and is highly divergent in terms of sequences and functions [12]. The only GH5_22 β-xylosidase characterized to date is from Phanerochaete chrysosporium [13]. Here we report the cloning and heterologous expression of a gene from Thermogemmatispora sp. T81 encoding a glycoside hydrolase from the GH5_22 family, designated T81Xyl5_22A. The enzyme’s thermostability in different buffers and pH conditions was evaluated using the thermofluor assay. In addition, the low-resolution envelope of the enzyme was determined using small angle X-ray scattering (SAXS) and changes in its tertiary structure monitored by tryptophan intrinsic fluorescence. Finally, the enzyme specificity activity was measured and the hydrolytic products generated were characterized, demonstrating that it is a thermostable β-D-xylosidase, which may have potential for biotechnological applications.
Protein stability assay Thermofluor assays [16,17] were conducted to identify the optimal buffer conditions for T81Xyl5_22A stability. The experiment was performed on the CFX96 Real-Time System (Bio-Rad). A SYPRO Orange stock solution in DMSO (Invitrogen) was diluted 1:300 in water to yield a working solution. 47 different conditions covering a pH range from 1.0 to 10.0 (Suppl. Table 1) were screened using a 96-well PCR microplate (Axygen). The total reaction volume was 20 μL and the plate was set up on ice: 10 μL of the screening solution, 5 μL of T81Xyl5_22A (7 μM final concentration) and 5 μL of SYPRO Orange working solution per well. Immediately following the addition of the dye, the measurements were carried out with excitation wavelength at 490 nm and emission at 530 nm. The samples were heated across a temperature gradient of 25–90 °C. Protein melting temperature (Tm) values were determined using Origin software (OriginLab, Northampton, MA) and experiments were performed in duplicate.
Materials and methods Cloning The T81Xyl5_22A gene product was identified by proteomic analyses of the Thermogemmatispora sp. T81 secretome [10]. T81Xyl5_22A (locus A4R35_07040) was one of four extracellular CAZymes identified in the secretome after growth of the bacterium on cellulose as a sole carbon source [10]. Bioinformatic analyses determined that T81Xyl5_22A was encoded by the gene located at locus tag A4R35_07040 in the sequenced genome of the thermophilic bacterium Thermogemmatispora sp. T81 (BioProject PRJNA317154) [10]. Ligase Independent Cloning (LIC) was used as previously described [14], and the set of oligonucleotides for gene amplification was synthesized (Exxtend Biotecnologia Ltda, Campinas Brazil); Forward: 5′-CAGGGCGCCATGACATCGGGTAT GCTCACTACG-3′ and Reverse: 5′-GACCCGACGCGGTTATTAGCGACAAGCATACTCCC TGAG-3′. The gene was cloned into vector pET Trx-1a/Lic, inserted into E. coli DH10B cells for propagation and expressed in E. coli Rosetta (DE3) pLysS cells.
Enzyme activity assays Enzyme activity was quantified using the 3,5-dinitrosalicylic acid (DNS) reagent method [18] by measuring absorbance at 535 nm in addition to assays measuring para-nitrophenol (pNP) absorbance measured at 405 nm. The experiments were performed in triplicate; xylose and glucose standard curves in the concentration range from 0.1 to 1 mM and a pNP standard curve in the range from 0 to 0.1 mM were used. Optimal temperature and pH assays for enzymatic activity
Protein expression, purification and gel electrophoresis analysis The effect of pH on T81Xyl5_22A activity was determined using 20 mM sodium citrate buffer at pHs ranging from 3.0 to 6.0. 0.4 mg of beechwood xylan (Sigma Aldrich, St Louis, MS) was incubated with 12.5μM T81Xyl5_22A for 2 h at 60 °C under continuous agitation at 1000 rpm. The optimal temperature was measured in the range of 55–80 °C. The reaction consisted of 40 μL 100 mM sodium citrate buffer, pH 5.0, 40 μL 1% beechwood xylan prepared in sodium citrate buffer, pH 5.0, 2.77 μM purified enzyme in a final reaction volume of 100 μL. The mixture was incubated for 60 min at 1000 rpm.
E. coli Rosetta (DE3) pLysS cells transformed with the recombinant plasmid were grown in Luria-Bertani (LB) medium supplemented with 1 μg/mL kanamycin and 20 μg/mL chloramphenicol at 37 °C, rotated at 160 rpm until A600 of approximately 0.6. The temperature was lowered to 18 °C and protein expression was induced by addition of 1 mM IPTG for 18 h. The cells were harvested by centrifugation (10,000 × g, 30 min, 4 °C) and resuspended in lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 5% glycerol, 0.2 mM PMSF, 4 mM β-mercaptoethanol, 0.8 mM DTT, 10 mM imidazole, and 0.1% N-lauroylsarcosine (Sigma Aldrich, St Louis, MS)). The cells were sonicated for 10 min (10 cycles of 30 s each) using a Branson Sonifier 450 (BRANSON Ultrasonics Corporation, Danbury, CT). A 40% duty cycle with the 4 microtrip power output was used and the cells were kept on ice. The resulting solution was clarified by centrifugation (30,000 × g, 30 min, 4 °C), followed by affinity chromatography of the supernatant using Ni-NTA resin. The supernatant was loaded onto a free resin previously washed with buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol), homogenized by stirring and kept on ice for 45 min. Next, the column was washed with binding buffer supplemented with 20 mM imidazole and the His-Thioredoxin (Trx) tagged protein was eluted using a step
Substrate specificity and products of hydrolysis To determine the substrate specificity for T81Xyl5_22A, the following were prepared at 1% (m/v) concentration in 100 mM sodium citrate buffer, pH 5.0: xyloglucan, mannan, liquenan, galactomannan, β-glucan, rye arabinoxylan, larch arabinogalactan and arabinan sugarbeet (Megazyme, Ireland), beechwood xylan, Sigmacell 20, low viscosity carboxymethyl-cellulose (CMC), microgranular cellulose, laminarin, Avicel PH-101 and pectin (Sigma Aldrich, St Louis, MS); filter paper n°1 (Whatman, Maidstone UK). The reaction mixture consisted of 40 μL 100 mM sodium citrate buffer, 40 μL of substrate and 20 μL of the 58
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water bath. T81Xyl5_22A fluorescence emission spectra at 3.7 μM in 100 mM sodium citrate buffer, pH 5.0 were recorded in a 300–450 nm range using an excitation wavelength of 295 nm and a 305 nm emission filter cutoff. Temperature variation was monitored from 30 to 90 °C with 2 min of protein incubation under readout temperature. After each measurement the spectrum was corrected for buffer contribution and the data analysis performed using Origin (OriginLab, Northampton, MA) software.
purified enzyme (1.85 μM final concentration). Reactions were conducted under the optimal temperature and pH conditions (65 °C and pH 5.0) for T81Xyl5_22A for 1 and 6 h under constant agitation at 1000 rpm. Thermostability assay For the thermostability assay, a mixture of purified enzyme and sodium citrate buffer, pH 5.0, were incubated for 24 h at 50–80 °C. 20 μL of the incubated enzyme (1.85 μM final concentration), 40 μL of 100 mM sodium citrate buffer and 40 μL of 1% (m/v) beechwood xylan solution were then added to the reaction and incubated for 1 h at optimum temperature (65 °C). Residual activity was quantified as described.
Circular dichroism (CD) measurements CD data were acquired on a Jasco J-720 CD spectrometer (Jasco, Tokyo, Japan). T81Xyl5_22A at 3.7 μM was equilibrated in 20 mM Na2HPO4, 20 mM NaCl, pH 7.5 and applied at 25 °C to 0.1-cm path length cuvette. CD spectra were recorded in the 190–260 nm wavelength range with 100 nm.min-1 increment step, 10 s averaging time, 1 nm bandwidth and a response time of 0.5 s. Solvent contributions were subtracted from the original spectral curves and an estimation of the secondary structure elements was obtained using the Dichroweb webserver [21].
Determination of T81Xyl5_22A kinetic parameters To determine T81Xyl5_22A kinetic parameters, KM and Vmax, beechwood xylan was used as substrate in a concentration range from 1 to 16 mg/mL, under enzyme optimum conditions of pH and temperature (pH 5.0 and 65 °C). After 30 min, the product was quantified using the 3,5-dinitrosalicylic acid (DNS) reagent method [18]. Kinetic parameters were also assayed using 4-nitrophenyl β-D-xylopyranoside (pNPX, Sigma Aldrich, St Louis, MS) as substrate, at concentrations from 0.04 to 4 mM at 65 °C. After 10 min, the reaction was stopped with 500 mM sodium carbonate and released p-nitrophenol (pNP) was quantified by measuring absorbance at 405 nm. Data analysis was performed using Origin (OriginLab, Northampton, MA) software and the determination of the kinetic parameters was calculated assuming that 1 unit of enzyme activity (U) releases 1 μmol per minute of xylose or pNP.
