Cloning and characterization of the constitutively expressed chitinase C gene from a marine bacterium, Salinivibrio costicola strain 5SM-1

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 96, No. 6, 529–536. 2003

Cloning and Characterization of the Constitutively Expressed Chitinase C Gene from a Marine Bacterium, Salinivibrio costicola Strain 5SM-1 RATCHANEEWAN AUNPAD1 AND WATANALAI PANBANGRED1* Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand1 Received 7 July 2003/Accepted 9 September 2003

The chitinase C gene (chiC) encoding chitinase C (ChiC) from Salinivibrio costicola 5SM-1 was cloned and the nucleotide sequence was determined. S. costicola ChiC was expressed constitutively and repressed by glucose. A single operon composed of two complete open reading frames organized in the order of chiB, chiC and one partial open reading frame of chiA was found in the same transcriptional direction. chiC was composed of 2610 bp encoding for 870 amino acids with a calculated molecular mass of 94 kDa including a signal peptide. Analysis of the deduced amino acid sequence alignment revealed a domain structure consisting of an N-terminal catalytic domain, followed by a putative cadherin-like domain and two type 3 chitin-binding domains located at the C terminus. Mutation of three highly conserved amino acid residues, two aspartic acids (Asp-313 and Asp-315) and one glutamic acid (Glu-317) resulted in a complete loss of chitinase activity against colloidal chitin substrate. This suggests that these amino acid residues which reside in the putative catalytic domain play an important role in catalysis. chiB classified as a chitinbinding protein with C-terminal type 3 chitin-binding domain was composed of 390 amino acids with the molecular mass of 43 kDa and does not have any detectable chitinase activity. Chitinase C was identified as an exo-type chitinase releasing chitobiose as a major product from colloidal chitin hydrolysis. [Key words: chitinase, cloning, site-directed mutagenesis, Salinivibrio costicola]

Chitin, an insoluble linear biopolymer with b-(1,4) linkage of N-acetylglucosamine, is the second most abundant renewable biomass in the environment besides cellulose and possibly the most plentiful biopolymer in the marine environment (1). The excess chitin synthesized in the aquatic biosphere is completely hydrolyzed by chitinolytic bacteria in order to restore the usable carbon and nitrogen resource to the marine ecosystem (2). Chitin is also present in the wall of higher fungi, in the major exoskeleton components of insects and crustaceans as well as many groups of invertebrates and as an extracellular polymer of some bacteria (3). The eradication and recycling of chitin-containing waste particularly in the seafood industry by using chitinase enzymes, especially marine chitinase, assumes commercial and economic importance (4). Chitinase (EC 3.2.1.14) is a glycosyl hydrolase which catalyzes the enzymatic degradation of chitin polymer (5). Chitinases have been detected in a vast array of organisms including bacteria, insects, viruses, plants and animals and may have various functions in differ-

ent organisms. Bacterial chitinases are produced to meet nutritional needs, so that chitin can be used as carbon and nitrogen sources (3). Plant chitinases function in self-defense against chitin-containing pathogens whereas yeast and fungal chitinases are required for development and growth (3). Chitinase genes from various marine bacteria have been cloned and characterized (6, 7). Typically chitinase enzymes are composed of at least three functional domains, a catalytic domain, chitin-binding domain, and cadherin-like domain or fibronectin type III like domain (8, 9). Amino acid sequence alignment and site-directed mutagenesis suggest that an aspartate and a glutamate, separated by three residues, are directly involved in the catalytic action of chitinase A1 (10). In this study, we describe the cloning of a single operon encoding a part of the chitinase A (chiA) gene, and the complete chitinase B (chiB) and C (chiC) genes, that encode a 43-kDa chitin-binding protein and a 94-kDa chitinase. We furthermore identified amino acid residues involved in the catalytic activity of ChiC, which is constitutively expressed in Salinivibrio costicola.

* Corresponding author. e-mail: [email protected] phone: +66-2-201-5927 fax: +66-2-201-5926 The costs of publication of this article were supported in part by Grants-in-Aid for Publication Scientific Research Results from the Ministry of Education, Culture, Sports, Science and Technology, Japan (no. 143050).

