Takuya NIHIRA,2 Yasuhiro YAMADA,2 Tomohisa KUZUYAMA,3 Haruo SETO,3 ... 2Department of Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, ...
Biosci. Biotechnol. Biochem., 67 (9), 2002–2005, 2003
Note
Nucleotide Sequences of Genes Encoding Allosamidin-sensitive and -insensitive Chitinases Produced by Allosamidin-producing Streptomyces Hiroshi MATSUURA,1 Susumu OKAMOTO,1 Sarintip ANAMNART,2 Qiuqi WANG,2 Ze-Yang ZHOU,2 Takuya NIHIRA,2 Yasuhiro YAMADA,2 Tomohisa KUZUYAMA,3 Haruo SETO,3 Jiro NAKAYAMA,1 Akinori SUZUKI,1 Hiromichi NAGASAWA,1 and Shohei SAKUDA1,† 1Department
of Applied biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871, Japan 3Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan Received February 26, 2003; Accepted May 18, 2003
Allosamidin is a strong inhibitor of family 18 chitinases. We previously reported the presence of allosamidin-sensitive and -insensitive chitinases (chitinase S and IS) in the culture ˆltrate of the allosamidinproducing strain, Streptomyces sp. AJ9463. In this study, we cloned and sequenced the genes encoding the two chitinases, which clariˆed that chitinase S and IS belong to the family 18 and 19 chitinase, respectively. Key words:
allosamidin; family 18 chitinase; family 19 chitinase; Streptomyces
Allosamidin is a strong inhibitor of family 18 chitinases. It has a unique pseudotrisaccharide structure and shows interesting biological activities toward a variety of chitin-containing organisms.1–3) From the viewpoint of a physiological role of allosamidin in its producing strain, we focused on chitinases produced by an allosamidin-producing strain, Streptomyces sp. AJ9463, and found that allosamidin-sensitive and -insensitive chitinases, named chitinase S and IS, respectively, are mainly produced by the strain when cultured in a Bennet medium.4) Since no microbial chitinase insensitive to allosamidin had been reported before our ˆnding of chitinase IS, we expected that chitinase IS would have some speciˆc characteristics as a chitinase produced by an allosamidin-producing strain. In this paper, we describe the analysis of the nucleotide sequences of genes encoding chitinases S and IS, which veriˆed that they are family 18 and 19 chitinases, respectively. We reported the puriˆcation of the two chitinases.4) Analysis of the N-terminal amino acid sequence of each of the puriˆed chitinases clariˆed the sequence (GTDPEPTIPSAPAGL) of chitinase S, but that of chitinase IS was not veriˆed. Therefore, †
puriˆcation of chitinase IS was tried again. When strain AJ9463 was cultured with spores from a stocked slant of the strain under conditions previously used for the production of chitinase IS,4) only a trace amount of chitinase IS was detected in the culture ˆltrate. To obtain the chitinase in su‹cient amount for analysis of its N-terminal amino acid sequence, a screening search for a suitable medium for the production of chitinase IS was done, and medium containing chitin powder was selected. Strain AJ9463 was cultured with the medium for 96 h, and the culture ˆltrate was used for the puriˆcation of chitinase IS. After ammonium sulfate precipitation, the crude enzyme was chromatographed on DEAE-cellulose and Superdex G-75 columns, successively, and a reverse-phase HPLC on TSK gel was used for the ˆnal step. Allosamidin-insensitive chitinase activity was detected in the neighboring two fractions of peaks 1 and 2 on the chromatogram of the HPLC (Fig. 1a), but the activity involved in peak 1 was much higher than that in peak 2. Figure 1b shows SDS-PAGE of the samples from the two peaks. A main band at 44 kDa was observed for peak 2, while two bands at 44 kDa and 31 kDa were observed for peak 1. Chitinase activity of the peak 1 fraction was detected by the activity staining after SDS-PAGE. As shown in Fig. 1c, chitinase activity was observed only at the 31-kDa band on the gel, conˆrming the position of chitinase IS on SDS-PAGE. This molecular mass of chitinase IS (＝31 kDa) on SDS-PAGE was not identical to that (＝44 kDa) we reported previously.4) In the previous puriˆcation of the chitinase using the same HPLC column, the active fraction might contain 44-kDa protein in large excess over 31-kDa protein, leading to a misunderstanding of what the molecular mass of chitinase IS. The Nterminal amino acid sequence of the 31-kDa protein
To whom correspondence should be addressed. Fax: ＋81-3-5841-8022; E-mail: asakuda＠mail.ecc.u-tokyo.ac.jp
Genes Encoding Chitinases from Allosamidin-producing Streptomyces
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Fig. 1. HPLC (a), SDS-PAGE (b) and Activity Staining (c) of Chitinase IS. (a) HPLC conditions: column, TSK-Gel Phenyl-5PW RP (7.5 mm×7.5 cm); mobile phase, gradient elution of 0–70z acetonitrile in 10 m M ammonium acetate in 30 min; ‰ow rate, 1 ml W min. (b) Lane 1, peak 1 of HPLC; lane 2, peak 2 of HPLC. (c) Lane 1, peak 1 of HPLC.
