Appl Biochem Biotechnol (2015) 175:573–588 DOI 10.1007/s12010-014-1295-2
Molecular and Biochemical Characterization of a Novel Multidomain Xylanase from Arthrobacter sp. GN16 Isolated from the Feces of Grus nigricollis Junpei Zhou & Jidong Shen & Rui Zhang & Xianghua Tang & Junjun Li & Bo Xu & Junmei Ding & Yajie Gao & Dongyan Xu & Zunxi Huang
Received: 23 July 2014 / Accepted: 10 October 2014 / Published online: 21 October 2014 # Springer Science+Business Media New York 2014
Abstract A novel glycosyl hydrolase family 10 (GH 10) xylanase (XynAGN16), consisting of five domains, was revealed from the genome sequence of Arthrobacter sp. GN16 isolated from the feces of Grus nigricollis. XynAGN16 and its truncated derivatives XynAGN16L (GH 10 domain at N-terminus) and XynAGN16Lpd (GH 10 domain at N-terminus and polysaccharide deacetylases domain) were expressed in Escherichia coli and characterized. Biochemical characterizations and hydrolysis products analyses of recombinant XynAGN16L and XynAGN16Lpd showed similar features, including showing catalytic activities at 0 °C, thermolabilities at temperatures of more than 50 °C, and similar substrate specificity. However, the polysaccharide deacetylases domain improved the affinity and catalytic efficiency towards xylans of the recombinant XynAGN16Lpd. The Km and kcat/Km values of recombinant XynAGN16L towards birchwood xylan were 2.6 mg/mL and 19.5 mL/mg/s, respectively,
Junpei Zhou and Jidong Shen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-1295-2) contains supplementary material, which is available to authorized users.
J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People’s Republic of China J. Zhou e-mail: [email protected] J. Zhou : J. Shen : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Y. Gao : D. Xu : Z. Huang College of Life Sciences, Yunnan Normal University, No.1 Yuhua District, Chenggong, Kunming, Yunnan 650500, People’s Republic of China J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, People’s Republic of China J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang (*) Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming 650500, People’s Republic of China e-mail: [email protected]
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while the two values of recombinant XynAGN16Lpd were 1.2 mg/mL and 42.7 mL/mg/s, respectively. Towards beechwood xylan, the Km and kcat/Km values of recombinant XynAGN16L were 1.8 mg/mL and 27.1 mL/mg/s, respectively, while the two values of recombinant XynAGN16Lpd were 1.0 mg/mL and 35.3 mL/mg/s, respectively. Compared with three thermophilic endoxylanases, XynAGN16L has a surface loop from A57 to Y77 and a decreased number of salt bridges. Keywords Multidomain . Xylanase . Polysaccharide deacetylases . Low-temperature-active . Kinetic . Grus nigricollis
Introduction Xylan is the major component of the plant cell wall and the most abundant renewable hemicellulose. The backbone of xylan is built of β-1,4-linked xylopyranosyl residues [1]. Endoxylanases (endo-β-1,4-xylanases; EC 3.2.1.8) hydrolyze xylan backbone to produce short chain xylo-oligosaccharides of varying lengths, therefore they play a key role in the biodegradation of xylan [2]. Most endoxylanases are classified in the family 10 or 11 of glycoside hydrolases (GH) based on the sequence similarities of their respective catalytic domains [3]. Besides endoxylanases, the complete breakdown of xylan requires β-Dxylosidases and five debranching enzymes [1]. Endoxylanases have received a great deal of attention mainly due to their extensive applications in many fields, such as bioconversion of biomass materials, pre-bleaching of kraft pulps, and improving the quality of food and feed [1]. Basic studies on endoxylanases can give insights into catalytic mechanisms and improve the performances of these enzymes. Multidomain xylanases consist of a catalytic GH10 domain with one or more carbohydratebinding domain (CBM), and/or fibronectin type 3 domain, and/or S-layer-homologous domain [4–9]. These studies revealed that non-catalytic domains could be responsible for the binding and hydrolysis of insoluble polysaccharides [6], the thermoactivity and thermostability [7], the pH stability [5], and the cell surface localization [8] of multidomain xylanases. However, the polysaccharide deacetylases (PD) domain has rarely been present in multidomain xylanases (GenBank accession no. CAA54145, CAA90745, and ADG73550), not to mention its effect on multidomain xylanases. Grus nigricollis, also named black-necked cranes, are categorized as Vulnerable on the IUCN Red List because their population is only approximately 8000 on Earth. The majority of G. nigricollis live their life on high plateaus of China, such as Qinghai–Tibet Plateau and Yunnan–Guizhou Plateau of China [10]. Unlike other gastrointestinal microorganisms and their enzymes, which have been given considerable research interests [11–15], microorganisms and their enzymes from the feces of G. nigricollis have rarely been reported [16, 17]. We previously identified and characterized the low-temperature-active exo-inulinase and αgalactosidase from Sphingobacterium sp. GN25 [16] and Arthrobacter sp. GN14 [17] isolated from the feces of G. nigricollis, respectively. In this study, a novel multidomain xylanase (XynAGN16) was revealed from Arthrobacter sp. GN16 isolated from the G. nigricollis feces. The xylanase consists of a catalytic domain of GH 10 (XynAGN16L), a PD domain, a carbohydrate-binding domain (CBM_4_9), a catalytic domain of GH 10 (XynAGN16R), and a hyalin repeat (HYR) domain, in order from the Nterminus (Fig. 1). The whole xylanase and four truncated derivatives were expressed in E. coli (Fig. 1). Biochemical characterizations and hydrolysis products analyses of recombinant
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Fig. 1 Schematic diagram of XynAGN16 and its truncated derivatives generated by Pfam online tool (http:// pfam.sanger.ac.uk/). Glyco_hydro_10 family 10 domain of glycoside hydrolases, Polysacc_deac_1 polysaccharide deacetylases domain, CBM_4_9 carbohydrate-binding domain; HYR hyalin repeat domain
XynAGN16L and XynAGN16Lpd (GH 10 domain at N-terminus and PD domain) showed similar features while the PD domain improved the affinity and catalytic efficiency towards xylans of XynAGN16Lpd. Furthermore, the potential mechanism for adaptation to low temperatures of XynAGN16L was also proposed.
Materials and Methods Vectors and Reagents E. coli BL21 (DE3) (TransGen, Beijing, China) and the pET-28a(+) vector (Novagen, San Diego, CA, USA) were used for gene expression. Restriction endonucleases, T4 DNA ligase, DNA polymerase (Taq and Pyrobest), and dNTPs were purchased from TaKaRa (Otsu, Japan). The His-tagged protein was purified using Ni2+-NTA agarose (Qiagen, Valencia, CA, USA). The substrates birchwood xylan, beechwood xylan, oat-spelt xylan, barley β-glucan, pullulan (from Aureobasidium pillulans), carboxymethyl cellulose sodium salt, p-nitrophenol (pNP), pNP-acetate (C2), pNP-butyrate (C4), pNP-caproate (C6), pNP-caprylate (C8), pNP-caprate (C10), and phenyl acetate were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH, USA). Silica gel G plate was purchased from Merck KGaA (Darmstadt, Germany). Methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose were gifts from Prof. James F. Preston (Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA). Other reagents were of analytical grade and were commercially available.
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Microorganism Isolation and Identification The feces were collected from G. nigricollis wintering in Dashanbao National Nature Reserve as previously described [16]. Two grams of the feces were suspended in 0.7 % (w/v) NaCl and spread onto the screening agar plates containing 0.5 % (w/v) hulless barley meal, 0.1 % (w/v) peptone, and 1.0 % (w/v) NaCl. The pure culture of the strain GN16 was obtained through repeated streaking on the screening agar plates at room temperature. The taxon of GN16 was identified based on 16S rDNA sequence PCR-amplified using primers 27F (AGAGTTTGAT CCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The strain was deposited in the China General Microbiological Culture Collection Center under CGMCC 1.10976. Genome Sequencing Genome sequencing is becoming both faster and cheaper. Some enzymes showing important values for basic research and industrial application were obtained based on genome sequencing [18–20]. In this study, the genome of GN16 was sequenced in the authors’ labs, as follows: Library Preparation: Genomic DNA of GN16 was extracted using the Tiangen (Beijing, China) genomic DNA Isolation Kit, assessed using NanoDrop-2000 (Thermo Scientific, Waltham, MA, USA), quantified using Qubit DNA Quantification Kit (Invitrogen, Carlsbad, CA, USA), randomly fragmented using the Bioruptor sonicator (Diagenode, Liège, Belgium), and purified using Zymo Genomic DNA Clean & Concentration Kit (Orange, CA, USA). Then, the DNA library was prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer’s instruction (San Diego, CA, USA). After adapter ligation, a library fragment size of appropriately 500 bp was chosen and PCR-enriched (10 PCR cycles). The library quality and quantity were confirmed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing: The MiSeq Reagent Kit V2 provided reagents for the cluster amplification and sequencing on a Miseq sequencer (Illumina). Data analysis: Real-time image analysis and base calling were performed using the compatible sequencing software RTA (Illumina). FASTAQ files were generated using CASAVA (Illumina) and loaded into Velvet 1.2.07 [21] for sequence assembly performed on a NF supercomputing server (Inspur, Shandong, China) in the authors’ labs.
