Indian J ournal of Biotechnology

Vol 5 (Suppl), July 2006, pp 357-363

Characterization of chitinases from microorganisms isolated from Lonar lake

Vijay B Bansode and Shyam S Bajekal*
Postgraduate and Undergraduate Department of Microbiology, Yashwantrao Chavan College of Science
Vidyanagar, Karad 415 124, India
Chitinases, the enzymes that breakdown chitinthe second most abundant polysaccharide in natureare known to
have numerous uses. Alkaline chitinases, in particular are considered to have a greater potential in this respect. Fifteen
chitinolytic microbial strains were isolated from the alkaline soil of Lonar lake in Buldhana district of Maharashtra. In this
paper, the characters of chitinases produced by the five most potent isolates are presented. All the enzymes exhibited
maximum activity in the neutral to alkaline range of pH (from 7.0 to 9.6) with a wide stability range from pH 4 to 11. The
temperature optima for all the enzymes were slightly on the higher side, between 35 and 60C, with a stability range from 25
to 60C. All the enzymes showed a typical response to substrate concentration. None of the enzymes had any specific
requirement for any particular metal ion, though a considerable stimulatory effect of Ca
2+
was observed on all the enzymes.
Minor effects of Cu
2+
, Mn
2+
and Na
1+
were also observed on some of the enzymes.
Keywords: alkaline, biocontrol, chitinases, fungal protoplasting, seafood waste
IPC Code: Int. Cl.
8
A01N63/04
Introduction
Chitinases are a group of enzymes that decompose
chitin, the second most abundant polymer in nature
and the most abundant in the marine environment
1
.
They are produced by a diverse range of life forms,
such as snails, crustaceans, insects, vertebrates, plants
and microorganisms. Among the microorganisms,
approximately 90 to 99 per cent of the chitinolytic
populations are actinomycetes. Only a fraction of
them are bacteria and less than one per cent are
fungi
1,2
.
Chitinases breakdown chitin into a variety of
products that include the deacylated oligomer
chitosan (GlcNAc)
n
, the disaccharide chitobiose
(GlcNAc)
2
and the monomer N-acetylglucosamine.
These derivatives are increasingly finding use in
diverse fields, such as biomedicine
3
, agriculture and
even in cosmetics
4,5
. The enzymes themselves have
been found to be antifungal and nematicidal agents
for agricultural biocontrol methods
6,7
.
The seafood industry is a major source of chitinous
wastes. The recycling of which is extremely important
to retain the carbon nitrogen balance in the
ecosystem
8
. Chitinases are considered to have the
potential for use in the management of these wastes
through the process of composting
9,10
.
Chitinases with alkaline pH optima are reported to
have greater applicability in their uses and those with
greater thermo-stability were most suitable in the
composting process
10
. This study, thus, concentrated
on such enzymes that are produced by
microorganisms isolated from the lake in the Lonar
crater, having extreme environment. Lonar lake,
situated in Buldhana district of Maharashtra, is the
only lake in the world formed by meteorite impact in
basalt rock. The water of this lake and, consequently,
the soil in its littoral zone are characterised by very
highly alkaline pH of 8.0 to 9.0. The microorganisms
in this environment would, therefore, be unique and
their biochemical activities would also be expected to
be so. Chitinases produced by microorganisms in this
environment would, therefore, be adapted to the
alkaline pH. Fifteen chitinase producing
microorganisms were isolated from this environment
and they were screened for chitinase production. Five
of themtwo species of Streptomyces, one of
Nocardia, one of Bacillus and a fungal isolate were
found to be the most potent producers.
In the present paper, we report the results of our
preliminary study on the characteristics of chitinases
produced by these five isolates.

