purification, characterization and gene cloning of two forms of a thermostable endo-xylanase from...
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Process Biochemistry 46 (2011) 2255–2262
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Process Biochemistry
jo u rn al hom epage: www.elsev ier .com/ locate /procbio
urification, characterization and gene cloning of two forms of a thermostablendo-xylanase from Streptomyces sp. SWU10
arin Deesukona, Yuichi Nishimurab, Naoki Haradab, Tatsuji Sakamotob,∗,asana Sukhumsiricharta,∗∗
Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, ThailandDivision of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 599-8531, Japan
r t i c l e i n f o
rticle history:eceived 25 June 2011eceived in revised form 8 September 2011ccepted 9 September 2011vailable online 16 September 2011
eywords:treptomyces sp., Endo-xylanases,haracterization, Gene cloning, Glycosylydrolase family 10
a b s t r a c t
Two forms of an endo-xylanase were isolated from the culture filtrate of Streptomyces sp. SWU10 thatgrew on rice straw. The molecular masses of both forms of an enzyme were 31 kDa (XynSW2A) and 44 kDa(XynSW2B). Analysis of internal amino acid sequences of the proteins by liquid chromatography/ion-trap/time-of flight mass spectrometer (LC/IT/TOF MS) revealed that XynSW2A may be the proteolyticfragment of XynSW2B. Optimal temperature and pH of XynSW2A and XynSW2B were 60 ◦C and 6.0,respectively. Both forms of the enzyme were stable in a wide pH ranges. More than 80% of the initialactivities remained at pH 3–9 (XynSW2A) and 2–9 (XynSW2B) after 16 h of incubation at 4 ◦C. XynSW2Aand XynSW2B were stable up to 80 ◦C and 60 ◦C, respectively. Both forms of the enzyme were stronglyinhibited by Hg2+ ions. Birch wood xylan, which has no arabinofuranosyl side chains, was the mostpreferred substrate for both forms. The xynSW2 gene encoding XynSW2B was isolated by in vitro cloning.
The coding sequence of xynSW2 gene was 1434 bp in length and encode a polypeptide of 477 amino acidresidues. Pfam analysis revealed Glyco hydro 10 and Ricin � lectin domains in XynSW2B. The deducedamino acid sequence of XynSW2B exhibited the highest identity with that of a xylanase A of Streptomycescoelicolor A3(2) belong to glycoside hydrolase (GH) family 10. Because of their pH and thermal stabilities,XynSW2A and XynSW2B may have potential application in biofuel industry by using rice straw and canbe applied in food, textile industries, and waste treatment.. Introduction
Cellulose and hemicelluloses are the major components of plantell walls including rice straw, hull, and bran. Rice straw predomi-antly contains cellulose (32–47%), hemicelluloses (19–27%) and
ignin (5–24%) [1–3]. It is ecologically essential to convert low-ost biomass to value-added products such as ethanol or industrialhemicals by biotechnological strategies. Xylan is one of the majoromponents of hemicelluloses and comprises up to 39% of thery weight of terrestrial plant. It is a heterogeneous polysaccha-ide with linear backbone composed of �-1,4-d-xylopyranosideesidues to which �-l-arabinofuranose units are attached at the
and/or 3 position as side-chains [4].
Bioethanol production from pentoses has been established bysing genetically modified yeasts which are constructed by intro-uction of initial metabolism and sugar transport, and changing
∗ Corresponding author. Tel.: +81 722549456; fax: +81 722549921.∗∗ Corresponding author. Tel.: +66 26495366; fax: +66 26495834.
E-mail addresses: [email protected] (T. Sakamoto),[email protected] (W. Sukhumsirichart).
359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.09.004
© 2011 Elsevier Ltd. All rights reserved.
the intracellular redox balance, although further optimization isneeded for their industrial use [5,6]. Xylooligosaccharides could beused in food, feed, pharmaceutical, and agriculture fields. For foodapplications, xylobiose has been used as a food ingredient to mod-ulate the intestinal function since xylooligosaccharides could beselectively used by the beneficial gastrointestinal microflora andsuppress the growth of pathogenic bacteria [7]. Therefore, effec-tive degradation of xylans in biomass is an important process fortheir utilization.
