ISOLATION, PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM AIRBORNE-BACTERIA

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Shivaji Bole et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION, PURIFICATION AND CHARACTERIZATION OF

AMYLASE FROM AIRBORNE-BACTERIA

Shivaji Bole1*, Anindita Maji1, Ankita Dey1, Ashutosh Acharya1, Siddharth Dubey1,

Rakeshlal1

Dept. of Biotechnology, The Oxford College of Science, 19th Main, 17th B cross, HSR Layout,

Bangalore.

ABSTRACT

Among different types of enzymes obtained from microbial sources,

amylases are the most widely used in industries. In the present study,

bacteria were isolated from air exposure and screened for the

production of amylase. Among four bacterial isolates, one isolate

produced maximum zone of starch hydrolysis. The bacterial isolate

was identified as Bacillus sp. and was later used for further

characterization. Maximum yield of amylase was obtained after 48hrs

of incubation. The optimum pH for enzyme activity was found to be at

pH 6.8 and the optimum temperature for the activity was found to be at

37 ºC.

Keywords: Amylase, Starch Hydrolysis, Cultural characterization,

Amylase activity, pH, Temperature.

INTRODUCTION

Microorganisms are the most important sources for enzyme production. Selection of the right

organism plays a key role in high yield of desirable enzymes. For production of enzymes for

industrial use, isolation and characterization of new promising strains using cheap carbon and

nitrogen source is a continuous process. Microorganisms have become increasingly important

as producer of industrial enzymes. Due to their biochemical diversity and the ease with which

enzyme concentrations may be increased by environmental and genetic manipulation,

attempts are now being made to replace enzymes, which traditionally have been isolated from

complex eukaryotes. Starch degrading amylase enzymes are most important in the

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Article Received on 28 August 2013, Revised on 19 Sept 2013, Accepted on 22 November 2013

*Correspondence for

Author:

*Shivaji B Bole

Associate Professor,

PG Dept of Biotechnology,

The Oxford College of Science,

Bangalore. India.

[email protected]

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biotechnology industries with huge application in food, fermentation, textile and paper.

Amylases are obtained from various origins like plant, animal, bacterial and fungal.

Amylases, glycoside hydrolases, break starch into glucose, maltose, maltotriose and dextrin

by hydrolysis of glycosidic bonds. Therefore they are also called digestive enzymes. The first

enzyme produced industrially was an amylase from a fungal source in 1894, which was used

as a pharmaceutical aid for the treatment of digestive disorders [1]. There are about 3000

enzymes known today only few are industrially exploited. These are mainly extracellular

hydrolytic enzymes, which degrade naturally occurring polymers such as starch, proteins,

pectin’s and cellulose.

In the production of glucose syrup the α-amylase is used in the first step of enzymatic

degradation yielding a mixture of glucose and fructose with high fructose content. The

amylases can be derived from several sources such as plants, animals and microbes [2, 3].

The microbial amylases meet industrial demands because it is economical when produced in

large quantities. Amylase has been derived from several fungi, yeasts, bacteria and

actinomycetes, however, enzymes from fungal and bacterial sources have dominated

applications in industrial sectors.

The microbial source of amylase is preferred to other sources because of its plasticity and

vast availability. Microbial amylase has almost surpassed the synthetic sources in different

industries. Amylolytic enzymes are widely distributed in bacteria and fungi [4].

In the present study, we report the isolation and optimization of novel amylase producing

bacteria from the soil samples collected from different field conditions. Production conditions

were optimized (temperature, pH, metal ions etc.) to achieve high enzyme production and

better enzyme activity.

MATERIALS & METHODS

Isolation of Amylase Producing Microorganisms

Soil samples were collected from different environment sources. Serial dilution was made by

One gram of soil sample was serially diluted in sterilized distilled water to get a

concentration range from 10-1 to 10-6 and volume of 0.1 ml of each dilution was transferred

aseptically to starch agar plates. The sample was spread uniformly. The plates were incubated

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at 37°C for 24 hrs. The bacterial isolates were further sub cultured to obtain pure culture.

Pure isolates on starch agar slants were maintained at 4ºC.

