Journal of Cluster Science

https://doi.org/10.1007/s10876-019-01520-z (0123456789().,-volV)(0123456789().,-volV)

ORIGINAL PAPER

Production, Optimization and Characterisation of Chitosanase

of Bacillus sp. and its Applications in Nanotechnology
Antony V. Samrot1,2 • N. Shobana1 • S. Suresh Kumar3,4 • G. Narendrakumar1

Received: 6 December 2018

 Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Chitosanases is a class of enzymes which hydrolyse chitosan, a natural biopolymer consisting of D-glucosamine in various
degrees. In this study, chitosanase producing Bacillus sp. was isolated from soil sample. Chitosanase production was
optimized using response surface methodology and the produced chitosanase was characterized. The crude enzyme was
found to possess antibacterial and antifungal activity. Chitosanase enzyme was used for trimming chitosan based polymeric
nanoparticles produced using sodium trimetaphosphate chelator. Chitosanase enzyme was also utilized for synthesis of
silver nanoparticles which were then characterized by UV–Vis, FTIR, SEM, TEM and AFM. The produced nanoparticles
were checked for antibacterial and antifungal activity.

Keywords Chitosanase  Optimization  Characterization  Silver nanoparticles production

Introduction prawns [4]. Chitosanase cleaves the b-1, 4-glycosidic

There are several enzymes isolated from microorganisms,
as they have got much applications in industry and ana-
lytical methods [1, 2]. Chitosanase (E.C 3.2.2.14) belongs
to the category of Glycosidases (O- and S- glycosyl
hydrolases) [3]. Chitosanases are particular class of
hydrolytic enzymes that display the capability of degrading
chitosan, a copolymer consisting of variable number of
N-acetyl-D-glucosamine and D-glucosamine in linear fash-
ion, which is commonly present on shell of crabs and
& Antony V. Samrot
antonysamrot@gmail.com
& S. Suresh Kumar
sureshkudsc@gmail.com
1
Department of Biotechnology, School of Bio and Chemical
Engineering, Sathyabama Institute of Science and
Technology, Chennai, Tamil Nadu 600119, India
2
Department of Biomedical Sciences, Faculty of Medicine and
Biomedical Sciences, MAHSA University, Jalan SP2, Bandar
Saujana Putra, 42810 Jenjarom, Selangor, Malaysia
3
Department of Parasitology, Universiti Putra Malaysia,
UPM Serdang, 43400 Seri Kembangan, Selangor, Malaysia
4
Institute of Bioscience, Universiti Putra Malaysia,
UPM Serdang, 43400 Seri Kembangan, Selangor, Malaysia
linkages present in the chitosan and produces chitosan—
oligosaccharides [5]. Chitosanase is commonly isolated
from microorganisms like fungi [6–8], actinomycetes
[9, 10], and bacteria [11–13]. Recent years, use of chi-
tosanase enzyme got increased as it is used as biocontrol
agent against fungi [14], waste decomposition [15] etc.
There are reports stating that enzymes are used to produce
silver nanoparticles, for example peroxidase was reported
to reduce Ag? to silver nanoparticles [16, 17].
In this study, a chitosan degrading Bacillus sp. was
isolated from soil sample collected from Fish market,
Besant Nagar, Chennai, Tamil Nadu, India. Chitosanase
production by Bacillus sp. was optimized, thus produced
chitosanase enzyme was characterized. The produced
enzyme was used to trim crab shell derived chitosan
nanoparticles, even the chitosanase enzyme was used to
produce silver nanoparticles. Chitosanase mediated silver
nanoparticles were also checked for its bioactivities.
Materials and Methods
Sample Collection and Isolation of Chitosanolytic
Bacteria
Soil sample was collected from Fish market, Besant Nagar,
Chennai, Tamil Nadu, India. Soil sample was serially

