Journal of Genetic Engineering and Biotechnology (2013) 11, 39–46

Academy of Scientific Research & Technology and

National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology
www.elsevier.com/locate/jgeb

ARTICLE

Isolation of novel chitinolytic bacteria

and production optimization of extracellular chitinase
Saima, M. Kuddus *, Roohi, I.Z. Ahmad

Protein Research Laboratory, Department of Bioengineering and Biotechnology, Integral University, Lucknow 226026, India

Received 27 November 2012; revised 5 February 2013; accepted 9 March 2013

Available online 1 April 2013

KEYWORDS Abstract Chitin is one of the most abundant biopolymers widely distributed in the marine and ter-
Microbial chitinase; restrial environments. Chitinase enzyme has received increased attention due to its wide range of
Aeromonas; biotechnological applications, especially in agriculture for biocontrol of phytopathogenic fungi
Colloidal chitin; and harmful insects. In the present study, 58 bacterial isolates were screened for chitinolytic activity
Production optimization and on the basis of chitin hydrolysis zone 6 isolates were selected for chitinase production in broth
media. Based on enzyme production, two most potent isolates identified as Aeromonas hydrophila
HS4 and Aeromonas punctata HS6 were selected for further study. The effects of media composition
and various fermentation conditions for optimization of chitinase production were studied. The
maximum chitinase production was obtained at 37 C and pH 8.0 after 24–48 h of incubation by
HS4; and at 37 C and pH 7 after 48 h incubation by HS6. Among the substrates colloidal chitin
was the best for both the strains. Regarding carbon sources, starch (1%) was the best for both
strains; while malt and yeast extract (1%) was found as the best nitrogen source for HS4 and
HS6, respectively. Out of metal ions Mn2+ and Cu2+ enhanced enzyme production in the case
of HS6. However, Co2+ was the most appropriate for HS4.
ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research &
Technology.

1. Introduction groups of each glucoside residue is replaced by an acetylated

or deacetylated amino group. Chitin is the second most abun-
Chitin is nitrogen containing polysaccharide consisting of dant natural polymer and widely distributed as a structural
b-1,4-linked N-acetyl-D-glucosamine which is chemically component of crustaceans, insects, and other arthropods, as
analogous to the cellulose, except that one of the hydroxyl well as a component of the cell walls of most fungi and some
algae. Approximately 75% of the total weight of shellfish, such
as shrimp, crabs and krill are considered as waste, and chitin
* Corresponding author. Tel.: +91 9415392163. comprises 20–58% of the dry weight of the said waste [49].
E-mail address: kuddus_biotech@yahoo.com (M. Kuddus). About 1011 tons of chitin is alone produced annually in the
Peer review under responsibility of National Research Center, Egypt. aquatic biosphere [32,36]. Chitinase (EC 3.2.11.14) enzyme is
capable of hydrolyzing insoluble chitin to its oligo and
monomeric components found in a variety of organisms
Production and hosting by Elsevier including viruses, bacteria, fungi insects, higher plants and

1687-157X ª 2013 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
http://dx.doi.org/10.1016/j.jgeb.2013.03.001
40 Saima et al.