Screening of potent amylase producing bacteria by starch hydrolysis test

Bacterial isolates were screened for amylolytic activity by starch hydrolysis test on starch

agar plate. The microbial isolates were streaked on the starch agar plate and incubated at

37°C for 48 hrs. After incubation iodine solution was flooded with dropper for 30 seconds on

the starch agar plate. Presence of blue colour around the growth indicates negative result and

a clear zone of hydrolysis around the growth indicates positive result. The isolates produced

clear zones of hydrolysis were considered as amylase producers and were further

investigated.

Morphological and Biochemical Characteristics

Gram staining, motility, indole production, methyl red, Vogues Proskauer's, citrate

utilization, triple sugar iron, nitrate reduction, catalase, oxidase, gelatin liquefication, urease,

hydrolysis of casein, hydrolysis of starch were carried out.

Biochemical Characterization Result Gram staining positive Indole production negative Methyl red negative Vogues proskauer positive Citrate utilization positive Triple sugar ion negative Nitrate reduction negative Catalase test positive Oxidase test positive Gelatin liquefication positive Urease test positive Hydrolysis of casein positive Hydrolysis of starch positive

Enzyme production media

Production medium contained (g/l) Trypticase 10gm, peptone 5gm, (NH4)2SO4 3gm, K2HPO4

2gm, L-Cysteine HCl 0.5gm, MgSO4 0.2gm.10 ml of medium was taken in a 100 ml conical

flask. The flasks were sterilized in autoclave at 1210 C for 15 min and after cooling the flask

was inoculated with overnight grown bacterial culture. The inoculated medium was incubated

at 37oC in shaker incubator for 24 hr. At the end of the fermentation period, the culture

medium was centrifuged at 5000 rpm for 15 min to obtain the crude extract, which served as

enzyme source.

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PURIFICATION OF ENZYME

The crude enxyme was purified using organic solvent and ammonium sulphate precipitation

method.

Acetone method

The crude extract is treated with different concentration of acetone. In this 30% and 50%

acetone was used for purification. Acetone is slowly added to the extract to precipitate out

the enzyme. This is done on an ice bath and kept for 1hr under continuous stirring. The

mixture is the centrifuged at 3000 rpm for 10 min. The supernatant and pellet are separated

and checked for enzyme activity.

Ammonium sulphate precipitation

The crude extract is treated with different concentration of Ammonium sulphate. In this 30%

and 50% Ammonium sulphate was used for purification. Ammonium sulphate is slowly

added to the extract to precipitate out the enzyme. This is done on an ice bath and kept for 1hr

under continuous stirring. The mixture is the centrifuged at 3000 rpm for 10mins. The

supernatant and pellet are separated and checked for enzyme activity.

CHARACTERIZATION OF AMYLASE

Standard curve for Maltose

A standard maltose curve is prepared using standard as 1mg/ml. Different aliquots of

standard maltose solution is taken and made up to mark by adding distill H2O. Then add 1ml

of D.N.S reagent to each tube and incubated it in boiling water bath for 15 min. After cooling

8 ml of distilled water was added and mix it thoroughly and read the absorbance at 540 nm in

a UV spectrophotometer.

Observation table

Test tube

Std Maltose (ml)

H2O (ml)

Conc. Of std maltose(µg /ml)

DNS (ml) Incubate H2O

(ml) OD at

540 nm 1. 0.0 1.0 0 1.0 in boiling 8.0 0.0 2. 0.2 0.8 200 1.0 water 8.0 0.07 3. 0.4 0.6 400 1.0 bath for 8.0 0.14 4. 0.6 0.4 600 1.0 15min 8.0 0.21 5. 0.8 0.2 800 1.0 8.0 0.28 6. 1.0 0.0 1000 1.0 8.0 0.35

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Enzyme activity

Enzyme activity is determined by DNS method. 0.5ml of isolated enzyme is incubating for 10

min at 370 C with 0.5 ml of substrate (ET) & 0.5 ml of substrate for (EC). After 10 min arrest

the reaction by adding 1 ml of DNS to both the tube followed by 0.5 ml of enzyme to control

tube only. Then kept the tubes in boiling water bath for 15 min. The solution is then dilute

with 8 ml of distilled water & read the absorbance at 540 nm.