Small angle X-ray scattering (SAXS) Synchrotron radiation X-ray scattering data were collected at the D02A-SAXS2 beamline of the Brazilian National Synchrotron Laboratory (LNLS, Campinas, Brazil) using a bi-dimensional detector (MarResearch, EUA). A sample to detector distance of 890 mm was used, thus covering the range of momentum transfer 0.012 Å−1 < q < 0.405 Ǻ−1 (q = 4π sin(θ)/λ), where 2θ was the scattering angle and λ = 0.148 nm. The protein solutions at 1, 2 and 3 mg/mL (which corresponds to 18, 28 and 56 μM of protein, respectively) in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 were manually loaded into the sample holder installed in the vacuum sample chamber. The data were normalized to the beam intensity using the FIT2D program [22]. The scattering of the buffer was subtracted and the difference curves were scaled for concentration. The radius of gyration (Rg) was computed by Guinier approximation [23] and distance distribution functions p(r), radius of gyration Rg and maximum diameters Dmax of the scattering objects were calculated using the GNOM program [24]. Ab initio shape models for T81Xyl5_22A were obtained by superposition of 10 independent models using the DAMMIN program [25]. These results were compared with the T81Xyl5_22A homology model obtained from the I-TASSER server [26] using the structure of endoglucanase Cel5A from Pseudomonas stutzeri (PDB id: 4LX4) as template. A theoretical SAXS curve from the predicted structure model was generated and compared with the experimental curve through Debye formula using FoXS web tool [27]. Finally, T81Xyl5_22A molecular mass was estimated from SAXS experimental data using SAXS MoW calculator [28,29].
Effect of ions and additives The effects of different metal ions (Mg2+, Ni2+, Ca2+, Cu2+, K+, Na+, Zn2+, Mn2+, Fe2+, Ag+, Co2+, Li+, Sr2+) and additives (ethylenediaminetetraacetic acid (EDTA, Synth, São Paulo, Brazil) and dithiothreitol (DTT) (Sigma Aldrich, St Louis, MS)) [19] on T81Xyl5_22A hydrolytic activity were tested in a reaction mixture of 40 μL of 1% beechwood xylan, 35 μL of 100 mM citrate buffer pH 5.0 and 20 μL of the purified enzyme (1.85 μM final concentration). Reaction without addition of salts, EDTA or DTT was used as a control. All reaction mixtures were incubated under agitation at 65 °C for 60 min. Analysis of the hydrolytic products To identify the products released by T81Xyl5_22A hydrolytic activity, 400 μL of 1% beechwood xylan or rye arabinoxylan, and 100 μL of purified enzyme (for a final concentration of 1.85 μM) were incubated for 15, 30, 60 and 120 min, at agitation rate of 1000 rpm under enzymatic optimum conditions (65 °C and pH 5.0). The products of hydrolysis were determined by ion chromatography (Dionex ICS-5000, Sunnyvale, CA), using a CarboPac PA100 column according to an adapted method [20]. The column was operated at a flow of 1 mL/min and maintained at 30 °C. The following elution gradient was applied: 0.1 M NaOH for 5 min, then a linear gradient from 0.1 M NaOH to 0.1 M NaOH with 0.2 M NaOAc in 27.5 min, followed by 0.1 M NaOH/1.0 M NaOAc in 2.5 min, then a linear decrease from 0.1 M NaOH/1.0 M to 0.1 M NaOH in 2 min. and to reconditioned the column 0.1 M NaOH were applied for 3 min.
Results Sequence analysis and recombinant protein expression Sequence analysis indicated that the gene encoded by locus A4R35_07040 falls in the GH5 CAZyme family [10]. Members of this family are mainly endocellulases [30]. BLASTp analysis revealed that T81Xyl5_22A enzyme shares the highest amino acid sequence identity (95 and 94%) with GH5 enzymes from Thermogemmatispora onikobensis (accession WP_069801996.1) and T. carboxidivorans (accession WP_052888460.1), respectively. Subsequent amino acid sequences with identities of 67% or less were mostly described as uncharacterized glycoside hydrolases from different organisms. The same analysis also indicated 49% identity between the amino acid sequence of T81Xyl5_22A and the only other characterized GH5 β-xylosidase from
Intrinsic fluorescence spectroscopy assay Intrinsic fluorescence measurements of T81Xyl5_22A were conducted in a quartz 10 × 2 mm cuvette using ISS-PC1 spectrofluorimeter (ISS, Inc., Champaign, IL) equipped with a temperature-controlled 59
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the fungus Phanerochaete chrysosporium (accession AHL69750.2; [13]), multiple sequence alignment shows the consensus region (Supplementary Fig. 1). The gene encoding for T81Xyl5_22A was amplified from genomic DNA of Thermogemmatispora sp. T81 and heterologously expressed in E. coli. The recombinant enzyme was purified by affinity chromatography using Ni-NTA resin. The purified T81Xyl5_22A protein appeared as a single band of approximately 54 kDa on SDS-PAGE gel (not shown). Thermofluor assay The effects of the composition of the buffer and pH on thermal stability for T81Xyl5_22A were assayed using the Thermofluor technique: the highest melting points (85 °C) were observed using Tris buffers at pHs 7–8. However, another 25 conditions among the 47 evaluated showed melting points higher than 80 °C (Supplementary Table 1) demonstrating the enzyme thermostability to different buffers and pH compositions, making possible the selection of any of 26 conditions as possible working buffer for T81Xyl5_22A characterization. Enzymatic assays Optimal temperature and pH assays for enzymatic activity The effect of temperature and pH on T81Xyl5_22A enzymatic activity were determined using sodium citrate buffer identified as optimum for thermal stability in Thermofluor assays and beechwood xylan as a substrate. The recombinant enzyme showed optimal activity at 65 °C (Supplementary Fig. 2A) and pH 5.0 (Supplementary Fig. 2B). Substrate specificity and products of hydrolysis Of all the assayed substrates, the enzyme was only active on beechwood xylan and rye arabinoxylan, with a clear preference for beechwood xylan, the activity on rye arabinoxylan being only half that observed for beechwood xylan after 1 h incubation. However, after 6 h, the cumulative enzymatic activities of T81Xyl5_22A against the two substrates were similar. Analyses of the products of hydrolysis of beechwood xylan (Fig. 1A) and rye arabinoxylan (Fig. 1B) showed that T81Xyl5_22A hydrolyzed these substrates to xylose exclusively, without release of arabinose residues, eliminating evidence of α-L-arabinofuranosidase activity for this enzyme. Moreover, the mode of action of T81Xyl5_22A was assessed using xylotriose as substrate (Fig. 2) and results showed that xylotriose was hydrolyzed to xylose, confirming the enzyme classification as a β-D-1-4-xylosidase. The hydrolytic release of xylose from beechwood xylan and rye arabinoxylan was quantified after 60 min reaction. In order to do so, a xylose calibration curve was built and showed good linearity (R2 ≥ 0.9989) in the concentration range of 3–50 mg/L. The rates of hydrolysis from beechwood xylan and rye arabinoxylan were 0.08 μg xylose/min and 0.02 μg xylose/min, respectively (Fig. 1A & B), i.e. T81Xyl5_22A was 4 times more active on beechwood xylan than on rye arabinoxylan.
Fig. 1. Products of beechwood xylan and rye arabinoxylan hydrolysis. A) Chromatogram of reaction products of beechwood xylan after ¼ h, ½ h, 1 h and 2 h incubations with purified enzyme. The single product detected was xylose (X1). B) Chromatogram of reaction products of rye arabinoxylan after 1 h and 6 h incubations with purified enzyme. The single product detected was xylose (X1). Standards ARA (arabinose), X1–X6 (X1 xylose, X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose and X6 xylohexaose). The increased xylose concentration, coupled with the absence of xylooligosaccharides, allowed the characterization of the enzyme as a β-D-xylosidase.
Effects of salts, EDTA and DTT T81Xyl5_22A enzymatic activity was enhanced in the presence of Ca2+, Sr2+ and DTT (at 33%, 18% and 42%, respectively) but was not affected by the presence of K+, Na+ and Li+ (Fig. 4). All other salts and EDTA inhibited enzymatic activity.
Intrinsic fluorescence spectroscopy assay Changes in T81Xyl5_22A tertiary structure were monitored by tryptophan intrinsic fluorescence. The enzyme has 12 tryptophan residues (Supplementary Table 2) of which approximately one third are buried within the protein structure (Supplementary Fig. 3). The emission spectrum of the indole side chain group showed high sensitivity to the polarity of local environment [31] providing information about the tertiary structure and allowing differentiation between native and unfolded forms. Upon excitation at 295 nm, the T81Xyl5_22A fluorescence emission spectra showed maxima of intensity at 350 nm when incubated at 30 and 50 °C. The maximum fluorescence shifted to 346 nm and a decrease in intensity was observed in the spectra recorded at 60 and 70 °C (Fig. 5), indicating changes of the local environments surrounding the various tryptophans. Due to the visible enzyme precipitation and aggregation, the maximum fluorescence shifted to 340 nm, at 80 °C.
