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Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC21721
FEMS Microbiology Letters 151 (1997) 197^204
Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172 Sang Wan Gal 1 a , Ji Young Choi a , Cha Young Kim a , Yong Hwa Cheong b , Young Ju Choi c , Jeong Dong Bahk a b , Sang Yeol Lee a b , Moo Je Cho a b * ;
;
a
;
;
;
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, South Korea
b c
Department of Biochemistry, Gyeongsang National University, Chinju 660-701, South Korea Department of Food and Nutrition, Pusan Women's University, Pusan 616-060, South Korea
Received 30 December 1996; revised 20 March 1997; accepted 24 March 1997
Abstract
A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 WM (min)31 mg31 and 17 WM (min)31 mg31 on the natural swollen chitin, respectively. Keywords :
Chitinase; Cloning;
Serratia marcescens
1. Introduction
Chitinases (EC 3.2.1.14), enzymes that degrade chitin, have been detected in a wide variety of organ* Corresponding author. Tel.: +82 (591) 751-5188; fax: +82 (591) 759-9363; e-mail: [email protected] 1
Present address: Department of Microbiological Engineering, Chinju National University, Chinju 660-758, South Korea. The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number L38484.
isms. However, the functions of these enzymes are believed to be di¡erent in the biological world. Chitinases in higher plants are part of a defense mechanism against fungal pathogens [1]. Many fungi contain chitin as a major component of their cell walls and produce chitinases to modify chitin as a structural component [2]. In the yeast Saccharomyces cerevisiae, chitin constitutes only about 1% of the cell wall but is found in rich deposits around the septum between mother and daughter cell [3]. In this case, chitinase was shown to be required for cell separation during proliferation.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 5 9 - 6
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Serratia marcescens is a Gram-negative soil bacterium and a well known chitin decomposer characterized by ¢ve types of chitinolytic activities [4]. Two genes, ChiA and ChiB, have been cloned from Serratia marcescens and sequenced [5,6]. Several other bacterial chitinase genes have also been cloned, e.g. from Altermonas [7], Bacillus circulans [8], and Vibrio vulni¢cus [9]. Still, our understanding of the regulatory mechanism of chitinase synthesis, the enzymatic characteristics and biological functions of the individual isoforms of chitinases is minimal. We describe in this report the cloning and expression of two sequential genes of Serratia marcescens KCTC2172 that encode a 54-kDa and a 22-kDa chitinase. We, furthermore, expressed each enzyme in E. coli and characterized its chitinase properties.
199
scribed by Sambrook et al. [10]. The DNA was partially digested with Sau3AI, treated with bacterial alkaline phosphatase, and size-fractionated on 5^ 25% linear NaCl gradient. Fractions containing fragments of 20^35 kb were pooled and ligated to pLAFR3 cosmid vector digested with BamHI. The recombinant plasmids were packaged in vitro by phage lambda packaging extracts (Stratagene) and transfected into E. coli DH5K. Transfectants were selected by resistance to tetracycline (10 Wg ml31 ), then the transfectants were transferred to LB agar plates containing 1% swollen chitin. The colonies forming clear halos by hydrolyzing chitin were selected as putative clones containing genomic insert DNA encoding chitinases. 2.3. Nucleotide sequence analysis
2. Materials and methods
2.1. Bacterial strains, plasmids, and culturing conditions Serratia marcescens KCTC2172, obtained from the Korean Type Culture Collection (Daejon, South Korea), was used as the source for chromosomal DNA. The bacterium was grown at 30³C in Luria Broth (LB) with 50 Wg ml31 ampicillin and 25 Wg ml31 tetracycline. E. coli DH5K was used as the host strain for recombinant plasmids and grown at 37³C on LB medium with 50 Wg ml31 ampicillin during the selection of transformants. Plasmids pLAFR3 and pBluescript SKII(3) were used as cloning vectors. 2.2. Genomic library construction and screening
Chromosomal DNA was prepared from S. marKCTC2172 according to the method de-
cescens
The recombinant plasmid DNA of pCHI5422 was isolated using a plasmid puri¢cation kit (Promega). Restriction enzymes (Promega) were used according to the manufacturer's directions. Fragments of appropriate sizes were generated as deletion derivatives of pCHI5422 and sequenced by the dideoxy-chaintermination method [11] using Sequenase (U.S. Biochemical) and the Taq Dye Primer Cycle Sequencing Kit with the Automatic DNA Sequencer ABI373A. Database searches were performed with the BLASTP-nr program at the NCBI network service [12]. 2.4. Isolation of the subcloned chitinases E. coli transformants carrying pCHI54 or pCHI22 were cultured up to the early stationary phase at 37³C with vigorous shaking in 200 ml of LB broth containing 0.1% glycol chitin. The 54-kDa and 22kDa chitinases were puri¢ed from the culture super-
6
Fig. 1. Physical map (A) and complete nucleotide sequence of pCHI5422 (B). Putative ribosomal binding sites and the promoter sequences (310 and 335) are underlined. The N-terminal amino acid sequences of the mature 54-kDa and 22-kDa chitinases as determined by Edman degradation are underlined. N-terminal peptide cleavage sites are indicated by vertical arrows. The inverted repeat sequences are indicated by horizontal arrows facing each other. The white boxes contain the sequences highly conserved in several bacterial chitinases. The circle indicates the C-terminal amino acid of the mature 54-kDa chitinase as determined by hydrazinolysis. SphI and NcoI are the sites of subcloning for pCHI54 and the two Sau3AI sites for pCHI22. The nucleotide sequence of pCHI5422 has been entered into the Genbank and EMBL data bases under accession number L38484.