MATERIALS AND METHODS Bacterial strains and plasmids A halophilic chitinase producer, strain 5SM-1, identified by Accugenix (Newark, DE, USA) as S. costicola from the 16S rRNA gene sequence data 529

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(Genbank/EMBL Database accession no. AF057016), was screened from salted mud in Samutsongkram, Thailand. It was grown in LB–NaCl (1% tryptone, 0.5% yeast extract and 3% NaCl) at 30°C for 4–6 h and used for chromosomal extraction and enzymatic assay at designated time points. Plasmid pGEM7Zf(+) was used as a cloning vector. Escherichia coli DH5a was used as a cloning host. E. coli DH5a and E. coli transformants were grown in LB-medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl) supplemented with ampicillin (50 mg/ml) when needed. Assay of chitinase activity S. costicola 5SM-1 was grown in LB–NaCl, LB–NaCl supplemented with either 0.1% colloidal chitin, 0.1% colloidal chitin and 1% glucose, 1 mM N-acetylglucosamine (GlcNAc), or 1 mM N,N¢-diacetylchitobiose. Over time, viable cell count was determined by direct spreading of the proper diluted culture broth (100 ml) on LB–NaCl agar, and aliquots were also removed and centrifuged at 6000 ´g (Sorvall, Newton, CT, USA). Chitinase in the supernatant was then assayed as described below. Glucose concentration (g/l) was determined using a biochemistry analyzer (YSI 2700 SELECT; YSI, Yellow Springs, Ohio, USA) with a YSI 2365 glucose membrane. SDS–PAGE and chitinase activity detection SDS–PAGE was performed in a 10% (w/v) polyacrylamide gel as described previously (11). Chitinolytic activity was detected using glycol chitin as a substrate according to the method described by Trudel and Asselin (12) with minor modification. Casein (1%) was incorporated in gel washing buffer (13). Cloning and sequencing of chitinase gene Chromosomal DNA of S. costicola was prepared by phenol chloroform extraction. Cloning of the chitinase gene was performed by digestion of chromosomal DNA with BamHI and then followed by ligation to BamHI-linearized and dephosphorylated pGEM7Zf(+). The ligated products were used to transform competent E. coli DH5a and the transformants were plated on LB-agar containing ampicillin (50 mg/ml), previously spread with 40 ml of 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal, 20 mg/ml). White colonies were then transferred to LB-agar plate overlayed with LB-soft agar (1% agar) containing 1% colloidal chitin supplemented with 50 mg/ml ampicillin. Among the approximate 2000 white colonies, four colonies forming a clear halo were picked for further analysis. The plasmids from the four clones were analyzed and found to have the same 6-kb insert, which was designated as pCHISI. Analysis of the cloned chitinase gene A restriction map was constructed using a single or multiple enzyme digestion of recombinant plasmid. The nucleotide sequence was determined using an automated DNA sequencer (PE Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using DNASIS (Hitachi Software America, Cascade, CO, USA). Homology searches in Genbank were carried out with a BLAST program. Comparison of amino acids was carried out using the CLUSTAL W program. N-terminal amino acid sequence analysis The N-terminal amino sequences of ChiC from S. costicola and a recombinant E. coli were analyzed using a protein sequencer (Applied Biosystems protein sequencer model 476A). Enzyme preparation and assay The culture supernatant (200 ml) from S. costicola and E. coli transformants harboring plasmids with wild type chiC (pCHISI) or mutated chiC gene was precipitated using 65% (NH4)2SO4. The precipitate was dissolved in 8 ml of 25 mM Tris–HCl buffer (pH 8.0) and dialyzed against the same buffer (dialysis tubing, M.W. cut off at 12,000; Sigma, St. Louis, MO, USA) at 4°C for 24 h with three changes of buffer. The dialyzed samples were recentrifuged and the supernatant was adjusted to 10 ml of total volume of the same buffer. Chitinase activity was determined by the colorimetric method (14) using 2.5% colloidal chitin (15) as a substrate. Each enzymatic assay was performed in triplicate. One unit of enzyme was defined as the