Among the various xylosidic enzymes, endo �-1,4-xylanases(EC 3.2.1.8) are crucial for depolymerization of the main xylaninto short xylooligosaccharides [8]. Two key reactions proceed dur-ing hydrolysis of xylan; endo-�-xylanases cleave internal linkagesin xylan backbone to yield oligosaccharides, which are furtherhydrolyzed by �-xylosidases (EC 3.2.1.37) to xylose. Xylanases havebeen used for enzymatic production of xylooligosaccharides andhave potential applications in food, animal feed, textile, wastetreatment, paper, and biofuel industries [9]. Many fungi, bacte-
ria, and actinomycetes secrete multiple isoenzymes of xylanase asdistinct gene products. Based on amino acid sequence similarities,xylanases are mainly classified into GH family 10 and 11 [10–12].The two families have different molecular structures, molecular
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eights, and catalytic properties. Family 10 generally consists ofigher molecular weight proteins (>30 kDa) with a (�/�)8 barreltructure, whereas family 11 consists of lower molecular weightroteins (20–30 kDa) with a �-jelly-roll structure. Streptomycesas been recognized as the dominant xylanolytic species of acti-omyces that produce enzymes involved in the degradation ofemicelluloses, which have important industrial applications [4].
In this study, two forms of a xylanase were isolated from Strep-omyces sp. selected from ruined rice straw. The enzymes wereurified and characterized for their stability and activity in degrada-ion of rice straw, the substrate for ethanol production. In addition,he gene was cloned, sequenced, and analyzed.
. Materials and methods
.1. Materials and chemicals
Mono Q HR 5/5, Resource PHE 6 ml, and Superdex 75 HR 10/30 were pur-hased from GE Healthcare UK Ltd. (Buckinghamshire, UK). DEAE-Toyopearl 650 Mas obtained from Tosoh Corp. (Tokyo, Japan). Wheat arabinoxylan (low viscos-
ty) was obtained from Megazyme International Ireland Ltd. (Wicklow, Ireland). Oatpelt xylan and birchwood xylan were from Sigma–Aldrich Co. (St. Louis, MO, USA).ll other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan)nless otherwise stated, and were of certified reagent grade.
.2. Microorganism screening and identification
To isolate microorganisms, soil and rotten rice straw collected from Lamphun,he northern province, of Thailand were suspended in sterilized water, then spreadn agar plates of Rice Straw medium composed of 1.0% ground rice straw, 0.2%aNO3, 0.1% K2HPO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.001% FeSO4, pH 6.0, and
ncubated at 37 ◦C for 1 week. The isolated organisms were cultivated in test tubes15 mm ID) containing 5 ml of Rice Straw liquid medium, at 37 ◦C for 3 days in aeciprocating shaker (120 strokes/min). The culture filtrates, which had been dia-yzed against 20 mM potassium phosphate buffer (KPB), pH 6.0, were analyzed fornzyme activities using oat spelt xylan as the substrate.
The microorganism that produced highest amount of xylanase was selectednd identified by analysis of 16S ribosomal DNA sequence. Briefly, the 16S rDNAas amplified from the genomic DNA using 16S DNA specific primers, 27F (5′-GAGTTTGATCCTGGCTCAG-3′) and 1492R (5′ -GGCTACCTTGTTACGACTT-3′). TheCR product was directly sequenced and the data was analyzed for its similarityy using BLAST program and phylogenetic tree was constructed.
For phylogenetic analysis, several of 16S rDNA sequences retrieved from theenBank database were multi-aligned using Clustal X program. Aligned sequencesere analyzed using Molecular Evolutionary Genetics Analysis Software Version 4.0
MEGA 4) [13] for phylogenetic inference. Phylogenetic tree was constructed usinghe reference strains by Neighbor-Joining Method [14] based on Maximum Com-osite Likelihood (MCL) model and bootstrap analysis of 10,000 replications waserformed. The selected microorganism was identified and named as Streptomycesp. SWU10.