animals and play important physiological roles depending on fuged at 10000 rpm for 15 min at 4 C and the crude was used
their origin [8,10,16]. Chitinases are constituents of several for chitinase assay.
bacterial species; some of the best known genera include
Aeromonas, Serratia, Vibrio, Streptomyces and Bacillus [7]. 2.4. Assays of chitinase activity and protein estimation
Chitinases can be classified as endochitinases or exochitinases.
Endochitinases cleave chitin at internal sites to generate The chitinase activity was assayed by measuring reducing
multimers of GlcNAc. Exochitinases catalyze the hydrolysis sugar released from colloidal chitin as per the modified method
of chitin progressively to produce GlcNAc, chitobiose or of Toharisman et al. [46]. Briefly, crude enzyme (150 ll) was
chitotriose. Chitinase has a wide-range of applications such added to the mixture consisting of 300 ll of 0.1% colloidal
as preparation of pharmaceutically important chitooligosac- chitin and 150 ll of 0.1 M phosphate buffer pH 7.0. After incu-
charides and N-acetyl D-glucosamine, preparation of single-cell bation at 55 C for 10 min, the reaction mixture was subjected
protein, isolation of protoplasts from fungi and yeast, control to a refrigerated centrifugation at 10,000 rpm for 5 min. The
of pathogenic fungi, treatment of chitinous waste, and control resulting supernatant (200 ll) was added with 500 ml of DW
of malaria transmission [9]. Chito-oligomers produced by and 1000 ml of Schales reagent then boiled for 10 min. After
enzymatic hydrolysis of chitin are used in various fields like cooling, the absorbance of the mixture was measured at
in medical, agricultural and industrial applications, such as 420 nm. One unit of the chitinase activity was defined as the
antibacterial, antifungal, antihypertensive and as a food amount of enzyme which yields 1 lmol of reducing sugar as
quality enhancer [5]. N-acetyl-D-glucosamine (GlcNAc) equivalent per minute.
Optimization of media is very important to maximize the
yield and productivity, and minimize the product cost [1]. 2.5. Characterization of bacterial isolates
The aim of this study was to isolate the most prominent chitin-
olytic bacteria from different soil samples with unique proper- 2.5.1. Identification of chitinolytic bacterium
ties and optimize their fermentation conditions for maximum
chitinase production. The identification of bacterial isolates HS4 and HS6 was
carried out according to the methods described in Bergey’s
Manual of Systematic Bacteriology [21].
2. Materials and methods
2.5.2. Isolation of genomic DNA for 16S rRNA and PCR
2.1. Sample collection and isolation of chitinolytic bacteria
amplification
Total genomic DNA was extracted from the cells by using the
A total of 15 different soil samples were aseptically collected phenol–chloroform method [35]. Isolated DNA was checked
from different regions of Lucknow, India. The location of col- for its quality and concentration by agarose gel electrophoresis
lected soil was rhizosphere of maize, wheat and rice, fish market on UV transilluminator. 16S rRNA region was amplified
and pond. For screening of chitinase producing bacteria, the with universal forward and reverse bacterial primers. The
agar medium amended with colloidal chitin was used. The med- PCR amplification was performed using a PTC-150 Mini
ium consists of (g L 1): Na2HPO4, 6; KH2PO4, 3; NH4Cl, 1; cycler (MJ Research), with a primary heating step for 2 min at
NaCl, 0.5; yeast extract, 0.05; agar, 15 and colloidal chitin 95 C, followed by 30 cycles of denaturation for 20 s at 95 C,
1% (w/v). The colonies showing clearance zones on a creamish annealing for 60 s at 55 C, and extension for 2 min
background were considered as chitinase-producing bacteria. at 72 C, then followed by a final extension step for 7 min at
72 C. Each 25 lL reaction mixture contained 2 lL of genomic
2.2. Preparation of colloidal chitin DNA, 14.25 lL of MilliQ water, 2.5 lL of 10· buffer (100 mM
Tris–HCl, pH 8.3; 500 mM KCl), 1.5 lL of MgCl2 (25 mM),
Colloidal chitin was prepared from the chitin (Hi Media) by 2.5 lL of dNTP’s mixture (dATP, dCTP, dGTP, dTTP at
the modified method of Hsu and Lockwood [20]. In brief, chi- 10 mM conc.), 1.0 lL of each primer (20 pmol/mL), and
tin powder (40 g) was slowly added with 600 ml of concen- 0.25 lL of Taq DNA polymerase. The PCR-amplified product
trated HCl and kept for 60 min at 30 C with vigorous was analyzed on 1% agarose gel containing ethidium bromide
stirring. Chitin was precipitated as a colloidal suspension by (0.5 mg/mL) and 1 kb DNA molecular weight marker and doc-
adding it slowly to 2 l of water at 4–10 C. The suspension umented using a gel documentation system. The PCR amplicon
was collected by filtration with suction on a coarse filter paper for the partial 16S rRNA gene was further processed for
and washed by suspending it in about 5 l of DW. Washing was sequencing. Sequencing was carried out using the same set of
repeated 3 times until the pH of the suspension was 3.5. After primers in both the directions to check the validity of the
the above treatment, the loose colloidal chitin was used as a sequence. Sequencing was done by Ocimum Biosolutions, India.
substrate [50].
2.6. Optimization of enzyme production
2.3. Screening of chitinase producing bacteria
2.6.1. Effect of media and incubation time on chitinase
Screening was performed with bacterial isolates on the colloi- production
dal chitin agar medium incubated at 37 C. Bacterial isolates Six different broth media viz. nutrient broth (g L 1: yeast
were selected on the basis of a larger hydrolysis zone after extract, 1.5; NaCl, 5; beef extract, 1.5; amended with 1%
96 h of incubation and further screened for maximum enzyme colloidal chitin), luria bertaini broth (g L 1: tryptone, 10; yeast
production in nutrient broth media. The cultures were centri- extract, 5; NaCl, 5; amended with 1% colloidal chitin), M1
Isolation of novel chitinolytic bacteria 41

