By:

Bhagyashree Ganatra

Under guidance of Mr.Uttam Bodake

Department of Biotechnology

Fergusson college, Pune.

 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. When they have broken down the insoluble starch, the soluble end products such as (glucose or maltose) are absorbed into their cells.

Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger

 Autoclave or pressure cooker Nutrient Agar powder Soluble starch Weighing scales Spectrophotometer or colorimeter Water bath (Temperature controlled)

 Sterile pipettes (One each of 10 mL, 5 mL and 1 mL) Sterile Glass Petri dishes Wire loop Bunsen burner and matches 95% ethanol.

i.

ii.

iv. v.

Sweep off the debris from the top of the soil, (about 10 grams), into a "Ziploc" bag Suspend about 1 gram soil in 10 mL sterile distilled water, mix properly iii. Allow the soil to settle down. Collect the supernatant in another test tube. Meanwhile, prepare 5 tubes containing 5ml sterile water.

The amylase activity was assayed by DNSA method(Plummer, Practical Biochemistry, 2005)
Three tubes were prepared. Tube 1 containing only DNSA reagent was used as blank. Tube 2 contained DNSA reagent + starch + pure amylase

Tube 3 contained DNSA reagent + starch + crude amylase

TABLE 1. CALCULATION OF SELECTION RATIO

SOIL SAMPLE DIAMETER OF THE ZONE (mm) DIAMETER OF THE COLONY (mm) 5 6 5 5 6 SELECTION RATIO

SAMPLE 1 SAMPLE 2 SAMPLE 3 SAMPLE 4 SAMPLE 5

18 17 20 17 16

3.60 2.83 4.00 3.40 2.66

TABLE 2. OPTICAL DENSITY OF AMYLASE AT 540nm

ENZYME PURE AMYLASE CRUDE AMYLASE

O.D. AT 540nm 0.493 0.613

342.3g/mol of maltose in 1lit water = 1M 342.3 g maltose liberates = 1 unit of enzyme Therefore, 4.7 g will liberate = 4.7/342.3 = 0.014 units of crude enzyme and 3.8g will liberate = 3.8/342.3 = 0.011 units of pure enzyme.

0.014units/ml of crude enzyme is liberated. Hence for 100ml = 0.014x100 = 1.4units 0.011 units/ml of pure enzyme = 0.011x100 = 1.1 units.

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10

Conc of maltose

O.D at 540nm

A total of 40 bacterial strains which produced clear zones in the starch- nutrient agar medium were isolated and purified. Among the 40 bacterial strains, one strain was selected as best amylase producer. As the strain was able to produce maximum amylase activity at 24 h, its activity was assayed using the DNSA reagent and the concentration of enzyme was evaluated using the standard maltose graph.

Mishra, S. and Behera, N. (2008). Amylase activity of a starch degrading bacteria isolated from soil receiving kitchen wastes. African J. of Biotechnology. Abe, J., Bergman, F.W., Obata, K. and Hikuri, S. (1988). Production of raw starch digesting amylase by Aspergillus K-27. Applied Microbiology and Biotechnology 27, 447-450. Bahadure R.B., Agnihotri U.S. and Akarte S.R. Department of Zoology, Vidya Bharati Mahavidyalaya Amravati.444 602

Bernfeld, P. (1951). Enzymes of starch degradation and synthesis. Advances in enzymology 12, 379-481
Brook, Elizabeth J., Stanton, W.R. and Wallbridge, Ann. (1969). Fermentation Methods for protein enrichment of cassava. Biotechnology & Bioengineering. 11:12711284 Rosario, E. J., and Wong, R. L. (1984). Conversion of dextrinized cassava starch to ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae. Enzymes and Microbial Technology, 6: 60-64