Journal of Bioscience and BioengineeringVOL. 109 No. 2, 115–117, 2010

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Characterization of Aspergillus oryzae glycoside hydrolase family 43 β-xylosidaseexpressed in Escherichia coli

Satoshi Suzuki,1 Mari Fukuoka,1 Hikaru Ookuchi,1 Motoaki Sano,2 Kenji Ozeki,2 Emi Nagayoshi,3 Yukio Takii,3

Mayumi Matsushita,1 Sawaki Tada,1 Ken-Ichi Kusumoto,1 and Yutaka Kashiwagi1,⁎

⁎ CorrespondE-mail add

1389-1723/$doi:10.1016/

National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan1 Genome Biotechnology Laboratory, Kanazawa Institute ofTechnology, Yatsukaho 3-1, Hakusan, Ishikawa 924-0838, Japan2 and Department of Food Science and Nutrition, School of Human Environmental Science,

Mukogawa Women's University, Nishinomiya, Hyogo 663-8558, Japan3

Received 15 January 2009; accepted 30 July 2009Available online 12 September 2009

This is the first report of glycoside hydrolase family 43 β-xylosidase from Aspergillus oryzae. To characterize this enzyme,the recombinant enzyme was expressed in Escherichia coli. Unlike known β-xylosidases from fungal origins, the enzyme didnot show substrate ambiguity and was stable at alkaline pH.

© 2009, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Aspergillus oryzae; β-Xylosidase; Glycoside hydrolase family 43; Recombinant enzyme; Wheat bran]

The effective degradation of xylan is a key process for theutilization of hemicellulose biomass. Aspergillus oryzae is a safemicro-organism that secretes many kinds of enzymes, which couldbe used in the degradation of refractory biomass such as hemi-celluloses from agricultural wastes. Wheat bran contains a highproportion of xylan; therefore, it was expected that A. oryzae wouldexpress many xylanolytic enzymes in wheat bran culture. Severalstudies have reported that A. oryzae produces several xylanolyticenzymes, some of which have been purified and characterized (1–4).Kitamoto et al. reported that the xylA gene encodes β-xylosidase in A.oryzae (3). The amino acid sequence of XylA exhibited a significantsimilarity to A. nidulans XlnD (70%), A. niger XlnD (64%) and Tricho-derma reesei BxlI (63%). These fungal β-xylosidases belong toglycoside hydrolase (GH) family 3 (3). Additionally, Hashimoto etal. have reported that A. oryzae has three extracellular and two cellwall-binding type β-xylosidases (4).

This investigation aims to identify the enzymes that are induced inwheat bran cultures of A. oryzae and to study their usefulness in thedigestion and utilization of agricultural wastes.

In this study,we found a gene encoding a novelβ-xylosidase,whichis a member of GH family 43, among the several xylanolytic genesinduced inwheat branmedium. Althoughmostmembers of GH family43have a bacterial origin, 139 kinds of eukaryoticDNA sequenceswereregistered to database (http://www.cazy.org). Many species offilamentous fungi possess DNA sequences that encode putative GHfamily 43 β-xylosidases (Fig. 1). The eukaryotic β-xylosidases of GHfamily 43 have not yet been studied, except for β-xylosidase from

ing author. Tel.: +81 0 29 838 8015; fax: +81 0 298 38 7996.ress: ykswg@affrc.go.jp (Y. Kashiwagi).

- see front matter © 2009, The Society for Biotechnology, Japan. Allj.jbiosc.2009.07.018

Penicillium herquei (5) and Cochliobolus carbonum (6). In order tocharacterize this novel β-xylosidase of A. oryzae, the recombinantenzyme was expressed in Escherichia coli.

A. oryzae RIB40, E. coli DH5α and E. coli Rosetta-gami™ (DE3)pLysS (Novagen, San Diego, CA, USA) were used in this study. Weperformed amicroarray analysis to compare gene expression patternsof wheat bran and wheat flour (unpublished data). The full details ofthe microarray analysis will be published elsewhere; in this study, wefocus on xylanolytic genes based on preliminary data. From the genesthat are induced in wheat bran, we chose a gene (A. oryzae genomedatabase; database number AO090005000698, http://www.bio.nite.go.jp/dogan/ProteinInformation?ORF_ID=AO090005000698) that en-codes a xylosidase-like protein. We named the gene xylB as the secondβ-xylosidase gene of A. oryzae. In A. oryzae genome database, there are10 candidates for xylosidase genes; however, both xylA and xylB werenot included in the 10 candidates. 6 of 10 candidates possessedpredicted signal peptide at their amino termini.

