Springer_CurrentMicrobiol_Mukherjee_2006

Purification, Characterization, and Antifungal Activity of Chitinase
from Streptomyces venezuelae P10
G. Mukherjee, S. K. Sen
Division of Microbiology, School of Life Sciences, Visva-Bharati (Central University), Santiniketan 731235, India
Received: 5 November 2005 / Accepted: 20 April 2006
Abstract. Streptomyces venezuelae P10 could produce extracellular chitinase in a medium containing
0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent
homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and
DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was
characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal
amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetyl-
glucosamine and N, NÕ-diacetylchitobiose.
Chitin is an insoluble linear polymer of b-1, 4)linked
N-acetylglucosamine (GlcNAc) residue and is the most
abundant renewable natural resource next to cellulose.
It is a major constituent not only of fungal cell walls
(22% to 44%) but also of insect exoskeletons and
crustacean shells (25% to 58%) [15, 18]. Worldwide
annual recovery of chitin from the processing of mar-
ine invertebrates is 3.7 · 104
metric tons [22].
Chitinases (EC 3.2.1.14) are glycosyl hydrolases
and are present in a wide range of organisms that may
not contain chitin but still play an important ecophysi-
ologic role [27]. Enzymatic hydrolysis of chitin to free
b-1, 4-linked N-acetylglucosamine (GlcNAc) is per-
formed by a chitinolytic system consisting of chitinase
and chitobiase, the actions of which are synergistic and
consecutive [3, 18, 22]. Micro-organisms, particularly
Streptomyces [6] and Serratia [24], are good chitin
hydrolysers.
Because many streptomycetes can use chitin as sole
source of carbon, chitin can be used as an enrichment
medium for the isolation of Streptomyces from soil [21].
Cell wall–degrading enzymes are involved in the bio-
logic control of phytopathogenic fungi by Trichoderma
harzianum and by streptomycetes. The production of
inexpensive chitinolytic enzymes is an important ele-
ment in the use of chitinous wastes, which not only solve
environmental problems but do with added value in
certain cases [26]. This article deals with characteriza-
tion, antifungal activity, and partial sequencing chitinase
from S. venezuelae P10 MTCC 4218 [14].
Materials and Methods
Organism and fermentation conditions. S. venezuelae P10 was
isolated from soil of coastal region of India [14]. The fermentation
medium [14] was inoculated with 4% spore suspension (1.5 · 106
spores mL)1
) of 96-hour-old seed culture and fermented for 4 days
under stirred conditions (120 rpm) at 30°C. The biomass-separated
culture broth was used as source of crude enzyme. Colloidal chitin was
used as substrate and was prepared from crab shell chitin (Sigma) [13,
14].
Chitinase assay. Chitinase activity was determined by hydrolyzing
colloidal chitin and measuring the amount of decreasing sugar
produced as estimated from a standard curve of GlcNAc [13]. The
assay mixture contained 1 mL colloidal chitin (0.6%) in 10 mM
phosphate buffer (pH 7.5) and 0.5 mL enzyme solution. Chitinase
activity is expressed as the amount of enzyme required to produce
1 lmol GlcNAc min)1
. Protein content was measured using Lowry
assay [12] using bovine serum albumin as standard.
Enzyme purification and chitin affinity chromatography. For
isolation of substrate specific chitinases, dialyzed protein was
charged by affinity binding to chitin (chitin binding) [8]. Cell-free
culture broth (1 L) was precipitated with ammonium sulfate (80%).Correspondence to: S. K. Sen; email: sksenvb@rediffmail.com
CURRENT MICROBIOLOGY Vol. 53 (2006), pp. 265–269
DOI: 10.1007/s00284-005-0412-4 Current
Microbiology
An International Journal
ª Springer Science+Business Media, Inc. 2006

Pellets were dissolved in phosphate buffer (10 mM, pH 7.5) and
dialyzed overnight. A 10 ml volume of colloidal chitin (20 mg/ml) and
15 ml 1 M sodium chloride were added and increased to 35 ml using
the same buffer. The mixture was transferred to small glass tray, left on
ice for 1 hour, and centrifuged (20,000 rpm for 15 minutes) to remove
unbound protein. The pellet was washed extensively with same
phosphate buffer and suspended in 15 ml of the same buffer. The
suspension was incubated for 6 hours at 35°C under gentle shaking (90
rpm; Orbitek) to digest colloidal chitin with the absorbed chitinase.
