Biochemical and Biophysical Research Communications 307 (2003) 620625

BBRC
www.elsevier.com/locate/ybbrc

A constitutively expressed 36 kDa exochitinase from

Bacillus thuringiensis HD-1
Naresh Arora, Tarannum Ahmad, R. Rajagopal, and Raj K. Bhatnagar*
International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, P.O. Box 10504, New Delhi 1100 67, India
Received 5 June 2003

Abstract

A 36 kDa chitinase was puried by ion exchange and gel ltration chromatography from the culture supernatant of Bacillus
thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced
even in the presence of glucose. The puried chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed
maximum activity at 65 C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)2 most
eciently and was therefore classied as an exochitinase. The sequence of the tryptic peptides showed extensive homology with
Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplied with primers based on
the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-
terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18
chitinases were mapped at positions 105109 and 138145 of Chi36. The recombinant chitinase was expressed in a catalytically
active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal eect of the veg-
etative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.
2003 Elsevier Inc. All rights reserved.

Keywords: Bacillus thuringiensis; Chitinase; Synergism

Bacillus thuringiensis is a gram positive, spore form-
ing, soil bacterium that forms parasporal crystals during
sporulation. A diverse range of crystal proteins is pro-
duced by dierent B. thuringiensis strains that are highly
specic for dierent insect larvae [1]. These features meet
the criteria of an ideal biocontrol agent and B. thurin-
giensis has been used as an insecticidal agent for decades
[2]. However, resistance to crystal toxins by targetted
pests has been observed and various methods are being
employed to increase the ecacy of the insecticidal
crystal proteins and in controlling agricultural pests
[3,4].
The insecticidal crystal proteins ingested by the insect
and undergo site-specic proteolysis from the N and the
C terminus to generate active fragments. These activated
polypeptides bind to the receptors in the midgut epi-
thelium and form ion channels, inducing osmotic lysis of
*
Corresponding author. Fax: +91-11-2616-2316.
E-mail address: raj@icgeb.res.in (R.K. Bhatnagar).
the epithelium that consequently kills the larvae [5]. The
midgut lumen is separated from the epithelium by a
protective structure consisting of a network of chitin
and proteins, called the peritrophic membrane [6]. It is
suggested that the accessibility of the toxins to the
midgut epithelium is restricted by the peritrophic ma-
trix. It is believed that chitinases disrupt the integrity of
the peritrophic membranes, facilitating the contact be-
tween the activated toxins and receptors in the midgut
epithelium [7].
A synergistic action between insecticidal crystal pro-
teins and chitinase, which hydrolyzes the b-1,4 linkage in
chitin, has been demonstrated to occur during co-ap-
plication of insecticidal protein containing spore sus-
pension along with chitinase [8]. Co-expression of
heterologous chitinase genes in B. thuringiensis has also
been demonstrated to increase the insecticidal activity of
the bacterium [911]. B. thuringiensis itself produces
chitinases and the role of these endogenous chitinases
has recently come under investigation. The involvement

