Research J. Pharm. and Tech.

8(3): March 2015

ISSN

0974-3618 (Print)
0974-360X (Online)

www.rjptonline.org

RESEARCH ARTICLE

Production and optimization of extracellular fungal chitinase produced by

Metarhizium anisopliae (M.) Sorokin through Submerged and Solid State
Fermentation
G. Narendrakumar*, S. Karthick Raja Namasivayam, R. Arul luca sunder singh
Department of Biotechnology, Faculty of Bio and Chemical Engineering, Sathyabama University, Chennai,
Tamil Nadu, India
*Corresponding Author E-mail: gnaren22@gmail.com

ABSTRACT:
Chitinase, one of the major metabolite produced by many organisms for the degradation of chitin. Chitinases also play
a role in the utilization of waste from crustacean and other insects resulting in the hydrolysis of chitin to its monomer
N-acetyl-D-glucosamine. The production of Chitinase can be enhanced by changing the constituents in the medium. In
present study chitin production from Metarhizium anisopliae isolated from the dead insect showed production with
different concentration of chitin in submerged media and solid state fermentation using fish waste.

KEYWORDS: Chitin, Chitinase, Metarhizium anisopliae, Submerged fermentation, Solid state fermentation.
INTRODUCTION:
Chitinases are used extensively in biological research for
the generation of fungal protoplasts due to its ability to
degrade fungal cell wall. The hydrolytic property of
chitinases makes it an attractive alternative as an
environmentally safe bio control agent. There are a variety
of instances where chitinase producing organisms are used
to inhibit the growth of phytopathogens. Chitinase is known
to process diverse characteristics worthy of detailed
enzymatic studies related to their biological role and
structural elucidation8-10.A wide range of microorganisms
are known to produce chitinase which have a lot of
industrial and environmental applications.Among the
microbes,fungi play avital role in chitinase production
because of the ease of cultivation on a wide range of media
and high yield. Metarhizium anisopliae (M.) Sorokin is an
important fungal biopesticidal agent against economic
important pests. The fungal organism produces diverse
metabolites during the pathogenesis of the insects.[8].These
entomopathogenic fungi produces extracellular chitinases
only during host penetration and degrade chitin. It has the
ability to grow in wide range of pH5.

Enzymes are among the most important sequels obtained

for human needs through plant, animal and microbial
sources. Many of the enzymes have the lot of applications
in industry, including food processing, brewery and
baking1-4. Chitin forms the exoskeleton of many
invertebrates and is a major component of the cell wall of
fungi. There are enormous chitin result in environmental
problem all round the world. Different microorganism are
capable of producing chitinase that degrades chitin directly
to low-molecular-weight products. Almost all of the
reported chitinase-producing strains will use chitin or
colloidal chitin as a carbon source. Chitinases a group of
enzymes catalyse the hydrolysis of chitin to its monomer Nacetyl-D-Glucosamine. Chitinases may find important
industrial applications to the treatment of chitin, especially
derived from sea-food-processing units5-7.

Received on 02.01.2014
Accepted on 20.01.2014

Solid state fermentation (SSF) has grown importance

recently in the production of microbial enzymes obtaining
economic advantages over conventional submerged culture
(SF). Several groups of microorganisms have been used in
SSF, especially the filamentous fungi that have been
exploited for their abilities to produce a wide range of
extracellular enzymes and to grow on solid complex

Modified on 09.01.2014
RJPT All right reserved

Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 316-321

DOI:

Research J. Pharm. and Tech. 8(3): March 2015

substrates. Several enzymes including amylases, cellulases, Production and optimization

pectinases, proteases and glucoamylasas have been The cultures under Solid State Fermentation were obtained
produced in solid state fermentation18-,21.
by inoculating of 1 ml of spore suspension (105 spores/mL)
on 4 g of crushed fish waste as carbon source moistened
In the present study, production and optimization of with distilled water (Different concentration), yeast extract
chitinase through submerged and solid state fermentation by solution (1% v/v), salt solution. After incubation, the
Metarhizium anisopliae (M.) Sorokin was studied.
cultures were added with 50 ml of autoclaved distilled
water15-17.