The resulting clear supernatant was again precipitated with 80%
ammonium sulfate, centrifuged, and dialyzed.
DEAE–cellulose ion exchange column. Ion-exchange chromatography
was done using a DEAE-cellulose column (2.5 · 7 cm). The column was
packed with overnight-swollen DEAE-cellulose in 10 mM phosphate
buffer (pH 7.5) and eluted stepwise with 0.1 to 0.6 M NaCl. The pooled
protein fractions were dialyzed against 10 mM phosphate buffer (pH
7.5), stored at –20°C, and used as purified enzyme.
Enzyme characterization and determination of molecular
weight. SDS-PAGE was prepared using the buffer system of
Laemmli [10], i.e., 10% acrylamide separating gel and 5%
acrylamide stacking gel containing 0.1% SDS. The sample was
compared with a standard protein marker (P7702S, New England
BioLabs) after staining with Coomassie Brilliant Blue R250.
Gels were analysed using Gel Documentation (MiniBis, Bio-
Imaging System, Germany) software (GelCapture, version 2.5).
Quantification, molecular weight determination, and Rf values were
determined (GelQuant, version 2.7 and TotalLab version 2003, 2004
DNR/Bio-Imaging Systems Ltd.).
Temperature and pH optimization. To determine temperature and
pH tolerance, the enzyme, in 10 mM phosphate buffer (pH 7.5), was
treated for 10 minutes at a temperature range of 20°C to 50°C and in
pH from 5.0 to 9.0.
Antifungal activity. Antifungal activity was observed by hyphal
extension–inhibition [20]. Test fungal cultures were inoculated on malt
yeast extract agar (ISP-2) plates and incubated at 28°C. After 24 hours,
discs containing sample enzyme (10 lg) were placed over the growth
and incubated at 28°C for up to 7 days.
Chitin hydrolysis. Chitin hydrolysis was done using colloidal chitin
as substrate [13]. The hydrolysate was analyzed by thin layer
chromatography (TLC) using n-butanol, acetic acid, and water
(4:1:5) as mobile phase, and products were detected by spraying
with aniline phthalate.
Amino-acid sequencing chitinase. Proteins from SDS-PAGE were
electrotransferred to polyvinylidene-difluoride (PVDF) membrane
(Immobilion-P; Millipore, Bedford, CA) using electroblotting
apparatus (Bio-Rad, Richmond, CA) with N-cyclohexyl-3-
aminopropane sulfonic acid (CAPS) transfer buffer (1 mM CAPS
and 20% methanol, pH 10.5). The membrane was stained with Ponceu
S for visualization. The blotted band was cut and subjected to N-
terminal amino-acid sequence analysis (Applied Biosystems, Model
491). The data were analysed using data from the National Center for
Biotechnology Information Protein Data Bank.
Results
Enzyme production. The isolate S. venezuelae P10
could produce extracellular chitinase under standard
conditions. Cell-free broth (96 hours old) was used as
crude enzyme. Crude protein content was measured (180
mg/L) with specific activity 8.35 U mg)1
(Table 1).
Enzyme purification. The ammonium sulfate
precipitated and dialyzed protein was measured as 32
mg with specific activity of 15.71 U mg)1
. The protein
was purified 2.47-fold with 16.1% recovery through
affinity chromatography (Table 1). The semipurified
enzyme was further purified through the DEAE-
cellulose column. Chitinase activity was detected in
the 0.3 to 0.4 M NaCl step-elution fraction. The active
fractions were pooled, resulting in 3.19-fold total
purification with a specific activity of 26.67 U mg)1
and 2.1% recovery of protein (Table 1).