Thermostability assay The enzyme was thermostable and maintained activity when incubated below 70 °C for up to 24 h. Above 70 °C, T81Xyl5_22A activity quickly decreased, displaying about 50% residual activity after 1 h (Fig. 3). At the optimum temperature of 65 °C, a residual activity of approximately 70% was observed after four d incubation. Enzyme kinetic parameters The KM, Vmax, kcat and kcat/KM kinetic parameters of T81Xyl5_22A were determined using beechwood xylan and pNPX as substrates. For xylan, KM, Vmax, kcat and kcat/KM were 9.42 ± 0.42 mM, 0.83 ± 0.02 U/mg, 2.24 s−1 and 0.106 mM−1 s−1 respectively. For pNPX, KM, Vmax, kcat and kcat/KM were 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s−1 and 156.8 mM−1 s−1, respectively. 60
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Fig. 2. Products of xylotriose hydrolysis. Chromatogram of reaction products after 0 (control), 1, 5 and 10-min incubations of purified enzyme and xylotriose. The single product detected was xylose (X1) after 10 min incubation. Standards X1–X6 (X1 xylose, X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose and X6 xylohexaose).
Fig. 4. Effects of ions and modulators on activity of T81Xyl5_22A. The effects of 5 mM of cations and modulators are shown. Enzymatic activity without additives was defined as 100%.
Fig. 5. T81Xyl5_22A intrinsic fluorescence emission spectra at different temperatures. Emission spectra of T81GH5_22 in the range of 300–450 nm were recorded using an excitation wavelength of 295 nm, with a 1 nm scan interval. Experiments were carried out in a 10 × 2 mm fluorescence cuvette and corrected for buffer contribution. Fig. 3. Thermostability for T81Xyl5_22A. The enzyme showed optimum activity at 65 °C, and was thermostable at temperatures ≤70 °C for 24 h.
and less sensitive to β-sheets [52]. Small angle X-ray scattering (SAXS) data The small-angle X-ray scattering curves obtained for T81Xyl5_22A are practically identical at different protein concentrations (1, 2, 3 mg/ mL), indicating an absence of spatial correlation effects within the applied concentration range (Supplementary Fig. 4). For this reason, subsequent analyses were performed using SAXS data collected from samples at 3 mg/mL. The radius of gyration (Rg) value obtained by Guinier analysis (Fig. 7A, insert) showed a good correlation with Rg obtained from the p(r) analysis (Fig. 7B; Table 1). The distances frequency distribution p(r) of the protein showed Dmax of 115 Å (Fig. 7B). The Rg, Dmax values were calculated for a range of q between 0.012 Å−1 and 0.235 Å−1 with a resolution of 30 Å. The SAXS-based molecular mass estimated for T81Xyl5_22A, obtained using SAXS MoW2 server, was 106 kDa. This value is very close to the theoretical molecular mass of T81Xyl5_22A dimer (relative error of 1.4%), which confirms a notion that it exists in a dimeric state in solution. Dimerization of proteins may have an effect on their stability
Circular dichroism (CD) measurements The CD spectra (Fig. 6) exhibited a maximum peak at 190 nm due to π–π* transitions and minimum peaks around 208 nm due to π–π* and 225 nm due to n–π* transitions, which are in agreement with α-helical and β-sheet conformations [52]. The secondary structure composition, calculated from the T81Xyl5_22A CD spectrum (Fig. 6) using the algorithm CDSSTR with the SP175 dataset [33] resulted in an estimate of 26% α-helices, 28% β-sheets, 46% turns and disordered elements as the secondary structure with an average error in determination of secondary structure elements of 5.1%. The secondary structure prediction program, GOR4, [32] estimated that T81Xyl5_22A had 34% α-helices, 15% β-sheets and 51% of turns and disordered secondary structures. Thus, the observed percentages of the secondary structure elements were grossly consistent between theoretical and experimental data obtained by CD spectroscopy. It is important to mention that CD experiments are generally more precise in determination α-helical content 61
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Table 1 SAXS parameters determined for T81GH5_22. Data collection parameters Beam line Wavelength (Å) ̊ q range (Å− 1) Exposure time per frame (s) Concentration range (mg/mL) Temperature (°C) Structural parameters Rg ( ± SE, Å) (from Guinier)) Rg ( ± SE, Å) (from GNOM) Dmax (Å) Resolution ( ± SE, Å) (from SASRES) Molecular mass MWSAXS (kDa) MWTheo (kDa) Oligomeric state Ab initio modelling Number of models Normalized spatial discrepancy (NSD) XDAM Homology model XHomology
Fig. 6. Structural characterization of T81Xyl5_22A by CD spectroscopy. The farUV spectrum was recorded in 20 mM Na2HPO4, 20 mM NaCl, pH 7.5 using an optical path length of 1 mm.
LNLS D02A-SAXS2 1.488 0.012 Å−1 < q < 0.405 300 1, 2 & 3 25 35.18 ± 1.47 34.58 ± 0.81 115 30 ± 2 123 54 Dimer 10 0.48 ± 0.01 1.3 2.12
and result in enhanced specificity, proximity and orientation of substrates and changes in their kinetic process [34]. An experimental X-ray scattering curve and two theoretical SAXS curves (one simulated from an average dummy atom model (DAM) and another one computed from the homology model (PDB id: 4LX4) closely overlap (Fig. 7A). The difference between the experimental curve and the simulated curve derived from the homology model could be attributed to the quality of a homology model for T81Xyl5_22A dimer. The putative T81Xyl5_22A dimer interface is shown in Supplementary Fig. 3. As a following step the Subcomb program was used to superpose the DAM with a high-resolution homology model (Fig. 8). The latter fitted well into the low-resolution SAXS molecular envelope reconstructed for T81Xyl5_22A, further confirming its dimeric quaternary structure. Discussion Thermogemmatispora sp. T81 grows on mono and di-saccharide carbon sources, lignocellulosic wood pulps and several others polysaccharide compounds [9]. We have cloned, expressed, and characterized a GH5 enzyme, and determined that it is a β-D-1,4-xylosidase. The enzyme described is the first GH5 β-xylosidase from subfamily 22 of the GH5 family isolated from a bacterium. This subfamily has 61 members
Fig. 7. A) Small-angle X-ray scattering curve for T81Xyl5_22A (log I (q) X q). Experimental curve (circle); High-resolution homology model (dashed line); and Simulated scattering DAM (Dammin) (solid line). The insert shows the Guinier adjustment for obtaining the Rg. B) Distance-distribution p(r) plot.
Fig. 8. Superposition of the homology model of T81Xyl5_22A with the lowresolution model generated by DAMMIN. Center and right models were rotated 90° around y-axis and 90°around x-axis from the orientation shown on the left panel. 62
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residues for the temperatures higher than 60 °C. This effect could be induced by the protein aggregation. The CD studies of T81Xyl5_22A demonstrate that it has similar content of α-helix and β-sheet structures. This is also consistent with the secondary structure estimates, based on the homology-based 3D model of the enzyme. Finally, SAXS analysis further confirmed that T81Xyl5_22A forms dimers in solution, and allowed us to compute low-resolution molecular envelope of the enzyme that fits nicely the homology-based model of the β-xylosidase.
