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European Journal of Experimental Biology, 2014, 4(3):588-594

ISSN: 2248 9215

CODEN (USA): EJEBAU

Isolation and medium optimization for amylase producing bacterial strain

isolated from potato field of Bhatinda, Punjab, India
Tulika Mishra*, Sneh Ahluwalia and Mahavir Joshi
Department of Biotechnology, Chandigarh University, Gharuan, Mohali, Punjab India
_____________________________________________________________________________________________
ABSTRACT
Alpha amylases are one of the most important and widely used enzymes whose application spectrum has widen in
many sectors and so is its demand. In the present study the organism has been isolated from the soil sample of
potato field of Bhatinda, Punjab, India. Out of fifty seven strains twelve strains showed the amylolectic activity. The
strain showing the maximum zone of clearance was further subjected to morphological and biochemical
characterization, showing the presence of Baciillus sp. Isolated strain was than used further for medium
optimization for amylase production in shake flask culture. The maximum enzyme activity was observed after
incubation of 48hrs at 7.5 pH and 45C. The inoculum concentration was also optimized with maximum potency at
1.5%. Of the various carbon sources used corn flour maximally promoted the enzyme activity. Tryptone as organic
nitrogen source and ammonium nitrate as Inorganic nitrogen source enhanced the enzyme activity to 32.63 2.3
U/ml and 48.17 1.3 U/ml respectively. Other optimum parameters include potassium chloride as Chloride salt and
Manganese sulphate as sulphate source for amylase production. The purpose of present investigation was isolation,
Partial characterization and media optimization with regard to -amylase production.
Key words: Amylase production, Bacillus Species, Amylolectic activity, media optimization.
_____________________________________________________________________________________________
INTRODUCTION
Microorganisms are the most targeted and economical source of Industrial enzymes. For the higher yield of
desirable enzymes, isolation and characterization of right organism using cheap source of Carbon & Nitrogen is a
continuous and rigorous process. Due to the immense diversity and ease of production of enzymes by
microorganisms they are now replacing many conventional techniques. Amylases are starch degrading enzymes and
captured approx 25-30% of enzyme market [1]. Starch degrading amylases has many applications in various food,
fermentation, textile and paper industries [2]. Usage of microbial enzymes for industrial process has been increased
duer to their low cost, large productivity, chemical stability, environmental protection, plasticity and vast availability
[3].
Out of vast variety of organisms Bacillus species are well documented bacterial workhorses for industrial purposes
and for fine biochemicals. Due to the large quantity production of extracellular enzymes Bacillus strains are most
significant industrial enzymes producers[3,4,5]
Amylase enzymes hydrolyses -1,4, glycosidic linkage in polysaccharides including maltose, glucose and dextrin. It
randomly acts on -1,4, linkages in starches, glycogens and oligosaccharides generating groups. These are widely
distributed in plants, animals, and microbes. As amylases are widely used in various Industries and due to its
increasing demand there is enormous interest in finding new strains showing better economical aspects. Keeping
above in view the present study was designed to isolate and partially characterize amylase producing strain from the

