APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2003, p. 894900 0099-2240/03/$08.00 0 DOI: 10.1128/AEM.69.2.894900.

2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.

Vol. 69, No. 2

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

Hiroshi Tsujibo,* Takahiro Kubota, Mitsugu Yamamoto, Katsushiro Miyamoto, and Yoshihiko Inamori
Department of Microbiology, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan
Received 8 July 2002/Accepted 6 November 2002

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB , in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a signicant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed signicant similarity to Streptomyces family 19 chitinases. ChiB was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efcient hydrolysis of chitin and stronger antifungal activity than ChiB , indicating that the ChtBD type 3 of ChiB plays an important role in the efcient hydrolysis of chitin and in antifungal activity. Furthermore, the nding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.

Chitin, an insoluble linear -1,4-linked polymer of N-acetylglucosamine (GlcNAc), is the second most abundant polymer in nature. This polysaccharide is found in the cell walls of fungi and in the exoskeletons of insects and crustaceans. Chitinases (EC 3.2.1.14) are produced by many organisms, such as viruses, bacteria, higher plants, and animals, and play important physiological and ecological roles (7). Chitinases hydrolyze the -1,4 linkages in chitin, yielding predominately N-N -diacetylchitobiose, which is further degraded by N-acetylglucosaminidases to the GlcNAc monomer. Actinomycetes are gram-positive mycelial soil bacteria with high G C contents. In addition to having the ability to synthesize a wide variety of antibiotics and biologically active compounds, they produce extracellular hydrolytic enzymes to obtain nutrients and energy by solubilizing polymeric compounds in soil. These enzymes include proteases, nucleases, lipases, and a variety of enzymes that hydrolyze different types of polysaccharides, such as chitin and cellulose (14). Among actinomycetes, Streptomyces species make up one group regarded as well-known decomposers of chitin. Chitinases have been puried and characterized from various Streptomyces species, and the corresponding genes have been cloned and sequenced. The multiple genes for chitinases were cloned from Streptomyces plicatus (22, 23), Streptomyces lividans 66 (6, 18, 19), Streptomyces thermoviolaceus OPC-520 (3335), and Streptomyces coelicolor A3 (2) (24). However, no work has been done on the molecular cloning and analysis of chitinase genes
* Corresponding author. Mailing address: Department of Microbiology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan. Phone: (81-726) 90-1057. Fax: (81726) 90-1057. E-mail: tsujibo@gly.oups.ac.jp. 894

from the nonstreptomycete group. It has already been reported that an alkaliphilic actinomycete, Nocardiopsis prasina OPC131, produces two types of chitinases (ChiA and ChiB) (32). The most characteristic properties of ChiA and ChiB indicated that the enzyme activities remained well within the alkaline pH range compared with those of Streptomyces species. In particular, even at pH 9.0, ChiA and ChiB showed 50% of the activity at the optimum pH (32). In this paper, we describe the cloning and sequencing of the chiA and chiB genes. Furthermore, we have expressed these enzymes in Escherichia coli and investigated the characterization of the cloned enzymes.
MATERIALS AND METHODS Bacterial strains and culture conditions. N. prasina OPC-131, which was isolated from soil, was used as the source of chromosomal DNA (32). The strain was grown at 27C for 6 days in a medium containing (in grams per liter) colloidal chitin, 5.0; yeast extract, 2.5; K2HPO4, 1.0; MgSO4 7H2O, 0.2; Na2CO3, 10.0 (pH 10.0). E. coli JM109 and TOP10 (Invitrogen Co., Carlsbad, Calif.) were grown at 37C in Luria-Bertani (LB) broth. For agar medium, Luria-Bertani broth was solidied with 1.5% (wt/vol) agar (Nacalai Tesque, Kyoto, Japan). Purication of chitinases. Chitinases were puried from the culture ltrate by successive chromatographies of DEAE-Toyopearl 650 M, Sephadex G-100, and UnoQ as described previously (32). N-terminal amino acid sequence, SDS-PAGE, and protein assay. The amino acid sequence was analyzed with a Procise 491 HT protein sequencer (Applied Biosystems) that was connected to an online phenylthiohydantoin derivative analyzer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was done by the method of Laemmli (11). After electrophoresis, activity staining of chitinases in polyacrylamide gel was carried out by the method of Wolfgang et al. (42). Protein was assayed by the method of Bradford with bovine serum albumin as a standard (2). Enzyme assay. Chitinase activity was measured, as described previously, with ethylene glycol chitin (Seikagaku Co., Tokyo, Japan) or colloidal chitin (crab shell; Nacalai Tesque) as a substrate (32, 34, 36). One unit of chitinase was dened as the amount of enzyme that liberated reducing sugar corresponding to 1 mol of GlcNAc.