123

diluted and spread plate was performed over the medium
containing (g L-1): Na2HPO4—6; KH2PO4—3; NH4Cl—
2; NaCl—5; MgSO4—1; peptone—5, Agar—20 and chi-
tosan 20 (dissolved with 10 mL of 10% acetic acid). Plates
were incubated at room temperature for 48 h. The colonies
with highest chitosan hydrolyzing ability was picked up
and purified by repeated streaking on chitosan rich media
plates. Obtained pure culture was maintained on chitosan
rich media and stored at 4 C.
Identification of Bacteria
The isolated organism was identified by its microscopic
analysis, biochemical tests and 16S rRNA sequencing. 16S
rRNA sequence was submitted in GENBANK.
Production and Optimisation of Enzyme
Production
Production and Extraction of Crude Enzyme
10 6 CFU/mL culture was allowed to grow on minimal media
containing (g L-1): Na2HPO4—6; KH2PO4—3; NH4Cl—2;
NaCl—5; MgSO4—1; peptone—5 and chitosan 20 (dis-
solved with 10 mL of 10% acetic acid). After appropriate
days of incubation, the broth was filtered using no. 2 What-
mann (Maidstone, UK) filter paper to remove the partially
dissolved chitosan residues. The filtrate was then centrifuged
at 5000g for 15 min. The pellet was discarded, and super-
natant was used for further studies. In order to prevent
enzyme autolysis, all procedures were carried out at 4 C.
Enzyme Activity
Chitosanase activity was estimated using chitosan as the
standard substrate using the method followed by Wee et al.
[18]. 940 lL of 2% (w/v) chitosan was added to 10 mmol/L
sodium citrate buffer (pH 6.0). To this, 30 lL of crude
enzyme was added. The reaction was carried out at 60 C for
10 min. After incubation, the reaction was stopped by adding
30 lL of 10 M NaOH. The solution was then centrifuged at
10,000g for 15 min. The amount of reducing sugar present in
the supernatant was measured at 520 nm using dinitrosali-
cylic acid method, where D-glucosamine was used as a
standard. One unit (1 U) of chitosanase activity is defined as
Table 1 Actual and coded
Factor Name
values used in response surface
methodology for chitosanase A Incubation time
enzyme production
B pH
C Temperature
D Agitation
123
A. V. Samrot et al.
the amount of enzyme required to liberate 1 lmol of D-glu-
cosamine per min under the assay conditions.
Optimization of Enzyme Production
To determine the optimal incubation time, crude enzyme
was extracted from the above mentioned media for every
5th day and enzyme activity was checked using dinitros-
alicylic acid method for a period of 30 days [18]. Likewise,
optimal NaCl concentration (0.5–2.5%), pH (4–9), Tem-
perature (25, 30, 35, 37 and 40 C) and Agitation were
determined by incubating the inoculated media at optimal
incubation time. Enzyme activity was determined by
aforementioned method [18].
Response Surface Methodology
Response Surface Methodology was used for biomass
production and optimization of chitosanase enzyme pro-
duction [19–21]. The optimal conditions required for the
biomass production and production of chitosanase enzyme
were determined and the effects of these variables have
been assessed using RSM. Four parameters such as incu-
bation time, pH, temperature and agitation were used for
designing the experiment (Tables 1, 2).
The general polynomial equation was used
Equation ðYÞ ¼ a0 þ a1 X1 þ a2 X2 þ a3 X3 þ a4 X4 þ a11 X21
þ a22 X22 þ a33 X23 þ a44 X24 þ a12 X1 X2 þ a13 X1 X3
þ a14 X1 X4 þ a23 X2 X3 þ a24 X2 X4 þ a34 X3 X4
where Y is predicted response; X1, X2, X3, X4 are inde-
pendent variables; a0 is an offset term; a1, a2, a3, a4 are
coefficients of linear effects; a11, a22, a33, a44 are coeffi-
cients of squared effects; a12, a13, a14, a23, a34 are coeffi-
cients of interaction terms.
Characterization of Crude Enzyme
Chitosanase Activity
Maximum chitosanase activity was determined at various
pH and temperatures using chitosan as the standard sub-
strate. The reactions were carried out by incubating 2%
substrate and crude enzyme at different pH using Citrate
Low actual High actual Low coded High coded Mean Std. Dev.
10 30 -1 1 20 6.432675
5 9 -1 1 7 1.286535
20 40 -1 1 30 6.432675
0 100 -1 1 50 32.16338
Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…