and includes only two enzymes that have been characterized biochemically: one cellulase [35] and one β-xylosidase from P. chrisosporium [13]. In the context of polysaccharide degradation within class Ktedonobacteria, the presence of highly homologous genes in closely related T. onikobensis and T. carboxidivorans is consistent with their capability to grow on xylan [36,37]. Conversely, Ktedonobacter racemifer, which does not possess a gene homologous to T81Xyl5_22A has been shown not to grow on xylan under the conditions tested [38]. Compared with the only characterized fungal β-xylosidase from GH5_22, PcGH5, T81Xyl5_22A showed a similar molecular weight and pH optimum (5.0), but considerably higher temperature optimum (65 °C vs 50 °C), as well as better temperature stability at the optimal temperature. When compared to other recently characterized β-xylosidases from thermophilic bacteria, the molecular weight of T81Xyl5_22A was smaller than for some other described xylanases [39–41], but it has a similar temperature optimum and thermal stability as, for example, XylA4 from Alicyclobacillus sp. A4 [41]. The enzyme exhibited catalytic activity against only two of the polysaccharides tested in current study, beechwood xylan and rye arabinoxylan. Beechwood xylan is composed of over 90% xylose, while rye arabinoxylan contains 62% xylose and 38% arabinose. The higher degree of rye arabinoxylan decorations might explain the longer reaction time necessary to depolymerize this substrate, which is less accessible to the enzymatic action of T81Xyl5_22A. The synergistic use of β-xylanases in association with β-xylosidases helps to overcome steric hindrance and rate-limiting inhibition by the end product [42] and results in higher depolymerization of xylan substrates [43]. In general, β-xylosidases tend to be more efficient on soluble synthetic substrates and xylo-oligosaccharides. Catalytic activity against beechwood xylan is generally observed among xylanases, but among xylosidases it is uncommon [44]. Xylosidase activity against beechwood xylan is usually small [45,46] consistent with the present results, or not observed at all in the absence of xylanases [47]. With xylan as a substrate, the enzyme displayed Michaelis-Menten kinetics, and the determined KM was higher than that reported for BxTW1 (β-xylosidase from Talaromyces amestolkiae), 9.4 ± 0.4 mM vs 7.0 ± 0.2 mM, whereas Vmax and kcat were considerably lower: 0.83 ± 0.03 U mg−1 vs 68.7 ± 0.6 U mg−1 and 2.24 s−1 vs 229 s−1, respectively [44]. On the other hand, when pNPX was used as a substrate, positive cooperativity was observed with a Hill coefficient of 1.6 and KM of 0.2 mM. The measured KM for T81Xyl5_22A had the same order to that determined for β-xylosidase from Talaromyces amestolkiae, Talaromyces thermaru and mAlicyclobacillus sp., against the same substrate. The obtained Vmax for T81Xyl5_22A was 889.47 U/mg, comparatively higher than Vmax values determined for Talaromyces amestolkiae, Thermotoga thermarum and Alicyclobacillus sp.: 52, 223.3 and 341.6 U/mg, respectively [7,44]. The concentration of substrate which permits T81Xyl5_22A to achieve half Vmax for xylan (KM) was 37 times higher than for pNPx and the efficiency (kcat/KM) to convert xylan to product substrate was 1470 times lower than to convert pNPX to product. The efficiency of T81Xyl5_22A against pNPX was in an intermediate range compared to other β-xylosidases reported in the literature. The obtained (kcat/KM) for T81Xyl5_22A was 156.8 mM−1 s−1 while one of the highest reported values was 3900 mM−1 s−1 for Humicola insolens [48] and one of the lowest reported values was 0.02 mM−1 s−1 for Trichoderma reesei [49]. Ion chromatography was used to determine the products of enzymatic hydrolysis. For both beechwood xylan and rye arabinoxylan, the only detected reaction product was xylose (Fig. 1), thus allowing classification of the enzyme as a β-xylosidase [50]. Similarly to the β-xylosidase from Paecilomyces thermophile [42], but unlike that from Geobacillus thermodenitrificans [40], the enzymatic activity of T81Xyl5_22A was enhanced when Ca2+ was added to the reaction mix. Strong inhibition was observed with Zn2+, as previously reported for XylA4 [41] and Xyl455 [51]. Intrinsic fluorescence studies of T81Xyl5_22A revealed a shift in the maxima emission for tryptophan
Acknowledgments This research was supported by São Paulo Research Foundation, (FAPESP) grant 2015/13684-0, and by National Council for Scientific and Technological Development (CNPq) grants 405191/2015-4, 303988/2016-9, 423693/2016-6 and 440977/2016-9. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.nbt.2019.07.002. References [1] Saha BC. Lignocellulose biodegradation and applications in biotechnology. Lignocellulose diodegradation vol. 1. American Chemical Society; 2004. p. 2–34. [2] Dodd D, Cann IK. Enzymatic deconstruction of xylan for biofuel production. Glob Change Biol Bioenergy 2009;1:2–17. [3] Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—factors affecting enzymes, conversion and synergy. Biotechnol Adv 2012;30:1458–80. [4] Shallom D, Shoham Y. Microbial hemicellulases. Curr Opin Microbiol 2003;6:219–28. [5] Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014;42:D490–5. [6] van den Brink J, de Vries RP. Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol Biotechnol 2011;91:1477–92. [7] Bosetto A, Justo PI, Zanardi B, Venzon SS, Graciano L, Dos Santos EL, et al. Research progress concerning fungal and bacterial beta-xylosidases. Appl Biochem Biotechnol 2016;178:766–95. [8] Yeoman CJ, Han Y, Dodd D, Schroeder CM, Cann IK. Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 2010;70:1–55. [9] Stott MB, Crowe MA, Mountain BW, Smirnova AV, Hou S, Alan M, et al. Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand. Environ Microbiol 2008;10:2030–41. [10] Tomazini Jr. A, Lal S, Munir R, Stott M, Henrissat B, Polikarpov I, et al. Analysis of carbohydrate-active enzymes in Thermogemmatispora sp. strain T81 reveals carbohydrate degradation ability. Can J Microbiol 2018;12:1–12. [11] Huang CH, Sun Y, Ko TP, Chen CC, Zheng HC, Chan HC, et al. The substrate/ product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Biochem J 2012;3:401–7. [12] Liang PH, Lin WL, Hsieh HY, Lin TY, Chen CH, Tewary SK, et al. A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5. Biochim Biophys Acta 2018;3:513–21. [13] Huy ND, Nguyen CL, Seo JW, Kim DH, Park SM. Putative endoglucanase PcGH5 from Phanerochaete chrysosporium is a beta-xylosidase that cleaves xylans in synergistic action with endo-xylanase. J Biosci Bioeng 2015;119:416–20. [14] Camilo CM, Polikarpov I. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC). Protein Expr Purif 2014;99:35–42. [15] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [16] Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J, Weiss MS. Optimization of protein buffer cocktails using Thermofluor. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2013;69:209–14. [17] Miotto LS, de Rezende CA, Bernardes A, Serpa VI, Tsang A, Polikarpov I. The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on beta-glucan. PLoS One 2014;9. [18] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [19] Kumar S, Haq I, Prakash J, Singh SK, Mishra S, Raj A. Purification, characterization and thermostability improvement of xylanase from Bacillus amyloliquefaciens and its application in pre-bleaching of kraft pulp. 3 Biotech 2017;7:20. [20] Cannella D, Hsieh CW, Felby C, Jorgensen H. Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels 2012;5:26. [21] Whitmore L, Wallace BA. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 2004;32.
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Full length Article
A novel thermostable GH5 β-xylosidase from Thermogemmatispora sp. T81 Atilio Tomazinia,1, Paula Higasia,1, Livia R. Manzinea, Matthew Stottb, Richard Sparlingc, ⁎ David B. Levind, Igor Polikarpova, a
Sao Carlos Institute of Physics, University of Sao Paulo, São Carlos, São Paulo, Brazil School of Biological Sciences, University of Canterbury, New Zealand Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada d Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, Canada b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Thermogemmatispora sp. T81 Heterologous gene expression Thermostability Enzyme kinetics Enzyme structure
A glycoside hydrolase family 5 (GH5) subfamily 22 gene, designated T81Xyl5_22A, was identified in the genome of the aerobic thermophilic bacterium, Thermogemmatispora sp. T81 (locus A4R35_07040). The gene was cloned and heterologously expressed in Escherichia coli and the gene product characterized biochemically. The recombinant enzyme had an optimal catalytic activity at pH5.0 and 65 °C, and was active against beechwood xylan and rye arabinoxylan. It yielded only xylose molecules as products of beechwood xylan hydrolysis, indicating that it is a GH5 family β-D-xylosidase. Using 4-nitrophenyl β-D-xylopyranoside (pNPX) as a substrate, the KM, Vmax, kcat and kcat/KM kinetic parameters were determined as 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s−1 and 156.8 mM−1 s−1, respectively. Small-angle X-ray scattering (SAXS) data enabled reconstruction of the enzyme’s low-resolution molecular envelope and revealed that it formed dimers in solution. As far as we are aware, this is the first description of a thermostable bacterial GH5 family β-D-xylosidase.
Introduction Hemicellulose is the second most abundant heteropolysaccharide in nature and its main component is xylan [1]. Xylan has a backbone of covalently linked β-D-1-4-xylopyranose units, which can be decorated by L-arabinofuranose, 4-O-methyl-D-glucuronic acid, p-cumaric acid and ferrulic acid residues [1,2]. The ratio and degree of these decorations depend on xylan origin. Complete degradation of xylan into monosaccharide units requires a set of enzymes acting in synergy, known as a xylanolytic complex [3]. The core enzymes that depolymerizing the xylan backbone are β-D-1,4xylanases (EC 3.2.1.8) and β-D-1,4-xylosidases (EC 3.2.1.37). The xylanase hydrolyzes the β-1,4 linkages in an endo fashion, yielding xylooligosaccharides, which are degraded into xylose monomers by exoacting β-D-xylosidases [1,4]. To date, according to the CarbohydrateActive enZymes database (CAZy; www.cazy.org) [5], xylosidases are classified into glycoside hydrolase families (GHs) 1, 3, 5, 30, 39, 43, 51, 52, 54, 116, and 120, representing enzymes from all the kingdoms of life. All β-xylosidases hydrolyze their substrate with retention of the anomeric configuration, except for GH43 members, which are inverting
GHs. The GH3 and GH43 Families are the most prevalent, accounting for more than two thirds of the currently known β-xylosidases [6]. To date, nine β-xylosidase structures have been deposited in the PDB database (http://www.rcsb.org/), distributed between four different GH families (GH3 (1), GH39 (4), GH43 (3), GH52 (1)). Eight are of bacterial enzymes, of which Geobacillus stearothermophilus enzymes are the most extensively studied, with 4 structures determined (GH39 (2), GH43 (1) and GH52 (1)). No structural information is currently available for GH5 β-xylosidases. β-xylosidases have a number of biotechnological applications, including in the pulp and paper industry, the food industry and in bioethanol production [7]. However, enzymes intended for industrial application must optimally have specific characteristics such as thermostability, high activity and appropriate pH range, which translate into reduced amounts of enzyme required and a lower risk of microbial contamination [8]. Thermogemmatispora sp. T81 (Phylum Chloroflexi, Family Thermogemmatisporaceae) is an aerobic, thermophilic bacterium isolated from a geothermal hotspring [9], possessing a filamentous morphology with hyphaed mycelium and aerial spores capable of growth in
Abbreviations: CAZy, Carbohydrate-Active enZymes database; CMC, carboxymethyl-cellulose; GH, glycoside hydrolase family; SAXS, small-angle X-ray scattering; Trx, thioredoxin; DNS, 3,5-dinitrosalicylic acid; pNPX, 4-nitrophenyl β-D-xylopyranoside ⁎ Corresponding author. E-mail address: [email protected] (I. Polikarpov). 1 Equally contributing authors. https://doi.org/10.1016/j.nbt.2019.07.002 Received 7 April 2018; Received in revised form 9 June 2019; Accepted 6 July 2019 Available online 09 July 2019 1871-6784/ © 2019 Elsevier B.V. All rights reserved.