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Fig. 2. N-terminal sequence comparison between the 22-kDa chitinase of S. marcescens KCTC2172 (SMCHI22) and the 20.5kDa chitinase of S. olivaceoviridis (SOCHI20.5).
natants according to the method of Roberts and Cabib [13]. 2.5. Enzymatic characterization
Chitinolytic activity was determined either by colorimetric assay [14] with chitohexaose and swollen chitin or by £uorometric assay [15] with 4-methylumbelliferyl N,NP-diacetylchitobiose (4-Mu(GlcNAc)2 . The speci¢c activity of the puri¢ed 54- and 22-kDa chitinases on the swollen chitin were determined by a colorimetric procedure with some modi¢cation [14]. The reaction mixture which contained 700 Wl of 1% swollen chitin in 20 mM sodium acetate bu¡er (pH 5.0) and 700 Wl of enzyme solution containing 10 Wg of protein was incubated at 50³C for 30 min. Chitinase speci¢c activity was expressed as WM of reducing sugar formed per min per mg of protein. Km and Kcat for the puri¢ed chitinases were determined with chitohexaose (Sigma) as substrate. Also, kinetic studies of 4-MU-(GlcNAc)2 were performed by £urometric assay [15]. Puri¢ed chitinases (10 Wg) were incubated at 37³C in 20 mM sodium acetate bu¡er (pH 5.0) containing 200 mM 4-Mu(GlcNAc)2 . The reactions were terminated by the addition of 0.1 M potassium phosphate bu¡er (pH 11.0). The amount of 4-MU released was measured spectro£uorometrically with excitation at 360 nm and emission at 450 nm using a Fluorometer TKO 100 (Hoefer Scienti¢c Instruments). The optimal temperature for the enzyme reaction was determined by measuring chitinase activity for a 30 min reaction between 20³C and 90³C in 20 mM sodium acetate bu¡er (pH 5.0) with swollen chitin as substrate. The optimum pH for the enzymatic reaction, 50 Wl enzyme solution (10 Wg) was added to 400 Wl of 1% swollen chitin in bu¡ers ranging from pH 3 to pH 11. Sodium acetate, sodium bicarbonate, and sodium carbonate bu¡ers (20 mM) were used for the pH adjustments between pH 3 and 6, 7 and 9, and 10
and 11, respectively. After the mixtures were incubated for 30 min at 37³C, the chitinase activities were determined. 2.6. SDS-polyacrylamide gel electrophoresis and detection of chitinolytic activity
SDS-polyacrylamide gel electrophoresis (PAGE) was performed in a 12% (w/v) polyacrylamide gel according to the method of Laemmli [16]. Detection of chitinolytic activity in the gel was done according to Trudel and Asselin [17]. 2.7. Analysis of the chitinolytic product
To assess enzymatic speci¢city, the enzymatic re-
Fig. 3. SDS-polyacrylamide gel electrophoresis of 54-kDa and 22-kDa chitinases. The 54-kDa and 22-kDa chitinases were puri¢ed from culture supernatants of E. coli harboring pCHI54 (A) and pCHI22 (B). Lanes 1 and 2: Coomassie blue stained gels. Lanes 3 and 4: substrate activity stained gels containing 0.01% (w/v) glycol chitin. Lanes 1 and 3: 80% ammonium sulfate precipitates of culture supernatant of transformed E. coli. Lanes 2 and 4: the chitinase puri¢ed by chitin a¤nity chromatography from the culture supernatant of transformed E. coli.
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3. Results and discussion
3.1. Cloning of 54-kDa and 22-kDa chitinase genes
Fig. 4. HPLC pro¢le of reaction products of 54-kDa and 22-kDa chitinases. (A) Standard oligosaccharide mixture. (B) Reaction products of the 54-kDa chitinase. (C) Reaction products of the 22-kDa chitinase.
action products of chitohexaose were analyzed by high performance liquid chromatography (Waters Model 590) equipped with a carbohydrate analysis column (3.9U300 mm). Ten Wg of enzymes were mixed with 100 mM chitohexaose (Sigma) in 50 Wl of sodium acetate bu¡er (pH 5.0). The reaction mixtures were incubated for 1 h at 37³C and injected into the HPLC column. The samples were eluted with 75% (v/v) acetonitrile in water with a £ow rate of 1.0 ml per min. The elution pro¢les were then compared to those of standard GlcNAc monomer, dimer, trimer, tetramer and hexamer pro¢les. 2.8. N-terminal and C-terminal amino acid sequencing
N-terminal amino acid sequences of the 54-kDa and 22-kDa chitinases puri¢ed from the culture broth of E. coli transformants harboring the respective genes were determined by Edman degradation using an automatic peptide sequencer (ABI 373A). The C-terminal amino acid residue of the 54-kDa chitinase was cleaved by hydrazinolysis [18] and the liberated amino acid was analyzed with an automatic amino acid analyzer (Hitachi 835).
