Volume 8, Issue 3, March – 2023 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Isolation, Identification and Characterization of

Amylase Producing Microorganism from Soil
Amrutha Philip1, Smitha Mathews2, Dona Thomas3 and Shradha Sanil4
1
Department of Zoology, Assumption College, Changanacherry, Kottayam, Kerala, India

Abstract:- The enzyme Amylase catalyses the hydrolysis economical bulk production capacity and the fact that
of starch into oligosaccharides and sugars. Bacteria microbes are easy to manipulate to obtain enzymes of
isolated from the soil are the most common sources of desired characteristics [[1.]]. Soil acts as a major source of
amylase. In this study, soil samples (n=30) collected amylolytic microorganisms. The above study was aimed at
from different sites of Changanacherry were taken for isolating extracellular amylase producing organisms from
screening amylase-producing bacteria. The samples soil that can be further employed for waste degradation as a
were serially diluted and representative bacterial flora monoculture or in a consortium.
was isolated by plating on the nutrient agar media. The
amylase production of these isolates was assessed using II. MATERIALS AND METHODS
starch agar. Thirteen isolates were selected for further
investigations. The growth conditions of these isolates A. Collection of samples
were optimized and they were analysed for antagonistic The samples were collected aseptically from
activity. Two strains were selected for assessing Changanacherry (9⁰26′47.4”N76⁰32′24.8”E). They were
antibacterial resistance patterns by the well diffusion carried to the lab in sterile polythene bags for further
method and to check the antibiotic sensitivity against 12 analysis and stored at 5ͦC.
antibiotics using the disk diffusion method. The
B. Screening and Isolation of Amylolytic Organisms
microbiological and biochemical characteristics of these
strains were also determined. They were tested for The samples were serially diluted upto the dilution of
10⁻6 and 0.1mL of last two dilutions were plated in nutrient
extracellular amylase activity at elevated temperatures
agar plates fortified with starch. These plates were
using the DNS assay method. The strain showing
incubated at 37⁰C for 24 hours. The plates were then
maximum amylase activity (2.395 units/ml) was
flooded with Lugol’sIodine . The colonies that have a clear
identified as Alcaligenes faecalis using 16S rDNA-based
zone around it against the dark background are selected.
method. The selected thermophilic strains can be
These colonies are then purified using quadrant streaking.
employed to degrade starchy waste materials in
The pure cultures were subcultured in Nutrient Agar slants;
monoculture or in a consortium, as well as for the
incubated at 28⁰C to achieve vigorous growth and then
industrial production of amylase. The study highlights
that there is an increased scope for identifying preserved in 20% glycerol vials at -20⁰C [[3.]].
industrially important bacteria from soil.
C. Determination of moisture content and pH of the soil
Keywords: Soil, Bacterial strain, Antagonism, The petri plates were weighed and their weights noted
Extracellular amylase, Isolates. down. 40g of soil was weighed and placed in the petri dish
and were reweighed. The soil was placed in the hot air
I. INTRODUCTION oven and dried overnight at 106℃. The samples were taken
out from the oven and allowed to cool. The plates were
With the increase in population during recent years, weighed again with the dry soil. The moisture content was
the problem of waste management has become an area of calculated using the following equation % moisture content
great concern. The eco-friendly management of the waste = weight of moist soil (M)-weight of dry soil (D) x 100
produced has become the major problem faced by Weight of dry soil (D)
environmentalists. Most of the waste produced is either
dumped in a landfill or subjected to incineration. The To test the pH of the soil samples. 5g of each soil
organic waste produced can be degraded using bacteria sample was taken into tube number 1 and transferred to
which help in the bioremediation of the organic waste. tube number 2. 2mL of pH reagent (pH-1) was added to it
Bioremediation is a process where biological organisms are and shaken for 5-10 minutes. One drop of decolouriser (D-
used to remove or neutralize an environmental pollutant by 1) was added and mixed well. The sample solution was
metabolic process. The process of bioremediation is filtered into bottle number 3 and 2 drops of pH reagent
catalysed by microbial enzymes. The microorganisms, (pH-2) were added and shaken for 1-2 minutes until the
using these enzymes, degrade the waste and use this energy colour developed. The colour was then compared with the
for their metabolic activities and growth. Amylase is a standard pH chart.
hydrolytic enzyme that degrades starch into simple sugars
and is mainly produced by bacteria that use starch as their
primary source of energy. Of the many uses of bacterial
amylase, it also can be used to degrade organic waste such
as kitchen and food waste. The major advantage of using
microorganisms for the production of amylases is the