.3. Enzyme assay
Standard assay for xylanase activity was performed by measuring the release ofeducing groups in a reaction mixture containing 190 �l of 0.1% oat spelt xylan in0 mM KPB, pH 6.0, and 10 �l of enzyme sample at 60 ◦C for 10 min. In the screeningxperiment, xylanase activity was assayed at 37 ◦C. Reducing sugars released in theeaction mixture were measured by Somogyi–Nelson method using xylose as thetandard [15]. All activity measurements were performed at least three times andata were expressed as mean ± SD. One unit of xylanase activity was defined as themount of enzyme that forms reducing groups corresponding to 1 �mol of xylosen 1 min under the above conditions.
.4. Purification of xylanases
For large scale production of xylanases, 10 ml of pre-cultured medium of thetreptomyces sp. SWU10 (approximately 450 mg biomass by wet weight measure)as transferred to a 3-l shaking flask containing 1 l of Rice Straw liquid medium and
ultured at 37 ◦C for 3 days in a rotary shaker at 200 rpm. The culture supernatant (2 l)as concentrated by ultrafiltration (10-kDa cutoff), dialyzed against 20 mM KPB, pH
.0, and applied onto a DEAE-Toyopearl column (3 cm × 25 cm) equilibrated with theialysis buffer. The bound proteins were eluted by a linear gradient of NaCl (from
to 0.5 M) in the same buffer. Xylanase activities were found in both the unboundnd bound fractions, termed XynSW2A and XynSW2B, respectively. Both enzymesere further purified separately.
mistry 46 (2011) 2255–2262
2.4.1. Purification of XynSW2AThe fraction that did not bind to DEAE-Toyopearl column was adjusted to a 30%
saturation ammonium sulfate, and loaded onto a 6 ml Resource PHE column equi-librated with 20 mM KPB, pH 6.0, containing ammonium sulfate at a concentrationof 30% saturation. The adsorbed proteins were eluted by a linear gradient of ammo-nium sulfate (80 ml, from 30 to 0% of saturation). The XynSW2A-containing fractionswere pooled, concentrated using a 10-kDa cut off filter (Nanosep 10 K Omega) (PallLife Sciences, Ann Arbor, MI, USA), and loaded onto a Superdex 75 HR 10/30 columnequilibrated with 100 mM NaCl in 20 mM KPB, pH 6.0. Proteins were eluted with thesame buffer.
2.4.2. Purification of XynSW2BThe XynSW2B fraction from the DEAE-Toyopearl column was dialyzed against
20 mM KPB, pH 6.0, and loaded onto a Mono Q HR 5/5 column equilibrated with thedialysis buffer. The bound proteins were eluted by a linear gradient of NaCl (35 ml,from 0 to 0.3 M). The active fractions were collected, adjusted to 10% saturationammonium sulfate, and loaded onto a 6 ml Resource PHE column equilibrated with20 mM KPB, pH 6.0, containing ammonium sulfate at a concentration of 10% satu-ration. Elution was performed by a linear gradient of ammonium sulfate (100 ml,from 10 to 0% of saturation).
The protein concentration in each fraction was determined by measuring anabsorbance at 280 nm. Purified xylanases were stored at 4 ◦C for biochemical char-acterization.
2.5. Analysis of proteins by electrophoresis and LC/IT/TOF MS
The molecular masses of the purified proteins were determined by sodium dode-cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [16]. Protein bands in thegel were visualized by Coomassie Brilliant Blue staining.
Internal amino acid sequences of purified XynSW2A and XynSW2B wereanalyzed using LC/IT/TOF MS after trypsin digestion according to the methodof Sakamoto et al. [17]. Protein identification was performed by compar-ing the collected LC/MS/MS data with the NCBI sequence databank. Databanksearches were performed using the Mascot server 2.3 program (Matrix Science;http://www.matrixscience.com). The Mascot search parameters were set as fol-lows: type of search (MS/MS ion search), taxonomy (bacteria), enzyme (trypsin),fixed modifications [carbamidomethyl (C)], variable modifications [oxidation (M)],fragment mass tolerance (±0.6 Da).