Test tube Enzyme Substrate Incubation DNS

(ml) Enzyme Incubation for 10 min

H2O (ml)

OD at 540 nm

EC ------ 0.5 for 10 min 1.0 0.5 at boiling 8.0 0.0 ET 0.5 0.5 at RT 1.0 ----- Water bath 8.0 0.10

Specific activity of enzyme

The amount of protein content in the culture filtrate was assayed by Lowry method. The

enzyme is incubated along with buffer, with 5ml of freshly prepared alkaline solution and

incubated for 10min at room temperature.0.6ml of FC reagent and incubated for 30 minutes

and the absorbance is read at 660nm. The protein content is estimated using calibration curve

of standard BSA protein. Then the specificity of enzyme is calculated by the amount of

protein in mg present per ml of sample.

Observation table

Sl.no.

Std BSA soln.(ml) H2O(ml) Conc.

BSA (µg) Alkaline Cu reagent(ml) F.C.

reagent(ml) O.D at 660nm

1 0.0 1.0 0 5 Incubation 0.6 Incubation 0.0 2 0.2 0.8 40 5 At 0.6 At 0.06 3 0.4 0.6 80 5 Room 0.6 30mins 0.12 4 0.6 0.4 120 5 temperature 0.6 At 0.18 5 0.8 0.2 160 5 For 0.6 Room 0.24 6 1.0 0.0 200 5 10mins 0.6 temperature 0.30

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Specific activity of amylase

Sl.no. Enzyme

H2O (ml)

Alkaline cu++ reagent (ml)

Incubation

F.C. reagent (ml) incubation O.D at

660nm Blank

0.0 0.5 5.0 At RT for 0.6 At RT for 0.0

Direct 0.5 0.5 5.0 10mins 0.6 30mins 0.29 CHARACTERIZATION STUDY OF AMYLASE ACTIVITY BY DIFFERENT

Effect of substrate concentration

Different concentration of substrate (1%, 2% and 3% starch) added to 0.5 ml of enzyme, all

the tubes were incubated at 370C for 5 min. Then 1ml of DNS was added and determines

optical activity at 540 nm.

Test tube

Substrate (ml)

Enzyme (ml)

Concentration of starch (mg/ml)

Incubation DNS (ml)

Enzyme (ml) Boil H2O OD at

540 nm

1% starch

Control 0.5 ---- 5 mg/ml 1.0 0.5 8.0 0.02 Test 0.5 0.5 1.0 ---- 8.0 0.10 2%

starch

Control 0.5 ---- 10 mg/ml 1.0 0.5 8.0 0.20 Test 0.5 0.5 1.0 ---- 8.0 0.24 3%

starch

Control 0.5 ---- 15 mg/ml 1.0 0.5 8.0 0.26 Test 0.5 0.5 1.0 ---- 8.0 0.28

Determination of optimum pH 1% Starch was used as a substrate. Substrate solution was prepared in sodium phosphate

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buffer at pH 6.2, 6.4, 6.6, 6.8, 7.0 & 7.2 in different test tubes. 1 ml each of crude enzyme

solution was added into buffer tubes. Then the mixture was incubate at 35°C for 10 min,

reactions were terminated by adding 1 ml DNS reagent and the mixture was incubated in

boiling water for 10 min. After cooling at room temperature, final volume was made to 8ml

with distilled water and the activity of enzymes was determined by taking the absorbance at

540nm.

Observation table

Test tube pH Substrate

(ml) Enzyme

(ml) Incubation DNS (ml)

Enzyme (ml) incubation H2O

(ml) OD at

540 nm

Activity (micro

mole/ml/min) EC 5.8 0.5 for 1.0 0.5 for 8.0 0.0 ET 0.5 0.5 1.0 8.0 0.04 0.088

15 min 15 min EC 6.2 0.5 1.0 0.5 8.0 0.0 ET 0.5 0.5 at 1.0 at 8.0 0.08 0.177

EC 6.6 0.5 room 1.0 0.5 room 8.0 0.0 ET 0.5 0.5 1.0 8.0 0.13 0.288

temp - temp- EC 6.8 0.5 1.0 0.5 8.0 0.0 ET 0.5 0.5 erature 1.0 erature 8.0 0.24 0.533