Table 2 Actual and coded values used in response surface methodology

Factor Name Type Low High Low High Mean Std. Dev.
actual actual coded coded

A Incubation Numeric 10 30 -1 1 20 6.432675

time
B pH Numeric 5 9 -1 1 7 1.286535
C Temperature Numeric 20 40 -1 1 30 6.432675
D Agitation Numeric 0 100 -1 1 50 32.16338
Response Name Obs Analysis Minimum Maximum Mean Std. Dev. Ratio Trans Model

Y1 Biomass 29 Polynomial 6.27 24.33 15.37966 4.623778 3.880383 None Quadratic

Y2 Enzyme 29 Polynomial 24.034 94.428 59.63697 17.92148 3.928934 None Quadratic
production

buffer (pH 4), Sodium Acetate buffer (pH 5 and 6),

Phosphate buffer (pH 7 and 8), Glycine—NaOH buffer (pH
9). The enzyme activities were also determined at different
temperatures ranging from 20 to 70 C in the presence of
sodium acetate buffer (pH 6.0) containing 2% substrate
[18].

90
80
Enzyme Activity (U/ml)

70
60
50
40
30
20
10
0
4 5 6 7 8 9
pH

Fig. 1 Chitosanase producing organism on plate Fig. 3 Enzyme production at various pH

25
Enzyme Activity (U/ml)

20

15

10

0
25 30 35 37 40
Temperature (0C)

Fig. 2 Enzyme production at various incubation time Fig. 4 Enzyme production at various temperatures

123
 A. V. Samrot et al.

80 resuspended in distilled water and dialyzed overnight. The

70 dialysed sample was then made to run in 12% polyacry-
Enzyme Activity (U/ml)

60 lamide gel, stained using Coomassie brilliant blue R-250

50 and molecular weight was determined by comparing with
40
known molecular weight markers [22, 23].
30
Antimicrobial Activity of Chitosanase
20
10
Antimicrobial activity of the crude enzyme was performed
0 against Gram positive bacteria—Bacillus subtilis, Gram
0 10 20 30 40 50 60 70 80 90 100
negative bacteria—Pseudomonas aeruginosa and yeast—
Fig. 5 Enzyme production at various agitation speed Candida albicans using agar well diffusion assay [24, 25].

Molecular Weight Determination Size Reduction of Chitosan Nanoparticles Using

Crude Chitosanase
Chitosanase enzyme was precipitated using saturated
ammonium sulphate solution after which it was centrifuged 50 mg of the lyophilized chitosan nanoparticles were
to collect the pellet and the pellet was immediately treated with 1 mL of the crude chitosanase enzyme for a

Table 3 Design of experiments

Std Run Factor 1 Factor 2 Factor 3 Factor 4 Response 1 Response 2
with responses
A:Incubation time B:pH C:Temperature D:Agitation Biomass Enzyme
production

21 1 20 5 30 0 15.5 15.1 59.66 59.58

3 2 10 9 30 50 7.9 8.2 30.99 31.84
26 3 20 7 30 50 20.7 22.7 80.15 87.89
7 4 20 7 20 100 14.9 14.8 60.02 58.42
1 5 10 5 30 50 9.5 9.4 35.01 35.24
2 6 30 5 30 50 18.3 18.0 71.13 70.35
17 7 10 7 20 50 9.3 9.5 36.11 36.42
10 8 30 7 30 0 17.8 18.2 72.96 71.84
19 9 10 7 40 50 13.5 13.5 51.00 50.55
25 10 20 7 30 50 21.9 22.7 84.06 87.89
6 11 20 7 40 0 18.0 18.0 70.15 71.84
9 12 10 7 30 0 12.5 12.7 51.85 50.45
8 13 20 7 40 100 18.7 18.0 67.95 65.60
16 14 20 9 40 50 13.5 14.0 53.92 54.76
15 15 20 5 40 50 13.5 13.8 51.36 51.38
22 16 20 9 30 0 12.6 11.6 48.31 46.79
23 17 20 5 30 100 15.2 16.3 59.29 61.39
11 18 10 7 30 100 13.5 13.1 47.46 47.93
14 19 20 9 20 50 6.3 5.9 24.03 23.36
12 20 30 7 30 100 18.7 18.4 71.37 72.12
18 21 30 7 20 50 15.2 15.4 60.02 61.04
13 22 20 5 20 50 15.3 14.8 59.66 58.17
24 23 20 9 30 100 10.6 11.1 42.09 42.74
27 24 20 7 30 50 24.3 22.7 94.43 87.89
5 25 20 7 20 0 13.5 14.1 51.97 54.41
29 26 20 7 30 50 23.8 22.7 92.48 87.89
4 27 30 9 30 50 10.3 10.4 42.46 42.31
20 28 30 7 40 50 18.5 18.5 71.25 71.51
28 29 20 7 30 50 22.7 22.7 88.33 87.89

