Bioorganic Chemistry 77 (2018) 494–506

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Development of thermostable amylase enzyme from Bacillus cereus for

potential antibiofilm activity
Ramalingam Vaikundamoorthy a,1, Rajaram Rajendran a,⇑,1, Ananth Selvaraju b, Kaviyarasan Moorthy b,
Santhanam Perumal b
a
DNA Barcoding and Marine Genomics Laboratory, Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India
b
Marine Planktonology and Aquaculture Laboratory, Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The marine bacterial strain Bacillus cereus was used to produce amylase enzyme and has excellent alkali-
Received 6 December 2017 stable and thermostable enzymatic activity. The combined effects of pH, temperature and incubation
Revised 5 February 2018 time on amylase activity were studied using response surface methodology. The amylase enzyme activity
Accepted 10 February 2018
was also determined in the presence of various metal ions, chelating agents, detergents and the results
Available online 12 February 2018
showed that the maximum enzyme activity was observed in the presence of calcium chloride (96.1%),
EDTA (63.4%) and surf excel (90.6%). The amylase enzyme exhibited excellent antibiofilm activity against
Keywords:
marine derived biofilm forming bacteria Pseudomonas aeruginosa and Staphylococcus aureus in microtiter
Bacillus cereus
Thermostable amylase enzyme
plate assay and congo red assay. Light and confocal laser scanning microscopic (CLSM) analysis were also
Optimization used to confirm the potential biofilm activity of amylase enzyme. The CLSM analysis showed the inhibi-
Antibiofilm activity tion of complete biofilm formation on amylase enzyme treated glass surface. Further in vivo toxicity anal-
In vivo toxicity ysis of amylase enzyme was determined against marine organisms Dioithona rigida and Artemia salina.
The results showed that there is no morphological changes were observed due to the minimal toxicity
of amylase enzyme. Overall these findings suggested that marine bacterial derived amylase enzyme could
be developed as potential antibiofilm agent.
Ó 2018 Elsevier Inc. All rights reserved.

1. Introduction ever, Bacillus sp. produces a large variety of extracellular enzymes

Amylase enzyme is one of the most important industrial
enzymes which hold the supreme market share of enzyme sales
with major applications in starch industry, baking, analytical
chemistry, detergents, textile desizing, medicine, pulp and paper
industries [1]. These amylase enzymes originate from different
sources like plants, animals and microorganisms; among them,
microbial amylases are the most produced and used in industries
due to their high productivity and thermostability [2]. Because of
the industrial importance of amylase, there is ongoing interest in
the isolation of new bacterial strains producing amylase that suit-
able for new industrial applications [3].
Studies on bacterial amylase in developing countries are con-
centrating mainly on Bacillus sp. probably because of simple nature
and cheaper nutritional requirements of these organisms [4]. How-
⇑ Corresponding author at: DNA Barcoding and Marine Genomics Laboratory,
Department of Marine Science, Bharathidasan University, Tiruchirappalli 620 024,
Tamil Nadu, India.
E-mail address: rajaramdms@bdu.ac.in (R. Rajendran).
1
These authors contributed equally to this work.
such as amylases, proteases, and lipases which have significant
industrial importance [5]. The amylases produced by different
Bacillus species vary not only in their types (saccharifying or lique-
fying) but also in the varying range of pH and temperature [6]. The
bacterial source of the enzyme is usually either from Bacillus amy-
loliquefaciens or Bacillus licheniformis, the latter now being of
greater industrial importance [7].
The optimal production of a microbial enzyme and its growth
depends on the nature of strain involved as well as various envi-
ronmental parameters such as dissolved oxygen, agitation, temper-
ature, pH, substrate and nutrients [8]. Although production of
amylase has been implemented on agricultural and industrial
wastes and its co-products such as starch materials to solve pollu-
tion problems and obtain a low cost medium [9]. Moreover, the
rice husk, wheat bran and starchy potato wastes were used as
low-cost carbon substrates for amylase activity by B. subtilis [10].
However, the optimum conditions to produce the enzyme or
metabolite are not always the same as that for growth [11]. There-
fore, statistical and experimental factorial designs combined with
Response Surface Methodology (RSM) could have much impact

