Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442

Biochimica et Biophysica Acta 1774 (2007) 1099 – 1107 www.elsevier.com/locate/bbapap

Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442 Shiro Itoi a,⁎, Yuna Kanomata a , Yuki Koyama a , Kazunari Kadokura b , Shinsuke Uchida a , Toshiyuki Nishio b , Tadatake Oku b , Haruo Sugita a a

Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-8510, Japan b Department of Agriculture and Biological Chemistry, Nihon University, Kanagawa 252-8510, Japan Received 7 February 2007; received in revised form 14 June 2007; accepted 19 June 2007 Available online 29 June 2007

Abstract Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85–90% identical to the 22nd–41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78–80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597 and K598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600 and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and α-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins. © 2007 Elsevier B.V. All rights reserved. Keywords: Chitinase; Chitinolytic activity; Marine bacteria; Vibrio proteolyticus

1. Introduction Chitin is a highly insoluble β-(1,4)-linked polymer composed primarily of N-acetyl-glucosamine and some glucosamine residues, and is believed to be the second most abundant biomaterial after cellulose. As chitin is a structural material in many marine invertebrates, such as cuttlefish, crab, and lobster as well as fungi and algae, the annual production in marine water alone is estimated to be more than 100 billion tons [1]. Large quantities of chitin in marine waters are rapidly consumed as marine snow. Ocean sediment contains only traces of chitin despite the constant rain of marine snow to the ocean floor; marine snow rarely reaches the ocean bottom because it is degraded as it slowly settles in the water column. ZoBell and Rittenberg [2] reported that many marine bacteria are capable of ⁎ Corresponding author. Tel.: +81 466 84 3679; fax: +81 466 84 3679. E-mail address: [email protected] (S. Itoi). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.06.003

chitin depolymerization. Such chitinolytic processes in marine snow play an important role in the carbon cycle. Chitin is degraded by a sequential process with chitinases (EC 3.2.1.14) followed by N-acetyl-hexosaminidase (EC 3.2.1.52) [3]. Chitinases have been detected in various microorganisms, plants, insects, crustaceans, and vertebrates [4]. They play an important role in normal life cycle functions such as morphogenesis and cell division, or in defence against pathogens. Among these organisms, only chitinivorous bacteria can use chitin as the sole source of carbon and nitrogen. Enzymatic degradation of chitins can effectively depolymerize chitin and may produce novel saccharides. In order to use chitin commercially as a nutrient source and industrial material, we need to obtain an extracellular chitinase with high α-chitin cleavage activity. Austin [5] reported that marine bacteria are excellent sources of chitinases, with bacteria from the Vibrionaceae family being particularly well suited as sources of chitinolytic enzymes. Although many studies concerned

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with chitinases from marine bacteria such as Vibrio furnissii [6], Alteromonas sp. strain O-7 [7], Vibrio anguillarum and Vibrio parahaemolyticus [8], Salinivibrio costicola [9], and Microbulbifer degradans [10] have been reported, endo-type chitinases have not typically been observed in marine bacteria. Recently, Suginta et al. [11, 12] clearly showed that the endochitinase A of Vibrio carchariae, a synonym of Vibrio harveyi, expressed high chitinolytic activity following proteolytic processing of the C terminus of the chitinase. They reported that the proteolytic processing was required for expression of the highly chitinolytic activity of the V. carchariae enzyme [12]. We recently found that the Vibrio proteolyticus isolated from an intestinal tract of Japanese flounder Paralichthys olivaceus [13] showed high-chitinolytic activity against α-chitin from crab shells. In the present report, we describe the highly specific chitinase produced by V. proteolyticus strain No. 442, which rapidly degraded α-chitin, and the characterization and heterologous expression of the V. proteolyticus chitinase encoding gene in wild-type and truncated forms. 2. Materials and methods 2.1. Bacterial strain and growth condition V. proteolyticus was grown and kept on peptone-yeast extract-beef extractglucose (PYBG)-agar plate [14] at 25 °C. Chitinolytic activity of V. proteolyticus was observed as the clear zone of chitin by incubation at 25 °C on a PYBG plate containing 1% α-crystal chitin from crab shells (Funakoshi, Tokyo, Japan). For induction of chitinase expression, the bacterium was cultured in PYBG broth containing 1% α-chitin in shaking flasks at 30 °C.