S. marcescens KCTC2172 secretes chitinases of ¢ve distinct molecular masses of 58, 54, 52, 35, 22 kDa into the culture broth. A genomic DNA library for S. marcescens KCTC2172 constructed in a cosmid vector, pLAFR3, was screened on LB plates containing 1% swollen chitin. Out of 4U103 colonies screened, 15 clones produced clear chitinolytic zones after 3 days of growth at 37³C and were isolated. Among those chitinase-positive clones, two clones were identi¢ed as each producing both 54-kDa and 22-kDa chitinases, indicating that either these clones each contained both genes or that the 22-kDa chitinase resulted from proteolytic cleavage of the 54kDa chitinase after translation. The insert sizes in both clones were about 20 kb. To characterize the clones, one of them was selected and a 4.5 kb EcoRI DNA fragment was subcloned into pBluescript IISK(3) where it produced chitinolytic zones of similar intensity as the original cosmid clone. It was designated pCHI5422. The E. coli transformants carrying pCHI5422 were con¢rmed to secrete 54-kDa and 22-kDa chitinases into the culture broth. A transformant containing a plasmid with the 4.5 kb EcoRI DNA fragment in reverse orientation as compared to pCHI5422 was also able to degrade swollen chitin indicating that the cloned chitinase genes were expressed under the control of their own promoters. Complete nucleotide sequencing of pCHI5422 (Fig. 1) revealed that the cloned sequence was 4581 bp in length and contained the two genes that encode the 54-kDa and the 22-kDa chitinases in the same orientation. The 54-kDa chitinase gene had an open reading frame (ORF) of 1497 bp encoding 499 amino acids with a calculated molecular mass of 55 442 Da. A possible ribosome binding site was found upstream of the ATG start codon of the ORF. Computer analysis of the region upstream of the coding region revealed a putative prokaryotic promoter region with relatively weak homology to the consensus sequence of the corresponding genes in E. coli. A typical rho-independent transcription termination signal was identi¢ed eight nucleotides downstream from the translation termination codon as shown in Fig. 1. The deduced amino acid sequence of the
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54-kDa chitinase gene exhibits 95.6% similarity to ChiB of S. marcescens BJL200 [19] and 97.2% similarity to the ChiB of S. marcescens QMB1466 [6]. Thus, the 54-kDa chitinase of the S. marcescens KCTC2172 can be considered the ChiB homolog in this Serratia strain. The gene coding for the 22-kDa chitinase is located 1422 bp downstream from the TAA termination codon of the 54-kDa chitinase gene in tandem. The gene consists of 681 bp of ORF encoding 227 amino acids with a calculated molecular mass of 25 157 Da, which is slightly higher than the apparent molecular mass in SDS-PAGE, indicating that it may contain signal sequences (see below). The gene has a putative Shine-Dalgarno sequence which confers the ability to bind ribosomes upstream of the ATG start codon. The putative 310 and 335 regions are in the A+T rich region ahead of the Shine-Dalgarno sequence. The inverted repeat sequence for rho-independent transcriptional termination is located ¢ve nucleotides downstream of the TGA stop codon. Although the 22-kDa chitinase de¢nitely has chitinolytic activity and binds chitin (see below), the deduced amino acid sequence of the 22-kDa chitinase gene does not exhibit any sequence homology with other cloned chitinases so far identi¢ed including bacteria, fungi and plants. Recently, Romaguera et al. [20] puri¢ed a 20.5kDa chitinase from Streptomyces olivaceoviridis and sequenced 22 N-terminal amino acid sequences by Edman degradation without cloning the gene (Fig. 2). Comparison of the 22 N-terminal amino acids of 20.5-kDa chitinase from S. olivaceoviridis with the corresponding ones of the 22-kDa chitinase reveals 36% sequence similarity, which suggests that the 22kDa chitinase is possibly a homolog of the 20.5-kDa chitinase of S. olivaceoviridis. They assumed that the 20.5-kDa chitinase was derived from a 70-kDa chitinase by post-translational proteolytic cleavage. However, our results suggest that the 20.5-kDa chitinase from S. olivaceoviridis might be encoded by a separate gene. 3.2. Puri¢cation of the 54-kDa and 22-kDa chitinases
To purify the 54-kDa and 22-kDa chitinases, the individual genes encoding each protein were ligated into pBluescript IISK(3) vector and introduced into
. With the information obtained from sequence of pCHI5422, we could subclone a 1.7 kb SphI/NcoI fragment containing the gene that codes the 54-kDa chitinase into the SmaI site of the pBluescript IISK(3) vector. The E. coli transformants with the resulting recombinant plasmid, pCHI54, secreted 54-kDa chitinase into the culture broth during incubation at 37³C in LB broth supplemented with 0.1% glycol chitin. The chitinolytic activity could also be observed in the activity stained SDS-PAGE gel as shown in Fig. 3A,B (lanes 1 and 2). A 1.7 kb DNA fragment of a partial Sau3AI digest containing the 22-kDa chitinase gene was subcloned into the BamHI site of pBluescript IISK(3) and designated pCHI22. The secretion of 22-kDa chitinase in the culture supernatant of the E. coli transformants was also con¢rmed by SDS-PAGE with activity staining as shown in Fig. 3A,B (lanes 3 and 4). The 54- and 22-kDa chitinases were isolated from the culture broth of E. coli transformants containing pCHI54 and pCHI22 by chitin a¤nity chromatography (Fig. 3). Since the N-terminal 20 amino acid sequence of the secreted 54-kDa chitinase, STRKVIGYYFIPTNQINNY, coincided with the deduced amino acid sequence of the gene starting with Ser-2 of the coding sequence, it indicated that the secreted protein did not contain the N-terminal signal sequence. Still, the 54-kDa chitinase synthesized in the E. coli transformants was secreted into the culture broth very e¤ciently after culturing to early stationary phase. To clarify C-terminal processing, the C-terminal amino acid residue of the 54-kDa chitinase puri¢ed from culture broth of E. coli transformants (pCHI54) was determined by hydrazinolysis and found to be Ile-482. Ile residues are located at position 450 and 482 in the C-terminal region of the 54kDa chitinase. A polypeptide from Ser-2 to Ile-482 would result in a deduced molecular mass of 53 610 Da which corresponds to the molecular mass of the secreted 54-kDa chitinase as determined by SDSPAGE. A polypeptide from Ser-2 to Ile-450 would result in a molecular mass of only 50 056 Da. This suggests that the 54-kDa chitinase is C-terminally processed between Ile-482 and Thr-483. Thus, the missing 17 amino acids of the C-terminus may be involved in secretion of the 54-kDa chitinase enzyme as a signal sequence. Further evidence is needed, E. coli
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203
though, to clarify whether the C-terminal sequence is
As mentioned in the previous section, the coding
involved in the secretion of the protein or whether it
sequence of the 22-kDa chitinase does not contain a
is cleaved by proteolysis.
sequence homologous to the consensus sequence for the
the catalytic domain of other chitinases, including
22-kDa chitinase, HGYVESPASRAYQCKLQLNT-
the 54-kDa chitinase. However, the enzymatic reac-
QXGXXVQYEPQ, determined by automatic Edman
tion product of the 22-kDa chitinase is the same as
degradation, coincides with the sequence starting at
that of the 54-kDa chitinase. Thus, the results sug-
His-28 of the deduced amino acid sequence of the
gest that the 22-kDa chitinase may have a di¡erent
cloned 22-kDa chitinase gene. Thus, cleavage of a
catalytic domain for chitinase activity. Further stud-
signal peptide must have occurred between Ala-27
ies will be necessary to characterize the active do-
and His-28 of the precursor 22-kDa chitinase. The
mains in the 22-kDa chitinase by deletion or site-
deduced molecular mass of the peptide from His-28
directed mutagenesis.