amount of enzyme that released 1 mmole of GlcNAc equivalent in 1 min under the above conditions. Protein concentration was measured by the method of Bradford (16) using bovine serum albumin as a standard. Thin-layer chromatography (TLC) of sugar products Concentrated chitinase enzyme (150 ml containing 15 mU of chitinase) from E. coli transformant (pCHISI) was incubated with 150 ml of 5% colloidal chitin at 37°C for 0.5–24 h. The hydrolysis products were analyzed on silica gel 60 (Merck, Darmstadt, Germany) using butanol: glacial acetic acid:water (12: 3:5) (4). TLC plates were run twice and the products were visualized by spraying with freshly prepared 20% H2SO4 in ethanol, followed by heating at 150°C for 10 min. Site-directed mutagenesis The site-directed mutagenesis was carried out according to the procedure described previously (17). The PCR mutagenesis method relies on an overlap extension of the ends of PCR-amplified DNA strands. pCHISI was used as a template for mutagenesis. The synthetic oligonucleotide primers used for site-directed mutgenesis were as follows: 5¢-GCATTGT CACTTTTCCCTA-3¢ (M1), 5¢-GTTGACCATCCTGTGCTA-3¢ (M4), 5¢-GATGGGGTCGGCATTGACT-3¢ (D313G2 for Asp313 ® Gly), 5¢-GTCGACATTGGCTGGGAATA-3¢ (D315G2 for Asp315 ® Gly) and 5¢-ATTGACTGGGGATACCCAG-3¢ (E317G2 for Glu317 ® Gly). The numbers indicate the position of the substituted amino acid residues of the deduced amino acid sequence starting from methionine. The underlined sequences in each primer were targeted for glycine substitution. PCR was carried out with a thermal cycler 2400 (PE Applied Biosystem) using Pfu DNA polymerase (Promega, Madison, WI, USA). The final PCR products were digested with NotI and EcoRV to generate the cassette containing the mutagenized nucleotide and ligated to pCHISI cut with the corresponding restriction enzymes. Three mutant plasmids harboring a single amino acid substitution by glycine in the chitinase gene at position Asp-313, Asp-315 or Glu-317 were designated as pD313G, pD315G and pE317G, respectively. The mutant plasmids were confirmed by restriction mapping and nucleotide sequencing. Nucleotide sequence accession number The nucleotide sequences of chiB and chiC genes for S. costicola strain 5SM-1 were submitted to the GenBank Database under the accession nos. AY207003 and AF261749, respectively.

RESULTS Chitinase activity in S. costicola 5SM-1 S. costicola 5SM-1 showed almost identical growth curves in all media with or without inducer or glucose (Figs. 1A and 2A). Chitinase activity was detected in the culture supernatant of S. costicola 5SM-1, and was produced constitutively in LB– NaCl medium (Fig. 1B) without requiring any inducers. However, the addition of N,N¢-diacetylchitobiose at 1 mM could increase the enzyme production by around 20% (Fig. 1B) when compared to other medium. Neither colloidal chitin nor N-acetylglucosamine could act as an inducer of this S. costicola chitinase (Fig. 1B). When the cells were cultured in LB–NaCl, the strain produced a high level of chitinase activity comparable to that of the cultures grown on media containing either colloidal chitin or GlcNAc (10– 11 mU/ml). However, cells grown in LB–NaCl supplemented with colloidal chitin showed a slight decrease in chitinase activity. Chitinase activity gel staining of the concentrated culture supernatant of S. costicola growing in LB–NaCl medium, with or without colloidal chitin (Fig. 1C), showed

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FIG. 1. Growth and chitinase activity of S. costicola 5SM-1 grown on different media. Cells were grown in LB–NaCl (closed circles), LB– NaCl with 0.1% (w/v) colloidal chitin (open circles), LB–NaCl with 1 mM N,N¢-diacetylchitobiose (closed triangles) and LB–NaCl with 1 mM N-acetylglucosamine (open triangles). The viable cell count (A) and chitinase activity (B) are shown as CFU/ml and mU/ml of culture broth, respectively. The enzyme activity was the average of triplicate determinations. Bar indicates SD. (C) SDS–PAGE of 65% ammonium sulfate precipitates of culture supernatant of S. costicola growing in LB–NaCl medium with (lanes 1 and 3) and without 0.1% colloidal chitin (lanes 2 and 4), respectively. Lane M, Standard protein marker in kDa; lanes 1 and 2, Coomassie Blue stained gels; lanes 3 and 4, chitinase activity gel staining.