Materials and Methods

Preparation of Colloidal and Swollen Chitin
Colloidal chitin was prepared from practical grade
crab shell chitin (Loba Chemie) as described by Hsu
______________
*Author for correspondence:
Tel/Fax: 91-2164-271356
E-mail: str_yccskarad@sancharnet.in
INDIAN J BIOTECHNOL, J ULY 2006


358
and Lockwood
11
. Swollen chitin was prepared from
the same practical grade chitin by the method of
Hackman
12
described in Gohel et al
6
.

Isolation and Selection of Chitin Utilizers
Isolation of chitin utilizers was done through
enrichment in colloidal chitin agar medium
12
of
following composition (g L
-1
): colloidal chitin (4),
MgSO
4
.7H
2
O (0.5), K
2
HPO
4
(0.7), KH
2
PO
4
(0.3),
FeSO
4
.7H
2
O (0.01), MnCl
2
(0.001) and agar (20);
0.03% NaCl, and 0.03 and 0.02% yeast extract. By
visual analysis, well-separated colonies that showed a
zone of clearance around them were transferred onto
slants as pure cultures. Colonies showing large zones
of clearance were selected as potent chitinase
producers.

Production of Chitinases
Colloidal chitin broths (100 mL) in 250 mL
capacity Erlenmeyer flasks were inoculated with 1.0
mL spore suspensions (adjusted to 1.0 OD
600
) of the
isolates and incubated in a rotary incubator (Lab
Hosp) at 150 rpm and 37-40C for 12 d. The culture
broths were centrifuged at 8000g for 20 min and the
cell free supernatant saturated with ammonium
sulphate to 60-70% levels and kept at 4C overnight
to extract the enzymes. The precipitates were
dissolved in 50 mM phosphate buffer (pH 7.0) and
dialysed against distilled water at 4C overnight
through a dialysis membrane (Hi Media dialysis
membrane-70) with a molecular weight cut off at
12,000 Da. The stocks thus obtained were preserved
at 0-4C in PVC bottles.

Chitinase Assay and Characterization
The assay system of Monreal and Reese
13
,
estimating reducing sugars released by enzyme action,
was adapted for the study. In 2.5 mL buffer, 2.5 mL
1% substrate (swollen chitin) was added, followed by
0.5 mL crude enzyme preparation. The tubes were
incubated at appropriate temperature for 1 h. After
which the reaction was stopped by adding 3.0 mL
10% dinitrosalicylic acid (DNSA), followed by
heating in a boiling water bath. The coloured solution
was then centrifuged at 8000g for 5 min and the
absorption of supernatant was measured at 540 nm
wavelength in a Systronics spectrophotometer. The
reducing sugar was estimated from a standard curve
of glucose. One unit of enzyme is defined as the
amount of enzyme that catalyses the release of 1 M
of reducing sugar per minute under assay
condition
5,14
.

Effect of pH on Enzyme Stability and Activity
Four different buffers of 50 mM strength, glycine-
HCl (pH 2.2-3.4), acetate (pH 3.6-5.6), phosphate (pH
5.8-8.0) and glycine-NaOH (pH 8.6-11.0) were used
to create pH values from 2.2-11.0 for this study.
The stability of enzyme at different pH was tested
by keeping a suitable amount of enzyme in buffers of
various pH values ranging from 2.2 to 11.0 at 37C
for 1 h, after which their residual activities were
determined. pH values within the stability range of
the enzymes (4.0-11.0) were created in the respective
enzyme reaction systems and the effect on reaction
velocity (M/min of reducing sugars released) was
analyzed after 1 h of reaction at 37C, using
appropriate substrate controls.

Effect of Temperature on Enzyme Stability and Activity
Thermal stability was investigated by incubating
the enzymes in buffers of their optimum pH at
temperatures 0, 15, 28, 37, 45, 60, 80 and 100C for 1
h, after which those from higher temperatures were
cooled rapidly by immersing in ice before the residual
activities of each were measured at 37C.
Appropriate buffers to maintain optimum pH of
each enzyme were used in the system and the enzyme
substrate reaction was carried out at the different
temperatures within the stability range of the enzymes
for 1 h, after which the velocity of the enzyme
reaction was measured in each employing appropriate
substrate controls.

