Journal of Biochemistry and Molecular Biology, Vol. 36, No. 2, March 2003, pp.

185-189

© KSBMB & Springer-Verlag 2003

Purification and Characterization of Chitinase from Streptomyces sp. M-20

Kyoung-Ja Kim*, Yong-Joon Yang† and Jong-Gi Kim‡

Department of Life Science, Soonchunhyang University, Asan-si 336-745, Korea
†
Department of Horticultural Science, Sangmyung University, Cheonan 330-180, Korea
‡
Department of Horticultural Science, Jungang University, Anseong 456-756, Korea

Received 16 September 2002, Accepted 29 October 2002

Chitinase (EC 3.2.1.14) was isolated from the culture have been isolated (Han et al., 2000; Baek et al., 2001), and
filtrate of Streptomyces sp. M-20 and purified by several genes that encode these enzymes have also been
ammonium sulfate precipitation, DEAE-cellulose ion- cloned from a variety of bacteria (Robbins et al., 1992;
exchange chromatography, and Sephadex G-100 gel Tsujibo et al., 1993). During the last decade, chitinases have
filtration. No exochitinase activity was found in the culture received increased attention because of their wide range of
filtrate. The molecular mass of the purified chitinase was applications (Rast et al., 1991; Oppenheim and Chet, 1992).
20 kDa, estimated by a sodium dodecyl sulfate- The enzyme could either be used directly in the biological
polyacrylamide gel electrophoresis, and was confirmed by control on microorganisms, indirectly using purified proteins,
activity staining with Calcofluor White M2R. Chitinase or through gene manipulation (Oppenheim and Chet, 1992).
was optimally active at pH of 5.0 and at 30oC. The enzyme This paper deals with the purification and antifungal activity
was stable from pH 4 to 8, and up to 40oC. Among the of chitinase from Streptomyces sp. M-20.
metals and inhibitors that were tested, the Hg+, Hg2+, and
p-chloromercuribenzoic acid completely inhibited the
enzyme activity. The chitinase activity was high on Materials and Methods
colloidal chitin, chitotriose, and chitooligosaccharide. The
purified chitinase showed antifungal activity against Chemicals The p-nitropheny-β-N-acetylglucosaminide, glycol
Botrytis cinerea, and lysozyme activity against the cell wall chitosan, lysozyme, amylose, carboxymethyl cellulose, cellobiose,
of Botrytis cinerea. caucofluor white M2R, chitobiose, chitotriose, laminarin were
purchased from Sigma Chemical Co. (St. Louis, USA). All of the
Keywords: Antifungal activity, Botrytis cinerea, Chitinase other chemicals and reagents that were used were of highest grade
commercially available.

Organism and cultural conditions The sample that was used for
this experiment was obtained from Mongolian soil. Five grams of
Introduction
soil were added into 50 ml distilled water. After the sample was
incubated at 30oC for 1 h, it was spread onto a chitinase detection
Lysis of the host structure by secretion of extracellular lytic
medium that contained the following per liter; 1 g colloidal chitin,
enzymes is one of the important mechanisms that are involved
0.7 g KH2PO4, 0.3 g K2HPO4, 4 g NaCl, and 0.5 g MgSO4 · 7H2O
in the antagonistic activity of biocontrol agents (Saksirirat and
that was solidified with 2% agar. Clear halos were observed around
Hoppe, 1991; Mathivanan et al., 1997; Kim et al., 2001). 7 colonies after incubation for 7 d at 30oC. Among the 7 colonies,
Among these, chitinase (EC 3.2.1.14) plays a vital role in the one colony that showed predominant chitinase activity was selected
biological control of many plant diseases by degrading the and grown in a chitinase production medium that contained the
chitin polymer in the cell walls of fungal pathogens (Haran et following per liter; 1 g colloidal chitin, 0.7 g KH2PO4, 0.3 g
al., 1993). It affects fungal growth through the lysis of cell K2HPO4, 4 g NaCl, 0.5 g MgSO4 · 7H2O, 1 mg FeSO4 · 7H2O,
walls (Kunz et al., 1992), hypal tips, and germ tubes 0.1 mg ZnSO4 · 7H2O, and 0.1 mg MnSO4 · 7H2O at 30oC for 7 d.
(Gunarantna and Balasubramanian 1994). Many chitinases The culture was harvested, filtered, and centrifuged. The
supernatant was used for further chitinase purification.
*To whom correspondence should be addressed.
Tel: 82-41-530-1352; Fax: 82-41-530-1350 Purification of chitinase The proteins from the culture were
E-mail: kyoungjakim@hotmail.com precipitated by ammonium sulfate (75%). The precipitate was
186 Kyoung-Ja Kim et al.