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soil sample of potato field of Bhatinda district (Punjab, India). The efforts were also made to optimize the culture
conditions of isolated strain.
MATERIALS AND METHODS
Microrganism
Amylase producing strain was isolated from the soil samples collected from potato field of Dist Bhatinda (Punjab,
India) as described by [2] Mishra & Behera 2008. Briefly, 1 % soil solution was prepared in 2% of starch broth.
After 24 hrs fresh inoculum was taken for batch culture at various temperatures 25C, 37 C, 45 C & 50 C followed
by plating on starch agar medium for 7 days.
Starch broth media and starch agar media was supplemented with peptone 0.05%, KCl 0.01% (w/v), MgSO4.7H2O
0.05% (w/v), (NH4)2SO4 0.01% (w/v), NaH2PO4 0.01%, and 1% starch [6].Addition to this starch media consist of
0.9% of Agar. Various colonies were then streaked on starch agar plates for pure culture.
Amylolectic Activity
Amylolectic activity was determined as described by [7] Bertrand et al., 2004 with slight modification. Briefly,
plates with bacterial colonies were then flooded with Grams Iodine reagent (0.01 M I2-KI solution). Amylolectic
strains hydrolyze the starch present and showed the zone of clearance. While, the non amylolectic strain does not
hydrolyze the starch & hence no zone of clearance was observed.
Morphological & Biochemical Characterization
The strain showing the maximum zone of clearance was then subjected to Morphological & Biochemical
Characterization as described in Bergeys Manual of Determinative bacteriology [8]
Enzyme Production Media
The isolated strain showed the characteristics of the Bacillus sp. and was then inoculated in enzyme production
media (0.1% KH2PO4, 0.25% Na2HPO4, 0.1% NaCl, 0.2 % (NH4)2SO4, 0.005% MgSO4.7H2O, 0.005% CaCl2, 0.2%
tryptone and 1% soluble starch, pH 7.0) as described by [3] Deb et al., 2013 and incubated for 48 hr at 37C at
150rpm. After incubation the medium was centrifuged at 5000 rpm for 15-20 min at 4C. The supernatant was
collected and used to quantify enzyme activity. Enzyme activity was assessed by the standard method of [9]
Bernfeld 1995 using dinitrosalicylic acid (DNS) regent at 540 nm and soluble protein was assessed by standard
method of Lowry et al[10] .
To increase the enzyme production from isolated strain various parameters were optimized at shake flask level.
Process optimization for enzyme production
pH
The effect of pH on amylase production was assessed by culturing the bacterium in a production medium with
different pH range. The experiment was conducted at various pH ranging from 5 to 9.5 and incubated for 24 hrs,
37C, 150 rpm. Enzyme activity was assessed as described earlier.
Temperature
The effect of temperature on enzyme production was assessed by incubating the inoculated production media at
various temperatures ranging from 30C-50C, and enzyme activity was assessed after 48 hrs
Incubation period
Production media inoculated with selected isolated strain was incubated for 24, 48, 72 and 95 hrs at 37 C, 150 rpm.
The enzyme assay was carried out after every 24 hrs.
Inoculum Concentration
Enzyme production by selected microorganism was determined by adding inoculum at various concentrations
ranging from 1%-4% and incubated for 48hrs at 37 C, 150 rpm and enzyme activity was quantified at 540 nm.
Carbon sources
The effect of various carbon sources viz. Wheat bran, wheat flour, corn flour, rice bran, rice flour and soluble starch
at the concentration of 1% (w/v) on enzyme production was investigated using 1.5 % inoculum (w/v) in production
medium followed by incubation at 37 C, 150 rpm for 48 hrs. After 48hrs the enzyme activity was assessed at
540nm.

