Appl Microbiol Biotechnol (2005) 66: 434442

DOI 10.1007/s00253-004-1664-9
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Frank Hoster
.
Jessica E. Schmitz
.
Rolf Daniel
Enrichment of chitinolytic microorganisms: isolation
and characterization of a chitinase exhibiting antifungal activity
against phytopathogenic fungi from a novel Streptomyces strain
Received: 3 March 2004 / Revised: 15 May 2004 / Accepted: 21 May 2004 / Published online: 29 July 2004
# Springer-Verlag 2004
Abstract Thirteen different chitin-degrading bacteria
were isolated from soil and sediment samples. Five of
these strains (SGE2, SGE4, SSL3, MG1, and MG3)
exhibited antifungal activity against phytopathogenic
fungi. Analyses of the 16S rRNA genes and the substrate
spectra revealed that the isolates belong to the genera
Bacillus or Streptomyces. The closest relatives were
Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B.
ehimensis (MG1), and Streptomyces griseus (MG3). The
chitinases present in the culture supernatants of the five
isolates revealed optimal activity between 45C and 50C
and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6
(SGE4), and 57 (MG3). The crude chitinase preparations
of all five strains possessed antifungal activity. The
chitinase of MG3 (ChiIS) was studied further, since the
crude enzyme conferred strong growth suppression of all
fungi tested and was very active over the entire pH range
tested. The chiIS gene was cloned and the gene product
was purified. The deduced protein consisted of 303 amino
acids with a predicted molecular mass of 31,836 Da.
Sequence analysis revealed that ChiIS of MG3 is similar
to chitinases of Streptomyces species, which belong to
family 19 of glycosyl hydrolases. Purified ChiIS showed
remarkable antifungal activity and stability.
Introduction
Chitin, a homopolymer of -1,4-linked N-acetyl-D-glu-
cosamine residues, is the most abundant renewable
resource after cellulose. It is widely distributed in nature
as a structural component of crustacea, fungi, protozoa,
and insects. Chitinases (EC 3.2.1.14) are glycosyl hydro-
lases, which catalyze the degradation of chitin. These
enzymes are present in a wide range of organisms such as
bacteria, fungi, insects, plants, and animals. Chitinases
belong to either family 18 or family 19 of glycosyl
hydrolases (Davis and Henrissat 1995; Henrissat and
Bairoch 1993). The enzymes of the two families do not
share similarities in amino acid sequence or three-dimen-
sional structure. Family 18 encompasses chitinases found
in bacteria, fungi, viruses, and animals, and class III or V
of plant chitinases. Family 19 includes class I, II, or IV
chitinases of plants and chitinases present in some
Streptomyces and other Actinobacteria strains (Kawase
et al. 2004; Watanabe et al. 1999).
The chitinases of the above-mentioned organisms play
important physiological and ecological roles. Invertebrates
require chitinases for partial degradation of their old
exoskeletons (Ruiz-Herrera and Martinez-Espinosa 1999)
and plants as a defense mechanism against fungal
pathogens (Honee 1999). Chitinases are constituents of
several bacterial species; some of the best known include
the Aeromonas, Serratia, Myxobacter, Vibrio, Streptomy-
ces, and Bacillus genera (Cody et al. 1990). Bacteria
produce chitinases mainly to degrade chitin and utilize it
as an energy source. In addition, some chitinases of
chitinolytic bacteria, such as the chiA gene products from
Serratia marcescens and Enterobacter agglomerans, are
potential agents for the biological control of plant diseases
caused by various phytopathogenic fungi (Chernin et al.
1997; Downing and Thomson 2000). The latter enzymes
inhibit fungal growth by hydrolyzing the chitin present in
the fungal cell wall. Antifungal proteins such as chitinases
are of great biotechnological interest because of their
potential use as food and seed preservative agents and for
engineering plants for resistance to phytopathogenic fungi
(Dempsey et al. 1998).
In this report, we isolated and classified chitinolytic
bacteria from soil and sediment samples. The recovered
species were examined for the production of chitinases
with antifungal activities against several phytopathogenic
fungi. The goal of this study was to identify and
F. Hoster
.
J. E. Schmitz
.
R. Daniel (*)
Abteilung Angewandte Mikrobiologie, Institut fr
Mikrobiologie und Genetik der Georg-August-Universitt
Gttingen,
Grisebachstrasse 8,
37077 Gttingen, Germany
e-mail: rdaniel@gwdg.de
Tel.: +49-551-393827
Fax: +49-551-393808
characterize from among the recovered chitinases con-
ferring antifungal activity, the chitinase most suitable for
application as a biocontrol agent.