https://doi.org/10.1016/j.bioorg.2018.02.014
0045-2068/Ó 2018 Elsevier Inc. All rights reserved.
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
on experimental designs which tests one factor at a time and time
consuming as it requires many experiments [8].
On other hand, amylase enzymes have potential applications
other than industries such as pharmaceuticals, medical, clinical
applications etc. Recently a-amylase enzyme has been found as a
good antibiofilm agent against biofilm forming clinical pathogens
Vibrio cholera, S. aureus and P. aeruginosa [12]. Biofilms are less sus-
ceptible to host inflammatory and immune responses and have
higher antibiotic resistance than free-living planktonic cells [13].
Production of new antibiofilm agents against biofilms requires an
understanding of bacterial biofilm-specific functional characters.
Several studies have been initiated to clarify the complicated
mechanisms underlying biofilm development and many aspects
of these mechanisms are still poorly understood [14]. There are
very less studies were reported that the antibiofilm activity of
amylase enzyme with inadequate methods including but limited
with spectrometric and agar plate method. In the present study,
we dealt with optimization and characterization of amylase
enzyme produced by Bacillus cereus isolated from deep sea water
sample. Further, the potential antibiofilm efficiency of amylase
enzyme against marine biofilm forming bacteria and its toxicity
against marine copepod Dioithona rigida and brine shrimp Artemia
salina was evaluated.
2. Materials and methods
2.1. Isolation and identification of microorganisms
The deep seawater samples were collected from Indian Ocean
Equator region (1°0. 1370 N 82°59.9040 E) at 1000 m depth during
the onboard cruise of Sagar Kanya 289 (SK 289, NCAOR, Goa, India).
One ml of water sample was taken and transferred to an Erlen-
meyer’s flask containing 9 ml of sterile 50% seawater and the sam-
ple suspension was further diluted up to 10 folds. One ml of the
diluted suspension was spread over the surface of nutrient agar
medium prepared in 50% seawater to enhance the isolation of
microorganisms. The colonies found on the plates were transferred
onto agar plates and incubated at 28 °C. The colonies were
observed from the second day and purified by repeated streak on
agar plates. The purified colonies were stored at
80 °C in nutrient
broth containing 30% glycerol. For taxonomic identification, the
isolates were subjected to a series of morphology characterization
[15] and biochemical tests [16] such as indole test, nitrate reduc-
tion, Voges-Proskauer (VP), and degradation of starch, The
amylase-producing bacterial colonies were selected after flooding
the plates with iodine solution. The strain (88DSB10) produced
the high level of amylase enzyme was selected for further experi-
ments. Further, the isolated strain was identified by amplification
of 16S rDNA and sequencing, the sequences were submitted to
NCBI database. The sequences were aligned using ClustalX 2.0 soft-
ware and the phylogenetic tree was constructed using neighbor
joining method (Mega 6.0 software) with bootstrap confidence of
1000 replicates.
2.2. Production and partial purification of amylase enzyme
The selected strain (88DSB10) was cultivated in minimal med-
ium (M9) containing 10% NaCl and 1% soluble starch and pH of
the medium was adjusted to 10.5 after autoclaving with 10% Na2-
CO3. Cultures were grown for 20 h at 37 °C with shaking at 200g.
After the removal of cells by centrifugation at 10,000g for 20 min.
at 4 °C and the supernatant was used for further work [17]. Previ-
ously chilled (
20 °C) ethanol was added dropwise to the clear
supernatant for continuous stirring to the final concentration of
75% and the solution was stored at
20 °C for 24 h. The precipitate
495
was recovered by centrifugation at 13,000g for 20 min. at 4 °C and
resuspended in 100 mM phosphate buffer solution (pH 7) [18].
Further 1 mg/ml concentration of enzyme was dialyzed with molec-
ular weight cut-off of 30 kDa. The concentrated enzyme solution
was precipitated by drop-wise addition of two volumes of chilled
absolute ethanol. The precipitate was recovered in 100 mM phos-
phate buffer solution (pH 7) and dialyzed overnight against the
same buffer at 4 °C.
2.3. SDS page
The molecular weight of the crude and partially purified amy-
lase was estimated using sodium dodecyl sulphate-
polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentra-
tion was determined according to Lowry’s method with bovine
serum albumin as standard. The purified enzyme (1 mg) was loaded
onto 1 mm thick 10% polyacrylamide gel together with molecular
size markers. The relative molecular mass of amylase was deter-
mined by comparing with known standard molecular weight
markers (97.4–29.0 kDa). After completion of electrophoresis, the
protein gel was stained with Coomassie Brilliant Blue R-250.
2.4. Enzyme assay
The activity of the amylase enzyme was assayed by adding 0.5
ml of the enzyme to 0.5 ml soluble starch (1% v/v) in 100 mM
glycine-NaOH buffer at pH 10.5 and incubated at 37 °C for 60
min. The 10 ml of 0.1 N HCl was added to stop the reaction and
A540 was measured [2] in double beam spectrophotometer (Shi-
madzu model 1800). The amount of reducing sugars released dur-
ing the starch hydrolysis was measured and using the glucose
standard curve. A unit of enzyme activity was defined as the
amount of enzyme required to release 1 mmol of sugar reduced
per minute under enzyme assay conditions by dinitrosalicylic acid
(DNS) method.
2.5. Effects of pH and temperature on activity and stability
The enzyme activity at different temperature was determined
by incubating the enzyme with glycine-NaOH buffer between 40
and 100 °C for 30 min [19]. The effect of different pH on amylase
activity was carried out by assessing the enzyme in citrate-
phosphate buffer (pH 4–6), Sodium-phosphate buffer (pH 6.5–8),
Glycine-NaOH buffer (pH 8.5–10.5) and Borax-NaOH buffer (pH
11–13) for 20 min [20]. The stability of the enzyme at different
temperature was determined by incubating the enzyme between
50 °C and 100 °C and for the measurement of pH stability, the
enzyme was pre-incubated at 37 °C for 8 h [21–23].
2.6. Optimization of amylase activity
To study the effect of four main variables such as starch, pH,
temperature and NaCl on enzyme activity was obtained through
optimization; based on five levels (
2,
1, 0, +1, +2) a second-
order quadratic model was used [24]. The design consisted of 30
experiments with four factorial points, four axial points to make
a central composite design and five center points for replication
in order to determine the experimental error. The enzyme activity
under the optimized conditions was obtained from the Central
Composite Design (CCD). Design Expert 7.1.5 (State-Ease, MN,
USA) was used for regression analysis and plotting the response
surface.
A reduced cubic polynomial model was calculated to estimate
the response of the dependent variable for the production of amy-
lase enzyme and its activity.
496 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506

Y ¼ b0 þ b1 A þ b2 B þ b3 C þ b4 D þ b12 AB þ b13 AC þ b14 AD þ b23 BC enzyme to determine the activity of amylase enzyme against bio-
2 2 3 4 film formation.
þ b24 BD þ b34 CD þ b11 A þ b22 B þ b33 C þ b44 D

