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10/28/2009
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Extraction of amylase from bacillus subtillis

Microbial cells produce a variety of enzymes. These enzymes are the biological catalysts for the biochemical reactions, leading to microbial growth and respiration, as well as to produce fermentation products. Enzymes are used for a variety of purposes. They are employed in three major fields.
They are:
• Laboratory
• Industrial
• Clinical
Amylase is an enzyme, which is produced by the microorganisms, which has many applications in today’s market. Amylase is required in digestion of carbohydrates into smaller units and eventually converting them into even smaller units such as glucose. It is also involved in inflammatory reaction, such as those caused by the release of Histamine and similar substances. A number of digestive enzymes including amylase are required to produce Fructose in large quantities. Many detergents also contain enzymes such as Amylase to remove stains.

[...] Our aim was to collect the sample rich in Bacillus sp., for this soil sample was collected from the basement of our lab. Sample was collected by using sterile gloves, scalpel and placed in sterile container. SERIAL DILUTION AGAR-PLATING METHOD After collecting the samples serial dilution was carried out to isolate bacteria from soil. For this 10ml sterile distilled water was taken in a test tube. To this 1gm of finely pulverized, air dried soil was added. The tube was vigorously vortexed for 3 minutes to obtain uniform suspension of microorganisms. [...]


[...] The slides were rinsed with distilled water and decolouriser was slowly applied drop by drop from one end of the smear for 1 min. The slides were washed with distilled water and counter stained with saffranin for 30 seconds. The slides were rinsed in distilled water, air-dried and observed under oil immersion (100x) objective of the microscope. Pure culture: After identification, Bacillus subtilis was sub cultured on starch agar medium to get a pure culture. Approximately 15ml of molten Starch Agar media was poured into sterilized Petri plates and allowed to solidify. [...]


[...] To this g of Ammonium Sulphate was added respectively). No precipitation was observed in case of submerged fermentation sample. Therefore 70% cut was given by adding extra 21.11 g of ammonium Sulphate. Ammonium Sulphate was added very slowly with continuous stirring of the solution on a magnetic stirrer in cold conditions. The solutions were centrifuged at 10000 rpm for 10 minutes at 40C. The pellet was collected and dissolved in 10 ml of 50 mM Tris HCl solution. This solution contains the enzymes precipitated by Ammonium Sulphate DIALYSIS: Requirements: Dialysis bag Water bath Distilled water Sodium bicarbonate solution Beaker Glass rod The dialysis bag was first processed, to activate it. [...]

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