Sequence and Structure Analyses of Endoxylanases Genes were predicted by tools combined GeneMark.hmm 2.8 (http://exon.gatech.edu/ GeneMark/gmhmm2_prok.cgi) with local BLAST 2.2.25 [22], using the genomic sequence of Arthrobacter phenanthrenivorans Sphe3 (accession no. NC_015145) as the local database. The signal peptide and domains in XynAGN16 were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/) and Pfam online tool (http:// pfam.sanger.ac.uk/), respectively. The tertiary structures of XynAGN16L and the thermophilic endoxylanases TmXyn (accession no. AAD35164) [23], TxXyn (CAA76420) [24], and BsXyn (ABI49951) [25] were predicted by homology modeling using SwissModel (http://swissmodel.expasy.org/; Table 1). The tertiary structures were aligned using Discovery Studio v2.5 software (Accelrys, San Diego, CA, USA) for structure comparison. Salt bridges (distances ≤4 Å), and hydrogen bonds were predicted with VMD 1.8.6 [26] and UCSF Chimera 1.2540 [27],
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respectively. Accessible surface area (ASA) and total packing volume (TPV) were calculated using VADAR [28]. Expression of XynAGN16 in E. coli The coding sequence of the mature protein (without the predicted signal peptide) of XynAGN16 was amplified by PCR using Pyrobest DNA polymerase and the primer set XynAHF and XynANR (Fig. 1). The PCR product was gel-purified, digested with HindIII and NotI, and cloned into the corresponding site of pET-28a(+) vector. The recombinant plasmid, pET-xynAGN16, was transformed into E. coli BL21 (DE3) competent cells. Transformants were identified by PCR analysis and confirmed by DNA sequencing. A positive transformant harboring pET-xynAGN16 was inoculated into LB medium supplemented with 100 μg/mL kanamycin and grown overnight at 37 °C. The culture was transferred into fresh LB medium (1:100 dilution) containing 100 μg/mL kanamycin and grown aerobically at 37 °C to an A600 of 0.8. IPTG was then added to a final concentration of 0.7 mM for further induction at 20 °C for 20 h. The cells were harvested by centrifugation at 8000×g for 5 min at 20 °C. Purification and Identification of the Recombinant XynAGN16 To purify the recombinant His6-tagged XynAGN16 (rXynAGN16), the harvested cells were washed and re-suspended with sterilized ice-cold buffer A (20 mM Tris–HCl, 0.5 M NaCl, pH 7.2), and then were disrupted by sonication (5 s, 130 W) on ice. After centrifugation at 12,000×g for 10 min at 20 °C, the supernatant was applied to a Ni2+NTA agarose gel column for purification with a linear imidazole gradient of 20–300 mM in buffer A. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12 % running gel). To verify that the purified protein was rXynAGN16, the in-gel band was cut and sent to Tianjin Biochip (Tianjin, China) which provides analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The Bradford method [29] was used to determine protein concentration, using bovine serum albumin as a standard. Enzyme Assay and Substrate Specificity Xylanase activity was determined by measuring the release of reducing sugars from substrate using the 3,5-dinitrosalicylic acid (DNS) reagent. The standard reaction contained 0.1 mL of diluted enzyme and 0.9 mL of McIlvaine buffer (pH 6.5) containing 0.5 % (w/v) xylan as the substrate. After incubation at 37 °C for 10 min, the reaction was stopped with 1.5 mL of DNS reagent and then boiled for 5 min. The absorption at 540 nm was measured when the above mixture was cooled down to room temperature. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1.0 μmol of reducing sugars equivalent to xylose per minute. Esterase activity assay was performed using pNP-esters as the substrates [30]. Reactions containing 50.0 μL of diluted enzyme, 30.0 μL of 10.0 mM substrate, and 420.0 μL of McIlvaine buffer (pH 8.0) were incubated at 37 °C for 10 min. After adding 2.0 mL of 1.0 M Na2CO3, the absorption at 405 nm indicating the amount of liberated pNP was measured. One unit of esterase activity was defined as the amount of enzyme that released 1.0 μmol of pNP per minute.
31
Number of salt bridges (≤4 Å)
AAD35164 Thermotoga maritima
[23]
Total packing volume (103 Å3)
Accession number Organism
Reference
NR not reported, ASA accessible surface area
7.2
47.4
Exposed nonpolar ASA (103 Å2)
354
4.0
Proline (%)
13.1
5.2 4.3
Glycine (%) Arginine (%)
Total ASA (103 Å2)
ca. 40 at 90 °C
Half-life (min)
Number of hydrogen bonds
NR
NR
90 ca. 10 % at 30 °C
Optimum temperature (°C) Relative xylanase activity at 20 °C
Relative xylanase activity at 0 °C
6.1
Optimum pH
Relative xylanase activity at 10 °C
TmXyn
Parameter
Table 1 Characterizations of the thermophilic endoxylanases and XynAGN16L
[24]
CAA76420 Thermobacillus xylanilyticus
45.3
8.0
14.4
294
27
4.6
6.0 8.2
30 at 75 °C
NR
NR
75 ca. 25 % at 30 °C
6.5
TxXyn
[25]
ABI49951 Bacillus stearothermophilus
52.1
9.1
15.6
394
32
5.6
5.6 4.1
ca. 360 at 65 °C
NR
NR
75 28 % at 45 °C
6.5
BsXyn
This study
JQ863105 Arthrobacter sp.
48.3
8.6
14.9
303
19
5.8
7.7 6.0
ca. 2 at 50 °C
17.3 %
26.4 %
45 37.5 %
5.5
XynAGN16L
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Arylesterase activity was determined by using phenyl acetate as the substrate [30]. Reactions containing 0.5 mL of diluted enzyme, 0.03 mL of 600.0 mM substrate, and 3.0 mL of McIlvaine buffer (pH 8.0) were incubated at 37 °C for 10 min. After adding 0.1 mL of 1.0 M HCl, the absorption at 270 nm indicating the amount of liberated phenol was measured. One unit of arylesterase activity was defined as the amount of enzyme that released 1.0 μmol of phenol per minute. A solution containing inactivated (90 °C for 5 min) enzyme instead of active enzyme was used as an assay blank to set the absorption to zero. A solution containing water instead of enzyme served as a negative control. For identifying the substrate specificity of rXynAGN16, oat-spelt xylan, beechwood xylan, birchwood xylan, barley β-glucan, pullulan (from A. pillulans), carboxymethyl cellulose sodium salt, C2, C4, C6, C8, C10, or phenyl acetate was individually added to the reaction solutions. Biochemical Characterization of Xylanase The optimal pH for the xylanase activity of purified rXynAGN16 was determined at 37 °C in buffers with pH 4.0–12.0. The enzyme stability at different pH values was estimated by measuring the residual enzyme activity after incubating the enzyme solution in various buffers at 37 °C for 1 h. The buffers used were McIlvaine buffer for pH 4.0–8.0 and 0.1 M glycine– NaOH for pH 9.0–10.0. The temperature-dependent activity of rXynAGN16 was determined over the range of 0– 70 °C in McIlvaine buffer (pH 6.5). The thermostability of rXynAGN16 was determined after pre-incubation of the enzyme for 60 min in McIlvaine buffer (pH 6.5) at 37, 50, or 60 °C without substrate for various periods. To investigate the effects of different metal ions and chemical reagents on the rXynAGN16 activity measured in McIlvaine buffer (pH 6.5) at 37 °C, 10.0 mM (final concentration) NaCl, KCl, CaCl2, CoCl2, NiSO4, FeSO4, CuSO4, MgSO4, ZnSO4, Pb(CH3COO)2, HgCl2, FeCl3, EDTA, β-mercaptoethanol, or SDS was individually added to the reaction solution. The values of Michaelis constant (Km), maximum rate of reaction (Vmax), and turnover number (kcat) for rXynAGN16 were determined using beechwood xylan or birchwood xylan as the substrate. The reaction contained 0.1 mL of diluted enzyme (appropriately 0.2 nmol) and 0.9 mL of McIlvaine buffer (pH 6.5) containing 0.5–10.0 mg/mL xylan as the substrate. The mixture was incubated in a water bath at 45 °C for 5 min. The rate of reaction was calculated using the value of absorption at 540 nm. Double-reciprocal plots were constructed according to the Lineweaver–Burk method [31]. Hydrolysis Products Analysis The hydrolysis of 0.5 % (w/v) birchwood xylan was carried out with rXynAGN16 (1.0 U/mL reaction system) for 24 h at 45 °C and pH 6.5. The hydrolysis products were analyzed by thinlayer chromatography (TLC) performed as previously described [32]. Birchwood xylan with the inactivated rXynAGN16 (90 °C for 5 min) was used as a control. Construction and Characterization of the Truncated Derivatives To study the effect of domains on the enzyme performance, four truncated derivatives of XynAGN16 were constructed, including XynAGN16L, XynAGN16Lpd, XynAGN16R, and XynAGN16Rhyr (GH 10 domain at C-terminus and HYR domain) (Fig. 1). The expressions,
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purifications, biochemical characterizations, and hydrolysis products analyses of the recombinant derivatives were performed as that of rXynAGN16. Biochemical characterizations and hydrolysis products analyses of the recombinant XynAGN16L (rXynAGN16L) and recombinant XynAGN16Lpd (rXynAGN16Lpd) were performed at pH 5.5 unless otherwise noted. Nucleotide Sequence Accession Numbers The nucleotide sequences for the Arthrobacter sp. GN16 16S rDNA and endoxylanase gene (xynAGN16) were deposited in GenBank under the accession numbers JQ863104 and JQ863105, respectively.
