Novel GH10 Xylanase, with a Fibronectin Type 3 Domain, fromCellulosimicrobium sp. Strain HY-13, a Bacterium in
the Gut of Eisenia fetida�
Do Young Kim,1 Mi Kyoung Han,1 Doo-Sang Park,2 Jong Suk Lee,3 Hyun-Woo Oh,1 Dong-Ha Shin,4Tae-Sook Jeong,1 Sung Uk Kim,1 Kyung Sook Bae,2 Kwang-Hee Son,1* and Ho-Yong Park1*
Industrial Bio-Materials Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806,South Korea1; Biological Resources Center, KRIBB, Daejeon 305-806, South Korea2; Industrial Biotechnology and
Bioenergy Research Center, KRIBB, Daejeon 305-806, South Korea3; and Insect Biotech Co. Ltd.,Daejeon 305-811, South Korea4
Received 11 May 2009/Accepted 12 September 2009
The gene encoding a novel modular xylanase from Cellulosimicrobium sp. strain HY-13 was identified andexpressed in Escherichia coli, and its truncated gene product was characterized. The enzyme consisted of threedistinct functional domains, an N-terminal catalytic GH10 domain, a fibronectin type 3 domain, and C-terminal carbohydrate-binding module 2.
Most known microbial xylanases, which decompose primar-ily �-1,4-xylosic polysaccharides in an endo fashion, are cur-rently affiliated with the two glycoside hydrolase (GH) families10 and 11. Compared to GH11 xylanases, GH10 xylanasesgenerally have a molecular mass of �30 kDa and an acidic pI.In addition, GH10 xylanases are frequently found in nature asmodular enzymes that consist of a catalytic GH10 domain withone or more substrate-binding domains, such as a cellulose-binding domain, carbohydrate-binding module (CBM), or xy-lan-binding domain (1, 5, 12). However, no modular xylanasewith a fibronectin type 3 (Fn3) domain has been characterizedto date, even though Fn3 modules are often found in bacterialcarbohydrolases such as cellulases, amylases, pullulanases,polygalacturonidases, and chitinases. It is now believed that theFn3 domains in bacterial carbohydrolases participate in pro-motion of the hydrolysis of carbohydrate substrates by modi-fying their surfaces (10, 17).
Gut microorganisms from invertebrates have recently at-tracted a great deal of attention as sources of novel fibrolyticenzymes with unique molecular structures and distinct sub-strate specificities (2–4, 7, 15). However, no study has beenconducted to evaluate xylanolytic enzymes from the gut micro-organisms of earthworms that may participate in the digestionof cellulosic or hemicellulosic foods taken up by the hosts.Here, we report a novel GH10 xylanase (XylK1) with an Fn3domain from Cellulosimicrobium sp. strain HY-13 KCTC11302BP (11), which was isolated from the digestive tract ofthe earthworm Eisenia fetida.