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Cloning of chiA and chiB genes. General recombinant DNA techniques were performed as described by Sambrook and Russel (25). Two sets of primers (A-1, 5 -GCTACACGCACAACTTCGAT-3 , and A-2, 5 -AGATCTATGTCGACGC CGTC-3 ; and B-1, 5 -GGCCCAGTTCAACCAGATGT-3 , and B-2, 5 -AGTA CCACAGGCCGGTCTTC-3 ) were synthesized based on the N-terminal amino acid sequences of ChiA and ChiB and the conserved sequences of family 18 and 19 chitinases. PCR amplication was performed by KOD-Plus-DNA polymerase (Toyobo, Osaka, Japan) for 30 cycles consisting of 97C for 15 s, 51C for 30 s, and 68C for 1 min. The amplied fragments were phosphorylated by T4 DNA polynucleotide kinase. Each of the resulting fragments (pCHIA1 and pCHIB1) was cloned into the dephosphorylated SmaI site of pUC19, sequenced, and used as a probe. Chromosomal DNA was digested with BamHI and SalI or BamHI alone and electrophoresed on a 0.6% agarose gel. The fragments in the range of 4.0 to 5.0 kb were excised from the gel, puried with a GenElute gel extraction kit (Sigma), and then ligated into the corresponding sites of pUC19. The recombinant plasmids were inserted into competent E. coli JM109. Each of the libraries was screened by colony hybridization with alkaline phosphatase-labeled pCHIA1 or pCHIB1 as a probe (AlkPhos DIRECT; Amersham Bioscience). Hybridization and washing were performed according to the suppliers instructions. DNA sequencing. Nucleotide sequencing was carried out by the dideoxy chain termination method by using the DYEnamic ET terminator cycle-sequencing premix kit (Amersham Bioscience) on a DNA sequencer (Prism 310 genetic analyzer; Applied Biosystems) (26). Sequence data were analyzed by using the GENETYX-WIN program (Software Development Co., Ltd.). Construction of expression plasmids and purication of recombinant proteins. The expression plasmids pThioHis-ChiA, pThioHis-ChiB, and pThioHisChiB , coding for ChiA, ChiB, and the truncated form of ChiB, respectively, were constructed as follows. The primers for ChiA (5 -CCCGTCATGACCCA GCAGACCAGCC-3 and 5 -GCGGGCTCGAGGGGTTACTGCAGTC-3 ), ChiB (5 -CTCGTTACCCTCCCCATGGCCGCCG-3 and 5 -TGCCTCACTG CAGCCGGGGTTCAGC-3 ), and ChiB (5 -GACCAGGTACCCTGCGGCG ACGGC-3 and 5 -TGCCTCACTGCAGCCGGGGTTCAGC-3 ), which were modied to contain BspHI and XhoI (pThioHis-ChiA), NcoI and PstI (pThioHisChiB), and KpnI and PstI (pThioHis-ChiB ) recognition sites to facilitate cloning in frame into the thioredoxin fusion protein expression vector pThioHisB (Invitrogen Co.), were synthesized. PCR amplication was performed with the full-length chiA or chiB gene as a template for 30 cycles consisting of 97C for 15 s, 51C for 30 s, and 68C for 1 min. The amplied DNAs were digested by BspHI and XhoI, NcoI and PstI, and KpnI and PstI, respectively, and the resulting fragments were inserted into the corresponding sites of pThioHisB. The nucleotide sequences of pThioHis-ChiA, pThioHis-ChiB, and pThioHis-ChiB were conrmed by DNA sequencing. E. coli TOP10 cells harboring pThioHis-ChiA, pThioHis-ChiB, and pThioHisChiB were induced with 1 mM IPTG (isopropyl- -D-thiogalactopyranoside) at the mid-exponential growth phase and further incubated for 3 h at 27C. The cells were harvested by centrifugation, washed, and resuspended with 20 mM phosphate buffer (pH 7.8) containing 0.5 M NaCl. The cells were disrupted by sonication, and the lysates were centrifuged at 10,000 g for 10 min. The fusion proteins were puried from the supernatant by afnity chromatography with a nickel-charged Sepharose resin (ProBond resin; Invitrogen Co.). The puried fusion proteins were treated with enterokinase (Invitrogen Co.) for 16 h at 37C to obtain ChiA, ChiB, and ChiB . The N-terminal amino acid sequences of these proteins were conrmed by protein sequencing. Binding study and antifungal activity. Chitin-binding assays were carried out by adding 1.5 g of puried ChiB or ChiB to 5 mg each of -chitin, -chitin, and crystalline cellulose (Avicel) in 0.1 ml of 20 mM HEPES-KOH buffer (pH 7.0) in 1.5-ml microcentrifuge tubes. Samples were permitted to stand for 20 min on ice and then centrifuged at 24,650 g for 5 min. The chitinase in the supernatant was measured using colloidal chitin as a substrate, and the activity lost from the supernatant was assumed to be the activity bound. Antifungal activity was estimated using the hyphal extension-inhibition assay as described previously (35). The mycelium of Trichoderma reesei was directly inoculated onto the center of a dish containing 10 ml of potato dextrose agar (Eiken Chemical Co., Ltd., Tokyo, Japan). After incubation at 27C for 24 h, paper disks were placed around the edge of the T. reesei culture, and 30 l of solution containing 80 g of ChiA, ChiB, or ChiB was put onto the disks. The plate was incubated at 27C for 30 h, and the inhibition of hyphal extension around the disks was examined. Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession numbers AB086831 (chiA) and AB086832 (chiB).

