Chitinase Isolated from Water and Soil Bacteria in Shrimp farming Ponds

Abstract

Chitinases have received attention because of their wide applications in the medicine, biotechnology, agriculture, waste management and industrial applications such as food quality enhancer and biopesticide. Excessive use of insecticides has led to several problems related to pollution and environmental degradation. In this study, isolation and identification of native bacterial strains with chitin hydrolysis activity, took place from water and soil of shrimp culture ponds in Bushehr and Abadan. To investigate the capacity of our chitinase for using in insecticide, biochemical properties of selected chitinase obtained in this research were compared to that of produced by Bacillus cereus p-1, isolated from an insecticide tablet. In this research, three mesophilic strains containing: Serratia marcescens B4A, Citrobacter freundii B1A and Bacillus cereus B3R were isolated. Results showed a 1600 bp band corresponding to chitinase gene. The similarity between temperature and pH profile and stability of chitinase was extracted from native bacteria and ones was obtained from Bacillus cereus p-1 implied that chitinase extracted from Serratia marcescens B4A has potential application in industry.

Full text

Iranian Journal of Fisheries Sciences 11(4) 911-925 2012
Chitinase Isolated from Water and Soil Bacteria in Shrimp
farming Ponds
Zarei M.1, 2; Aminzadeh S.* 1; Ghoroghi A.3; Motalebi A. A.3; Alikhajeh J.4;
Daliri M. 1
Received: June 2011 Accepted: March 2012
Abstract
Chitinases have received attention because of their wide applications in the medicine,
biotechnology, agriculture, waste management and industrial applications such as food
quality enhancer and biopesticide. Excessive use of insecticides has led to several problems
related to pollution and environmental degradation. In this study, isolation and identification
of native bacterial strains with chitin hydrolysis activity, took place from water and soil of
shrimp culture ponds in Bushehr and Abadan. To investigate the capacity of our chitinase for
using in insecticide, biochemical properties of selected chitinase obtained in this research
were compared to that of produced by Bacillus cereus p-1, isolated from an insecticide tablet.
In this research, three mesophilic strains containing: Serratia marcescens B4A, Citrobacter
freundii B1A and Bacillus cereus B3R were isolated. Results showed a 1600 bp band
corresponding to chitinase gene. The similarity between temperature and pH profile and
stability of chitinase was extracted from native bacteria and ones was obtained from Bacillus
cereus p-1 implied that chitinase extracted from Serratia marcescens B4A has potential
application in industry.
Keywords: Chitinase, Chitin, Shrimp pond, Bacteria
_______________
1-Department of Animal & Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology
(NIGEB), Shahrak-e Pajoohesh, km 17, Tehran-Karaj Highway, Tehran, Iran.
2-Department of marine biotechnology, Persian Gulf University, Bushehr, Iran.
3-Iranian Fisheries Research Organization, Tehran-Karaj High way, Sarve Azad Ave. Nation herbarium,
Tehran. Iran.
4-Department of Chemistry, City College of New York, New York, NY 10031.
* Corresponding author’s email: aminzade@nigeb.ac.ir