Materials and methods
Sample sites, strains, and plasmids
For the enrichment of chitinolytic microorganisms, soil
from a meadow near Gttingen (Germany), a sediment
sample with a salinity of approximately 20% from the
Solar Lake (Egypt), and sediment from the Gulf of Eilat
(Israel) were collected. The Escherichia coli strains DH5
(Ausubel et al. 1987) and BL21 (Invitrogen, Karlsruhe,
Germany) were used as hosts for cloning experiments and
production of chitinases, respectively. The plasmids
pCR2.1-TOPO and pET101/D (Invitrogen) were em-
ployed as vectors for the cloning experiments. The
phytopathogenic fungi Guignardia bidwellii DSM 3513,
Sclerotia sclerotiorum DSM 1946, Fusarium culmorum
DSM 1094, and Botrytis cinerea DSM 877 were obtained
from the Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ, Braunschweig, Germany). Aspergil-
lus nidulans FGSC A4 was acquired from the Fungal
Genetics Stock Center (Kansas City, Kan.). The isolated
strain MG3 has been deposited with the DSMZ under
accession number DSM 16384.
Media and growth conditions
For enrichment of chitinase-producing microorganisms, a
mineral medium containing colloidal chitin as sole carbon
and energy source was used and incubated at 30C.
Colloidal chitin was prepared by the method of Wiwat et
al. (1999). The medium for enrichment cultures contained
the following (g/l): colloidal chitin, 7.0; KH
2
PO
4
, 3.0;
K
2
HPO
4
, 2.0; NH
4
Cl, 1.0; MgSO
4
7H
2
O, 0.2; CaCl
2
2-
H
2
O, 0.01; vitamin solution according to Wolin et al.
(1964), 2.0 ml; and trace element solution SL4 (Pfennig
and Lippert 1966), 1 ml; pH 6.8. The mineral medium was
supplemented with 40 g/l sea salt when organisms derived
from marine samples were grown. The use of different
growth substrates was tested by replacing chitin with other
carbon sources. Enrichments were performed under aer-
obic conditions. The enrichment was initiated by adding
0.5 g (wet weight) soil or sediment to enrichment medium
(25 ml). In order to obtain pure cultures, samples of chitin-
degrading enrichment cultures were streaked on chitin
agar. This agar contained enrichment medium supplemen-
ted with agar (25 g/l). Colonies showing zones of
clearance against the creamy background were regarded
as chitinase-producing and restreaked until pure cultures
were obtained.
E. coli was routinely grown in LB medium at 30C
(Ausubel et al. 1987). All growth media for E. coli strains
harboring plasmids contained 100 g/ml ampicillin. All
fungi were cultivated in Aspergillus medium as described
by Bennett and Lasure (1991).
Preparation of E. coli cell extracts
Cells from the stationary growth phase from 500-ml
cultures were harvested by centrifugation at 6,000 g for
20 min, washed once with 100 mM potassium phosphate
buffer (pH 8.0), and resuspended in 23 ml of the same
buffer. The cells were disrupted by French pressing
(1.3810
8
Pa) and the extract was cleared by centrifuga-
tion at 32,000 g and 4C for 30 min.
Monitoring of cell growth
Cell growth was followed by measuring the increase in
turbidity at 600 nm. Optical density was quantified
directly by insertion of culture tubes into a Spectronic
20 photometer (Bausch and Lomb, Rochester, N.Y.) or by
measuring the turbidity of 1 ml culture in a Jasco
spectrophotometer V-550 (Jasco, Jena, Germany). All
growth experiments were performed in triplicate and
results represent mean values.
Enzyme concentration
The cells were removed from the culture medium by
centrifugation at 16,300 g for 15 min and the enzyme was
concentrated by adding 561 g solid ammonium sulfate per
1,000 ml cell-free supernatant. Precipitated protein was
collected by centrifugation at 16,300 g for 30 min. The
resulting pellet was dissolved in a minimal amount of
0.02 M potassium phosphate buffer (pH 6.0) and dialyzed
extensively at 4C against the same buffer. The dialysate
was centrifuged at 13,000 g for 30 min to remove
insoluble material. The clear supernatant was used for the
characterization of chitinase activity.
Enzyme assay
Chitinase activity was assayed with colloidal chitin as the
substrate. Enzyme solution (0.5 ml) was added to 1 ml
substrate solution, which contained 0.3% colloidal chitin
in a potassium phosphate buffer (20 mM, pH 6.0). The
mixture was incubated at 45C for 1 h. After centrifuga-
tion, the amount of reducing sugar produced was
measured by the method of Imoto and Yagashita (1971).
Reducing sugar levels were determined relative to N-
acetyl--D-glucosamine standards of 10150 g/ml. One
unit of chitinase activity is defined as the amount of
enzyme producing 1 mol reducing sugar per minute at
45C. Substrate and enzyme blanks were also prepared in
which enzyme or substrate were replaced with buffer. All
measured values of absorbency were corrected for the
appropriate blank absorbencies.