only a single 92-kDa chitinase band. N-terminal amino acid sequence analysis revealed the sequence of APSTPSL. There was no other activity band detected under this condition in which glycol chitin could serve as a substrate for both endo- and exo-type chitinase (8, 18). This result indicated that a 92-kDa chitinase was constitutively expressed in S. costicola and was a major chitinase produced by this bacterium. Glucose (1%) in the presence of 0.1% colloidal chitin seemed to inhibit overall enzyme production, but the activity rapidly increased after 20 h due to glucose depletion (Fig. 2A). Chitinase production increased rapidly during 4 to 20 h of cultivation in all media without glucose (Fig. 1B). Addition of 1% glucose to the cell culture at mid log phase (8 h) affected the increase in chitinase production. Chitinase activity was maintained at 9 mU/ml at 8–16 h and increased to a maximum level when glucose in the media was completely consumed (Fig. 2B). Cloning and sequencing of chitinase genes E. coli transformant harboring the chitinase gene displayed a clear halo around the colony within 2 d. The plasmid from the clone harbored a 6-kb insert and was designated as pCHISI (Fig. 3). The cloned chitinase gene and enzyme were designated as chiC and chitinase C (ChiC), respectively. Upstream of chitinase C, there was a single reading frame encoding chitinase B (ChiB), which was classified as a chitinbinding protein by sequence alignment. A single open reading frame of 2610 bp coding for 870

amino acids with a characteristic signal peptide sequence of 22 amino acids was identified starting from the ATG start codon. The cloned chitinase C was secreted into the culture medium of E. coli transformant, indicating that the signal peptide is functional in E. coli. The calculated molecular mass of chitinase was 94 kDa including the signal peptide and 92 kDa for the mature enzyme. The N-terminal amino acid sequence of the cloned chitinase C from the E. coli transformant was APSTPSL, which was the same as that of the 92 kDa chitinase from S. costicola (Fig. 1C). The results of the N-terminal amino acid sequences suggested that the putative cleavage site was between alanine residues 22 and 23 in both bacteria. The results from cloning and activity detection (Fig. 1C) in addition to the N-terminal sequence analysis therefore suggested that ChiC (a 92 kDa chitinase) was constitutively expressed, and might be a major chitinase produced by this marine bacterium. The potential ribosome-binding site for the chiC gene, AAGGAG, was found upstream of the start codon. There are only nine nucleotides between the TAA termination codon of chiA and chiB and the start codon ATG of chiB and chiC, respectively. This result suggested that the three genes were in the same operon and might be controlled by the promoter upstream of the chiA gene. However, another putative functional promoter driven by E. coli RNA polymerase, located in the region within the PstI-BstXI fragment, was found by deletion analysis (pCHISII and III) (Fig. 3). The inverted repeat se-

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FIG. 2. Effect of glucose on chitinase production of S. costicola 5SM-1. Cells were grown in LB–NaCl with 0.1% (w/v) colloidal chitin in the presence of 1% (w/v) glucose from 0 h (A) or addition of 1% (w/v) glucose after 8 h of cultivation (B). The viable cell count (closed circles), chitinase activity (open circles), and glucose concentration (triangles) are shown as CFU/ml, mU/ml, and g/l of culture broth, respectively. The enzyme activity and glucose concentration were the average of triplicate determinations. Bar indicates standard deviation. Arrow indicates time when glucose was added to cell culture.

quence, which is composed of a 6-bp stem and a 13-bp loop, was located downstream of the termination codon (TGA) of chitinase. This sequence is a putative rho-independent transcription terminator. Deduced amino acid sequence analysis The deduced amino acid sequence of ChiC was compared with available protein sequences from the Genbank and SWISS-PROT databases. The chitinase enzyme (ChiC) from S. costicola 5SM-1 showed the highest homology (74% homology with 64% identity) with chitinase C from Pseudoalteromonas sp. strain S91 (6) and showed high homology (72% homology with 60% identity) with chitinase A from Alteromonas sp. strain O-7 (7). Amino acid sequence comparison showed that ChiC from strain 5SM-1 is composed of three major domains, i.e., a catalytic domain, a cadherin-like domain, and a chitin-binding domain. The deduced amino acid sequence at a position 257 to 326 from the start codon was homologous to the catalytic region of the chitinase enzyme in family 18 of the glycosyl hydrolases, including fungal, animal and bacterial chitinases and plant chitinases of classes III and V (5), as shown in Fig. 4. In particular, the region showed sequence similarity to Alteromonas sp. strain O-7 ChiA (78% identity), Aeromonas caviae ChiA (71% identity), B. circulans ChiA1 (42% identity), B. thuringienesis ChiA71 (37% identity), Serratia marcescens ChiA (71% identity) and Kurthia zopfii

