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Cloning and Sequencing of the xynC Gene Encoding Acid Xylanase of Aspergillus kawachii a

a

a

Kiyoshi Ito , Kazuhiro Iwashita & Kimio Iwano a

National Research Institute of Brewing, 2–6–30, Takinogawa, Kita-ku, Tokyo 114, Japan Published online: 12 Jun 2014.

To cite this article: Kiyoshi Ito, Kazuhiro Iwashita & Kimio Iwano (1992) Cloning and Sequencing of the xynC Gene Encoding Acid Xylanase of Aspergillus kawachii, Bioscience, Biotechnology, and Biochemistry, 56:8, 1338-1340, DOI: 10.1271/bbb.56.1338 To link to this article: http://dx.doi.org/10.1271/bbb.56.1338

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Biosci. Biotech. Biochem., 56 (8), 1338-1340, 1992

Note

Cloning and Sequencing of the xynC Gene Encoding Acid Xylanase of Aspergillus kawachii Kiyoshi

ITO,

Kazuhiro

IWASHITA,

and Kimio

IWANO

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National Research Institute of Brewing, 2-6-30, Takinogawa, Kita-ku, Tokyo 114, Japan Received February 19, 1992

Xylanase (l,4-fi-D-xylan-xylanohydrolase; EC 3.2.1.8) is one of the hemicellulases, and microbial xylanases have received considerable attention in industrial processes. Xylanolytic microorganisms often produce multiple xylanases, which can be derived either from the processing of a single gene productl) or from the direct product of multiple xylanase genes. 2 ) Aspergillus kawachii is a common fungus used in the fermentation of shochu (Japanese traditional spirit), and this strain makes a large quantity of citric acid and simultaneously produces many interesting acid stable enzymes including several xylanases and cellulases, among which we have purified three xylanases and characterized their properties. 3) We are now cloning the gcnes encoding these xylanases and studying the structures of the genes and proteins, from which we have reported the cloning of the xynA gene encoding xylanase A of A. kawachii. 4 ) Xylanase C is one of the xylanase family of A. kawachii and it is an interesting acid xylanase, whose optimum pH is 2.0, and it is stable at pH 1.0. In this paper, we describe the xynC gene encoding xylanase C. The purification and properties of xylanase C protein have been reported in another paper?) The N-terminal amino acid sequence of the protein was identified by automated Edman degradation using Applied Biosystem's 47lA protein sequencer and the following sequence, S-AG-I-N-Y-V-, was found. The methods for the construction of cDNA and genomic DNA library, for the screening, and other general methods were almost the same as in our previous paper. 4) A. kawachii was grown in xylan medium to induce xylanase production and poly(A) + RNA was prepared from the mycelia. A cDNA library was constructed in Agtll vector with cDNA which was synthesized from poly(A) + RNA. About 50,000 individual plaques were screened with the antibody against xylanase C protein, and nine positive clones were obtained. Each clone was purified to a single plaque by a second screening, then the inserted cDNAs were amplified by polymerase chain reaction (PCR) using Agt11 forward and reverse primers (Takara Shuzo), and the lengths of inserted cDNA were measured by gel electrophoresis. The lengths of inserted cDNA were about 0.9 kbp and their restriction maps were the same, so the longest clone was subcloned into pUC118. A genomic DNA library was constructed in the EMBL3 vector and about 50,000 individual plaques were screened with the 32P-Iabelled probes derived from cDNA. Eight positive clones were obtained and they had the same restriction maps in part. Since it was suggested that the coding region was in a 2.2-kbp PstI-PstI fragment by the hybridization

p

s Fig. 1.

K

with cDNA, this fragment was subcloned into pUC119. The restriction map is shown in Fig. 1. The nucleotides of the genomic DNA and cDNA inserts were sequenced by the dideoxy methodS) and the intervening sequence was located by the comparison of both sequences. The result is shown in Fig. 2 with the deduced amino acid sequence. The xynC genomic DNA contained only one relatively short (49 nucleotides) intron in contrast with the large number (nine) of introns in xynA. The cDNA insert comprises 895 nudeotides, and contains an open reading frame coding for 211 amino acid residues. The N-terminal amino acid sequence was recognized at position 28-35 in Fig. 2, consequently the putative signal peptide was 27 amino acid residues from Metl to Arg27. Though the Arg27 residue is unusual as a cleavage site by signal peptidase,6) the same cleavage site was also shown in xynA of A. kawachii, and in the other genes of Aspergillus species. 7 ) The molecular weight of the putative mature protein was 19,888 and the carbohydrate content of xylanase C protein is 8.7%.3) Thus the molecular weight of glycosylated protein is about 22,000, while the molecular weight estimated by SDSPAGE was 29,000. 3 ) We consider that this may be caused by the peculiar electrophoretic mobility of xylanase C protein in SDS-PAGE. A protein homology search was done by the method of Lipman and Pearson 8 ) using SWISS-PROT data base (DNASIS-DBREF50; Hitachi Software Engineering). Comparison of amino acid sequences showed that xylanase C had about 40-50% homology with xylanases from Bacillus pumilus,9) Clostridium acetobutylicum,10) Bacillus subtilis,l1) and Bacillus circulans. 12 ) Since all of these four enzymes are classified into the same cellulase subfamily (subfamily G) by the hydrophobic cluster analysis of putative catalytic domains,13,14) it may be presumed that xylanase C also belongs to the same subfamily. However Gilkes et al. 13 ) described how subfamily G contains only bacterial enzymes. The amino acid sequence alignments of the conserved segments are shown in Fig. 3 Akatsuka et al. 15 ) identified two glutamic acid residues as the active site of xylanase of B. pumilus, and these residues (Glu107 and Glu197 in xylanase C) are conserved in all xylanases. No significant homology was found with xylanase A from A. kawachii, cellulase from A. kawachii, 16) cellulase from Aspergillus aculeatus, 17) and other glucanases from filamentous fungi. Yeast Saccharomyces cerevisiae DBY747 was transformed with the xynC cDNA by the method described in a previous paper.4) The transformed yeast produced xylanase and its optimum pH was 2.0 similarly to that of A. kawachii. The produced xylanase was also identified by Western blot analysis (data not shown).