collected by centrifuging at 8,000 × g for 20 min, and resuspended Properties of chitinase

in an acetate buffer (50 mM, pH 5.0). It was dialyzed against the
same buffer and freeze-dried. The sample was then loaded on a pre- Effect of pH and temperature on chitinase activity and stability
equilibrated DEAE-cellulose column (2 × 20 cm), and washed with The pH effect was determined by incubating the purified chitinase
the acetate buffer. The proteins were eluted in a stepwise gradient (10 µg as protein) at different pH levels (4-11) under standard assay
on NaCl (0-1.0 M) at a flow rate of 24 ml/h. Fractions of 3 ml were conditions using colloidal chitin as the substrate. Acetate buffer
collected, and the absorbance was read at 280 nm in a (50 mM) was used for pH 3-6, phosphate buffer (50 mM) for pH 7,
spectrophotometer. The fractions with chitinase activity were Tris-HCl buffer (50 mM) for pH 8-9, and glycine-NaOH buffer for
combined, dialyzed against the acetate buffer, and concentration by pH 10-11. The enzyme stability was determined after preincubation
lyophilization. The concentrated sample was passed through a at various pHs without the substrate for 16 h.
Sephadex G-100 column (2 × 40 cm) and eluted with the acetate The optimum temperature for the chitinase activity was
buffer at the rate of 15 ml/h. The fractions of 3 ml were collected, determined by performing the standard assay in the range of 20-
and the absorbance and chitinase activity were measured. 80oC. Thermal stability was determined by assaying the residual
chitinase activity after incubation for 1 h at the previous
Chitinase assay Colloidal chitin was used as a substrate to assay temperature without the substrate.
chitinase activity; 0.2 g in a 2 ml acetate buffer (50 mM, pH 5.0)
was incubated with 1 ml of enzyme at 30oC for 1 h. The product Effect of metals and inhibitors on chitinase activity The
was measured in 1 ml of filtrate by the dinitrosalicylic acid (DNS) enzyme was preincubated with a 10 mM concentration of different
method (Miller, 1959). One unit of enzyme activity was defined as metals and inhibitors. After 30 min, the remaining chitinase activity
the amount of enzyme that catalyzed the release of 1 µmol of N- was measured using the standard assay. The relative inhibition of
acetylglucosamine per ml in 60 min. Exochitinase (N-acetyl-β- the calculated enzyme was based on the release of N-
glucosaminidase) activity was assayed by mixing 0.1 ml of the acetylglucosamine.
aliquot of the appropriately-diluted enzyme with 0.5 ml of 1.75 mM
p-nitropheny-β-N-acetylglucosaminide in a 50 mM acetate buffer Activity of chitinase on different substrates The purified
(pH 6.0). After incubation at 30oC for 30 min, the reaction was chitinase was incubated separately with different substrates using
terminated by adding 0.9 ml of 0.2 M Na2CO3 and p-nitrophenol standard assay methods. Following incubation, the release of N-
was measured at 405 nm. acetylglucosamine was measured. The relative activity was
calculated using colloidal chitin as a control.
Inhibition of fungal growth by the purified-enzyme extract
A spore suspension of Botrytis cinerea was uniformly spread on Lysozyme activity Lysozyme activity was determined by
plates of PDA (potato dextrose agar). Discs that were soaked with measuring the decrease in optical density at 660 nm. The mixture
the purified enzyme extract (5 U, 10 U) were laid on the seeded contained 1.5 ml of a Botrytis cinerea cell suspension (optical
plates; the control was disc-soaked in boiled-enzyme extract. density of 1.7) in 50 mM phosphate buffer (pH 7.0) and 5 U of the
Fungal growth was observed over 2 d of incubation at 30oC. enzyme solution. The mixture was incubated at 37oC for 30 min.
Purity and molecular mass Homogeneity and molecular mass of (Murao et al., 1990).
the purified chitinase were determined by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gel,
adopting the method of Laemmli (1970). The protein molecular Results and Discussion
markers that were used were lactalbumin (14,200), trypsin inhibitor
(20,000), carbonic anhydrase (29,000), glyceraldehydes 3- Purification of chitinase Table 1 summarizes the results of
phosphate dehydrogenase (36,000), ovalbumin (45,000), and the purification of chitinase from Streptomyces sp. M-20.
bovine serum albumin (66,000). The gel was stained in 0.15% w/v Extracellular chitinase from Streptomyces M-20 was purified
Coomassie brilliant blue R-250 in methanol-acetic acid-water (40: to 18.9-fold. The proteins from the culture were precipitated
10: 50, by volume) for 4 h and destained in the same solvent
without the dye.
Table 1. Purification steps of chitinase from Streptomyces sp. M-
Glycol chitin synthesis Glycol chitin was obtained by acetylation 20
of glycol chitosan (Truel, J. and Asselin, A., 1989). Total Total Specific
Purification
Step protein activity activity
fold
Activity staining SDS-PAGE was carried out with the 12% gel (mg) (Unit) (U/mg)
that contained 0.05% glycol chitin. After electrophoresis, the gels
Culture filtrate 425 3,078 0007.24 1.0
were incubated for 2 h at 37oC in 100 mM sodium acetate buffer,
Ammonium sulfate 220 3,130 014.2 1.9
pH 5.0 containing 1% (v/v) Triton X-100. The gels were then
Precipitate
stained with 0.01% Caucofluor white M2R in 0.5 M Tris-HCl, pH
8.9, for 7 min, and destained with water. The lytic zones were DEAE-cellulose 042 1,260 030.0 4.1
photographed under the UV-transilluminator. Sephadex G-100 006 0,822 137.0 60.
 Chitinase from Streptomyces sp. M-20 187