The gene is located on chromosome 1. There is no paralogue geneor similar sequence in the genome of A. oryzae. The coding region ofxylB consists of 999 bp and contains no introns. The coding sequencecomprises 332 amino acids. There are no potential secretion signalpeptides in the whole amino acid sequence. One potential N-glycosylation site was found at N-243. The deduced amino acidsequence showed 72% identity with P. herquei cell wall xylosidase S2,which is a member of GH family 43 (5). The molecular mass and pI,calculated from the amino acid sequence, are 37.4 kDa and 4.81,respectively. In the 5′ non-coding region of xylB, a potential CCAATsequence was found at −620 and two CreA consensus binding sites(7) were found at −167 and −180. The XlnR consensus binding siteGGCTAAA sequence (8) was found at −125. These findings suggest

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FIG. 1. Phylogenetic tree of fungal GH family 43 with known family 3 fungal xylosidase XylA as the outgroup. Phylogenetic analyses were conducted in MEGA4 (12). An asterisk (⁎)marks the enzymes with confirmed activity and numbers in brackets indicate the DDBJ/EMBL/GenBank accession numbers.

FIG. 2. The enzymatic properties of purified xylosidase. (A), Effect of temperature onenzyme stability; (B), effect of pH on enzyme stability; (C), effect of temperature onactivity; (D), effect of pH on activity. The enzyme activity on p-nitrophenyl-β-D-xylopyranoside was determined by 10 min reaction.

116 SUZUKI ET AL. J. BIOSCI. BIOENG.,

that xylB is under the control of XlnR induction and the glucoserepression system.

To confirm the expression of xylB in wheat bran culture, real-timeRT-PCR was performed. mRNAs were purified from total RNAextracted from the mycelia of RIB40 cultured in wheat bran contain-ing Czapek-Dox (CD) medium (3% wheat bran, 0.1% K2HPO4, 0.05%KCl, 0.05% MgSO4, 0.001% FeSO4·7H2O) or flour containing CDmedium (3% flour instead of wheat bran) by Oligotex™-dT30bSuperNmRNA Purification Kit (Takara Bio, Shiga) according to the manufac-turer's instructions. Reverse transcription and real-time PCR wereperformed with a SuperScript III Platinum Two-Step qRT-PCR Kit withSYBR Green® (Invitrogen, Carlsbad, CA, USA). Thermal cycling wasperformed with an ABI 7500 sequence detection system (PE AppliedBiosystems, Tokyo). The RT-PCR thermal cycling conditions consistedof an initial step at 50 °C for 2 min, followed by 95 °C for 10 min. Thenext stage involved 45 cycles at 95 °C for 15 s and 60 °C for 1 min.Relative quantification was performed using the 2−ΔΔCt technique(ABI User Bulletin 2). Each sample was analyzed three times. Thestatistical analysis of the real-time RT-PCR data indicated that xylBwas induced more in the wheat bran culture than in the flour culture(data not shown).

The vector for expression of the recombinant enzyme in E. coliwasconstructed as follows: The coding region of the gene was amplifiedby PCR from the genomic DNA of RIB40 with the primer set(698infusion5 [5′-GACGACAAGGCCATGATGTCCAATTCCAAC-CCCCCTCTAATAACC-3′] and 698infusion3 [5′-TCCGATATCGCCATGT-TAGTCCACCAAATAAATGTCCCCCTGATC-3′]). The PCR product wascloned into the NcoI/XhoI site of pET32b (Novagen) by In-FusionPCR Cloning Kit (Takara Bio) according to themanufacturer's protocol.The host cell E. coli Rosetta-gami™ (DE3)pLysS (Novagen) wastransformed with resulting expression vector named pET698. Theenzyme was expressed with N-terminal thioredoxin tag, His-tag andS-tag. The E. coli cells harboring pET698 were induced with 1 mMisopropyl β-D-thiogalactopyranoside at the mid-log phase for 7 h at30 °C. The cells were harvested by centrifugation and suspended inHis-tag binding buffer (20 mM phosphate buffer at pH 7.4, 0.5 M NaCland 40 mM imidazole). The E. coli cells were disrupted by sonicationand the lysate was centrifuged at 23,000 ×g for 20 min. Therecombinant enzyme in the crude extract was purified using a HisTrap FF crude column (GE Healthcare Bio-sciences KK, Tokyo), Ni-IMAC Profinity (Bio-Rad, Hercules, CA) and Thrombin Purification Kit(Novagen). The protein was detected as a single band with SDS-polyacrylamide gel electrophoresis after staining with Coomassiebrilliant blue R-250.

The xylosidase activity of the purified enzymewasmeasured usingp-nitrophenyl-β-D-xylopyranoside (Nacalai Tesque, Kyoto) as asubstrate. A sample of 100 μl of reaction mixture containing 0.5 mMof the substrate, 100 mM citric acid – 200 mM phosphate buffer or

100 mM glycine–NaOH buffer (pH 7.0 or the given pH for optimal pHdetermination) and an appropriate amount of enzyme was incubatedat 30 °C for 10 min. To stop the reaction, 100 μl of 0.2 M NaOH wasadded, and the p-nitrophenol released was measured at 415 nm. Oneunit of enzyme activity was defined as the amount of enzyme thatliberated 1 μmol of p-nitrophenol per min under the above conditions.The kinetic parameters of the purified enzymewere determined for p-nitrophenyl-β-D-xylopyranoside at concentrations ranging from 0.05to 25 mM. The initial velocities were determined and used to estimateKm and Vmax from a Lineweaver–Burk plot.