Results Strain Identification The comparison of the 16S rDNA sequence from GN16 (1368 bp; JQ863104) with that in GenBank showed nucleotide identities of 99.6 % with Arthrobacter citreus DSM 20133 (accession no. NR_026188) and of 99.5 % with Arthrobacter agilis TP-Snow-C61 (KC986991). Thus, the strain GN16 was classified into the genus Arthrobacter. Genome Sequencing and Sequence Analyses Genome sequencing generated appropriately 550 Mbp of sequence data. After sequence assembly, these data yielded appropriately 3.4 Mbp. Local BLAST analysis revealed the gene xynAGN16 (JQ863105) was homologous to the xylanase-encoding gene from A. phenanthrenivorans Sphe3 (ADX74784). The full-length xynAGN16 (3,639 bp) starts with the putative codon ATG, ends with TGA, and encodes a 1,212-residues polypeptide with a calculated mass of 131.5 kDa. The signal peptide was predicted from M1 to A25, followed by a catalytic domain of GH 10 from T41 to L368, a PD domain from A404 to L532, a CBM_4_9 domain from V610 to V741, a catalytic domain of GH 10 from G764 to A1117, and a HYR domain from D1133 to V1203 (Fig. 1). A xylanase showing similar multidomain structure has not been recorded in GenBank. BLASTp analysis showed that: XynAGN16L shared the highest identities of 71.6 % with the predicted xylanase from A. phenanthrenivorans Sphe3 (ADX74784) and of 66.2 % with the experimentally verified GH 10 xylanase from Lechevalieria sp. HJ3 (AFE82289); the PD domain of XynAGN16 shared the highest identities of 70.1 % with the predicted xylanase/chitin deacetylase from Nocardioidaceae bacterium Broad-1 (EGD41782) and of 33.1 % with the identified chitooligosaccharide deacetylase or nodulation protein B from Rhizobium galegae HAMBI 1174 (P50354); the CBM_4_9 domain of XynAGN16 shared the highest identities of 70.2 % with the predicted CBM_4_9 domain from Clavibacter michiganensis subsp. michiganensis NCPPB 382 (CAN01726) and of 50.4 % with the identified CBM_4_9 domain from Paenibacillus terrae HPL-003 (ADX97440); XynAGN16R shared the highest identities of 73.3 % with the predicted xylanase from C. michiganensis NCPPB 382 (CAN01726) and of 38.1 % with the identified GH 10 xylanase from P. terrae HPL-003 (ADX97440); and the HYR domain of XynAGN16 shared the highest identity of 62.5 % with the predicted HYR domain from C. michiganensis NCPPB 382 (CAN01726).
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Multiple Sequence Alignment and Structure Analysis Compared with the thermophilic endoxylanases TmXyn, TxXyn, and BsXyn, the multiple sequence alignment revealed increased amino acid residues from A57 to Y77 of XynAGN16L, including three increased glycines G58–G60 (Fig. 2). The tertiary structure of amino acid residues from A57 to Y77 of XynAGN16L consists of β-sheets and a loop, while the corresponding structures of TmXyn, TxXyn, and BsXyn are α-helixes (Fig. 2). Compared with TmXyn, TxXyn, and BsXyn, XynAGN16L has less salt bridges and more glycines. However, other parameter values were similar or showed irregularity, including the numbers of arginine, proline, and hydrogen bonds, and the total ASA, exposed nonpolar ASA, and total packing volume (Table 1).
Fig. 2 Homology model structures and partial multiple sequence alignment of XynAGN16L and thermophilic endoxylanases. Homology models and sequence names are shown with accession numbers except XynAGN16L, as follows: TmXyn from Thermotoga maritima MSB8 (AAD35164) [23], TxXyn from Thermobacillus xylanilyticus D3 (CAA76420) [24], and BsXyn from Bacillus stearothermophilus T-6 (ABI49951) [25]. The circles indicate the structure built by amino acid residues from A57 to Y77 of XynAGN16L and its corresponding structures of TmXyn, TxXyn, and BsXyn. The putative catalytic E177 and E285 of XynAGN16L are detailed in ball-and-stick form
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Expression, Purification, and Identification of rXynAGN16 and its Truncated Derivatives XynAGN16, XynAGN16L, and XynAGN16Lpd were expressed in E. coli BL21 (DE3) (Fig. 3). However, XynAGN16R and XynAGN16Rhyr were neither detected on SDS-PAGE (Fig. 3) nor released reducing sugars using xylans as substrates. Then, further studies on XynAGN16R and XynAGN16Rhyr were not performed. rXynAGN16 could not be purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, ammonium sulfate precipitation, ion exchange chromatography, and molecular exclusion chromatography. A band showing appropriately 130 kDa was detected from the cell lysate of an induced transformant harboring pET-xynAGN16 (Fig. 3). The calculated value of rXynAGN16 is 135.0 kDa, which is approximately equal to the molecular mass of the band. The cell lysate of E. coli harboring the empty pET-28a(+) was neither showed the band nor xylanase activity (Fig. 3). Consequently, the crude rXynAGN16, not the purified rXynAGN16, was used for further studies. rXynAGN16L and rXynAGN16Lpd were purified to electrophoretic homogeneity by Ni 2+ -NTA metal chelating affinity chromatography (Fig. 3). The purified rXynAGN16L and rXynAGN16Lpd migrated as two single bands on SDS-PAGE with molecular masses of appropriately 48 and 69 kDa, respectively, which is close to the calculated values (rXynAGN16L, 46.9 kDa; rXynAGN16Lpd, 68.7 kDa). After tryptic digest, the two bands of rXynAGN16L and rXynAGN16Lpd were individually analyzed using the MALDI-TOF MS. Results revealed that the MALDI-TOF MS spectrums matched the molecular masses of the known internal peptides of rXynAGN16L
Fig. 3 SDS-PAGE analysis of XynAGN16 and its truncated derivatives. Lanes M1, M2, and M3 protein molecular weight markers SM1851, SM0671, and SM0431 from Generay Biotech, respectively (Shanghai, China), lane CK cell extract from an induced transformant harboring the empty plasmid pET-28a(+), lane Xyn cell extract from an induced transformant harboring the positive transformant pET-xynAGN16, lanes L and Lpd rXynAGN16L and rXynAGN16Lpd purified by Ni2+-NTA chelating affinity chromatography, respectively, lanes R and Rhyr cell extracts from induced transformants harboring the positive transformants pET-xynAGN16R and pET-xynAGN16Rhyr, respectively. Arrow indicates rXynAGN16
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and rXynAGN16Lpd (Fig. S1), confirming that the purified enzymes were rXynAGN16L and rXynAGN16Lpd. Substrate Specificity The activities of the crude rXynAGN16 towards substrates of 0.5 % (w/v) beechwood xylan, birchwood xylan, and oat-spelt xylan were detected. Determined at pH 5.5 and 45 °C, the activities of the purified rXynAGN16L towards substrates of beechwood xylan, birchwood xylan, and oat-spelt xylan were 1234.1 ± 83.1, 1081.8 ± 20.9, and 1242.4±69.7 U/μmol, respectively; the activities of the purified rXynAGN16Lpd towards beechwood xylan, birchwood xylan, and oat-spelt xylan were 1266.2 ± 47.0, 1177.8±65.5, and 819.0±85.5 U/μmol, respectively. Determined at pH 8.0 and 37 °C, the activities of the purified rXynAGN16L towards phenyl acetate, C4, and C6 were 21.1±0.5, 14.8±1.8, and 4.7±1.2 U/μmol, respectively; the activities of the purified rXynAGN16Lpd towards phenyl acetate, C4, and C6 were 26.6±0.4, 19.2±3.1, and 3.1 ±1.6 U/μmol, respectively. However, the activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were not detected towards substrates of 0.5 % (w/v) barley β-glucan, pullulan, and carboxymethyl cellulose sodium salt. No activity of rXynAGN16L or rXynAGN16Lpd was detected towards C2, C8, or C10. Enzyme Characterization When assayed at 37 °C, the crude rXynAGN16 showed apparently optimal xylanase activity at pH 6.5 and retained 29.2 % of the maximum activity at pH 5.5, while the purified rXynAGN16L and rXynAGN16Lpd showed apparently optimal xylanase activities at pH 5.5 (Fig. 4a). rXynAGN16, rXynAGN16L, and rXynAGN16Lpd showed close relative activities at pH 8.0–12.0. After incubation in buffers ranging from pH 7.0–12.0 at 37 °C for 1 h, the three xylanases exhibited more than 30 % of their initial activities (Fig. 4b). The activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were all apparently optimal at 45 °C. When assayed at 0 °C, the three xylanases still remained appropriately 15 % of their maximum activities (Fig. 4c). The three xylanases are thermolabile. After preincubation for 60 min at 37 °C, rXynAGN16, rXynAGN16L, and rXynAGN16Lpd exhibited 52.4, 71.2, and 70.9 % of their initial activities. The enzymes stabilities decreased rapidly at temperatures of more than 50 °C (Fig. 4d). The activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were completely inhibited by 10.0 mM Hg2+ or SDS. The addition of Zn2+ and EDTA slightly inhibited (retaining appropriately 65 % activities) rXynAGN16 and rXynAGN16Lpd, respectively. In the presence of 10.0 mM Fe2+, the purified rXynAGN16L and rXynAGN16Lpd remained appropriately 75 % of activities. The activities of the purified rXynAGN16L and rXynAGN16Lpd were enhanced by appropriately 0.5and 0.7-fold by 10.0 mM β-mercaptoethanol, respectively. Other reagents had little or no effect on the three xylanases. Because rXynAGN16 was not purified to electrophoretic homogeneity, kinetic experiment of the enzyme was not performed. The Km, Vmax, and kcat values of the purified rXynAGN16L and rXynAGN16Lpd towards xylans were determined in McIlvaine buffer (pH 5.5) at 45 °C, and were shown in Fig. 4. The Km and kcat/Km values of the purified rXynAGN16L towards birchwood xylan were 2.6 mg/mL and 19.5 mL/mg/s, respectively, while the two values of the purified rXynAGN16Lpd were 1.2 mg/mL and 42.7 mL/mg/s, respectively (Fig. 4e). Towards beechwood xylan,