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ISSN No:-2456-2165
punched on the surface of the agar using a sterile cork
D. Determination of physico chemical properties of soil borer. 10μl of the isolated sample was inoculated in the
The analysis of nitrogen, phosphorus and potassium well using a micropipette. The samples were incubated at
content was done.For nitrogen analysis 5g of each soil 37ºC for 24 hours and the result was obtained.
sample was taken in tube number 1 and transferred to tube Antimicrobial activity of the isolates were tested against six
number 2. 2mL of nitrogen reagent was added to it and pathogenic organisms namely Pseudomonas, Klebsiella,
shaken for 5-10 minutes. One drop of decolourizer (D-1) is Escherichia coli, Staphylococcus aureus, Vibrio cholera
added and mixed well. The sample solution was filtered to and Vibrio parahaemolyticus.
bottle 3 and two drops of nitrogen reagent (N2) was added
and shaken for 1-2 minutes until colour developed. The I. Microbiological and Biochemical Identification of the
colour developed was compared with the standard nitrogen Isolated Bacterial Strains
chart. The same was repeated for phosphorus and The bacterial colonies were checked for colony
potassium using their respective reagents. morphology and gram staining properties. Various
biochemical tests were also carried out to identify the
E. Test for Antagonistic Activity characteristics of the isolated strains. The biochemical tests
The antagonistic activity of the isolated strains was include Carbohydrate Fermentation Test for Glucose,
tested using cross streak. In this method, the strain to be Lactose and Sucrose, Indole test, MRVP Test, Citrate Test,
tested is streaked along the middle of the agar plate. The Urease Test, Catalase Test, Oxidase Test and TSIA Test.
indicator strain is streaked by a single streak perpendicular
to the test strain and incubated at 37˚C for 24 hours. All J. Enzyme Assay-DNSA Assay Method
isolates were tested against each other for antagonistic The enzyme production media was prepared and
activity. The strains that showed antagonism were autoclaved. The selected isolate was inoculated into
separated. fermentation media and incubated in a shaker incubator at
120 rpm, 37°C for 3-4 days. After the incubation, the
F. Optimization of growth conditions - pH and culture centrifuged at 12,000 rpm for 15 min to get cell free
Temperature culture filtrate. This is used as an enzyme to determine
The growth conditions of all the isolates were amylase activity. The amount of reducing sugar, produced
determined by growing them in different pH in different by the hydrolysis of starch by amylase, can be estimated by
temperatures. Nutrient broth of different pH ranging from DNS (3,5-dinitrosalicylic acid) method, thus can measure
2, 5.5, 7 and 9 were used. The isolate was inoculated into the activity of amylase. Two test tubes were taken, one as
nutrient broth of all pH. The cultures from each pH were test and other as control. The enzyme assay is performed in
incubated in five different temperatures ranging from 8ºC, tubes containing 0.5 ml phosphate buffer (100 mM. pH 7)
21ºC, 32ºC, 37ºC and 60ºC for 24 hours. The same and 0.5 ml substrate ( 1% starch). 0.5ml. of the enzyme is
procedure was followed for all the ten isolates. added to the reaction tube labelled test and incubated for 1
hour at 30ͦ C. The reaction is terminated at the end of
G. Antibiotic Susceptibility using Disc Diffusion Test incubation by adding 1 ml. of DNSA reagent to both test
The disc diffusion method for antibiotic susceptibility and control. To the tube labelled control 0.5 ml. of the
was done [[5.]]. The strains to be tested are plated on enzyme is added. Then the assay tubes were kept in a
Muller-Hinton Agar plate using the lawn culture method. boiling water bath for 10 minutes and 1 ml of distilled
The antibiotic discs of different antibiotics are placed at water was added to each tube. The absorbance of the red
different corners of the agar plates. These plates are coloured complex developed is recorded at 540 nm using
incubated at 35ºC for 24 hrs. The plates are studied for UV-Visible spectrophotometer. A standard curve is
inhibition zones and the zones are measured in nearest prepared using glucose standard solution (0.5 mg/mL).
millimetre to study the susceptibility of the organisms. The Enzyme unit (U) is defined as the amount of enzyme
susceptibility to the following antibiotics were checked - required to catalyse 1µmol of substrate per minute under
Gentamicin, Tetracycline, Penicillin, Norfloxacin, the assay conditions [[7.]].
Ceftriaxone, Ciprofloxacin, Co-trimoxazole, mezlocillin,
Amikacin, Carbenicillin, Tobramycin. The zones were Enzymatic activity u/ml/min = mg of glucose
observed after 24 hours of incubation. liberated × total assay volume × df ml of enzyme used ×
time of incubation (min)
The MAR Index for each isolate was calculated using
the formula: where:
df = dilution factor
MAR Index = Antibiotic Resistance Shown ml. enzyme = ml. of enzyme added
time of incubation: 30 min
Total number of antibiotics used
K. Identification
H. Test For Antimicrobial Activity Against Pathogenic
The strain which was found to have maximum amylase
Organisms
activity was identified using molecular analysis. Genomic
Well Diffusion Method was employed for the analysis
DNA was isolated from the samples provided using Sigma
of antimicrobial activity [[6.]]. The agar plates were Aldrich DNA extraction Kit. The quality was evaluated on
inoculated with the pathogenic strains using a sterile swab 0.8% Agarose Gel. The 16s gene fragment was amplified
on the entire agar surface. Holes of diameter 6-8mm were