In this equation, Y is the predicted response, A, B, C and D are inde-
pendent variables, b0 is a constant term, b1, b2, b3 and b4 are linear
effects, b11 b22 b33 and b44 are squared effects, and b12, b13, b14, b23,
b24 and b34 are interaction terms.
2.7. Effect of metal ions and chelating agent
The effect of metal ions on amylase enzymatic activity was
determined by adding 5 mM of each metal ion solutions to enzyme
using standard enzyme assay. The activity of amylase was deter-
mined against metals ions (magnesium chloride, sodium chloride,
copper sulphate, cadmium chloride, potassium chloride, calcium
chloride, manganese sulphate and zinc chloride) and chelating
agents (EDTA and 2-mercaptoetanol). The enzyme was incubated
with inhibitors for 15 min. at 37 °C and performing the activity
in the presence of same inhibitor concentration at the optimum
temperature for 60 min [25]. The activity of the sample without
metal ions and chelating agents was considered as 100%.
2.10. Light microscopic analysis
The effect of amylase enzyme against biofilm formation of P.
aeruginosa and S. aureus on glass surfaces was tested as described
earlier [27]. Biofilm formation was visualized using a bright field
microscope under 40 magnification and images were captured
using a digital camera attached with to microscope.
2.11. Confocal laser scanning microscopy (CLSM) analysis
The effect of amylase enzyme on the 3-D architecture of P.
aeruginosa and S. aureus biofilm was observed using CLSM (CLSM,
LSM 710, Carl Zeiss, Jena, Germany). The test cultures were grown
on glass surfaces as mentioned above. After washing with PBS and
staining with 0.01% acridine orange, biofilms were observed under
the CLSM. A 488 nm Ar laser and a 500–640 nm bandpass emission

2.8. Evaluation of enzyme for use in detergent formulation

The effect of different detergents on amylase enzymatic activity

was determined by assaying the enzyme (1 mg/ml) with detergent
solution and incubated at 50 °C for 60 min. Before, the detergents
like Surf excel, Rin, Ariel and Tide were diluted in double distilled
water to a final concentration of 7 mg/ml. The detergent solution
was heated at 100 °C for 15 min. to inactivate the enzymes present
in detergents. The reaction of detergents with reaction mixture
was determined by assaying under standard enzyme conditions.
The stability of amylase enzyme against different detergents was
determined by the addition of 1 mg/ml enzyme in detergent and
incubated at 50 °C for 60 min. The results were compared with
the control which was incubated without any detergent at 50 °C
[25].

2.9. Anti-biofilm activity of amylase enzyme

The biofilm forming bacteria P. aeruginosa (JQ989348) and S.

aureus (KF554244) was inoculated on the brain heart infusion agar
supplemented with 5% sucrose and congo red for the formation of
biofilm [26]. Initially, the aqueous solution of congo red was auto-
claved at 121 °C for 15 min. and the autoclaved solution was added
to pre-cooled agar medium. The biofilm forming organisms were
inoculated and incubated at 37 °C for 24–48 h. In the meantime,
congo red agar plates were also inoculated with 200 ml of amylase

KU991849 | Bacillus subtilis B-1

KY672899 | Bacillus sp. BAB-6056
KY672901 | Bacillus sp. BAB-6054
KY907008 | Bacillus sp. K-93-2
KY085971 | Bacillus thuringiensis WG20
KY085987 | Bacillus thuringiensis CMS20
KY910170 | Bacillus cereus PNA2
MG491525 | Bacillus cereus GOACSMMS10
KJ683733 | Bacillus cereus DMS/RR/88DSB10
KR132554 | Bacillus cereus GRI-SD-LC

Fig. 1. The phylogenetic tree for 16S rRNA gene sequences of Bacillus cereus Fig. 2. The molecular weight of the crude and partially purified amylase was
88DSB10 (KJ683733) isolated from deep seawater sample and the tree was estimated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-
constructed by neighbor joining method from the Mega software (version 6.0) PAGE). Lane 1: Molecular marker; Lane 2: Crude amylase enzyme; Lane 3: Partially
with bootstrap confidence of 1000 replicates. purified amylase enzyme.
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506 497

filter was used to excite and detect the stained cells and the images for Grams staining, Voges Proskauer, gelatin liquefaction, catalase
were acquired and processed using Zen 2011 software (Carl Zeiss) test, oxidase test, nitrate reduction, citrate utilization and starch
[28]. Meanwhile, the biofilm formation of P. aeruginosa and S. aur- hydrolysis and negative for indole test, methyl red rest, urease test
eus on amylase enzyme coated glass surface was also tested using and hydrogen sulfide test. Further, the comparative 16S rDNA gene
CLSM. Initially, amylase enzyme was coated on the glass surface sequence (1172 bp) analysis of 88DSB10 showed 97% homology
using magnetic coater at 5000g. Briefly, 2 ml of overnight cultures with Bacillus cereus and sequence was submitted to NCBI GenBank
of P. aeruginosa and S. aureus (cultured in LB medium) was inocu- (Accession No. KJ683733). The phylogenetic tree was constructed
lated in 6 well microtiter plate and incubated at 37 °C for 48 h. using neighbor joining method and the result confirmed that the
B. cereus has 97% similarity with its related species (Fig. 1). The
2.12. In vivo toxicity analysis crude amylase enzyme present in the supernatant of culture and
partially purified amylase enzyme was subjected to ethanol precip-
In vivo toxicity of marine bacterial derived amylase enzyme was itation. The molecular weight of amylase was 13 kDa, as estimated
determined against marine copepod D. rigida and brine shrimp by SDS-PAGE (Fig. 2).
Artemia salina. For toxicity analysis against copepod, 10 healthy
copepods were inoculated in a beaker containing 25 ml of sterile
3.1. Effect of incubation time
seawater. Mortality of copepods was monitored every day. 100
ml/5 ml of amylase enzyme was used to assess the toxicity and
The effect of incubation time in the range of 24–96 h for the
treatment was continued for 4 days. For toxicity against A. salina,
production of amylase enzyme by Bacillus cereus KJ683733 was
nauplii were hatched in 2 L plastic tank stocked at 0.5 g/l cyst in fil-
tested and the highest and lowest enzyme activity was observed
tered and autoclaved seawater with 35‰ salinity, 8–8.5 of pH and
at 24 h and 96 h respectively (Fig. 3). Initially, the activity of amy-
proper aeration. After overnight incubation for hatching, the aera-
lase enzyme was increased with the increase of incubation time up
tion was stopped, the nauplii were trapped using light and trans-
to 24 h and enzyme activity reduced at maximum incubation time
ferred to fresh filtered autoclaved seawater and were used for
(96 h). However, the variation of amylase enzyme activity between
toxicity assessment. Briefly, 10 Artemia nauplii were inoculated
into the beaker containing 25 ml of sterile seawater and proper
growth conditions (pH, salinity, temperature, etc.) were provided.
For control experiments, we used micro algae as feed for both
Dioithona rigida and Artemia salina. After treatment, the number
of live and dead animals (copepod and brine shrimp) was calcu-
lated and morphological changes were observed under an inverted
microscope (Micros, Austria). All the experiments were conducted
in triplicate and mean value of survival rate was determined.