0006-291X/03/$ - see front matter 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0006-291X(03)01228-2
 N. Arora et al. / Biochemical and Biophysical Research Communications 307 (2003) 620625
of endogenous chitinases during B. thuringiensis aizawai
infection of Spodoptera littoralis larvae was demon-
strated by the addition of a chitinase inhibitor, allos-
amidin that increased the LC50 values of the toxin [12].
Chitinases from two dierent strains of B. thuringi-
ensis, kenyae and pakistani, have been cloned and they
are very similar to each other in gene structure and
properties of the encoded protein [13,14]. We report here
the purication of a novel chitinase from B. thuringiensis
sp. HD-1, cloning of the corresponding gene, and its
heterologous expression in Escherichia coli. The syner-
gistic action of this chitinase along with the vegetative
insecticidal protein (Vip) against Spodoptera litura lar-
vae is also demonstrated.
Materials and methods
Bacterial strains and growth conditions. B. thuringiensis HD1 was
obtained from Bacillus Genetic Stock Centre, OH, USA. For chitinase
production, the bacterium was grown in G-medium [15] with 0.2%
glucose or 0.2% colloidal chitin at 30 C with shaking. Colloidal chitin
was prepared according to Roberts and Selitrenniko (1988) [16]. For
chromosomal DNA extraction, bacteria were grown in LuriaBertani
(LB) medium. E. coli DH5a and M15 carrying the pGEMT-chi and
pQE-chi plasmids, respectively, were grown at 37 C in LB medium for
expression and plasmid preparations.
Purication of Chi36 from the culture uid of Bt-HD1. Protein from
the supernatant of a 500 ml culture grown in G-medium supplemented
with glucose was concentrated by ammonium sulphate fractionation at
the following saturation concentration, 035%, 3555%, and 5595%.
Protein in each fraction was dissolved in 5 ml of 50 mM TrisHCl, pH
7.5, and dialyzed overnight against the same buer. A glycol-chitin
activity gel was done in each fraction to locate the chitinase activity
[16]. The active fraction from ammonium sulphate precipitation was
further resolved on a DE-52 anion exchange column previously
equilibrated with 50 mM TrisHCl buer, pH 7.5. After washing un-
adsorbed proteins with the same buer, chitinase was eluted with a
linear gradient of 00.75 M KCl in same buer at a ow rate of 1 ml/
min. The active fractions were pooled, dialyzed and concentrated by
passing through amicom PM-30 membrane. The concentrated sample
was resolved on a Sephacryl S-100 4R gel ltration column. Elution
was performed at a ow rate of 1 ml/min in 50 mM TrisHCl, pH 7.5,
containing 100 mM NaCl. One milliliter fractions were collected and
individual fractions were analyzed for chitinase activity on a non-
denaturing PAGE.
Amino-acid sequence of Chi36. Sequencing of the tryptic digests of
the 36 kDa major protein of the active fraction after gel ltration was
performed at Biotechnology Research Laboratory, Medical University
of South Carolina, Charleston, USA.
Chitinase assays. Chitinase was assayed on a 10% non-denaturing
gel containing 0.01% glycol chitin by the method of Trudel and Asselin
[17], with minor modications. Fluorometric substrates 4-methylum-
belliferyl b-D -N ,N 0 ,N 00 -triacetylchitotrioside and 4-methylumbelliferyl
b-D -N ,N 0 -diacetylchitobioside were obtained from Sigma. The reaction
mixture contained in a total volume of 100 ll, 50 lM of the substrate,
50 mM phosphate buer, pH 7.0, and an appropriate amount of the
enzyme. To nd out the optimum pH of the reaction, the following
range of buers were used: 50 mM sodium acetate buer, pH 4.05.5,
50 mM sodium phosphate buer, pH 6.07.5, 50 mM TrisHCl, pH
8.09.0, and sodium carbonate buer, pH 1011. The reaction mixture
was incubated at 30 C for 20 min and stopped by adding 2.4 ml of
150 mM glycineNaOH buer, pH 10.5. The release of free 4-meth-
621
ylumbelliferone was monitored by uorescence spectroscopy with ex-
citation at 360 nm and measuring emission at 450 nm. The uorimetric
intensity was converted to nanomolar of 4-methylumbelliferone re-
leased by preparing a standard curve of 4-methylumbelliferone. The
activity has been expressed as nmol of 4-methylumbelliferone released
min1 mg1 protein.
Insect bioassays. The insecticidal eect of Chi36 in combination
with the Vip was tested against S. litura neonate larvae by feeding the
larvae leaves coated with required concentrations of Vip and Chi36, or
Vip alone. Six neonate larvae were released on the leaves at each set of
concentration. The larvae on the leaves were incubated under con-
trolled conditions of temperature 25 C, 70% relative humidity, and a
photoperiod of 12 h light:12 h dark. Observations were recorded after
48 h and the dose combination was replicated in ve independent sets
of experiments.
Cloning and expression of chi36. Forward (50 GATGTTAA
ACAGGTTCAA 30 ) and reverse primers (50 TTATTTTTGCAAGG
AAAG 30 ) were designed based on homology to Bacillus cereus 36 kDa
chitinase, and the gene encoding for Chi36 was amplied by PCR using
Bt-HD1 genomic DNA as a template. The amplied product was
cloned into the PCR cloning vector, pGEMT easy, and was sequenced
completely. To express the polypeptide in E. coli, the gene was sub-