Amplification of a partial sequence of the Cellulosimicro-bium sp. strain HY-13 xylanase gene from the genomic DNAwas conducted using the degenerate primers designed on thebasis of conserved regions (WDVVNE and ITELDI) in the
GH10 xylanases. The upstream primer (KF) was 5�-TGGGACGTCSTCAACGAG-3�, and the downstream primer (KR)was 5�-GATGTCGAGCTCSGTGAT-3�, which produced a342-bp DNA fragment. Cloning of the full xylK1 gene wasperformed by repeated genomic walking and nested-PCRmethods using a DNA Walking SpeedUp premix kit (See-gene). To overproduce mature XylK1, its encoding gene wascloned into the NdeI/HindIII sites of a pET-28a(�) vector(Novagen). Likewise, a partial sequence containing the GH10domain (Ala34 to Leu345) of XylK1 was amplified using theprimers tKF (5�-CATATGGCCACCGAGCCGCTCG-3�) andtKR (5�-AAGCTTTCAGGACCTCGGCGATCGC-3�) andsubsequently also cloned into the same expression vector.When overexpressed in recombinant Escherichia coli BL21cells harboring pET-28a(�)/xylK1, most recombinant proteins(rXylK1) were produced as inactive inclusion bodies. There-fore, after solubilization of the isolated inclusion bodies, on-column refolding and purification of rXylK1 was conductedusing a HisTrap HP (GE Healthcare, Sweden) (5-ml) columnattached to a fast-performance liquid chromatography (LC)system (Amersham Pharmacia Biotech, Sweden) according tothe manufacturer’s instructions. The active rXylK1 proteinswere then purified to electrophoretic homogeneity by gel per-meation chromatography using a HiLoad 26/60 Superdex 200prep-grade (Amersham Biosciences, Sweden) column, as pre-viously described (11). Like rXylK1, the recombinant proteins(rXylK1�Fn3) without both an Fn3 domain and CBM 2 werealso purified by the method described above because they wereproduced as insoluble inclusion bodies. The relative molecularmass of the denatured rXylK1 was evaluated by sodium dode-cyl sulfate-polyacrylamide gel electrophoresis on a 12% gel,and the protein concentrations were assayed by using the Brad-ford reagent (Bio-Rad). Matrix-assisted laser desorption ion-ization–time-of-flight mass spectrometry (MALDI-TOF MS)analysis was conducted using an Ultraflex III MALDI-TOFmass spectrometer (Bruker Daltonics, Germany) at the KoreaBasic Science Institute (Daejeon, South Korea). The bindingcapacity of recombinant enzymes with/without the Fn3 domain
* Corresponding author. Mailing address: Industrial Bio-MaterialsResearch Center, KRIBB, Daejeon 305-806, South Korea. Phone:82-42-8604650. Fax: 82-42-8604659. E-mail for Ho-Yong Park: [email protected]. E-mail for Kwang-Hee Son: [email protected].
to carbohydrate polymers was determined as described else-where (4). Xylanase activity was routinely assayed by measur-ing the amount of reducing sugars released from birch woodxylan by using the 3,5-dinitrosalicylic acid reagent. The stan-dard assay mixture (0.5 ml) consisted of birch wood xylan(1.0%) or p-nitrophenyl (PNP)–sugar derivatives (5 mM) withsuitably diluted enzyme solution (0.05 ml) in 50 mM sodiumphosphate buffer (pH 6.0), and the catalytic reaction was per-formed at 55°C for 10 min. One international unit of xylanaseactivity for xylans or PNP-sugar derivatives was defined as theamount of enzyme required to produce 1 �mol of reducingsugar or PNP, respectively, per min under standard assay con-ditions. Enzymatic hydrolysis of birch wood xylan (10 mg)(Sigma Co.), xylooligosaccharides (1 mg each) (Megazyme In-ternational Ireland, Ireland), and cellooligosaccharides (1 mgeach) (Seikagaku Biobusiness Co., Japan) was conducted usingpurified rXylK1 (2 �g) in 0.1 ml of 50 mM sodium phosphatebuffer (pH 6.0) for 3 or 6 h at 37°C, during which time theenzyme remained fairly stable. The reaction mixture was thenheated to 100°C for 5 min to stop the enzyme reaction. Thehydrolysis products were identified by LC-MS, as previouslydescribed (11).