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FIG. 3. Restriction map of insert fragment in pCHISI harboring chiA, chiB and chiC genes. The domain structure of ChiB, ChiC and deletion analysis of 6-kb insert are shown in pCHISI. The box indicates the coding sequence and the domain structure of chitinase B and C. Solid squares, Signal peptide; upward diagonal square, catalytic domain; dotted square, cadherin-like domain; checker board squares, chitin-binding domain. Arrow indicates direction of transcription. The abilities or inabilities of deletion clones to produce a clear zone on colloidal chitin agar plates are indicated to the right hand of each clone (+, having chitinase activity; -, no chitinase activity).

FIG. 4. Alignment of putative catalytic domain. Sco ChiC, S. costicola 5SM-1 chitinase C; Alt ChiA, Alteromonas sp. strain O-7 chitinase A (A40633) (7); Aer ChiA, Aeromonas caviae chitinase A (U09139) (34); Bcl ChiA1, Bacillus circulans chitinase A1 (P20533) (9); Btp ChiA71, B. thuringenesis chitinase A71 (AAB58579) (24); Ser ChiA, S. marcescens chitinase A (AB015996) (29); Kuz Chi, Kurthia zopfii chitinase (D63702) (unpublished). Amino acid residues conserved in at least five of the seven sequences are outlined in black boxes. Asterisks indicate putative amino acid sequences needed for catalytic activity and targeted for mutagenesis.

chitinase (41% identity). Two conserved amino acid residues, aspartic acid (Asp-313) and glutamic acid (Glu-317), were found, corresponding to Asp-200 and Glu-204 which are essential for the chitinase activity in ChiA1 of B. circulans (Fig. 4) (10). In addition, one more aspartic acid residue (Asp-315) was also found to be conserved in most chiti-

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FIG. 6. Thin-layer chromatography of products from colloidal chitin hydrolysis by ChiC from E. coli transformant. Lane S, a 10 ml mixture of 0.5% each of N-acetylglucosamine (GlcNAc), chitobiose (GlcNAc)2 and chitotriose (GlcNAc)3; lanes 1–6, 10 ml of product from ChiC colloidal chitin hydrolysis for 0.5, 1, 2, 3, 6 and 24 h, respectively; lane 7, a control, spotted with 10 ml of 2% colloidal chitin only. FIG. 5. (A) Alignment of the putative cadherin-like domain. Sc_ChiCR1, S. costicola 5SM-1 reiterated segment 1 of ChiC; Cp_ChiAR1 and Cp_ChiAR2, reiterated cadherin-like domain segments 1 and 2 of Clostridium paraputrificum chitinase A (AB012764) (19); Cp_ChiBR1 and Cp_ChiBR2, reiterated Cadherin-like domain segments 1 and 2 of Clostridium paraputrificum chitinase B (AB001874) (8); Vh_ChiAR1 and Vh_ChiAR2, reiterated Cadherin-like domain segments 1 and 2 of V. harveyi chitinase A (U81496) (22). Amino acid residues conserved in at least four of the seven sequences are shaded in black. Aspartic residue identified as Ca2+-binding site of uvomorulin, a member of the cadherin superfamily, is indicated by an asterisk. (B) Alignment of putative chitin-binding domain. Sco ChiC1, S. costicola 5SM-1 chitinase C fragment 1; Sco ChiC2, S. costicola 5SM-1 chitinase C fragment 2; Vbh ChiA, V. harveyi chitinase A (U81496) (22); Alt ChiA, Alteromonas chitinase A (A40633) (7); Ser ChiA, S. marcescens chitinase A (AB015996) (29); Btp ChiA71, B. thuringenesis subsp. pakistani chitinase A71 (AAB58579) (24); Sco ChiB, S. costicola 5SM-1 chitinase B. Amino acid residues conserved in at least four out of the six sequences are boxed.

