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    Alpha Amylase

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    Yogananth Nagarajan
    Estimation of alpha amylase Bacillus cereus DNS Method Dinitrosalicylic Acid Reagent

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    © All Rights Reserved
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    Alpha Amylase

    Uploaded by

    Yogananth Nagarajan
    233 views3 pages
    Estimation of alpha amylase Bacillus cereus DNS Method Dinitrosalicylic Acid Reagent

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    © © All Rights Reserved

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    DOC, PDF, TXT or read online from Scribd

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    Estimation of alpha amylase Bacillus cereus DNS Method Dinitrosalicylic Acid Reagent

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    233 views3 pages

    Alpha Amylase

    Uploaded by

    Yogananth Nagarajan
    Estimation of alpha amylase Bacillus cereus DNS Method Dinitrosalicylic Acid Reagent

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    of 3

    Experiment No: 5

    ESTIMATION OF AMYLASE ACTIVITY


    Aim: To determine amylase activity in the given sample.
    Principle: -Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked
    polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is the
    major form of amylase found in humans and other mammals. It is also present in seeds
    containing starch as a food reserve, and is secreted by many fungi.
    Starch + H2O a-Amylase
    Reducing Groups (Maltose)
    Reagents required:
    1. 20 mM sodium phosphate buffer with 6.7 mM Sodium Chloride, pH 6.9 at 20C:
    Prepare 100 ml in deionized water using anhydrous monobasic sodium phosphate.
    Adjust to pH 6.9 at 20C with 1 M NaOH.)
    2. 1.0% (w/v) Soluble Starch Solution (Starch): Prepare 25 ml in sodium phosphate
    buffer with 6.7 mM Sodium Chloride, pH 6.9 at 20C using starch. Facilitate
    solubilization by heating the starch solution in a glass beaker directly on a
    heating/stir plate using constant stirring. Bring to boil and maintain the solution
    at this temperature for 15 minutes. Allow the starch solution to cool to room
    temperature with stirring. Return the starch solution to its original volume (25
    ml) by the addition of water and dispense samples for assay while stirring.
    3. Dinitrosalicylic Acid Reagent (DNS Reagent): Dissolve by stirring 1g
    Dinitrosalicylic acid, 200mg crystalline phenol and 50mg sodium sulphite in
    100mL 1% NaOH.
    4. 40% Rochelle salt solution (Potassium sodium tartrate)
    5. Standard maltose solution: 0.1% maltose solution.
    6. -Amylase Solution: Prepare a solution containing 1 unit/ml of -Amylase in cold
    deionized water.
    Procedure:
    1. Pipette out 1ml of starch solution into test tubes labelled test and blank.
    2. Mix by swirling and equilibrate to 20C. Then add 1ml of enzyme solution.
    3. Mix by swirling and incubate for exactly 3.0 minutes at 20C.
    4. Add 2mL of DNS reagent.
    5. Heat the contents in a boiling water bath for 5-10 minutes.
    6. When the contents of the tubes are still warm, add 1mL of 40% Rochelle salt
    solution.
    7. Mix by inversion and measure the OD at 540nm for both the test and blank using
    a suitable spectrophotometer. One unit of amylase activity is defined as 1.0 mg of
    maltose liberated from starch in 3 minutes at pH 6.9 at 20C.
    Standard Curve Preparation:
    1. Label test tubes 0, 1, 2,....10 and add 0,0.1,0.2,....1ml of standard glucose solution
    in the test tubes respectively.
    2. Make up the volume to 5ml using distilled water.

    3. Take 2ml from each tube and follow the procedure from step-4 as described
    above.
    4. Plot a standard curve with sucrose concentration along X-axis and O.D against Yaxis.

    CALCULATIONS:
    Units/ml enzyme =

    (mg of Maltose released)(Dilution factor)


    Volume of enzyme taken

    OBSERVATIONS:
    1
    2
    3
    4
    5

    REAGENTS
    Starch solution
    Enzyme
    DNS reagent
    Rochelle solution
    Enzyme
    O.D at 540nm

    BLANK
    1ml
    2ml
    1ml
    1ml

    TEST
    1ml
    1ml
    2ml
    1ml
    -

    STANDARD CURVE DETERMINATION:


    Volume of
    Standard
    Maltose(m
    l)
    0
    0.2
    0.4
    0.6
    0.8
    1.0

    Volume
    of
    distilled
    water
    (ml)
    5
    4.8
    4.6
    4.4
    4.2
    4

    Maltose
    Sample
    Concentrati volume
    on (mg)
    (ml)
    0
    0.2
    0.4
    0.6
    0.8
    1.0

    2
    2
    2
    2
    2
    2

    DNS
    Cap
    Reagen the
    t (ml)
    tube
    s
    and
    boil
    2
    for
    2
    5-10
    2
    min
    2
    2
    2

    Rochelle
    salt
    solution
    (ml)
    1
    1
    1
    1
    1
    1

    Report:
    -Amylase activity in the given sample is found to be = __________ Units/ml.

    Optical
    Density
    at
    540nm

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