2.6. Characterization of xylanases
The optimum pH was determined by measuring the activity at 60 ◦C for 10 minover the pH range 1–12 using 20 mM sodium acetate/HCl (pH 1–5), 20 mM KPB(pH 6–8), 20 mM sodium carbonate/bicarbonate (pH 9–10), and 20 mM sodiumphosphate/NaOH (pH 11–12). The pH stability was studied by pre-incubation ofthe enzyme at 4 ◦C for 16 h at various pHs in 100 mM the above buffers described.The remaining activity was calculated taken the activity of the enzyme solutionkept in 20 mM KPB, pH 6.0, at 4 ◦C for 16 h as 100%. The optimum temperature wasdetermined by assaying the enzyme activity in 20 mM KPB, pH 6.0, at different tem-peratures (30–70 ◦C) for 10 min. The thermal stability was evaluated by measuringthe residual activity after 1 h pre-incubation of the enzyme at temperatures between30 ◦C and 95 ◦C in 20 mM KPB, pH 6.0. The remaining enzyme activity was calculatedtaken the activity of enzyme solution kept in 20 mM KPB, pH 6.0, at 4 ◦C for 1 h as100%.
Substrate specificity was studied using oat spelt xylan, birch wood xylan, andwheat arabinoxylan. Each substrate was dissolved in 20 mM KPB, pH 6.0, at a con-centration of 0.46%. The enzyme activity was assayed as described above.
The sensitivity of the enzyme to metals and EDTA was examined by assayingxylanase activity under standard conditions in the presence of each compound at aconcentration of 1 mM.
2.7. Analysis of enzymatic products and sugar contents in arabinoxylans byhigh-performance anion-exchange chromatography (HPAEC)
XynSW2A or XynSW2B (10 mU) was incubated with 0.2 ml of 0.1% oat speltxylan in 20 mM KPB, pH 6.0, at 60 ◦C for 16 h. After incubation, the mixture wasboiled for 5 min to inactivate the enzyme. The products were analyzed by HPAECusing a Dionex DXc-500 system with a CarboPac PA-1 column (4 mm × 250 mm;Dionex, CA, USA). Sugars were eluted at a flow rate of 1 ml/min with 0.1 M NaOHfor 5 min followed by a linear gradient from 0 M to 0.45 M sodium acetate in 0.1 MNaOH for 30 min. The effluent was monitored with pulsed amperometric detection.Xylose, xylobiose, and xylotriose were used as standards.
The arabinose and xylose contents in various arabinoxylans were determined
by HPAEC after hydrolysis of the soluble fractions of 1% polysaccharides with 1 Msulfuric acid at 100 ◦C for 2 h. HPAEC was performed using a Carbopac PA-1 column(2 mm × 250 mm) at the flow rate of 0.25 ml/min with 16 mM NaOH for 30 min. Theeffluent was monitored with pulsed amperometric detection. Experiments wereperformed in triplicate.
W. Deesukon et al. / Process Biochemistry 46 (2011) 2255–2262 2257
Fig. 1. Phylogenetic analysis of 16S rDNA sequences of Streptomyces sp. SWU10 and related species. The phylogenetic tree was constructed using the neighbor-joining methodw
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.8. Cloning and sequencing of the xynSW2 gene encoding XynSW2B
The partial fragment of the xynSW2 gene from Streptomyces sp. SWU10as amplified using genomic DNA and degenerate primers (Sxyn10F: 5′-
GGGACGTSGTSAACGAG-3′ and Sxyn10R: 5′- GGATGTCSAGYTCSGTGA-3′) whichesigned on the basis of highly conserved amino acids among GH family 10 xylanasesrom Streptomyces species [18]. The full-length gene of xynSW2 was then ampli-ed using degenerate primers (SxynFde: 5′- ATGGGYTYNYAYGCCCTYCBCRGA-3′
nd SxynRde: 5′- TCAGGYGCGGGWCCASCGYTGGYTG-3′) which designed based onhe alignment result between the partial fragment of the xynSW2 gene and otherylanase genes from Streptomyces including Streptomyces fradiae (GenBank acces-ion no. EF429086.1), Streptomyces lividans (GenBank accession no. M64551.1),treptomyces sp. S27 (GenBank accession no. EU660498.1), S. coelicolor A3(2) (NCBIccession no. NP 733679), and Streptomyces sviceus ATCC 29083 (NCBI accessiono. YP 002208602). The amplified product was purified, ligated into pGEM-T Easyector, and sequenced.