MATERIALS AND METHODS:

The free cell filtrate, identified as extracellular crude
extract, was dialyzed against distilled water at 4C overnight and used for chitinase activity determination8.

Fungal strain
Chitinase producing Metarhizium anisopliae was used in
the present study was isolated from dead larval instars of
Spodopteralitura by the modified method of Sahayaraj and
Karthick Raja Namasivayam15.

Analytical methods
Enzyme assay
Monreal and Resse method18 was used to determine
chitinase enzyme quantification. The reaction mixture,
which consisted of 1.5 ml enzyme solution, 2.5 ml of 1 %
colloidal chitin in 0.075 M, pH 7 sodium phosphate buffer,
was incubated at 50, 100 RPM for 10 min in a water bath.
After the enzymatic reaction, 1 ml of DNS reagent (3, 5dinitrosalicylic acid 10 g/l, phenol 2 mg/l, Na2SO30.5 g/l,
and NaOH 10 g/l) was added, followed by heating of the
mixture at 100 for 5 min. Then the solution was cooled for
5 min, diluted with l ml of distilled water, and centrifuged
at 3000 g for 15 min to remove the precipitate. The
absorbance was measured in a spectrophotometers at 540
nm. One unit (U) of enzyme activity was defined as the
amount of enzyme required to produce 0.5 mol of Nacetyl-D-glucosamine for 1 h.

The identification of isolated fungi was performed by

macroscopic (colony morphology) and microscopic (conidia and conidiophores using a compound microscope)8.
Inocula preparation
Fungal inocula was prepared from Potato Dextrose Agar
slant culture of M. anisopliae by scrapping the slant with
Tween 20 using glass rod. Slurry thus obtained was filtered
through crude filter paper, collected filtrate suspension was
used as source of inocula
Chitinase production through submerged fermentation
Production and optimization
Production of chitinase was carried out by the modified
method20.Five millilitre of fungal spore suspension was
inoculated into one litre of production media consist of
colloidal chitin at different concentration, peptone 1 g/l,
MgSO47H2O 1 g/l, (NH4)2SO42 g/l,K2HPO41g/l, NaCl 1
g/l,. Inoculated flasks were incubated under shaking
condition at 300 RPM for 48 hours. After the incubation
period, the media was filtered through cheese cloth to
remove mycelia, filtered broth was centrifuged at 10,000
rpm and collected filtrate was used for enzyme assay.

RESULTS AND DISCUSSION:

Identification:
Microbiological analysis (macroscopic and microscopic
analysis) was performed to characterize the screened strain.

Macroscopic observation
Colonies grow rather gradually on PDA and pigmentation
ranging from light to dark yellow diffusing into the
medium. Conidia color may differ in colony size and
Optimization
Optimization of nutrient factors of chitinase production condition. After 7 days of incubation, the culture produces a
media was studied by changing the compositions of white mycelial margin with clumps of more or less
chitin11-14.
verticillate branching conidiophores.
Chitinase production through Solid state fermentation
Estimation of Moisture content
The sample was weighed along with moisture and recorded
as wet weight of sample Followed by the drying at 90oC
using hot air oven until the sample is dried. The sample was
allowed to cool. The sample was weighed again and
recorded as the dry weight of sample

Microscopic observation
Conidial chains were round, columnar phialides in a dense
parallel arrangement, and conidia were cylindrical to oval.
The above Macroscopic and Microscopic observation
suggest the isolated organism was Metarhizium anisopliae.
Cynthia Barbosa Rustiguel et al 2012 8used Metarhizium
anisopliae was used as the culture organism for the
production of chitinase. Many report suggest the usage of
The moisture content of the sample is calculated using the fungi and bacteria for the production.
following equation:
Effect of chitin:
Different concentration of chitin was inoculated in the
minimal media and the enzyme activity was calculated in
Where: %W = Percentage of moisture in the sample, A = different time intervals (12, 24, 36, 48, 60 and 72) (Table-1
Weight of wet sample (grams), and B = Weight of dry & Figure -1). In the concentration of 5% (w/v) the enzyme
sample (grams)
activity was comparatively higher than the other