Fig. 2. Alignments of Amino Acid Sequences between Chitinase S and Chitinase A from S. lividans (a), and between Chitinase IS and Chitinase C from S. griseus (b). Amino acids obtained by N-terminal analysis are indicated with asterisks. Identical residues are boxed. ChiS, chitinase S; ChiA, chitinase A; ChiIS, chitinase IS; ChiC, chitinase C.
was analyzed after SDS-PAGE, and the sequence (ADXAAPWNSSSVYTGGGTAXYN) was obtained. It had a high similarity with the N-terminal amino acid sequence of chitinase C, which is a family 19 chitinase produced by Streptomyces griseus HUT6037.5) Base on the N-terminal amino acid sequences obtained above, the genes encoding chitinases S and IS were cloned as follows. In the case of chitinase S (ChiS), an oligonucleotide probe designed from the amino acid sequence was used for screening of a genomic library of Streptomyces sp. AJ9463, and a 1.9-kbp SmaI fragment containing one open reading frame (chiS, AB104620) was obtained. On the other hand, an internal fragment of the chitinase IS gene (chiIS, AB104621) was ampliˆed by PCR with
oligonucleotide primers designed based on the N-terminal amino acid sequence and the conserved amino acid sequence found within the family 19 chitinases. After sequence analysis, the oligonucleotide primers were designed and used for PCR-based screening of a genomic library of strain AJ9463. Finally, a clone (pCIS2) containing the complete chiIS gene was obtained. The amino acid sequence of chitinase S deduced from the nucleotide sequence indicated that it belongs to the family 18 chitinases (Fig. 2a). It has a high similarity with chitinase A (77z) produced by Streptomyces lividans.6) Family 18 chitinases are popularly produced by Streptomyces as extracellular chitinases.7) The N-terminal position of chitinase S obtained by N-terminal analysis in the amino acid
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H. M ATSUURA et al. Table 1. Puriˆcation of Chitinase IS
Step
Protein (mg)
Total activity (units)
Speciˆc activity mg) (units W
Fold
Yield (z)
Culture ˆltrate 80z Ammonium sulfate precipitation DEAE cellulose Superdex G-75 Phenyl 5PW RP
397.8 217.5 21.1 12.5 0.023
131.0×103 108.3×103 68.7×103 8.6×103 2.3×102
3.29×102 4.98×102 3.26×103 6.84×102 1.22×105
1.0 1.5 9.9 2.1 371
100 83 52 6.6 0.2
sequence was similar to the case of ChiI of Streptomyces griseus.8) On the other hand, the amino acid sequence of chitinase IS has a high similarity with chitinase C (78z) of S. griseus throughout the sequence (Fig. 2b).5) Since allosamidin has not inhibited any of the family 19 chitinases tested, the case of chitinase IS is not an exception. And the recent fact that genes encoding family 19 chitinases are widely distributed in Streptomyces 9) shows that chitinase IS is not a speciˆc chitinase for allosamidinproducing strains. The nucleotide sequences of chitinases S and IS could not aŠord any information on a physiological role of allosamidin in an allosamidin-producing strain. However, we have recently found that the production of chitinases by strain AJ9463 is strongly accelerated by addition of allosamidin to its culture exogenously.10) The two chitinase genes obtained in this study may be useful probes to investigate the molecular mechanism of this phenomenon.