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Organic nitrogen source
Various nitrogen sources at concentration of 0.2% w/v was added to production medium inoculated with selected
strain and incubated at 37 C, 150 rpm for 48 hrs. Tryptone, gelatine, peptone, yeast extract, beef extract and casein
were added to production medium as a nitrogen source and subjected to quantify enzyme activity after 48 hrs at
540nm.
Inorganic nitrogen source
In addition to organic sources various inorganic sources at the concentration of 0.2 % w/v was also tested for
enhanced enzyme production. NH4H2PO4, KNO3, NH4Cl, NH4NO3, (NH4)2SO4 and urea was added as a Inorganic
nitrogen source in a production medium and incubated at 37 C, 150 rpm for 48 hrs. The enzyme activity of various
samples was tested at 540nm.
Chlorides
Enzyme production by the selected bacterium was assessed by inoculating bacterium in production medium
containing various chloride sources such as Potassium chloride, Sodium chloride, Magnesium chloride, Calcium
chloride, Barrium chloride and Ferric chloride. The enzyme activity was quantified after 48 hrs of incubation at 37
C and 150 rpm.
Sulphates
Various salts of sulphates (Ammonium sulphate, Manganese sulphate, Sodium sulphate, Potassium sulphate,
Ferrous sulphate, Magnesium sulphate) were added to production medium and incubated at 37 C, 150 rpm for 48
hrs. Thereafter the enzyme activity was measured at 540nm as described earlier.
All the experiments were done in triplicates and the results are expressed as Standard error mean (SEM).
RESULTS AND DISCUSSION
In the present study strain of Bacillus was isolated from the soil sample of potato field of Bhatinda district (Punjab,
India). Genus Bacillus has been reported for the production of various extracellular enzymes of which amylases are
of a particular interest due to their industrial importance. As the potato is a good source of starch so it was assumed
that we may be able to find more potential strain producing -amylase enzyme.
The amylolectic activity was done to assess the number of amylase producing bacteria. The amylolectic strain
hydrolyze the starch present in the medium and thus no blue colour formation is observed [11]. It was observed that
among the various colonies 12 1.2 were found to be amylase positive colony. The colony showing maximum zone
was then selected and was subjected to morphological & biochemical characterization. Our results showed the
colonies were found to be translucent & cremish in colour. Their shape appeared to be similarly like that of Bacilli
with positive motality. All other characters observed were also found to be that of Bacilli (Table 1).
The isolated strain showing maximum zone of clearance was than used further for extracellular amylase production
in shake flask culture using production medium. The enzyme activity after 48 hrs was found to be 29.76 0.68
U/ml. To enhance the enzyme production optimized culture conditions plays a very important role [12]
pH of the medium plays vital role in enzyme secretion. Earlier reports, showed that bacteria requires either acidic or
neutral ph for higher enzyme production [13,14]
Table1: Morphological and Biochemical Characterization of isolated strain
Test
Gram Staining
Spore Formation
Cell Shape
Motility
Citrate Utilization
Oxidase
Urea hydrolysis
Gelatin hydrolysis

Response of Strain
+
+
+
+
+
+
+

Test
Catalase test
Nitrate reduction
Starch hydrolysis
Indole
Anaerobic growth
Voges Proskauer
Methyl red

Response of Strain
+
+
+
+
-

Thus, enzyme activity was assessed from pH 5-9.5. Our results showed lower activity at acidic medium while the
activity was increased at neutral pH and again declined in more alkaline medium. The maximum activity 38.3
0.794 U/ml was observed at pH 7.5 (Figure 1). This might be due to the fact that at extreme conditions may be the
enzyme was inactive. Our results are in corroboration with [15] Nusrat and Rehman 2007.

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Euro. J. Exp. Bio., 2014, 4(3):588-594
_____________________________________________________________________________
45

60

40

50
Activity (U/ml)

Activity (U/ml)

35
30
25
20
15

30
20
10

10
5

0
5

5.5

6.5

7.5
pH

8.5

30

9.5

Figure 1: Effect of pH on Amylase activity

35

40

45
50
Temperature

55

60

65

Figure 2: Effect of temperature on Amylase activity

40

45

35

40
35
Activity (U/ml)

30
Activity (U/ml)

40

25
20
15

30
25
20
15

10

10

5
0

0
12

24

48

72

96

1.5

2.5

Inoculum Volume (% v/v)

Time (hrs)

Figure 3: Effect of incubation period on Amylase activity

Figure 4: Effect of Inoculum concentration on Amylase

activity

Temperature is another important factor that affects the microbial growth and thus the enzyme production.
Suitability of temperature for enzyme production varied for various organisms. There are many reports showing
wide range of temperature for amylase production by Bacillus sp. [16,17]. Our study showed that of the varied
temperature range (30C- 65C), the maximum enzyme activity was observed at 45C (Figure 2) Verma et al [11]
has reported maximum enzyme production at 40C from Bacillus
Our study revealed that enzyme production was increased with increase in fermentation period but after 48 hrs the
enzyme activity was found to be declined down (Figure 3). These findings are similar to the results of Nurullah,
2011[18] for Bacillus licheniformis; Riaz et al., 2003 [19] in case of Bacillus subtilis. This may be due to the
accumulation of other by products after certain time period of fermentation (Riaz et al., 2003)[19]. Inoculum
concentration is one of the important factors to be considered while optimizing the enzyme production [20]. In our
study 1.5% of inoculum concentration showed the maximum enzyme activity. It was observed that as the inoculum
concentration was increased above 1.5% the enzyme activity declined down from 39.83 1.72 to 33.64 2.28
U/ml (Figure 4).Which could be contributed to the fact that at higher inoculum concentration the growth of bacteria
is very fast & the nutrient available is insufficient to fulfil the requirement of culture and thus, resulting in lower
enzyme production. Our results are in corroboration with Riaz et al., 2003 [19].
In addition to above physical parameters, other parameters also affected the economical enzyme production.
Keeping economical source in view various natural carbon sources was considered as readily available raw material.
The starch has been reported as best carbon source for extra cellular enzyme production [21,22]. Our study showed
that of the used natural carbon sources in addition to starch to production media Corn flour gave the maximum
enzyme activity (Figure 5). Earlier, Nurullah Akcan, 2011 [18], also reported cornflour as best carbon source.