nases (Fig. 4). The middle region (residues 693–782) showed sequence similarity to the cadherin-like domain in ChiA and ChiB of Clostridium paraputrificum and ChiA of Vibrio harveyi (8, 19), as shown in Fig. 5A. An aspartate (Asp-720) residue identified as the Ca2+-binding site of uvomorulin, a member of the cadherin superfamily (20), was also found in this region of ChiC. There is only one cadherin-like segment found in ChiC of S. costicola 5SM-1. The C-terminal region of ChiC (residues 784 to 826) is suggested to be the putative chitin-binding domain (Fig. 5B). There are two putative chitin-binding domains (fragments 1 and 2) found in the C terminus of ChiC (Fig. 5B). Conserved aromatic amino residues important for chitin binding activity, principally tryptophan and tyrosine, were also found in the C-terminal part of ChiC, suggesting the existence of two chitin-binding domains in the C terminus of ChiC from S. costicola 5SM-1. ChiB from S. costicola showed similarity with chitinase B from Pseudoalteromonase sp. strain S91 (65% homology with 52% identity) (6), chitin-binding protein 1 or cpb1 from Alteromonas sp. strain O-7 (67% homology with 50% identity) (21). They all lack a catalytic domain for chitinase activity. Therefore, the three encoding proteins for these three genes did not have chitinase activity. Furthermore, cloning of the chiB gene in the same orientation with lacZ promoter

did not show any detectable chitinase activity (pCHISV and VI) (Fig. 3). On the other hand, the C-terminal region of ChiB (residue 345 to 387) and the two chitin-binding domains of ChiC showed significant similarity with type 3 chitin-binding domain (Fig. 5B), as classified by the National Center for Biotechnology Information conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/ cdd.shtml). According to the database of Carbohydrate Active enZYmes (http://afmb.cnrs-mrs.fr/CAZY), these C-terminal regions of both ChiB and ChiC were classified as family 5 of carbohydrate binding module (CBM). Product analysis of colloidal chitin digestion by ChiC The chitinolytic products from digestion of colloidal chitin as a substrate by the cloned ChiC were analyzed by TLC. As visualized by time-dependent incubations (0.5–24 h) and followed by TLC analyses (Fig. 6), the main product from enzymatic reaction was N,N¢-diacetylglucosamine (GlcNAc)2. A small amount of N¢-acetylglucosamine (GlcNAc) was detected after 24 h. This result suggested that this constitutively produced ChiC was an exochitinase releasing the dimer or chitobiose as a major product, but could release small amounts of monomer after prolonged incubation. A single amino acid substitution in the catalytic domain abolished ChiC activity Three conserved amino acid residues (Asp-313, Asp-315 and Glu-317) residing in the putative catalytic site of ChiC were selected for site-directed mutagenesis in order to verify their significance in the catalytic activity of ChiC. Enzymatic hydrolysis of colloidal chitin as a substrate was performed in order to assess the effect of the mutations. The three mutant clones harboring the mutated chiC gene did not show clear zones on the selective medium containing colloidal chitin (data not shown). E. coli transformants harboring pCHISI, pD313G, pD315G, and pE317G grew similarly (data not shown) and produced similar amounts of protein (Table 1). Assays of chitinase activity in 20-fold concentrates from the culture supernatant using colloidal chitin did not show any detectable activity in any of the three mutants (Table 1). These results agreed well with the fact that conserved Asp and Glu residues separated by three amino acids play a role in catalysis activity (10). However, in our study, another conserved amino acid (Asp-315) separated from the other two residues by one amino acid was

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TABLE 1. Chitinase activity of E. coli transformants harboring plasmid wild type (pCHISI) and mutant chitinase C (pD313G, pD315G and pE317G) Activitya Proteina Specific activity (mU/ml) (mg/ml) (mU/mg) pCHISI 55.32 2.61 21.19 pD313G 0 2.65 0 pD315G 0 2.63 0 pE317G 0 2.93 0 a The chitinase activity and protein determination were calculated as milliunits and milligrams per milliliter of crude enzyme, respectively. Plasmid