The
N-terminal
amino
acid
sequence
of
to Tyr-227 is 22 307 Da which agrees with the mass of the processed 22-kDa chitinase as determined by SDS-PAGE. This signal sequence of 27 amino acid
Acknowledgments
residues has characteristic features of a signal sequence, that is, a positively charged amino terminal
This work was supported by a grant from the
segment followed by a hydrophobic sequence as seen
Korea Science and Engineering Foundation to the
in Fig. 1.
Plant
Molecular
Biology
and
Biotechnology
Re-
search Center, the Korea Research Foundation and
3.3. Enzymatic properties
a Genetic Engineering Grant from the Ministry of Education.
The catalytic speci¢cities of the 54-kDa and 22kDa chitinases were assessed with chitohexaose as substrate. The major product of the enzymatic reaction of both the 54-kDa and the 22-kDa chitinase was a (GlcNAc)2 dimer with a monomer as a minor product as shown in Fig. 4.
chitinase against natural swollen chitin was determined. The speci¢c activity of the 54-kDa and 221 kDa chitinase was estimated to be 300 M (min) 1 1 1 mg and 17 M (min) mg . When the kinetic
3
W
parameters,
Km
and
Kcat ,
3
W
3
of the enzymes were deter-
mined with chitohexaose as substrate, the
Km
values
W
for the 54-kDa and 22-kDa chitinases were 34 M 1 and and 700 M, and the Kcat values were 26.80 s 3 1 s , respectively. Also, Km and Kcat val7.44 10
U
W 3
3
3
ues of the enzymes were determined with 4-Mu(GlcNAc)2 as substrate. The
Km
values for the 54-
W
U
W
kDa and 22-kDa chitinases were 66 M and 710 M, 1 2 and 4.46 10 and the Kcat values were 28.59 s 1 s , respectively. The 54-kDa chitinase has, thus, a
3
[1] Boller, T., Gehri, A., Mauch, F. and Vogeli, U. (1983) Chitinase in bean leaves, induction by ethylene, puri¢cation, prop-
The speci¢c activity of the 22-kDa and 54-kDa
3
References
3
3
much higher a¤nity for the substrate and stronger catalytic activity than the 22-kDa protein, although the catalytic speci¢city is similar. The optimal pH and temperature for the enzymatic reactions of the chitinases were pH 5.0 and 55³C for both enzymes.
erties, and possible function. Planta 157, 22^31. [2] Gooday,
G.W.,
Humphreys,
A.M.
and
McIntosh,
W.H.
(1986) Roles of chitinases in fungal growth. In : Chitin in Nature and Technology (Muzzarelli, R., Jeuniaux, C. and Gooday, G.W., Eds), pp. 83^91. Plenum Press, New York. [3] Bulawa, C.E. (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu. Rev. Microbiol. 47, 505^534. [4] Fuchs, R.L., Mcpherson, S.A. and Drahos, D.J. (1986) Cloning of a
Serratia marcescens
gene encoding chitinase. Appl.
Environ. Microbiol. 51, 504^509. [5] Jones, J.D.G., Grady, K.L., Suslow, T.V. and Bedbrook, J.R. (1986) Isolation and characterization of genes encoding two chitinase enzymes from
Serratia marcescens.
EMBO J. 5, 467^
473. [6] Harpster, M.H. and Dunsmuir, P. (1989) Nucleotide sequence of the chitinase B gene of
Serratia marcescens
QMB1466. Nu-
cleic Acids Res. 17, 5395. [7] Tsujibo, H., Orikoshi, H., Tanno, H., Fujimoto, K., Miyamoto, K., Imada, C., Okami, Y. and Inamori, Y. (1993) Cloning, sequence, and expression of a chitinase gene from a marine bacterium,
Altermonas
sp. strain O-7. J. Bacteriol. 175, 176^
181. [8] Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K. and Tanaka, H. (1990) Gene cloning of chitinase A1 from
FEMSLE 7587 2-9-97
Bacillus
S.W. Gal et al. / FEMS Microbiology Letters 151 (1997) 197^204
204
circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of ¢bronec[9] Wortman, A.T., Somerville, C.C. and Colwell, R.R. (1986)
Vibrio vulni¢cus :
gene cloning and
applications of a chitinase probe. Appl. Environ. Microbiol. 52, 142^145.
Streptomyces plicatus chitinase (chitinaseEscherichia coli. J. Biol. Chem. 263, 443^447.
and expression of a 63) in
tin. J. Biol. Chem. 265, 15659^15665.
Chitinase determinants of
[15] Robbins, P.W., Albright, C. and Ben¢eld, B. (1988) Cloning
[16] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680^685. [17] Trudel, J. and Asselin, A. (1989) Detection of chitinase activ-
[10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
ity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 362^366. [18] Akabori, S., Ohno, K. and Narita, K. (1952) On the hydra-
[11] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA se-
zinolysis of proteins and peptides : a method for the character-
quencing with chain-terminating inhibitors. Proc. Natl. Acad.
ization of carboxyl-terminal amino acids in proteins. Bull.
Sci. USA 74, 5463^5467.
Chem. Soc. Jpn. 25, 214^218.
[12] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lip-
[19] Brurberg, M.B., Eijsink, V.G.H., Haandrikman, A.J., Vene-
man, D.J. (1990) Basic local alignment search tool. J. Mol.
ma, G. and Nes, I.F. (1995) Chitinase B from
Biol. 215, 403^410.
cescens
[13] Roberts, R.L. and Cabib, E. (1982)
Serratia marcescens
chiti-
nase : one-step puri¢cation and use for the determination of chitin. Anal. Biochem. 127, 402^412.
ing. Microbiology 141, 123^131. [20] Romaguera, A., Menge, U., Breves, R. and Diekmann, H. (1992) Chitinases of
[14] Imoto, T. and Yagishita, K. (1971) A simple activity measurement of lysozyme. Agric. Biol. Chem. 35, 1154^1156.
Serratia mar-
BJL200 is exported to the periplasm without process-
Streptomyces olivaceoviridis
and signi¢-
cance of processing for multiplicity. J. Bacteriol. 174, 3450^ 3454.