Effect of EDTA and Metal Ions
One-millimole solutions of EDTA and the chloride
salts of 10 metals, Na
+
, K
+
, Ag
+
, Ca
2+
, Cu
2+
, Hg
2+
,
Mg
2+
, Mn
2+
, Zn
2+
, and Fe
3+
were used in the study.
Varying volumes of each solution were added into the
assay systems to obtain different final concentrations
from 0.2-2.0 mM for the reaction. The enzyme
substrate reactions were allowed to proceed for 1 h at
their respective optimum pH and temperature and the
relative activities were calculated with respect to the
controls where the reactions were carried out in the
absence of any additive.

Effect of Substrate Concentration
Varying volumes of 1% swollen chitin were added
in the assay systems of each enzyme and the reactions
with fixed enzyme volumes (0.5 mL) were carried out
BANSODE & BAJ EKAL: CHITINASES FROM MICROORGANISMS


359
at their respective optimum pH and temperature for
1 h before velocities of each enzyme were measured.

Results

Effect of pH on Enzyme Stability and Activity
The effect of pH on the stability and activity of the
chitinases from the three actinomycete isolates, two
Streptomyces spp. (SB1 and VB3) and one Nocardia
sp. (SB4) is shown in Figs 1-3. While SB4 and VB3
showed stability up to pH 10 with an optimum pH 9,
SB1 showed a tolerance of up to just pH 8 with its
optimum activity at pH 6. The Bacillus (SB5)
chitinase showed a broad range of pH tolerance from
7 to 11 with maximum activity at pH 9 (Fig. 4), while
the fungal chitinase (VB2) showed a broad range of
pH tolerance from 4.0 to 9.0 and an optimum of pH 7
(Fig. 5). The optimum pH determined for each
enzyme in this experiment was used in future
experiments.

Effect of Temperature on Enzyme Stability and Activity
Observations noted in Figs 6-10 indicate the
temperature stability of all the chitinases to be on the
higher side, between 60 and 80C. Among the
chitinases from actinomycetes, it was observed that
the enzymes from Strptomyces had their optimum
temperature of activity at 37C, while the Nocardia
chitinase showed its maximum activity at 45C. The
Bacillus chitinase also had an optimum temperature of
37C, whereas the fungal enzyme had between 50 and
60C, which is the highest optimum temperature
range among all the five isolates. The optimum
temperature determined for each enzyme was used in
subsequent studies.

Effect of Metal Ions
Only 5 of the 10 metals tested showed any
significant stimulatory effect on the enzymes
(Table 1). As expected, Hg
2+
inhibited all enzymes at
even the lowest concentration, while EDTA had no
effect on any of the enzymes.
Calcium was the only metal showing a consistent
stimulating effect on all the enzymes, resulting in a
2.5 to 3.4-fold increase in their activity. Copper also
stimulates all the enzymes, though slightly (1.2-1.7).
Magnesium enhanced the enzyme activity of only
VB2 and VB3 by 3.5 and 2.3 times, respectively,
while it showed no significant effect on the other
three enzymes. Manganese caused a 1.7 to 2.8-fold
increase in the activity of four enzymes, SB1, SB5,
VB2 and VB3. Sodium also showed stimulatory
effect on all the enzymes, but the extent of stimulation
range from 1.2 to 2.1.

Effect of Substrate Concentration
All the enzymes showed a typical relationship with
their substrate (Figs 11-13), in which all of them are
saturated at low concentrations and reach to a
maximum velocity fairly soon, which is indicative of
a good catalytic efficiency.