912 Zarei et al., Effect of different levels of dietary Betaine on growth…
Introduction
Chitin is the second source of natural
organic compound on the earth after
cellulose. This long biopolymer contains
N-acetyl D-glucosamine (GLcNAc)
monomer from covalent ß-1, 4 linkages.
Chitin occurs mainly as a structural
component in the exoskeleton of
crustaceans, mollusk shells and insects. It
is also found to lesser extents in other
animals, plants, fungi and bacteria (Ikeda
et al., 2009; Lee et al., 2009). Shrimp
culture was started in Iran in 1994
(Kakoolaki et al., 2010) and Indian white
shrimp, has been considered as the main
endemic culture species so shrimp culture
ponds are one of the most plentiful chitin
resources (Emadi et al., 2010; Zhang et al.,
2010). Production of chitin from shell of
shrimp, crab, lobster and Artemia urmiana
has been reported in Iran (Asadpour et al.,
2003). Chitinases, which hydrolyze chitin,
are largely found in nature, for example, in
stomachs of fish (Gutowska et al., 2004;
Molinaria et al., 2007) and the livers of
squid ( Matsumiya et al., 1998; Matsumiya
et al., 2002). Recently, the different
applications for chitinase have been
discovered, such as: biocontrol of fungal
diseases in plants ( Demarco et al., 2000;
Chang et al., 2003), using in biopesticides
(Mendonsa et al., 1996), production of
single cell protein from shellfish waste
(Raveh and Carrod, 1981; Vyas and
Deshpande, 1991), isolation of protoplast
from fungi (Dahiya et al., 2005),
production of chitooligosaccharides,
glucosamine and GLcNAc by chitinase
extracted from Burkholderia cepacia
TU09 for the hydrolysis of chitin
(Pichyangkura et al., 2002) and medical
application (Dahiya et al., 2006). The
chitinase has been extracted from number
of microorganisms such as Trichoderma
harzianum 8 (Seyedasli et al., 2004),
Bacillus subtilis SG2 (Khorramzadeh et
al., 2005) and Trichoderma atroviride
PTCC5220 (Harighi et al., 2006) in Iran.
The ability of chitinase for
digesting insect chitin raises the idea of
using it for controlling insects (Mendonsa
et al., 1996).
The aim of this research was a
comparison between biochemical
properties of native bacterial strains
isolated, with commercial ones in terms of
their chitinolytic activities. These species
with chitinolytic activity were isolated
from soil and water; the desired samples
that hydrolyze chitin effectively, were
belong to Bacillus sp., Enterobacter sp.,
Aeromonas sp. and Serratia sp.
respectively. These bacteria can be used to
control plant pathogenic fungi and
biopesticides (Dahiya et al., 2006).
Materials and methods
Chemical compounds such as 3, 5-
dinitrosalicylic acid (DNS), N-acetyl D-
glucosamine and Bovine Serum Albumin
(BSA) were obtained from sigma (St.
Louis, Mo. USA). Taq DNA polymerase
and 1 kb DNA ladder were obtained from
Cinna Gene and Fermentas, respectively.
All other chemicals were purchased from
Merck (Darmstadt, Germany) and were
reagent grade.
The modified method of Takiguchi
( 1991) was used for obtaining chitin
powder (Fig. 1). Shrimps (Penaeus

Iranian Journal of Fisheries Sciences, 11(4), 2012 913
indicus) were obtained from the fresh
hunting and then shells were isolated,
cleaned, washed and dried. For elimination
of mineral, dried shells were kept in HCl
10% for 24 h. After washing, NaOH was
added for 24 h. Then shells were dried
again and powdered (Fig. 1).
Figure 1: Chitin powder extracted from shrimp shells.
Commonly, colloidal chitin is used as a
water insoluble substrate for studying of
chitinase. Colloidal chitin was prepared by
the modified method of Roberts and
Selitrennikoff (1998). 12 g of chitin
powders that obtained from shrimp shells
was poured quietly into 380 ml of HCl and
kept at 4 ◦C overnight on stirrer. Then 4 L
of ice-cold 95% ethanol was added to
mixture and left at 4 ◦C with vigorous
stirring overnight. Centrifugation at 5000 g
for 20 min at 4 ◦C was done to collect the
sediment. The precipitate washed with
sterile distilled water until the colloidal
chitin became neutral (pH 7.0).
Microorganisms isolated from
shrimp farming soils, water and
wastewater at different locations in
southern part of Iran. All morphological
contrasting colonies were purified by
streaking in Animal & Marine
Biotechnology Lab in National Institute of
Genetic Engineering and Biotechnology
(NIGEB). Sampling was accomplished in
June and October 2008. (Fig. 2a).
At the time of sampling, the
important physical and chemical
parameters of water such as salinity,
temperature, dissolved oxygen and pH
were measured (YSI MODEL 63) at three
part of each pond in morning and evening.
Some of the isolated microorganism from
water and wastewater of shrimp culture
ponds was shown in Fig. 2b.