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gradient of 50–500 mM of imidazole. The eluted protein containing fractions were pooled, dialyzed (buffer A) to remove the imidazole and concentrated using Vivaspin 20 (GE Healthcare). The recombinant protein was incubated overnight with TEV protease (1 mg TEV protease per 500 mg of recombinant protein) and submitted to a second affinity chromatography step using Ni-NTA resin (1 mL of resin per 50 mg of recombinant protein) to separate the HisTrx tag from the target enzyme. The purified enzyme was concentrated and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using a 15% (v/v) gel [15], and the protein concentration determined by extinction coefficient (Ɛ = 101,885 M−1 cm−1) at 280 nm using a Nanodrop 1000 spectrophotometer (Thermo Scientific). SDS-PAGE gels were stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories) and destained with 10% (v/v) acetic acid.
mostly acidic and neutral pHs ranging from 3.1 to 7.5, and temperatures varying from 40 °C to 75 °C. The organism exhibits halotolerance and is able to use different substrates as a source of nutrients [9]. Genome analysis has identified two GH5 genes (A4R35_16780 and A4R35_07040) in the Thermmogematispora sp. T81 genome [10], of which the latter was annotated as a β-xylosidase. Given that most βxylosidases used in industrial applications are thermophilic enzymes [11], its biochemical characterization is a subject of interest. The β-xylosidase from Thermmogematispora sp. T81 belongs to subfamily 22 of GH5 [10]. The GH5 is one of the largest among 149 GH families and is highly divergent in terms of sequences and functions [12]. The only GH5_22 β-xylosidase characterized to date is from Phanerochaete chrysosporium [13]. Here we report the cloning and heterologous expression of a gene from Thermogemmatispora sp. T81 encoding a glycoside hydrolase from the GH5_22 family, designated T81Xyl5_22A. The enzyme’s thermostability in different buffers and pH conditions was evaluated using the thermofluor assay. In addition, the low-resolution envelope of the enzyme was determined using small angle X-ray scattering (SAXS) and changes in its tertiary structure monitored by tryptophan intrinsic fluorescence. Finally, the enzyme specificity activity was measured and the hydrolytic products generated were characterized, demonstrating that it is a thermostable β-D-xylosidase, which may have potential for biotechnological applications.
Protein stability assay Thermofluor assays [16,17] were conducted to identify the optimal buffer conditions for T81Xyl5_22A stability. The experiment was performed on the CFX96 Real-Time System (Bio-Rad). A SYPRO Orange stock solution in DMSO (Invitrogen) was diluted 1:300 in water to yield a working solution. 47 different conditions covering a pH range from 1.0 to 10.0 (Suppl. Table 1) were screened using a 96-well PCR microplate (Axygen). The total reaction volume was 20 μL and the plate was set up on ice: 10 μL of the screening solution, 5 μL of T81Xyl5_22A (7 μM final concentration) and 5 μL of SYPRO Orange working solution per well. Immediately following the addition of the dye, the measurements were carried out with excitation wavelength at 490 nm and emission at 530 nm. The samples were heated across a temperature gradient of 25–90 °C. Protein melting temperature (Tm) values were determined using Origin software (OriginLab, Northampton, MA) and experiments were performed in duplicate.
Materials and methods Cloning The T81Xyl5_22A gene product was identified by proteomic analyses of the Thermogemmatispora sp. T81 secretome [10]. T81Xyl5_22A (locus A4R35_07040) was one of four extracellular CAZymes identified in the secretome after growth of the bacterium on cellulose as a sole carbon source [10]. Bioinformatic analyses determined that T81Xyl5_22A was encoded by the gene located at locus tag A4R35_07040 in the sequenced genome of the thermophilic bacterium Thermogemmatispora sp. T81 (BioProject PRJNA317154) [10]. Ligase Independent Cloning (LIC) was used as previously described [14], and the set of oligonucleotides for gene amplification was synthesized (Exxtend Biotecnologia Ltda, Campinas Brazil); Forward: 5′-CAGGGCGCCATGACATCGGGTAT GCTCACTACG-3′ and Reverse: 5′-GACCCGACGCGGTTATTAGCGACAAGCATACTCCC TGAG-3′. The gene was cloned into vector pET Trx-1a/Lic, inserted into E. coli DH10B cells for propagation and expressed in E. coli Rosetta (DE3) pLysS cells.
Enzyme activity assays Enzyme activity was quantified using the 3,5-dinitrosalicylic acid (DNS) reagent method [18] by measuring absorbance at 535 nm in addition to assays measuring para-nitrophenol (pNP) absorbance measured at 405 nm. The experiments were performed in triplicate; xylose and glucose standard curves in the concentration range from 0.1 to 1 mM and a pNP standard curve in the range from 0 to 0.1 mM were used. Optimal temperature and pH assays for enzymatic activity
Protein expression, purification and gel electrophoresis analysis The effect of pH on T81Xyl5_22A activity was determined using 20 mM sodium citrate buffer at pHs ranging from 3.0 to 6.0. 0.4 mg of beechwood xylan (Sigma Aldrich, St Louis, MS) was incubated with 12.5μM T81Xyl5_22A for 2 h at 60 °C under continuous agitation at 1000 rpm. The optimal temperature was measured in the range of 55–80 °C. The reaction consisted of 40 μL 100 mM sodium citrate buffer, pH 5.0, 40 μL 1% beechwood xylan prepared in sodium citrate buffer, pH 5.0, 2.77 μM purified enzyme in a final reaction volume of 100 μL. The mixture was incubated for 60 min at 1000 rpm.
E. coli Rosetta (DE3) pLysS cells transformed with the recombinant plasmid were grown in Luria-Bertani (LB) medium supplemented with 1 μg/mL kanamycin and 20 μg/mL chloramphenicol at 37 °C, rotated at 160 rpm until A600 of approximately 0.6. The temperature was lowered to 18 °C and protein expression was induced by addition of 1 mM IPTG for 18 h. The cells were harvested by centrifugation (10,000 × g, 30 min, 4 °C) and resuspended in lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 5% glycerol, 0.2 mM PMSF, 4 mM β-mercaptoethanol, 0.8 mM DTT, 10 mM imidazole, and 0.1% N-lauroylsarcosine (Sigma Aldrich, St Louis, MS)). The cells were sonicated for 10 min (10 cycles of 30 s each) using a Branson Sonifier 450 (BRANSON Ultrasonics Corporation, Danbury, CT). A 40% duty cycle with the 4 microtrip power output was used and the cells were kept on ice. The resulting solution was clarified by centrifugation (30,000 × g, 30 min, 4 °C), followed by affinity chromatography of the supernatant using Ni-NTA resin. The supernatant was loaded onto a free resin previously washed with buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol), homogenized by stirring and kept on ice for 45 min. Next, the column was washed with binding buffer supplemented with 20 mM imidazole and the His-Thioredoxin (Trx) tagged protein was eluted using a step
Substrate specificity and products of hydrolysis To determine the substrate specificity for T81Xyl5_22A, the following were prepared at 1% (m/v) concentration in 100 mM sodium citrate buffer, pH 5.0: xyloglucan, mannan, liquenan, galactomannan, β-glucan, rye arabinoxylan, larch arabinogalactan and arabinan sugarbeet (Megazyme, Ireland), beechwood xylan, Sigmacell 20, low viscosity carboxymethyl-cellulose (CMC), microgranular cellulose, laminarin, Avicel PH-101 and pectin (Sigma Aldrich, St Louis, MS); filter paper n°1 (Whatman, Maidstone UK). The reaction mixture consisted of 40 μL 100 mM sodium citrate buffer, 40 μL of substrate and 20 μL of the 58
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water bath. T81Xyl5_22A fluorescence emission spectra at 3.7 μM in 100 mM sodium citrate buffer, pH 5.0 were recorded in a 300–450 nm range using an excitation wavelength of 295 nm and a 305 nm emission filter cutoff. Temperature variation was monitored from 30 to 90 °C with 2 min of protein incubation under readout temperature. After each measurement the spectrum was corrected for buffer contribution and the data analysis performed using Origin (OriginLab, Northampton, MA) software.
purified enzyme (1.85 μM final concentration). Reactions were conducted under the optimal temperature and pH conditions (65 °C and pH 5.0) for T81Xyl5_22A for 1 and 6 h under constant agitation at 1000 rpm. Thermostability assay For the thermostability assay, a mixture of purified enzyme and sodium citrate buffer, pH 5.0, were incubated for 24 h at 50–80 °C. 20 μL of the incubated enzyme (1.85 μM final concentration), 40 μL of 100 mM sodium citrate buffer and 40 μL of 1% (m/v) beechwood xylan solution were then added to the reaction and incubated for 1 h at optimum temperature (65 °C). Residual activity was quantified as described.