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Fig. 4 Characterizations of the crude rXynAGN16 and the purified rXynAGN16L and rXynAGN16Lpd. a Effects of pH on the three xylanases determined at 37 °C from pH 4.0 to 12.0. b pH stabilities assays. After preincubation of the enzymes at pH 5.0–12.0 and 37 °C for 60 min, the enzymes activities were determined at 37 °C. c Thermoactivities of the three xylanases. d Thermostabilities assays. The enzymes were pre-incubated at 37, 50, or 60 °C, and aliquots were removed at specific time points for the measurements of residual activities at 37 °C. e Lineweaver–Burk plots and kinetic values of the purified rXynAGN16L and rXynAGN16Lpd determined using 0.5–10 mg/mL birchwood xylan as the substrate at 45 °C. f Lineweaver–Burk plots and kinetic values of the purified rXynAGN16L and rXynAGN16Lpd determined using 0.5–10 mg/mL beechwood xylan as the substrate at 45 °C. Characterizations of the crude rXynAGN16 were performed at pH 6.5, while characterizations of the purified rXynAGN16L and rXynAGN16Lpd were performed at pH 5.5 unless otherwise noted. The error bars represent the means±SD (n=3)
the Km and kcat/Km values of the purified rXynAGN16L were 1.8 mg/mL and 27.1 mL/mg/s, respectively, while the two values of the purified rXynAGN16Lpd were 1.0 mg/mL and 35.3 mL/mg/s, respectively (Fig. 4f). The results implied that rXynAGN16Lpd has a higher affinity and catalytic efficiency towards xylans than rXynAGN16L: the affinities towards birchwood xylan and beechwood xylan were enhanced by appropriately 0.5- and 0.4-fold, respectively; the catalytic efficiencies
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towards birchwood xylan and beechwood xylan were enhanced by appropriately 1.2and 0.3-fold, respectively. Hydrolysis Products The hydrolysis products of 0.5 % (w/v) birchwood xylan by rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were analyzed by TLC (Fig. 5). Xylose and some xylo-oligosaccharides, including potential xylobiose and methylglucuronoxylotetraose, were released from beechwood xylan by the three xylanases, while xylotriose, xylotetraose, methylglucuronoxylobiose, and methylglucuronoxylotriose were not clearly detected. The hydrolysis analysis indicated the endo-acting nature of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd.
Discussion The occurrence of additional domains to the catalytic domain in an enzyme might be the result of mutation and domain shuffling during the course of evolution, which in turn has yielded a multiplicity of enzymes [33]. The common additional domains in many modular endoxylanases are CBM domains [4–9]. To the best of the authors’ knowledge, only three
Fig. 5 Thin layer chromatography (TLC) analysis of the hydrolysis of 0.5 % (w/v) birchwood xylan. Lanes CKXyn, CK-L, and CK-Lpd birchwood xylan with the inactivated (90 °C for 5 min) rXynAGN16, rXynAGN16L, and rXynAGN16Lpd, respectively, lane S-Xyn birchwood xylan hydrolyzed by the crude rXynAGN16 (1.0 U/ml reaction system) for 24 h at 45 °C and pH 6.5, lanes S-L and S-Lpd birchwood xylan hydrolyzed by the purified rXynAGN16L and rXynAGN16Lpd, respectively (1.0 U/ml reaction system, 45 °C, pH 5.5, 24 h), lane M1 xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose marker, lane M2 methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose marker
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multidomain xylanases have the PD domains (CAA54145, CAA90745, and ADG73550), only three multidomain xylanases have the HYR domains (CAN01726, CCE75600, and ABS02556), and no xylanase has both the PD and HYR domains. Thus, xynAGN16 encodes a xylanase with a previously undescribed combination of domains. The non-catalytic domains contribute to the function of multidomain enzymes in cooperation with catalytic domains [4–9]. The functions of CBM domains in many modular endoxylanases have been studied [4–9]. Polysaccharide deacetylases, members of carbohydrate esterase family 4, include chitin deacetylases, acetyl-xylan esterases, rhizobial NodB chitooligosaccharide deacetylases, and peptidoglycan deacetylases [3]. However, the effect of PD domain on multidomain xylanases has not been reported. In this study, the kinetic values of rXynAGN16L and rXynAGN16Lpd reveal that the PD domain improves the affinity and catalytic efficiency towards xylans of the xylanase rXynAGN16Lpd. High catalytic efficiency is one of the major factors for widespread applications of enzymes [34]. Over 80 % of the Earth’s biosphere is permanently cold. The largest proportion of biomass on the Earth is generated at low temperatures [35, 36]. Low-temperature-active enzymes show high catalytic activity at low temperatures and thermolability at intermediate and high temperatures [36]. The two low-temperature-active single-domain xylanases XynA19 and XynGR40 were isolated from the gastrointestinal tracts of Batocera horsfieldi larvae [37] and Boer goat [14], respectively. Many Arthrobacter strains can produce low-temperature-active enzymes, such as the glycerol dehydrogenase from Arthrobacter sp. MS-7 [38] and the chitobiase from Arthrobacter sp. TAD20 [39], where as Arthrobacter sp. MTCC 5214 produces a thermophilic xylanase [40]. rXynAGN16L showed catalytic activity at 0 °C and thermolability at temperatures of more than 50 °C. This report is the first to present the cloning, heterologous expression, and characterization of a low-temperature-active xylanase from an Arthrobacter strain isolated from the feces of G. Nigricollis. Compared with thermophilic counterparts, low-temperature-active enzymes generally have a decreased number of proline and/or arginine residues, and/or disulfide and/ or hydrogen bonds, and/or salt bridges, and an increased number of glycine residues, and/or a larger total accessible surface area and/or exposed hydrophobic accessible surface area, and/or longer surface loops [36]. Compared with TmXyn, TxXyn, and BsXyn, the surface loop from A57 to Y77 of rXynAGN16L might increase the amplitude of the movement between secondary structures; the decreased salt bridges might reduce the stabilization energy of rXynAGN16L. The two factors in combination might lead to the reduced stability and enhanced flexibility of the structure of rXynAGN16L and consequently have a key role in maintaining the catalytic activity of rXynAGN16L at low temperatures. In conclusion, this study identified and characterized a novel xylanase XynAGN16 consisting of a previously undescribed combination of domains. The GH 10 catalytic domain at N-terminus of XynAGN16 plays a key role in catalysis towards xylans. The PD domain of XynAGN16 improves the affinity and catalytic efficiency towards xylans of the truncated xylanase rXynAGN16Lpd. The structural adaptation to low temperatures of the truncated xylanase rXynAGN16L might be ascribed to the surface loop from A57 to Y77 and the decreased salt bridges. Furthermore, this study is the first to present the cloning, heterologous expression, and characterization of a lowtemperature-active xylanase from an Arthrobacter strain isolated from the feces of G. nigricollis.
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Acknowledgments This work was supported by the Key Technologies Research and Development Program of China (2013BAD10B01), National Natural Science Foundation of China (31260215), and Applied and Basic Research Foundation of Yunnan Province (2011FB048). Thanks for the kind gifts (methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose) from Prof. James F. Preston from University of Florida.