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Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…

Table 4 ANOVA and

Source Sum of Mean F p value
regression of analysis for
biomass production Squares df Square Value Prob [ F

Model 606.6136382 14 43.32954559 45.31479 \ 0.0001 Significant

A-Incubation time 88.4547 1 88.4547 92.50746 \ 0.0001
B-pH 57.11603333 1 57.11603333 59.73294 \ 0.0001
C-Temperature 37.84300833 1 37.84300833 39.57688 \ 0.0001
D-Agitation 0.273008333 1 0.273008333 0.285517 0.6015
AB 10.1124 1 10.1124 10.57572 0.0058
AC 0.2116 1 0.2116 0.221295 0.6453
AD 0.0025 1 0.0025 0.002615 0.9599
BC 20.385225 1 20.385225 21.31922 0.0004
BD 0.765625 1 0.765625 0.800704 0.3860
CD 0.1156 1 0.1156 0.120896 0.7332
A^2 134.7788288 1 134.7788288 140.954 \ 0.0001
B^2 285.197518 1 285.197518 298.2645 \ 0.0001
C^2 99.14492005 1 99.14492005 103.6875 \ 0.0001
D^2 41.17816329 1 41.17816329 43.06484 \ 0.0001
Residual 13.38665833 14 0.956189881
Lack of fit 4.632858333 10 0.463285833 0.211696 0.9788 Not significant
Pure error 8.7538 4 2.18845
Cor total 620.0002966 28
Std. Dev. 0.97785 R-Squared 0.97841
Mean 15.3797 Adj R-Squared 0.95682
C.V.% 6.35807 Pred R-Squared 0.9349
PRESS 40.3631 Adeq Precision 23.8252

period of 10 min at pH 6 and 40 C. The enzyme hydrol-
ysis reaction was stopped via heat fixing by boiling for
5 min. After cooling, samples were centrifuged at
3500g for 10 min and subsequently lyophilized. The
enzyme treated chitosan nanoparticles were analysed by
Scanning Electron Microscopy (Zeiss Ultra Plus, Ger-
many) for the size reduction caused by the enzyme on the
nanoparticles.
Synthesis of Silver Nanoparticles
5 mL of crude chitosanase enzyme was mixed with 25 mL
of 3 mM silver nitrate and kept in dark for the synthesis of
silver nanoparticles at room temperature for 24 h [26, 27].
After 24 h, the solution was centrifuged at 6000g to collect
silver nanoparticles and was lyophilised and stored in a
black container at 4 C.
Characterisation of Silver Nanoparticles
The silver nanoparticles produced using the crude chi-
tosanase enzyme was characterized using Fourier Trans-
form Infrared Spectroscopy (FTIR) (Shimadzu, Japan),
Scanning Electron Microscopy (SEM) (Zeiss Ultra Plus,
Germany), Atomic Force Microscopy (AFM) (Bruker,
Germany) and Transmission Electron Microscopy (TEM)
(TEECNAI G2 Spirit Biotwin—120 kV).
Bioactivity Studies
Antibacterial Activity of Silver Nanoparticles
Bactericidal activity of silver nanoparticles was evaluated
using agar well diffusion method. The antibacterial activity
of different concentrations of silver nanoparticles (2, 4, 6
and 8 lg/mL) was performed against gram positive
Bacillus subtilis and Gram negative Pseudomonas aerugi-
nosa [28, 29].
Swarming Motility
Swarming plates supplemented with 50 lg/mL of silver
nanoparticles were inoculated with Gram positive Bacillus
subtilis and Gram negative Pseudomonas aeruginosa using
the procedure as reported earlier [27, 30, 31].

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 A. V. Samrot et al.

