Activity of Salivary Amylase
Domingo, Guray, Hugo, Lorenzo, Mohammad Isa
Because everything has a start
The process of increasing the rate of reaction with the use of a catalyst. Catalyst – any substance that increases rate of reaction upon addition to a certain reaction
Act on substrates in a reaction
Breaks down complex macromolecules, synthesizes compounds essential for the cell
Speeds up reaction rates
http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg Page 5
Require cofactors for activity
Classified according to the types of reaction they catalyze
An enzyme that breaks down starch into oligosaccharides through hydrolysis
Secreted by the human’s parotid glands and the pancreas α-Amylase
Factors that may affect catalysis rates
Amount of substrate
Materials and Methods
Saliva was collected.
1 ml of saliva was diluted to 10 ml with distilled water. 10 % salivary amylase solution
Estimation of salivary amylase activity
A mixture of 5.0 mL 1% starch, 2 mL 1% NaCl solution and 2 mL phosphate buffer put in a test tube and then placed in a water bath At 38oC, 1 mL salivary enzyme solution added to the solution. A drop from the digestion mixture mixed with 1 drop of iodine for every minute.
Achromic point was determined.
Effect of enzyme concentration
The salivary amylase solution diluted to five lower concentrations: 2.5%, 2.0%, 1.5%, 1.0%, 0.75% and 0.5%.
The same procedure done as previous using 1% concentration of starch solution.
Reaction rates observed for each dilution.
Effect of amount of substrate
Six percent starch prepared from which five other dilutions were prepared: 5%, 4%, 3%, 2%, and 1%.
The same procedure for Estimation of salivary amylase activity used using 2.5% salivary amylase solution.
Reaction rates for each substrate dilution recorded.
Effect of pH
A mixture of 0.2 M sodium biphosphate (Na2HPO4) and 0.1 M citric acid prepared to obtain different buffer solutions with pH varying from 3.0 to 8.0.
Similarly, procedures from the estimation of enzymatic activity were applied, recording all notable reaction rates for each pH setup.
Effect of temperature
Test tube with 2.5% salivary enzyme was placed on water baths maintained at 4oC, 10oC, 38oC, 58oC, 78oC and 100oC.
Reaction rates recorded.
Results and Discussion
Estimation of amylase activity
Achromic point is the time it takes for
the enzyme to completely hydrolyze the
enzyme-starch mixture is not able to
produce a blue to violet color with
iodine -> absence of starch
Estimation of amylase activity
Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva and Their Corresponding Time to Reach the Achromic Points, Amylase Units and Enzyme Activity Effect of Enzyme Concentration
time to achromic point
Amylase units - amount of enzyme necessary to digest 5 ml 1% starch to reach the achromic point within 10 minutes
Enzyme activity - mg starch hydrolyzed per minute per unit enzyme Page 19
Effect of Enzyme Concentration
enzyme + substrate enzyme-substrate complex enzyme + product
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