2.2. Electrophoretic analysis and N-terminal amino acid sequencing of chitin binding protein (CBP) CBPs secreted by V. proteolyticus were recovered batch-wise by addition of α-chitin as follows. V. proteolyticus was cultured in PYBG broth containing 1% α-chitin, and after 48 h, the CBP-bound chitin left without shaking at 20 °C for 5 min. It was then collected and washed five times with 0.1 M Tris–HCl buffer (pH 6.8). The buffer was removed by centrifugation at 12,000×g for 1 min and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer containing 2% SDS, 2% β-mercaptoethanol, 20 mM Tris–HCl (pH 6.8), 40% glycerol, 4 mM EDTA and 0.015% bromophenol blue was added to the resultant pellet. After boiling for 5 min, the suspension was centrifuged at 12,000×g for 15 min, and the resultant supernatant was applied to SDS-PAGE. SDS-PAGE was carried out by the method of Laemmli [15] using 7.5, 10.0 and 12.5% polyacrylamide slab gels containing 0.1% SDS. Gels were stained with 0.1% Coomassie brilliant blue R250 in 50% methanol and 10% acetic acid after electrophoresis. The N-terminal amino acid sequence was determined by the method of Matsudaira [16] as follows. CBPs of V. proteolyticus separated on SDS-PAGE were electrically transferred onto an Immobilon PVDF membrane (Millipore, Billerica, MA, USA) and stained with Coomassie brilliant blue R250. A part of the membrane carrying the blotted protein was cut out with a clean razor. Several membranes bearing the same protein were applied to an ABI Procise 492 protein sequencer (Applied Biosystems, Foster City, CA, USA) in our institute.

2.3. Cloning of a V. proteolyticus gene encoding a full-length chitinase A Genomic DNA was prepared from V. proteolyticus cells according to Ausubel et al. [17], and PCR amplification was performed with a GeneAmp PCR

System 9700 (Applied Biosystems) as follows. The reaction mixture contained genomic DNA as a template, 2 μl of 10× Ex Taq buffer supplied with the kit, 1 μl of 10 μM primers, 1.6 μl of 2.5 mM dNTP and one unit of Takara Ex Taq DNA polymerase (Takara, Otsu, Japan), and the total volume was brought up to 20 μl with sterilized water. PCR consisted of initial denaturation at 95 °C for 1 min followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s extension at 72 °C for 1 min, with a final extension step at 72 °C for 2 min. The conditions of PCR for 5′- and 3′-region of the gene were performed using a DNA Walking SpeedUp Kit (Seegene, Seoul, Korea) according to the manufacturer's instructions. PCR products were cloned into the TA site of pGEM T-Easy vector (Promega, Madison, WI, USA) according to Marchuk et al. [18] using the Escherichia coli DH5α strain as a host bacterium. Sequencing was performed for both strands with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Similarity search was carried out by BLAST search [19] using the DDBJ/EMBL/GenBank databases. Comparison of the deduced amino acid sequences for chitinase A of V. proteolyticus with that appearing in the DDBJ/EMBL/GenBank databases was performed by CLUSTAL W [20].

2.4. Construction of expression vector of a chitinase A from V. proteolyticus V. proteolyticus chitinase A encoding gene was amplified by PCR from V. proteolyticus genomic DNA with primers VpChiEx_Nde_F, containing a NdeI restriction endonuclease site and VpChiEx_Xba_R2, containing an XbaI site (Table 1). The PCR product was ligated into the NdeI/XbaI site of pCold IV DNA (Takara) to yield a chitinase A-expression plasmid, pVPchiAfull. A truncated chitinase-encoding gene was amplified by PCR from V. proteolyticus genomic DNA with primers VpChiEx_Nde_F and VpChiEx_Xba_R1, containing an XbaI site (Table 1). The PCR product was ligated into the NdeI/XbaI site of pCold IV DNA (Takara) to yield a C-terminal deletion chitinase A-expression plasmid, pVPchiAR600. The plasmid constructs pVPchiAfull and pVPchiAR600 were transformed into E. coli DH5α to get the following two recombinant strains: dhFULL and dhR600, respectively.