3. Results

Totally 13 strains were isolated from deep sea water sample

and all 13 strains were incubated on starch agar plates to isolate
the colony which exhibits the high level of amylase enzyme pro-
duction. Among 13 strains, the strain 88DSB10 showing maximum
amylase enzymatic activity and was selected for further study of
optimization and production of amylase enzyme. Initially, the
selected strain was identified using various biochemical tests and
molecular techniques. The isolated strain 88DSB10 was positive

Fig. 4. a. Effect of temperature on the amylase enzyme activity and thermostability

of enzyme activity produced from Bacillus cereus. b. Effect of pH on the amylase
Fig. 3. Effect of various incubation time on activity of amylase enzyme produced enzyme activity and pH stability of enzyme activity (error bars are represents mean,
from marine bacteria Bacillus cereus (Error bars are represents mean, n = 3). n = 3).
498 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506

24 and 48 h was not significant and the enzymatic activity was sig- natants were determined. The optimum temperature of the amy-
nificantly reduced at 72 h. lase enzyme activity was determined at different temperatures
between 30 and 110 °C and the high enzyme activity was obtained
3.2. Effect of temperature at 80 °C (Fig. 4a). The activity of amylase enzyme was gradually
decreased when the temperature was increased. Meanwhile, the
To characterize these variations in amylase production and thermostability of the enzyme was determined by incubating the
activity, the effect of temperature and pH of the culture super- enzyme between 30 and 110 °C for 60 min. and results showed

a) b)
80
92
76.5

R elativ e activ ity

85.25
R elativ e activ ity

73
78.5
69.5
71.75
66
65

8.00 4.00

42.00 4.00 7.50 3.50

37.50 3.50 7.00 3.00

6.50 2.50
33.00 3.00 C: pH A: Starch
6.00 2.00
28.50 2.50
B: Temperature A: Starch
24.00 2.00

c) d)
79 92

73.75 85
R elativ e activ ity

R elativ e activ ity

68.5 78

63.25 71

58 64

8.00 42.00
72.00 4.00
7.50 37.50
60.00 3.50
7.00 33.00
48.00 3.00
6.50 28.50
D: Incubation time 36.00 2.50
A: Starch C: pH B: Temperature
6.00 24.00
24.00 2.00

e) f)
88 79

79.5 73.75
R elativ e activ ity

R elativ e activ ity

71 68.5

62.5 63.25

54 58

72.00 42.00 72.00 8.00

60.00 37.50 60.00 7.50
48.00 33.00 48.00 7.00
36.00 6.50
D: Incubation time 36.00 28.50
B: Temperature D: Incubation time C: pH
24.00 24.00 24.00 6.00

Fig. 5. Response surface plot showing the influence of starch, pH, temperature and incubation on amylase enzyme activity.
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506 499

that amylase enzyme was highly stable at a higher temperature y ¼ 76:00
3:17A
10:05B
3:25C
1:75D þ 1:75AB
1:00AC
(100 °C) showed up to 90% of activity.
þ 0:63AD þ 2:13BC þ 1:50BD þ 1:50CD
0:32A2 þ 1:13B2
3.3. Effect of pH
1:32C2
12:45D2

In other hands, the optimum pH for amylase enzyme produc-

tion was obtained at different pH between pH 4–13. The results
where A, B, C and D are independent variables representing the
showed that the enzymatic activity was high at alkali pH (9–13)
starch, temperature, pH and incubation time respectively and y rep-
reveal amylase has broad pH activity range (Fig. 4b). The lowest
resents the amylase activity as a response variable. The ANOVA
enzymatic activity was observed at pH 4 and activity was increased
results for the quadratic model indicated to fit for the design and
with an increase of pH from 4 to 10 and slowly decreases after pH
also significant at 95% confidence level (p < 0.05). The various
10. In the interim, the pH stability of the enzyme was also tested by
parameters evaluated and the subsequent p values recommended
incubating the enzyme between pH 4–13 for 8 h and results exhib-
that starch, temperature, pH and incubation time are act as inde-
ited that enzyme was highly stable in alkali pH (10) over the period
pendent variables and having the significant effect on enzyme activ-
of time showed 83% activity.
ity. The negative coefficient for A, B, C and D (from the quadratic
equation) reveals the existence of linear effect that reduces the
3.4. Optimization of amylase activity
amylase activity. Meanwhile, the presence of positive coefficients
(AB, AD, BC, BD, CD and B2) indicates cubic interactions and also
To optimize the amylase enzyme activity, 30 experiments were
increases the amylase enzyme activity. From these results, we con-
designed using CCD by varying the concentration of starch, tem-
clude that the four variables significantly affect the amylase enzyme
perature, pH and incubation time. The results showed that the
activity individually as well as interactions. Through RSM (Fig. 5),
amylase enzyme activity of B. cereus has concurred with predicted
3% starch, 33 °C temperature, pH 7 and 48 h incubation could signif-
and experimental values and the coefficient of determination (R2)
icantly affect amylase activity and thus selected for further produc-
for enzyme activity was 0.6943. The R2 was quantitatively con-
tion, characterization and antibiofilm activity.
firmed using the correlation between experimental (0.862) and
predicted responses (0.943), is indicated a good agreement
between the experimental and predicted values for the response.
The following quadratic model equation for the response enzy-
matic activity (y) was

