Isolation and screening of amylase producing microorganisms from soil

 Aim

To isolate bacteria producing amylase, capable of degrading the polysaccharide starch from the soil

Principle

Polysaccharides are large molecular weight polymers of monosaccharide units joined by glycosidic bonds. Polysaccharides can be structural and nutritional in nature. Nutritional polysaccharides are formed by both plant and animal cells and they are used as reserve food supplies. Glycogen is the reserve polysaccharide of animal cells and starch serves a similar function in plant cells.

Starch is a high molecular weight polysaccharide containing single monomeric units of glucose. Starch contains two forms of glucose polymers- amylose and amylopectin. Amylose consists of long unbranched chains of D-glucose units connected by α-1,4 glycosidic linkages. Amylopectin is branched with one branch for every thirty glucose units. The monomeric glucose units are linked by α-1,4 glycosidic linkages in the unbranched region and α-1,6 glycosidic linkages in the branched regions in amylopectin.

Starch is not capable of permeating the cell membrane because of its high molecular weight. To assimilate starch for energy and catabolic reactions, it must be degraded into basic glucose units by starch hydrolyzing enzyme, amylase. Starch is converted to dextrins and then maltose, which is broken down to soluble glucose molecules that can be transported into the cell for energy production, through glycolysis. Since enzymes are catalysed and are not consumed in chemical reactions, a few molecules of enzymes are used over and over to break down a great deal of starch.

Amylases are commercially used in bread making, brewing, for production of sugar syrups, for mash preparations, in spot removers, for the removal of silver from used photographic films, in instant hot breakfast cereals and also in dry cleaning process.

Amylases give a deep blue colour and amylopectin gives a red to brown colour when they react with iodine depending on the source of starch. Once starch is completely hydrolyzed the oligo and disaccharides do not form a colour complex with iodine. The zone of hydrolysis is evident by the lack of colour.

Materials Required

Starch agar medium, Gram’s Iodine solution, sterile petridish, inoculation loop, Bunsen burner

Procedure

1. 1 gm soil was suspended in 100 ml sterile distilled water in a conical flask

2. Serial dilution was performed in tubes containing 9 ml sterile distilled water.

2. Aliquots of  0.1 ml was transferred from 10^-5 and 10^-6 into starch agar plates and spread plate performed

3. The inoculated plates were incubated at 37^oC for 24-48 hours

4. After incubation, the plates were analyzed with Gram’s Iodine solution and then observed for starch hydrolysis

Observation

Presence of colorless hydrolysis zone around the colony after treatment with Gram’s Iodine solution is positive evidence for degradation of starch. Absence of clear zone indicate a negative reaction.

Result

A clear zone around amylase producing colonies indicated the presence of amylase producers which were isolated from the soil sample.

 

Media Composition (dont write in the record sheet)

 

Starch Agar (pH 7.0)

Peptone 5 g

Beef extract 3 g

Soluble Starch 2 g

Agar 15 g

Distilled water 1000 ml