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by polymerase chain reaction (PCR) from the above
isolated genomic DNA. The size and quality were III. RESULTS AND DISCUSSION
evaluated on 1.5% Agarose Gel. The PCR amplicon was
purified by column purification to remove contaminants. A. Isolation and characterization of Physico-Chemical
DNA sequencing of PCR amplicon was performed. The properties of Amylolytic Bacteria
sequences were then used for similarity searches using the A total of 8 soil samples were taken. These samples
Basic Local Alignment Search Tool for identifying the were serially diluted (till 10⁻⁶ dilution) and plated on starch
sample. agar plates. After an incubation of about 24- 48 hours, the
amylolytic (starch degrading) bacterial isolates were
identified using the appearance of a clear zone around the
streaked lines(Figure: 1).

Fig. 1: Growth of amylolytic bacteria on Starch Agar Media and presence of zone of clearance

The best isolate with highest amylase activity and Nitrogen was 151-200kg/acre, that of Phosphorus was
different colony morphology were selected (13 isolates >15kg/acre and Potassium had a concentration of 50-
viz.ST M₁, ST M₂, ST M₃, ST M₄, ST Ms, ST Ms₃, ST D₁, 80kg/acre. Soil pH and mineral Nitrogen availability appears
ST K₁, ST WRS₁, ST WRS₂, ST P, ST Vc & ST Mo). The to have a cooperative effect on bacterial abundance with soil
moisture content and pH of the soils from which the samples pH being the key influencing factor [[8.]].
where isolated was tested. The sample from which STM₃
was isolated had a moisture content of 9.001% and pH-6. A B. Antagonistic Activity of the Selected Strains
pH of 6 and moisture content of 9.46% was exhibited by the The first step in screening of isolates was testing for
soil from which STMS₃ was isolated. The other samples Antagonistic activity. Each isolate was tested (streaked on
have a varying pH from 4 to 9 and a moisture content of nutrient agar plates) with all of the other isolates to
4%.The physicochemical properties like the Nitrogen, determine antagonistic activity (Table 1). Out of the 13
Phosphorus, and Potassium content of the isolated soil isolates tested for the antagonistic activity ST M₄, ST WRS₁,
samples were analysed. The soil from which the isolate ST ST WRS₂ showed maximum antagonistic activity against the
M₃ was obtained, the concentration of Nitrogen was 151- various strains tested. These three isolates were separated
200kg/acre, that of Phosphorus was 8-10 kg/acre and and the remaining 10 isolates were subjected to further