435
Assay for antifungal activity
Antifungal activity was estimated using the hyphal
extension-inhibition assay (Roberts and Selitrennikoff
1986). An actively growing fungal culture (0.2 ml) was
placed on a central disc on an agar plate containing
solidified Aspergillus medium. Subsequently, cell suspen-
sions of the isolated chitinolytic bacteria, concentrated
culture supernatants, or solutions containing purified
chitinases (0.2 ml each) were added to perimeter discs.
Subsequently, the plates were incubated at 30C for 3
30 days. Fungal hyphae grew outward from the central
disc as a circle, unless an effective concentration of
inhibitor was contacted in a perimeter disc. In the latter
case, a crescent of growth inhibition was observed around
the disc.
Protein assay
Protein concentrations were determined by the method of
Bradford (1976) with bovine serum albumin as standard.
Electrophoresis
Analytical sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was carried out by the
procedure of Schgger and von Jagow (1987) using a
Mini Protean II vertical tank apparatus (Bio-Rad, Munich,
Germany). A commercial low-molecular-mass calibration
kit of standard proteins (Amersham Pharmacia, Freiburg,
Germany) served as molecular mass standards. Protein
bands were visualized in gels by staining with Coomassie
brilliant blue.
Purification of His
6
-tagged proteins
His
6
-tagged proteins were purified from cell-free extracts
by nickel affinity chromatography on Ni/nitrilotriacetic
acid agarose (Qiagen, Hilden, Germany) according to the
manufacturers instructions. After the column (15 cm, 3-
ml bed volume) was loaded with 5 ml cell-free extract
(approximately 20 mg/ml), it was washed with 15 ml
buffer (300 mM KCl in 50 mM potassium phosphate,
pH 8.0) containing 10 mM imidazole followed by 15 ml of
the same buffer containing 20 mM imidazole. Proteins
bound to the resin were eluted with buffer containing
250 mM imidazole. Fractions harboring homogeneous
His
6
-tagged proteins were pooled and used for the
experiments.
Molecular procedures
Manipulations of DNA, PCR and transformation of
plasmids into E. coli were done according to routine
procedures (Ausubel et al. 1987) unless otherwise
specified. The Gttingen Genomics Laboratory (Gttin-
gen, Germany) determined the DNA sequences. Sequence
analysis was performed with the Genetics Computer
Group (GCG) program package (Devereux et al. 1984).
DNA of the chitinolytic strains was isolated by using a
Qiagen RNA/DNA kit according to the manufacturers
instructions.
Identification of the isolated microorganisms
Identification of the isolated microorganisms was by 16S
rDNA sequence analysis. The 16S rRNA genes of the
isolated microorganisms were amplified by PCR using two
oligonucleotides: 5-AGAGTTTGATCATGGC-3 (posi-
tion 823 relative to E. coli 16S rDNA) and 5-
TACCTTGTTACGACTT-3 (positions 1,5071,492 rela-
tive to E. coli 16S rDNA). The PCR reactions were
initiated at 94C (1 min), followed by 30 cycles of 95C
(1 min), annealing at 55C (1 min), 72C (1 min), and
ended with incubation at 72C for at least 5 min. The PCR
products obtained during the amplification step were
cloned by the TA method, which takes advantage of the
terminal transferase activity of Taq polymerase. This
enzyme added a single 3-A overhang to each end of the
PCR product. Subsequently, the PCR products were
directly cloned into pCR2.1-TOPO by using the TOPO
TA cloning kit (Invitrogen) according to the manufac-
turers instructions. Subsequently, the inserts of the
resulting plasmids were sequenced and analyzed. The
classification of strain MG1 was performed by the DSMZ.
Cloning and high-level expression of the chitinase
gene derived from MG3
To clone the chitinase gene derived from genomic DNA of
MG3 by PCR, two oligonucleotides (5-ACGTGTATC-
GACGTGTCATGAGTC-3 and 5-TACGACATCAGCA
GCTCAGGTTCG-3) were employed, which were de-
duced from the 5- and 3-region of the chiC gene
encoding a chitinase in Streptomyces griseus (Ohno et al.
1996). Subsequently, the PCR products were cloned by the
TA method. PCR reactions and TA cloning were
performed as described for cloning of 16S rRNA genes.