(%: chitin, 1; K2HPO4, 0.1; MgSO4Æ7H20, 0.05; at pH 7), M2

Table 1 Production of chitinase at 37 C after 24 h
(%: colloidal chitin, 2; MgSO4Æ7H20, 0.05; NaH2PO4, 0.5),
incubation.
M3 (g L 1: chitin, 15; Urea, 0.32; CaCl2, 0.1; MgSO4Æ7H20,
0.08) and M4 (%: colloidal chitin, 0.3; K2HPO4, 0.1; Culture No. Chitinase activitya (Unit/ml)
MgSO4Æ7H20, 0.01; NaCl, 0.1; (NH4)2SO4Æ0.7) were used to HS-2 3272
determine the growth of bacteria and chitinase production. HS-4 5946
The culture was inoculated (1%) and incubated at 37 C for HS-6 6072
24 h in a rotary shaker (120 rpm). After 24 h of incubation, HS-8 4393
the cultures were harvested, centrifuged at 10,000 rpm for HS-9 4876
HS-12 5091
15 min and the supernatant used for chitinase assay [46]. For
a
optimum incubation time, the bacterial culture was grown for Values are mean of 3 replications.
5 days and chitinase production was estimated every day.

2.6.2. Effect of temperature and pH on chitinase production

The effect of temperature on enzyme production was Table 2 Characterization of isolates.
determined by incubating inoculated medium at different
Colony properties HS-4 HS-6
temperatures (18, 22, 37, 40, 50 and 55 C) for optimized
period of time. The effect of the initial pH value on the Colony shape and size Irregular , 2–3 mm Circular, 2 mm
chitinase production was investigated by varying the initial Elevation Convex Convex
Margin Undulate, Entire edge,
pH of the culture medium from 4 to 12 and at optimized
wrinkled surface smooth surface
temperature and incubation period. Chitinase assay was Gram reaction Negative Negative
performed as per standard protocol. Cellular morphology Rod Rod
Color Creamish white Creamish
2.6.3. Effect of different substrates and their concentration on Utilization of lactose, +ve +ve
chitinase production maltose, glucose, sucrose
To find out the best substrate for enzyme production, the chi- Spore ve ve
Citrate utilization +ve ve
tinase production was carried out by using different substrates
Motility +ve +ve
(1%) in medium viz. fish shell powder (FS), colloidal chitin Urease production ve ve
(CC) and chitin powder (CP) at previously optimized condi- Catalase test +ve +ve
tions. Different concentrations of substrates (0.1–1.2%) were Starch hydrolysis +ve +ve
applied in optimized media and condition to determine the Triple ion sugar ve ve
best substrate concentration. Methyl red +ve +ve
Voges–Proskauer reaction ve +ve
2.6.4. Effect of carbon and nitrogen sources on chitinase Growth at pH 6–7 5–9
production Growth at temperature (C) 22–40 22–37
Growth in NaCl 2–8% 2–6%
The effects of various carbon and nitrogen sources (1%) were
used as additional supplement in media for maximum enzyme
production. The supplemented media were inoculated with 1%
inoculums and fermented at an optimized condition. Simulta- neously media without any carbon and nitrogen source were
used as control.

2.6.5. Effect of metal ions on chitinase production

Influence of various metal ions on chitinase production was
determined by inoculating medium with different metal ions
such as Mn2+, Co2+, Ca2+, Fe2+, Mg2+ and Hg2+ in their
chloride form except Cu2+ (copper sulfate). Chitinase assay
was then performed as per standard protocol.

3. Results and discussion

A total of 58 morphologically different chitinolytic bacteria

were isolated from 15 soil samples collected from different
habitats of Lucknow, India. On the basis of colloidal chitin
degradation (Fig. 1) and zone of clearance (>0.2 cm) on
CCA plate, six colonies were selected for secondary screening
in broth media and tested for enzyme activity. Based on max-
imum chitinase production after 24 h of incubation, two po-
tential isolates HS4 and HS6, isolated from the soil of rice
rhizosphere and fish market respectively, were selected for fur-
Figure 1 Bacterial colony showing clear hydrolysis zone on ther studies (Table 1).
colloidal chitin agar.
42 Saima et al.

Figure 2 Phylogenetic tree of strain HS4 showing the similarity with Aeromonas hydrophila.

Figure 3 Phylogenetic tree of strain HS6 showing the similarity with Aeromonas punctata.

