University:
High School
Course:
High School Chemistry
Academic year:

2023
Views:

18

Pages:

1
Author:

D Lathiya

EXPERIMENT NO - 16 Aim: Determination of Salivary amylase activity. Requirements: (a) Glasswares: Test tube, Flask, Pipette, Stir rod. (b) Chemicals: Starch, Sodium Chloride, NaOH, Sodium potassium tartrate, Maltose, Distilled Water. Reagent Preparation: Substrate (Starch): Mix 1 gm of soluble starch in 200 mL of 0.1M Phosphate buffer (pH 6.8) boil for 3 minutes and cool to room temperature and filter if necessary. Enzyme: Saliva is the best and easily available source of amylase. Collect some saliva in a beaker and dilute it to 1:20 dilution with distilled water. 1% Sodium chloride: It is necessary for enzyme activity. DNS (Dinitro Salicylic acid): Dissolve 1.6 gm of NaOH in 20 mL of distilled water. Take 1 gm of 3, 5 DNS in NaOH solution. In other beaker take 30 gm of Sodium potassium tartrate. Dissolve in 50 mL of distilled water. Mix this DNS solution and finally make the volume up to 100 mL with distilled water. Standard solution of Maltose: It is prepared by dissolving 200 mg Maltose in 100 mL of water (2 mg / 1 mL). Principle: Amylase is the hydrolytic enzyme which breaks down many polysaccharides like Starch, Amylose, dextrins and yields a disaccharide i.e., Maltose. Procedure: Take 0.5 mL of substrate and 0.2 mL of 1% NaCl in a test tube and pre-incubated at 37°C for 10 minutes then add 0.3 mL of diluted saliva and incubate for 15 minutes at 37°C. Stop the reaction by addition of 1 mL of DNS reagent mix well and keep the test tubes in boiling water bath for 10 minutes. Cool and dilute with 10 mL of distilled water. Read the colour developed at 520 nm. Simultaneously setup the colour developed at 520 nm. Simultaneously setup the blank as per the test by adding DNS prior to the addition of enzyme simultaneously. Set up the standards of different test tubes and repeat the experiment as per the test and measure the colour developed at 520 nm absorbance. Sr. No. Volume of Std. (ml) Volume of Distilled water (ml) Concentration of Standard (ml) Volume of DNS (ml) Procedural Step Volume of Distilled water (ml) OD at 520 nm 1. Blank 1.0 0.0 1.0 Boiling water bath for 10 mins and cool 10 2. 0.2 0.8 0.4 1.0 " 10 3. 0.4 0.6 0.8 1.0 " 10 4. 0.6 0.4 1.2 1.0 " 10 5. 0.8 0.2 1.6 1.0 " 10 6. 1.0 0.0 2.0 1.0 " 10 Calculation: 1.5 mg of Maltose formed / 0.3 mL / 15 minutes 1.5 x 4 mg of Maltose formed / 0.3 mL of Enzyme / 1 hour 1.5 x 4 x 3.3 mg of Maltose formed / 1 mL of Enzyme / 1 hour 1.5 x 4 x 3.3 x 100 mg of Maltose formed / 100 mL of Enzyme / 1 hour Result: The amount of Maltose in the given unknown sample is ______ grams of Maltose formed per 100 mL of enzyme per one hour.