Alpha Amylase Enzyme Production And Partial Purification Biology Essay

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ABSTRACT

The present work is aimed at investigation of enzyme media, in order to isolate Bacteria strains producing amylolytic enzymes, and to characterize the strain productivity and the enzyme produced, particularly their behavior toward temperature and pH. The aim is to reduce the cost of Alpha amylase enzyme.

INTRODUCTION

Enzymes can be defined a as soluble, colloidal, organic catalysts which are Produced by living cells but are Capable of acting independently of the cells. Most Enzymes are protein in nature and exhibit all properties of the proteins. They are water soluble, precipitated by the usual protein precipitated reagents like alcohol, ammonium sulfate and alkaloid reagent. They are non-dialyzable amphoteric, have isolectric and have a nitrogen content of above 16%. It is inactive 0oC & destroyed by moist heat at 1000C (13, 14, and 15).

Co-enzymes are heat stable, dialyzable, non protein organic molecules & the prosthetic groups of Enzyme. Many reaction of subtract are catalyzed by Enzyme only in the presence of a specific non-protein organic molecule called the Co-enzyme (15), e.g. ATP, NAD, NADP, FMN.

The Enzymes that occur in a number of different from & different Each other Chemically, immunological and Electrophoretically are called Isoenzymes are present in the serum and tissues of mammals, amphibians, birds, insects, Plants and unicellular Organism (15).

Starches degrading amylolytic Enzymes are of great significance in biotechnological applications ranging from food, fermentation, textile to paper industries. The amylases can be derived from several sources such as plants, animals and microbes. The microbial amylases meet industrial demands, a large number of them are available commercially, and, they have almost completely replaced chemical hydrolysis of starch processing industry. The major advantage of using microorganisms for production of amylases in economical bulk production capacity and microbes are also easy to manipulate to obtain enzymes of desired characteristics.

Alpha -Amylase has been derived from several fungi, yeasts, bacteria and actinomycete, however, enzymes from fungal and actinomycetes. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors.

One isolate produced extra cellular alpha-amylase which was confirmed by percentage blue value and be identification of the products obtained by starch hydrolysis (3). Amylases are enzymes that break down starch or glycogen. Amylases are produced by a variety of living organisms, ranging from bacteria to plants and humans. Bacteria and fungi secrete amylases to the outside of their cells to carry out extra cellular digestion (4). The genus Bacillus Produces a large range of extra cellular enzyme, of which amylases and is significant industrial importance (5).

The amylase exhibited activity at a wide pH range, with the optimum being pH 9, desirable characteristics which can lead to its application in detergents as additive and in textile desisting. The enzyme was also relatively thermo stable. Since this natural isolate produced very low concentration of amylase, attempts were made to increase the productivity by optimizing the nature and relative concentration of carbon and nitrogen source (1).

Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licnentiformis, Bacillus amyloliquifaciens and Aspergillus niger (4). One unit of activity was defined as the amount of enzyme that catalyzed the liberation of reducing sugar equivalent to 1 mmol of D-glucose per min under the assay conditions.

METHODOLOGY

The Soil contains a rich deposit of bacteria which produces amylases. Different soil sample were collected from different crops like cotton, corn, millet. Isolation of soil sweep of the debris or moist part of the soil used a hand trowel to collect a sample of the top soil into a Ziploc bag (4).

Area

Crop

Quantity

Madhya Pradesh

Cotton

100 mg

Maharashtra

Corn

100 mg

Gujarat

Millet

100 mg

Suspend about 1 gm soils (Cotton, corn, millet) in 10 ml sterile distilled water & mixed properly. Prepare the serial dilution for above standard media like 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6. Then three serial dilution 10-4, 10-5, 10-6 were prepared and prepared an Agar media. Then spread 0.1 ml from the dilution on the agar plates with a glass spreader. Spread 0.1 ml of the diluted samples on nutrient agar plates & incubated at 300C for 24 hours. After 24 hours starch producing colonies was have an area of clearing around them. Confirm by flooding plates with Gram’s iodine’s Parts of the plant still containing starch will stain ink-black. Then Clear zone determined the two plates Cotton (10-6) and Corn (10-5) (4, 5).

Components

Quantity

Soluble starch

1 gm

Beef extract

1gm

Peptone

1 gm

Sodium Chloride

0.5 gm

Agar

2-3 gm

Distilled water

100 ml

Sub culturing of screen out Bacterial species:

Prepare a slant Culture with the help of Agar Media. In this slant Culture spread above bacterial species under the Petri dish with the help of wire loops. The isolates were maintained on nutrients agar the slant at 400C in incubator for three days and after this slants at 4oC in the refrigerator for use in the present investigation (6, 10).

Components

Quantity

Soluble starch

1 gm

Beef extract

1gm

Peptone

1 gm

Sodium Chloride

0.5 gm

Agar

2-3 gm

Distilled water

100 ml

Characterization of Bacterial Species:

Methyl red test (MR):

This test was employed to detect the production of acid during the fermentation of glucose and the maintenance of a pH below 4.5 in an old culture five drops of 0.04% Solution of methyl- red are added to the culture in glucose phosphate medium which had been incubated at 300C for five days, mixed well and read at once. Read color is positive while yellow signifies a negative test (12).

