International Journal of Advanced Research in ISSN: 2278-6252

Engineering and Applied Sciences Impact Factor: 7.388

EFFECT OF SUB MERGED FERMENTATION ON PRODUCTION OF AMYLASE

THROUGH FRUIT ISOLATES.
VINIT SINGH BAGHEL1, 1Government Nagarjuna Post Graduate College of Science Raipur,
MP, India
CHITRANSHU PANDEY2, R&D, MRD LifeSciences Pvt. Ltd. Lucknow, UP, India.
PALLAVI SHARMA3, R&D, MRD LifeSciences Pvt. Ltd. Lucknow, UP, India.

ABSTRACT:

The isolation of amylase producing bacteria from the soil and identifying the gene
responsible was done. The gene was then mutated for the enhanced production of
amylase; the study was carried out using starch (2%) as the substrate of enzyme.
The production of enzyme was carried out under submerged fermentation. The best
condition for the production of enzyme were, incubation temperature 37oC, pH 6,
incubation period of 72 hours, the starch as carbon source and the ammonium
sulfate as nitrogen source. The amylase was purified using ammonium sulfate (40%)
and dialysis. The refined amylase had a maximum activityat pH 6. The enzyme was
active between pH range of 6-8 and temperature range of 30oC to 40oC.

1. INTRODUCTION:
Amylase is an enzyme that breaks (cleaves) starch. Total of 19 enzymes have been ranked
that relate to the microbial amylase: hydro lases such as glucoamylase,α-amylase,β-
amylase,α-glucosidase,debranchingenzymesand transferases (EC 2) such as CGT ase, 4-α-
glucanotransferase. First, the enhancing of break down properties of each of the nineteen
enzymes were explained to express the discrimination among them and their microbial
sources. Industrial usages of amylase were seen with an attention on the function of
α-amylase and gluco-amylase in the starch saccharification industry. There have
been recent developments in trehalose production and other cyclic-glucans. Many enzymes
work on starch or on the oligosaccharides derived from them, and manipulate them into
maltose, a disaccharide and few monosaccharide like glucose. These disaccharides and
monosaccharides moves into cytoplasm of the bacterial cell through semi permeable cell

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

membrane and are then employed by endo-enzymes. Starch is a composite carbohydrate

made up of two constituents – amylose, a straight chain polymer of 200 – 300 glucose sub-
units, and amylopectin, a larger branched polymer with phosphate groups.

Amylase exist in the saliva of humans and few other mammals, where it begin the chemical
process of digestion. The foods like rice and potatoes that include the starch in higher
quantity in contrast to other sugars may tastes sweet when chewed because the amylase
present in the saliva will break the starch into simple sugars. The amylase in the body of
humans is secreted by pancreases and salivary glands and is ranked as alpha amylase. Alpha
amylase help in the breakdown of the dietary starch and convert the in to disaccharides and
trisaccharides, which are further hydrolyzed into simpler glucose such as glucose by other
enzymes. Amylase production is known in some bacteria while well known in fungi. Amylase
commercially produced from various aspergillusare used in the initial steps in the several
food fermentation process to convert starch into fermentable sugars. They are also used to
partially predigest foods for young children, to clarify fruit juices and in the manufacture of
corn and chocolate syrups.

2. MATERIALS AND METHODOLOGY:

2.1. Collection of sample:

The soil sample was collected from the areas near to the fruit stalls, which are enriched in
starch presence.

2.2. Isolation of amylase producing bacteria:

The sample was serially diluted in 0.85% sterilized saline and then spread over the sterilized
nutrient agar plates. Then the cultures were incubated at 37°C for 24 hours. Once the
growth observed then these cultures were selected on the basis of different morphological
parameters and streaked over sterilized nutrient agar plates. Further the screening of
amylase producing bacteria was performed by the hydrolysis of starch and iodine test.

2.3. Strain identification:

The identification of positive culture was done by using biochemical test based on Bergey’s
manual.

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

2.4. Strain improvement by mutation:

The physical and chemical mutation was given for improving the production of amylase. The
sources for physical and chemical mutations are UV rays and Ethidium bromide.

2.5. Selection and optimization of media:

The best media was selected on the basis of highest growth of culture. Further each
component of the media was optimized by applying one factor at a time method.

Table1: Optimized media for amylase production by OFAT methods.