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Molecular and Biochemical Characterization of a Novel Multidomain Xylanase from Arthrobacter sp. GN16 Isolated from the Feces of Grus nigricollis Junpei Zhou & Jidong Shen & Rui Zhang & Xianghua Tang & Junjun Li & Bo Xu & Junmei Ding & Yajie Gao & Dongyan Xu & Zunxi Huang
Received: 23 July 2014 / Accepted: 10 October 2014 / Published online: 21 October 2014 # Springer Science+Business Media New York 2014
Abstract A novel glycosyl hydrolase family 10 (GH 10) xylanase (XynAGN16), consisting of five domains, was revealed from the genome sequence of Arthrobacter sp. GN16 isolated from the feces of Grus nigricollis. XynAGN16 and its truncated derivatives XynAGN16L (GH 10 domain at N-terminus) and XynAGN16Lpd (GH 10 domain at N-terminus and polysaccharide deacetylases domain) were expressed in Escherichia coli and characterized. Biochemical characterizations and hydrolysis products analyses of recombinant XynAGN16L and XynAGN16Lpd showed similar features, including showing catalytic activities at 0 °C, thermolabilities at temperatures of more than 50 °C, and similar substrate specificity. However, the polysaccharide deacetylases domain improved the affinity and catalytic efficiency towards xylans of the recombinant XynAGN16Lpd. The Km and kcat/Km values of recombinant XynAGN16L towards birchwood xylan were 2.6 mg/mL and 19.5 mL/mg/s, respectively,
Junpei Zhou and Jidong Shen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12010-014-1295-2) contains supplementary material, which is available to authorized users.
J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, Kunming 650500, People’s Republic of China J. Zhou e-mail: [email protected] J. Zhou : J. Shen : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Y. Gao : D. Xu : Z. Huang College of Life Sciences, Yunnan Normal University, No.1 Yuhua District, Chenggong, Kunming, Yunnan 650500, People’s Republic of China J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Yunnan, Kunming 650500, People’s Republic of China J. Zhou : R. Zhang : X. Tang : J. Li : B. Xu : J. Ding : Z. Huang (*) Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming 650500, People’s Republic of China e-mail: [email protected]
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while the two values of recombinant XynAGN16Lpd were 1.2 mg/mL and 42.7 mL/mg/s, respectively. Towards beechwood xylan, the Km and kcat/Km values of recombinant XynAGN16L were 1.8 mg/mL and 27.1 mL/mg/s, respectively, while the two values of recombinant XynAGN16Lpd were 1.0 mg/mL and 35.3 mL/mg/s, respectively. Compared with three thermophilic endoxylanases, XynAGN16L has a surface loop from A57 to Y77 and a decreased number of salt bridges. Keywords Multidomain . Xylanase . Polysaccharide deacetylases . Low-temperature-active . Kinetic . Grus nigricollis
Introduction Xylan is the major component of the plant cell wall and the most abundant renewable hemicellulose. The backbone of xylan is built of β-1,4-linked xylopyranosyl residues [1]. Endoxylanases (endo-β-1,4-xylanases; EC 3.2.1.8) hydrolyze xylan backbone to produce short chain xylo-oligosaccharides of varying lengths, therefore they play a key role in the biodegradation of xylan [2]. Most endoxylanases are classified in the family 10 or 11 of glycoside hydrolases (GH) based on the sequence similarities of their respective catalytic domains [3]. Besides endoxylanases, the complete breakdown of xylan requires β-Dxylosidases and five debranching enzymes [1]. Endoxylanases have received a great deal of attention mainly due to their extensive applications in many fields, such as bioconversion of biomass materials, pre-bleaching of kraft pulps, and improving the quality of food and feed [1]. Basic studies on endoxylanases can give insights into catalytic mechanisms and improve the performances of these enzymes. Multidomain xylanases consist of a catalytic GH10 domain with one or more carbohydratebinding domain (CBM), and/or fibronectin type 3 domain, and/or S-layer-homologous domain [4–9]. These studies revealed that non-catalytic domains could be responsible for the binding and hydrolysis of insoluble polysaccharides [6], the thermoactivity and thermostability [7], the pH stability [5], and the cell surface localization [8] of multidomain xylanases. However, the polysaccharide deacetylases (PD) domain has rarely been present in multidomain xylanases (GenBank accession no. CAA54145, CAA90745, and ADG73550), not to mention its effect on multidomain xylanases. Grus nigricollis, also named black-necked cranes, are categorized as Vulnerable on the IUCN Red List because their population is only approximately 8000 on Earth. The majority of G. nigricollis live their life on high plateaus of China, such as Qinghai–Tibet Plateau and Yunnan–Guizhou Plateau of China [10]. Unlike other gastrointestinal microorganisms and their enzymes, which have been given considerable research interests [11–15], microorganisms and their enzymes from the feces of G. nigricollis have rarely been reported [16, 17]. We previously identified and characterized the low-temperature-active exo-inulinase and αgalactosidase from Sphingobacterium sp. GN25 [16] and Arthrobacter sp. GN14 [17] isolated from the feces of G. nigricollis, respectively. In this study, a novel multidomain xylanase (XynAGN16) was revealed from Arthrobacter sp. GN16 isolated from the G. nigricollis feces. The xylanase consists of a catalytic domain of GH 10 (XynAGN16L), a PD domain, a carbohydrate-binding domain (CBM_4_9), a catalytic domain of GH 10 (XynAGN16R), and a hyalin repeat (HYR) domain, in order from the Nterminus (Fig. 1). The whole xylanase and four truncated derivatives were expressed in E. coli (Fig. 1). Biochemical characterizations and hydrolysis products analyses of recombinant
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Fig. 1 Schematic diagram of XynAGN16 and its truncated derivatives generated by Pfam online tool (http:// pfam.sanger.ac.uk/). Glyco_hydro_10 family 10 domain of glycoside hydrolases, Polysacc_deac_1 polysaccharide deacetylases domain, CBM_4_9 carbohydrate-binding domain; HYR hyalin repeat domain
XynAGN16L and XynAGN16Lpd (GH 10 domain at N-terminus and PD domain) showed similar features while the PD domain improved the affinity and catalytic efficiency towards xylans of XynAGN16Lpd. Furthermore, the potential mechanism for adaptation to low temperatures of XynAGN16L was also proposed.
Materials and Methods Vectors and Reagents E. coli BL21 (DE3) (TransGen, Beijing, China) and the pET-28a(+) vector (Novagen, San Diego, CA, USA) were used for gene expression. Restriction endonucleases, T4 DNA ligase, DNA polymerase (Taq and Pyrobest), and dNTPs were purchased from TaKaRa (Otsu, Japan). The His-tagged protein was purified using Ni2+-NTA agarose (Qiagen, Valencia, CA, USA). The substrates birchwood xylan, beechwood xylan, oat-spelt xylan, barley β-glucan, pullulan (from Aureobasidium pillulans), carboxymethyl cellulose sodium salt, p-nitrophenol (pNP), pNP-acetate (C2), pNP-butyrate (C4), pNP-caproate (C6), pNP-caprylate (C8), pNP-caprate (C10), and phenyl acetate were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH, USA). Silica gel G plate was purchased from Merck KGaA (Darmstadt, Germany). Methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose were gifts from Prof. James F. Preston (Department of Microbiology and Cell Science, University of Florida, Gainesville, FL, USA). Other reagents were of analytical grade and were commercially available.
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Microorganism Isolation and Identification The feces were collected from G. nigricollis wintering in Dashanbao National Nature Reserve as previously described [16]. Two grams of the feces were suspended in 0.7 % (w/v) NaCl and spread onto the screening agar plates containing 0.5 % (w/v) hulless barley meal, 0.1 % (w/v) peptone, and 1.0 % (w/v) NaCl. The pure culture of the strain GN16 was obtained through repeated streaking on the screening agar plates at room temperature. The taxon of GN16 was identified based on 16S rDNA sequence PCR-amplified using primers 27F (AGAGTTTGAT CCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). The strain was deposited in the China General Microbiological Culture Collection Center under CGMCC 1.10976. Genome Sequencing Genome sequencing is becoming both faster and cheaper. Some enzymes showing important values for basic research and industrial application were obtained based on genome sequencing [18–20]. In this study, the genome of GN16 was sequenced in the authors’ labs, as follows: Library Preparation: Genomic DNA of GN16 was extracted using the Tiangen (Beijing, China) genomic DNA Isolation Kit, assessed using NanoDrop-2000 (Thermo Scientific, Waltham, MA, USA), quantified using Qubit DNA Quantification Kit (Invitrogen, Carlsbad, CA, USA), randomly fragmented using the Bioruptor sonicator (Diagenode, Liège, Belgium), and purified using Zymo Genomic DNA Clean & Concentration Kit (Orange, CA, USA). Then, the DNA library was prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer’s instruction (San Diego, CA, USA). After adapter ligation, a library fragment size of appropriately 500 bp was chosen and PCR-enriched (10 PCR cycles). The library quality and quantity were confirmed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Sequencing: The MiSeq Reagent Kit V2 provided reagents for the cluster amplification and sequencing on a Miseq sequencer (Illumina). Data analysis: Real-time image analysis and base calling were performed using the compatible sequencing software RTA (Illumina). FASTAQ files were generated using CASAVA (Illumina) and loaded into Velvet 1.2.07 [21] for sequence assembly performed on a NF supercomputing server (Inspur, Shandong, China) in the authors’ labs.