The resulting construct (pMG3) was sequenced. In order
to achieve high-level expression and subsequent purifica-
tion of the cloned chitinase located on pMG3 by nickel
affinity chromatography, plasmids pMG3.1 and pMG3.2
were constructed. For this purpose the coding region of the
chitinase gene was amplified from pMG3 using the
following sets of oligonucleotides with synthetic sites
(underlined): pMG3.1, 5-CACCATGGTGTATC-
GACGTGT-3 and 5-GCAGCTCAGGTTCG-
GACCCGTGGT-3; pMG3.2 5-CACCATGGGC-
CACCTGCGCCACG-3 and 5-GCAGCTCAGGTTCG-
GACCCGTGGT-3. The synthetic sites allowed direc-
tional cloning of the chitinase genes into pET101/D by
436
employing the pET101 directional TOPO expression kit
(Invitrogen).
Nucleotide sequence accession numbers
The nucleotide sequences of the chiIS gene of MG3 and
the 16S rRNA gene sequences of the isolates have been
deposited with the GenBank database under accession
numbers AY553325 (chiIS), AY556408 (SGE1),
AY556409 (SGE2), AY556410 (MG5), AY556411
(SGE4), AY556412 (SSL1), AY556413 (SSL3),
AY556414 (SSL4), AY556415 (SGE3), AY556416
(SSL2), AY556417 (MG2), and AY556418 (MG3).
Results
Enrichment, isolation, and identification of
chitinolytic microorganisms
Enrichment of chitinolytic microorganisms was per-
formed with samples derived from three environments:
sediment of the Gulf of Eilat (Israel), soil from a meadow
collected near Gttingen (Germany), and sediment
collected from the Solar Lake (Egypt). These samples
were used to inoculate enrichment medium containing
colloidal chitin as sole carbon and energy source. Cultures
were incubated under aerobic conditions at 30C. After 8
transfers of 20 independent enrichments, further studies
concentrated on 13 cultures that exhibited an increase in
optical density.
The cultures were purified by plating on solidified
enrichment medium. Within 2496 h of incubation at
30C, the chitin-degrading organism formed colonies of
12 mm in diameter, surrounded by clear zones indicating
chitinase activity. The colonies served as inoculum for
liquid cultures. The procedure of growing the organisms
on agar plates and subsequently in liquid culture was
repeated four times and led to the isolation of 13 different
chitin-degrading strains. Five of these strains (MG1
MG5) were derived from soil of a meadow near Gttingen,
four (SGE1SGE4) from sediment of the Gulf of Eilat,
and four (SSL1SSL4) from sediment of the Solar Lake
(Table 1). 16S rRNA gene analyses revealed that most of
the recovered microorganisms were related to Paeniba-
cillus macerans (SSL1, SSL2, and SSL4), Bacillus
chitinolyticus (SSL3, SGE2, SGE4), and Sphingobacter-
ium multivorum (SGE3, MG2), which are all known for
their chitinolytic activities (Kuroshima et al. 1996;
Matsuda et al. 2001; Singh et al. 1998). The recorded
similarities of the 16S rRNA genes were 98.399.2%,
98.398.9%, and 99%, respectively, (Table 1). From a
soil-sample-isolated strain, MG4 was identified as Steno-
trophomonas maltophilia, since the 16S rRNA sequences
of both organisms were identical. S. maltophilia represents
a rhizosphere bacterial species of potential agronomic
importance (Kobayashi et al. 2002). The closest phyloge-
netic relatives of the other four isolates were chitin-
degrading bacterial species such as Alcaligenes xylosox-
idans (SGE1), Bacillus ehimensis (MG1), S. griseus
(MG3), and Flexibacter cf. sancti (MG5). Further studies
focused on the identification and characterization of
isolates among the recovered strains that exhibit antifungal
activities.
Identification and characterization of isolates
exhibiting antifungal activity
To identify isolates that exhibit antifungal activity, hyphal
extension-inhibition assays employing A. nidulans and the
fungal phytopathogens B. cinerea, F. culmorum, G.
bidwellii, and S. sclerotiorum as test strains were
performed (Fig. 1a). The latter four organisms cause
gray-mold rot, crown rot, black root rot, and stem rot,
Table 1 Isolation of chitinolytic microorganisms from environmental samples: determination of the closest phylogenetic relatives and
antifungal activities of the isolates. + Antifungal activity, no antifungal activity
Sample site Isolate
designation
Closest relative and identity of the
16S rRNA genes (%)
Antifungal activity of the isolates against the following fungi
Aspergillus
nidulans
Botrytis
cinerea
Fusarium
culmorum
Guignardia
bidwellii
Sclerotia
sclerotiorum
Sediment Gulf
of Eilat
SGE1 Alcaligenes xylosoxidans (98.9)
SGE2 Bacillus chitinolyticus (98.9) + +
SGE3 Sphingobacterium multivorum (99)
SGE4 B. chitinolyticus (98.3) + + + +
Sediment Solar
Lake
SSL1 Paenibacillus macerans (98.3)
SSL2 P. macerans (99.2)
SSL3 B. chitinolyticus (98.8) + + + +
SSL4 P. macerans (98.4)
Meadow Gt-
tingen
MG1 Bacillus ehimensis (98.3) + + + + +
MG2 S. multivorum (99)
MG3 Streptomyces griseus (98.4) + + + + +
MG4 Stenotrophomonas maltophilia (100)
MG5 Flexibacter cf. sancti (99)
437
respectively. The isolates SGE2, SGE4, SSL3, MG1, and
MG3 were found to suppress growth on plates of A.