914 Zarei et al., Effect of different levels of dietary Betaine on growth…
Figure 2: (a) Shrimp culture ponds and their wastewater as a sampling regions. (b) Some of
microorganisms isolated from soil, water and wastewater.
Nutrient agar (N.A) for isolation and
maintenance contained nutrient agar (2%
w/v) supplemented with 0.1% w/v chitin
(pH 7.5). For primary screening culture
medium contained chitin 5 g; peptone 0.3
g; yeast extract 0.3 g; K2HPO4 0.7 g;
KH2PO4 0.3 g; MgSO4 .7H2O 0.5 g; Agar
15 g; NH4NO3 2 g; NaCl 1 g in 1 lit H2O.
To determine the chitinase production on
agar plates, 1% of chitin was incorporated
in a buffered agar solution. Clear zone
reactions produced by chitinolytic bacteria
were measured after routine casting the
gels onto Petri dishes (Dingle et al., 1953).
For preculture, medium contained nutrient
broth 8 g; malt extract 10 g; peptone 10 g;
chitin 5 g; NaCl 1 g in 1 lit H2O. Liquid
culture medium for chitinase production
contained chitin 5 g; peptone 0.3 g; yeast
extract 0.3 g; K2HPO4 0.7 g; KH2PO4 0.3
g; MgSO4.7H2O 0.5 g; (NH4)2SO4 1 g;
NaCl 1 g; MnSo4.2H2O 16 mg;
ZnSo4.7H2O 14 mg; FeSo4.6H2O 50 mg;
Cacl2 20 mg in 1 lit H2O.Solutions that
used in measurement of chitinase activity
were (Miller et al., 1959): (1) Enzyme
solution: the selected strain was cultured in
the production medium for 48 h at 30 ◦C
and then solution was centrifuged and
supernatant was collected. (2) 1% w/v
colloidal chitin solution: for obtaining this
solution 50 ml colloidal chitin was added
to 50 ml phosphate buffer 20 mM (pH
7.2). (3) Phosphate buffer 20 mM (pH
7.2). (4) Color solution of 3, 5-
dinitrosalicylic acid (DNS): for preparing
this, 1 g of DNS was added to 50 ml
distilled water on stirrer and then, 30 g
potassium sodium tartrate was added
slowly. 1.6 g NaOH was dissolved in 20
ml water previously. This solution was
also added to the previous solution. For
(a)
(b)

Iranian Journal of Fisheries Sciences, 11(4), 2012 915
enzyme assay, 0.5 ml of enzyme solution
was added to 0.5 ml substrate (colloidal
chitin). In blank sample 0.5 ml phosphate
buffer was added instead of enzyme
solution. Then samples were placed in 45
◦C for 1 h. The reaction was stopped by
addition of 3 ml DNS. The reagents were
placed in boiled water for 5 min. After
centrifugation, the absorbance of the
supernatant was measured at 530 nm
(Miller, 1959). In Fig. 3 changing color of
DNS by enzymes was shown.
For determination of enzyme
activity standard curve was drawn. At first,
different and consecutive concentrations of
N-acetyl D-glucosamine (From 0 to 50
mM) were prepared. 1 ml DNS was added
to 1 ml of above solutions and placed in 45
◦C bathwater. Then enzyme unit was
calculated by using the below formula;
Unit = μ mol N- acetyl D-glucosamine
released / time (min)
By definition, one unit (U) of the
chitinase activity equals as an amount of
enzyme required to produce one mmol of
reducing sugar per minute.
The protein concentration of
unpurified and partially purified chitinase
was determined by Branford's method
(Bradford, 1976) using 1 mg/ml bovine
serum albumin (BSA) as a standard
solution (table 1). In this method, 100 mg
Coomassie Brilliant Blue G250 was
dissolved in 50 ml 96% ethanol and then
100 ml 85% phosphoric acid was added.
Final volume of the solution was received
to 1 lit by distilled water. The absorbencies
of solution were measured at 595 nm. The
following reagents were added to the PCR
tube for further reaction ( Table 2).
Figure 3: Enzyme assay with DNS method. Tube 4 was blank and 1
to 3 was samples. Chitinase extracted from selected strain
made strong color in tube 3.
\
4 3 2 1

916 Zarei et al., Effect of different levels of dietary Betaine on growth…
Table1: Solution which used to protein determination
Chitinase gene was amplified using below
primers:
Forward: (5'- ATG CGC AAA TTT AAT
AAA CCG CTG- 3') and
Reverse (5'- TTA TTG AAC GCC GGC
GCT ATT GCC- 3').
Table2: Reagents which used to PCR amplification
Reagent Volume /
Concentration
PCR buffer 2.5 μl
MgCL2 1.5-2 mM
dNTP 0.5 mM
Distilled water 10 μl
DMSO 2.5 μl
Forward primer 0.5 pmol/µl
Reverse primer 0.5 pmol/µl
Template DNA 1 µg
Taq DNA polymerase 2.5 unit
PCR amplification was conducted
according to the thermal cycle of; 1 cycle
of 94 ◦C for 5 min: 35 cycle of 94 ◦C for 30
s, 55 ◦C for 60 s and 72 ◦C for 1.5 min:
final extension for 5 min at 72 ◦C.
To determine temperature and pH
profile and stability of Bacillus cereus
isolated from an insecticide tablet, one
insecticide tablet was dissolved in
preculture medium for 18 h at 30 ◦C on a
shaker incubator (200 rpm).
Microorganisms that growth in preculture
medium, were cultured in nutrient agar
plates and agar plates contain 0.5% chitin,
0.03% peptone, 0.03% yeast extract,
0.07% K2HPO4, 1.5% agar, 0.1% NaCl
and 0.1% v/v trace elements. One strain
that growth on agar plate with chitin was
cultured in the production medium and
used for enzyme assay, temperature
profile, temperature stability, pH profile
and pH stability.
The results are the average of at
least three repeated experiments in a
typical run in order to confirm
reproducibility.
Results
In this research, nearly 300 g chitin
powders were extracted from 1 kg shrimp
Tube
Solution (μl) 1 2 3 4 5 6 7 8 9 Unknown
protein
Standard protein 10 20 30 40 50 60 70 80 90
Distilled water 90 80 70 60 50 40 30 20 10
Sample 100
Introducer (ml) 5 5 5 5 5 5 5 5 5 5
Protein concentration 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 ?