2.5. Overexpression and purification of recombinants in E. coli To confirm the chitinase activity of E. coli carrying the transgene, the resultant expression strains were cultured on Luria–Bertani (LB)-agar plates containing 1% colloidal chitin, 100 μg/ml ampicillin and 0.1 mM isopropyl-β-Dthiogalactopyranoside (IPTG) at 37 °C for 12 h followed by 15 °C for 24 h, and chitinolytic activity appeared as a clear zone with further incubation at 4 °C for 5 days. Colloidal chitin was prepared from α-crystal chitin using a modification of the method described previously [21]. To overexpress the recombinant proteins, these two strains were cultured in LB broth containing 100 μg/ml ampicillin at 37 °C for 12 h followed by 15 °C for 48 h after addition of 0.2 mM IPTG for full-length recombinant and for 72 h after addition of 0.1 mM IPTG for truncated recombinant. Purification of these recombinants in culture fluid was performed as follows. The recombinants in precipitate fraction with 60% saturated ammonium sulfate were dialyzed against 20 mM phosphate buffer (pH 7.0) containing 0.1 mM phenylmethanesulfonyl (PMSF). The dialysate containing recombinant was applied to a column of DEAE-Toyopearl 650M resin (Tosoh, Tokyo, Japan) equilibrated with 20 mM phosphate buffer (pH 7.0). Proteins were eluted with a linear gradient of 0–400 mM NaCl and fractions containing recombinant were collected.

2.6. Chitinase activity assay Chitinase activity was measured by a modification of the Schales' procedure [22] with colloidal or α-crystal chitin as a substrate. The standard assay was performed at 37 °C in a 1-ml reaction mixture containing 20 mM phosphate buffer (pH 7.0) and 0.5% chitin for 30 min. The reaction was terminated by boiling for 5 min, and the amount of reducing sugar generated was measured by the potassium ferricyanide reduction test. One unit of chitinase activity was defined as the amount of enzyme that produces 1 μmol of reducing sugar (di-Nacetyl-chitobiose) per min at 37 °C.

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Table 1 Primers used for amplifying Vibrio proteolyticus chitinase A gene Application

Partial gene

5′-Part

3′-Part

Full-length Expression

a b

Primer

VproChi_F1 VproChi_F2 Chi_Fw VproChi_R1 VproChi_R3 Chi_GSPR1 Chi_GSPR2 Chi_GSPR3 VproChiAGSPF1 VproChiAGSPF2 VproChiAGSPF3 VproChiFull_F1 VproChiFull_R1 VpChiEx_Nde_F VpChiEx_Xba_R1 VpChiEx_Xba_R2

Sequence a

Location b

5′-GCAGCTCCGACCGCACCAAG-3′ 5′-CCAAGTGTCGATATGTACGG-3′ 5′-GATATCGACTGGGAGTTCCC-3′ 5′-CCTGTCATTGGGTCGTTTGGATC-3′ 5′-CCACCATTTCGCTTCCCA-3′ 5′-AGATGTGAGCCATCAGTGTCG-3′ 5′-CGCACTGGCACTACAACC-3′ 5′-CTGGTATAAGCCACCTTTGC-3′ 5′-GCTGCAGTTCACGGTCACCGTG-3′ 5′-CGTCAACCACGGTGGTCGTTG-3′ 5′-TCAAGTTACTTGGGCAGCGAAG-3′ 5′-TACACTAACGATCATTTGGACTACATC-3′ 5′-CTCTCATTGATATGGTGTTATACAT-3′ 5′-GAACTCTAGATCAGTTGGTCGTGCAATC-3′ 5′-GAAGCTCTAGATCATCGATTGGCAGGAGGATCC-3′ 5′-GAACTCTAGATCAGTTGGTCGTGCAATC-3′

Reference

From

To

664 679 1,534 2,026 3,043 999 961 914 2,880 2,927 3,018 1 3,227 577 2,370 3,124

683 698 1,553 2,046 3,060 1,019 978 933 2,901 2,947 3,039 27 3,251 601 2,402 3,151

This study This study [23] This study This study This study This study This study This study This study This study This study This study This study This study This study

The underlined letters in the primer sequences indicate the recognition sites for the restriction endonucleases used in the construction of the expression plasmids. Location numbers indicate DNA nucleotides from the 5′ end of clone of V. proteolyticus chitinase A gene (AB252739).