Table 5 ANOVA and

Source Sum of Mean F p value
regression of analysis for
enzyme production Squares df Square Value Prob [ F

Model 9141.469 14 652.9621 52.92291 \ 0.0001 Significant

A-Incubation time 1558.654 1 1558.654 126.3297 \ 0.0001
B-pH 741.1351 1 741.1351 60.06938 \ 0.0001
C-Temperature 453.993 1 453.993 36.79636 \ 0.0001
D-Agitation 3.752008 1 3.752008 0.304102 0.5900
AB 151.8317 1 151.8317 12.30604 0.0035
AC 3.3489 1 3.3489 0.27143 0.6105
AD 1.968409 1 1.968409 0.159541 0.6956
BC 364.5426 1 364.5426 29.54637 \ 0.0001
BD 8.573184 1 8.573184 0.694861 0.4185
CD 26.25538 1 26.25538 2.128012 0.1667
A^2 1985.537 1 1985.537 160.9288 \ 0.0001
B^2 4203.399 1 4203.399 340.6877 \ 0.0001
C^2 1561.057 1 1561.057 126.5245 \ 0.0001
D^2 624.2108 1 624.2108 50.59261 \ 0.0001
Residual 172.7318 14 12.33799
Lack of Fit 34.2332 10 3.42332 0.098869 0.9985 Not significant
Pure Error 138.4986 4 34.62465
Cor Total 9314.201 28
Std. Dev. 3.512547 R-Squared 0.981455
Mean 59.63697 Adj R-Squared 0.96291
C.V. % 5.889882 Pred R-Squared 0.955596
PRESS 413.5873 Adeq Precision 25.54257

Statistical Analysis day. Depletion of nutrients and formation of inhibitory factors

All the experiments were carried thrice and mean value is
illustrated in this study.
Results and Discussion
Isolation and Identification of Microorganism
Chitosanase producing microorganism was confirmed by
the presence of a clear zone around the colony (Fig. 1).
Organism was isolated, sub cultured and used for further
studies. The strain was found to be gram positive rods and
the sequence stated that it was Bacillus sp. (sequence
submitted in GENBANK—Accession no. MH879837).
Optimisation and Characterization
of Chitosanase Enzyme
When the growth curve and enzyme production was plotted
(Fig. 2), it was observed that the organism showed a charac-
teristic sigmoidal growth curve with a long adaptation phase till
15th day which was followed by a rapid growth phase till 25th
might be the reason behind the steep downfall after 25th day.
Likewise, enzyme production also increased gradually and was
found to be maximum on 20th day (Fig. 2). Further incubation
resulted in gradual decrease in enzyme production by the
microorganism. Having incubation time as constant, the other
parameters have been standardized.
Chitosanase enzyme production was found to be high at
20th day, pH 7, 35 C and at 50 rpm agitation (Figs. 2, 3,
4, 5). Variation in the NaCl concentration did not show any
increase in chitosanase production. Similar results showing
the EPS production by Bacillus circulans has been reported
by Vidhyalakshmi et al. [20].
Response Surface Methodology
The effect of four variables on biomass and chitosanase
enzyme production was studied by Response Surface
Methodology. Table 3 gives the Central Composite design
matrix with experimental and predicted values for both the
responses such as biomass production and chitosanase
enzyme production. The regression equation shows the
Chitosanase enzyme production as an empirical function in
terms of coded factors as:

123
Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…

a b
Design-Expert® Software Design-Expert® Software

Biomass Biomass
24.33 24.33

6.27 6.27
25 25
X1 = A: Incubation time X1 = A: Incubation time
X2 = B: pH 20.5
X2 = C: Temperature
21

Actual Factors Actual Factors

C: Temperature = 30.00 16 B: pH = 7.00
Biomass

17

Biomass
D: Agitation = 50.00 D: Agitation = 50.00
11.5
13

7
9

30.00
40.00 30.00
5.00
25.00 35.00 25.00
6.00
20.00 30.00 20.00
7.00
A: Incubation time 15.00 8.00
C: Temperature
25.00 15.00
A: Incubation time
10.00 9.00
B: pH 20.00 10.00

c d
Design-Expert® Software
Design-Expert® Software
Biomass
Biomass 24.33
24.33
6.27
6.27
25
25 X1 = B: pH
X1 = A: Incubation time X2 = C: Temperature
X2 = D: Agitation 20
21.75 Actual Factors
Actual Factors A: Incubation time = 20.00
B: pH = 7.00 D: Agitation = 50.00 15