Research J. Pharm. and Tech. 8(3): March 2015

concentrations. According to Mandana Zarei et al., 5% in the medium where Serratia marcescens was
20106expressed that the concentration of chitin was about cultivated for the production of chitinase.
Table 1: Effect of Chitin on Enzyme Activity
S. No
Concentration of chitin (%)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Enzyme activity (unit per ml)

12
24
36
5.6
11.2
26.2
6
14.5
31.2
10
19
40.4
24
25
44.2
30.1
39.4
51.4
36.1
41.2
54.1
39.3
45.2
56.2
43.2
52.3
60.4
44.2
53.1
61.7
48.4
56.1
63.2
50.1
59.2
66.2
60.2
71.4
82.3
41.4
47.4
51.2
24.7
17.9
4
9.4
11.3
0.9

0.1
0.25
0.5
0.75
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6

48
30.1
40.1
53.4
59.1
62.4
66.1
69.3
72.2
73
76.4
79.4
90.1
60.2
1.75
0.4

72
51.3
60.1
69.2
71.4
78.1
81.4
78.7
81.4
82.2
89.4
91.4
97.4
56.1
0.4
0.07

0.1
0.25
0.5
0.75
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6

100

Enzyme activity (IU/ml)

60
42.4
50.1
61.4
64.1
74.1
79.2
73.1
76.2
78.4
81.4
84.2
101
69.1
0.7
0.2

50

0
12

24

36

48

60

72

Hours
Figure 1.Theenzyme activity in (IU/ml) in different concentration of chitin with respect to time in hours

Table 2: Effect of Solid State Fermentation using Fish Cell Waste

S.NO
Moisture content%
Enzyme activity (Unit per ml)
12
24
36
1
0.0
9.1
24.5
32.4
2
1.0
9.5
21.0
30.5
3
2.5
10.6
11.2
32.2
4
5.0
9.2
19.3
28.4
5
7.5
8.7
13.4
24.4
6
10.0
3.2
11.2
14.1
7
12.5
1.0
4.2
6.2
8
15.0
0.05
1.01
2.02
9
17.5
0.05
0.09
0.09
10
20.0
0.05
0.07
0.09
11
22.5
0.03
0.07
0.09
12
25.0
0.03
0.05
0.06

48
41.1
40.4
38.1
38.3
32.4
16.1
8.3
2.53
1.90
1.02
1.00
0.05

60
49.0
47.4
47.4
43.4
39.1
9.2
4.1
0.09
0.08
0.07
0.03
0.03

72
53.1
49.2
54.3
46.4
41.1
3.4
1.7
0.07
0.07
0.06
0.03
0.03

Research J. Pharm. and Tech. 8(3): March 2015

0
1
2.5
5
7.5
10
12.5
15
17.5
20
22.5
25

Enzyme activity (IU/ml)

60

40

20

0
12

24

36

48

60

72

Hours
Figure 2.The enzyme activity in (IU/ml) in different moisture content with respect to time in hours

The effect of solid state fermentation using fish cell

waste:
The fish waste was added with suitable moistening agents
to meet the water requirement and supplementary nutrients
to the growing cultures. The Fig. 2 and Table-2 shows that
the maximum enzyme production of ranges from 10.6
54.3 IU/ml with respect to the time was obtained using salt
solution and enzyme secreation was subjective by the
additional proportionate amount of mineral ions.

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CONCLUSION:

5.

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ACKNOWLEDGMENT:
The authors would like to thank the Management and 7.
Faculty of Bio and Chemical Engineering, Sathyabama
University, Chennai for their support.
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