FEMSLE 7587 2-9-97
Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172 Sang Wan Gal 1 a , Ji Young Choi a , Cha Young Kim a , Yong Hwa Cheong b , Young Ju Choi c , Jeong Dong Bahk a b , Sang Yeol Lee a b , Moo Je Cho a b * ;
;
a
;
;
;
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, South Korea
b c
Department of Biochemistry, Gyeongsang National University, Chinju 660-701, South Korea Department of Food and Nutrition, Pusan Women's University, Pusan 616-060, South Korea
Received 30 December 1996; revised 20 March 1997; accepted 24 March 1997
Abstract
A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)2 dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 WM (min)31 mg31 and 17 WM (min)31 mg31 on the natural swollen chitin, respectively. Keywords :
Chitinase; Cloning;
Serratia marcescens
1. Introduction
Chitinases (EC 3.2.1.14), enzymes that degrade chitin, have been detected in a wide variety of organ* Corresponding author. Tel.: +82 (591) 751-5188; fax: +82 (591) 759-9363; e-mail: [email protected] 1
Present address: Department of Microbiological Engineering, Chinju National University, Chinju 660-758, South Korea. The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number L38484.
isms. However, the functions of these enzymes are believed to be di¡erent in the biological world. Chitinases in higher plants are part of a defense mechanism against fungal pathogens [1]. Many fungi contain chitin as a major component of their cell walls and produce chitinases to modify chitin as a structural component [2]. In the yeast Saccharomyces cerevisiae, chitin constitutes only about 1% of the cell wall but is found in rich deposits around the septum between mother and daughter cell [3]. In this case, chitinase was shown to be required for cell separation during proliferation.
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 5 9 - 6
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Serratia marcescens is a Gram-negative soil bacterium and a well known chitin decomposer characterized by ¢ve types of chitinolytic activities [4]. Two genes, ChiA and ChiB, have been cloned from Serratia marcescens and sequenced [5,6]. Several other bacterial chitinase genes have also been cloned, e.g. from Altermonas [7], Bacillus circulans [8], and Vibrio vulni¢cus [9]. Still, our understanding of the regulatory mechanism of chitinase synthesis, the enzymatic characteristics and biological functions of the individual isoforms of chitinases is minimal. We describe in this report the cloning and expression of two sequential genes of Serratia marcescens KCTC2172 that encode a 54-kDa and a 22-kDa chitinase. We, furthermore, expressed each enzyme in E. coli and characterized its chitinase properties.
199
scribed by Sambrook et al. [10]. The DNA was partially digested with Sau3AI, treated with bacterial alkaline phosphatase, and size-fractionated on 5^ 25% linear NaCl gradient. Fractions containing fragments of 20^35 kb were pooled and ligated to pLAFR3 cosmid vector digested with BamHI. The recombinant plasmids were packaged in vitro by phage lambda packaging extracts (Stratagene) and transfected into E. coli DH5K. Transfectants were selected by resistance to tetracycline (10 Wg ml31 ), then the transfectants were transferred to LB agar plates containing 1% swollen chitin. The colonies forming clear halos by hydrolyzing chitin were selected as putative clones containing genomic insert DNA encoding chitinases. 2.3. Nucleotide sequence analysis
2. Materials and methods
2.1. Bacterial strains, plasmids, and culturing conditions Serratia marcescens KCTC2172, obtained from the Korean Type Culture Collection (Daejon, South Korea), was used as the source for chromosomal DNA. The bacterium was grown at 30³C in Luria Broth (LB) with 50 Wg ml31 ampicillin and 25 Wg ml31 tetracycline. E. coli DH5K was used as the host strain for recombinant plasmids and grown at 37³C on LB medium with 50 Wg ml31 ampicillin during the selection of transformants. Plasmids pLAFR3 and pBluescript SKII(3) were used as cloning vectors. 2.2. Genomic library construction and screening
Chromosomal DNA was prepared from S. marKCTC2172 according to the method de-
cescens
The recombinant plasmid DNA of pCHI5422 was isolated using a plasmid puri¢cation kit (Promega). Restriction enzymes (Promega) were used according to the manufacturer's directions. Fragments of appropriate sizes were generated as deletion derivatives of pCHI5422 and sequenced by the dideoxy-chaintermination method [11] using Sequenase (U.S. Biochemical) and the Taq Dye Primer Cycle Sequencing Kit with the Automatic DNA Sequencer ABI373A. Database searches were performed with the BLASTP-nr program at the NCBI network service [12]. 2.4. Isolation of the subcloned chitinases E. coli transformants carrying pCHI54 or pCHI22 were cultured up to the early stationary phase at 37³C with vigorous shaking in 200 ml of LB broth containing 0.1% glycol chitin. The 54-kDa and 22kDa chitinases were puri¢ed from the culture super-
6
Fig. 1. Physical map (A) and complete nucleotide sequence of pCHI5422 (B). Putative ribosomal binding sites and the promoter sequences (310 and 335) are underlined. The N-terminal amino acid sequences of the mature 54-kDa and 22-kDa chitinases as determined by Edman degradation are underlined. N-terminal peptide cleavage sites are indicated by vertical arrows. The inverted repeat sequences are indicated by horizontal arrows facing each other. The white boxes contain the sequences highly conserved in several bacterial chitinases. The circle indicates the C-terminal amino acid of the mature 54-kDa chitinase as determined by hydrazinolysis. SphI and NcoI are the sites of subcloning for pCHI54 and the two Sau3AI sites for pCHI22. The nucleotide sequence of pCHI5422 has been entered into the Genbank and EMBL data bases under accession number L38484.