Sequence and Structure Analyses of Endoxylanases Genes were predicted by tools combined GeneMark.hmm 2.8 (http://exon.gatech.edu/ GeneMark/gmhmm2_prok.cgi) with local BLAST 2.2.25 [22], using the genomic sequence of Arthrobacter phenanthrenivorans Sphe3 (accession no. NC_015145) as the local database. The signal peptide and domains in XynAGN16 were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/) and Pfam online tool (http:// pfam.sanger.ac.uk/), respectively. The tertiary structures of XynAGN16L and the thermophilic endoxylanases TmXyn (accession no. AAD35164) [23], TxXyn (CAA76420) [24], and BsXyn (ABI49951) [25] were predicted by homology modeling using SwissModel (http://swissmodel.expasy.org/; Table 1). The tertiary structures were aligned using Discovery Studio v2.5 software (Accelrys, San Diego, CA, USA) for structure comparison. Salt bridges (distances ≤4 Å), and hydrogen bonds were predicted with VMD 1.8.6 [26] and UCSF Chimera 1.2540 [27],
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respectively. Accessible surface area (ASA) and total packing volume (TPV) were calculated using VADAR [28]. Expression of XynAGN16 in E. coli The coding sequence of the mature protein (without the predicted signal peptide) of XynAGN16 was amplified by PCR using Pyrobest DNA polymerase and the primer set XynAHF and XynANR (Fig. 1). The PCR product was gel-purified, digested with HindIII and NotI, and cloned into the corresponding site of pET-28a(+) vector. The recombinant plasmid, pET-xynAGN16, was transformed into E. coli BL21 (DE3) competent cells. Transformants were identified by PCR analysis and confirmed by DNA sequencing. A positive transformant harboring pET-xynAGN16 was inoculated into LB medium supplemented with 100 μg/mL kanamycin and grown overnight at 37 °C. The culture was transferred into fresh LB medium (1:100 dilution) containing 100 μg/mL kanamycin and grown aerobically at 37 °C to an A600 of 0.8. IPTG was then added to a final concentration of 0.7 mM for further induction at 20 °C for 20 h. The cells were harvested by centrifugation at 8000×g for 5 min at 20 °C. Purification and Identification of the Recombinant XynAGN16 To purify the recombinant His6-tagged XynAGN16 (rXynAGN16), the harvested cells were washed and re-suspended with sterilized ice-cold buffer A (20 mM Tris–HCl, 0.5 M NaCl, pH 7.2), and then were disrupted by sonication (5 s, 130 W) on ice. After centrifugation at 12,000×g for 10 min at 20 °C, the supernatant was applied to a Ni2+NTA agarose gel column for purification with a linear imidazole gradient of 20–300 mM in buffer A. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12 % running gel). To verify that the purified protein was rXynAGN16, the in-gel band was cut and sent to Tianjin Biochip (Tianjin, China) which provides analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The Bradford method [29] was used to determine protein concentration, using bovine serum albumin as a standard. Enzyme Assay and Substrate Specificity Xylanase activity was determined by measuring the release of reducing sugars from substrate using the 3,5-dinitrosalicylic acid (DNS) reagent. The standard reaction contained 0.1 mL of diluted enzyme and 0.9 mL of McIlvaine buffer (pH 6.5) containing 0.5 % (w/v) xylan as the substrate. After incubation at 37 °C for 10 min, the reaction was stopped with 1.5 mL of DNS reagent and then boiled for 5 min. The absorption at 540 nm was measured when the above mixture was cooled down to room temperature. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1.0 μmol of reducing sugars equivalent to xylose per minute. Esterase activity assay was performed using pNP-esters as the substrates [30]. Reactions containing 50.0 μL of diluted enzyme, 30.0 μL of 10.0 mM substrate, and 420.0 μL of McIlvaine buffer (pH 8.0) were incubated at 37 °C for 10 min. After adding 2.0 mL of 1.0 M Na2CO3, the absorption at 405 nm indicating the amount of liberated pNP was measured. One unit of esterase activity was defined as the amount of enzyme that released 1.0 μmol of pNP per minute.
31
Number of salt bridges (≤4 Å)
AAD35164 Thermotoga maritima
[23]
Total packing volume (103 Å3)
Accession number Organism
Reference
NR not reported, ASA accessible surface area
7.2
47.4
Exposed nonpolar ASA (103 Å2)
354
4.0
Proline (%)
13.1
5.2 4.3
Glycine (%) Arginine (%)
Total ASA (103 Å2)
ca. 40 at 90 °C
Half-life (min)
Number of hydrogen bonds
NR
NR
90 ca. 10 % at 30 °C
Optimum temperature (°C) Relative xylanase activity at 20 °C
Relative xylanase activity at 0 °C
6.1
Optimum pH
Relative xylanase activity at 10 °C
TmXyn
Parameter
Table 1 Characterizations of the thermophilic endoxylanases and XynAGN16L
[24]
CAA76420 Thermobacillus xylanilyticus
45.3
8.0
14.4
294
27
4.6
6.0 8.2
30 at 75 °C
NR
NR
75 ca. 25 % at 30 °C
6.5
TxXyn
[25]
ABI49951 Bacillus stearothermophilus
52.1
9.1
15.6
394
32
5.6
5.6 4.1
ca. 360 at 65 °C
NR
NR
75 28 % at 45 °C
6.5
BsXyn
This study
JQ863105 Arthrobacter sp.
48.3
8.6
14.9
303
19
5.8
7.7 6.0
ca. 2 at 50 °C
17.3 %
26.4 %
45 37.5 %
5.5
XynAGN16L
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Arylesterase activity was determined by using phenyl acetate as the substrate [30]. Reactions containing 0.5 mL of diluted enzyme, 0.03 mL of 600.0 mM substrate, and 3.0 mL of McIlvaine buffer (pH 8.0) were incubated at 37 °C for 10 min. After adding 0.1 mL of 1.0 M HCl, the absorption at 270 nm indicating the amount of liberated phenol was measured. One unit of arylesterase activity was defined as the amount of enzyme that released 1.0 μmol of phenol per minute. A solution containing inactivated (90 °C for 5 min) enzyme instead of active enzyme was used as an assay blank to set the absorption to zero. A solution containing water instead of enzyme served as a negative control. For identifying the substrate specificity of rXynAGN16, oat-spelt xylan, beechwood xylan, birchwood xylan, barley β-glucan, pullulan (from A. pillulans), carboxymethyl cellulose sodium salt, C2, C4, C6, C8, C10, or phenyl acetate was individually added to the reaction solutions. Biochemical Characterization of Xylanase The optimal pH for the xylanase activity of purified rXynAGN16 was determined at 37 °C in buffers with pH 4.0–12.0. The enzyme stability at different pH values was estimated by measuring the residual enzyme activity after incubating the enzyme solution in various buffers at 37 °C for 1 h. The buffers used were McIlvaine buffer for pH 4.0–8.0 and 0.1 M glycine– NaOH for pH 9.0–10.0. The temperature-dependent activity of rXynAGN16 was determined over the range of 0– 70 °C in McIlvaine buffer (pH 6.5). The thermostability of rXynAGN16 was determined after pre-incubation of the enzyme for 60 min in McIlvaine buffer (pH 6.5) at 37, 50, or 60 °C without substrate for various periods. To investigate the effects of different metal ions and chemical reagents on the rXynAGN16 activity measured in McIlvaine buffer (pH 6.5) at 37 °C, 10.0 mM (final concentration) NaCl, KCl, CaCl2, CoCl2, NiSO4, FeSO4, CuSO4, MgSO4, ZnSO4, Pb(CH3COO)2, HgCl2, FeCl3, EDTA, β-mercaptoethanol, or SDS was individually added to the reaction solution. The values of Michaelis constant (Km), maximum rate of reaction (Vmax), and turnover number (kcat) for rXynAGN16 were determined using beechwood xylan or birchwood xylan as the substrate. The reaction contained 0.1 mL of diluted enzyme (appropriately 0.2 nmol) and 0.9 mL of McIlvaine buffer (pH 6.5) containing 0.5–10.0 mg/mL xylan as the substrate. The mixture was incubated in a water bath at 45 °C for 5 min. The rate of reaction was calculated using the value of absorption at 540 nm. Double-reciprocal plots were constructed according to the Lineweaver–Burk method [31]. Hydrolysis Products Analysis The hydrolysis of 0.5 % (w/v) birchwood xylan was carried out with rXynAGN16 (1.0 U/mL reaction system) for 24 h at 45 °C and pH 6.5. The hydrolysis products were analyzed by thinlayer chromatography (TLC) performed as previously described [32]. Birchwood xylan with the inactivated rXynAGN16 (90 °C for 5 min) was used as a control. Construction and Characterization of the Truncated Derivatives To study the effect of domains on the enzyme performance, four truncated derivatives of XynAGN16 were constructed, including XynAGN16L, XynAGN16Lpd, XynAGN16R, and XynAGN16Rhyr (GH 10 domain at C-terminus and HYR domain) (Fig. 1). The expressions,
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purifications, biochemical characterizations, and hydrolysis products analyses of the recombinant derivatives were performed as that of rXynAGN16. Biochemical characterizations and hydrolysis products analyses of the recombinant XynAGN16L (rXynAGN16L) and recombinant XynAGN16Lpd (rXynAGN16Lpd) were performed at pH 5.5 unless otherwise noted. Nucleotide Sequence Accession Numbers The nucleotide sequences for the Arthrobacter sp. GN16 16S rDNA and endoxylanase gene (xynAGN16) were deposited in GenBank under the accession numbers JQ863104 and JQ863105, respectively.