FIG. 1. SDS-PAGE and zymogram analyses of proteins in the culture supernatant of N. prasina OPC-131. (A) SDS-PAGE stained with Coomassie brilliant blue R-250. (B) Zymogram of chitinase activity. N. prasina OPC-131 was grown in medium supplemented with 0.5% chitin, 0.5% chitin plus 1% glucose, or 1% glucose. Samples were taken at 80 h for these experiments. The same volume of each culture supernatant was concentrated to 1/30 of its original volume by ultraltration with NanoSpin Plus. The concentrated samples were applied to SDS-PAGE. Lanes: M, marker proteins; 1, 0.5% chitin; 2, 0.5% chitin plus 1% glucose; 3, 1% glucose.

RESULTS Purication of chitinases. Before we began the purication of chitinases from N. prasina OPC-131, zymogram analysis was performed to investigate the multiplicity of chitinases. Zymogram analysis indicated that strain OPC-131 secreted four chitinases in the presence of chitin (Fig. 1). The molecular masses of ChiA and ChiB were 32 kDa but were overestimated as 35 kDa in a previous paper (32). The two additional chitinases, ChiB and ChiX, corresponding to proteins of 28 and 27 kDa, respectively, were detected. These enzymes and several unidentied proteins were induced in the presence of chitin; however, the production of these proteins was not repressed in the presence of glucose. Among these enzymes, ChiA, ChiB, and ChiB were puried, and their N-terminal amino acid sequences were determined. However, the production of ChiX was so low that we could not purify and characterize the enzyme. The BLAST search program revealed that the N-terminal amino acid sequence of ChiA (QTSQWLTG YWHNFDNGSTV) showed high homology with family 18 chitinases (8, 9), such as Bacillus circulans ChiD (39), S. thermoviolaceus Chi30 (34), Streptomyces griseus ChiI (10), and S. lividans ChiA (18). On the other hand, ChiB (ATACATA WSSSSVYTGGGQVSFEG) and ChiB (GDGGGGEQPG PNDFVVSEAQF) showed high homology with family 19 chitinases (8, 9), such as S. griseus ChiC (21) and S. thermoviolaceus Chi35 (35). These results suggest that ChiA belongs to family 18 chitinases and that ChiB and ChiB belong to family 19 chitinases. Cloning and sequencing of chiA and chiB. To isolate chiA and chiB genes from a genomic library of N. prasina OPC-131, PCR probes were synthesized on the basis of the N-terminal amino acid sequences of ChiA and ChiB and conserved sequences of family 18 and 19 chitinases. Two probes for the cloning of chiA (0.34 kb) and chiB (0.40 kb) were specically amplied. Southern hybridization using these probes against