cloned into the expression vector, pQE-32 (Qiagen) and expressed as
an N-terminal fusion protein with 6 His tag in M-15 expression host.
Expression of recombinant chitinase was induced by adding IPTG at a
nal concentration of 1 mM to the actively growing culture of E. coli.
The culture was further grown for 2 h and the cells harvested by
centrifugation. The cells were disrupted by sonication and the
induced proteins were analyzed by resolving the proteins on 10%
SDSPAGE.
Results
Production of Chi36 by B. thuringiensis HD1
The bacterium was grown in the following combi-
nation of carbon sources supplemented individually in
G-medium: glucose, glucose + chitin or only chitin.
Resolution of equal amounts of protein on a glycol-
chitin activity gel revealed that comparable level of
chitinase was produced in cultures independent of the
nature of the carbon source (Fig. 1). In all subsequent
experiments, the bacterium was grown in the presence of
glucose only. Time course for the accumulation of chi-
tinase was examined in cultures grown for 24, 36, 48, 60,
and 72 h in G-medium supplemented with glucose.
Highest chitinase activity was observed in the culture
grown for 72 h. Protein in the supernatant of 72 h grown
culture was fractionated by ammonium sulphate. Pro-
tein precipitating at 3565% ammonium sulphate satu-
ration contained nearly 80% of total chitinase activity.
Ammonium sulphate precipitated protein was subjected
to anion exchange chromatography and chitinase was
obtained in the wash fraction, whereas other proteins
eluted at higher salt concentrations. Further purication
of the protein was done by gel ltration chromatogra-
phy and a chitinase corresponding to a molecular mass
of 36 kDa was obtained (Fig. 2). To determine the
amino-acid sequence of the protein, tryptic digests of the
protein were generated and the obtained sequence was
622 N. Arora et al. / Biochemical and Biophysical Research Communications 307 (2003) 620625
Fig. 1. Chitinase production in the presence of glucose and chitin. B.
thuringiensis HD-1 was grown in the presence of only 0.2% glucose,
only 0.2% chitin, 0.2% glucose + 0.2% chitin, or without any carbon
source. Proteins in the culture supernatant were precipitated by am-
monium sulphate fractionation and equal amounts (5 lg) were loaded
on a glycol chitin activity gel, as described in Materials and methods.
The dark areas represent the clearing zones produced by the hydrolysis
of glycol chitin. (+) or ()) indicates the presence or absence of glucose
or chitin.
analyzed for homologies at the NCBI server using the
BLAST application. The sequence of the tryptic pep-
tides matched the exochitinase from B. cereus.
Fig. 2. (A) Gel ltration chromatography of DE-52 puried protein.
The solid line indicates absorbance at 214 nm and the dashed line in-
dicates the chitinase activity on a glycol chitin activity gel. The clearing
zones were subjected to densitometric analysis and plotted where
100,000 pixels is equivalent to 1 absorbance unit. (B) (a) Coomassie
brilliant blue staining of the gel ltration puried active fractions. (b)
Activity staining of the active fractions. The dark zones represent the
hydrolysis of glycol chitin in the activity gel.
Properties and kinetics of Chi36
Chi36 is active on both polymeric as well as oligo-
saccharide substrates. Higher specic activity was ob-
tained with the disaccharide substrate as compared to
the trisaccharide substrate, which classies chi36 as a
chitobiosidase or an exochitinase (Table 1). When tested
against the monomer, 4-MU N-acetylglucosaminide, no
activity was obtained showing that chi36 does not have
an N-acetylglucosaminidase activity. The Km and Vmax
for the oligosaccharide substrates were calculated to be
30 lM and 45 nmol of product formed min1 mg1
protein, respectively, for the hydrolysis of 4 MU-(Glc-
NAc)2 and 16 lM and 7.2 nmol of product formed
min1 mg1 protein for 4 MU-(GlcNAc)3 . To study the
eect of pH on chitinase activity, buers of dierent pH
were used. The results are shown in Fig. 3. The enzyme
was active at acidic pH with the highest activity at pH
6.5. At alkaline pH, considerable activity was lost. To
investigate the optimum temperature of chi36, the en-
zymatic reactions were performed at 2565 C in sodium
phosphate buer, pH 6.5, using 4 MU-(GlcNAc)3 . The
activity of chi36 increased with increase in temperature
(Fig. 4) and showed a high activity at 65 C.
Table 1
Substrate specicity and reaction rate of puried Chi36 from
B. thuringiensis HDI
Kinetic parameters Substrate
4-MU 4-MU 4-MU
(GlcNAc) (GlcNAc)2 (GlcNAc)3
Km (lM)a 0 30 16
Vmax (nmol of 4-MU 0 45 7.2
formed/minute)a
a
Reaction rate was measured with the oligosaccharide substrates
4-MU(GlcNAc)1-3 , nal concentration 50 lM, in sodium phosphate
buer, pH 6.5 at 30 C for 20 min.
Fig. 3. Optimum pH of Chi36: activity of chitinase was measured with
4-MU (GlcNAc)3 , nal concentration 50 lM, in 50 mM sodium acetate
buer, pH 4.05.0, 50 mM sodium phosphate buer, pH 6.07.5,
50 mM Trisacetate, pH 8.09.0, and 50 mM sodium carbonate buer,
pH 1011, at 30 C for 20 min.
 N. Arora et al. / Biochemical and Biophysical Research Communications 307 (2003) 620625 623