Fig. 6. (a) The observed activity of amylase enzyme versus the predicted activity of
amylase enzyme under the experimental conditions. (b) A plot of the internally Fig. 7. The effect of various metal ions (a) and detergents (b) on activity of amylase
predicted residuals versus the actual response. enzyme (Error bars are represents mean, n = 3).
500 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
The cluster point for correlation between experimental and pre-
dicted values is fall in and around the slopping line are reveals the
selected quadratic model is effectively fit model for optimization.
In this study, the values of operating variables represent the amy-
lase activity determined from acceptable variations between
experimental and predicted values (Fig. 6a). The determination of
normality assumption between residual and predicted responses
is another way to prove the selected quadratic model is effectively
fit for optimization. The random scatters of residual responses
indicate that the variance of the experimental responses is con-
stant for all amylase enzyme activity (Fig. 6b).
3.5. Effect of metals ions, chelating agents and detergents
In order to study the adaption of amylase enzyme to metal
elements of the marine environment and provide fundamental
research for the application of amylase, the effects of metal ions
and other chemicals on enzyme activity were analyzed. The
results presented in Fig. 7a and showed that high enzyme activ-
ity was obtained in the presence of sodium chloride (94.2%) and
calcium chloride (96.1%). The lowest amylase activity was
observed in the presence of copper sulphate (5.7%) and moderate
activity was observed in the presence of rest of the metals. In
another hand, moderate enzymatic activity was observed in the
presence of chelating agents such as EDTA (63.4%) and 2-
mercaptoethanol (58.3%). Further, the amylase enzyme activity
was estimated against different detergents like Surf excel, Rin,
Ariel and Tide. The maximum and minimum enzyme activity
were showed against Surf excel (90.6%) and Rin (28.1%) respec-
tively (Fig. 7b).
a) P. aeruginosa
b)
Fig. 8. Antibiofilm activity of amylase enzyme against P. aeruginosa and S. aureus determined using 96 microtiter plate assay (b) and congo red assay (error bars are represents
mean, n = 3).
3.6. Antibiofilm activity
The biofilm forming capability of isolates was determined using
96 well microtiter plate assay, congo red assay and further con-
firmed by microscopic analysis. In order to determine the amylase
enzyme concentration to obtain highest biofilm inhibitory effi-
ciency, 96 well microtiter plate assay was evaluated by varying
the concentration of enzyme. The results showed that 20 ml and
25 ml concentration of enzyme could effectively inhibit the biofilm
formation of P. aeruginosa and S. aureus respectively (Fig. 8a). The
antibiofilm efficiency was further confirmed by congo red agar
method and the results showed that amylase enzyme inhibits the
synthesis of exopolysaccharides of biofilm forming bacteria that
reveals the potential antibiofilm activity of amylase enzyme
(Fig. 8b).
3.7. Microscopic analysis of biofilm formation
The amylase enzyme was most efficacious at eliminating plank-
tonic populations, preventing biofilm formation and eradicating
established biofilms. The light microscopic as well as CLSM analy-
sis clearly shows the reduction of biofilm formation on glass sur-
face upon the treatment with amylase enzyme. The light
microscopic analysis showed that 20 ml and 25 ml of enzyme com-
pletely inhibit the biofilm formation of P. aeruginosa and S. aureus
respectively (Fig. 9). The CLSM 2D and 3D images confirmed the
anti-biofilm activity of amylase enzyme against both P. aeruginosa
and S. aureus showing a reduction of the biomass, which was not
observable in control sample (Fig. 10). Meanwhile, the 2D and 3D
rendering shows the thick mass of biofilm was adhered to the cov-
erslip in untreated sample, whereas in amylase treated coverslip
S. aureus
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
a) Control
P. aeruginosa
c)
S. aureus
Fig. 9. Light microscopic images for formation of biofilms by P. aeruginosa and S. aureus control and treated with amylase enzyme.
showed a drastic reduction in biofilm thickness and intensity
(Fig. 11).
3.8. In vivo toxicity analysis
The potential antibiofilm activity of marine bacterial derived
amylase enzyme was further examined in vivo toxicity against
marine microorganisms D. rigida and A. salina. The results showed
that there is no toxicity was observed after 24 h exposure of 100
ml/5 ml of amylase enzyme against marine copepod D. rigida
(Fig. 12a). About 85% of survival of D. rigida was observed after con-
tinuous exposure of amylase enzyme for 4 days and there is no
apparent morphological changes were observed in treated copepod
compared with control (Fig. 12b and c). In another hand, 90% of the
survival rate of A. nauplii was observed against 100 ml/5 ml of amy-
lase enzyme treatment after 24 h (Fig. 13a). There is no significant
morphological changes were observed in control and enzyme trea-
ted A. nauplii (Fig. 13b and c) is reveals that the amylase enzyme
has no toxicity against both organisms which are involved in the
marine food web.
4. Discussion
Amylase enzymes exemplify a second major group of industrial
enzymes which are used for the degradation of numerous natural
substances [29]. These enzymes can be produced from various
sources such as plants, animals and microorganisms. The signifi-
501
b) T r e at e d
d)
cant use of microorganisms for the production of amylases is the
economical bulk production capacity and the fact that microbes
are easy to employ to produce enzymes of preferred characteristics
[30]. Among the various Bacillus sp. used to produce the amylase
enzyme at the wide range of temperatures [31], Bacillus amyloliq-
uefaciens, Bacillus subtilis, Bacillus licheniformis and Bacillus
stearothermophilus are used to produce a-amylase at temperatures
of 37–60 °C [20,32–34]. In the present study Bacillus cereus isolated
from deep sea water sample was used for the production of ther-
mostable amylase enzyme and used for inhibition of biofilm
formation.
The applications and performance of enzymes are disturbed by
its microenvironment such as incubation time, temperature and
pH could affect the surface and residual charges on the solid matrix
[35]. In this study, the amylase enzyme activity gradually
decreased during increase the incubation time after 24 h. 72 h of
incubation was found to be the best incubation time for maximal
enzyme production by Bacillus cereus and further incubation time
decreases the yield of enzyme as well as the growth of the bacteria
[36].
The temperature and pH have the significant effect on the
microbial growth, enzyme production, enzyme catalysis and stabil-
ity [37]. The stability of amylase enzymes under the various
extreme conditions is highly significant and it could affect the
structural changes of enzyme molecule is an important aspect to
investigate [38]. Our results showed that enzyme activity remains
100% at 80 °C for 30 min and there is no loss of activity after
502 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
2D
Control
P. aeruginosa
Treated
Control
S. aureus
Treated
Fig. 10. CLSM shows the effect of amylase enzyme on inhibition of biofilm formation on glass surface.
incubating the enzyme at 80 °C for 90 min. Previously, a-amylase
from Bacillus subtilis showed highest enzymatic activity in the tem-
perature range of 65–75 °C, and the optimal activity was deter-
mined at 70 °C. However, the activity decreased dramatically
above 80 °C [39]. Oyeleke et al. [40] demonstrated that the maxi-