Circular dichroism (CD) measurements CD data were acquired on a Jasco J-720 CD spectrometer (Jasco, Tokyo, Japan). T81Xyl5_22A at 3.7 μM was equilibrated in 20 mM Na2HPO4, 20 mM NaCl, pH 7.5 and applied at 25 °C to 0.1-cm path length cuvette. CD spectra were recorded in the 190–260 nm wavelength range with 100 nm.min-1 increment step, 10 s averaging time, 1 nm bandwidth and a response time of 0.5 s. Solvent contributions were subtracted from the original spectral curves and an estimation of the secondary structure elements was obtained using the Dichroweb webserver [21].
Determination of T81Xyl5_22A kinetic parameters To determine T81Xyl5_22A kinetic parameters, KM and Vmax, beechwood xylan was used as substrate in a concentration range from 1 to 16 mg/mL, under enzyme optimum conditions of pH and temperature (pH 5.0 and 65 °C). After 30 min, the product was quantified using the 3,5-dinitrosalicylic acid (DNS) reagent method [18]. Kinetic parameters were also assayed using 4-nitrophenyl β-D-xylopyranoside (pNPX, Sigma Aldrich, St Louis, MS) as substrate, at concentrations from 0.04 to 4 mM at 65 °C. After 10 min, the reaction was stopped with 500 mM sodium carbonate and released p-nitrophenol (pNP) was quantified by measuring absorbance at 405 nm. Data analysis was performed using Origin (OriginLab, Northampton, MA) software and the determination of the kinetic parameters was calculated assuming that 1 unit of enzyme activity (U) releases 1 μmol per minute of xylose or pNP.
Small angle X-ray scattering (SAXS) Synchrotron radiation X-ray scattering data were collected at the D02A-SAXS2 beamline of the Brazilian National Synchrotron Laboratory (LNLS, Campinas, Brazil) using a bi-dimensional detector (MarResearch, EUA). A sample to detector distance of 890 mm was used, thus covering the range of momentum transfer 0.012 Å−1 < q < 0.405 Ǻ−1 (q = 4π sin(θ)/λ), where 2θ was the scattering angle and λ = 0.148 nm. The protein solutions at 1, 2 and 3 mg/mL (which corresponds to 18, 28 and 56 μM of protein, respectively) in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 were manually loaded into the sample holder installed in the vacuum sample chamber. The data were normalized to the beam intensity using the FIT2D program [22]. The scattering of the buffer was subtracted and the difference curves were scaled for concentration. The radius of gyration (Rg) was computed by Guinier approximation [23] and distance distribution functions p(r), radius of gyration Rg and maximum diameters Dmax of the scattering objects were calculated using the GNOM program [24]. Ab initio shape models for T81Xyl5_22A were obtained by superposition of 10 independent models using the DAMMIN program [25]. These results were compared with the T81Xyl5_22A homology model obtained from the I-TASSER server [26] using the structure of endoglucanase Cel5A from Pseudomonas stutzeri (PDB id: 4LX4) as template. A theoretical SAXS curve from the predicted structure model was generated and compared with the experimental curve through Debye formula using FoXS web tool [27]. Finally, T81Xyl5_22A molecular mass was estimated from SAXS experimental data using SAXS MoW calculator [28,29].
Effect of ions and additives The effects of different metal ions (Mg2+, Ni2+, Ca2+, Cu2+, K+, Na+, Zn2+, Mn2+, Fe2+, Ag+, Co2+, Li+, Sr2+) and additives (ethylenediaminetetraacetic acid (EDTA, Synth, São Paulo, Brazil) and dithiothreitol (DTT) (Sigma Aldrich, St Louis, MS)) [19] on T81Xyl5_22A hydrolytic activity were tested in a reaction mixture of 40 μL of 1% beechwood xylan, 35 μL of 100 mM citrate buffer pH 5.0 and 20 μL of the purified enzyme (1.85 μM final concentration). Reaction without addition of salts, EDTA or DTT was used as a control. All reaction mixtures were incubated under agitation at 65 °C for 60 min. Analysis of the hydrolytic products To identify the products released by T81Xyl5_22A hydrolytic activity, 400 μL of 1% beechwood xylan or rye arabinoxylan, and 100 μL of purified enzyme (for a final concentration of 1.85 μM) were incubated for 15, 30, 60 and 120 min, at agitation rate of 1000 rpm under enzymatic optimum conditions (65 °C and pH 5.0). The products of hydrolysis were determined by ion chromatography (Dionex ICS-5000, Sunnyvale, CA), using a CarboPac PA100 column according to an adapted method [20]. The column was operated at a flow of 1 mL/min and maintained at 30 °C. The following elution gradient was applied: 0.1 M NaOH for 5 min, then a linear gradient from 0.1 M NaOH to 0.1 M NaOH with 0.2 M NaOAc in 27.5 min, followed by 0.1 M NaOH/1.0 M NaOAc in 2.5 min, then a linear decrease from 0.1 M NaOH/1.0 M to 0.1 M NaOH in 2 min. and to reconditioned the column 0.1 M NaOH were applied for 3 min.
Results Sequence analysis and recombinant protein expression Sequence analysis indicated that the gene encoded by locus A4R35_07040 falls in the GH5 CAZyme family [10]. Members of this family are mainly endocellulases [30]. BLASTp analysis revealed that T81Xyl5_22A enzyme shares the highest amino acid sequence identity (95 and 94%) with GH5 enzymes from Thermogemmatispora onikobensis (accession WP_069801996.1) and T. carboxidivorans (accession WP_052888460.1), respectively. Subsequent amino acid sequences with identities of 67% or less were mostly described as uncharacterized glycoside hydrolases from different organisms. The same analysis also indicated 49% identity between the amino acid sequence of T81Xyl5_22A and the only other characterized GH5 β-xylosidase from
Intrinsic fluorescence spectroscopy assay Intrinsic fluorescence measurements of T81Xyl5_22A were conducted in a quartz 10 × 2 mm cuvette using ISS-PC1 spectrofluorimeter (ISS, Inc., Champaign, IL) equipped with a temperature-controlled 59
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the fungus Phanerochaete chrysosporium (accession AHL69750.2; [13]), multiple sequence alignment shows the consensus region (Supplementary Fig. 1). The gene encoding for T81Xyl5_22A was amplified from genomic DNA of Thermogemmatispora sp. T81 and heterologously expressed in E. coli. The recombinant enzyme was purified by affinity chromatography using Ni-NTA resin. The purified T81Xyl5_22A protein appeared as a single band of approximately 54 kDa on SDS-PAGE gel (not shown). Thermofluor assay The effects of the composition of the buffer and pH on thermal stability for T81Xyl5_22A were assayed using the Thermofluor technique: the highest melting points (85 °C) were observed using Tris buffers at pHs 7–8. However, another 25 conditions among the 47 evaluated showed melting points higher than 80 °C (Supplementary Table 1) demonstrating the enzyme thermostability to different buffers and pH compositions, making possible the selection of any of 26 conditions as possible working buffer for T81Xyl5_22A characterization. Enzymatic assays Optimal temperature and pH assays for enzymatic activity The effect of temperature and pH on T81Xyl5_22A enzymatic activity were determined using sodium citrate buffer identified as optimum for thermal stability in Thermofluor assays and beechwood xylan as a substrate. The recombinant enzyme showed optimal activity at 65 °C (Supplementary Fig. 2A) and pH 5.0 (Supplementary Fig. 2B). Substrate specificity and products of hydrolysis Of all the assayed substrates, the enzyme was only active on beechwood xylan and rye arabinoxylan, with a clear preference for beechwood xylan, the activity on rye arabinoxylan being only half that observed for beechwood xylan after 1 h incubation. However, after 6 h, the cumulative enzymatic activities of T81Xyl5_22A against the two substrates were similar. Analyses of the products of hydrolysis of beechwood xylan (Fig. 1A) and rye arabinoxylan (Fig. 1B) showed that T81Xyl5_22A hydrolyzed these substrates to xylose exclusively, without release of arabinose residues, eliminating evidence of α-L-arabinofuranosidase activity for this enzyme. Moreover, the mode of action of T81Xyl5_22A was assessed using xylotriose as substrate (Fig. 2) and results showed that xylotriose was hydrolyzed to xylose, confirming the enzyme classification as a β-D-1-4-xylosidase. The hydrolytic release of xylose from beechwood xylan and rye arabinoxylan was quantified after 60 min reaction. In order to do so, a xylose calibration curve was built and showed good linearity (R2 ≥ 0.9989) in the concentration range of 3–50 mg/L. The rates of hydrolysis from beechwood xylan and rye arabinoxylan were 0.08 μg xylose/min and 0.02 μg xylose/min, respectively (Fig. 1A & B), i.e. T81Xyl5_22A was 4 times more active on beechwood xylan than on rye arabinoxylan.