The enzyme showed good activity between tem-
peratures of 30°C to 40°C, with optimal activity at 35°C
(Fig. 1). The enzyme also maintained good activity in a
range of pHs from 6.0 to 8.0 (Fig. 1). Loss of enzyme
activity was 30% at pH 5.0 and 40% at pH 9.0. Purity of
the enzyme was estimated by SDS-PAGE (Fig. 2A).
The Rf value of purified chitinase was 0.26, corre-
sponding to an estimated molecular weight of 66 kDa.
Antifungal activity. Fungal hyphae grew outward from
the centre of the Petri plates (Fig. 2B through 2D). After
5 days of incubation, a crescent of growth inhibition was
observed around the perimeter of the discs containing
chitinase for Aspergillus niger, Alternaria alternate, and
Helminthosporium sativum; however, no inhibition was
seen with Penicillium chrysogenum (data not shown).
Maximum inhibition was observed with A. niger.
However, filters spotted with buffer did not show any
growth inhibition.
Chitin hydrolysis. The hydrolysate was analyzed by
TLC. Two compounds were detected with hRf value
of 30.13 and 83.86, corresponding to GlcNAc and
Table 1. Purification of chitinase from S. venezuelae P10
Purification step Total activity (U) Specific activity (Umg)1
) Purification fold Yield (%)
Crude enzyme (supernatant) 1503 8.35 1.00 100
80% Ammonium, sulfate precipitation 502 15.71 1.88 33.3
Affinity binding to chitin 247.56 20.63 2.47 16.1
DEAE-cellulose column 32.00 26.67 3.19 2.1
266 CURRENT MICROBIOLOGY Vol. 53 (2006)

Temperature (˚C)
15 20 25 30 35 40 45 50 55
Relativeactivity
Relativeactivity
50
60
70
80
90
100
110
50
60
70
80
90
100
110
pH
4 5 6 7 8 9 10
Relative activity (Temp)
Residual activity (Temp)
Relative activity (pH)
Residual activity (pH)
Fig. 1. Chitinase activity of isolate P10.
Temperature and pH tolerance relative
activities were assayed under standard
conditions, with 100% activity corresponding
to 2.4 U/ml. Residual activity was assayed
after exposure of the enzyme to different
temperatures and pHs.
kDa
200
97
66
43
29
14
Lane 1 2 3
A
B
C
D
Fig. 2. Growth against
filter paper disc containing
purified chitinase (10 lg
each) of isolate P10:
(A) Marker (Lane 1), crude
enzyme (Lane 2), purified
enzyme (Lane 3; also
used for B through D).
(B) A. niger. (C) A.
alternata. (D) H. sativum.
G. Mukherjee and S. K. Sen: Purification and Characterization of Chitinase 267

N,NÕ-diacetylchitobiose (CHBdiNAc), respectively
(data not shown). The hydrolysed products were
identified by cochromatography with authentic
GlcNAc and CHBdiNAc (data not shown).
Partial sequencing. N-terminal sequence analysis
revealed the first 15 amino acids as Glu, Gln, Pro,
Gly, Gly, Asp, Lys, Val, Asn, Leu, Gly, Tyr, Phe, Thr,
and Asn. Possible homology with other streptomycetes
of the sequenced amino acids is listed in Table 2.
Discussion
Chitinases from various sources showed bifunctional
chitinase and lytic activity [18]. Purification of chitin-
ases is preferred with ammonium sulfate precipitation
[29], including Streptomyces sp [1]. For the isolate P10,
affinity chromatography resulted in 16.1% chitinase
recovery with 2.47-fold purification. Further purification
was possible by DEAE-cellulose column to 3.19-fold
and recovery of 2.1%. Chitinase recovery was 27.4% in
S. marcescens [19] and 69.4% with 3.8-fold purification
in Pseudomonas sp. YHS-A2 [11]. Nawani and
Kapadnis [16] reported 5.1-fold purification of S. mar-
cescens NK1 chitinase with ammonium sulfate precipi-
tation followed by Sephadex G-100 gel filtration.
A single band on SDS-PAGE revealed that the P10
chitinase was purified to near homogeneity, with an
estimated molecular weight of 66 kDa. Microbial
chitinases weigh from 20 to 120 kDa, and fungi mostly
weigh 30 kDa [7]. The molecular weight of chitinase
from Streptomyces is between 30 [23] and 68 kDa [25].