nidulans and of at least one of the four fungal
phytopathogens tested, whereas the remaining eight
chitinolytic strains showed no antifungal activity under
the conditions employed (Table 1). The inhibition zones
between the tested fungi and the five microorganisms
exhibiting antifungal activity were up to 20 mm. MG1 and
MG3 had the largest inhibition zones of all isolates (data
not shown). In addition, both strains suppressed the
growth of all tested fungi (Table 1).
The five isolates possessing antifungal activity were
studied further. All five strains were rod-shaped and Gram-
positive. These results are consistent with those reported
for the closest relatives B. chitinolyticus (SGE2, SGE4,
and SSL3), B. ehimensis (MG1), and S. griseus (MG3)
(Kuroshima et al. 1996; Williams et al. 1983). At a growth
temperature of 30C the doubling times of SGE2, SGE4,
SSL3, MG1, and MG3 with colloidal chitin as substrate
were 3.7, 11.6, 2.4, 6.3 and 3.7 h, respectively. In addition,
all five isolates were able to grow with native powdered
crab shell chitin as sole carbon source, but the doubling
times were longer (data not shown). As shown in Table 2,
the substrate spectra of SGE2, SGE4, SSL3, MG1, and
MG3 comprised, in addition to chitin, a variety of carbon
sources, i.e., peptone, tryptone, and yeast extract. All
isolates exhibiting antifungal properties utilized starch as
substrate except SGE4. No growth was observed on
dextran and cellulose. The recorded substrate spectra of
SGE2, SGE4, SSL3, MG1, and MG3 were similar to those
reported for their closest relatives (see above). Thus, we
concluded, based on 16S rRNA gene analyses, morphol-
ogy, and substrate spectra, that the isolated microorgan-
isms showing antifungal activities represent new strains
within the genera Bacillus (MG1, SGE2, SGE4, and
SSL3) and Streptomyces (MG3).
Properties of the chitinases produced by SGE2, SGE4,
SSL3, MG1, and MG3
The chitinase activities in the unconcentrated culture
supernatants of chitin-grown SGE2, SGE4, SSL3, MG1,
Table 2 Substrate spectra of the isolates SGE2, SGE4, SSL3, MG1,
and MG3. For media and growth conditions, see Materials and
methods. + Growth, no growth
Substrate SGE2 SGE4 SSL3 MG1 MG3
Chitin + + + + +
Dextran
Starch + + + +
Cellulose
Peptone + + + + +
Yeast extract + + + + +
Tryptone + + + + +
Raffinose + + +
Cellobiose + + +
Maltose + + + + +
Sucrose + + + + +
Xylose + +
Lactose +
Arabinose + +
Glucose + + + + +
Fructose + + + + +
Mannitol + + + +
Tributyrin + + +
Fig. 1 Fungal growth inhibition by the isolated chitinolytic
microorganisms (a) or purified chitinase IS of MG3 (c) and
purification of Chitinase JS (b). a The chitinolytic isolates were
grown in enrichment medium containing colloidal chitin as carbon
source. Subsequently, cell suspensions of the stationary growth
phase (0.2 ml) were used for hyphal extension-inhibition assays.
The fungus Aspergillus nidulans was used as the test strain in this
case (for more results see Table 1). Discs: 1 MG3, 2 MG5, 3 MG2, 4
MG1. b Purification of chitinase IS produced by Escherichia coli
BL21/pMG3.2. Proteins were separated according to the procedure
of Schgger and von Jagow (1987) and stained with Coomassie
blue. Lanes: 1 Molecular mass marker, 2 purified chitinase IS of
MG3 produced by E. coli BL21/pMG3.2. c The phytopathogenic
fungus Guignardia bidwellii was used as the test strain in this case.
