Enzymes are the catalyst of biological system and are extremely
efficient and specific as catalysts. In fact, typically an enzyme accelerates
the rate of a reaction by factors of at least a million compared to the rate of
the same reaction in the absence of enzymes.
Michaelis - Menten equation:
Vmax = Maximum velocity
Km = Michaelis constant
[S] = Substrate concentration.
Enzyme catalyzed reaction model.
Km is the
characteristic of an enzyme and its particular substrate and reflects the
affinity of the enzymes for the substrate. Km is numerically equal
to the substrate concentration at which the reaction velocity is equal to ½ Vmax.
Km does not vary with the concentration of enzymes.
i.
Small Km – A numerically small
(low) Km reflects a high affinity of the enzymes for substrate
because a low concentration of substrate is needed to half saturate the
enzymes.
ii.
Large Km – A numerically large
(high) Km reflect a low affinity of the enzymes for substrate.
Material
Required:
1. Enzymes / Saliva
2.
Spectrophotometer
3.
Starch solution- 2% (stock), working – 0.1- 2%
4. 0.1 HCL
5. Iodine solution
6. Potassium phosphate buffer, pH – 7.0
Procedure
1.
Prepared the varied concentration of
soluble starch substrate of 0.01, 0.025,
0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution from
stock solution - total volume 8 ml.
2.
Add 1 ml of amylase solution to each of
the test tube to start the reaction.
3.
Incubate at 370C for exactly 10
min.
4.
Stop the reaction after exctly 10 min by
adding 1 ml of 0.1 N HCL.
5.
Add 0.1 ml iodine solution.
6.
Take absorbance at 546 nm.
7.
Plot the graph with absorbance or V in Y
axis and concentration of substrate in X-axis and determine Vmax and Km.
Or
By Lineweaver Burk double reciprocal plot taking 1/[S] in X-axis and 1/V
in Y-axis.
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