Biomass
18.5
Biomass

C: Temperature = 30.00
10
15.25

5
12
40.00

35.00
5.00
30.00 6.00
100.00 30.00
7.00
75.00 25.00
25.00 C: Temperature
8.00
50.00 20.00
B: pH 9.00 20.00
15.00
D: Agitation
25.00 A: Incubation time
0.00 10.00
e f
Design-Expert® Software Design-Expert® Software

Biomass Biomass
24.33 24.33

6.27 6.27
25 25

X1 = B: pH X1 = C: Temperature
X2 = D: Agitation X2 = D: Agitation 22
21.25

Actual Factors Actual Factors

A: Incubation time = 20.00 A: Incubation time = 20.00 19
Biomass

17.5
Biomass

C: Temperature = 30.00 B: pH = 7.00

16
13.75

13
10

100.00 100.00
5.00 40.00
75.00 75.00
6.00 35.00
7.00 50.00 50.00 30.00

B: pH
8.00 25.00
D: Agitation D: Agitation 25.00 25.00
C: Temperature
9.00 0.00 0.00 20.00

Fig. 6 Surface plot and Contour plot for biomass production. a Incubation time versus pH, b temperature versus incubation time, c agitation
versus incubation time, d pH versus temperature, e agitation versus pH, f agitation versus temperature

Enzyme activity ðY) ¼ þ 72:04 þ 9:34A  6:44B þ 5:04C ANOVA for response surface polynomial model in case
 0:46D  5:05AB  0:75AC þ 0:58AD þ 7:83BC of biomass production gave P values of the model
 1:20BD  2:10CD  14:34A2  20:87B2 (P \ 0.0001), implying its significance. The coefficient of
variation of the model was (CV = 6.358072%). The
 12:72C2  8:04D2

123
 A. V. Samrot et al.

a b
Design-Expert® Software Design-Expert® Software

Enzyme activity Enzyme activity

94.428 94.428
95
24.034 24.034
95

X1 = A: Incubation time X1 = A: Incubation time 80.25

X2 = C: Temperature

Enzyme activity
X2 = B: pH 78.75
65.5
Enzyme activity

Actual Factors Actual Factors

C: Temperature = 30.00 62.5 B: pH = 7.00
D: Agitation = 50.00 D: Agitation = 50.00 50.75

46.25
36

30
30.00

25.00
30.00
20.00
25.00
5.00 A: Incubation time
6.00 15.00
20.00 20.00
7.00 25.00
30.00
35.00
A: Incubation time 15.00 8.00
10.00 40.00

10.00 9.00 B: pH
C: Temperature
c d
Design-Expert® Software
Design-Expert® Software
Enzyme activity
Enzyme activity
94.428
94.428

24.034 24.034
95
95
X1 = A: Incubation time X1 = B: pH
X2 = D: Agitation X2 = C: Temperature 77
83

Enzyme activity
Actual Factors Actual Factors
Enzyme activity

B: pH = 7.00 A: Incubation time = 20.00 59

71
C: Temperature = 30.00 D: Agitation = 50.00

41
59

47 23

0.00 5.00
30.00
40.00
25.00 25.00
6.00
35.00
20.00 50.00
7.00
30.00
75.00
A: Incubation time
15.00 D: Agitation
B: pH 8.00 25.00
10.00 100.00
9.00 20.00
C: Temperature
e f
Design-Expert® Software Design-Expert® Software

Enzyme activity Enzyme activity

94.428 94.428

24.034 24.034
95
95
X1 = B: pH X1 = C: Temperature
X2 = D: Agitation X2 = D: Agitation 84
81.75
Actual Factors Actual Factors
Enzyme activity
Enzyme activity

A: Incubation time = 20.00 A: Incubation time = 20.00 73

C: Temperature = 30.00 68.5 B: pH = 7.00

62
55.25

51
42

100.00
100.00
40.00
5.00
75.00 75.00
6.00 35.00
50.00 50.00
7.00 30.00

8.00 25.00 D: Agitation D: Agitation 25.00 25.00

B: pH C: Temperature
9.00 0.00 0.00 20.00

Fig. 7 Surface plot and Contour plot for enzyme production. a Incubation time versus pH, b temperature versus incubation time, c agitation
versus incubation time, d pH versus temperature, e agitation versus pH, f agitation versus temperature

goodness of fit of the model was examined by determina- variables. The adjusted determination coefficient (Adj
tion coefficient (R2 = 0.978409) which implied that the R2 = 0.956817) was also satisfactory to confirm the sig-
sample variation of more than 97.1% was attributed to the nificance of the model (Table 4).