Fig. 5. The deduced amino-acid sequence of Bt Chi36: the signal

peptide is underlined. The conserved domains I and II are boxed and
the tryptic peptide is double underlined. Bold lettering indicates resi-
dues that are dierent from B. cereus 6E1 36 kDa exochitinase.

Fig. 4. Temperature dependency of Chi36: activity of chitinase was

measured with 4-MU (GlcNAc)3 , nal concentration 50 lM, in 50 mM
sodium phosphate buer, pH 6.5, at 2565 C for 1 h. The points
represent mean values of two independent measurements.

Cloning and heterologous expression of chi36

The homology search at NCBI BLAST revealed that

the amino-acid sequences of tryptic peptides matched
the exochitinase from B. cereus with 96% identity.
Forward and reverse primers were synthesized based on
the nucleotide sequence of B. cereus exochitinase and a
product of ca. 1000 bp was amplied from Bt-HD1 ge-
nomic DNA, and sequenced. BLAST search showed
that the cloned fragment had extensive homology with
B. cereus chitinase, the gene was therefore designated as Fig. 6. Expression of recombinant Chi36 in E. coli: M 15 cells trans-
chi36. Residues depicted in bold are unique to Chi36 formed with the plasmid pQE-Chi36 were induced with 1 mM IPTG
for 2 h. The induced and un-induced cultures were separated on 10%
being reported here. Chi36 is 1083 bp in length and en-
SDSPAGE and stained with Coomassie brilliant blue. M, marker; U,
codes a polypeptide of 39,407 Da, which is larger than un-induced; and I, induced.
the size of the native protein observed on SDSPAGE.
The Signal P site predicted a signal peptide from resi-
dues 127 and cleavage between residues Ala 27 and Ala Synergism of insecticidal activity
28. Therefore, the cleavage of the signal peptide gener-
ates a 36 kDa protein, which has alanine at the mature Preliminary analysis had shown that the LC50 for Vip
N-terminus. Conserved Domain Architecture Retrieval towards S. litura larvae was 20 ng cm2 . To investigate if
Tool (CDART) at NCBI identied a carbohydrate me- the insecticidal activity of Vip was potentiated by the
tabolism and transport domain containing the con- addition of chitinase, partially puried Chi36 alone,
served motifs characteristic of family 18 chitinases at 20 lg of Vip alone, and Vip in combination with dif-
residues 105109 and 138145 (Fig. 5). The entire open ferent concentrations of chitinase was fed S. litura ne-
reading frame was cloned in the prokaryotic expression onate larvae. A 30% decrease in the LC50 of vip
vector, pQE 32 as a NcoI/SphI fragment. A novel in- indicated that chi36 potentiated the toxic eect of vip
duced protein of 36 kDa was found in induced cultures against S. litura larvae.
transformed with the recombinant pQE-chi36 plasmid,
which was absent in the un-induced cultures (Fig. 6).
The recombinant chitinase was localised in both the Discussion
soluble and insoluble fractions of the cell lysate. The
expressed recombinant chitinase was catalytically active It is well established that chitinase production in most
against polymeric and oligomeric substrates. In addi- of the chitinase producing bacteria is inducible by chitin,
tion, the recombinantly expressed Chi36 showed iden- chito-oligosaccharides, or even N-acetylglucosamine
tical properties and kinetics to the native enzyme. [18]. Low levels of chitinase are observed in cultures
624 N. Arora et al. / Biochemical and Biophysical Research Communications 307 (2003) 620625
grown in the presence of glucose. However, in this study,
high levels of Chi36 were present when the bacteria were
grown in the presence of glucose, suggesting that the
production of Chi36 is independent of the presence of
chitin. Normally, the induction of chitinase in Strepto-
myeces is dependent on the presence of chitin in the