.9. Sequence analysis
The similarity analysis of the nucleotide and deduced amino acid
equence of xynSW2 gene were carried out using the NCBI (http://blast.ncbi.lm.nih.gov/Blast.cgi) and the UniProtKB (http://www.uniprot.org/blast/uniprot)last programs [19]. Multiple alignments of protein sequences were accomplishedsing ClustalW program (http://www.ebi.ac.uk/clustalW). The signal peptidesn the deduced amino acid sequence of the xynSW2 gene were predicted using
SignalP (http://www.cbs.dtu.dk/Sercices/SignalP) [20]. Module sequence analysis ofproteins was performed using the Pfam database (http://pfam.sanger.ac.uk/search).
3. Results and discussion
3.1. Selection of microorganisms that produce xylanases
Microorganisms from soil and rotten rice straw were screenedfor species that produce xylanases. Seventy-five microorganismsisolated on agar plates of Rice Straw medium were cultured inRice Straw liquid medium at 37 ◦C for 3 days and analyzed forxylanase activity in the culture filtrates. Among these microorgan-isms tested, the SWU10 strain isolated from rice straw showed thehighest xylanase activity. Thus, the SWU10 strain was selected asa potent producer of xylanases.
In a BLAST search, the 16S ribosomal DNA sequence of theSWU10 strain (GenBank accession no. AB604027) exhibited thehighest identity (99%) to the sequence of Streptomyces sp. A310
Ydz-XM (GenBank accession. no. EU368776). Based on this result,the strain SWU10 was identified as Streptomyces species and it wasnamed as Streptomyces sp. SWU10. Several xylanases from Strepto-myces sp. have been purified and characterized such as Streptomyces
2258 W. Deesukon et al. / Process Biochemistry 46 (2011) 2255–2262
Table 1Purification of XynSW2A and XynSW2B produced by Streptomyces sp. SWU10.
Procedure Activity (U) Protein (mg) Specific activity (U/mg) Yield (%) Purification (fold)
XynSW2AUltrafiltration 1300 3200 0.41 100 –DEAE-Toyopearl 180 73 2.5 14 6Resource PHE 56 1.7 33 4 80Superdex 75 7.4 0.2 37 1 90
XynSW2BUltrafiltration 1300 3200 0.41 100 –DEAE-Toyopearl 410 110 3.7 32 9Mono Q 160 28 5.7 12 14
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arabinose was found in birch wood xylan. Among the substratestested, both XynSW2A and XynSW2B showed the highest activ-ity on birch wood xylan, indicating that the enzymes preferredarabinose-free regions in �-1,4-xylans. Moreover, comparison of
Resource PHE 24 0.4
nzyme assay was performed using oat spelt xylan as the substrate at 60 ◦C.
yaneus SN32 [21], Streptomyces thermotrificans [22] and S. lividans23].
Phylogenetic tree analysis revealed the strain Streptomyces sp.WU10 fall with the radiation of the genus Streptomyces (Fig. 1). Thelosest relative within the Streptomyces cluster was Streptomycesp. A310 Ydz-XM (GenBank accession no. EU368776). Thermoacti-omyces dichotomicus (GenBank accession no. AF138733) was useds an outgroup.
.2. Purification of the xylanases
Xylanases were purified from 2 l of the culture filtrate of Strep-omyces sp. SWU10. In the first step of purification of the enzymes,wo xylanase activities were separated by anion-exchange chro-
atography using a DEAE-Toyopearl column. The activity that didot bind to this column was termed XynSW2A. The other xylanasectivity that eluted at around 0.1 M of NaCl on a DEAE-Toyopearlolumn was named XynSW2B. The purification procedure for theylnases is summarized in Table 1. XynSW2A and XynSW2B wereurified 90- and 146-fold from the culture filtrate, respectively. Theesulting specific activity of the purified XynSW2A and XynSW2Bere 37 and 60 U mg−1, with activity yield of 1% and 2%, respec-
ively. The enzymes were highly purified as judged by SDS-PAGEFig. 2).