Results Strain Identification The comparison of the 16S rDNA sequence from GN16 (1368 bp; JQ863104) with that in GenBank showed nucleotide identities of 99.6 % with Arthrobacter citreus DSM 20133 (accession no. NR_026188) and of 99.5 % with Arthrobacter agilis TP-Snow-C61 (KC986991). Thus, the strain GN16 was classified into the genus Arthrobacter. Genome Sequencing and Sequence Analyses Genome sequencing generated appropriately 550 Mbp of sequence data. After sequence assembly, these data yielded appropriately 3.4 Mbp. Local BLAST analysis revealed the gene xynAGN16 (JQ863105) was homologous to the xylanase-encoding gene from A. phenanthrenivorans Sphe3 (ADX74784). The full-length xynAGN16 (3,639 bp) starts with the putative codon ATG, ends with TGA, and encodes a 1,212-residues polypeptide with a calculated mass of 131.5 kDa. The signal peptide was predicted from M1 to A25, followed by a catalytic domain of GH 10 from T41 to L368, a PD domain from A404 to L532, a CBM_4_9 domain from V610 to V741, a catalytic domain of GH 10 from G764 to A1117, and a HYR domain from D1133 to V1203 (Fig. 1). A xylanase showing similar multidomain structure has not been recorded in GenBank. BLASTp analysis showed that: XynAGN16L shared the highest identities of 71.6 % with the predicted xylanase from A. phenanthrenivorans Sphe3 (ADX74784) and of 66.2 % with the experimentally verified GH 10 xylanase from Lechevalieria sp. HJ3 (AFE82289); the PD domain of XynAGN16 shared the highest identities of 70.1 % with the predicted xylanase/chitin deacetylase from Nocardioidaceae bacterium Broad-1 (EGD41782) and of 33.1 % with the identified chitooligosaccharide deacetylase or nodulation protein B from Rhizobium galegae HAMBI 1174 (P50354); the CBM_4_9 domain of XynAGN16 shared the highest identities of 70.2 % with the predicted CBM_4_9 domain from Clavibacter michiganensis subsp. michiganensis NCPPB 382 (CAN01726) and of 50.4 % with the identified CBM_4_9 domain from Paenibacillus terrae HPL-003 (ADX97440); XynAGN16R shared the highest identities of 73.3 % with the predicted xylanase from C. michiganensis NCPPB 382 (CAN01726) and of 38.1 % with the identified GH 10 xylanase from P. terrae HPL-003 (ADX97440); and the HYR domain of XynAGN16 shared the highest identity of 62.5 % with the predicted HYR domain from C. michiganensis NCPPB 382 (CAN01726).
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Multiple Sequence Alignment and Structure Analysis Compared with the thermophilic endoxylanases TmXyn, TxXyn, and BsXyn, the multiple sequence alignment revealed increased amino acid residues from A57 to Y77 of XynAGN16L, including three increased glycines G58–G60 (Fig. 2). The tertiary structure of amino acid residues from A57 to Y77 of XynAGN16L consists of β-sheets and a loop, while the corresponding structures of TmXyn, TxXyn, and BsXyn are α-helixes (Fig. 2). Compared with TmXyn, TxXyn, and BsXyn, XynAGN16L has less salt bridges and more glycines. However, other parameter values were similar or showed irregularity, including the numbers of arginine, proline, and hydrogen bonds, and the total ASA, exposed nonpolar ASA, and total packing volume (Table 1).
Fig. 2 Homology model structures and partial multiple sequence alignment of XynAGN16L and thermophilic endoxylanases. Homology models and sequence names are shown with accession numbers except XynAGN16L, as follows: TmXyn from Thermotoga maritima MSB8 (AAD35164) [23], TxXyn from Thermobacillus xylanilyticus D3 (CAA76420) [24], and BsXyn from Bacillus stearothermophilus T-6 (ABI49951) [25]. The circles indicate the structure built by amino acid residues from A57 to Y77 of XynAGN16L and its corresponding structures of TmXyn, TxXyn, and BsXyn. The putative catalytic E177 and E285 of XynAGN16L are detailed in ball-and-stick form
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Expression, Purification, and Identification of rXynAGN16 and its Truncated Derivatives XynAGN16, XynAGN16L, and XynAGN16Lpd were expressed in E. coli BL21 (DE3) (Fig. 3). However, XynAGN16R and XynAGN16Rhyr were neither detected on SDS-PAGE (Fig. 3) nor released reducing sugars using xylans as substrates. Then, further studies on XynAGN16R and XynAGN16Rhyr were not performed. rXynAGN16 could not be purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography, ammonium sulfate precipitation, ion exchange chromatography, and molecular exclusion chromatography. A band showing appropriately 130 kDa was detected from the cell lysate of an induced transformant harboring pET-xynAGN16 (Fig. 3). The calculated value of rXynAGN16 is 135.0 kDa, which is approximately equal to the molecular mass of the band. The cell lysate of E. coli harboring the empty pET-28a(+) was neither showed the band nor xylanase activity (Fig. 3). Consequently, the crude rXynAGN16, not the purified rXynAGN16, was used for further studies. rXynAGN16L and rXynAGN16Lpd were purified to electrophoretic homogeneity by Ni 2+ -NTA metal chelating affinity chromatography (Fig. 3). The purified rXynAGN16L and rXynAGN16Lpd migrated as two single bands on SDS-PAGE with molecular masses of appropriately 48 and 69 kDa, respectively, which is close to the calculated values (rXynAGN16L, 46.9 kDa; rXynAGN16Lpd, 68.7 kDa). After tryptic digest, the two bands of rXynAGN16L and rXynAGN16Lpd were individually analyzed using the MALDI-TOF MS. Results revealed that the MALDI-TOF MS spectrums matched the molecular masses of the known internal peptides of rXynAGN16L
Fig. 3 SDS-PAGE analysis of XynAGN16 and its truncated derivatives. Lanes M1, M2, and M3 protein molecular weight markers SM1851, SM0671, and SM0431 from Generay Biotech, respectively (Shanghai, China), lane CK cell extract from an induced transformant harboring the empty plasmid pET-28a(+), lane Xyn cell extract from an induced transformant harboring the positive transformant pET-xynAGN16, lanes L and Lpd rXynAGN16L and rXynAGN16Lpd purified by Ni2+-NTA chelating affinity chromatography, respectively, lanes R and Rhyr cell extracts from induced transformants harboring the positive transformants pET-xynAGN16R and pET-xynAGN16Rhyr, respectively. Arrow indicates rXynAGN16
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and rXynAGN16Lpd (Fig. S1), confirming that the purified enzymes were rXynAGN16L and rXynAGN16Lpd. Substrate Specificity The activities of the crude rXynAGN16 towards substrates of 0.5 % (w/v) beechwood xylan, birchwood xylan, and oat-spelt xylan were detected. Determined at pH 5.5 and 45 °C, the activities of the purified rXynAGN16L towards substrates of beechwood xylan, birchwood xylan, and oat-spelt xylan were 1234.1 ± 83.1, 1081.8 ± 20.9, and 1242.4±69.7 U/μmol, respectively; the activities of the purified rXynAGN16Lpd towards beechwood xylan, birchwood xylan, and oat-spelt xylan were 1266.2 ± 47.0, 1177.8±65.5, and 819.0±85.5 U/μmol, respectively. Determined at pH 8.0 and 37 °C, the activities of the purified rXynAGN16L towards phenyl acetate, C4, and C6 were 21.1±0.5, 14.8±1.8, and 4.7±1.2 U/μmol, respectively; the activities of the purified rXynAGN16Lpd towards phenyl acetate, C4, and C6 were 26.6±0.4, 19.2±3.1, and 3.1 ±1.6 U/μmol, respectively. However, the activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were not detected towards substrates of 0.5 % (w/v) barley β-glucan, pullulan, and carboxymethyl cellulose sodium salt. No activity of rXynAGN16L or rXynAGN16Lpd was detected towards C2, C8, or C10. Enzyme Characterization When assayed at 37 °C, the crude rXynAGN16 showed apparently optimal xylanase activity at pH 6.5 and retained 29.2 % of the maximum activity at pH 5.5, while the purified rXynAGN16L and rXynAGN16Lpd showed apparently optimal xylanase activities at pH 5.5 (Fig. 4a). rXynAGN16, rXynAGN16L, and rXynAGN16Lpd showed close relative activities at pH 8.0–12.0. After incubation in buffers ranging from pH 7.0–12.0 at 37 °C for 1 h, the three xylanases exhibited more than 30 % of their initial activities (Fig. 4b). The activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were all apparently optimal at 45 °C. When assayed at 0 °C, the three xylanases still remained appropriately 15 % of their maximum activities (Fig. 4c). The three xylanases are thermolabile. After preincubation for 60 min at 37 °C, rXynAGN16, rXynAGN16L, and rXynAGN16Lpd exhibited 52.4, 71.2, and 70.9 % of their initial activities. The enzymes stabilities decreased rapidly at temperatures of more than 50 °C (Fig. 4d). The activities of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were completely inhibited by 10.0 mM Hg2+ or SDS. The addition of Zn2+ and EDTA slightly inhibited (retaining appropriately 65 % activities) rXynAGN16 and rXynAGN16Lpd, respectively. In the presence of 10.0 mM Fe2+, the purified rXynAGN16L and rXynAGN16Lpd remained appropriately 75 % of activities. The activities of the purified rXynAGN16L and rXynAGN16Lpd were enhanced by appropriately 0.5and 0.7-fold by 10.0 mM β-mercaptoethanol, respectively. Other reagents had little or no effect on the three xylanases. Because rXynAGN16 was not purified to electrophoretic homogeneity, kinetic experiment of the enzyme was not performed. The Km, Vmax, and kcat values of the purified rXynAGN16L and rXynAGN16Lpd towards xylans were determined in McIlvaine buffer (pH 5.5) at 45 °C, and were shown in Fig. 4. The Km and kcat/Km values of the purified rXynAGN16L towards birchwood xylan were 2.6 mg/mL and 19.5 mL/mg/s, respectively, while the two values of the purified rXynAGN16Lpd were 1.2 mg/mL and 42.7 mL/mg/s, respectively (Fig. 4e). Towards beechwood xylan,