Optimum enzyme activity of chitin was observed at
35°C, and it maintained activity at temperatures up to
40°C. The relative enzyme activity beyond 35°C was
lower than the residual activity, thus, illustrating its tem-
perature-induced conformational changes. The optimum
reaction temperatures of chitinase from Streptomyces
were reported as 40°C [2, 5] (range 30°C to 55°C) [6].
pH influences the proton-donating or proton-
accepting groups (ionization) in the catalytic site.
Therefore, S. venezuelae P10 chitinase showed good
activity from pHs 6.5 to 8.0, with optimum activity at
pH 7.5. In other species of Streptomyces, the optimum
pH for chitinase activity was found to be from 3.3 to 7.5
[2, 5, 6].
Potential use of chitinases as biocontrol agents have
been reported [4]. Chitinases from S. griseus also
showed activity against fungi such as Aspergillus sp.,
Phycomyces blakesleeanus, and T. reesei [28]. Hoster
et al. [7] reported chitinase activity against A. nidulans
and phytopathogens such as Botrytis cinerea, Fusarium
culmorum, Gulgnardia bidwellii, and Sclerotia sclero-
tiorum. Mycelial growth of A. niger, H. sativum, and
A. alternata were inhibited with the application 10 lg
chitinase purified from isolate P10. However, growth
was not inhibited for Trichoderma and Phycomyces sp.
with 50 lg chitinase from S. griseus [7]. Family 19
chitinases, the plant chitinases, usually show antifungal
activity [27]. Antifungal activity with family 18 chitin-
ases, the bacterial chitinase, was first reported from
S. griseus HUT 6037 and seven more actinomycetes
[17], excluding the working isolate P10.
The chitinase from isolate P10 hydrolyzed colloidal
chitin to produce GlcNAc and CHBdiNAc. Ohno et al.
[17] observed that hydrolysis of b-1,4 bonds in chitin
produced GlcNAc and CHBdiNAc as sole reaction
products that were dominated by GlcNAc.
The enzyme from S. venezuelae P10 was electrob-
lotted to PVDF membrane for Edman degradation.
Generation of single amino acids in each cycle of deg-
radation confirmed purity of the enzyme. N-terminal
sequencing of P10 chitinase showed homology with the
deduced amino-acid sequence extending from 249 to
263 of chitinase of S. griseus chitinase [17]. The protein
sequence data of P10 is available in the Uniport knowl-
edge base under accession number P84754.
Thus, it is possible to purify the chitinase from
S. venezuelae P10 to apparent homogeneity with an
estimated molecular weight of 66 kDa. Thermal stability,
pH tolerance, and antifungal properties of the chitinase
may contribute to its biotechnologic potentiality. The
results of this study may be useful in the development of
genetically engineered micro-organisms with high
chitinase activity. Apart from manifold applications [18,
22], to our knowledge, no data have been available for
Table 2. Sequence analysis of chitinase of P10 and its homology with other species of Streptomyces
S. griseusa
TT–GGGGEQPG-GDKVNLGYFTNWGVYGRNYHVKNLVTSGSAAKITHIN 294
S. venezuelae EQPG GDKVNLGYFTN 15
S. peucetiusb
TTGADPGPGPGPGDKVKLGYFTNWGVYGRNYHVKNLVTSGSAQKITHIN 300
S. aureofaciensc
TG–TVKLGYFTNWGVYGRNYHVKNLVTSGSADKITHIN 87
DB source: www.ncbi.nlm.nih.gov/BLAST/
a
Accession AB081807.1
b
Accession AF206633.1
c
Accession AB106648.2
268 CURRENT MICROBIOLOGY Vol. 53 (2006)

chitinase purification and characterization from S. ven-
ezuelae before the publication of this article.
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support received from DBT,
Ministry of S & T, and UGC, Government of India. We are also
thankful to Dr. A. K. Ghosh, IICB, Kolkata, India, for technical
support.
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G. Mukherjee and S. K. Sen: Purification and Characterization of Chitinase 269

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