Salivary Amylase

Only available on StudyMode
  • Topic: Enzyme, PH, Enzyme kinetics
  • Pages : 15 (1593 words )
  • Download(s) : 68
  • Published : January 19, 2013
Open Document
Text Preview
Activity of Salivary Amylase
Domingo, Guray, Hugo, Lorenzo, Mohammad Isa

Intro
Because everything has a start

Catalysis

 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

Page  3

Enzymes

 Act on substrates in a reaction
 Highly specific
 Breaks down complex macromolecules, synthesizes compounds essential for the cell
 Active site
 Enzyme-substrate complex
 Speeds up reaction rates

Page  4

http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg Page  5

Enzymes

 Require cofactors for activity
 Classified according to the types of reaction they catalyze

– Oxidoreductase
– Transferase
– Hydrolase
– Lyase
– Isomerase
– Ligase

Page  6

Page  7

Amylase

 An enzyme that breaks down starch into oligosaccharides through hydrolysis
 Secreted by the human’s parotid glands and the pancreas  α-Amylase
 β-Amylase
 γ-Amylase

Page  8

Factors that may affect catalysis rates

 Temperature
 pH
 Enzyme concentration
 Amount of substrate

Page  9

Materials and Methods

Solution Preparation

Saliva was collected.
1 ml of saliva was diluted to 10 ml with distilled water.  10 % salivary amylase solution

Page  11

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.

Page  12

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.

Page  13

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.

Page  14

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.

Page  15

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.

Page  16

Results and Discussion

Estimation of amylase activity

 Achromic point is the time it takes for
the enzyme to completely hydrolyze the
starch solution.
 enzyme-starch mixture is not able to
produce a blue to violet color with
iodine -> absence of starch

Page  18

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

Salivary
Amylase (%)

time to achromic point
(min)

0.5

30

0.75

23

1

18

1.5

11

2

7

2.5

7

amylase units

Enzyme
activity

0.833333333

0.4000

1.630434783

0.2667

2.777777778

0.2000

6.818181818

0.1333

14.28571429

0.1000

17.85714286

0.0800

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

Page  20...
tracking img