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FIG. 2. Restriction maps of pCHIA629 and pCHIB377 and domain structures of ChiA and ChiB. (A and C) Restriction maps of PCHIA629 (A) and pCHIB377 (B). The arrows indicate the ORFs and the direction of transcription. (B and D). Domain structures of ChiA (B) and ChiB (D). Solid, signal peptide; hatched, ChtBD; open, catalytic domain.

total DNA digested with various restriction endonucleases showed that the probes for chiA and chiB hybridized strongly with a 4.6-kb BamHI-SalI fragment and a 4.1-kb BamHIBamHI fragment, respectively (data not shown). Thus, for library construction, the DNA fragments with sizes between 4.0 and 5.0 kb were ligated to the corresponding sites of pUC19. Positive clones (pCHIA629 and pCHIB377) which hybridized to the probe for chiA or chiB were isolated from the libraries by colony hybridization. Analysis by restriction endonuclease digestion showed that pCHIA629 and pCHIB377 contain 4.6and 4.1-kb insert DNAs, respectively (Fig. 2). The nucleotide sequences of 4.6- and 4.1-kb insert DNAs were determined. An open reading frame (ORF) of chiA was identied in a 1.8-kb KpnI-SalI fragment of pCHIA629, and that of chiB was identied in the central region of pCHIB377. The ORF of chiA consists of 1,011 nucleotides encoding a protein of 336 amino acids with a predicted molecular mass of 35,257 Da. The initiation codon (ATG) was preceded at a distance of 9 bp by a possible ribosome-binding site (AGGA), which was homologous to the consensus Shine-Dalgarno sequence (27). The possible promoter sequences TTGACT and TAAAGT were identied upstream of the initiation codon. On the other hand, the 891-bp chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. The putative initiation codon, GTG, was preceded at a distance of 9 bp by a potential ribosome-binding site (AGGAG). The possible promoter sequences TGGTCT and TAGACC were identied upstream of the initiation codon. We examined whether chiA and chiB possess a pair of 12-bp direct-repeat sequences that are commonly found in the promoter regions of Streptomyces chitinase genes (5, 20). However, we could not nd similar direct-repeat sequences in chiA and chiB. The deduced amino acid sequences of orf1, orf2, orf3, orf4, and orf5 showed homology with a hypothetical protein (accession number CAB88920), a putative oxide reductase (CAB88921), an Na -H antiporter (AAF06676), a transpepti-

dase (T36689), and an ABC transporter (CAB76002) from S. coelicolor, respectively. Structural features of ChiA and ChiB. BLAST search analysis of the deduced amino acid sequence of ChiA revealed that the enzyme is composed of a signal sequence and a catalytic domain. The N-terminal amino acid sequence of the puried ChiA from N. prasina OPC-131 coincided precisely with the sequence starting from the Gln45 residue of the deduced amino acid sequence encoded by the chiA gene. Thus, cleavage of the signal sequence must occur between Gln44 and Gln45, which is compatible with the 3- 1 rule of von Heijne (38). The catalytic domain of ChiA showed sequence homology with chitinases classied in glycosyl hydrolase family 18, subfamily B, such as Chi30 from S. thermoviolaceus (47% identity) (34), ChiA from S. lividans (43% identity) (18), ChiA from S. coelicolor (43% identity) (24), and ChiD from B. circulans (28% identity) (39). As shown in Fig. 3, family 18 chitinases contain the consensus sequences (SXGG and DXXDXDXE) (37). The sequences SXGG and DXXDXDXE indicate substratebinding and active sites, respectively. These consensus sequences were also perfectly conserved in the catalytic domain of ChiA. These results suggest that ChiA is classied among family 18 chitinases and that Glu165 of ChiA is involved as the proton donor in the catalytic double-displacement mechanism during hydrolysis (9, 37). On the other hand, ChiB is a modular enzyme consisting of three domains: the signal sequence (29 amino acids), the chitin-binding domain type 3 (ChtBD type 3) (41 amino acids), and the catalytic domain (203 amino acids). Between the ChtBD type 3 and the catalytic domain there was a small Gly- and Pro-rich sequence that seems to be a linker region. The N-terminal amino acid sequence of the puried ChiB coincided precisely with the sequence starting from the Ala30 residue of the deduced amino acid sequence encoded by the chiB gene. The domain from Ala36 to Gln76 showed sequence homology with ChtBD type 3, as classied by the National Center for Biotechnology Information conserved-do-