The isolated XylK1 gene (GenBank accession no. FJ859907)contained a 1,671-bp open reading frame that encodes a pro-tein of 556 amino acids with a deduced molecular mass of58,296 Da and a calculated pI of 4.59. It was predicted that thesignal sequence cleavage site of premature XylK1 was betweenAla33 and Ala34, which may generate a mature XylK1 of 523amino acids with a deduced molecular mass of 54,843 Da anda calculated pI of 4.49 (Fig. 1). The results of a protein BLASTsurvey revealed that XylK1 was a unique modular xylanasecomposed of an N-terminal catalytic GH10 (Leu38-to-Asp330)domain, an Fn3 (Pro359-to-Gly430) domain, and a C-terminalCBM 2 (Cys454 to Cys553), which was very comparable to thedomain architectures of other related GH10 enzymes (Fig. 2).To the best of our knowledge, no xylanase with domain archi-tecture identical to that of XylK1 with an Fn3 domain has beenreported to date, although an uncharacterized modular xyla-nase (GenBank accession no. ABQ06877) consisting of anN-terminal Fn3 domain and a C-terminal GH10 domain fromFlavobacterium johnsoniae UW101 was previously identifiedthrough a genome survey. As shown in Fig. 1, the catalyticGH10 domain of XylK1 showed the highest sequence identity(67%) with that of the Cellulomonas fimi xylanase (AAA56792) among other GH10 enzymes available in the NCBIdatabase. However, its CBM 2 was 64% identical to that ofCellulomonas fimi GH6 cellulase (AAC36898). The highestsequence identity (70%) of the Fn3 domain in XylK1 wasobtained when it was compared to that of the Acidothermuscellulolyticus 11B GH48 enzyme (ABK52390), which degradescellulose. The two conserved residues of Glu161 (acid/basecatalyst) and Glu266 (catalytic nucleophile) were predicted inthe active site of premature XylK1.
The molecular mass of the purified enzyme was estimated tobe approximately 42.0 kDa, which was smaller than the de-duced molecular mass (57,138 Da) of intact His-taggedrXylK1, as determined by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (data not shown). In addition,MALDI-TOF MS analysis revealed that the purified His-tagged rXylK1 with a calculated molecular mass of 45,169 Da
was a smaller protein than the intact rXylK1. These resultsindicate that rXylK1 was formed by proteolytic cleavage at theC terminus region because the enzyme was able to tightly bindto a His tag column. Based on the calculated molecular mass(45,169 Da) of the truncated rXylK1, it is assumed that theintact rXylK1 was processed at the Val439-Thr440 site in ahinge region between the Fn3 domain and the C-terminalCBM 2 of the premature XylK1. The deduced molecular mass(45,179 Da) of rXylK1 with the Val439 residue at the C-terminal extremity was very close to the molecular mass(45,169 Da) of the enzyme calculated by MALDI-TOF MSanalysis. A similar C-terminal processing of some modularxylanases with a cellulose-binding domain by proteases has alsobeen observed when they are expressed in E. coli (8, 14). It islikely that the C-terminal truncation does not induce a signif-icant alteration of the binding affinity of rXylK1 with an Fn3domain to carbohydrate polymers since the truncated enzymecould still bind to both Avicel and insoluble oat spelt xylan(Table 1). In this case, only weak catalytic activity of therXylK1 (�3% of its original activity [0.5 IU]) was recoveredfrom an enzyme solution after binding. It was of great interestthat no rXylK1�Fn3 was bound to Avicel, although the en-zyme could still not only bind to insoluble oat spelt xylan butalso catalyze the hydrolysis of xylosic polymers. The specificactivity (27 IU/mg) of rXylK1�Fn3 for birch wood xylan wasevaluated to be approximately 19% of that (143 IU/mg) ofrXylK1 with the Fn3 domain for the same substrate. Takentogether, the binding ability of the C-terminal CBM 2-lackingrXylK1 to Avicel and insoluble oat spelt xylan clearly suggeststhat the Fn3 domain plays an important role in enzyme-sub-strate binding because rXylK1�Fn3 was not bound to Avicel.A significant decrease in the catalytic activity of rXylK1�Fn3induced by deletion of the Fn3 domain also suggests that theFn3 domain may take part in the promotion of the catalytichydrolysis