2.7. Carbohydrate analysis To determine the reaction mechanism of the V. proteolyticus chitinase, purified enzyme solution (5 μg) was added to 100 μl of 20 mM sodium phosphate buffer (pH 7.0) containing 0.5 mg hexa-N-acetyl-chitohexaose, then the mixture incubated at 37 °C with stirring. Oligosaccharides produced from hexa-N-acetyl-chitohexaose were analyzed by Silica Gel thin-layer chromatography (TLC) using 5:4:3 (v/v/v) n-butanol/methanol/16% aqueous ammonia as the mobile phase. Silica Gel 60 TLC plates (0.25 mm) were obtained from E. Merck (Darmstadt, Germany). After developing the TLC plate being chromatographed twice, compounds were visualized by spraying with an aqueous solution of 2.4% (w/v) phosphomolybdic acid, 5% (v/v) sulfuric acid and 1.5% (v/v) phosphoric acid, followed by heating with a Hakko heating gun (Osaka, Japan) for a few minutes.

2.8. Determination of protein concentration The protein concentration was determined by the bicinchoninic acid (Pierce, Rockford, IL, USA) method using BSA as the standard.

3. Results

CBP (Fig. 2A). The N-terminal amino acid sequence of the 110kDa CBP was determined as APAAPSIDVYGSNNLQFSKI, which was 85 to 90% identical to 22nd–41st residues from the N terminus of chitinase A precursor from other Vibrio species (Fig. 2B). 3.3. Cloning of the V. proteolyticus chitinase A gene Primers to amplify the gene encoding the CBP of V. proteolyticus were constructed as shown in Table 1. Primers VproChi_F1 and VproChi_F2 were designed from N-terminal amino acid sequence of the CBP and referring to the nucleotide sequences reported for chitinase A from V. parahaemolyticus (BA000032), Vibrio alginolyticus (AJ292004) and V. harveyi (AF323180). On the other hand, primers VproChi_R1 and VproChi_R3 were synthesized referring to the nucleotide sequences reported for chitinase A from other Vibrio species described above. A DNA fragment of about 1400 bp was obtained from V. proteolyticus by PCR amplification with

3.1. Chitinolytic activity of V. proteolyticus Chitinolytic activity of V. proteolyticus was observed on PYBG plates containing 1% α-crystal chitin from crab shells. Strains pre-cultured on PYBG containing 1% chitin had higher chitinolytic activity than those pre-cultured without chitin (data not shown). The highest activity against α-chitin was observed following four passages on medium containing 1% chitin (Fig. 1). This strain also rapidly degraded chitin in PYBG broth containing 1% α-chitin (data not shown). 3.2. Determination of CBP SDS-PAGE of CBP yielded two bands of 110 kDa and 130 kDa (Fig. 2A). After SDS-PAGE, we were only able to obtain N-terminal amino acid sequence information for the 110-kDa

Fig. 1. Chitinase activity of Vibrio proteolyticus. The bacteria were inoculated onto the center of a PYBG plate containing 1% α-crystal chitin from crab shells and incubated at 25 °C for 4 days. Arrowheads indicate the position of the inoculation. Photographs A and B represent inoculated agar plates before and after incubation, respectively, showing that white particles (chitin powder) in the panel A disappear in the panel B. Bars represent scale for 10 mm.

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Fig. 2. SDS PAGE pattern and N-terminal amino acid sequence of chitin-binding protein (CBP) of Vibrio proteolyticus. (A) CBP on 7.5% polyacrylamide slab gels containing 0.1% SDS. CBPs were prepared from chitin in 48-h culture medium. Arrows indicate CBP with the N-terminal amino acid sequence. (B) Comparison of the N-terminal amino acid sequence of the V. proteolyticus 110-kDa CBP with those of chitinase A from other Vibrio species. Only different amino acids are indicated and hyphens mark deletions. Numbers in the sequence of the 110-kDa CBP indicate amino acids from the N terminus, whereas those of other Vibrio species represent amino acids from methionine encoded by the initiation codon.

primers VproChi_F1 and VproChi_R1 followed by nested PCR with primers VproChi_F2 and VproChi_R1. Subsequently, PCR with the primer set of Chi_Fw reported by Ramaiah et al. [23] and VproChi_R3 amplified about 1500 bp of DNA fragment and the DNA fragment overlapped with the 1400-bp DNA fragment obtained above. To obtain 5′- and 3′-regions of V. proteolyticus chitinase A by ACP technology (DNA Walking Speed Up Kit) with gene specific primers Chi_GSPR1, Chi_GSPR2 and Chi_GSPR3 for the 5′-part and VproChiAGSPF1, VproChiAGSPF2 and VproChiAGSPF3 for 3′-part, the two DNA fragment, about 800 bp for 5′-part and about 400 bp for the 3′-part, were obtained and overlapped with the nucleotide sequences of 5′and 3′-regions, respectively. Subsequently, to obtain a gene encoding a full-length V. proteolyticus chitinase A, oligonucleotide primers VproChiFull_F1 and VproChiFull_R1 were designed referring to the nucleotide sequence obtained (Table 1). PCR with these primers yielded a DNA fragment of 3251 bp containing putative initiation and termination codons. The coding region of 2550 nucleotides for 850 amino acids included a predicted short signal polypeptide of 26 amino acids and the N-terminal 20-amino acid-sequence of the CBP as described above (Fig. 3). The molecular mass of the mature protein was calculated to be 87,396 Da. The nucleotide sequence of V. proteolyticus chitinase A gene appears in the DDBJ/EMBL/GenBank databases with accession number AB252739.