3.1. Characterization of bacterial isolates A. hydrophila (Fig. 2) while HS6 having maximum homology
(99%) with A. punctata (Fig. 3).
3.1.1. Identification of bacteria
The isolates HS4 and HS6 were identified as Aeromonas hydro- 3.2. Optimization of enzyme production
phila and Aeromonas punctata, respectively. The identity was
further confirmed by 16S rRNA analysis. The strains were 3.2.1. Effect of media and incubation period
gram negative, motile, non-spore forming and facultative Among all the tested media, LB with colloidal chitin was
anaerobes (Table 2). more productive in both the strains such as A. hydrophila
HS4 (64.41 U/ml) and A. punctata HS6 (59.41 U/ml)
3.1.2. Analysis of DNA sequences (Fig. 4). Karunya et al. [22] also observed that presence of
The homology of the partial 16S rRNA gene sequence of the colloidal chitin in LB supports maximum chitinase produc-
isolates was analyzed using the BLAST algorithm in GenBank tion from Bacillus subtilis. The effects of incubation time
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analyses on chitinase production are shown in Fig. 5. A. hydrophila
were conducted using a multiple sequence alignment tool HS4 produced highest chitinase after 24 h and remains con-
(Clustral W). Only the highest-scored BLAST result was con- stant up to 48 h (80.9 U/ml) while A. punctata HS6 produced
sidered for phylotype identification. BLAST showed that the maximum chitinase (82.36 U/ml) after 48 h of incubation.
isolate HS4 linear DNA has maximum homology (99%) with Enzyme production gradually decreased in both strains after
Isolation of novel chitinolytic bacteria 43

80 60
HS4 HS6 HS4 HS6
70
50

Enzyme Activity (U/ml)

Enzyme Activity (U/ml)

60

50 40

40
30
30
20
20

10 10
0
LB NB M1 M2 M3 M4 0
Media 18 22 37 40 50 55
Temperature
Figure 4 Effect of media on chitinase production by HS4 and
HS6. Figure 7 Effect of temperature on chitinase production by HS4
and HS6.

90 HS4 HS6 60
HS4 HS6
80
50

Enzyme activity (U/ml)

Enzyme Activity(U/ml)

70

60 40

50
30
40
30 20

20
10
10

0 0
CP CC FS
12 24 48 72 96 120 Substrate (1%)
Incubation time (hr)

Figure 8 Effects of different substrates on chitinase production

Figure 5 Effect of incubation period on chitinase production by
by HS4 and HS6.
HS4 and HS6.

48 h. One of the reasons for decreased production may be 60

HS4 HS6
the lack of nutrients or production of toxic chemicals in 50
Enzyme Activity (U/ml)

the medium resulting in the inactivation of secretary machin-

ery of the enzymes [31]. Similar observations were also re- 40
ported by Nawani et al. [30] with Microbispora sp. Wang
30
and Hwang [48] reported that B. cereus, B. alvei and B. sph-
aericus produced highest chitinase after 48 h of incubation. 20
Although Faramarzi et al. [11] reported maximum chitinase
10
production at 36 h of incubation by M. timonae. However,
at 72 h of incubation, maximum chitinase production was 0
observed by Penicillium aculeatum [6] and Trichoderma har- 0.1 0.3 0.6 0.8 1 1.2
Colloidal chitin conc. (%)
zianum [37].
Figure 9 Effect of substrate concentration on chitinase produc-
tion by HS4 and HS6.
100
90
80 3.2.2. Effect of pH on chitinase production
Enzyme Activity (U/ml)

70 In order to evaluate the effect of pH of media on the chitinase

60 production, bacterial cultures were grown at different pH (4–
50 12). Among the tested pH, pH 8 for A. hydrophila HS4
40 (93.27 U/ml) and pH 7 for A. punctata HS6 (73.43 U/ml) sup-
30 ported the maximum chitinase production (Fig. 6). From the
HS4 HS6
20 above results, we can conclude that pH of media not only helps
10 in the production of chitinase but also plays an important role
0 in cell growth.
4 5 6 7 8 9 10 11 12
pH Like A. hydrophila HS4, previous reports also suggested
that Bacillus laterosporous [38], Micrococcus sp. AG84 [4],
Figure 6 Effect of pH on chitinase production by HS4 and HS6. Alcaligenes xylosoxydans [47], Serratia marcescens XJ-01 [51],
44 Saima et al.

Aeromonas sp. JK1 [2] and Bacillus pabuli [12] are capable of 120 HS4 HS6
producing a high amount of chitinase at alkaline condition.