of xylosic substrates by modifying their surfaces, asshown in other GHs (10, 17). The maximum catalytic activity ofrXylK1 toward birch wood xylan was observed at pH 6.0 and55°C, and it maintained over 80% of its highest activity at arelatively broad pH range of 5.0 to 9.0 during the reactionperiod of 15 min. These high activities of rXylK1 in alkaline pHranges suggest that it is a peculiar enzyme that can be distin-guished from xylanases of other invertebrate-symbiotic micro-organisms, which showed weak hydrolytic activities toward xy-lan at the same alkaline pHs (4, 11, 15). At 55°C, the half-lifeof rXylK1 was approximately 10 min, which indicates that it isa typical mesophilic enzyme. Compared to many other xyla-nases that were completely inhibited by Hg2� (11, 13), rXylK1was partially inhibited (by 40% relative to its original activity)by 1 mM Hg2�. In addition, the enzyme was relatively sup-pressed by �25%, relative to its original activity, in the pres-ence of some divalent cations at a concentration of 1 mM, inthe order of Ca2� � Cu2� � Ba2�. No significant alterationsof rXylK1 activity by Mn2� and Co2� were interesting to notebecause the xylanases from Streptomyces sp. strain S9 [12] andAeromonas caviae ME-1 [14] have been negatively affected bythe compound. In this study, the catalytic activity of rXylK1increased by approximately 1.4-fold when the reaction wasconducted in the presence of 1 mM Fe2�. The promotion ofrXylK1 activity by Fe2� was comparable to previous observa-tions of xylanase inhibition by the metal ion (6, 15). The
FIG. 1. Alignment of the deduced amino acid sequence of GH10 xylanase from Cellulosimicrobium sp. strain HY-13 with those of other GH10xylanases. Shown are sequences (GenBank accession numbers) of Cellulosimicrobium sp. strain HY-13 (Csp) xylanase (FJ859907), Cellulomonasfimi (Cfi) xylanase (AAA56792), Streptomyces coelicolor (Sco) A3 xylanase (CAB61191), Streptomyces ambofaciens (Sam) xylanase (CAJ88420),Acidothermus cellulolyticus 11B (Ace) xylanase (ABK51955), and Thermobifida alba (Tal) xylanase (CAB02654). The identical and similar aminoacids are shown by black and gray boxes, respectively. The predicted signal peptide is indicated by a black bar. The internal peptide sequences usedin the design of degenerate oligonucleotides for PCR are marked by arrows. Highly conserved amino acid residues that play an essential role inthe catalytic reaction are indicated by asterisks. GH10, Fn3, and CBM 2 domains are outlined by solid, dashed, and dotted lines, respectively.
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rXylK1 was relatively unaffected by sulfhydryl reagents (5 mM)such as sodium azide, iodoacetamide, and N-ethylmaleimide,while the enzyme lost 68% of its original activity when prein-cubated with 5 mM EDTA for 10 min. The complete inhibitionof rXylK1 by 5 mM N-bromosuccinimide was in good agree-ment with the fact that three Trp residues in the highly con-served region of the GH10 enzymes are critically involved inenzyme-substrate interaction, as shown for Streptomyces livi-dans (16) and Geobacillus stearothermophilus T-6 (18) GH10xylanases. It was predicted that the three residues Trp118,Trp306, and Trp314 in premature XylK1 might be responsiblefor catalysis and substrate binding of the enzyme. It is alsonoteworthy that the catalytic activity of His-tagged rXylK1increased significantly, by approximately 1.8-fold, when thereaction was conducted in the presence of Tween 80 or TritonX-100 at a concentration of 0.5%. It is assumed that the non-ionic-detergent-induced activation of His-tagged rXylK1 mightbe due to the direct interaction of the recombinant enzymewith the Tween 80 or Triton X-100 molecule, which may leadto an alteration of the enzyme-substrate interaction. Indeed,the stimulation of His-tagged rXylK1 activity was insignificantwhen the enzyme reaction was conducted in the presence ofthe detergents without preincubation with the same com-pounds for 10 min. As with rXylK1, it has been reported thatthe catalytic activity of a His-tagged esterase from Bacillusmegaterium 20-1 expressed in E. coli is greatly stimulated byvarious nonionic detergents (9).