Puriˆcation of chitinase IS. Cultivation of Streptomyces sp. AJ9463 was done according to the procedure previously described4) except for the medium used for the main culture. The medium consisting of chitin powder (0.2z), peptone (0.2z), yeast extract (0.1z), and meat extract (0.1z) (pH 7.2) was used in this study, and strain AJ9463 was cultivated in the medium for 96 h at 26.59 C and 137 rpm. The puriˆcation of chitinase IS, which is summarized in Table 1, was done by almost the same procedure as that previously reported.4) Chitinase activity was measured with glycol chitin as a substrate according to the method of Ohtakara et al.11) in the presence of ml. The allosamidin at the concentration of 10 mg W amount of enzyme which liberated 1 mmole of a reducing sugar from the substrate was deˆned as one unit of chitinase. Activity staining to detect the chitinase activity on the gel was done according to the method of Koga et al.12) Cloning of the chitinase S gene. Genomic DNA of strain AJ9463 was digested with several restriction enzymes and fractionated electrophoretically on a 1.0z agarose gel. The DNA was transferred to a nylon membrane, and hybridized with the synthetic oligonucleotide probe (probe S, 5?-GAYCCNGARCCNACNAT-3? ), which was designed based on the
N-terminal amino acid sequence (DPEPTI) and end-labeled with [ g-32P]ATP and T4 polynucleotide kinase. Hybridization was done in 5×SSPE containing 0.5z SDS, 5×Denhardt's solution, and 0.1 mg W ml of salmon sperm DNA for 18 h at 509 C, and washed twice with 2×SSC cintaining 0.1z SDS for 15 min at room temperature, once for 15 min at 609 C, once with 0.2×SSC containing 0.1z SDS for 15 min at room temperature, once for 15 min at 509 C, and ˆnally washed with 0.1×SSC containing 0.1z SDS for 15 min at 509C. By this hybridization, the chiS gene was localized on a 6-kb Bam HI fragment. After enrichment of the 6-kb fragment by preparative agarose gel electrophoresis, the DNA fragments were inserted into the Bam HI site of pUC19, and the ligation products were used to transform E. coli SURE (Stratagene) cells to create a Bam HI library. By screening the Bam HI library by colony hybridization using probe S as described above, 26 candidate-clones were obtained from 1000 transformants. These recombinant plasmids were recovered, cut by Bam HI, and hybridized again with probe S. Three clones were identiˆed as positive, and the plasmids recovered from these clones were found to be identical by restriction analyses. The chitinase S gene was further localized on the 1.9-kb SmaI fragment by southern hybridization.
Cloning of the chitinase IS gene. An internal fragment of the chitinase IS gene was ampliˆed by PCR. Oligonucleotide primers F1 (5?-GACTGYGCSGCSCCNTGGAA-3? ) and R3 (5?were GTASTTFAASTTCCAGCTSAGCTG-3? ) designed based on the N-terminal amino acid sequence (DXAAPWN) of the puriˆed protein and the conserved amino acid sequence (QLSWNFNY) found within the family 19 chitinases, respectively. The PCR reaction was done with AmpliTaq Gold (applied Biosystems) under the following conditions: 9 min at 959C, followed by 35 cycles of 30 s at C and 30 s at 559 C. The resulting PCR product 969 (¿500 bp) was cloned into pGEM-T Easy (Promega), and sequenced. On the basis of he nucleotide sequence of the cloned DNA fragment, PCR primers F3 (5?-AACTCCTCGTCCGTCATCACC-3? ) and R5 (5?-GTTCCAGCTGAGCTGGATCGG-3? ) were designed and used for isolation of the complete chitinase IS gene. A genomic library was constructed
Genes Encoding Chitinases from Allosamidin-producing Streptomyces
by using a Bam HI digest of the strain AJ9463 genomic DNA with the pGEM-3Zf (＋) vector (Promega) and sequenced by PCR under the following conditions: 9 min at 959C, followed by 35 cycles of 30 s at 969C and 30 s at 659C. One positive clone, designated pCIS2, was eventually identiˆed.
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