Fig. 1. Products of beechwood xylan and rye arabinoxylan hydrolysis. A) Chromatogram of reaction products of beechwood xylan after ¼ h, ½ h, 1 h and 2 h incubations with purified enzyme. The single product detected was xylose (X1). B) Chromatogram of reaction products of rye arabinoxylan after 1 h and 6 h incubations with purified enzyme. The single product detected was xylose (X1). Standards ARA (arabinose), X1–X6 (X1 xylose, X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose and X6 xylohexaose). The increased xylose concentration, coupled with the absence of xylooligosaccharides, allowed the characterization of the enzyme as a β-D-xylosidase.
Effects of salts, EDTA and DTT T81Xyl5_22A enzymatic activity was enhanced in the presence of Ca2+, Sr2+ and DTT (at 33%, 18% and 42%, respectively) but was not affected by the presence of K+, Na+ and Li+ (Fig. 4). All other salts and EDTA inhibited enzymatic activity.
Intrinsic fluorescence spectroscopy assay Changes in T81Xyl5_22A tertiary structure were monitored by tryptophan intrinsic fluorescence. The enzyme has 12 tryptophan residues (Supplementary Table 2) of which approximately one third are buried within the protein structure (Supplementary Fig. 3). The emission spectrum of the indole side chain group showed high sensitivity to the polarity of local environment [31] providing information about the tertiary structure and allowing differentiation between native and unfolded forms. Upon excitation at 295 nm, the T81Xyl5_22A fluorescence emission spectra showed maxima of intensity at 350 nm when incubated at 30 and 50 °C. The maximum fluorescence shifted to 346 nm and a decrease in intensity was observed in the spectra recorded at 60 and 70 °C (Fig. 5), indicating changes of the local environments surrounding the various tryptophans. Due to the visible enzyme precipitation and aggregation, the maximum fluorescence shifted to 340 nm, at 80 °C.
Thermostability assay The enzyme was thermostable and maintained activity when incubated below 70 °C for up to 24 h. Above 70 °C, T81Xyl5_22A activity quickly decreased, displaying about 50% residual activity after 1 h (Fig. 3). At the optimum temperature of 65 °C, a residual activity of approximately 70% was observed after four d incubation. Enzyme kinetic parameters The KM, Vmax, kcat and kcat/KM kinetic parameters of T81Xyl5_22A were determined using beechwood xylan and pNPX as substrates. For xylan, KM, Vmax, kcat and kcat/KM were 9.42 ± 0.42 mM, 0.83 ± 0.02 U/mg, 2.24 s−1 and 0.106 mM−1 s−1 respectively. For pNPX, KM, Vmax, kcat and kcat/KM were 0.25 ± 0.03 mM, 889.47 ± 28.54 U/mg, 39.20 s−1 and 156.8 mM−1 s−1, respectively. 60
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Fig. 2. Products of xylotriose hydrolysis. Chromatogram of reaction products after 0 (control), 1, 5 and 10-min incubations of purified enzyme and xylotriose. The single product detected was xylose (X1) after 10 min incubation. Standards X1–X6 (X1 xylose, X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose and X6 xylohexaose).
Fig. 4. Effects of ions and modulators on activity of T81Xyl5_22A. The effects of 5 mM of cations and modulators are shown. Enzymatic activity without additives was defined as 100%.
Fig. 5. T81Xyl5_22A intrinsic fluorescence emission spectra at different temperatures. Emission spectra of T81GH5_22 in the range of 300–450 nm were recorded using an excitation wavelength of 295 nm, with a 1 nm scan interval. Experiments were carried out in a 10 × 2 mm fluorescence cuvette and corrected for buffer contribution. Fig. 3. Thermostability for T81Xyl5_22A. The enzyme showed optimum activity at 65 °C, and was thermostable at temperatures ≤70 °C for 24 h.
and less sensitive to β-sheets [52]. Small angle X-ray scattering (SAXS) data The small-angle X-ray scattering curves obtained for T81Xyl5_22A are practically identical at different protein concentrations (1, 2, 3 mg/ mL), indicating an absence of spatial correlation effects within the applied concentration range (Supplementary Fig. 4). For this reason, subsequent analyses were performed using SAXS data collected from samples at 3 mg/mL. The radius of gyration (Rg) value obtained by Guinier analysis (Fig. 7A, insert) showed a good correlation with Rg obtained from the p(r) analysis (Fig. 7B; Table 1). The distances frequency distribution p(r) of the protein showed Dmax of 115 Å (Fig. 7B). The Rg, Dmax values were calculated for a range of q between 0.012 Å−1 and 0.235 Å−1 with a resolution of 30 Å. The SAXS-based molecular mass estimated for T81Xyl5_22A, obtained using SAXS MoW2 server, was 106 kDa. This value is very close to the theoretical molecular mass of T81Xyl5_22A dimer (relative error of 1.4%), which confirms a notion that it exists in a dimeric state in solution. Dimerization of proteins may have an effect on their stability
Circular dichroism (CD) measurements The CD spectra (Fig. 6) exhibited a maximum peak at 190 nm due to π–π* transitions and minimum peaks around 208 nm due to π–π* and 225 nm due to n–π* transitions, which are in agreement with α-helical and β-sheet conformations [52]. The secondary structure composition, calculated from the T81Xyl5_22A CD spectrum (Fig. 6) using the algorithm CDSSTR with the SP175 dataset [33] resulted in an estimate of 26% α-helices, 28% β-sheets, 46% turns and disordered elements as the secondary structure with an average error in determination of secondary structure elements of 5.1%. The secondary structure prediction program, GOR4, [32] estimated that T81Xyl5_22A had 34% α-helices, 15% β-sheets and 51% of turns and disordered secondary structures. Thus, the observed percentages of the secondary structure elements were grossly consistent between theoretical and experimental data obtained by CD spectroscopy. It is important to mention that CD experiments are generally more precise in determination α-helical content 61
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Table 1 SAXS parameters determined for T81GH5_22. Data collection parameters Beam line Wavelength (Å) ̊ q range (Å− 1) Exposure time per frame (s) Concentration range (mg/mL) Temperature (°C) Structural parameters Rg ( ± SE, Å) (from Guinier)) Rg ( ± SE, Å) (from GNOM) Dmax (Å) Resolution ( ± SE, Å) (from SASRES) Molecular mass MWSAXS (kDa) MWTheo (kDa) Oligomeric state Ab initio modelling Number of models Normalized spatial discrepancy (NSD) XDAM Homology model XHomology
Fig. 6. Structural characterization of T81Xyl5_22A by CD spectroscopy. The farUV spectrum was recorded in 20 mM Na2HPO4, 20 mM NaCl, pH 7.5 using an optical path length of 1 mm.
LNLS D02A-SAXS2 1.488 0.012 Å−1 < q < 0.405 300 1, 2 & 3 25 35.18 ± 1.47 34.58 ± 0.81 115 30 ± 2 123 54 Dimer 10 0.48 ± 0.01 1.3 2.12
and result in enhanced specificity, proximity and orientation of substrates and changes in their kinetic process [34]. An experimental X-ray scattering curve and two theoretical SAXS curves (one simulated from an average dummy atom model (DAM) and another one computed from the homology model (PDB id: 4LX4) closely overlap (Fig. 7A). The difference between the experimental curve and the simulated curve derived from the homology model could be attributed to the quality of a homology model for T81Xyl5_22A dimer. The putative T81Xyl5_22A dimer interface is shown in Supplementary Fig. 3. As a following step the Subcomb program was used to superpose the DAM with a high-resolution homology model (Fig. 8). The latter fitted well into the low-resolution SAXS molecular envelope reconstructed for T81Xyl5_22A, further confirming its dimeric quaternary structure. Discussion Thermogemmatispora sp. T81 grows on mono and di-saccharide carbon sources, lignocellulosic wood pulps and several others polysaccharide compounds [9]. We have cloned, expressed, and characterized a GH5 enzyme, and determined that it is a β-D-1,4-xylosidase. The enzyme described is the first GH5 β-xylosidase from subfamily 22 of the GH5 family isolated from a bacterium. This subfamily has 61 members
Fig. 7. A) Small-angle X-ray scattering curve for T81Xyl5_22A (log I (q) X q). Experimental curve (circle); High-resolution homology model (dashed line); and Simulated scattering DAM (Dammin) (solid line). The insert shows the Guinier adjustment for obtaining the Rg. B) Distance-distribution p(r) plot.