Fig. 1. Chromatogram of the chitinase from Streptomyces sp. M-

20 on DEAE-cellulose (2.0 × 20 cm). The column was eluted
with NaCl (0-1.0 M) at a flow rate of 24 ml/h. Fractions of 3 ml
were collected.

Fig. 3. SDS-PAGE of purified chitinase of Streptomyces sp. M-

20. Activity-stained chitinase (A) and Coomassie-stained
chitinase (B) and standard markers (C); lactalbumin (14,200),
trypsin inhibitor (20,100), carbonic anhydrase (29,000),
glyceraldehydes 3-phosphate dehydrogenase (36,000), ovalbumin
(45,000), and bovine serum albumin (66,000).

Fig. 2. Chromatogram of the chitinase from Streptomyces sp. M-

20 on a Sephadex G-100 column (2.0 × 40 cm). The column was
eluted with a 50 mM sodium acetate buffer (pH 5.0) at a flow
rate of 15 ml/h. Fractions of 3 ml were collected.

by ammonium sulfate (75%). The precipitate was adsorbed by

a DEAE-cellulose column, and eluted at 0.1-0.3 M NaCl in
the buffer (Fig. 1). The concentrated-active fractions were Fig. 4. Optimal pH (;) and stability of pH ( 0) of purified
chitinase from Streptomyces sp. M-20.
further purified by a Sephadex G-100 column
chromatography. The elution profiles of protein and chitinase
activity are shown in Fig. 2. most active between pH 5.0 and 6.0. It was relatively stable at
The purified chitinase from Streptomyces sp. M-20 revealed pHs between 4.0 and 8.0, when kept at 4oC. However, beyond
homogeneity of a single protein band on 12% native PAGE these pH ranges, it rapidly lost its activity (Fig. 4). Many
(data not shown). Its molecular weight was estimated as chitinases, including the present one, showed a pH optimum
20 kDa by SDS-PAGE (Fig. 3). Different molecular masses in the acidic range. The Alteromonas sp. strain O-7 (Tsujibo,
that ranged from 35-45 kDa have been reported for other H.Y. et al., 1993) was in the basic range and Bacillus sp. LJ-
fungal chitinases (Ulhoa and Peberby, 1992; Gunaratna and 25 was in the neutral range (Lee et al., 2000).
Balasubramanian, 1994; Sakurada et al., 1996). After SDS-
PAGE, the chitinase was regenerated by the removal of SDS Effect of temperature The chitinase activity was most
with purified Triton X-100. The gels were stained with active at 30oC. Above 40oC, the activity decreased and was
Calcofluor white M2R and destained. The hydrolytic zone at lost completely at 60oC. When the enzyme was kept at various
20 kDa was visualized by placing the gel on a UV- temperatures for 30 min in an acetate buffer (pH 5.0), it was
transilluminator and photographed (Fig. 3). significantly inactivated above 50oC and completely at 65oC
(Fig. 5).
Effect of pH The optimal pHs for chitinase activity and
stability of the chitinase were examined. The enzyme was Effect of metal ions and chemical reagents The inhibitory
188 Kyoung-Ja Kim et al.