B

x s

p

500 bp

Restriction Map of the Genomic DNA Encoding Xylanase C.

The protein coding region is indicated by the solid (exons) and open (intron) boxes. Abbreviations: B, BamHI; K, KpnI; P, Pst!; S, Sad; X, Xbal.

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xynC Gene of Aspergillus kawachii

399

315 252 -189 -126 -63

ctgcagggaaccggtgaagaa tccccacttccccgtctcccccaactgcagcccttttatccgtctgccgtgcatttagccaaa tgtagtgcatttagccaagtgcggtccatttagccaagagcagtggctagattggtggctaca gagcagacgcatgactgagacacaactataggactgtctctggaaataggctcgaggttgttc aagcgtttaaggtgatgcggcagaatgcatatgactaagctgcttcatcttgcagggggaagg gataaatagtctttttcgcagaatataaatagaggtagagtgggctcgcagcaatactgacca gcacagtgcttctcttccagttgcataaatccattcaccagcatttagctttcttcaatcatc

1 49 17 97 33 145 49 193 65 243

ATG AAG GTC ACT GCG GCT TCT GCG GGT CTA CTT GGT CAC GCA TTC GCC M K V T A A SAG L L G H A F A GCT CCT GTG CCG CAA CCT GTT CTG GTG TCG CGA AGT GCT GGT ATT AAC SAG I N TAC GTG CAA AAC TAC AAC GGC AAC CTT GCT GAT TTC ACC TAT GAC GAG Y V Q N Y N G N LAD F T Y D E AGT GCC GGA ACA TTT TCC ATG TAC TGG GAA GAT GGA GTG AGC TCC GAC SAG T F S M Y WED G V S S D TTT GTC GTT GGT CTG GGC TGG ACC ACT GGT TCT TCT AA gtgagtgactgt F V V G L G W T T G S S N attctttaaccagagtctaggatctaacgttttctag C GCT ATC AGC TAC TCT GCC

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378

78

A

S

Y

S

A

299 84 347 100 395 116 443 132 491 148 539 164 587 180 635 196

GAA TAC AGT GCT TCT GGC TCC TCT TCC TAC CTC GCT GTG TAC GGC TGG E Y S A S G S S S Y L A V Y G W GTC AAC TAT CCT CAG GCT GAA TAC TAC ATC GTC GAG GAT TAC GGT GAT V N Y P Q A E Y Y I V E D Y G D TAC AAC CCT TGC AGC TCG GCC ACA AGC CTT GGT ACC GTG TAC TCT GAT GGA G ATC I ACG T GCG A GTG V

683

taa gggataagtgccttggtagtcggaagatgtcaacgcggaactctgttctcagctggtgt

745 808 871 934 997 1060 1123 1186

gatgatcggatccggcctctggtggttacattgaggctgtataagttattctggggccgagct gtcagcggctgcgtttccaatttgcacagataatcagctttcgttttctatctcttgCgtttc cacgctgtttatcctatccatagatattattttgcccaatacatattatctatatacaacttg ttccgtcgcagtagtcactccgagcaaggcattgggaaattggaagatgcggggtgctgcgta cgctctaaggtagggcatttaaagggatatttagcctccagatattctatactaacagacttc taatgactgcggataatatagagggcaagaatttctacagttcgacgcagttcaacgcaatca gagagggaatactgatgagagtgcaatcagttagagaaggacaacatggcagtcttagtgtga acttacataacgatatggactctagaaaaaaggaaggagctc

Y

Fig. 2.

N

P

C

AGC ACC TAC STY ACG GGA ACA T G T CGC ACA TCT R T S CAG CAT GGG Q H G GAA GCA TGG E A W

S

SAT

CAA GTG TGC ACC Q V C T AGC ACG TTC ACG S T F T GGA ACG GTG ACT G T V T TTC GGA AAT AGC F G N S AGC GGT GCT GGC S GAG

S

L

G

GAC ACT CGA D T R CAG TAC TTC Q Y F GTT GCC AAC V A N GAC TTC AAT D F N AGC GCC AGT S A S

T

V

ACT T TCC S CAT H TAT Y GTC V

AAC N GTT V TTC F CAG

Y

S

D

GAA CCG TCC E P S CGA GAG AGC RES AAC TTC TGG N F W GTC ATG GCA Q V M A ACG ATC TCC TCT TIS S

*

Nucleotide Sequence and Deduced Amino Acid Sequence of the xynC Gene from A. kawachii.