also essential for the ChiC chitinase activity of S. costicola 5SM-1. Thus, it is suggested that Asp-313, Asp-315 and Glu-317 are all required and important for the activity of chitinase C from marine S. costicola 5SM-1. DISCUSSION S. costicola 5SM-1 is a moderately halophilic bacteria since it can grow in medium containing 1–15% NaCl, with the optimum concentration being 3% NaCl. On the other hand, chitinase from this strain can function well in the absence of NaCl or in the presence of 1–5% NaCl (data not shown). Chitinase from two other marine bacteria, V. harveyi and Alteromonas sp. strain O-7, can also function in the absence of salt (22, 23). Most bacterial chitinases such as B. thuringienesis subsp. pakistani (24), C. paraputrificum (19), Serratia (25) and S. lividans (26) are inducible enzymes. Even though Alteromonas sp. strain O-7 (23), Pseudoalteromonas sp. strain S91 (27) and S. viridificans (28) express chitinase constitutively, only low basal levels of chitinase are produced, and the maximum levels are only achieved on the addition of inducer. Since these bacteria produce several types of chitinases from different genes, the major classes of these chitinases are inducible enzymes. In S. costicola, the addition of either colloidal chitin or GlcNAc did not affect the overall chitinase production when compared to other medium without these two substances. Therefore, neither colloidal chitin nor GlcNAc acts as an inducer or repressor of chitinase production. Only a single chitinase activity band of 92 kDa was detected in SDS–PAGE containing glycol chitin as a substrate, after renaturation from 20-fold concentrated crude chitinase, grown in LB–NaCl with or without inducer. Though the substrate for chitinase activity detection in SDS–PAGE (glycol chitin) was different from that used in colorimetric assay (colloidal chitin), both substrates could be used as a substrate for either endoor exo-chitinase (8, 18). The 92-kDa chitinase was identified as chitinase C by gene cloning and N-terminal amino acid sequence analysis. ChiC, therefore, was regarded as constitutively expressed chitinase in S. costicola. The addition of chitobiose increased 20% production of the overall chitinase activity. Therefore, chitobiose may induce other types of chitinase rather than ChiC. Chitobiose was also reported to induce chitinase production in S. marcescens (29). Soluble degradation products were reported to repress chitinase production, e.g., N-acetylglucosamine represses chitinase production in Alteromonas (23), S. marcescens (25) and S. lividans (26), while chitobiose, another degradation

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product, also represses chitinase production in Alteromonas (23). Glucose seems to be an efficient repressor of chitinase in Streptomyces sp. (26, 28) and S. marcescens (25) but did not repress chitinase production in Pseudoalteromonas sp. (27) and Alteromonas sp. (23). In S. costicola 5SM-1, the study of overall chitinase production when colloidal chitin was used as a substrate showed that chitinase production is repressed by glucose and de-repressed when the cells were grown for 20 h, due to glucose depletion. Addition of glucose inhibits the increase in chitinase activity and the enzyme activity was maintained at the same level (~9 mU/ml) for more than 8 h. This finding suggested that chitinase in S. costicola is quite a stable enzyme since activity in the culture medium remained unchanged under the culture condition. ChiC, from a single cloned chiC gene in an E. coli transformant, digested the colloidal chitin substrate to produce chitobiose as a major product, with a trace amount of GlcNAc after prolonged incubation for at least 24 h. Moreover, the reaction of purified ChiC from E. coli transformant against five chito-oligosaccharides varied from 2–6 mers as substrates, produced only two types of end product, N-acetylglucosamine and chitobiose (manuscript in preparation). ChiC can be further classified as an exo-type chitinase. An invariant aspartate separated from an invariant glutamate are essential for lysozyme-type catalytic activity in chitinase, when analyzed by site-directed mutagenesis (10). In the chiA gene from Autographa californica nucleopolyhedrovirus, the mutagenesis of both aspartate (Asp-311) and glutamate (Glu-315) residues resulted in the attenuation of chitinolytic activity (30). Furthermore, the structure of chiA gene product from S. marcescens formed a complex with the substrate N,N¢,N¢¢,N¢¢¢-tetraacetylchitotetraose. This result suggested that Glu-315 might be the catalytic residue of the active site (31). Both mutagenesis and structural determination indicated that two conserved aspartic and glutamic acid residues, located in the catalytic site, are essential for chitinolytic activity. A single mutation of aspartate-313, aspartate-315 or glutamate-317 to glycine resulted in complete loss of chitinase activity. Therefore, these three conserved amino acid residues are probably directly involved in the catalytic mechanism and important for chitinase C activity. These mutations did not affect protein synthesis from the mutated gene because similar amounts of protein bands of mutated ChiC without chitinase activity were detected (data not shown). The amounts of proteins were comparable to those of wild type (pCHISI). Many other highly conserved residues located within the catalytic domain, such as Ser-273, Gly-275 and Gly-276 (Fig. 4), might also be essential for ChiC catalytic activity. The importance of these residues in enzyme activity requires further investigation. A single cadherin-like domain (8, 19) was found in the middle region linking the N-terminal catalytic domain and C-terminal chitin-binding part of ChiC. This domain is found in a few chitinases such as ChiA and ChiB from C. paraputrificum and ChiA from V. harveyi (8, 19). The function of the cadherin-like domain in chitinase is still unclear. The cadherin-like domain has no affinity toward chitin and is also not directly responsible for chitin binding (8). However, its location links the catalytic and chitin-binding domains so that the cadherin-like domain might function as a