Similar results were obtained by employing A. nidulans, Fusarium
culmorum, Sclerotia sclerotiorum, and Botrytis cinerea as test
strains (data not shown). Discs: 1 20 g ChiIS, 2 20 g boiled
ChiIS, 3 20 g crude extract of E. coli BL21 without pMG3.2, 4
0.2 ml H
2
O
438
and MG3 were detectable but difficult to quantify because
of the high dilution of the enzymes produced. Therefore,
proteins in the cell-free culture supernatant (500 ml) were
concentrated by ammonium sulfate precipitation. The
specific chitinase activities using colloidal chitin as the
substrate recorded in the concentrates of SGE2, SGE4,
SSL3, MG1, and MG3 were 0.30, 0.32, 0.37, 0.22, and
0.25 U/mg, respectively. The activity in the concentrated
culture supernatant fluids of all five isolates was
approximately 7080% of the total activity measured
after the cells were lysed by French pressing. This result is
consistent with an extracellular location of the chitinases
in SGE2, SGE4, SSL3, MG1, and MG3. The concentrated
culture supernatants exhibited the same antifungal proper-
ties as the corresponding strains during hyphal extension-
inhibition assays (data not shown). The largest inhibition
zones and growth suppression of all tested fungi were
again observed using supernatants derived from MG1 and
MG3 (see above). To demonstrate that the antifungal
activity is caused by enzymes such as chitinases, the
proteins in the concentrated supernatants derived from
SGE2, SGE4, SSL3, MG1, and MG 3 were inactivated by
incubation at 100C for 10 min or by treatment with
proteases. These treatments led to a strong reduction or
loss of the antifungal properties in all cases. This indicated
that proteins are responsible for the antifungal activity
present in the culture supernatants.
The effects of pH and temperature on the chitinase
activities were measured at pH values of 310 and
temperatures of 2070C. The crude chitinases of all
isolates were active at temperatures from 20C to 65C.
The highest activities were recorded between 45C and
50C (data not shown). The dependence of chitinase
activity on pH was measured after incubation at 45C for
30 min. The chitinases produced by SGE2, SGE4, SSL3,
MG1, and MG3 were active over a broad range of pH
values, with maximal values at pH 5, 6, 4, 6, and 57,
respectively, (Fig. 2). At pH 10, the remaining chitinase
activity of SGE2, SGE4, SSL3, and MG1 was 1020% of
the maximal observed activity, whereas that of MG3 was
45%. At the acidic pH of 3, the highest remaining
activities were recorded for the crude chitinases of SSL3
and MG3 (68% and 40%, respectively). Thus, the crude
chitinase produced by MG3 exhibited the best pH-stability
of all tested enzymes, since it maintained high activity
(40%) over the entire tested pH range. Taking the pH
profile and the antifungal properties into account, the
crude chitinase of MG3 exhibited the best properties of all
crude enzymes derived from the five isolates for
application as a biocontrol agent. In addition, the closest
phylogenetic relative of isolate MG3 is S. griseus, which is
known for its ability to produce a chitinase (ChiC) with
strong antifungal activity (Itoh et al. 2003; Ohno et al.
1996; Watanabe et al. 1999). In order to characterize the
gene encoding the chitinase of MG3 and the correspond-
ing gene product cloning, high-level expression, and
purification of the chitinase were performed.
Cloning and molecular analysis of the gene encoding
the chitinase of MG3
We cloned the chitinase of MG3 using isolated genomic
DNA as template and two primers, whose design had been
based on the above-mentioned chiC gene (885 bp) of S.
griseus. The PCR product obtained was inserted into
pCR2.1-TOPO by the TA method. The 912-bp insert of
the resulting construct (pMG3) was sequenced. The origin
of the cloned fragment was verified by Southern blot
analysis (data not shown).
Sequence analysis of the pMG3 insert revealed one
open reading frame (ORF; 912 bp). The deduced gene
product consists of 303 amino acids with a predicted
molecular mass of 31,836 Da. As shown in Fig. 3a,
homology searches revealed that the amino acid sequence
of the gene product encoded by pMG3 is 92% identical to
chiIS, which encodes an uncharacterized chitinase of
Streptomyces sp. AJ9463. Therefore, this ORF was also
designated chiIS. The chiIS gene product also revealed
significant homology to chitinase C of S. griseus HUT
6037 (79% identity; 82% similarity), and to other
chitinases belonging to family 19 of the glycosyl hydro-
lases, including class I, II, or IV chitinases of plants such
as class I chitinase from Pisum sativum (Chang et al. 1995)
and class IV chitinase from Vitis vinifera (Robinson et al.
1997).
The chiIS gene product of MG3 exhibited the typical
domain structure of bacterial family 19 chitinases such as
ChiC of S. griseus (Fig. 3b). The C-terminal region of the
deduced polypeptide (amino acids 89303) is considered
to be the catalytic domain (Fig. 3b). This region showed
high similarity to the catalytic domains of plant chitinases
Fig. 2 Effect of pH on chitinase activity. The crude chitinases of
SGE2, SGE4, SSL3, MG1, and MG3 were preincubated for 30 min
in 20 mM acetate buffer at pH 35, 20 mM phosphate buffer at pH
68, or 20 mM glycine-NaOH buffer at pH 910. Subsequently,
chitinase activity was determined under standard assay conditions
(see Materials and methods) but the buffer was replaced by the
above-mentioned buffers. Activities are expressed relative to the
maximal recorded specific activity: SGE2 0.3 U/mg, SGE4 0.3 U/
mg, SSL3 0.37 U/mg, MG1 0.22 U/mg, MG3 0.25 U/mg
439
of classes I, II, and IV, which form family 19 of glycosyl
hydrolases. In addition, two glutamate residues, which are
catalytic amino acid residues of family 19 chitinases
(Andersen et al. 1997; Kawase et al. 2004), were both
conserved in the sequence of ChiIS from MG3 (Fig. 3a).