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FIG. 3. Comparison of amino acid sequences of catalytic domains of ChiA and ChiB with those of other proteins. (A) Amino acid sequence of the active-site region of ChiA compared with those of family 18 chitinases. The SXGG and DXXDXDXE motifs are boxed. A Glu residue identied as a proton donor is marked with an asterisk. The residue number of the rst amino acid in each line is shown on the left. Residues that are identical are indicated by boldface letters. ChiA, N. prasina chitinase A; StChi30, S. thermoviolaceus chitinase 30; SlChiA, S. lividans chitinase A; ScChiA, S. coelicolor chitinase A; BcChiD, B. circulans chitinase D. (B) Amino acid sequence of the catalytic domain of ChiB compared with those of family 19 chitinases. ChiB, N. prasina chitinase B; SgChiC, S. griseus chitinase C; ScChiG, S. coelicolor chitinase G; ScChiF, S. coelicolor chitinase F; StChi35, S. thermoviolaceus chitinase 35; BnChi25, B. napus class I chitinase 25; HvChi26, H. vulgare class II chitinase 26. The residue number of the rst amino acid in each line is shown on the left. Residues that are identical are indicated by boldface letters.

main database. ChtBD of ChiB had similarities to ChiC from S. griseus (85% identity) (21), a family 19 chitinase from Aeromonas sp. strain 10S-24 (65% identity) (28), and ChiA from Arthrobacter sp. strain TAD20 (55% identity) (13). The catalytic domain of ChiB showed high sequence homology to family 19 chitinases, such as ChiC (76% identity) from S. griseus (21), ChiF (73% identity) and ChiG (71% identity) from S. coelicolor (24), Chi35 (70% identity) from S. thermoviolaceus (70% identity) (35), Chi25 (37% identity) from Brassicanapus (7a), and Chi26 (36% identity) from Hordiumvulgare (12). Furthermore, the Streptomyces family 19 chitinases so far reported have deletions of several amino acid residues at two positions compared with those of class I and II chitinases (21, 35). Thus, Streptomyces family 19 chitinases are classied among class IV chitinases. As shown in Fig. 3, the catalytic domain of ChiB also has deletions similar to those of Streptomyces family 19 chitinases. These results indicate that ChiB belongs to the family of class IV chitinases. The amino acid sequence of ChiB puried from N. prasina OPC-131 did not match any region near the N terminus of the deduced polypeptide. However, it coincided precisely with the sequence starting from Gly80 of the deduced amino acid sequence. These results appear to indicate that ChiB is generated by the action of a protease(s) present in the culture supernatant after the initial gene product is secreted into the culture medium. Expression and purication of recombinant proteins. We constructed the expression plasmid coding for ChiA, ChiB, or ChiB by using the thioredoxin fusion protein expression vector pThioHisB. When cell pellets from cultures grown under protein expression conditions were disrupted and centrifuged, the majority of the fusion proteins were found in the supernatant fraction. Each of the fusion proteins was digested with

enterokinase, and then the recombinant ChiA, ChiB, and ChiB were puried by afnity chromatography. The nal enzyme preparations were shown to be homogeneous by SDSPAGE (Fig. 4). The molecular mass of ChiA (32 kDa) calculated from the amino acid sequence without a signal peptide was in reasonable agreement with that estimated by SDSPAGE. However, the molecular masses of ChiB (32 kDa) and ChiB (28 kDa) estimated by SDS-PAGE were higher than their theoretical values (ChiB, 29 kDa; ChiB , 23 kDa) despite the conrmation of their nucleotide sequences and N-terminal amino acid sequences. The puried recombinant enzymes were characterized by using colloidal chitin as the substrate (Fig. 5). The optimum temperatures of ChiA, ChiB, and ChiB were

FIG. 4. SDS-PAGE of recombinant chitinases. Lanes: M, marker proteins; 1, ChiA; 2, ChiB; 3, ChiB .

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FIG. 5. Effects of pH and temperature on chitinase activities. (A) The following buffer systems were used: McIlvaine buffer (pH 3.0 to 6.0), 50 mM Tris-HCl buffer (pH 7.0 to 8.0), and 50 mM glycine-NaOH buffer (pH 9.0 to 11.0). The reaction mixtures were incubated at the optimum temperature for each enzyme (ChiA, 60C; ChiB, 60C; ChiB , 50C) for 10 min. The amount of each enzyme was 20 mU. (B) The reaction was carried out at various temperatures for 10 min at the optimum pH of each enzyme. The buffers used were 50 mM Tris-HCl buffer (pH 7.0) for ChiA and McIlvaine buffer (pH 6.0) for ChiB and ChiB . The amount of each enzyme was 20 mU. F, ChiA; , ChiB; , ChiB .