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Fig. 4 Characterizations of the crude rXynAGN16 and the purified rXynAGN16L and rXynAGN16Lpd. a Effects of pH on the three xylanases determined at 37 °C from pH 4.0 to 12.0. b pH stabilities assays. After preincubation of the enzymes at pH 5.0–12.0 and 37 °C for 60 min, the enzymes activities were determined at 37 °C. c Thermoactivities of the three xylanases. d Thermostabilities assays. The enzymes were pre-incubated at 37, 50, or 60 °C, and aliquots were removed at specific time points for the measurements of residual activities at 37 °C. e Lineweaver–Burk plots and kinetic values of the purified rXynAGN16L and rXynAGN16Lpd determined using 0.5–10 mg/mL birchwood xylan as the substrate at 45 °C. f Lineweaver–Burk plots and kinetic values of the purified rXynAGN16L and rXynAGN16Lpd determined using 0.5–10 mg/mL beechwood xylan as the substrate at 45 °C. Characterizations of the crude rXynAGN16 were performed at pH 6.5, while characterizations of the purified rXynAGN16L and rXynAGN16Lpd were performed at pH 5.5 unless otherwise noted. The error bars represent the means±SD (n=3)
the Km and kcat/Km values of the purified rXynAGN16L were 1.8 mg/mL and 27.1 mL/mg/s, respectively, while the two values of the purified rXynAGN16Lpd were 1.0 mg/mL and 35.3 mL/mg/s, respectively (Fig. 4f). The results implied that rXynAGN16Lpd has a higher affinity and catalytic efficiency towards xylans than rXynAGN16L: the affinities towards birchwood xylan and beechwood xylan were enhanced by appropriately 0.5- and 0.4-fold, respectively; the catalytic efficiencies
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towards birchwood xylan and beechwood xylan were enhanced by appropriately 1.2and 0.3-fold, respectively. Hydrolysis Products The hydrolysis products of 0.5 % (w/v) birchwood xylan by rXynAGN16, rXynAGN16L, and rXynAGN16Lpd were analyzed by TLC (Fig. 5). Xylose and some xylo-oligosaccharides, including potential xylobiose and methylglucuronoxylotetraose, were released from beechwood xylan by the three xylanases, while xylotriose, xylotetraose, methylglucuronoxylobiose, and methylglucuronoxylotriose were not clearly detected. The hydrolysis analysis indicated the endo-acting nature of rXynAGN16, rXynAGN16L, and rXynAGN16Lpd.
Discussion The occurrence of additional domains to the catalytic domain in an enzyme might be the result of mutation and domain shuffling during the course of evolution, which in turn has yielded a multiplicity of enzymes [33]. The common additional domains in many modular endoxylanases are CBM domains [4–9]. To the best of the authors’ knowledge, only three
Fig. 5 Thin layer chromatography (TLC) analysis of the hydrolysis of 0.5 % (w/v) birchwood xylan. Lanes CKXyn, CK-L, and CK-Lpd birchwood xylan with the inactivated (90 °C for 5 min) rXynAGN16, rXynAGN16L, and rXynAGN16Lpd, respectively, lane S-Xyn birchwood xylan hydrolyzed by the crude rXynAGN16 (1.0 U/ml reaction system) for 24 h at 45 °C and pH 6.5, lanes S-L and S-Lpd birchwood xylan hydrolyzed by the purified rXynAGN16L and rXynAGN16Lpd, respectively (1.0 U/ml reaction system, 45 °C, pH 5.5, 24 h), lane M1 xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose marker, lane M2 methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose marker
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multidomain xylanases have the PD domains (CAA54145, CAA90745, and ADG73550), only three multidomain xylanases have the HYR domains (CAN01726, CCE75600, and ABS02556), and no xylanase has both the PD and HYR domains. Thus, xynAGN16 encodes a xylanase with a previously undescribed combination of domains. The non-catalytic domains contribute to the function of multidomain enzymes in cooperation with catalytic domains [4–9]. The functions of CBM domains in many modular endoxylanases have been studied [4–9]. Polysaccharide deacetylases, members of carbohydrate esterase family 4, include chitin deacetylases, acetyl-xylan esterases, rhizobial NodB chitooligosaccharide deacetylases, and peptidoglycan deacetylases [3]. However, the effect of PD domain on multidomain xylanases has not been reported. In this study, the kinetic values of rXynAGN16L and rXynAGN16Lpd reveal that the PD domain improves the affinity and catalytic efficiency towards xylans of the xylanase rXynAGN16Lpd. High catalytic efficiency is one of the major factors for widespread applications of enzymes [34]. Over 80 % of the Earth’s biosphere is permanently cold. The largest proportion of biomass on the Earth is generated at low temperatures [35, 36]. Low-temperature-active enzymes show high catalytic activity at low temperatures and thermolability at intermediate and high temperatures [36]. The two low-temperature-active single-domain xylanases XynA19 and XynGR40 were isolated from the gastrointestinal tracts of Batocera horsfieldi larvae [37] and Boer goat [14], respectively. Many Arthrobacter strains can produce low-temperature-active enzymes, such as the glycerol dehydrogenase from Arthrobacter sp. MS-7 [38] and the chitobiase from Arthrobacter sp. TAD20 [39], where as Arthrobacter sp. MTCC 5214 produces a thermophilic xylanase [40]. rXynAGN16L showed catalytic activity at 0 °C and thermolability at temperatures of more than 50 °C. This report is the first to present the cloning, heterologous expression, and characterization of a low-temperature-active xylanase from an Arthrobacter strain isolated from the feces of G. Nigricollis. Compared with thermophilic counterparts, low-temperature-active enzymes generally have a decreased number of proline and/or arginine residues, and/or disulfide and/ or hydrogen bonds, and/or salt bridges, and an increased number of glycine residues, and/or a larger total accessible surface area and/or exposed hydrophobic accessible surface area, and/or longer surface loops [36]. Compared with TmXyn, TxXyn, and BsXyn, the surface loop from A57 to Y77 of rXynAGN16L might increase the amplitude of the movement between secondary structures; the decreased salt bridges might reduce the stabilization energy of rXynAGN16L. The two factors in combination might lead to the reduced stability and enhanced flexibility of the structure of rXynAGN16L and consequently have a key role in maintaining the catalytic activity of rXynAGN16L at low temperatures. In conclusion, this study identified and characterized a novel xylanase XynAGN16 consisting of a previously undescribed combination of domains. The GH 10 catalytic domain at N-terminus of XynAGN16 plays a key role in catalysis towards xylans. The PD domain of XynAGN16 improves the affinity and catalytic efficiency towards xylans of the truncated xylanase rXynAGN16Lpd. The structural adaptation to low temperatures of the truncated xylanase rXynAGN16L might be ascribed to the surface loop from A57 to Y77 and the decreased salt bridges. Furthermore, this study is the first to present the cloning, heterologous expression, and characterization of a lowtemperature-active xylanase from an Arthrobacter strain isolated from the feces of G. nigricollis.
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Acknowledgments This work was supported by the Key Technologies Research and Development Program of China (2013BAD10B01), National Natural Science Foundation of China (31260215), and Applied and Basic Research Foundation of Yunnan Province (2011FB048). Thanks for the kind gifts (methylglucuronoxylose, methylglucuronoxylobiose, methylglucuronoxylotriose, and methylglucuronoxylotetraose) from Prof. James F. Preston from University of Florida.
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