Potassium had a concentration >150kg/acre. The soil from studies.
which the isolate ST MS₃ was obtained, the concentration of

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Sl.no Test Target Strain

Strains

S S S S S S S ST ST ST ST ST S
T T T T T T T K₁ W WR P Vc T
M M M M M M D RS₁ S₂ M
₁ ₂ ₃ ₄ s s₃ ₁ o

1 ST M₁ - - - - - - - - - - - - -

2 ST M₂ - - - - - - - - - - - - -

3 ST M₃ - - - - - - - - - - - - -

4 ST M₄ - - - - - - - + - - - - -

5 ST Ms - - - - - - - - - - - - -

6 ST Ms₃ - - - - - - - - - - - - -

7 ST D₁ - - - - - - - - - - - - -

8 ST K₁ - - - - - - - - - - - - -

9 ST WRS₁ - - - + - - - - - + - - -

10 ST WRS₂ - - - + - - - - + - - - -

11 ST P - - - - - - - - - - - - -

12 ST Vc - - - - - - - - - - - - -

13 ST Mo - - - - - - - - - - - - -
Table 1: The Antagonistic property of the isolated strains

C. Optimization of Growth Conditions

The remaining isolates viz; ST M₁, ST M₂, ST M₃, ST
Ms, ST Ms₃, ST D₁, ST K₁, ST P, ST Vc, ST Mo were then D. Antibiotic Susceptibility using Disk Diffusion Test
optimised under different growth conditions- pH and The antibiotic susceptibility test of the isolates ST Ms₃
temperature. All isolates are transferred to a media with and ST M₃ to various antibiotics viz; Gentamicin, Penicillin,
different pH and temperatures mentioned in the procedure. Streptomycin, Tetracycline, Norfloxacin, Ceftriaxone,
Out of 10 isolates 2 of them were able to grow at high Ciprofloxacin, Co-trimoxazole, Mezlocillin, Amikacin,
temperature (60℃). It is desirable that α-amylase should be Carbenicillin and Tobramycin were tested using agar disc
active at high temperature of gelatinization and liquefaction diffusion method. The strain ST M₃ and ST Ms₃ showed
to economise the process [[9.]]. Therefore, there has been a resistant to mezlocillin, amikacin, carbenicillin, tobramycin.
need for thermophilic and thermostable alpha amylase. In addition, ST M₃ was found to be resistant to co-
Thus, the strains ST Ms₃ and ST M₃ were selected for trimoxazole and ST Ms₃ to ceftriaxone.ST M₃ was sensitive
further studies. to Gentamicin, Penicillin, Streptomycin, Tetracycline,
Norfloxacin, Ceftriaxone, and Ciprofloxacin. ST Ms₃ was
found to be sensitive to Gentamicin, Penicillin,
Streptomycin, Tetracycline, Norfloxacin, Co-trimoxazole
and Ciprofloxacin (Table: 2 & Figure 2). The acquisition of
an antibiotic resistance genotype may actually increase the
fitness of certain bacteria in the absence of antibiotic

