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Annals of Microbiology, 54 (3), 289-297 (2004)
Gene cloning and biochemical characterization
of chitinase CH from Bacillus cereus 28-9
C.-J. HUANG, C.-Y. CHEN*
Department of Plant Pathology and Microbiology, National Taiwan University, No. 1,
Sec. 4, Roosevelt Rd., Taipei, Taiwan 106, Republic of China
Abstract - Bacillus cereus 28-9 is a chitinolytic bacterium showing antagonistic activity
against several fungi. One chitinase of 37 kDa, named chitinase CH (ChiCH), was purified
by ammonium sulphate fractionation and anion exchange chromatography. The N-termi-
nal sequence of purified ChiCH was determined as ANNLGSKLLVGYWHNFD. The
chiCH (1,083 bp), cloned from the genomic DNA of B. cereus 28-9, encodes a polypep-
tide of 360 amino acids containing the N-terminal signal peptide and a catalytic domain.
ChiCH, partially purified from an Escherichia coli transformant harbouring chiCH, exhibit-
ed chitinase activity with an optimal pH of 6.0 and an optimal temperature of 40 °C. This
ChiCH was slightly inhibitory to conidial germination of Botrytis elliptica. It was suggested
that ChiCH is one of the factor involved in the antagonism of B. cereus 28-9 toward fungi.
Key words: chitinase, ChiCH, glycosyl hydrolase family 18, gene cloning.
INTRODUCTION
Bacillus cereus is a large, Gram-positive, endospore-forming bacterium that is
very common in soils and plants (Brunel et al., 1994; Martinez et al., 2002). For
plant disease control, B. cereus UW85 has been proven as a reliable biocon-
trol agent of Phytophthora damping off and root rot of soybean (Emmert and
Handelsman, 1999), and capable of producing two antibiotics responsible for
disease suppression (Silo-Suh et al., 1994). In addition, an endophytic B.
cereus strain 65 producing a chitobiosidase is effective against Rhizoctonia
solani in cotton (Pleban et al., 1997). However, the role of chitobiosidase in the
antagonism of B. cereus strain 65 toward fungal plant pathogens is not clearly
understood.
In this study, we analysed the chitinases produced by a chitinolytic strain of
B. cereus and found that this B. cereus strain excreted two chitinases. One of
them was partially purified and its encoding gene was cloned. In addition, this
chitinase was characterized and investigated on its antifungal activity toward
Botrytis elliptica, a fungal pathogen of lily leaf and blossom blight.
* Corresponding Author. E-mail: cychen@ntu.edu.tw
289
MATERIALS AND METHODS
Achitinolytic strain 28-9 was classified as Bacillus cereus / Bacillus thuringien-
sis according to carbon source utilization ability by using BIOLOG plate (Bacte-
ria & Yeast Identification System, Biolog, Inc., Hayward, CA) and identified as a
strain of B. cereus by PCR analysis of a gyrase gene (Yamada et al., 1999).
Chitinase activity was determined using a fluorometric substrate, 4-methy-
lumbelliferyl β-D-N, N’-diacetylchitobioside (Sigma, St. Louis, MO, USA), fol-
lowing the method of Morimoto et al. (1997). One unit of chitinase activity was
defined as the amount of enzyme required to release 1 µmol of 4-methylum-
belliferone per min. In addition, protein concentration was measured using
Bradford’s method (1976) and bovine serum albumin was used as a standard.
For the purification of ChiCH, all steps were carried out at 4 °C. Bacillus
cereus 28-9 was cultured in 500 mL of M9 broth that contained 0.4% GlcNAc at
37 °C on a rotary shaker at 175 rpm for three days. The culture supernatant
was collected by centrifugation at 10,000 ×gfor 15 min, and proteins in the su-
pernatant were precipitated with ammonium sulphate at 40-70% saturation. The
precipitate was dissolved in 0.1 M of Tris-HCl buffer (pH 8.0) and dialyzed
overnight in the same buffer. The dialysate was loaded onto a Hyper-D anion
exchange column (Sigma) and proteins were eluted with 0.1-0.5 M NaCl gradi-
ent in 0.1 M of Tris-HCl buffer (pH 8.0). ChiCH was eluted with 0.1 M NaCl and
fractions that exhibited chitinase activity were pooled, concentrated by ultrafil-
tration through a Centriplus YM-10 membrane (10 kDa MW cut-off, Millipore,
Bedford, MA, USA), and finally stored at –20 °C.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed following the method of Laemmli (1970) using Mini-Protein II ap-
paratus (Bio-Rad, Herculus, CA, USA). A separating gel (10%) containing
0.01% of glycol chitin was used for detection of chitinase activity. After elec-
trophoresis, separated proteins were renatured by soaking the gel in 0.1 M ac-
etate buffer (pH 5.0) containing 1% Triton X-100 at 37 °C with gentle shaking
for 2 h. The gel was stained with 0.01% Calcofluor White M2R (Sigma) in 0.5 M
Tris-HCl (pH 8.9). Protein bands exhibiting chitinolytic activities were visualized
under a UV transilluminator (Trudel and Asselin, 1989). Proteins in the poly-
acrylamide gel were stained with Coomassie Brilliant Blue G-250.