123
Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…

50
45
Enzyme Activity (U/ml)

40
35
30
25
20
15
10
5
0
4 5 6 7 8 9
pH

Fig. 8 Chitosanase activity at various pH

45
40
Enzyme Acvity (U/ml)

35
30
25
20
15
10
5
0
20 30 40 50 60 70
Temperature (OC)
Fig. 10 SDS-PAGE of crude chitosanase enzyme. a Molecular
Fig. 9 Chitosanase activity at various temperature marker, b crude chitosanase enzyme

In case of chitosanase enzyme production, the P value
for the model was found to be \ 0.0001 concluding that it
was significant. The coefficient of variation of the model
was (CV = 5.889882%). The goodness of fit of the model
was examined by determination coefficient
(R2 = 0.981455) which implied that the sample variation of
more than 98.1% was attributed to the variables. The
adjusted determination coefficient (Adj R2 = 0.96291) was
also satisfactory to confirm the significance of the model
(Table 5).
Response surface contours plots and three dimensional
graphs helps in understanding the type of interaction
between test variables and to obtain the optimum condi-
tions [32, 33] (Figs. 6a–f, 7a–f). A total of six response
surfaces were shown by considering all the possible com-
binations. A circular contour plot shows negligible inter-
action between the independent variables used, while
perfect interaction indicates elliptical contours. The maxi-
mum predicted value is represented by the surface confined
in the smallest ellipse in the contour diagram [34]. The
optimum value of each variable was identified based on the
hump in the three-dimensional plot, or from the central
point of the corresponding contour plot. Each contour
curve represents an infinite number of combinations of the
two tested variables, with the other two maintained at zero
levels [20]. Senthilkumar et al. [19] reported about the
production of Polyhydroxyalkanoates optimisation by
Pseudomonas aeruginosa SU-1 using RSM as pH 8, tem-
perature 40 C, and incubation time of 48 h. The maximum
production of PHA with these optimized conditions was
found to be around 1.3 times higher than the normal
optimization method.
Characterization of Crude Enzyme
Chitosanase Activity
The optimum pH for chitosanase activity (crude) was
observed at pH 6 (Fig. 8). Chitosanase enzyme produced
using Bacillus subtilis RKY3 also showed highest chi-
tosanase activity at pH 6 [18]. Optimum temperature for
chitosanase activity was found to be at 40 C (Fig. 9).
Similar results were found by Prakash and Gopal [35] with
chitosanase of Bacillus cereus CH12.

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 A. V. Samrot et al.

Table 6 Antimicrobial activity

Bacteria Zone of inhibition (cm)
of crude chitosanase enzyme
against different Positive control Negative control 2 (lg/mL) 4 (lg/mL) 6 (lg/mL) 8 (lg/mL)
microorganisms
B. subtilis 2.7 – – – 1.4 1.6
P. aeruginosa 3.4 – – 0.7 0.8 0.9
C. albicans 3.5 – 1.8 2.2 2.4 2.5

[36] produced chitosanase enzyme of molecular weight

28 kDa [36].

Antimicrobial Activity

The enzyme produced a zone of inhibition of diameter

1.6 cm and 0.9 cm against Bacillus subtilis and Pseu-
domonas aeruginosa respectively at the highest concen-
tration used (Table 6). Fernandes et al. [37] also showed
antibacterial activity of chitosanase enzyme against Sta-
phylococcus aureus and Escherichia coli.
Antifungal activity was found for crude chitosanase
enzyme against Candida albicans. A zone of about 2.5 cm
was observed at the highest concentration of enzyme added
indicating that the crude chitosanase enzyme can be used as
an effective antifungal agent (Table 6).