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selective pressure, possibly allowing rapid emergence and [[10.]]; [[11.]].
dissemination of antibiotic resistance on a worldwide scale

SR.NO ANTIBIOTIC SAMPLE

ST M₃ ST Ms₃

1. CO-TROMOXAZOLE RESISTANT SENSITIVE

2. MEZLOCILLIN RESISTANT RESISTANT

3. AMIKACIN RESISTANT RESISTANT

4. CARBENICILLIN RESISTANT RESISTANT

5. TOBRAMYCIN RESISTANT RESISTANT

6. CEFTRIAXONE SENSITIVE RESISTANT

7. CIPROFLOXACIN SENSITIVE SENSITIVE

8. GENTAMICIN SENSITIVE SENSITIVE

9. PENICILLINE SENSITIVE RESISTANT

10. STREPTOMYCIN SENSITIVE SENSITIVE

11. TETRACYCLINE SENSITIVE SENSITIVE

12. NORFLOXACIN SENSITIVE SENSITIVE

Table 2: Susceptibility of amylolytic bacteria to various antibiotics

Fig. 2: Antibiotic susceptibility of amylolytic bacteria

The MAR Index of STM3 was found to be 0.41 and E. Antimicrobial Activity using Well Diffusion test
that of STMs3 was found to be 0.5. The MAR index is an The antimicrobial activity of the two isolates to various
effective, valid and cost-effective method that is used in pathogenic strains such as Pseudomonas, Klebsiella,
source tracking of antibiotic resistant organisms Escherichia coli, Staphylococcus aureus, Vibrio cholera and
[[12.]].Bacteria having MAR index ≥ 0.2 originate from a Vibrio parahaemolyticus revealed the antimicrobial property
high-risk source of contamination where several antibiotics of sample ST M₃ towards Pseudomonas and ST M₃ & ST
are used.[[13.]][[13.]][[15.]]. Ms₃ towards S.aureus. The results are expressed as table in
Table: 3.

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SL. NO PATHOGENIC STRAIN ST M₃ STMs₃

1. Pseudomonas - +

2. Klebsiella - -

3. Escherichia. coli - -

4. Staphylococcus aureus + +

5. Vibrio cholera - -

6. Vibrio parahaemolyticus - -
Table 3: Test for Antimicrobial Activity Against Pathogenic Organisms

F. Biochemical Characteristics of Characteristics be a Gram-negative coccus while ST Ms₃ to be Gram-

Colony morphology of the selected strains indicated that negative rod-shaped bacteThe biochemical analyses of the
ST M₃ produced off-white, irregular, boil like, shrinked isolates were carried out and the results are summarized
colonies while ST Ms₃ produced whitish, feathery, thin, Table: 4.
flared, irregular edges. The Gram staining proved ST M₃ to

Sl. no sample Colour of colony Nature of colony Gram nature shape

1. ST M₃ Off -white Irregular, Boil like, Shrinked Gram negative Cocci

2. ST Ms₃ Whitish Feathery, thin flared, irregular edges Gram negative Rod
Table 4: Colony morphology and staining property

The strain ST M₃ was tested positive for Methyl red, the absence of carbohydrate fermentation. The strain was
citrate and oxidase test. It was also noted that the organism also tested positive for Voges Prausker test, citrate test,
produced an acid slant and acid butt indicating that it had the catalase production and oxidase production. ria. The results
capacity to ferment glucose. On The other hand, the strain are summarised in Table: 5.
ST Ms₃ showed an alkaline slant and alkaline butt indicating