No significant similarities to catalytic domains of bacterial
chitinases belonging to the family 18 of glycosyl hydro-
lases such as ChiC from Alteromonas sp. (Tsujibo et al.
1998) or ChiC from B. circulans WL12 (Alam et al. 1995)
were detected. The N-terminal region of ChiIS of MG3
(amino acids 3788) is probably responsible for binding of
chitin, since it showed significant similarity to chitin-
binding domains of family 18 chitinases such as chitinases
A1 and D of B. circulans (Ikegami et al. 2000). This
region is unrelated to chitin-binding domains of family 19
chitinases derived from plants. In addition, the N-terminal
region of ChiIS (amino acids 136) exhibited features
typical of a signal sequence, i.e., a hydrophilic segment
containing positively charged amino acids followed by a
stretch of hydrophobic amino acids. The putative signal
sequence of ChiIS from MG3 is longer than that of ChiC
of S. griseus HUT 6037 and the corresponding sequence
of Streptomyces sp. AJ9463 (Fig. 3a), but similar to the
signal sequence of ChiC from S. lividans (Fujii and
Miyashita 1993).
Purification and characterization of ChiIS from MG3
To facilitate purification of the MG3 chitinase, the coding
region of the corresponding gene, including the putative
signal sequence, was amplified from pMG3 and cloned
into the expression vector pET101/D, thereby placing the
chitinase gene under control of the isopropyl-1-thio--D-
galactoside (IPTG)-inducible T7 promoter and adding a
sequence encoding a His
6
tag. The fidelity of the PCR
product and the cloning step were confirmed by sequen-
cing of the resulting construct, pMG3.1. The plasmid was
transformed into E. coli BL21. Subsequently, E. coli
BL21/pMG3.1 was grown in LB medium and production
of chitinase induced by addition of IPTG. E. coli cells
harboring pMG3.1 produced the chiIS gene product, and
chitinase activity was detectable in crude extracts but the
amount produced was not sufficient to carry out
biochemical studies (data not shown). To achieve higher
production, the coding region of chiIS was cloned into
pET101/D without the region encoding the putative signal
sequence. E. coli BL21 cells containing the resulting
construct, pMG3.2, produced a level of chitinase IS
approximately 10-fold higher than E. coli BL21/pMG3.1
(data not shown). Subsequently, the chiIS gene product
was purified from cell-free extracts by affinity chroma-
tography on Ni/nitrilotriacetic acid agarose. SDS-PAGE
analysis revealed a molecular mass for purified ChiIS of
29,000 Da (Fig. 1b). The observed molecular mass is in
excellent agreement with that derived from the deduced
amino acid sequence without the signal sequence
(28,288 Da). The specific chitinase activity of the final
enzyme preparation with colloidal chitin as substrate was
1.8 U/mg. The purified enzyme was also active with native
powdered crab shell chitin as the substrate (0.8 U/mg).
The activity of the purified Chitinase IS exhibited the same
pH profile and optimal temperature as the chitinase
activity in the concentrated culture supernatant of MG3.
The antifungal activity of purified chitinase IS was tested
by hyphal extension-inhibition assays (Fig. 1c). The
enzyme inhibited the growth of all fungal pathogens
tested. An inhibitory effect of chitinase IS was detectable
using less than 4 g purified enzyme (data not shown).
The antifungal activity of chitinase IS was very stable, as
no significant loss of antifungal activity was observed
during incubation on agar plates at room temperature for
2 months.
Discussion
Detection of chitin-degrading bacteria from natural
sources such as soil and sediments is useful in the
isolation of bacteria that produce antifungal or other novel
Fig. 3 a Alignment of amino acid sequences of ChiIS from MG3,
ChiIS from Streptomyces sp. AJ9463, and ChiC from S. griseus
HUT 6037 and b putative domain structure of ChiIS from MG3. a
ChiIS MG3 Chitinase IS of MG3 (this study), ChiIS S. sp. chitinase
of Streptomyces sp. AJ9463 (AB104621), ChiC S. gr. chitinase C of
S. griseus (Ohno et al. 1996). The alignment was obtained using
CLUSTAL W. Dashed lines Gaps introduced to optimize alignment.