Fig. 8. Superposition of the homology model of T81Xyl5_22A with the lowresolution model generated by DAMMIN. Center and right models were rotated 90° around y-axis and 90°around x-axis from the orientation shown on the left panel. 62
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residues for the temperatures higher than 60 °C. This effect could be induced by the protein aggregation. The CD studies of T81Xyl5_22A demonstrate that it has similar content of α-helix and β-sheet structures. This is also consistent with the secondary structure estimates, based on the homology-based 3D model of the enzyme. Finally, SAXS analysis further confirmed that T81Xyl5_22A forms dimers in solution, and allowed us to compute low-resolution molecular envelope of the enzyme that fits nicely the homology-based model of the β-xylosidase.
and includes only two enzymes that have been characterized biochemically: one cellulase [35] and one β-xylosidase from P. chrisosporium [13]. In the context of polysaccharide degradation within class Ktedonobacteria, the presence of highly homologous genes in closely related T. onikobensis and T. carboxidivorans is consistent with their capability to grow on xylan [36,37]. Conversely, Ktedonobacter racemifer, which does not possess a gene homologous to T81Xyl5_22A has been shown not to grow on xylan under the conditions tested [38]. Compared with the only characterized fungal β-xylosidase from GH5_22, PcGH5, T81Xyl5_22A showed a similar molecular weight and pH optimum (5.0), but considerably higher temperature optimum (65 °C vs 50 °C), as well as better temperature stability at the optimal temperature. When compared to other recently characterized β-xylosidases from thermophilic bacteria, the molecular weight of T81Xyl5_22A was smaller than for some other described xylanases [39–41], but it has a similar temperature optimum and thermal stability as, for example, XylA4 from Alicyclobacillus sp. A4 [41]. The enzyme exhibited catalytic activity against only two of the polysaccharides tested in current study, beechwood xylan and rye arabinoxylan. Beechwood xylan is composed of over 90% xylose, while rye arabinoxylan contains 62% xylose and 38% arabinose. The higher degree of rye arabinoxylan decorations might explain the longer reaction time necessary to depolymerize this substrate, which is less accessible to the enzymatic action of T81Xyl5_22A. The synergistic use of β-xylanases in association with β-xylosidases helps to overcome steric hindrance and rate-limiting inhibition by the end product [42] and results in higher depolymerization of xylan substrates [43]. In general, β-xylosidases tend to be more efficient on soluble synthetic substrates and xylo-oligosaccharides. Catalytic activity against beechwood xylan is generally observed among xylanases, but among xylosidases it is uncommon [44]. Xylosidase activity against beechwood xylan is usually small [45,46] consistent with the present results, or not observed at all in the absence of xylanases [47]. With xylan as a substrate, the enzyme displayed Michaelis-Menten kinetics, and the determined KM was higher than that reported for BxTW1 (β-xylosidase from Talaromyces amestolkiae), 9.4 ± 0.4 mM vs 7.0 ± 0.2 mM, whereas Vmax and kcat were considerably lower: 0.83 ± 0.03 U mg−1 vs 68.7 ± 0.6 U mg−1 and 2.24 s−1 vs 229 s−1, respectively [44]. On the other hand, when pNPX was used as a substrate, positive cooperativity was observed with a Hill coefficient of 1.6 and KM of 0.2 mM. The measured KM for T81Xyl5_22A had the same order to that determined for β-xylosidase from Talaromyces amestolkiae, Talaromyces thermaru and mAlicyclobacillus sp., against the same substrate. The obtained Vmax for T81Xyl5_22A was 889.47 U/mg, comparatively higher than Vmax values determined for Talaromyces amestolkiae, Thermotoga thermarum and Alicyclobacillus sp.: 52, 223.3 and 341.6 U/mg, respectively [7,44]. The concentration of substrate which permits T81Xyl5_22A to achieve half Vmax for xylan (KM) was 37 times higher than for pNPx and the efficiency (kcat/KM) to convert xylan to product substrate was 1470 times lower than to convert pNPX to product. The efficiency of T81Xyl5_22A against pNPX was in an intermediate range compared to other β-xylosidases reported in the literature. The obtained (kcat/KM) for T81Xyl5_22A was 156.8 mM−1 s−1 while one of the highest reported values was 3900 mM−1 s−1 for Humicola insolens [48] and one of the lowest reported values was 0.02 mM−1 s−1 for Trichoderma reesei [49]. Ion chromatography was used to determine the products of enzymatic hydrolysis. For both beechwood xylan and rye arabinoxylan, the only detected reaction product was xylose (Fig. 1), thus allowing classification of the enzyme as a β-xylosidase [50]. Similarly to the β-xylosidase from Paecilomyces thermophile [42], but unlike that from Geobacillus thermodenitrificans [40], the enzymatic activity of T81Xyl5_22A was enhanced when Ca2+ was added to the reaction mix. Strong inhibition was observed with Zn2+, as previously reported for XylA4 [41] and Xyl455 [51]. Intrinsic fluorescence studies of T81Xyl5_22A revealed a shift in the maxima emission for tryptophan
Acknowledgments This research was supported by São Paulo Research Foundation, (FAPESP) grant 2015/13684-0, and by National Council for Scientific and Technological Development (CNPq) grants 405191/2015-4, 303988/2016-9, 423693/2016-6 and 440977/2016-9. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.nbt.2019.07.002. References [1] Saha BC. Lignocellulose biodegradation and applications in biotechnology. Lignocellulose diodegradation vol. 1. American Chemical Society; 2004. p. 2–34. [2] Dodd D, Cann IK. Enzymatic deconstruction of xylan for biofuel production. Glob Change Biol Bioenergy 2009;1:2–17. [3] Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes—factors affecting enzymes, conversion and synergy. Biotechnol Adv 2012;30:1458–80. [4] Shallom D, Shoham Y. Microbial hemicellulases. Curr Opin Microbiol 2003;6:219–28. [5] Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 2014;42:D490–5. [6] van den Brink J, de Vries RP. Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol Biotechnol 2011;91:1477–92. [7] Bosetto A, Justo PI, Zanardi B, Venzon SS, Graciano L, Dos Santos EL, et al. Research progress concerning fungal and bacterial beta-xylosidases. Appl Biochem Biotechnol 2016;178:766–95. [8] Yeoman CJ, Han Y, Dodd D, Schroeder CM, Cann IK. Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 2010;70:1–55. [9] Stott MB, Crowe MA, Mountain BW, Smirnova AV, Hou S, Alan M, et al. Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand. Environ Microbiol 2008;10:2030–41. [10] Tomazini Jr. A, Lal S, Munir R, Stott M, Henrissat B, Polikarpov I, et al. Analysis of carbohydrate-active enzymes in Thermogemmatispora sp. strain T81 reveals carbohydrate degradation ability. Can J Microbiol 2018;12:1–12. [11] Huang CH, Sun Y, Ko TP, Chen CC, Zheng HC, Chan HC, et al. The substrate/ product-binding modes of a novel GH120 β-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485. Biochem J 2012;3:401–7. [12] Liang PH, Lin WL, Hsieh HY, Lin TY, Chen CH, Tewary SK, et al. A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5. Biochim Biophys Acta 2018;3:513–21. [13] Huy ND, Nguyen CL, Seo JW, Kim DH, Park SM. Putative endoglucanase PcGH5 from Phanerochaete chrysosporium is a beta-xylosidase that cleaves xylans in synergistic action with endo-xylanase. J Biosci Bioeng 2015;119:416–20. [14] Camilo CM, Polikarpov I. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC). Protein Expr Purif 2014;99:35–42. [15] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [16] Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J, Weiss MS. Optimization of protein buffer cocktails using Thermofluor. Acta Crystallogr Sect F: Struct Biol Cryst Commun 2013;69:209–14. [17] Miotto LS, de Rezende CA, Bernardes A, Serpa VI, Tsang A, Polikarpov I. The characterization of the endoglucanase Cel12A from Gloeophyllum trabeum reveals an enzyme highly active on beta-glucan. PLoS One 2014;9. [18] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426–8. [19] Kumar S, Haq I, Prakash J, Singh SK, Mishra S, Raj A. Purification, characterization and thermostability improvement of xylanase from Bacillus amyloliquefaciens and its application in pre-bleaching of kraft pulp. 3 Biotech 2017;7:20. [20] Cannella D, Hsieh CW, Felby C, Jorgensen H. Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels 2012;5:26. [21] Whitmore L, Wallace BA. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 2004;32.
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