The protein was electroblotted onto a polyvinylidene difluoride (PVDF)
membrane (Millipore), using a Mini-Electroblot apparatus (Bio-Rad). Proteins
on the membrane were stained with 0.1% amido black. The protein band cor-
responding to that exhibiting chitinase activity was cut out from the membrane
and subjected to N-terminal amino acid sequencing by automated Edman
degradation using the Applied Biosystems model 477A protein sequencer (Ap-
plied Biosystems, Perkin Elmer, Foster City, Calif., USA).
The N-terminal amino acid sequence of ChiCH and the conserved se-
quence of family 18 chitinases were used to design degenerated primers.
Primer dchf (5’-TAITGGCAIAACTTTG-3’) corresponding to the amino acid se-
quence YWHNF and primer dchr (5’-TTCITCITCIATITCTATTCC-3’) correspon-
ding to the amino acid sequence G(L/I)D(L/I)DXE were used in polymerase
chain reaction (PCR). “I” refers to inosine. For DNA amplification, PCR was
done with melting at 94 °C for 10 min, followed by 30 cycles of 94 °C 1 min, 54
°C 1.5 min, and 72 °C 1 min, with final extension at 72 °C for 10 min after the
290 C.-J. HUANG and C.-Y. CHEN
last cycle. Amplified DNA fragments were cloned into pGEMT-easy vector and
sequenced. The insert of recombinant plasmid, encoding amino acid sequence
of chitinase was used as a probe in Southern blot analysis and subsequent
colony hybridisation. Probe was prepared using a PCR DIG Probe Synthesis
Kit (Roche Molecular Biochemicals, Mannheim, Germany) following the method
described by the manufacturer. A subgenomic library of B. cereus 28-9 was
constructed in pBluescript II KS(-) and transformed into E. coli TOP10F’. After
colony hybridisation, the insert DNA from a selected clone was sequenced
using the ABI-310 autosequencer (Applied Biosystems).
The DNA fragment carrying the chiCH gene and 17-bp upstream region was
amplified by PCR with primer chf, 5’-GTATAGGAGTGTTGATAATGTTAAA
CAAG-3’, and primer chr, 5’-GTTATTTTTCGAAGGAAAGACCATC-3’. The am-
plified chiCH-containing fragment was cloned into pGEMT-easy vector to cre-
ate recombinant plasmid, pGH51, and transformed into E. coli DH5α. The re-
sulting E. coli DH5α(pGH51) was cultured in LB broth containing 50 µg/ml
ampicillin under constant shaking at 37 °C for 20 h. The periplasmic protein of
E. coli DH5α(pGH51) was extracted following the method of Manoil and Beck-
with (1986). Purification of ChiCH from the periplasmic fraction of B. cereus 28-
9 was performed by the same procedures used for purification of ChiCH from
culture supernatant of B. cereus 28-9.
ChiCH purified from the periplasmic fraction of E. coli DH5α(pGH51) was
used to determine the effects of pH and temperature on chitinase activity of
ChiCH. Glycol chitin was used as a substrate. Chitinase activity was analysed
by a procedure described by Imoto and Yogishita (1971). Hydrolysis reaction of
ChiCH was performed at 37 °C for 25 min in the following buffers of 0.1 M: sodi-
um citrate (pH 3-5), potassium phosphate (pH 6-7), Tris-HCl (pH 8), and
glycine-NaOH (pH 9-11) buffers. Temperature effect on chitinase activity of
ChiCH was measured in 0.1 M potassium phosphate buffer (pH 6.0) from 20 °C
to 80 °C. In addition, substrate specificity of ChiCH was examined on soluble
substrates, namely glycol chitin (Sigma), glycol chitosan (Sigma), car-
boxymethylcellulose (Hayashi, Osaka, Japan), laminarin (Sigma), and soluble
starch (Hayashi).
For antifungal assay, conidial suspension of Botrytis elliptica, at a final con-
centration of 4 × 105conidia/ml, was mixed with purified ChiCH and incubated
at room temperature for 12 h. After incubation, the percentage of germinated
spores of Botrytis elliptica was calculated and the inhibition rate was used as
an indication of antifungal activity. The experiment was repeated for three times
and analysed statistically by Duncan’s Multiple Range Test.