Enzyme Activity (U/ml)

The result of HS6 strain is also supported by other publica- 100

tions [2,25,26,29,33,40,41]. Furthermore, Hiraga et al. [19] re- 80

ported chitinase production in a broad range of pH-value
60
(5–8) by A. hydrophila H-2330.
40

3.2.3. Effect of temperature for chitinase production 20

Temperature affects various biological processes, therefore the 0

growth of bacteria and enzyme production are also affected
with the change in incubation temperature. To evaluate the
optimum growth temperature for chitinase production, the Carbon Source
cultures of A. punctata HS6 and A. hydrophila HS4 were grown
at 18–55 C. Chitinase production was maximum at 37 C with Figure 10 Effect of carbon sources on chitinase production by
both strains A. hydrophila HS4 (43.08 U/ml) and A. punctata HS4 and HS6.
HS6 (53.22 U/ml) (Fig. 7). The enzyme production was de-
creased above 40 and 50 C by A. punctata HS6 and A. hydro-
phila HS4, respectively. Narayana et al. [28] and Sudhakar and 100
HS4 HS6
Nagarajan [44] also reported maximum chitinase production at

Enzyme Activity (U/ml)

90
35 C by Streptomyces sp. ANU6277 and T. harzianum, 80
70
respectively. Other reports also concluded maximum enzyme 60
production from Streptomyces sp. in between 30 and 40 C 50
[15,18,24,38,43]. 40
30
20
3.2.4. Effect of different substrates on chitinase production 10
0
Among the various substrates like fish shell (FS), chitin pow-
der (CP) and colloidal chitin (CC), colloidal chitin was found
to be a best substrate for chitinase production by both the
strains (40.74 and 49.6 U/ml by A. hydrophila HS4 and A. punc-
Nitrogen source
tata HS6, respectively) (Fig. 8). Similar observation has also
been reported with Streptomyces viridificans [18], Streptomyces Figure 11 Effect of nitrogen sources on chitinase production by
lydicus WYEC108 [25] and Acremonium obclavatum [17]. Fara- HS4 and HS6.
marzi and coworkers [11] also showed that colloidal chitin acts
as a sole carbon and nitrogen source for chitinase production.
A study on chitinase production from Streptomyces sp. con-
firmed that the presence of colloidal chitin along with sucrose Table 3 Effect of metal ions on chitinase production by HS4
doubled the enzyme production [43]. Karunya et al. [22] and and HS6.
Andronopoulou and Vorgias [3] also reported a similar result Metal ions Chitinase activitya (Unit/ml)
with Bacillus subtilis and Thermococcus chitonophagus, HS-4 SH-6
respectively.
Control 51.77 48.22
Mn+2 84.67 92.71
3.2.5. Effect of substrate concentration on chitinase production Co+2 87.05 48.54
Different concentrations of colloidal chitin were used to eluci- Ca+2 40.2 49.93
date the best concentration for maximum chitinase production Cu+2 72.81 85.43
which can be exploited at the industrial level. The results Fe+2 75.24 40.12
showed both the strains produced enzyme maximally at Mg+2 45.63 45.14
0.3% of colloidal chitin (Fig. 9). Accordingly A. hydrophila Hg+2 59.22 29.28
a
HS4 and A. punctata HS6 produced 52.8 and 43.4 U/ml of en- Values are mean of 3 replications.
zyme, respectively. Our results are also supported by the find-
ings of Souza et al. [42] and Karunya et al. [22] who reported
the maximum chitinase production at 0.3% colloidal chitin. aureofaciens CMUAc130 [45], S. marcescens [40] and
Streptomyces sp. ANU [28].
3.2.6. Effect of carbon sources on chitinase production
A total of seven different carbon sources (1%) namely manni- 3.2.7. Effect of nitrogen source for chitinase production
tol, glucose, fructose, sucrose, lactose, starch and dextrose As per the results, the addition of malt extract, peptone, geletin
were tested for maximum chitinase production. Among the and casein in A. hydrophila HS4 and yeast extract, malt extract
carbon sources, starch supported maximum chitinase produc- and casein in A. punctata HS6 had a significant effect on chi-
tion for A. hydrophila HS4 (97.35 U/ml) and A. punctata HS6 tinase production among the various tested nitrogen sources
(89.87 U/ml) followed by lactose and fructose (Fig. 10). The (Fig. 11). Malt extract in A. hydrophila HS4 (86.01 U/ml)
same findings were also reported with Streptomyces and yeast extract in A. punctata HS6 (82.64 U/ml) were the
Isolation of novel chitinolytic bacteria 45

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