The putative signal peptide is underlined.

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K. ITO, K. IWASHITA, and K. IWANO

! 96 VYGWVN YP Q A E Y Y I VE DYGDYNpes SAT S L G T VYS DG STY Q veT DT R T N E PSI T G

XynC

XynBp 110 VYGWT Q S P L A E Y Y I V DS WGT YR P - - T GAY KG S F YA DGGT YDIY E T T R VNQ PSI I G XynCA 142 VYGWT SSP L VE Y Y I VDS WG S WR P - P GGT S KGTIT VDGG I YDIY E T T R I N Q PSI Q G XynBS

96 L YGWT R S P LIE YYVVDS WGT YR P - - T GT YKGT VKS DGGT YDIY T T TRY NAP SID G

XynBC

96 L YGWT R S P LIE YYVV DS WGT YR P -

T GT YKGT VKS DGGT YDIY T T TRY NAP SID G

~

XynC

151 - T S T F T Q Y F S VRES T R T S G- - - T VT VA N HF NF WA Q HGF G- NS DF NY Q VMAVE AWS

XynBP 165 - I AT F K Q YW S VR Q T K R T S G- - - T VS VS A HF R KW E S L GMP - MGKMYET AFT VE G Y Q XynCA 197 - NT T F K Q YWS VR R T KR T S G - - TIS VS K HF A AWE S KGMP - L G KMH ETA F N lEG YQ Downloaded by [University of Illinois Chicago] at 17:50 14 November 2014

XynBS 149 DR T T F T Q YWS VR Q S KR P T G S NAT I T F S N HVNAWKS HGMNL G S NWAY

Q VMAT

E G YQ

XynBC 149 DR T T F T Q YWS VR Q S KR P T G S NAT I T F T N HVNAWKS HGMNL GS NWAY Q VMAT E G YQ Fig. 3. Amino Acid Sequence Alignments for Xylanases from A. kawachii (XynC; this study), Bacillus pumilus (XynBP),9) Clostridium acetobutylicum (XynCA), 10) B. subtilis (XynBS), 11) and B. circulans (XynBC).12) Identical amino acid residues are enclosed within a box. The arrows indicate Glu residues at the putative active site in XynBP as described by Akatsuka et alY)

References 1) P. Biely, Trends Biotechnol., 3, 286-290 (1985). 2) T. R. Whitehead and R. B. Hespell, J. Bacteriol., 172, 2408-2412 (1990). 3) K. Ito, H. Ogasawara, T. Sugimoto, and T. Ishikawa, Biosci. Biotech. Biochem., 56, 547-550 (1992). 4) K. Ito, T. Ikemasu, and T. Ishikawa, Biosci. Biotech. Biochem., 56, 906-912 (1992). 5) F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. U.S.A., 72, 3961-3965 (1977). 6) G. Heijne, Eur. J. Biochem., 133, 17-21 (1983). 7) I. Shibuya, K. Gomi, Y. Iimura, K. Takahashi, G. Tamura, and S. Hara, Agric. BioI. Chem., 54, 1905-1914 (1990). 8) D. J. Lipman and W. R. Peason, Science, 227, 1435-1441 (1985). 9) E. Fukusaki, W. Panbangred, A. Shinmyo, and H. Okada, FEBS Lett., 171, 197-201 (1984). 10) H. Zappe, W. A. Jones, and D. R. Woods, Nuc!. Acids Res., 18, 2179 (1990).

11) 12) 13) 14) 15)

16)

17)

M. G. Paice, R. Bourbonnais, M. Desrochers, L. Jurasek, and M. Yaguchi, Arch. Microbiol., 144, 201-206 (1986). R. C. A. Yang, C. R. MacKenzie, and S. A. Narang, Nucl. Acids Res., 16, 7187 (1988). N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, and R. A. J. Warren, Microbiol. Rev., 55, 303-315 (1991). B. Henrissat, M. Claeyssens, P. Tomme, L. Lemesle, and J. P. Momon, Gene, 81, 83-95 (1989). H. Akatsuka, P. K. Elizabeth, H. Moriyama, S. Negoro, A. Shinmyo, H. Okada, Y. Hata, and Y. Katsube, Abstracts of Papers, the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, November, 1989, p. 14. S. Sakamoto, G. Tamura, K. Ito, and T. Ishikawa, Abstracts of Papers, the Annual Meeting of the Society of Fermentation Technology, Japan, Hiroshima, November, 1991, p. 186. T. Ooi, A. Shinmyo, H. Okada, S. Hara, T. Ikenaka, and S. Murao, Curro Genet., 18, 217-222 (1990).

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Cloning and sequencing of the xynC gene encoding acid xylanase of Aspergillus kawachii.

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