The nucleotide sequence data of chiCH have been submitted to Gen-
Bank/EMBL DNA Databases under accession numbers AF510723.
RESULTS AND DISCUSSION
Three chitinolytic B. cereus strains isolated from Israel (Pleban et al., 1997),
United States (Wang et al., 2001), and Japan (Mabuchi et al., 2000), have been
reported. These B. cereus strains produce chitinases, such as chitobiosidase
of strain 65 (Pleban et al., 1997), Chi36 of strain 6E1 (Wang et al., 2001), and
ChiA of strain CH (Mabuchi and Araki, 2001). In this report, we found that
Ann. Microbiol., 54 (3), 289-297 (2004) 291
ChiCH produced by the chitinolytic B. cereus 28-9 from Taiwan was similar to
these chitinases. However, the biological function of this kind of chitinases from
different B. cereus strains has not yet been studied. Therefore, we cloned the
ChiCH-encoding gene and investigated the biological function of ChiCH of B.
cereus 28-9 herein.
B. cereus 28-9 produced at least two chitinases and secreted both enzymes
into culture medium. Fig. 1 shows the zymogram of partially purified chitinases
produced by B. cereus 28-9. One chitinase with estimated molecular mass of
37 kDa was named ChiCH and its N-terminal amino acid sequence was deter-
mined as ANNLGSKLLVGYWHNFD.
The N-terminal amino acid sequence of ChiCH and the conserved amino
acid sequence of catalytic domains of family 18 chitinases were used to design
degenerate primers, dchf and dchr. A DNA fragment of about 300 bp was am-
plified and determined as the partial sequence of a chitinase gene based on se-
quence analysis data. This fragment was subsequently used as a probe in
Southern blot analysis and colony hybridisation.
Figure 2 shows the result of Southern blot analysis using the 300-bp DNA
probe. EcoRI-digested genomic DNA of B. cereus 28-9 yielded a single band at
the 1.7 kb-position. Single bands were also detected in the EcoRV- and PvuII-
digested B. cereus 28-9 genomic DNA at the positions of about 4.0 kb and 8.0
kb, respectively. Therefore, EcoRI fragments (1.5-3 kb) of B. cereus 28-9 were
used to construct a subgenomic library. One clone, screened from this subge-
nomic library, harboured a recombinant plasmid carrying a 1.7-kb EcoRI insert.
Sequence analysis revealed an open reading frame (ORF) in this region. The
N-terminal amino acid sequence, ANNLGSKLLVGYWHNFD, of excreted
ChiCH coincided with that deduced from the nucleotide sequence of the pre-
dicted ORF as shown in Fig. 3.
292 C.-J. HUANG and C.-Y. CHEN
FIG. 1 – SDS-PAGE and zymogram analysis. (A) Gel was stained with Coomassie
Brilliant Blue G-250. (B) Chitinase activity was detected by staining the gel
containing 0.01% glycol chitin with 0.01% Calcofluor. Lanes: 1, proteins par-
tially purified from the culture supernatant of B. cereus 28-9; 2, ChiCH par-
tially purified from E. coli DH5α(pGH51). ChiCH with estimated molecular
mass of 37 kDa is indicated by an arrow.
Ann. Microbiol., 54 (3), 289-297 (2004) 293
FIG. 2 – Southern blot analysis of the genomic DNA of B. cereus 28-9. The genomic
DNA was digested with EcoRI (lane 1), EcoRV (lane 2) and PvuII (lane 3).
The Southern blot showed signals of hybridization with the 300-bp DNA probe
corresponding to chiCH sequence. The estimated DNA fragment size of each
signal is indicated beside the arrow.
FIG. 3 – Nucleotide and the deduced amino acid sequences of chiCH. Amino acid
residues corresponding to those determined by N-terminal sequencing are in
bold type. The putative active site of ChiCH is underlined. The stop codon of
chiCH is indicated by an asterisk. The putative Shine-Dalgarno sequence,
AGGAG, is italicized.
Sequence analysis indicated that chiCH gene is 1,083 bp in length with an
ATG start codon and a TAA stop codon (Fig. 3). The putative Shine-Dalgarno
sequence, AGGAG, was located 8 nucleotides upstream of the start codon. The
deduced protein (ChiCH precursor) consisted of 360 amino acid residues with a
calculated molecular weight of 39,372 and isoelectric point of 6.21.
Alignment of the deduced amino acid sequence of ChiCH precursor with the
N-terminal amino acid sequence of excreted ChiCH of B. cereus 28-9 showed
that the deduced amino acid sequence of ChiCH precursor contained a signal
peptide which was cleaved off between Ala-27 and Ala-28 by the signal pepti-
dase. The signal peptide of ChiCH produced by B. cereus 28-9 had 27 amino
acid residues with the common characteristics of a signal peptide, including a
positive-charged region, a hydrophobic central core and a signal peptidase
recognition site, Ala-X-Ala (Perlman and Halvorson, 1983).