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50

Activity (U/ml)

45
40
35
30
25
20
15
10
5
0
Wheat
Bran

Wheat
Flour

Corn
Flour

Rice Bran Rice Flour Soluble

Starch

Carbon Source (1.5% w/v)

Figure 5: Effect of Various carbon sources on Amylase activity

40

60
50

30

Activity (U/ml)

Activity (U/ml)

35

25
20
15
10

40
30
20
10

Organic Nitrogen Source (0.2% w/v)

H4
)2
SO
4

O3

(N

NH

4N

4C
l
NH

Casein

O3

Beaf
Extract

KN

4
Yeast
Extract

2P
O

Peptone

4H

Gelatin

NH

Tryptone

Ur
ea

0
0

Inorganic Nitrogen Sources (0.2% w/v)

Figure 6: Effect of Various Organic Nitrogen sources on

Figure 7: Effect of Various Organic Nitrogen sources on

Amylase activity

Amylase activity

50

60

45

50

40

Activity (U/ml)

Activity (U/ml)

35
30
25
20
15
10

40
30
20
10

0
KCl

NaCl

MgCl2

CaCl2

BaCl2

FeCl2

NH4)2SO4 MnSO4

Chloride Salts

Na2SO4

K2SO4

FeSo4

MgSO4

Sulphate salts

Figure 8: Effect of Various Chloride salts on Amylase activity.

Figure 9: Effect of Various Sulphate salts on Amylase activity

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Different organic and inorganic nitrogen sources are equally important for synthesis of enzyme and microbial
growth [23]. Lower level or higher level of nitrogen is equally detrimental for microbial growth and inhibits the
enzyme production [24]. Our study showed the use of tryptone resulted in maximum enzyme activity, which has
also been reported earlier by Okalo et al., 1996 [25] as one of the best organic nitrogen source for maximum enzyme
production. Followed by this, casein, beef extract and yeast extract also showed interesting effects on amylase
production (Figure 6). Earlier, casein [26] beef extract [27] and yeast extract [28] reported these to be helpfull for
higher amylase production from Bacillus. Of the various inorganic nitrogen sources ammonium nitrate promoted
maximum microbial activity and thus, the enzyme activity was observed (Figure 7). Mahmood and Rahman 2008;
Coleman and Elliott 1962, [29,30] has also reported ammonium to be one of the best stimulators for Bacillus
subtilis.
Enzyme production is greatly affected by the various chloride or sulphate salts. Our results have shown that addition
of potassium chloride showed the maximum enzyme activity (Figure 8) and addition of Manganese sulphate has also
promoted enzyme activity (Figure 9). Similar results were also observed by Patel et al., 2005; Francis et al., 2003
[31, 32].
CONCLUSION
Amylases are one of the integral part of food, paper, pulp and textile industries. With increase in its application
spectrum, the demand of the enzyme has also been increased. In the present study, the cultural conditions of the
media have been optimized for the enzyme production from the soil isolate. Enzyme synthesis was affected by
various carbon sources where Corn flour gave the maximum activity. The inorganic nitrogen source promoted the
activity more than any other source. Addition to above, the activity was found to be maximum at pH 7.5, 45 C after
48 hrs of incubation with 1.5% inoculum concentration. Potassium chloride and Manganese sulphate also enhanced
the enzyme production. The strain with further identification (work is going on in our lab) can be use as a potential
producer of extracellular amylase which could find appropriate application in industry.
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