Of the evaluated xylosic materials, oat spelt xylan was mostefficiently hydrolyzed by rXylK1; however, the enzyme was notcapable of degrading glucose-based polysaccharides, which isindicative of a lack of other GH activities (Table 2). It shouldalso be noted that the catalytic activity of rXylK1 toward PNP-cellobioside was approximately 1.7-fold higher than that (193IU/mg) of the enzyme toward oat spelt xylan. In this study, thecleavage activity of rXylK1 for PNP-cellobioside was approxi-mately 48 IU/mg, which is much higher than the activity (�10IU/mg) of other known xylanases for the same substrate (6,11). However, LC-MS analysis revealed that cellooligosaccha-rides of cellobiose to cellotetraose were not susceptible torXylK1 (data not shown). Taken together, these results indi-cate that rXylK1 is a true endo-�-1,4-xylanase that lacks cel-lulase activity. Interestingly, rXylK1 was found to have tran-sxylosylation activity (approximately 7.5% of its maximumhydrolytic activity for oat spelt xylan) that enabled the cleavageof PNP-xylopyranoside (Table 2). A similar transxylosylationreaction by rXylK1 was also observed when xylotriose (X3) andxylotetraose (X4) were subjected to hydrolytic reaction by theenzyme (Table 3). Specifically, a series of xylooligosaccharides(X4 to X7) were produced after the enzymatic hydrolysis of X3
for 3 h at 37°C, although X2 and X3 were identified as themajor products. Similarly, the hydrolysis of X4 by rXylK1 re-sulted in the production of a mixture that contained longerxylooligosaccharides (42.3%) of X5 to X8, which suggests thatthese xylooligomers were produced by an rXylK1-catalyzedtransxylosylation reaction. However, no X1 was detected as thehydrolysis product of X2, X3, or X4. The ability of rXylK1 tocatalyze the synthesis of longer xylooligosaccharides from X3
or X4 was of interest because microbial xylanases generallyproduced shorter xylooligosaccharides, such as X2 and/or X3,from the same substrates (2, 15). Additionally, rXylK1 primar-
FIG. 2. Domain architectures of Cellulosimicrobium sp. strainHY-13 xylanase and the following related bacterial GH10 xylanases(GenBank accession numbers): Cellulosimicrobium sp. strain HY-13xylanase (FJ859907) (A), Streptomyces thermocarboxydus HY-15 xyla-nase (EU880430) (B), Cellulomonas fimi xylanase (AAZ76373) (C),Streptomyces thermoviolaceus xylanase (BAD02382) (D), Pseudomonasfluorescens xylanase (P23030) (E), and Cellvibrio japonicus xylanase(YP 001982932) (F).
TABLE 1. Binding of rXylK1 and rXylK1�Fn3 tohydrophobic polysaccharides
ily degraded birch wood xylan to X2 (65.1%) and X3 (29.5%)together with small amounts of X4 (5.4%) when the enzymereaction was conducted for 6 h at 37°C.
In conclusion, the novel gene (xylK1) encoding a modularGH10 xylanase that consists of three putative functional domains(an N-terminal GH10 domain, an Fn3 domain, and C-terminalCBM 2) was identified from an earthworm-symbiotic bacterium,Cellulosimicrobium sp. strain HY-13. The molecular architectureof XylK1 indicates that it is a unique GH10 enzyme with an Fn3domain that has not previously been reported. In addition, therelatively high cleavage activity of rXylK1 toward PNP-cellobio-side and its transxylosylation activity that enables it to producelonger xylooligosaccharides from X3 or X4 differentiate it fromother known GH10 xylanases.
This work was supported by a grant from the KRIBB ResearchInitiative Program (KGS2330911) and the 21C Frontier MicrobialGenomics and Applications Center Program (MGM0900837) of theKorean Ministry of Education, Science, and Technology.
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