mum amylase enzyme was produced by Bacillus megaterium at
the temperature of 60 °C and the thermostability of the enzyme
was at 75 °C. Among the various physical parameters, pH of the
growth medium plays a vital role by inducing morphological
changes in the organism and in enzyme production and it also
serves as a valuable indicator of the initiation and end of enzyme
synthesis [41]. Our results also showed that amylase enzyme has
excellent activity over the pH 10 and it also never loses its activity
when incubating the enzyme at pH 10 for 8 h. Earlier, the amylase
enzyme produced from Bacillus sp. showed higher than 85% rela-
tive activity in the pH range 3–10, with the optimum pH at 9
[42]. Effect of pH was studied by incubating the enzyme substrate
mixture in different pH (3.0–9.0) levels using the buffer and max-
imum a-amylase activity was found at pH 7.0 [43].
Ortho 3D Intensity
Starch is the important carbon source in the liquid medium
available for strain growth and moreover the amount of starch in
the medium plays a crucial role in the production of amylase.
Therefore, the influence of starch on amylase production should
be evaluated by changing the concentration of starch [42]. In con-
nection to the statement, the production of amylase enzyme was
optimized by varying the concentrations of starch, temperature,
pH and incubation time and the optimized conditions for maxi-
mum amylase production were obtained using Design Expert (Ver-
sion 7.1.5, State-Ease, MN, USA). Mahdavi et al. [44] demonstrated
that the optimum temperature of the amylase enzyme produced
by Bacillus cereus strain was 50 °C, with a loss of activity over 70
°C. The optimum temperature for the production of amylase
enzyme produced by Bacillus sp. isolated from the soil was at 90
°C showing 100% activity and thermostability was at 80 °C showing
82% activity [45]. Our results showed that high amylase activity
was obtained at 3% starch, 33 °C temperature, pH 7 and 48 h incu-
bation time after 30 runs of optimization using RSM. Yandri et al.
[46] revealed that the ideal temperature for amylase enzyme
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
2D
Control
P. aeruginosa
Treated
Control
S. aureus
Treated
Fig. 11. CLSM shows the biofilm formation of P. aeruginosa and S. aureus on amylase enzyme coated glass surface.
production was at 60 °C and the enzyme was stable at 70 °C show-
ing 80% activity.
The temperature and the pH are the important factors to deter-
mine the production of amylase enzyme from microorganisms. The
maximum amylase enzyme production was obtained at the pH of
11 and the optimum pH was at 10.5. The production of amylase
enzyme was decreased when the pH was increased above 11 and
the pH stability of the enzyme was showing high alkali at 13 with
92% activity. The similar results were shown by Burhan et al. [2].
Carvalho et al. [45] demonstrate that the optimum pH for amylase
enzyme produced by Bacillus sp. at 8.5 and the pH stability was at
pH 11 showing 80% activity. The optimum pH for the production of
amylase enzyme from the Halobacillus sp. MA-2 was at 8 and the
stability of the enzyme was at pH 9.5 shows 80% activity [47].
The enzyme activity also determined against different metal
ions and the maximum enzyme activity was showed against CaCl2
and the minimum was CuSO4 as 96% and 5% respectively. Goyal
et al. [48] reported that the Ca2+ ions could bind to the enzyme
so as to confer stability but does not interfere with the activity.
Calcium-independent amylase has received increasing attention
503
Ortho 3D Intensity
in recent years because calcium requirement is one of the signifi-
cant problems in the starch industry for the production of amylase
enzyme during the formation of calcium oxalate [2,49]. The amy-
lase enzyme activity was estimated in this study against different
detergents and maximum and minimum enzyme activity was
showed against Surf excel (90.6%) and Rin (28.1%) respectively.
Further amylase enzyme also showed moderate enzymatic activity
in the presence of the chelating agent such as EDTA (63.4%) and 2-
mercaptoethanol (58.3%). The chelating agent EDTA has not inhib-
ited the enzymatic activity of Bacillus sp. RM16 [50] and Alicy-
clobacillus sp. A4 [51].
In another hand, the applications of amylase enzyme were
determined by analyzing its antibiofilm potential against marine
microorganisms such as P. aeruginosa and S. aureus. Literally, most
of the antimicrobial agents are unable to penetrate the biofilm due
to the exopolysaccharides (EPS) which acts as a barrier that pro-
tecting the bacterial cells within the biofilm. Recently microbial
enzymes have been proven as an effective antibiofilm agent for
degradation of EPS of microbial biofilms [52]. In the present study,
amylase enzyme from B. cereus effectively inhibits the biofilm
504 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
Fig. 12. Effect of amylase enzyme (100 ml/5 ml) on marine copepod D. rigida (a) and effect of enzyme on morphological changes in control (b) and treated copepod (c) (error
bars are representing mean, n = 3; Image magnification - 40).
formation of P. aeruginosa and S. aureus as evidenced by congo red
assay, CLSM and light microscopic analysis. The mechanism of
antibiofilm activity underlies that enzymes initially weakened
the proteins, carbohydrates and lipids and then destroy the physi-
cal structure of EPS and initiates degrading process [12].
The antibiofilm potential of amylase enzyme was further used
for toxicity analysis against marine copepod D. rigida and brine
shrimp Artemia salina. There are many reports stated that copepod
could be used as appropriate toxicity testing [53–54] and many
international organizations standardized the procedure for toxicity
test in copepods [55–58]. Previously, Bielmyer et al. [59] reported
that marine copepods are very sensitive and its survival depends
upon the concentration and exposure time, moreover the continu-
ous treatment may lethal to the host. In contrast, our present
results showed that 100% of survival was observed after 24 h with
100 ml/5 ml amylase enzyme treatment using acute toxicity and
increasing the treatment time up to 4 days could affect only 15%
of survival of copepod.
Further, the toxicity of amylase enzyme was also determined
against Artemia nauplii and the results showed that there is no
apparent toxicity and morphological changes were observed after
continuous exposure of amylase enzyme (100 ml/ml) for 24 h. The
short life cycle and reliable culture practices can make Artemia
nauplii used as a model organism for assessing the toxicity of
pathogenic bacteria. Since copepod and Artemia nauplii play a vital
role in food web between phytoplankton and higher tropical ani-
mals, by analyzing the toxicity of amylase enzyme against copepod
could much help in developing amylase enzyme as potential bio-
film inhibitor.
5. Conclusion
In conclusion, the present study described the production and
purification of amylase enzyme from marine bacterium Bacillus
cereus. The thermostable and pH stable amylase enzyme produc-
tion was optimized using RSM and results showed that nutrients
and environmental factors could play a vital role in amylase
enzyme activity. Further, the light microscopic as well as CLSM
images clearly show the reduction of biofilm formation on covered
surface area upon treatment with amylase enzyme. Since amylase
enzyme did not affect survival and morphology of marine copepod
and Artemia, this amylase enzyme could be developed as effective
biofilm inhibitor. In future, analysis the expression of gene and
protein in amylase enzyme treated biofilm forming bacteria could
prove the novelty of amylase enzyme.
 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506 505