Size Reduction of Chitosan Nanoparticles Using

Crude Chitosanase

When chitosanase enzyme was made to react with chitosan

nanoparticles, the nanoparticles underwent a hydrolytic
size reduction from 20–25 to 15–19 nm, which was con-
firmed by SEM (Fig. 11a, b).

Production and Characterisation of Silver

Nanoparticles

The UV absorption spectrometric analysis of crude chi-

Fig. 11 SEM analysis of chitosanase enzyme treated chitosan
nanoparticles. a Untreated, b treated
Molecular Weight Determination
The apparent molecular weight of crude chitosanase
enzyme was 59 kDa (Fig. 10). An extracellular chitosanase
producing Bacillus cereus CH12 was isolated by Prakash
and Gopal [35] whose molecular weight was 30 kDa.
Pseudomonas geniculata strain isolated by Kassem et al.
tosanase enzyme mediated synthesized silver nanoparticles
showed absorbance spectra at 259 nm suggesting the bio
reduction of silver nitrate into silver nanoparticles
(Fig. 12b), where the surface plasmon resonance of silver
nanoparticle showed a sharp peak at 259 nm confirmed the
formation of silver nanoparticles [38]. Silver nanoparticles
produced using crude chitosanase enzyme was of size
range between 31 and 40 nm (Fig. 13) but the particles
were bit aggregated which was also confirmed by AFM
(Fig. 14). Transmission Electron Microscopy revealed the
size range of 27–35 nm (Fig. 15).

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Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…
Fig. 12 UV–Vis analysis of silver nanoparticles. a AgNO3, b silver nanoparticles
Fig. 13 Scanning electron microscopy analysis of silver nanoparticles
Bioactivity Studies
Antibacterial Activity of Silver Nanoparticles
Silver nanoparticles synthesized using crude chitosanase
enzyme showed antibacterial activity against Bacillus
subtilis and Pseudomonas aeruginosa. Highest zone of
inhibition of 1.3 and 1.5 cm was visualised against Bacillus
subtilis and Pseudomonas aeruginosa respectively at the
highest concentration of 8 lg/mL (Table 7). Senthilkumar
et al. [39] and Samrot et al. [40] reported plant derived
silver nanoparticle to have antibacterial activity against
Pseudomonas aeruginosa.
Swarming Motility of Bacteria Against Silver Nanoparticles
Swarming motility for Bacillus subtilis and Pseudomonas
aeruginosa against the synthesised silver nanoparticles was
checked. It showed complete inhibition at the concentra-
tion of 50 lg/mL (Fig. 16b, d)
Summary and Conclusions
The chitosanase producing microorganism was isolated
from soil and it was identified as Bacillus sp. Chitosanase
production was optimized and maximum enzyme produc-
tion was found to be on 20th day, at pH—7, temperature of
35 C and 50 rpm agitation. Using RSM, the optimal
123
 A. V. Samrot et al.

Fig. 14 Atomic force

microscopy analysis of silver
nanoparticles

condition for chitosanase production was determined.

Chitosanase enzyme activity was optimal at pH 6 and
40 C and its molecular weight was around 59 kDa. The
crude enzyme had shown antibacterial activity and anti-
fungal activity. Crude enzyme was utilized for size
reduction of chitosan nanoparticles. 27–40 nm sized silver
nanoparticles were synthesized using crude chitosanase
enzyme and it was showing antibacterial activity against
Bacillus subtilis and Pseudomonas aeruginosa.

Fig. 15 Transmission electron microscopy analysis of silver

nanoparticles

Table 7 Antibacterial activity

Bacteria Zone of inhibition (cm)
of silver nanoparticles against
different bacteria Positive control Negative control 2 (lg/mL) 4 (lg/mL) 6 (lg/mL) 8 (lg/mL)

B. subtilis 2.6 – 0.5 0.7 0.8 1.3

P. aeruginosa 3.7 – – – 0.5 1.5

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Production, Optimization and Characterisation of Chitosanase of Bacillus sp. and its…
Fig. 16 Swarming Motility of
bacteria. a Control—Bacillus
subtilis, b plate with silver
nanoparticles and B. subtilis,
c Control—Pseudomonas
aeruginosa, d plate with silver
nanoparticles and P. aeruginosa
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest.
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