Materials and Methods

Collection of soil sample:

Soil sample collected near sewage.

Isolation of organism:

Starch agar plate ( peptone—5g; Nacl—5g/L; HM peptone—1.5g/L; soluble starch—2g/L; Agar —

20g/L; pH—7.4+0.2) is a selective medium was used for isolation of Bacillus subtilis about 1.0gm of
soil sample was serially diluted in sterile physiological saline and dilution was done upto 10^-7 by
through mixing using vortex. 0.1ml of sample from 10^-4 dilution was spread on sterile petri dishes
containing starch agar with the help of L-rod and plates were incubated at 37°cfor24—48hrs. After
incubation the plates were observed for the growth of bacteria

Determination of amylase activity

All the Bacillus subtilis isolates were tested for amylase production by starch agar. Starch agar
medium innoculated with the organism and after incubation, plates were subsequently flooded with
iodine solution for 1-2mins and washed gently .cleared zone around amylase producing colonies are
seen that indicates the production and presence of extra cellular enzymes

Sub culture of enzyme producing bacteria

Colonies showing positive results were subcultured in plates containing starch agar medium
and incubated at 37°C overnight. These cultured colonies are inoculated in culture broth and
incubated overnight for enzyme reaction occurs.

Enzyme preparation

Culture broth was centrifuged as 12000 rpm for 5 mins. The supernatant was separated and
was assayed for Alpha-Amylase activity (We got total of 9 culture broth)

Enzyme assay

Amylase activity were assayed by measuring the amount of reducing sugars released from
starch using dinitrosalicylic acid method

Estimation of glucose by DNS

Pipette out 0.1ml from culture into a clean test tube. Make up the volume to 1ml with H2o.(Sterile)
To this add 1 ml of1% starch then incubate it for 30mins after add 1ml of NaoH and 2 ml of DNS
reagent. Place the tube in boiling water for 30 mins and cool them. Read the intensity at 580nm.Plot
a graph for series of the standard glucose solution.

Calculator the amount of reducing sugar glucose present in the sample using standard graph

Estimation of protein by Lowrys method:

Solution A-2% sodium carbonate in 0.1N NaoH

Solution B-2% Cuso4

Solution C-2% sodium potassium tartarate

Solution D-0.5 ml solution B+0.5ml solution C +99ml solution A

Folin ciocalteau reagent-1:1dilution.

BSA stock: 1mg/1ml.

Pipette out 0.1ml of culture into a test tube. Make up the final volume to1ml with Sterile
water. To this add 1 ml solution D, 0.5ml of foline ciocalteau reagent incubate the tube at dark
condition for 30 mins. As a result, it develop blue colour. Read the intensity of colour at
610nm.Calulate the amount of protein present in the sample using standard graph

Estimation of specific activity:

Specific activity = EA/mg of protein

Enzyme activity (EA) was calculated as = uh of product released * 1000 /molecular weight of
product.

mg of protein was calculated by unknown sample with standard graph (BSA)