Fig. 13. Effect of amylase enzyme (100 ml/5 ml) on A. salina (a) and effect of enzyme on morphological changes in control (b) and treated A. salina (c) (error bars are
representing mean, n = 3; Image magnification - 40).

Acknowledgement [9] A.K. Mukherjee, M. Borah, S.K. Rai, Biochem. Eng. J. 43 (2009) 149–156.
[10] Z. Baysal, F. Uyar, C. Aytekin, Proc. Biochem. 38 (2003) 1665–1668.
[11] H.K. Sodhi, K. Sharma, J.K. Gupta, S.K. Soni, Proc. Biochem. 40 (2005) 525–534.
The authors gratefully acknowledge the authorities of Bharathi- [12] B.J. Kalpana, S. Aarthy, S. Karuthapandian, Appl. Biochem. Biotechnol. 167 (6)
dasan University for providing CLSM facility under the DST- (2012) 1778–1794.
[13] F.G. Vital-Lopez, J. Reifman, A. Wallqvist, PLoS Comput. Biol. 11 (10) (2015)
PURSE scheme (SR/FT/LS-113/2009).
e1004452.
[14] C.T. O’Loughlin, L.C. Miller, A. Siryaporn, K. Drescher, M.F. Semmelhack, B.L.
Bassler, PNAS 110 (44) (2013) 17981–17986.
Competing financial interest
[15] J.H. Woo, K.M. Kang, T.H. Kwon, N.H. Park, E.Y. Lee, J. Ind. Eng. Chem. 28 (2015)
225–228.
Authors declares that there is no competing financial interest. [16] Y. Liu, Y. Lei, X. Zhang, Y. Gao, Y. Xiao, H. Peng, Mar. Biotechnol. 14 (3) (2012)
253–260.
[17] S. Shanmughapriya, G. Seghal Kiran, J. Selvin, R. Gandhimathi, T. Bastin Baskar,
References Aseer Manilal, S. Sujith, Biotechnol. Bioprocess Eng. 14 (2009) 67–75.
[18] M.A. McTigue, C.T. Kelly, E.M. Doyle, W.M. Fogarty, Enzyme Microb. Technol.
[1] R. Gubta, P. Gigras, H. Mohapatra, V.K. Goswami, B. Chauhan, Proc. Biochem. 38 17 (1995) 570–573.
(2003) 1599–1616. [19] H.F. Lo, L.L. Lin, H.L. Chen, W.H. Hsu, C.T. Cheng, Proc. Biochem. 36 (2001) 743–
[2] A. Burhan, U. Nisa, C. Gokhan, C. Omer, A. Ashabil, G. Osman, Proc. Biochem. 38 750.
(2003) 1397–1403. [20] S. Afrisham, A. Badoei-Dalfard, A. Namaki-Shoushtari, Z. Karami, J. Mol. Catal.
[3] R.E. Ghorbel, S. Maktouf, E.B. Massoud, S. Bejar, S.E. Chaabouni, Appl. Biochem. B: Enzym. 132 (2016) 98–106.
Biotechnol. 157 (1) (2009) 50–60. [21] B. Karakas, M. Inan, M. Certel, J. Mol. Catal. B: Enzym. 64 (2010) 129–134.
[4] P. Deb, S.A. Talukdar, K. Mohsina, P.K. Sarker, S.M. Abu Sayem, Springerplus 2 [22] M.C.V. Egas, M.S. da Costa, D.A. Cowan, E.M.V. Pires, Extremophiles 2 (1998)
(2013) 154. 23–32.
[5] C.A.M. Cordeiro, M.L.L. Martinas, A. Lucaino, Braz. J. Microbiol. 33 (2003) 1–3. [23] E. Leveque, S. Janecek, B. Haye, A. Belarbi, Enzyme Microbiol. Technol. 26
[6] B.T. Fossi, F. Tavea, L.A. Fontem, R. Ndjouenkeu, S. Wanji, Biotechnol. Rep. 4 (2000) 3–14.
(2014) 99–106. [24] V. Ramalingam, K. Varunkumar, V. Ravikumar, R. Rajaram, RSC Adv. 6 (2016)
[7] N. Declerck, M. Machius, G. Wiegand, R. Huber, C. Gaillardin, J. Mol. Biol. 31 64087–64096.
(2000) 1041–1057. [25] U.C. Banerjee, R.K. Sani, W. Azmi, R. Soni, Proc. Biochem. 35 (1999) 213–219.
[8] M. Hashemi, S.M. Mousavi, S.H. Razavi, S.A. Shojaosadati, Ind. Crops Prod. 43 [26] V. Ramalingam, R. Rajaram, C. PremKumar, P. Santhanam, P. Dhinesh, S.
(2013) 661–667. Vinothkumar, K. Kaleshkumar, J. Basic Microbiol. 54 (9) (2014) 928–936.
506 R. Vaikundamoorthy et al. / Bioorganic Chemistry 77 (2018) 494–506