S no. Factors Media composition Quantity
1 Production media 1 Peptone 6 g/l
MgSO4.7H2O 0.5 g/l
KCl 0.5 g/l
Starch 10 g/l
2. Production media 2 Starch 10 g/l
Nutrient broth 8 g/l
(NH4)2SO4 2.25 g/l
NaCl 0.85 g/l
MgSO4.7H2O 0.25 g/l
3 Production media 3 Starch 10 g/l
K2HPO4 9 g/l
KH2PO4 2 g/l
(NH4)2SO4 5 g/l
Sodium citrate 1 g/l
MgSO4.7H2O 0.2 g/l
FeSO4.7H2O 0.1 g/l
ZnSO4.7H2O 0.1 g/l
4 Production media 4 Yeast extract 5 g/l
Starch 10 g/l
MgSO4.7H2O 0.5 g/l
K2HPO4 1 g/l
5 Starch 0.5%, 1%, 2.5%, 2%
6 Nutrient broth 1%, 1.3%, 2%, 2.3%
7 (NH4)2SO4 0.1%, 0.2%, 1%, 1.2%
8 NaCl 0.1%, 0.8%, 1%, 1.8%
9 MgSO4.7H2O 0.02%, 0.1%, 1%, 1.25
10 pH 6,7,8,9

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

2.6. Bacterial growth curve:

The bacterial culture was inoculated in sterilized optimized media and incubated at 37ºC for
one week. Then OD was taken in spectrophotometer, at 620 nm, after 1 hour of time
interval.

2.7. Fermentation and downstream processing:

The fermentative media was prepared by using optimized components and then culture was
[Link] fermentation and shake flask method was used for the production of
amylase. Purification was carry out by using salt precipitation with 40% ammonium sulfate
and then dialysis for obtaining the partial pure enzyme.

2.8. Enzyme assay:

The estimation of enzyme was performed by using DNS method. 1 ml of perfectly diluted (in
acetic acid buffer solution; pH=5) enzyme sample are hatched to 15 min at T=37°C with 1 ml
of soluble solution of starch 1 % w/v. Following, the created quantity of reducing sugars
discharged from starch is determined with the help of DNS standard graph. In the way that a
unit of activity (unit, U) of the enzyme amylase, is promptly appointed, the amount of the
enzyme needed for the production of 1 μmole of maltose in 1 min, when the enzyme is
hatched along with the substrate at pH=5 and Τ=37 °C.
3. RESULTS:
3.1. Sample Collection:

Soil sample containing rotten fruits was collected from fruit stall, Gomti Nagar, Lucknow.
Sample was taken from 2-2.5 inches below ground level in sterile polybag and was brought
to laboratory. Sample was blackish brownish in color.

3.2. Isolation amylase producing bacteria:

The white colonies were observed representing the bacterial growth, which were then
streaked to sterilized nutrient agar media to make the pure culture. Further these cultures
was streaked over minimal salt agar media supplemented with 1% starch and then
incubated at 37ºC for 48 hours. Iodine flood was carrying out to visualize the zone of

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

hydrolysis. On the basis of the bacterial growth and enzyme production, primary and
secondary screening was performed.

a. Bacterial culture after serial dilution

b. Pure culture by streaking method

c. Screening for amylase producing bacteria.

Figure 1: isolation of bacteria from fruit sample by serial dilution, its purification and
screening for amylase enzyme production.

Table 2: Primary and Secondary screening for amylase producing bacteria

S no. Isolated cultures Primary screening Secondary screening
1 CPVS19001 + +
2 CPVS1900 2 + ++
3 CPVS1900 3 + +
4 CPVS1900 4 + +
5 CPVS19005 - -
6 CPVS1900 6 ++ +++

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

3.3. Strain identification:

Table 3: Biochemical test for strain (CPVS19006) identification.
S no. Isolated cultures Results
1 Gram’s staining Positive, Cocci
2 Endospore’s staining Negative
3 Catalase test Positive
4 Glucose fermentation test Positive
5 Mannitol test Positive
6 MR-VP test Negative

a. Gram’s staining b. Endospore staining c. Catalase test

d. Mannitol test e. MRVP test f. Glucose fermentation test

Figure 2: Results of biochemical tests

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 International Journal of Advanced Research in ISSN: 2278-6252
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3.4. Strain improvement:

a. Chemical mutation:

1
0.8
OD at 620 nm

0.6
0.4
0.2
0
1 µl 2 µl 3 µl 4 µl 5 µl
Ethidium bromide

Figure 3: effect of EtBr on isolated strain CPVS19006,Maximum growth of strain CPVS19006

was seen in 1µl EtBr
b. Physical mutation:

Control 2min 4min

6min 8min 10min

Figure 4: Effect of UV treatment

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

3.5. Screening of mutates strains:

a. Ethidium bromide mutant b. 10 min. UV mutant

Figure 5: Comparison between uv mutated (6min) and EtBr mutated (1µl) stain CPVS19006

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

3.6. Media selection and its optimization:

OD versus media components

pH 9
pH 8
pH 7
pH 6
MgSO4.7H2O 1.2%
MgSO4.7H2O 1%
MgSO4.7H2O 0.1%
MgSO4.7H2O 0.02%
NaCl 1.8%
NaCl 1%
NaCl 0.8%
media components

NaCl 0.1%
(NH4)2SO4 1.2%
(NH4)2SO4 1%
(NH4)2SO4 0.2%
(NH4)2SO4 0.1%
Nutrient broth2.3%
Nutrient broth2%
Nutrient broth1.3%
Nutrient broth1%
Starch 2.5%
Starch 2%
Starch 1.5%
Starch 1%
Production Media 4
Production Media 3
Production Media 2
Production Media 1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

OD at 620 nm

Figure 6: media optimization for production of amylase through uv mutated strain

CPVS19006

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Engineering and Applied Sciences Impact Factor: 7.388

3.6. Effect of temperature:

Table 4: Plates were incubated at different temperatures for overnight, Plate at 37o C shows
best result.
[Link]. Temperature Growth
1. 4ºC -
2. Room temperature +
3. 37 ºC +++
4. 50 ºC -

3.7. Bacterial growth curve:

OD versus hours
0.5
0.4
OD at 620 nm

0.3
0.2
0.1
0
1 2 3 4 5 6 7 8 9 1011121314151617181920212223
Hours

Figure 7: Growth curve of strain CPVS19006 detected by looking the OD with respect to
time.