3.4. Comparison of deduced amino acid sequence of V. proteolyticus chitinase A To investigate the novelty of the V. proteolyticus chitinase based on the primary structure level, the amino acid sequence of chitinase from V. proteolyticus was compared with those of various chitinase A from closely and distantly related bacterial species. Comparison of the amino acid sequence for V. proteolyticus chitinase A precursor with those appearing in the DDBJ/EMBL/GenBank databases revealed that the V. proteolyticus protein was 80, 80, 80, 78, 53, 52 and 46% identical to those of V. alginolyticus (protein database accession no. CAC29091), V. parahaemolyticus (BAC61398), Vibrio splendidus (ZP_00992475), V. harveyi (Q9AMP1), Alteromonas sp. (P32823), Serratia plymuthica (P97034) and Aeromonas hydrophila (DNA database accession no. AF251793), respectively. The glycoside hydrolase family 18 domain containing active site was highly conserved among all bacteria used in this study, whereas the overall amino acid sequence of chitinase A, including the N-terminal domain, the fibronectin type III like domain, and the carbohydrate-binding domain (CBD) were highly conserved among members of the genus Vibrio (Fig. 3). The CBD was classified into family 5 by CAZy database search (http://afmb.cnrs-mrs.fr/CAZY/). In addition, small α + β domain for specific to chitinase subfamily A was highly conserved among members of the genus Vibrio (Fig. 3). The polycystic kidney disease (PKD) domain observed

Fig. 3. Comparison of the amino acid sequence of Vibrio proteolyticus chitinase A precursor protein with other bacterial homologues. Numbers start from the putative N-terminal amino acid of the precursor protein from V. proteolyticus, Vibrio alginolyticus (protein database accession no. CAC29091), Vibrio parahaemolyticus (BAC61398), Vibrio harveyi (Vibrio carchariae, Q9AMP1), Vibrio splendidus (ZP_00992475), Alteromonas sp. (P32823), Aeromonas hydrophila (DNA database accession no. AF251793) and Serratia plymuthica (protein database accession no. P97034). Identical amino acids are indicated by periods. Dashes denote gaps introduced to maximize similarity. Motif structures were searched using the program MOTIF search (http://motif.genome.jp/) with the Pfam database. Chitinase-family 18-active site is indicated by asterisks under the sequence. Small α + β domain is showed by white letters in gray box for glycoside hydrolase family 18 domain. Amino acid residues for proteolytic processing reported by Suginta et al. [12] in endochitinase A from V. harveyi (V. carchariae) and the corresponding position in chitinase A from V. proteolyticus and other Vibrio species are shown by black boxes with white letters. PKD, polycystic kidney disease.

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Fig. 4. Levels of chitinase activity of recombinants secreted from the three Escherichia coli DH5α strains carrying pCold (Control), full-length chitinase A gene (dhFULL) and truncated gene (dhR600). All bacterial colonies were cultured on the same Luria–Bertani plate containing 1% colloidal chitin, 100 μg/ml ampicillin and 0.1 mM isopropyl-β-D-thiogalactopyranoside at 37 °C for 12 h followed by incubation at 15 °C for 24 h. Control strain was generated by transformation with pCold IV DNA plasmid, whereas dhFULL and dhR600 strains were generated by transformation with pVPchiAfull containing a full-length gene encoding chitinase A precursor protein and pVPchiAR600 containing a truncated gene encoding 600 amino acids from the initiation codon of Vibrio proteolyticus, respectively. Black and white arrowheads indicate colony of E. coli carrying expression plasmid and clear zone, respectively. Bar represent scale for 10 mm.