Gram Staining: - These stains impart different colors to different bacterial structure. Primary starting with a pararosaniline dye such as crystal violet. Apply dilute solution of iodine and decolorize with ethanol and acetone. Counter staining with a neutral red dye (12).

Hanging Drop techniques: A wet mount is made by placing a drop of fluid containing the organism into a glass slide & Covering the drop with a cover slip. To reduce the rate of evaporation and Exclude the effect of air current. The drop may few ringed with petroleum jelly and circular concave depreation is sometimes used for examination of wet preparation and determined the moving of bacteria (11).

Starch hydrolysis test: - In this test perform only absence or presence of starch in the medium by using iodine solution as an indicator. Starch in the presence of iodine produces a dark blue color of the medium and yellow zone around a colony in otherwise blue medium indicates amylolytic activity (12, 15).

Production of alpha amylase Enzyme: - Grow bacteria in nutrient Agar slants. Add a loopful of bacterial culture into the amylase production.

Ingredient

Quantity (gm)

Bacteriological Peptone

6

MgSO47H2O

0.5

Potassium- chloride

0.5

Starch

1

Distilled Water

100 ml

Mix and autoclave at 121oC for 15 minutes. The organism was propagated at 42oC for 3 days in 100 ml of medium with shaking on a shaker 120 rpm. Collected the supernatant of the culture after centrifugation at 5000 rpm for 30 min. and 4oC was used to for further study and obtained the crude enzyme extract. (4, 5)

Partial purification of alpha amylase Enzyme:

The supernatant was brought to 89% ammonium sulfate saturation by adding solid ammonium sulfate to an ice bath and stirred by the mechanical stirrer for 30 min. After the supernatant was left standing over night at 4oC. Then the

precipitate was collected by centrifuging 15,000 rpm for 30 min. and dissolved in 10 mm. Sodium- Phosphate buffer solution (pH-8.0). The Enzyme solution was dialyzed at 4OC against the same buffer for 1 day (9).

Enzyme assay: -Saccharolytic activity was determined. The reaction mixture contained 1 ml of substrate solution [2% soluble starch in 40 mm potassium phosphate buffer (pH6) including 1 mm CaCl2] and 1 ml of the enzyme solution. After 10 min of incubation at 700C, the reaction was stopped by the addition of 2 ml of di-nitro-salicylic-acid solution. The mixture was heated at 100oC for 5 min and measured at 540 nm. The enzyme activities were calculated using a calibration curve prepared with D-glucose as standard by following the same procedure above. One unit of activity was defined as the amount of enzyme that catalyzed the liberation of reducing sugar equivalent to 1 mmol of D-glucose per min under the assay conditions. (5)

Protein Assay: The protein concentrations determined using bovine serum albumin as standard (5).

Amylase assay: Amylase was assayed be adding 0.2 ml of enzyme to 0.5 ml of 1% soluble starch and incubated for 30 min at 370C. The reaction was stopped by adding 1 ml of 3.5 dinitrosalicylic acid, followed by boiling for 10 min and to develop brown color. The final volume was made to 5 ml with distilled water and the absorbency measured at 540 nm with a spectrophotometer. A calibration curve of absorbency and concentration of maltose was established with known amount of maltose.

RESULT AND DISCUSSION

Different bacterial strain were recovered from the soil sample collected from the regions root of cotton tree of Madhya Pradesh and root of corn tree of Maharashtra and root of millet tree of Gujarat. Bacterial strain was producing alpha amylase enzyme was identified starch hydrolysis surrounding the bacterial colonies.

Bacterial strain

Starch hydrolysis

C-1

NOT

C-2

NOT

C-3

MORE CLEAR

CR-1

NOT

CR-2

CLEAR

CR-3

NOT

Characterization of isolated bacterial strain:

Bacterial Strain

Gram Staining

Methyl-red test

Hanging Drop Test

Starch hydrolysis

VP test

C-1

+

-

+

-

+

C-2

+

-

+

-

+

C-3

+

+

+

+

+

CR-1

+

+

+

-

-

CR-2

-

+

-

+

-

CR-3

-

-

-

-

+

Production of alpha amylase enzyme

Only C-3 bacterial strain produced bacterial growth. Collection of supernatant of the culture & after centrifugation at 5000 rpm for 30 min. and 4OC enzyme obtained & stored.

Partial purification of enzyme: The supernatant was brought 80% ammonium sulfate stirred for 15,000 rpm for 30 min. with the help of centrifugation and enzyme was collected and stored on 4OC.

Enzyme assay:

Components

S-1

S-2

S-3

S-4

S-5

Soluble Starch

2%

2%

2%

2%

2%

Potassium phosphate Buffer

40mm

40mm

40mm

40mm

40mm

CaCl2

1 mm

1mm

1mm

1mm

1mm

Enzyme solution

1ml

1ml

1ml

1ml

1ml

Dinitro salicyclic Acid

2ml

2ml

2ml

2ml

2ml

D-Glucose

1%

1%

1%

1%

1%

Absorbance at 520nm

+0.591

+0.591

+0.591

+0.591

+0.591

CONCLUSION

The present work was aimed at investigation; these media in order to isolated bacteria strains Producing amylolytic Enzyme and to characterize the strain productivity and the enzyme produced particularly their behavior toward temperature and pH; the active principle which is responsible for those activity has to be established in future.

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