Iranian Journal of Fisheries Sciences, 11(4), 2012 917
shells. The average of important physical
and chemical parameters of water such as
salinity, pH, temperature and dissolved
oxygen was tabulated in Table 3. ANOVA
statistical test didn't show any significant
differences between water physical and
chemical parameters in 2 sampling areas.
Biochemical and microbiological analysis
was performed to characterize some
screened strains (Table 4). Qualitative cup-
plate assay for chitinase production
indicated that three strains: Serratia
marcescens B4A, Citrobacter freundii
B1A and Bacillus cereus B3R were the
most active strains (Fig. 4). In the other
words the above-mentioned strains
consumed chitin at a higher rate, and
produced a clear zone with larger
diameters (Fig. 4). Therefore some of them
were selected for further studies.
Table 3: Physicochemical parameters of ponds water
Bushehr
(Helleh)
Abadan
(Choebdeh)
Place of sampling
Physical - chemical index 49.2 35.5 Salinity of No. 1 pool (ppt) 48.6 36.0 Salinity of No. 2 pool (ppt)
48.2 32.8 Salinity of No. 3 pool (ppt) 33.5 25.8 Temperature of No.1 pool (◦C) 33 25.5 Temperature of No.2 pool (◦C) 33.5 25.9 Temperature of No.3 pool (◦C)
3.7
5.4
3.8
5.5
Oxygen of No.1 pool (ppm)
Morning
Evening
3.3
5.9
3.5
5.8
Oxygen of No.2 pool (ppm)
Morning
Evening
3.4
5.6
3.6
5.8
Oxygen of No.3 pool (ppm)
Morning
Evening
8.4
8.5
8.4
8.6
pH of No.1 pool
Morning
Evening
8.4
8.6
8.3
8.5
pH of No.2 pool
Morning
Evening
8.5
8.6
8.3
8.6
pH of No.3 pool
Morning
Evening

918 Zarei et al., Effect of different levels of dietary Betaine on growth…
Table 4: Biochemical properties of
some
screened bacteria
Bacillus
cereus B3R
Citrobacter
freundii B1A
Species
Biochemical
properties
* * Indole production
* + Methyl red (MR)
*
-Voges proskauer (VP)
-
+ Citrate consumption
*
-H2S
-
-Hydrolysis of urea
+
+ Motion in 36 ◦C
+
-Hydrolysis of gelatin at 22 ◦C
+
+ Glucose (Formation of gas) *
+ Fermentation of lactose
*
* Fermentation of sucrose
*
+ Fermentation of mannitol
*
-Fermentation of dulcitol
*
+ Fermentation of sorbitol
*
+ Fermentation of arabinose
-
* Fermentation of xylose
-
* Fermentation of galactose
*
* Ortho-nitrophenyl-β-D-
galactopyranoside (ONPG) *
-Lysine decarboxylase
*
* Ornitin decarboxylase
+
* Haemolysis
+
* Growth at 37 ◦C
+
* Growth at 50 ◦C
-
* Growth in 10% w/v NaCl
–: 10% strains were positive
+: 90%-100% strains were positive
*: this test didn't do.

Iranian Journal of Fisheries Sciences, 11(4), 2012 919
0
10
20
30
40
1 2 3 4 5 6 7 8
Clear zone diameter (mM)
Strains
Figure 4: Clear zone diameter (mm). Qualitative cup-
plate assay for chitinase production in
some isolated strains was detected that 3
strains: number 2 (Citrobacter freundii
B1A), 3(Serratia marcescens B4A) and
4(Bacillus cereus B3R) that showed much
more activity from others (1, 5, 6, 7 and 8
are unknown bacteria).
Figure 5: Comparison of diameters of clear zone produced
by some isolated chitinolytic bacteria (Citrobacter
freundii B1A and Bacillus cereus B3R).
Temperature, pH profile and temperature,
pH stability of Serratia marcescens B4A
chitinase (Zarei et al., 2010) were
compared by chitinase produced by
Bacillus cereus p-1 that isolated from an
insecticide tablet for probable capacity of
S. marcescens chitinase for using in
insecticide. The commercialized chitinase
had the optimum temperature 50 ◦C. The
enzyme was completely inactivated at 75
◦C (Fig. 6a). This chitinase was stable
under 50 ◦C for 20 min (Fig. 6b). The
optimum pH for activity of the enzyme
was measured 5 (Fig. 6c). Chitinase was
stable at pH 3 to 10 for 90 min at 25 ◦C
(Fig. 6d). Analysis of amplified Serratia
marcescens B4A chitinase DNA on 1%
agarose gel was shown in Fig. 5. As we
see in Fig. 7, size of chitinase gene was
about 1600 bp.
Citrobacter freundii B1A Bacillus cereus B3R