60, 60, and 50C, respectively. The optimum pHs of ChiA, ChiB, and ChiB were 7.0, 6.0, and 6.0, respectively. Even at pH 9.0, ChiA and ChiB showed 50% of the activities at their optimum pHs. However, ChiB showed signicantly lower activity, in the range of pH 7.0 to 10.0, than ChiA and ChiB. The native ChiA, ChiB, and ChiB showed pH and temperature proles similar to those of the recombinant enzymes (data not shown). Binding study and antifungal activity. To evaluate the role of ChtBD type 3 of ChiB, binding assays were carried out by adding the recombinant ChiB and ChiB to -chitin, -chitin, and Avicel. As shown in Fig. 6, ChiB showed strong binding activity to -chitin and -chitin but weak afnity for Avicel; however, ChiB bound to these polysaccharides much more

weakly than ChiB. These results indicate that ChtBD type 3 of ChiB functions as a chitin-binding domain. When glycol chitin was used as a substrate, the specic activities of ChiB and ChiB were 58.0 and 23.4 U/nmol, respectively. On the other hand, when colloidal chitin was used, ChiB had a 3.9-fold hydrolytic activity against the insoluble substrate compared with ChiB . The recombinant ChiB and ChiB were tested for antifungal activity as indicated by the ability to inhibit the hyphal extension of T. reesei. As shown in Fig. 7, growth of T. reesei was clearly inhibited by 80 g of ChiB, whereas ChiB showed reduced inhibitory activity at the same concentration. ChiA showed no effect on the hyphal extension of T. reesei. These results indicate that ChtBD type 3 of ChiB plays an important

FIG. 6. Binding assays and hydrolytic activities of ChiB and ChiB . (A) Binding assay mixtures contained 1.5 g of ChiB (open bars) or ChiB (solid bars) and 5 mg each of insoluble polysaccharides in 20 mM HEPES-KOH (pH 7.0). The mixtures were permitted to stand for 20 min on ice. The chitinase in the supernatant was measured, and the activity lost from the supernatant was assumed to be the activity bound. (B) Hydrolytic activities of ChiB (open bars) were measured at 60C for 10 min using McIlvaine buffer (pH 6.0), and those of ChiB (solid bars) were measured at 50C for 10 min using the same buffer. Colloidal chitin or glycol chitin was used as a substrate. The error bars represent standard deviations.

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FIG. 7. Antifungal activities of ChiA, ChiB, and ChiB . The mycelium of T. reesei was directly inoculated onto the center of a potato dextrose agar plate. After 24 h at 27C, paper disks were placed around the T. reesei colony, and samples were put onto the disks. 1, blank disk; 2, 80 g of ChiA; 3, 80 g of ChiB; 4, 80 g of ChiB .

role both in the efcient hydrolysis of chitin and in antifungal activity. DISCUSSION In this paper, we describe for the rst time the cloning, sequencing, and characterization of two chitinase genes from an alkaliphilic actinomycete, N. prasina OPC-131. Originally, members of the genus Nocardiopsis were isolated from mildewed grain (3), but the natural habitat of Nocardiopsis is soil, and many of the Nocardiopsis species grow best under moderately alkaline conditions (1, 15, 16, 43). On the other hand, the addition of chitin or cell walls of fungi to acidic soil increases the total number of Streptomyces species and leads to suppression of the growth of root-pathogenic fungi (30, 41). The suppression of root-pathogenic fungi is considered to be due to the action of family 19 chitinases produced by Streptomyces (21, 35). However, whether the amendment of alkaline soil with chitin also leads to an increase in the abundance of Streptomyces species remains to be elucidated. Thus, we isolated a number of alkaliphilic actinomycetes from soil samples using an alkaline medium containing colloidal chitin (32). One of the interesting alkaliphilic actinomycetes, N. prasina OPC-131, which produced two types of chitinases in the presence of chitin (32), was chosen for further investigation. The study of chitinases produced by actinomycetes has been energetically pursued in Streptomyces species; however, the genus Nocardiopsis has not been considered a potential source of chitinolytic enzymes. The chitinases so far reported can be classied in two families, named 18 and 19, based on amino acid sequence similarity (8, 9). Family 18 contains chitinases from virus, bacteria, fungi, and animals and class III and V chitinases from plants. On the other hand, family 19 chitinases, consisting of classes I, II, and IV, have been identied mostly