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3.8. Bradford’s Standard:

OD versus concentration
y = 0.0842x - 0.096
1 R² = 0.9831
0.8
OD at 680 nm

0.6
0.4
0.2
0
-0.2 1 2 3 4 5 6 7 8 9 10 11
Concentration (mg/ml)

Figure 8: Standard graph between OD and known protein concentration

3.9. Fermentation and downstream processing:

3.9.1- Estimation of enzyme by Bradford’s method:

Table 5: Concentration of protein (Enzyme) after fermentation, detected by Bradford’s

method.

S. Sample OD at Concentrati
no. 680nm on (mg/ml)
1. Blank 0 0
2. Amylas 0.40 0.14
e

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3.11. DNS Standard:

y = 0.0256x - 0.0338
OD versus concentration R² = 0.9639
0.35
0.3
0.25
OD at 540 nm

0.2
0.15
0.1
0.05
0
-0.05 1 2 3 4 5 6 7 8 9 10 11
Concentration (mg /ml)

Figure 9: Standard graph between known concentration of maltose and OD at 540 nm.

3.12. Estimation of enzyme activity by DNS method:

Table 6: Activity of extracted Amylase enzyme.
S. no. Sample OD at Concentration Activity
680nm U/ML
1. Blank 0 0 00
2. Amylase 0.59 0.14 5.41 U/mL

4. DISCUSSION:
Microorganism was isolated from the soil containing rotting fruits by serial dilution
and agar plating methods KHAN J.A. 2015. Isolated bacteria were further purified
which were named as CPVS19006. The culture were grown on minimal agar media
(MAM) at pH 7 supplement with 1% starch. The culture grown on MAM were
flooded with iodine solution and zone of hydrolysis were obtained in the plate
showing starch hydrolysis method has been used earlier by Gupta, S.K., 2015 in
order to screen the microorganism for amylase production.

Morphological properties and the taxonomical characteristics of the isolated

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 International Journal of Advanced Research in ISSN: 2278-6252
Engineering and Applied Sciences Impact Factor: 7.388

bacteria were studied according to Bergey’s manual as earlier done by the Khan, et
al. 2015 and the isolated bacteria were identified as Staphylococcus aureus. Strain
improvement was done to develop high amylase producing bacteria by treatment
with uv and EtBr and media optimization and CPVS19006 was improved by UV
mutation. The bacterial strain was identified by the help of various physical
characteristics, staining and biochemical activities.

Production media for the amylase production was optimized according to the
isolated requirements. The optimized media contains 2% starch, Nutrient Broth
2.3%, AmmoniumSulfate1%,Sodium Chloride 0.8%andMagnesiumSulfate0.02%.The
selected PM applies for the production of amylase under submerged fermentation.
Partial purification of the crude amylase was done by ammonium Sulfate
precipitation and dialysis technique as used earlier by Yandriet al., 2010.

The enzyme activity was assayed by incubating the 1 ml of the enzyme with 1%
starch in the 100mM tris of the pH5 at 37oC for 15 min after the incubation the
reaction was stopped by adding 1 ml of DNS reagent and reducing sugar were
assayed using colorimeter. Protein concentration was measured by Bradford
method using BSA as done earlier by Khan, et al., 2015 and total protein content of
pure enzyme was 0.14mg/ml.

[Link]:

This research shows that the isolated and uv mutated strain CPVS19006 can be a
fine source for amylase production for commercial uses. The amylase produced
here was found to be steady in the pH range of 6 to 8 and temperature range of
30oC to 40oC. The efficiency of amylase produced here was similar to that of diverse
researchers. This study can prove to be a foundation to the future commercial
production of amylase from bacterial source. This study can help relieve the stress
over fungal source for the commercial amylase and enhance the production
through different optimization techniques at the same time.

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6. ACKNOWLEDGEMENT:
I wish to express my immense gratitude to Mr. Manoj Verma, Director, MRD LifeSciences
Pvt. Ltd. Lucknow. I am very grateful and my heartiest thanks to Ms. Shraddha Prakash
(Research Scientist), Mr. Raj Shekhar Mishra (Research Assistant) and Ms. Pragya Srivastava
(Research Assistant) MRDLS, Lucknow, for their kind support throughout the research work,
I am also thankful to the almighty without whose blessings nothing was possible.

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