Fig. 1Effect of pH on stability and activity of chitinase from
actinomycete isolate SB1



Fig. 2Effect of pH on stability and activity of chitinase from
actinomycete isolate SB4

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360
Discussion
Chitinases with alkaline pH optima and stability are
considered to have a major potential in biological
control of insect pests. The peritrophic gut lining of
insects is chitinous and has an alkaline pH. Chitinases
with a better stability and activity in these conditions
can be used in synergism with other biocontrol
agents
10
. In the current study, four chitinases, isolated
from Streptomyces, Nocardia, Bacillus and a fungal
isolate (showing characters similar to members of
genus Penicillium), are found to have their pH optima
in the alkaline range. The enzymes from Bacillus and
Streptomyces hold the most promise as they are stable
up to pH 10 and are also the most efficient. Use of
alkaline chitinases from S. albidoflavous,
Nocardiopsis albus and Bacillus sp. for such purposes
has earlier been reported
15-17
. However, one of the
enzymes in the current study (SB1) has its pH optima
in the acidic range and could possibly be exploited for
control of fungal plant pathogens
18
and for fungal
protoplasting as reported for similar enzymes
earlier
10
.


Fig. 3Effect of pH on stability and activity of chitinase from
actinomycete isolate VB3



Fig. 4Effect of pH on stability and activity of chitinase from
bacterial isolate SB5



Fig. 5Effect of pH on stability and activity of chitinase from
fungal isolate VB21



Fig. 6Effect of temperature on stability and activity of chitinase
from actinomycete isolate SB1

BANSODE & BAJ EKAL: CHITINASES FROM MICROORGANISMS


361


Fig. 7Effect of temperature on stability and activity of chitinase
from actinomycete isolate SB4



Fig. 8Effect of temperature on stability and activity of chitinase
from actinomycete isolate VB3



Fig. 9Effect of temperature on stability and activity of chitinase
from bacterial isolate SB5
Table 1Effect of metal ions and EDTA on activity of chitinases

Relative activity Component
SB1 SB4 SB5 VB2 VB3

None 1 1 1 1 1
EDTA 1.1 1 0.098 1.1 0.86
Na
+
1.2 1.5 2.1 2 1.5
K
+
1 0.96 0.9 0.84 0.87
Ag
+
0.79 0.68 0.84 0.76 0.86
Ca
2+
3.2 3.4 2.8 2.5 3.2
Cu
2+
1.43 1.2 1.5 1.2 1.7
Hg
2+
0.01 0.1 0.02 0.01 0
Mg
2+
1 1.5 1 3.5 2.3
Mn
2+
1.7 1.2 2.8 2.1 1.9
Zn
2+
0.95 0.89 0.88 0.93 0.94
Fe
3+
1.1 0.98 0.93 1 0.87




Fig. 10Effect of temperature on stability and activity of
chitinase from fungal isolate VB21




Fig. 11Effect of substrate concentration on activity of chitinases
from actinomycete isolates (SB1, SB4 & VB3)
INDIAN J BIOTECHNOL, J ULY 2006


362
Alkaline chitinases are also considered useful in
management of chitinous wastes, such as those
generated by seafood manufacturing industries
9,10
. As
the process applied here is one of composting,
enzymes with stability at higher temperatures are
considered more useful
1
. The enzymes reported in this
study are in consonance with earlier reports
19,20
with
optimum temperature around 45-55C and retention
of 50% activity (stability) at 60C.
None of the enzymes showed requirement of any
specific metal ions for their activity, though Ca
2+
and
Na
+
did showed a consistent stimulatory effect on all
enzymes. The other metals, Mg
2+
,

Mn
2+
and Cu
2+

stimulated some enzymes. Ca
2+
is a known stimulator
of extracellular enzymes; especially helping them to
withstand high temperatures
21,22
and could be of use in
composting of seafood waste.

Acknowledgement
The authors are thankful to the management and
administration of Yashwantrao Chavan College of
Science, Karad for providing the necessary facilities
in the Microbiology Department for this work.

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Fig. 12Effect of substrate concentration on activity of chitinase
from bacterial isolate (SB5)



Fig. 13Effect of substrate concentration on activity of chitinase
from fungal isolate (VB2)

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