Table 3. Substrate specificity of purified chitinase of

Streptomyces sp. M-20
Substrate Relative activity (%)
Colloidal chitin 100
Glycol chitosan 0
p-nitropheny-β-N-acetylglucosaminide 0
Chitibiose 0
Chitotriose 250
Chitooligosaccharide* 310
Amylose 0
Fig. 5. Optimal temperature (;) and stability of temperature ( 0) Carboxymethyl cellulose 0
of purified chitinase from Streptomyces sp. M-20. Cellobiose 0
Laminarin 0
Table 2. Effect of some metals and chemical reagents on
chitinase activity *Chitooligosaccharides are mixture of chitotetraose, chitopen-
taose, and chitohexaose.
Relative Relative
Material Material
activity (%) activity (%)
None 100 CuSO4 96
HgNO3 0 SnCl2 98
HgCl2 0 EDTA 90
AgNO3 35 ρ-CMB 0
CaCl2 95 SDS 95
MgCl2 98 FeCl2 80
FeCl3 80 CoCl2 89
MnCl2 87 2-ME 170

p-CMB: p-chloromercuric benzoic acid

2-ME: 2-mercaptoethanol
The chitinase activities were measured at 30oC in the presence of
1 mM of various ions or reagents as the standard assay method,
described in Materials and Methods.
Fig. 6. Agar diffusion test with purified chitinase on Botrytis
cinerea. The Agar diffusion test was performed on Botrytis
cinerea agar plates with paper discs that were coated with the
effects of some metal ions and chemical reagents on chitinase purified-chitinase enzyme (A, 5 U; B, 10 U enzyme; C, boiled
activity were investigated in an assay system where 1 mM of enzyme).
each ion or reagent was present. As shown in Table 2, the
chitinase activity was strongly inhibited by Ag+ and
completely by Hg+, Hg2+, and p-chloromercuribenzoic acid. activity was determined by measuring the decrease in optical
The increase in activity with mercaptoethanol indicated the density at 660 nm. In a 50 mM phosphate buffer (pH 7.0)
presence of sulfyhdryl groups on the active site of the enzyme, solution, 5U of the purified chitinase showed lysozyme
which was confirmed by the total inhibition by Hg2+. Similar activity against Botrytis cinerea cell suspension (optical
inhibition and mercaptoethanol enhancement was reported by density of 1.7). A 50% decrease in OD 660 nm was observed
Ueno et al. (1990). at 5 U of the purified chitinase from Streptomyces sp. M-20.

Substrate specificity Table 3 shows the results of the Inhibition of fungal growth by purified chitinase The
hydrolysis of various substrates with the enzyme. The enzyme inhibition of the Botrytis cinerea growth was observed on agar
hydrolyzed the colloidal chitin, chitotriose, and plates with paper discs that were coated with the purified-
chitooligosaccharides, but did not hydrolyze chitobiose, p- chitinase enzyme (Fig. 6). Antifungal activity of the purified
nitrophenyl-N-acetylglucosaminide, or any other substrate chitinase of Streptomyces sp. M-20 was confirmed. The
(Shon et al., 2001) that was composed of glucosidic linkage. inhibition diameter of the 10 U enzyme was greater than that
This indicates the absence of chitobiase and glycosidases, of the 5 U enzyme, and the boiled enzyme had no inhibitory
other than chitinases. activity against Botrytis cinerea.

Lysozyme activity of purified-chitinase activity Lysozyme Acknowledgments This work was supported in part by the
 Chitinase from Streptomyces sp. M-20 189

2001 Research Fund from the Soonchunhyang University and determination of reducing sugars. Anal. Chem. 31, 426-428.
Korea Science and Engineering Foundation (2000-1-22100- Murao, S., Kato, M., Wang, S. L., Hoshino, M. and Shin, T.
002-3). (1990) Isolation and characterization of a novel hen egg white
lysozyme inhibitor from Bacillus subtilis I-139. Agri. Biol.
Chem. 54, 119-1136.
Oppenheim, A. B. and Chet, I. (1992) Cloned chitinase in fungal
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