920 Zarei et al., Effect of different levels of dietary Betaine on growth…
Figure 6: (a) Temperature profile of enzyme (produced by Bacillus cereus P-1 in insecticide tablet) in 20
mM phosphate buffer, pH 5.0. (b)Temperature Stability of enzyme (produced by Bacillus cereus
P-1) at 10 (♦), and 20 (■) minute from 10 to 60 ºC. (c) Effect of pH on enzyme (produced by
Bacillus cereus P-1) activity at 25 ºC. A mixture of glycine, acetate, and phosphate buffer at a
concentration of 50 mM was used. (d) pH stability of enzyme (produced by Bacillus cereus P-1)
at pH 3.0-11.0 for 90 min at 25 ºC.
(a)
0
50
100
020 40 60 80
Temperature (0C)
%Avtivity
(b)
0
50
100
020 40 60 80
Temperatre (0C)
%Activity
(c)
0
50
100
0 5 10 15
pH
%Activity
(d)
0
50
100
0 5 10
pH
%Activity

Iranian Journal of Fisheries Sciences, 11(4), 2012 921
Figure 7: Analysis of amplified DNA on 1% agarose gel.
Discussion
In this research, 3 mesophilic strains,
Serratia marcescens B4A, Citrobacter
freundii B1A and Bacillus cereus B3R was
isolated from Bushehr and Abadan ponds.
Considering Table 3 and the fact that
sampling from Bushehr ponds was done in
June and temperature of water was
relatively high, it seems that sampling
season has not relation to finding the
thermostable strains.
The number of chitin scientific
reports in the 1990s were very high, for
example, in 1998 more than 260 articles
about chitin has been published in science
direct (Khor, 2002). This can confirm the
ever-increasing importance of this
biopolymer. Approximately, 75% of the
total weights of shellfish, such as shrimp,
crab and krill is considered waste, and
chitin comprises 20 to 58% of the dry
weight of the said waste (Wang and
Chang, 1997). There is a great supply of
crustacean's shells in Iran, which often
burned or reminded in nature useless
(Asadpour et al., 2003). Extraction of
chitin from shrimp shells has been done
previously in Iran but its efficiency to
change to colloidal state has not been
investigated. Therefore, the performance
for extraction of chitin from crustacean's
shells in a semi-industrial scale, in addition
to environmental advantages, the
fisherman in south of Iran can improve
their economical condition.
As it is shown in Fig. 7, the size of
chitinase gene was almost 1600 bp.
250
500
750
1000
1500
2000
DNA Ladder (bp)
Serratia marcescens B4A
amplified chitinase gene

922 Zarei et al., Effect of different levels of dietary Betaine on growth…
Molecular weight of partial purified
chitinase was estimated by SDS-PAGE to
be almost 54 kDa (Zarei et al., 2011). This
was shown the homogeneity of partial
purified chitinase and synthesis of
chitinase gene.
Based on Fig. 6c, the optimum pH
for activity of the Bacillus cereus p-1
chitinase is 5 and for Serratia marcescens
B4A is also 5 (Zarei et al., 2010). Both
chitinases were stable at pH 3 to 10 for 90
min at 25 ◦C (Fig. 6d). The optimum
temperature of Bacillus cereus p-1 and
Serratia marcescens B4A for chitinase
production was 50 ◦C (Fig. 6a) and 45 ◦C
(Zarei et al., 2010), respectively.
The similarity between temperature
and pH profile and stability of these two
strains determined that chitinase extracted
from Serratia marcescens B4A has
potential use in insecticide. Further work
on the application of this enzyme and its
economic/commercial feasibility is
currently underway.
Acknowledgements
This work was supported by National
Institute of Genetic Engineering and
Biotechnology (NIGEB), Grants No. 404,
ministry of science, Research and
Technology of Iran.
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