in plants. Recently, it was demonstrated that family 19 chitinases classied in class IV are distributed widely in Streptomyces species (40). However, it has not been claried whether actinomycetes other than the genus Streptomyces produce family 19 chitinases. We have demonstrated that N. prasina OPC131, which is phylogenetically distant from the genus Streptomyces, produces a family 19 chitinase (ChiB) classied in class IV, together with a family 18 chitinase (ChiA), in the presence of chitin. Furthermore, the strain secretes an 18-kDa chitinbinding protein in the presence of chitin (unpublished data). Recently, it was reported that the alkaliphilic Nocardiopsis sp. strain TOA-1 also produces alkaline hydrolytic enzymes, such as chitinase and protease (17). These results allow us to assume that family 19 chitinases are widely distributed not only in the genus Streptomyces but also in the non-Streptomyces group of actinomycetes. Chitinolytic bacteria usually possess multiple chitinase genes, although the contribution of individual enzymes to chitin degradation has not yet been elucidated in detail. N. prasina OPC-131 possesses at least two types of chitinase genes, encoding ChiA and ChiB. ChiA is a family 18 chitinase consisting of only a catalytic domain. On the other hand, ChiB is a family 19 chitinase consisting of ChtBD type 3 and a catalytic domain. ChtBDs of chitinases have usually been grouped according to the classication of cellulose-binding domains (CBDs) postulated by Tomme et al. (31). Recently, ChtBDs have been seen as independent from CBDs and have been classied into types 1, 2, and 3 in the National Center for Biotechnology Information conserved-domain database (http: //www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). Types 1, 2, and 3 consist of ChtBDs from fungi and plants, viruses and insects, and bacteria, respectively. Among them, ChtBD type 3 possesses a consensus sequence (AKWWTQ) that is well conserved in bacterial ChtBDs. The consensus sequence was also found in the ChtBD of ChiB, which is 41 amino acid residues long and shows homology with ChtBDs classied in type 3. The crystal structure of endoglucanase Z showed that two Trp residues of the CBD belonging to ChtBD type 3 are involved in stacking against the pyranose rings of glucose in cellulose (4, 29). By analogy with the CBD of endoglucanase Z, two similar Trp residues in the AKWWTQ sequence of the ChtBD of ChiB might participate in stacking against the pyranose rings of GlcNAc in chitin. Thus, to investigate the effects of ChtBD of ChiB on hydrolytic and antifungal activities, the activities of ChiB and ChiB were compared. Removal of the ChtBD from ChiB signicantly decreased the enzyme activity of ChiB toward both colloidal chitin and glycol chitin. These results suggest that the ChtBD of ChiB, unlike other ChtBDs, is necessary for the efcient hydrolysis of soluble, as well as insoluble, chitin. Furthermore, the ChtBD of ChiB was also essential for efcient antifungal activity. In Streptomyces species, chitinase production is induced in the presence of chitin and repressed by the addition of glucose to the medium containing chitin. These phenomena are regulated at the transcriptional level. Delic et al. and Ni and Westpheling demonstrated that family 18 chitinase genes of Streptomyces contain similar pairs of direct-repeat sequences in the promoter regions, which have been suggested to be involved in both chitin induction and glucose repression (5, 20). Recently, it was reported that there are similar direct-repeat sequences

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in the upstream regions of several family 19 chitinase genes, such as chi35 and chi25 of S. thermoviolaceus OPC-520, chiC of S. griseus HUT6037, and chiF of S. coelicolor A3 (2, 34). These results suggest that family 18 and 19 chitinase-encoding genes of Streptomyces might be regulated by common regulatory factors or mechanisms. However, we could not nd similar directrepeat sequences in chiA and chiB, and these genes were not subjected to catabolite repression in the presence of glucose. These results indicate that the expression of these chitinase genes is regulated by mechanisms which are different from the regulatory mechanisms of Streptomyces chitinase genes. We are examining the distribution of family 19 chitinase genes in a non-Streptomyces group and the physiological role of actinomycete family 19 chitinases.
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