.3. Characterization of xylanases
SDS-PAGE analysis of the enzymes showed a single protein bandith a molecular mass of 31 kDa for XynSW2A and 44 kDa forynSW2B, respectively (Fig. 2). Internal amino acid sequences ofynSW2A and XynSW2B were analyzed using LC/IT/TOF MS. SixynSW2A and seven XynSW2B peptides were obtained (Fig. 3And B). A Mascot search found that both of XynSW2A and XynSW2Bit with the xynSW2 gene which encodes an endo-�-1,4-xylanase
rom Streptomyces sp. SWU10 (GenBank accession no. AB601446)ith a Probability Based Mowse Score of 526 for XynSW2A and
77 for XynSW2B. From these results, XynSW2A seems to be theroteolytic product of XynSW2B.
The effect of pH on xylanase activity was determined by usingat spelt xylan as the substrate. Enzyme activities at 60 ◦C wereighest at around pH 6.0 for both enzymes (Fig. 4A). More than 80%f the initial XynSW2A activity remained after 16 h of incubationt pH from 3 to 9 at 4 ◦C. On the other hand, XynSW2B was stablet pH from 2 to 9 (Fig. 4B). Both forms of a xylanase exhibited wideanges of pH stability. The optimum temperature of XynSW2A andynSW2B was 60 ◦C at pH 6.0 (Fig. 5A). After incubation of both
orms of enzyme at 80 ◦C and 60◦ for 1 h, more than 80% of the
nitial activity remained. XynSW2A still retained its activity until0 ◦C and more than 50% activity remained (Fig. 5B). Comparinghe properties of xylanases from Streptomyces spp. (Table 2), it wasound that the XynSW2A exhibited the highest thermal stability.60 2 146
XynSW2B and recombinant xylanase, XynAS9 from Streptomycessp. S9 showed similar thermal stability [18]. The recombinantXynAS27 of Streptomyces sp. S27 showed high pH stability over thepH range 2.2–12.0 when incubated at 37 ◦C only for 1 h [24,25].
We examined the sensitivity of the enzymes to 13 metal ions(Ag+, Ba2+, Cd2+, Co2+, Cu2+, Fe2+, Fe3+, Hg2+, K+, Mn2+, Na+, Ni2+, andZn2+) and metal chelator (EDTA). It was found that both enzymeswere strongly inhibited by Hg2+ (Table 3). Inactivation of xylanasesby Hg2+ has been reported by other studies [26–28]. No effect onactivity was detected with the other metals and EDTA.
3.4. Mode of action of XynSW2A and XynSW2B
The specificity of both forms of enzyme towards xylans preparedfrom different sources is summarized in Table 4. Both enzymesdegraded oat spelt xylan, birch wood xylan and wheat arabinoxy-lan with different specificities. Analysis of sugar composition ofthe soluble fractions of these xylans revealed that they possesseddifferent degrees of arabinofuranose residues as side-chains. No
Fig. 2. SDS-PAGE analysis of the purified XynSW2A and XynSW2B from Strepto-myces sp. SWU10. M, protein marker.
W. Deesukon et al. / Process Biochemistry 46 (2011) 2255–2262 2259
Fig. 3. Analysis of internal amino acid sequences of XynSW2A (A) and XynSW2B (B) by LC/IT/TOF MS. The peptides matched with the deduced amino acid sequence of theendo-�-1,4-xylanase from Streptomyces sp. SWU10 are shown in bold.
Fig. 4. Effect of pH on enzyme activity (A) and stability (B) of XynSW2A and XynSW2B. The experimental conditions are described in the text.
Fig. 5. Effect of temperature on enzyme activity (A) and stability (B) of XynSW2A and XynSW2B. The experimental conditions are described in the text.
2260 W. Deesukon et al. / Process Biochemistry 46 (2011) 2255–2262
Table 2Some properties of xylanases from Streptomyces sp. SWU10 and other Streptomyces species.