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Fig. 2. N-terminal sequence comparison between the 22-kDa chitinase of S. marcescens KCTC2172 (SMCHI22) and the 20.5kDa chitinase of S. olivaceoviridis (SOCHI20.5).
natants according to the method of Roberts and Cabib [13]. 2.5. Enzymatic characterization
Chitinolytic activity was determined either by colorimetric assay [14] with chitohexaose and swollen chitin or by £uorometric assay [15] with 4-methylumbelliferyl N,NP-diacetylchitobiose (4-Mu(GlcNAc)2 . The speci¢c activity of the puri¢ed 54- and 22-kDa chitinases on the swollen chitin were determined by a colorimetric procedure with some modi¢cation [14]. The reaction mixture which contained 700 Wl of 1% swollen chitin in 20 mM sodium acetate bu¡er (pH 5.0) and 700 Wl of enzyme solution containing 10 Wg of protein was incubated at 50³C for 30 min. Chitinase speci¢c activity was expressed as WM of reducing sugar formed per min per mg of protein. Km and Kcat for the puri¢ed chitinases were determined with chitohexaose (Sigma) as substrate. Also, kinetic studies of 4-MU-(GlcNAc)2 were performed by £urometric assay [15]. Puri¢ed chitinases (10 Wg) were incubated at 37³C in 20 mM sodium acetate bu¡er (pH 5.0) containing 200 mM 4-Mu(GlcNAc)2 . The reactions were terminated by the addition of 0.1 M potassium phosphate bu¡er (pH 11.0). The amount of 4-MU released was measured spectro£uorometrically with excitation at 360 nm and emission at 450 nm using a Fluorometer TKO 100 (Hoefer Scienti¢c Instruments). The optimal temperature for the enzyme reaction was determined by measuring chitinase activity for a 30 min reaction between 20³C and 90³C in 20 mM sodium acetate bu¡er (pH 5.0) with swollen chitin as substrate. The optimum pH for the enzymatic reaction, 50 Wl enzyme solution (10 Wg) was added to 400 Wl of 1% swollen chitin in bu¡ers ranging from pH 3 to pH 11. Sodium acetate, sodium bicarbonate, and sodium carbonate bu¡ers (20 mM) were used for the pH adjustments between pH 3 and 6, 7 and 9, and 10
and 11, respectively. After the mixtures were incubated for 30 min at 37³C, the chitinase activities were determined. 2.6. SDS-polyacrylamide gel electrophoresis and detection of chitinolytic activity
SDS-polyacrylamide gel electrophoresis (PAGE) was performed in a 12% (w/v) polyacrylamide gel according to the method of Laemmli [16]. Detection of chitinolytic activity in the gel was done according to Trudel and Asselin [17]. 2.7. Analysis of the chitinolytic product
To assess enzymatic speci¢city, the enzymatic re-
Fig. 3. SDS-polyacrylamide gel electrophoresis of 54-kDa and 22-kDa chitinases. The 54-kDa and 22-kDa chitinases were puri¢ed from culture supernatants of E. coli harboring pCHI54 (A) and pCHI22 (B). Lanes 1 and 2: Coomassie blue stained gels. Lanes 3 and 4: substrate activity stained gels containing 0.01% (w/v) glycol chitin. Lanes 1 and 3: 80% ammonium sulfate precipitates of culture supernatant of transformed E. coli. Lanes 2 and 4: the chitinase puri¢ed by chitin a¤nity chromatography from the culture supernatant of transformed E. coli.
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201
3. Results and discussion
3.1. Cloning of 54-kDa and 22-kDa chitinase genes
Fig. 4. HPLC pro¢le of reaction products of 54-kDa and 22-kDa chitinases. (A) Standard oligosaccharide mixture. (B) Reaction products of the 54-kDa chitinase. (C) Reaction products of the 22-kDa chitinase.
action products of chitohexaose were analyzed by high performance liquid chromatography (Waters Model 590) equipped with a carbohydrate analysis column (3.9U300 mm). Ten Wg of enzymes were mixed with 100 mM chitohexaose (Sigma) in 50 Wl of sodium acetate bu¡er (pH 5.0). The reaction mixtures were incubated for 1 h at 37³C and injected into the HPLC column. The samples were eluted with 75% (v/v) acetonitrile in water with a £ow rate of 1.0 ml per min. The elution pro¢les were then compared to those of standard GlcNAc monomer, dimer, trimer, tetramer and hexamer pro¢les. 2.8. N-terminal and C-terminal amino acid sequencing
N-terminal amino acid sequences of the 54-kDa and 22-kDa chitinases puri¢ed from the culture broth of E. coli transformants harboring the respective genes were determined by Edman degradation using an automatic peptide sequencer (ABI 373A). The C-terminal amino acid residue of the 54-kDa chitinase was cleaved by hydrazinolysis [18] and the liberated amino acid was analyzed with an automatic amino acid analyzer (Hitachi 835).
S. marcescens KCTC2172 secretes chitinases of ¢ve distinct molecular masses of 58, 54, 52, 35, 22 kDa into the culture broth. A genomic DNA library for S. marcescens KCTC2172 constructed in a cosmid vector, pLAFR3, was screened on LB plates containing 1% swollen chitin. Out of 4U103 colonies screened, 15 clones produced clear chitinolytic zones after 3 days of growth at 37³C and were isolated. Among those chitinase-positive clones, two clones were identi¢ed as each producing both 54-kDa and 22-kDa chitinases, indicating that either these clones each contained both genes or that the 22-kDa chitinase resulted from proteolytic cleavage of the 54kDa chitinase after translation. The insert sizes in both clones were about 20 kb. To characterize the clones, one of them was selected and a 4.5 kb EcoRI DNA fragment was subcloned into pBluescript IISK(3) where it produced chitinolytic zones of similar intensity as the original cosmid clone. It was designated pCHI5422. The E. coli transformants carrying pCHI5422 were con¢rmed to secrete 54-kDa and 22-kDa chitinases into the culture broth. A transformant containing a plasmid with the 4.5 kb EcoRI DNA fragment in reverse orientation as compared to pCHI5422 was also able to degrade swollen chitin indicating that the cloned chitinase genes were expressed under the control of their own promoters. Complete nucleotide sequencing of pCHI5422 (Fig. 1) revealed that the cloned sequence was 4581 bp in length and contained the two genes that encode the 54-kDa and the 22-kDa chitinases in the same orientation. The 54-kDa chitinase gene had an open reading frame (ORF) of 1497 bp encoding 499 amino acids with a calculated molecular mass of 55 442 Da. A possible ribosome binding site was found upstream of the ATG start codon of the ORF. Computer analysis of the region upstream of the coding region revealed a putative prokaryotic promoter region with relatively weak homology to the consensus sequence of the corresponding genes in E. coli. A typical rho-independent transcription termination signal was identi¢ed eight nucleotides downstream from the translation termination codon as shown in Fig. 1. The deduced amino acid sequence of the