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joint to keep the appropriate distance and orientation between the catalytic and chitin-binding domains of bacterial chitinases V (8). The chitin-binding domain of chitinases helps not only in hydrolyzing different forms of chitin but also in determination of enzyme movement along the substrate (22). The substrate binding ability of ChiA from V. harveyi is lost if the chitin-binding domain (CBD) was deleted (22). The threedimensional structure of ChiA from S. marcescens showed three exposed aromatic residues, Trp-33 and Trp-69 in the N-terminal CBD and Trp-245 in the catalytic domain, which are indispensable for chitin binding activity (32). The interaction of multiple aromatic residues with the chitin chain results in tight binding of substrate by the enzyme (32). Trp788 and Trp-824 were found in the putative C-terminal chitin-binding domain of ChiC, corresponding to Trp-33 and Trp-69 of S. marcescens ChiA. The two residues are exactly 35 amino acid residues apart from each other in both enzymes. Since the chiB gene from S. costicola 5SM-1 did not contain a catalytic domain, ChiB did not have any detectable chitinase. Analysis of the nucleotide sequence 1400 bp upstream of the chiB gene did not show any putative promoter sequence, while the chiB gene in the same orientation to lacZ promoter (pCHISV and VI) did not produce chitinase activity on a colloidal chitin plate assay. The deduced amino acid sequence of ChiB can be classified as chitin-binding protein with a C-terminal type 3 chitin-binding domain, which belongs to family 5 of carbohydrate binding module (CBM). The role of ChiB in chitin hydrolysis is not yet known. The partial chiA gene located upstream of chiB and chiC was found in the same orientation. The single chitinase operon composed of three open reading frames in the same transcriptional direction found in S. costicola was similar to that of Pseudoalteromonas sp. strain S91 chiA, chiB and chiC (6) and Alteromonas sp. strain O-7 chiD, cpb1 and chiA (21). Though the chiA, chiB and chiC in S. costicola was in the same operon, subcloning of a fragment encoding chiC with the appropriate length of upstream sequence could allow chiC expression in E. coli. The putative promoter sequences -10 (TATAAC) and -35 (TTGGGA), which were 711 and 739 nucleotides upstream of the chiC start codon within the ChiB encoding gene, were found and showed significant similarity to the consensus sequences -10 (TATAAT) and -35 (TTGACA) of E. coli promoter (33). The expression of chiC in pCHISIII and pCHISIV was not controlled by lacZ promoter, since subcloning of the fragments encoding chiC from both plasmids in an opposite orientation to that of lacZ promoter still showed chitinase activity (data not shown). In addition, E. coli harboring pCHISII, which contained the complete chiC gene in the same orientation with lacZ promoter, did not have chitinase activity (Fig. 3). Sequencing of the upstream region encoding the chiA gene and promoter sequence is still in progress. ACKNOWLEDGMENTS R. Aunpad received support from the Institutional Strengthening Program of the Faculty of Science, Mahidol University. This work was partially supported by the National Research Council

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of Thailand (NRCT). We thank Prof. MR Jisnuson Svasti from Mahidol University for his critical reading of the manuscript.

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