Source (enzyme name) Mr pH Temperature (◦C) Inhibitor Gene Reference
Optimum Stability Optimum Stability
Streptomyces sp.SWU10 (XynSW2A) 31 6.0 3.0–9.0 (4 ◦C, 16 h) 60 80 (80%)b Hg2+ – Present workSWU10 (XynSW2B) 44 6.0 2.0–9.0 (4 ◦C, 16 h) 60 60 (80%)b Hg2+ 1434 Present workS9 (XynAS9)a 46 6.5 4.0–12.0 (37 ◦C, 1 h) 60 60 (80%)b Hg2+, Ag+ 1395 [18]S27 (XynAS27)a 47 6.5 2.2–12.0 (37 ◦C, 1 h) 60 65 (25%)b Hg2+, SDS 1434 [24,25]Ab106 ND 6.0 ND 60 60 (70%)b ND ND [29]K37 26.4 6.0 ND 60 ND ND ND [30]S. cyaneus SN32 20.5 6.0 4.0–9.5 (37 ◦C, 1 h) 60–65 65 (half-lives 50 min) ND ND [21]S. thermonitrificans (STXF10)a 26 6.0 4.0–9.0 (50 ◦C, 30 min) 50 70 (50%)b ND ND [22]S. fradiae var. k11 (SfXyn10)a 47 7.8 4.0–10.0 (37 ◦C, 1 h) 60 60 (40%)b Hg2+, SDS 1437 [31]S. olivaceoviridis A1 (XYNB)a 20.8 5.2 4.0–8.8 (37 ◦C, 1 h) 60 60 (51%)b K+, Ca2+, Zn2+ Cu2+, EDTA 576 [32]S. matensis DW67 21.2 7.0 4.5–8.0 (55 ◦C, 30 min) 65 ND Hg2+ ND [33]SU9 ND 9.0 6.0–9.0 (50 ◦C, 30 min) 80 60 (65%)b ND ND [34]7b 30 6.0 5.0–9.0 50 60 (73%)b ND ND [35]
ND means no data.a Recombinant enzyme.b Remaining activity.
Table 3Influence of metals and EDTA on enzyme activity of xylanases from Streptomyces sp.SWU10.
Compound Relative activity (%)
XynSW2A XynSW2B
None 100 100AgNO3 90 ± 2 104 ± 2BaCl2 94 ± 6 77 ± 3CdCl2 94 ± 4 86 ± 3CoCl2 93 ± 2 91 ± 2CuCl2 88 ± 1 102 ± 8FeSO4 75 ± 6 83 ± 4FeCl3 65 ± 1 75 ± 4HgCl2 4 ± 0 6 ± 1KCl 95 ± 5 89 ± 3MnCl2 93 ± 4 83 ± 10NaCl 82 ± 4 90 ± 4NiCl2 84 ± 0 101 ± 3ZnCl2 93 ± 4 90 ± 4EDTA 95 ± 5 93 ± 3
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the signal peptide, the calculated molecular mass of XynSW2Bwas 47.131 Da that somewhat larger than the apparent molec-ular mass in SDS-PAGE analysis (44 kDa; Fig. 2). The internalamino acid sequences of XynSW2B estimated by LC/IT/TOF MS was
Xylose
Xylooligosacc harides
X 1
X 2 X 3
elative activities were calculated taken the activities in the absence of metals as00%. The experimental conditions are described in the text. Enzyme assays wereerformed in triplicate and data were expressed as mean ± SD.
he specificities of the enzymes revealed that XynSW2A was moreensitive to arabinofuranosylation in the substrates.
To determine whether the enzymes are endo- or exo-actingnzymes, the products formed were analyzed by HPAEC usingat spelt xylan as the substrate. Generally, in the case of exo-cting enzymes, only monomer or dimer accumulates throughout
he enzyme reaction. In the present study, xylose and sev-ral xylo-oligosaccharides were produced by the action of bothorms of enzyme (Fig. 6), demonstrating that XynSW2A andynSW2B are endo-type xylanases. The data also demonstrate thatable 4ugar content of arabinoxylans and substrate specificities of xylanases from Strep-omyces sp.SWU10 towards them.