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54-kDa chitinase gene exhibits 95.6% similarity to ChiB of S. marcescens BJL200 [19] and 97.2% similarity to the ChiB of S. marcescens QMB1466 [6]. Thus, the 54-kDa chitinase of the S. marcescens KCTC2172 can be considered the ChiB homolog in this Serratia strain. The gene coding for the 22-kDa chitinase is located 1422 bp downstream from the TAA termination codon of the 54-kDa chitinase gene in tandem. The gene consists of 681 bp of ORF encoding 227 amino acids with a calculated molecular mass of 25 157 Da, which is slightly higher than the apparent molecular mass in SDS-PAGE, indicating that it may contain signal sequences (see below). The gene has a putative Shine-Dalgarno sequence which confers the ability to bind ribosomes upstream of the ATG start codon. The putative 310 and 335 regions are in the A+T rich region ahead of the Shine-Dalgarno sequence. The inverted repeat sequence for rho-independent transcriptional termination is located ¢ve nucleotides downstream of the TGA stop codon. Although the 22-kDa chitinase de¢nitely has chitinolytic activity and binds chitin (see below), the deduced amino acid sequence of the 22-kDa chitinase gene does not exhibit any sequence homology with other cloned chitinases so far identi¢ed including bacteria, fungi and plants. Recently, Romaguera et al. [20] puri¢ed a 20.5kDa chitinase from Streptomyces olivaceoviridis and sequenced 22 N-terminal amino acid sequences by Edman degradation without cloning the gene (Fig. 2). Comparison of the 22 N-terminal amino acids of 20.5-kDa chitinase from S. olivaceoviridis with the corresponding ones of the 22-kDa chitinase reveals 36% sequence similarity, which suggests that the 22kDa chitinase is possibly a homolog of the 20.5-kDa chitinase of S. olivaceoviridis. They assumed that the 20.5-kDa chitinase was derived from a 70-kDa chitinase by post-translational proteolytic cleavage. However, our results suggest that the 20.5-kDa chitinase from S. olivaceoviridis might be encoded by a separate gene. 3.2. Puri¢cation of the 54-kDa and 22-kDa chitinases
To purify the 54-kDa and 22-kDa chitinases, the individual genes encoding each protein were ligated into pBluescript IISK(3) vector and introduced into
. With the information obtained from sequence of pCHI5422, we could subclone a 1.7 kb SphI/NcoI fragment containing the gene that codes the 54-kDa chitinase into the SmaI site of the pBluescript IISK(3) vector. The E. coli transformants with the resulting recombinant plasmid, pCHI54, secreted 54-kDa chitinase into the culture broth during incubation at 37³C in LB broth supplemented with 0.1% glycol chitin. The chitinolytic activity could also be observed in the activity stained SDS-PAGE gel as shown in Fig. 3A,B (lanes 1 and 2). A 1.7 kb DNA fragment of a partial Sau3AI digest containing the 22-kDa chitinase gene was subcloned into the BamHI site of pBluescript IISK(3) and designated pCHI22. The secretion of 22-kDa chitinase in the culture supernatant of the E. coli transformants was also con¢rmed by SDS-PAGE with activity staining as shown in Fig. 3A,B (lanes 3 and 4). The 54- and 22-kDa chitinases were isolated from the culture broth of E. coli transformants containing pCHI54 and pCHI22 by chitin a¤nity chromatography (Fig. 3). Since the N-terminal 20 amino acid sequence of the secreted 54-kDa chitinase, STRKVIGYYFIPTNQINNY, coincided with the deduced amino acid sequence of the gene starting with Ser-2 of the coding sequence, it indicated that the secreted protein did not contain the N-terminal signal sequence. Still, the 54-kDa chitinase synthesized in the E. coli transformants was secreted into the culture broth very e¤ciently after culturing to early stationary phase. To clarify C-terminal processing, the C-terminal amino acid residue of the 54-kDa chitinase puri¢ed from culture broth of E. coli transformants (pCHI54) was determined by hydrazinolysis and found to be Ile-482. Ile residues are located at position 450 and 482 in the C-terminal region of the 54kDa chitinase. A polypeptide from Ser-2 to Ile-482 would result in a deduced molecular mass of 53 610 Da which corresponds to the molecular mass of the secreted 54-kDa chitinase as determined by SDSPAGE. A polypeptide from Ser-2 to Ile-450 would result in a molecular mass of only 50 056 Da. This suggests that the 54-kDa chitinase is C-terminally processed between Ile-482 and Thr-483. Thus, the missing 17 amino acids of the C-terminus may be involved in secretion of the 54-kDa chitinase enzyme as a signal sequence. Further evidence is needed, E. coli
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203
though, to clarify whether the C-terminal sequence is
As mentioned in the previous section, the coding
involved in the secretion of the protein or whether it
sequence of the 22-kDa chitinase does not contain a
is cleaved by proteolysis.
sequence homologous to the consensus sequence for the
the catalytic domain of other chitinases, including
22-kDa chitinase, HGYVESPASRAYQCKLQLNT-
the 54-kDa chitinase. However, the enzymatic reac-
QXGXXVQYEPQ, determined by automatic Edman
tion product of the 22-kDa chitinase is the same as
degradation, coincides with the sequence starting at
that of the 54-kDa chitinase. Thus, the results sug-
His-28 of the deduced amino acid sequence of the
gest that the 22-kDa chitinase may have a di¡erent
cloned 22-kDa chitinase gene. Thus, cleavage of a
catalytic domain for chitinase activity. Further stud-
signal peptide must have occurred between Ala-27
ies will be necessary to characterize the active do-
and His-28 of the precursor 22-kDa chitinase. The
mains in the 22-kDa chitinase by deletion or site-
deduced molecular mass of the peptide from His-28
directed mutagenesis.