In addition to the N-terminal signal peptide, the deduced ChiCH contained a
catalytic domain as that shown in the conserved domain database of National
Center for Biotechnology (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).
The conserved amino acid sequence of the catalytic domain, from Gly-139 to
Leu-150, was homologous to a number of family 18 chitinases (Henrissat and
Bairoch, 1993) (Fig. 3). This region included two aspartate residues, Asp-141
and Asp-143, and one glutamate residue, Glu-145. These residues have also
been found in ChiA1 of Bacillus circulans WL-12 (Watanabe et al., 1993) and
ChiA of Serratia marcescens (Perrakis et al., 1994). Furthermore, Glu-145 in
the deduced ChiCH seemed to correspond to Glu-315 of S. marcescens ChiA,
which has been reported to be involved in the catalysis of chitinase (Perrakis et
al., 1994).
The amino acid sequence of the deduced ChiCH showed 97.5% homology
to that of the ChiA of B. cereus CH (Mabuchi and Araki, 2001) and 94.7% ho-
mology to that of the Chi36 of B. cereus 6E1 (Wang et al., 2001) as analysed by
the comparison program in the GCG package (Fig. 4). In addition, Wang et al.
(2001) have speculated that the chitobiosidase of B. cereus strain 65 is similar
to Chi36 of strain 6E1 (Pleban et al., 1997; Wang et al., 2001). Although ho-
mologous chitinases are distributed in different B. cereus strains from different
countries, it is unclear whether this kind of family 18 chitinase gene is species-
specific in B. cereus. However, a species-specific family 19 chitinase gene has
been found in Burkholderia gladioli (Kong et al., 2001). Therefore, the distribu-
tion of chiCH-like genes in B. cereus strains and other Bacillus species be-
comes a subject to study.
ChiCH was expressed and purified from the periplasmic fraction of E. coli
DH5α(pGH51) as shown by SDS-PAGE and in-gel activity assay (Fig. 1, lane
2). A 33-kDa protein without chitinase activity as shown by in-gel activity assay
was consistently co-purified with ChiCH in our procedure. Therefore, the par-
tially purified ChiCH was used for further characterization. The results indicated
that ChiCH had an optimal pH of 6 (Fig. 5) and an optimal temperature of 40 °C
(Fig. 6). It retained over 75% of optimal activity between pH 5.0–7.0. Further-
more, pH and temperature stabilities of ChiCH could be maintained in the
ranges of pH 3-8 (Fig. 5) and 40-50 °C (Fig. 6), respectively.
The ability of ChiCH to hydrolyse various carbohydrates was examined. The
result showed that glycol chitin was efficiently hydrolysed among five soluble
substrates. In addition, when glycol chitosan was used as a substrate, chitinase
294 C.-J. HUANG and C.-Y. CHEN
Ann. Microbiol., 54 (3), 289-297 (2004) 295
FIG. 4 – Sequence comparison of three chitinases from B. cereus strains. ChiCH (this
study); BcchiA, chitinase A of B. cereus CH (accession number AB041931);
Bcexo, exochitinase (Chi36) of B. cereus 6E1 (accession number AF275724).
The asterisks indicate conserved amino acid residues, two Asp and one Glu,
which have been identified as essential amino acid residues.
ChiCH exhibited 10% of relative enzyme activity. However, null effect on other
substrates, including laminarin (β-1,3-glucan), carboxymethyl cellulose (β-1,4-
glucan), and soluble starch (β-1,4/1,6-glucan), was observed.
ChiCH of 17 µunit inhibited about 10% of the conidial germination of Botry-
tis elliptica and the inhibition level was not augmented by increasing chitinase
activity from 17 to 66 µunit. On the other hand, inhibition of the conidial germi-
nation of Botrytis elliptica was much stronger (55.2% of inhibition) by the cul-
ture supernatant of B. cereus 28-9 at 20 µunits than by the partially purified
ChiCH from E. coli DH5α(pGH51).
According to the study of Pleban et al. (1997), a chitobiosidase is present in
the endophytic B. cereus strain 65. They have suggested that chitobiosidase
activity is important for antifungal activity of strain 65. Our present work indicat-
ed that ChiCH possibly has antifungal activity of mild strength. Since this anti-
fungal level was much lower than that exhibited by the culture supernatant of
B. cereus 28-9, we presume that not only ChiCH but also other antifungal fac-
tors were produced by B. cereus 28-9 and exhibited synergistic or combined ef-
fect against target fungi.
Acknowledgements
This work was financially supported by National Science Council, Taiwan, Re-
public of China.
296 C.-J. HUANG and C.-Y. CHEN
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