Substrate Sugar content (mol%) Relative activity (%)
Arabinose Xylose XynSW2A XynSW2B
Oat spelt xylan 11 ± 0 89 ± 2 72 ± 0 94 ± 6Birch wood xylan 0 ± 0 100 ± 0 100 100Wheat arabinoxylan 35 ± 1 65 ± 2 46 ± 2 80 ± 3
oluble fractions of the substrates were used in enzyme assay. Concentration of theubstrates were 0.46%. Enzyme activity was measured by Somogyi–Nelson method.etailed of the methods used for the enzyme assay are described in the text. Enzymessays were performed in triplicate and data were expressed as mean ± SD.
xylooligosaccharides were considerably resistant to degradationwith XynSW2B. Because less amounts of xylose were detectedwhen compared to XynSW2A. Similar hydrolysis products havebeen reported from many xylanases, which have been used asactive ingredients of functional foods [7].
3.5. Cloning and sequence analysis of the xynSW2 gene encodingXynSW2B
The coding sequence of xynSW2 gene (GenBank accession no.AB601446) was 1434 bp in length and encodes a polypeptide of477 amino acid residues. SignalP predicts that XynSW2B has aN-terminal signal peptide of 41 amino acids. After cleavage of
0 10 20 30
Xylan hydrolyzate of Xyn SW2A
Xylan hydrolyzate of XynSW2 B
Retenti on time (mi n)
Fig. 6. Analysis of the enzymatic products of oat spelt xylan obtained with XynSW2Aand XynSW2B. Authentic samples X1–X3 represent xylose to xylotriose, respec-tively. The experimental conditions are described in the text.
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ound in the deduced amino acid sequence as shown in Fig. 3B,hich demonstrated that the cloned gene encoded XynSW2B.
C/IT/TOF MS analysis of XynSW2A and XynSW2B indicated thatynSW2B might be cleaved at Lys–Lys at positions 330 and31 to produce XynSW2A (Fig. 3). The polypeptide consisting ofesidues 42–330 has a calculated molecular mass of 31.908 Da,hich was in good agreement with that of the native XynSW2A
31 kDa).Pfam analysis revealed Glyco hydro 10 (PF00331) and
icin � lectin (PF00652) domains at positions 45–341 and52–474 of XynSW2B, respectively. Ricin � lectin domains con-ain three homologous subdomains which bind to carbohydrates.t has been found in various kinds of glycosidases includingndo-1,3-�-glucanase [36], galactosidase [36], and endo-xylanaserom Streptomyces sp. S27 [24], S. lividans [37], and Streptomyceslivaceoviridis E-86 [38]. The domain has been reported to serveiverse functions such as enzymatic activity, inhibitory toxicity,nd signal transduction [24]. From the results of LC/IT/TOF MS,he Ricin � lectin domain seem to absent in XynSW2A. Usinghe deduced amino acid sequence of XynSW2B as a query, theest hits in a UniProtKB blast search were a xylanase A (Q8CJQ1:2% identity) of S. coelicolor A3(2) and a xylanase A (D6EN39:2% identity) of S. lividans TK24 which belong to GH family 10.herefore, both of XynSW2A and XynSW2B are classified into GHamily 10.
. Conclusions
The new strain of Streptomyces sp. isolated from rice straw pro-uced two forms of an endo-1,4-�-xylanase namely XynSW2A andynSW2B, respectively. Temperature and pH optima of XynSW2And XynSW2B were 60 ◦C and 6.0, respectively. Both forms ofnzyme are stable in wide range of pH (pH 3–9 and pH 2–9)nd high temperature (80 ◦C and 60 ◦C). They were inhibited byg2+ ions. The most preferred substrate was xylan which has norabinofuranosyl side-chains such as birch wood xylan. The genencoding XynSW2B contain 1434 nucleotides which correspond to77 amino acids. XynSW2A seem to be the proteolytic degradationroduct of XynSW2B. The absent part of the polypeptide may playole in thermal stability of XynSW2A. XynSW2A and XynSW2B arelassified as GH family 10 based on the amino acid sequence sim-larities. Because of the pH and thermal stabilities, XynSW2A andynSW2B may have potential application in biofuel industry bysing rice straw. Moreover, both forms of a xylanase can be applied
n food, textile industries and waste treatment. We are presentlyttempting to overexpress the xynSW2 gene in yeast systems.
cknowledgments
This work was supported by grants from The Royal Goldenubilee Ph.D Program of Thailand Research Fund (PHD/0257/2549)nd Srinakharinwirot University (315/2551). The authors thank Dr.lfredo Villarroel for English proof.
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