The specific activity of the purified enzyme was 6.1 U/mg protein.The crude cell extract of E. coli which possesses vector pET32(b)indicated no significant activity of xylosidase. The enzymaticproperties of purified xylosidase are shown in Fig. 2. The thermaland pH stabilities were determined by incubation of the enzyme atvarious temperatures (4–70 °C) for 1 h and at various pH values(3.0–10.0) for 24 h at 4 °C, respectively. The enzyme was stable at4 °C, inactivated gradually up to 30 °C and inactivated drasticallyabove 40 °C. The enzyme retained over 60% activity in the pH rangeof 7.0–9.0. The optimal temperature for enzyme activity determined by10 min reaction was approximately 30 °C. Themaximum activity of the

FIG. 3. TLC analysis for hydrolysis products of natural substrate by XylB. St, standards;X1, xylose; X2, xylobiose; X3, xylotriose. Bx, birchwood xylan; Ox, oat spelt xylan. Thereaction mixture consisting of 1.0% (w/v) solution of a substrate and enzyme wasincubated 1 h or 12 h in optimal condition.

NOTE 117VOL. 109, 2010

enzyme was observed at pH 7.0. The calculated Km and Vmax values ofthe enzyme were found to be 0.48 mM and 42.6 μmol min−1 mg−1 ofprotein. The Km value of the enzyme was significantly lower than thefive xylosidases described by Hashimoto et al. (4). The substratespecificity of the enzyme was investigated as follows. The enzymeactivity toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-α-D-galactopyranoside (Sigma-Aldrich Japan, Tokyo), p-nitrophenyl-α-L-arabinofuranoside (Sigma), p-nitrophenyl-α-L-arabinopyranoside(Sigma), p-nitrophenyl-β-L-arabinopyranoside (Sigma), p-nitrophenyl-β-D-galactopyranoside (Sigma), p-nitrophenyl-β-D-glucopyranoside(Sigma) and p-nitrophenyl-β-D-mannopyranoside (Sigma) was deter-mined following the method of Margolles-Clark et al. (9). Similar to theP. herquei xylosidase S2, XylB did not act on all of the substrates tested,except for p-nitrophenyl-β-D-xylopyranoside. Thin-layer chromatogra-phy (TLC) was used tomonitor the degradation of the xylobiose (Wako,Osaka), xylotriose (Wako), birchwood xylan (Sigma) and oat spelt xylan(Sigma). The reaction mixture consisting of a 1.0% (w/v) solution ofsubstrate and enzymewas incubated in optimum assay conditions. Thereaction was stopped by incubating at 100 °C for 10 min, and 2 μlportions of each reaction mixture were analyzed for TLC on silica gelplates (Merck, Darmstadt, Germany). The TLC plates were developedand stained as described by Wakiyama et al. (10). TLC analysis showedthat the purified enzyme hydrolyzed xylobiose and xylotriose (Fig. 3).Though we could not detect the activity of XylB to either xylan by TLCanalysis (Fig. 3), we confirmed that concentrated XylB could liberatexylose from birchwood xylan but not xylo-oligosaccharides (data notshown).

In this study, we expressed and purified GH family 43 β-xylosidasefrom A. oryzae in E. coli. This is the first report of β-xylosidasebelonging to this GH family from the genus Aspergillus. P. herqueixylosidase S2 (5) and xylosidase Xyp1 from the plant pathogen C.carbonum (6) also belong to this family. Interestingly, although P.herquei xylosidase S2 binds tightly to the cell wall while C. carbonumxylosidase Xyp1 is secreted, both proteins have no predicted signalpeptides. Known β-xylosidases from fungal origins such as A. niger

and T. reesei show substrate ambiguity (9), whereas the enzyme inthis study only acted on p-nitrophenyl-β-D-xylopyranoside, xylobioseand xylotriose in the several substrates tested. The enzymaticproperties of XylB were similar to P. herquei xylosidase S2. Themaximum activities of both enzymes were observed around neutralpH. Both enzymes were stable at weak alkaline pH. On the other hand,other xylosidases of A. oryzae were acidophilic (3, 4). Though thereare several reports of alkaline endo-xylanase which is industriallyimportant enzyme in pulp bleaching (11), there is no report of thefungal alkaline xylosidase except of XylB and P. herquei xylosidase S2.Besides, molecular weights of other xylosidases of A. oryzae werehigher than XylB (3, 4). These results suggested that XylB may bedifferent from all known β-xylosidases of A. oryzae. We hope toinvestigate the physiological significance and application of thisenzyme in future studies.

ACKNOWLEDGMENT

This work was supported by a grant from the Iijima KinenFoundation.

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