EC 7.2 0.5 1.0 0.5 8.0 0.0 ET 0.5 0.5 1.0 8.0 0.10 0.222

EC 7.4 0.5 1.0 0.5 8.0 0.0 ET 0.5 0.5 1.0 8.0 0.06 0.133

EC 7.6 0.5 1.0 0.5 8.0 0.0 ET 0.5 0.5 1.0 8.0 0.02 0.044

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EFFECT OF TEMPERATURE

1 ml of substrate was taken into six different test tubes and 1 ml of phosphate buffer pH 7

was added in each test tubes. Tubes were marked with different temperature (at 10, 20, 30,

50, 60°C). 1 ml of crude enzyme solution was added in each tube. Then tubes were incubated

at specific temperature for 10 minutes. Reactions were terminated by adding 1 ml DNS

reagent and the mixture incubated in boiling water for 10 min. After cooling at room

temperature, final volume was made to 8 ml with distilled water and the activity of enzymes

were determined by taking the absorbance at 540nm.

Test tube

Substrate (ml)

Enzyme (ml)

D.N.S (ml)

Enzyme (ml)

H2O (ml)

O.D. at 540nm

Activity (µmol/min/ml)

100C I I Control 0.5 - N 1.0 0.5 N 8.0 0.01 Test 0.5 0.5 Cu 1.0 - Cu 8.0 0.03 0.066 200C Ba Ti Control 0.5 - Ti 1.0 0.5 On 8.0 0.01 Test 0.5 0.5 On 1.0 - At 8.0 0.06 0.133 300C At bio Control 0.5 - R 1.0 0.5 Ling 8.0 0.01 Test 0.5 0.5 O 1.0 - w 8.0 0.1 0.222 400C O A Control 0.5 - M 1.0 0.5 Ter 8.0 0.01 Test 0.5 0.5 Tem 1.0 - Ba 8.0 0.08 0.177 500C Pera T Control 0.5 - Ture 1.0 0.5 h 8.0 0.01 Test 0.5 0.5 F 1.0 - F 8.0 0.06 0.133 600C O O Control 0.5 - R 1.0 0.5 R 8.0 0.01 Test 0.5 0.5 10

mins 1.0 - 15mins 8.0 0.04 0.088

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RESULT & DISCUSSION

Bacillus sp. Isolated from soil was found as an effective producer of amylases evident from

the findings. The enzyme together helps in the stain removal having varied applications in

detergent industries. A bacterial isolates were obtained from soil samples. They were initially

screened for amylase production based on zone of clearance on starch agar plates.

While characterizing the bacteria based on Bergey’s manual of systemic bacteriology, the

isolated strain was found to be gram positive, rod shaped, spore forming, and mobile

bacterium. The effect of the initial pH of the production medium was tested at varying pH 5–

11. Considerably less production of enzymes was obtained at acidic pH and comparably

increased enzymes production was obtained at weak alkaline pH. The maximum production

of protease was obtained at pH of 9.0. At pH of 7.0, high amylase activity was obtained. The

results indicated that the isolated Bacillus sp. was a alkaline organism. The weak alkaline

conditions were found to be optimum for the production of both the enzymes.

The culture conditions and media components were optimized for better production of both

the enzymes. It is important to produce both the enzymes together as they have combined

application in detergent formulation. The production medium was found to be cost effective,

convenient and easier to scale up than the conventional method of blending enzymes. Further

experiments will be carried out to obtain high yield of amylase and protease.

ASSAY OF AMYLASE ENZYME

Enzyme activity of crude enzyme was performed by using DNS reagent. And the enzyme

activity observed for this strain was found to be 9 U/ml. Among physical parameters, pH of

the growth medium plays an important role by inducing morphological changes in microbes

and inenzyme secretion. The pH change observed during the growth of microbes also

affected product stability in the medium. As shown in table 2 the isolate was able to grow in

the pH range of 5–8, but pH 7.0 was the optimum for the growth of the cultures. Temperature

also plays the significant role in the stability in enzyme activity. 35°C was found to be

optimum temperature at which enzyme activity was found to be higher.

ACKNOWLEDGMENT

We are sincerely grateful to the oxford college of Science, Bangalore, Karnataka, India, for

allowing us to use all facilities for our work, and their encouragement and support.

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ISOLATION, PURIFICATION AND CHARACTERIZATION OF AMYLASE FROM AIRBORNE-BACTERIA

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