in chitinase A of Alteromonas sp. and A. hydrophila was not detected in chitinase A of the genus Vibrio (Fig. 3). The V. proteolyticus chitinase A appears to lack the Kex-proteolytic cleavage site observed in the V. carchariae endochitinase A [12], similar to chitinase A of Alteromonas sp. and A. hydrophila (Fig. 3). Moreover, amino acid sequences of chitinase lacking Kex-proteolytic cleavage site were found in several Vibrio species such as Vibrio cholerae (NP_232428) and Vibrio vulnificus (NP_763124) on the protein database, but these have not been analyzed. 3.5. Purification and specific activity of the recombinant chitinases As shown in Fig. 3, chitinase A from V. proteolyticus lacks the proteolytic cleavage site. In contrast, the mature protein of V. carchariae chitinase A was produced following C-terminal processing [12]. Therefore, to elucidate the effect of the C terminus of V. proteolyticus chitinase A on the chitinolytic activity, the three E. coli DH5α strains, pCold (control), dhFULL and dhR600, were produced by transformation with expression plasmids, pCold IV DNA, pVPchiAfull and pVPchiAR600, respectively. Incubation of these strains on LB plates containing 1% colloidal chitin showed that clear zones

formed by chitin degradation of dhFULL strain were apparently larger than those of dhR600 strain (Fig. 4). The recombinant proteins expressed in E. coli DH5α were purified as follows. These recombinant chitinases were purified in two steps from 1 litter of culture fluid (Table 2). The column chromatographic step for both recombinants produced a single peak showing chitinase activity. The purified enzymes, which correspond to molecular mass of 87,396 Da for full-length and 61,988 Da for truncated enzymes calculated from the primary structure of the V. proteolyticus chitinase A, gave a single band on SDS-PAGE gel (Fig. 5), indicating that these recombinants are in a high state of purity. The full-length recombinant was purified 584-fold with 1.85% recovering of initial total activity in culture fluid (Table 2). The truncated recombinant was purified 7.1-fold with 29.0% recovering of initial total activity in a fraction of ammonium sulfate precipitation (Table 2). The specific activity of the full-length recombinant, normalized as units per milligram protein, was calculated to be 2.92 ± 0.13 against colloidal and 2.37 ± 0.23 against α-chitins, whereas that of the truncated recombinant was calculated to be 0.20 ± 0.02 against colloidal and 0.20±0.02 against α-chitins (Table 3). Deletion of the C terminus of the V. proteolyticus chitinase A resulted in about 95% decreases in the specific activity against colloidal and α-chitins, normalized as units per μmol protein (Table 3).

Table 2 Purification of recombinants from culture fluid of the Escherichia coli DH5α strain carrying the Vibrio proteolyticus chitinase gene Recombinanta

Purification stepb

Total activity (U)

Specific activity (U/mg protein)c

Yield (%)

Fold

Full-length

Culture fluid (NH4)2SO4 precipitation DEAE Toyopearl 650M (NH4)2SO4 precipitation DEAE Toyopearl 650M

20.5 3.40 0.40 3.24 0.94

0.005 0.032 2.92 0.028 0.20

100 16.6 1.85 100 29.0

1 6.4 584 1 7.1

Truncated a

Recombinants were purified from 1 L culture medium. Chitinase activity was not detected in the culture fluid of the Escherichia coli carrying the truncated gene. c Specific activity assay were carried out against colloidal chitin at 37 °C and 20 mM phosphate buffer (pH 7.0). One unit of chitinase activity was defined as the amount of enzyme that produces 1 μmol of reducing sugar (di-N-acetyl-chitobiose) per min. b

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Fig. 5. SDS-PAGE patterns of purified chitinase recombinants. Proteins were purified from culture fluid of the Escherichia coli DH5α strains carrying fulllength and truncated chitinase genes. SDS-PAGE was carried out using 10% polyacrylamide gel containing 0.1% SDS. Gels were stained with 0.1% Coomassie brilliant blue R-250 after electrophoresis. Lane M, molecular weight marker; lane 1, 0.2 μg of full-length recombinant; lane 2, 0.2 μg of C terminus truncated recombinant.