growth medium. Interestingly, a single point mutation in
the promoter of chitinase shifts the synthesis from in-
ducible to constitutive mode [19].
Of the various chitinases described from related Ba-
cillus sp., Bt Chi36 displayed extensive similarities to B.
cereus 6E1 exochitinase [20] as the two proteins shared
98% amino-acid identity (Fig. 5). But the two proteins
do not have identical properties. B. cereus exochitinase
activity for the trisaccharide was only 4.4% of the ac-
tivity with the disaccharide, whereas in Bt Chi36, 15%
relative activity was obtained with the trisaccharide.
Also, B. cereus exochitinase lost activity at high tem-
peratures but Bt Chi36 was active upto 65 C. Another
36 kDa chitinase, ChiA [20], from B. cereus CH shared
95% identity of amino acids but it diered from Bt
Chi36 in being an endochitinase. Amongst the Bacillus
circulans chitinases, A1, A2, B1, B2, C, and D [21], Bt
Chi36 shared 53% amino acid similarity with B. circu-
lans ChiD, which had a FnIII domain and a catalytic
domain. Bt Chi36 is distinct from the 74 kDa chitinases
characterized from B. thuringeinsis kenyae that showed
homology to B. cereus ChiB [13]. Therefore, it is possible
that the Bt Chi36 (present study) and 74 kDa chitinases
(B. thuringiensis kenyae) in B. thuringiensis correspond
to B. cereus ChiA and ChiB, respectively.
Btchi36 contains the conserved region characteristic
of family 18 chitinases that have the consensus sequence
[DN]G[LIVMF][DN][LIVMF][DN]xE, where E
is the catalytic residue. A signal peptide is present at the
N-terminus that may be responsible for the secretion of
the protein. Btchi36 consists of a single catalytic domain
and lacks the chitin binding or the bronectin domain
involved in binding chitin. In spite of the lack of the
chitin-binding domain, a high activity on a polymeric
substrate, glycol chitin, was obtained demonstrating
that the presence of the chitin binding domain is not an
absolute requirement for the hydrolysis of chitin. That
the absence of chitin binding domain does not aect the
catalytic eciency of chitinase has been from other
bacterial chitinases also. The 36 kDa chitinase homolog
from B. cereus also lacks the chitin binding domain and
is also active on the polymeric substrate, CM-chitin-
RBV [21]. Also, ChiA1 from B. circulans, retained some
activity for chitin on deleting the chitin binding domain,
although considerable activity was lost [22].
The observed potentiation of insecticidal activity of
Vip in the presence of chitinase oers another tool to
enhance the application of Bt proteins. Chi36, when
used in combination with Vip enhanced the insecticidal
activity of Vip against neonate larvae of S. litura. Vip,
like the cry toxins, also targets the midgut of the insect
and has to penetrate the peritrophic matrix to reach the
midgut epithelium. Scanning electron microscopy of
midguts of S. litura larvae fed on recombinant ChiAII
showed that the peritrophic membrane was perforated,
and when these larvae were fed ChiAII in combination
with CryIC, an increase in toxicity was obtained [7].
Similar synergistic eect of chitinase with cry toxins has
been demonstrated in other studies as well. The present
study suggests that the mechanism of the synergistic
eect is similar when Vip is used instead of cry toxins.
Chitinases in B. thuringiensis are likely to assist
pathogenesis of insects by hydrolyzing the chitin present
in the gut. The bacterium has probably adapted to a
constitutive, rather than inducible expression of chiti-
nase to meet this requirement.
Acknowledgments
We are grateful to the Bacillus Genetic Stock Center for providing
the strain Bacillus thuringiensis HD-1. This project was funded by the
Indo-Swiss grant.
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