[27] C. Nithya, M. Farzana Begum, S. Karuthapandian, Appl. Microbiol. Biotechnol.
88 (2010) 341–358.
[28] F. LewisOscar, D. MubarakAli, C. Nithya, R. Priyanka, V. Gopinath, M.S. Alharbi,
N. Thajuddin, Biofouling 31 (4) (2015) 379–391.
[29] S. Shoda, H. Uyama, J. Kadokawa, S. Kimura, S. Kobayashi, Chem. Rev. 116 (4)
(2016) 2307–2413.
[30] K.R. Mihajlovski, N.R. Radovanovic, D.N. Veljovic, Ind. Crops Prod. 80 (2016)
115–122.
[31] X. Li, H.Y. Yu, J. Ind. Microbiol. Biotechnol. 38 (11) (2011) 1837–1843.
[32] A. Straksys, T. Kochane, S. Budriene, Food Chem. 211 (2016) 294–299.
[33] D. Gangadharan, S. Sivaramakrishnan, K.M. Nampoothiri, R.K. Sukumaran, A.
Pandey, Bioresour. Technol. 99 (11) (2008) 4597–4602.
[34] F. Gashtasbi, G. Ahmadian, K.A. Noghabi, Enzyme Microb. Technol. 65 (2014)
17–23.
[35] T.N. Nwagua, B. Okolo, H. Aoyagi, S. Yoshida, Proc. Biochem. 48 (2013) 1031–
1038.
[36] H. Anto, U. Trivedi, P. Patel, Food Technol. Biotechnol. 44 (2006) 241–245.
[37] B.A. Kikani, S.P. Singh, Int. J. Biol. Macromol. 81 (2015) 450–460.
[38] H. Lund, S. Kaasgaard, L. Jorgensen, C.I. Jorgenson, M. Weert, J. Surfactants
Deterg. 15 (2011) 9–21.
[39] M. Tuzlakoglu Ozturk, N. Akbulut, S. Issever Ozturk, F. Gumusel, Appl.
Biochem. Biotechnol. 171 (2013) 263–278.
[40] S.B. Oyeleke, S.H. Auta, E.C. Egwim, J. Microbiol. Antimicrob. 2 (7) (2010) 88–
92.
[41] C. Suganthi, A. Mageswari, S. Karthikeyan, K.M. Gothandam, Microbiology 84
(2) (2015) 210–218.
[42] S. Wang, J. Jeyaseelan, Y. Liu, W. Qin, Appl. Biochem. Biotechnol. 180 (1) (2016)
136–151.
[43] N. Manoj Kumar, S. Karthikeyan, G. Jayaraman, Biocatal. Agric Biotechnol. 2
(2013) 38–44.
[44] A. Mahdavi, R.H. Sajedi, M. Rassa, V. Jafarian, Iran J. Biotechnol. 8 (2) (2010)
103–111.
[45] R.V. Carvalho, T.L.R. Correa, J.C.M. Silva, R.C.O. Mansur, M.L.L. Martins, Braz. J.
Microbiol. 39 (2008) 102–107.
[46] A.S. Yandri, S. Tati, H. Sutopo, Eur. J. Sci. Res. 39 (1) (2010) 64–74.
[47] M.A. Amoozegara, F. Malekzadeha, K.A. Malik, J. Microbiol. Methods 52 (2003)
353–359.
[48] N. Goyal, J.K. Gupta, S.K. Soni, Enzyme Microbiol. Technol. 37 (2005) 723–734.
[49] B.A. Kikani, S.P. Singh, Int. J. Biol. Macromol. 48 (2011) 676–681.
[50] S.A. Hassan, S.A. Ali, A. Abbasi, M. Kamal, Afr. J. Biotechnol. 10 (2011) 6082–
6089.
[51] Y. Bai, H. Huang, K. Meng, Food Chem. 131 (2012) 1473–1478.
[52] Y. Lequette, G. Boelsb, M. Clarissea, C. Faille, Biofouling 26 (2010) 421–431.
[53] R.A. van Dam, A.J. Harford, M.A. Houston, A.C. Hogan, A.P. Negri, Aust. J.
Ecotoxicol. 14 (2008) 55–88.
[54] F. Gissi, M.T. Binet, M.S. Adams, Ecotoxicol. Environ. Saf. 97 (2013) 86–93.
[55] ISO, International Organization for Standardization, Geneva, Switzerland,
1992.
[56] USEPA, third ed. United States Environmental Protection Agency, Office of
Water, Washington, DC (EPA-821-R-02-014), 2002.
[57] ASTM, American Society for Testing Materials International, West
Conshohocken, PA, USA, 2004.
[58] OECD, Organization for Economic Cooperation and Development, Brussels
Belgium (79. ENV/JM/MONO), 2007.
[59] G.K. Bielmyer, M. Grosell, K.V. Brixti, Environ. Sci. Technol. 40 (6) (2006) 2063–
2068.