The amino acids conserved in all three enzymes are shaded. The two
catalytic glutamate residues of family 19 chitinases are boxed. The
bar indicates the signal sequences. b Numbers indicate amino acid
positions of the putative domains
440
compounds. A high correlation between chitinolysis and
production of bioactive compounds has been reported
(Chen et al. 1991; Pisano et al. 1992). In this report, we
isolated 13 different chitinolytic bacterial strains from soil
and marine sediments. Almost all isolates were related to
bacterial species that are known for their chitinolytic
activity. The majority of the isolated microorganisms
(SGE2, SGE4, MG1, SSL1SSL4) were affiliated to the
phylogenetic class Bacilli. This was expected, since
members of this class have been frequently recovered
during enrichment of chitin-degrading microorganisms
from soil and marine samples (Ivanova et al. 1999;
Kuroshima et al. 1996). Five of the isolates (SGE2, SGE4,
SSL3, MG1, MG3), and crude chitinase preparations
derived from them, exhibited antifungal activity against
phytopathogenic fungi. The closest phylogenetic relatives
of these strains were B. chitinolyticus (SGE2, SGE4,
SSL3), B. ehimensis (MG1), and S. griseus (MG3). The
specific chitinase activities, using colloidal chitin as the
substrate, recorded in the concentrated culture superna-
tants of the five isolates (0.220.37 U/mg) were in the
same range as those described for other crude bacterial
chitinases (Wang and Chang 1997; Wiwat et al. 1999). In
addition, the optimal temperatures for chitinase activity
(4550C) were in the same range as those reported for
other chitinases derived from mesophilic Streptomyces or
Bacillus species (Watanabe et al. 1999; Wiwat et al. 1999).
Most microbial chitinases, including the enzymes pro-
duced by the five isolates, show optimal activity and
stability at acidic pH values (Kim et al. 2003; Koga et al.
1999). In contrast to the majority of characterized
chitinases, chitinase IS of MG3 showed high activity
over a wide range of pH, including alkaline and acidic pH
values (Fig. 2). This property, together with the ability of
the crude enzyme to inhibit the growth of all phytopath-
ogenic fungi tested (Table 1), make chitinase IS of MG3
potentially very useful for application as a biocontrol
agent. Therefore, the chitinase produced by the Strepto-
myces strain MG3 and the corresponding gene (chiIS)
were chosen for further molecular and biochemical
characterization.
Sequence analyses of the deduced chiIS gene product
revealed high similarity to bacterial chitinases such as
ChiC from S. griseus and ChiF of S. coelicolor (Ohno et
al. 1996; Saito et al. 1999; Watanabe et al. 1999) and to
plant chitinases, which belong to family 19 of glycosyl
hydrolases. The catalytic domain of ChiIS from MG3 is
related to family 19 plant chitinases, whereas the putative
chitin-binding domain is affiliated to bacterial family 18
chitinases and not to plant chitin-binding domains
(Fig. 3b). Similar results were reported for family 19
chitinases of Streptomyces species (Watanabe et al. 1999).
Considering the sequence similarities and the domain
structure, it can be concluded that the chiIS gene of MG3
encodes a chitinase that belongs to family 19 of glycosyl
hydrolases. Some chitinases of plants and bacteria
belonging to family 19 are known for their strong
antifungal activity (Itoh et al. 2003; Watanabe et al.
1999). The E. coli-produced and subsequently purified
chiIS gene product of MG3 also revealed strong and stable
antifungal activity (Fig. 1c). The molecular masses of
microbial chitinases range from 20,000 Da to 120,000 Da,
with little consistency. The molecular masses of chitinases
derived from actinomycetes, such as chitinase C from S.
griseus (Watanabe et al. 1999) and the chitinase from
Streptomyces sp. M-20 (Kim et al. 2003), are mostly
around 30,000 Da or lower. The molecular mass of
chitinase IS from MG3 is approximately 29,000 Da (by
SDS-PAGE), which in accordance with those of the
actinomycetes.
In conclusion, we confirmed that some chitinolytic
bacterial species produce chitinases with antifungal
properties. We were able to clone and to express a family
19 chitinase-encoding gene (chiIS) derived from a novel
Streptomyces strain in E. coli. Like the original strain,
MG3, the purified gene product exhibited remarkable
antifungal activity against several fungal phytopathogens,
and was active over a wide range of pH values. The results
indicated that chiIS is a good candidate for the develop-
ment of a biocontrol agent for fungal diseases of plants.
Further studies, including greenhouse assays, will now be
undertaken to test the suitability of the enzyme or the
original strain in such biocontrol applications.
Acknowledgements We thank Gerhard Gottschalk for generous
support and helpful discussions. This work was supported by the
Deutsche Bundesstiftung Umwelt.
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