3.6. Carbohydrate analysis The oligosaccharide produced by the action of full-length and truncated recombinants purified from culture fluid of E. coli on hexa-N-acetyl-chitohexaose was analyzed using TLC. Both recombinants showed similar hydrolyzing patterns for hexa-Nacetyl-chitohexaose with various reaction times of 0, 0.5, 1, 2 and 8 h. Time course experiments with full-length and truncated recombinants were conducted to investigate the hydrolysis pattern of hexa-N-acetyl-chitohexaose. Fig. 6 shows that after incubating a mixture containing hexa-N-acetyl-chitohexaose and a full-length or truncated recombinant for 8 h, the amount of di-N-acetyl-chitobiose is increased at each time point from 0.5 to 8 h. At 8 h, hexa-N-acetyl-chitohexaose was hydrolyzed to di-Nacetyl-chitobiose for the most part, while tri-N-acetyl-chitotriose was slightly detected (Fig. 6). The recombinant chitinase seemed to hydrolyze hexa-N-acetyl-chitohexaose producing di-Nacetyl-chitobiose and tetra-N-acetyl-chitotetraose, followed by rapid hydrolysis of tetra-N-acetyl-chitotetraose to di-N-acetylchitobiose (Fig. 6). 4. Discussion Marine bacteria, especially members of the Vibrionaceae, are excellent sources of chitinases [5,24], and α-crystal chitindegrading Vibrionaceae bacteria possess endo-type chitinase gene(s) [24]. Suginta et al. [11] reported that V. carchariae, a synonym of V. harveyi, is the best source of endochitinase out of the fourteen Vibrio species including Vibrio aestuarianus, V. alginolyticus, Vibrio campbellii, V. fisheri and V. harveyi that were screened in their study. In our study, chitinolytic activity was higher in V. proteolyticus cultures after passage on 1%

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α-crystal chitin-containing PYBG agar medium (data not shown). Cells passaged four times on this medium degraded most of the αchitin in the PYBG agar medium in 4 days at 25 °C (Fig. 1), suggesting endo-type chitinase production by V. proteolyticus. The overall primary structure of the 110-kDa CBP secreted by V. proteolyticus in this study was highly identical to that of V. carchariae endochitinase A precursor [12], suggesting that the V. proteolyticus protein is an endo-type chitinase (Fig. 3). Additionally, the recombinant chitinases expressed in E. coli exhibited endochitinase activity with producing tri-N-acetylchitotriose from hexa-N-acetyl-chitohexaose (Fig. 6). Suginta et al. [11,12] reported that the molecular mass of V. carchariae endochitinase A was 63–66 kDa on SDS-PAGE and mass spectrometry following proteolytic processing of the C terminus from a 95-kDa-precursor protein. However, the V. proteolyticus chitinase A we obtained did not appear to be processed because of its higher apparent molecular weight on SDS-PAGE gel (Fig. 1). The amino acid sequence of the Kexproteolytic cleavage site (R597 K598) in the V. carchariae chitinase A was substituted by R600A601 in the V. proteolyticus chitinase A (Fig. 3). In this position, chitinase A of other Vibrio species have the sequence RK (Fig. 3), suggesting proteolytic processing at this site as in the case of V. carchariae endochitinase A. On the protein database, amino acid sequence of chitinases having Kex-proteolytic cleavage site could be found in the several Vibrio species, but the processing manner and functional study of those have not been reported. In addition, amino acid sequences of chitinase A from other bacterial species such as Alteromonas sp. [7] and Aeromonas hydrophila [25], which are not subject to C-terminal processing, the RK sequence is not conserved (Fig. 3) and the apparent molecular mass of these chitinases is 85–92 kDa on SDS-PAGE, corresponding to the mass estimated from the deduced amino acid sequences. Alternatively, the molecular mass of the protein secreted by V. proteolyticus (Fig. 3) was larger than that calculated from the deduced amino acid sequence (87,396 Da) and shown in SDSPAGE pattern of the full-length recombinant (Fig. 5). The result suggested that the V. proteolyticus chitinase A might be due to posttranslational modifications such as glycosylation, but further studies along this line are required. The C terminus of V. proteolyticus chitinase A corresponding to the processed C-terminal polypeptide of V. carchariae Table 3 Specific activity of recombinant chitinases, full-length and truncated proteins of Vibrio proteolyticus, expressed in the Escherichia coli DH5α strains carrying pCold constructs a Recombinant

Substrate

Specific activity b, c (units/mg)

(units/μmol)

Full-length

Colloidal chitin α-Crystal chitin Colloidal chitin α-Crystal chitin

2.92 ± 0.13 2.37 ± 0.23 0.20 ± 0.02 0.20 ± 0.02

255.2 ± 11.4 207.1 ± 20.1 12.4 ± 1.2 12.4 ± 1.2

Truncated

a Enzyme activity assays were carried out at 37 °C and 20 mM phosphate buffer (pH 7.0). b One unit of chitinase activity was defined as the amount of enzyme that produces 1 μmol of reducing sugar (di-N-acetyl-chitobiose) per min. c Means of three replicates ± SD.