The
N-terminal
amino
acid
sequence
of
to Tyr-227 is 22 307 Da which agrees with the mass of the processed 22-kDa chitinase as determined by SDS-PAGE. This signal sequence of 27 amino acid
Acknowledgments
residues has characteristic features of a signal sequence, that is, a positively charged amino terminal
This work was supported by a grant from the
segment followed by a hydrophobic sequence as seen
Korea Science and Engineering Foundation to the
in Fig. 1.
Plant
Molecular
Biology
and
Biotechnology
Re-
search Center, the Korea Research Foundation and
3.3. Enzymatic properties
a Genetic Engineering Grant from the Ministry of Education.
The catalytic speci¢cities of the 54-kDa and 22kDa chitinases were assessed with chitohexaose as substrate. The major product of the enzymatic reaction of both the 54-kDa and the 22-kDa chitinase was a (GlcNAc)2 dimer with a monomer as a minor product as shown in Fig. 4.
chitinase against natural swollen chitin was determined. The speci¢c activity of the 54-kDa and 221 kDa chitinase was estimated to be 300 M (min) 1 1 1 mg and 17 M (min) mg . When the kinetic
3
W
parameters,
Km
and
Kcat ,
3
W
3
of the enzymes were deter-
mined with chitohexaose as substrate, the
Km
values
W
for the 54-kDa and 22-kDa chitinases were 34 M 1 and and 700 M, and the Kcat values were 26.80 s 3 1 s , respectively. Also, Km and Kcat val7.44 10
U
W 3
3
3
ues of the enzymes were determined with 4-Mu(GlcNAc)2 as substrate. The
Km
values for the 54-
W
U
W
kDa and 22-kDa chitinases were 66 M and 710 M, 1 2 and 4.46 10 and the Kcat values were 28.59 s 1 s , respectively. The 54-kDa chitinase has, thus, a
3
[1] Boller, T., Gehri, A., Mauch, F. and Vogeli, U. (1983) Chitinase in bean leaves, induction by ethylene, puri¢cation, prop-
The speci¢c activity of the 22-kDa and 54-kDa
3
References
3
3
much higher a¤nity for the substrate and stronger catalytic activity than the 22-kDa protein, although the catalytic speci¢city is similar. The optimal pH and temperature for the enzymatic reactions of the chitinases were pH 5.0 and 55³C for both enzymes.
erties, and possible function. Planta 157, 22^31. [2] Gooday,
G.W.,
Humphreys,
A.M.
and
McIntosh,
W.H.
(1986) Roles of chitinases in fungal growth. In : Chitin in Nature and Technology (Muzzarelli, R., Jeuniaux, C. and Gooday, G.W., Eds), pp. 83^91. Plenum Press, New York. [3] Bulawa, C.E. (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu. Rev. Microbiol. 47, 505^534. [4] Fuchs, R.L., Mcpherson, S.A. and Drahos, D.J. (1986) Cloning of a
Serratia marcescens
gene encoding chitinase. Appl.
Environ. Microbiol. 51, 504^509. [5] Jones, J.D.G., Grady, K.L., Suslow, T.V. and Bedbrook, J.R. (1986) Isolation and characterization of genes encoding two chitinase enzymes from
Serratia marcescens.
EMBO J. 5, 467^
473. [6] Harpster, M.H. and Dunsmuir, P. (1989) Nucleotide sequence of the chitinase B gene of
Serratia marcescens
QMB1466. Nu-
cleic Acids Res. 17, 5395. [7] Tsujibo, H., Orikoshi, H., Tanno, H., Fujimoto, K., Miyamoto, K., Imada, C., Okami, Y. and Inamori, Y. (1993) Cloning, sequence, and expression of a chitinase gene from a marine bacterium,
Altermonas
sp. strain O-7. J. Bacteriol. 175, 176^
181. [8] Watanabe, T., Suzuki, K., Oyanagi, W., Ohnishi, K. and Tanaka, H. (1990) Gene cloning of chitinase A1 from
FEMSLE 7587 2-9-97
Bacillus
S.W. Gal et al. / FEMS Microbiology Letters 151 (1997) 197^204
204
circulans WL-12 revealed its evolutionary relationship to Serratia chitinase and to the type III homology units of ¢bronec[9] Wortman, A.T., Somerville, C.C. and Colwell, R.R. (1986)
Vibrio vulni¢cus :
gene cloning and
applications of a chitinase probe. Appl. Environ. Microbiol. 52, 142^145.
Streptomyces plicatus chitinase (chitinaseEscherichia coli. J. Biol. Chem. 263, 443^447.
and expression of a 63) in
tin. J. Biol. Chem. 265, 15659^15665.
Chitinase determinants of
[15] Robbins, P.W., Albright, C. and Ben¢eld, B. (1988) Cloning
[16] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680^685. [17] Trudel, J. and Asselin, A. (1989) Detection of chitinase activ-
[10] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning : A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
ity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 362^366. [18] Akabori, S., Ohno, K. and Narita, K. (1952) On the hydra-
[11] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA se-
zinolysis of proteins and peptides : a method for the character-
quencing with chain-terminating inhibitors. Proc. Natl. Acad.
ization of carboxyl-terminal amino acids in proteins. Bull.
Sci. USA 74, 5463^5467.
Chem. Soc. Jpn. 25, 214^218.
[12] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lip-
[19] Brurberg, M.B., Eijsink, V.G.H., Haandrikman, A.J., Vene-
man, D.J. (1990) Basic local alignment search tool. J. Mol.
ma, G. and Nes, I.F. (1995) Chitinase B from
Biol. 215, 403^410.
cescens
[13] Roberts, R.L. and Cabib, E. (1982)
Serratia marcescens
chiti-
nase : one-step puri¢cation and use for the determination of chitin. Anal. Biochem. 127, 402^412.
ing. Microbiology 141, 123^131. [20] Romaguera, A., Menge, U., Breves, R. and Diekmann, H. (1992) Chitinases of
[14] Imoto, T. and Yagishita, K. (1971) A simple activity measurement of lysozyme. Agric. Biol. Chem. 35, 1154^1156.
Serratia mar-
BJL200 is exported to the periplasm without process-
Streptomyces olivaceoviridis
and signi¢-
cance of processing for multiplicity. J. Bacteriol. 174, 3450^ 3454.
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