SL. No BIOCHEMICAL TEST RESULTS

ST M₃ ST Ms₃

1. Carbohydrates Fermentation

a) Glucose - -

b) Fructose - -

c) Sucrose - -

2. Indole Production - -

3. MRVP Test

a) Methyl Red (MR) + -

b) Voges Proskauer (VP) - +

4. Citrate + +

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5. Urease - -

6. Catalase - +

7. Oxidase + +

8. TSIA Test A/A K/K

H2S
production
Table 5: Biochemical characteristics of the amylolytic bacteria

A/A : Acid slant Acid Butt K/K : Alkaline slant Alkaline Butt

G. Ezyme Assay Units/mL and enzyme activity of ST Ms₃ was 2.395

The quantitative assay of the enzyme production of each Units/mL. The quantitative assay helped in the identification
strain was carried out using the DNSA method. The of more efficient amylase producing arterial strain, which
absorbance of the colored compound was measured using a can be used for industrial purposes.
colorimeter. ST M₃ had an enzyme activity of 2.1726

SAMPLE ENZYME ACTIVITY (Units/mL)

ST M₃ 2.176

ST Ms₃ 2.395
Table 6: Enzyme activity of the isolates

Fig. 3: Absorbance of isolates at 600nm

H. Molecular Identification showed 99.77% identity with Alcaligenes faecalis (NCBI

The strain showing maximum amylase activity was Accession No: CO048039.1) with query coverage of 100%.
identified using molecular methods. Based on the BLAST The following is the BLAST sequence of the bacterial
analysis done for the sequencing data, the sample (STMs3) isolate (861bp).

ACCGCGTTAGCTGCGCTACTAAGGCCTAACGGACCCCAACAGCTAGTTGACATC
GTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTG
TCTGAGCGTCAGTATTATCCCAGGGGGCTGCCTTCGCCATCGGTATTCCTCCACA
TATCTACGCATTTCACTGCTACACGTGGAATTCTACCCCCCTCTGACATACTCTAG
CTCGGCAGTTAAAAATGCAGTTCCAAGGTTGAGCCCTGGGATTTCACATCTTTCT
TTCCGAACCGCCTACACACGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACC
CTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCTGCAGATA
CCGTCACCAGTATCCCGTATTAGGGGATACCTTTTCTTCTCTGCCAAAAGTACTTT
ACAACCCGAAGGCCTTCATCATACACGCGGGATGGCTGGATCAGGGTTTCCCCCA
TTGTCCAAAATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTC
CCAGTGTGGCTGGTCGTCCTCTCAAACCAGCTACGGATCGTTGCCTTGGTGAGCC
TTTACCCCACCAACTAGCTAATCCGATATCGGCCGCTCCAATAGTGAGAGGTCTT
GCGATCCCCCCCTTTCCCCCGTAGGGCGTATGCGGTATTAGCCACTCTTTCGAGT
AGTTATCCCCCGCTACTGGGCACGTTCCGATATATTACTCACCCGTCCGCCACTCGCCGCCAAGAGAGCAAGCTCTC
TCGCGCTGCCGTTCGACTTGCATGTGTAAAGCA TCCCGCTAGCGTTCAATCTGAGCCAGGATCAAAC

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Fig. 4: Blast result of STMs3

IV. CONCLUSIONS

The present study was conducted to isolate and ACKNOWLEDGMENTS

identify the potential microorganisms that produce
extracellular enzymes. Out of the 30 soil samples screened, We would like to express our gratitude to Research
13 isolates producing extracellular amylase were recognised. Promoting Council of Assumption College Autonomous,
Based on the physicochemical properties (pH, moisture, Changanacherry, Kottayam, Kerala, India for financially
nitrogen, phosphorus and potassium content), antagonistic supporting the project and the Department of Zoology,
activity, and antimicrobial activity, tolerance towards Assumption College, Changanacherry for providing access
different pH (2.2-9) and temperature (37℃-60℃) two to the Microbiology lab for the completion of the research.
strains were selected. They were identified using We are also grateful to Dr. Sudha K, Department of
morphological and biochemical and molecular Biotechnology, St. Peter’s College, Kolencherry for
characteristics. The strain ST Ms₃ showed maximum providing the necessary guidance throughout the research.
enzyme production. We also thank Ms. Sabina Sabu for her assistance in
completing the work.
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