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Fig. 6. TLC analysis of oligosaccharide produced by incubating hexa-N-acetyl-chitohexaose with recombinant chitinases expressed in Escherichia coli DH5α. Purified enzyme solution (5 μg) was added to 100 μl 20-mM sodium phosphate buffer (pH 7.0) containing 0.5 mg hexa-N-acetyl-chitohexaose, then the mixture incubated at 37 °C with stirring. After developing the TLC plates, reaction products produced after various incubation times were visualized using the phosphomolybdic acid reagent. Lane S, N-acetylchitooligosaccharide standards. G1, N-acetyl-glucosamine; G2, di-N-acetyl-chitobiose; G3, tri-N-acetyl-chitotriose; G4, tetra-N-acetyl-chitotetraose; G5, penta-N-acetyl-chitopentaose; G6, hexa-N-acetyl-chitohexaose.

chitinase A contained fibronectin type III-like domain and CBD (Fig. 3). Wu et al. [26] showed that multiple CBDs in the C terminus of A. hydrophila chitinase Chi92 were important for substrate binding and catalytic activity. Therefore, chitinase activity of the full-length recombinant V. proteolyticus chitinase A was compared with a C-terminal truncated version containing residues 1–600, corresponding to mature chitinase A produced by V. carchariae [12]. The chitinase activity of the full-length recombinant protein was higher than that of the truncated recombinant (Table 2), suggesting that the 250 amino acids of the C-terminal region of the V. proteolyticus chitinase A are required for hydrolytic activity against insoluble substrates such as α-crystal and colloidal chitins but do not appear to be as important for degrading soluble substrates such as tetra-Nacetyl-chitotetraose and hexa-N-acetyl-chitohexaose. This result is similar to a previous report for A. hydrophila chitinase Chi92 [26]. In addition, fibronectin type III-like domains are commonly found in chitinase A of various Vibrio species, suggesting that the domain facilitates the interaction of chitinase A with chitin [27]. However, Suginta et al. [12] reported that the expression of chitinase A activity in V. carchariae required C-terminal processing. The difference in chitinase activity expression following C-terminal processing of chitinase A from V. proteolyticus and V. carchariae is interesting, but remains unexplained. Further analysis of the unknown-function domain between the fibronectin type III-like domain and the CBD in the C-terminal region would be also required to reveal the role of the domain. The three-dimensional structure of Serratia marcescens chitinase has been reported [27] and the sequences and expression patterns of various S. marcescens strains have also been investigated [28,29]. However, crystallographic analysis of Vibrio chitinases has not been reported except for V. carchariae

chitinase A [30]. There have been no studies on the importance of the C-terminal region of Vibrio chitinases corresponding to the C-terminal 250 amino acids of V. proteolyticus chitinase A, whereas the C terminus has been shown to have functional importance in chitinases from other species [31–34]. The truncated chitinase in this study lacks about one third of the C-terminal molecule. It could affect the structure of the active site and surrounding areas, resulting in the lower activity of the truncated enzyme. The enzymes from other sources are folded before processing, but in the case of this truncated recombinant, it had to be folded without the C terminus, resulting in improper folding. On the other hand, the discrete domains, fibronectin type III-like domain and CBD, might not affect the tertiary structure of catalytic domain in the V. proteolyticus chitinase A. However, the difference in the tertiary structure of the N-terminal two thirds of the molecule between full-length and truncated enzymes could not be investigated in this study. In conclusion, a novel chitinase A with high specific activity secreted by V. proteolyticus has been identified and cloned, and both full-length and C-terminal truncated recombinant proteins were expressed in E. coli and characterized. We showed that the C terminus of the V. proteolyticus chitinase A was important for expression of high specific activity. V. proteolyticus and V. carchariae are known to be pathogens to Artemia [35] and prawn [36,37], respectively. Chitinases of V. proteolyticus and V. carchariae might be required for virulence, since Artemia and prawn possess chitin exoskeletons. Further studies along this line are required. Acknowledgments This study was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture,

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Sports, Science and Technology, a Nihon University Research Grant for 2005, and the Open Research Center Project of the Ministry of Education, Culture, Sports, Science and Technology.

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