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Expt 5

The document outlines an experiment to determine the deactivation kinetics of the enzyme α-amylase, focusing on factors affecting enzyme stability such as temperature and pH. It includes a detailed procedure for enzyme assays, reagent preparation, and the method for calculating kinetic constants and activation energy of deactivation. The experiment aims to analyze enzyme activity at various temperatures and incubation times to understand the first-order reaction kinetics of enzyme deactivation.

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14 views4 pages

Expt 5

The document outlines an experiment to determine the deactivation kinetics of the enzyme α-amylase, focusing on factors affecting enzyme stability such as temperature and pH. It includes a detailed procedure for enzyme assays, reagent preparation, and the method for calculating kinetic constants and activation energy of deactivation. The experiment aims to analyze enzyme activity at various temperatures and incubation times to understand the first-order reaction kinetics of enzyme deactivation.

Uploaded by

jsaswini6
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Experiment No.

Determination of Deactivation Kinetics of Soluble Enzymes

Objective:

To determine the kinetic constant of deactivation of a soluble enzyme, α - amylase.

Introduction:

An important criterion when using enzymes in technical processes for material


conversion or in analytical procedures is the stability of the enzyme during the process.
The stability of the enzyme dictates the length of time for which the enzyme is active
under the reaction conditions. Depending on the type of enzyme, its purity, enzyme
pretreatment and reaction conditions, the enzyme activity can change detectably over
periods of time ranging from minutes to years. The main factors affecting enzyme
stability are: temperature, pH, concentrations of substrate and product, chemical effects
(inhibitors, heavy metals, impurities) breakdown caused by microbes.

Temperature dependency of the catalytic activity and stability of an enzyme

If the enzyme reaction follows a Michaelis - Menten relation

CS
r = rmax
K S + CS

then the maximum velocity of the reaction increases initially with temperature according
to the Arrhenius equation

⎛ E ⎞
rmax = ACE exp ⎜ − A ⎟
⎝ RT ⎠

where A is the pre-exponential factor, CE is the enzyme concentration, EA is the


activation energy and R is the universal gas constant. At higher temperatures the
conformation of the enzyme is altered because of stronger rotational forces and
movement of the molecules and this in turn has an adverse effect on the ability of the
enzyme to catalyse the reaction. The optimal reaction temperature for technical enzymes
lies between 200oC and 60oC. If this temperature is exceeded only minimally then the
conformational changes are reversible and the deactivation can be described
thermodynamically.

Irreversible deactivation processes are described by kinetic expressions. Enzyme


deactivation is a reaction of the first order:

E N → ED
where EN is native enzyme and ED is deactivated enzyme with the rate equation

da
= −kd a
dt

where a is the activity and kd is rate of deactivation. Integration results:

a = a0 e − kd t

deactivation is extremely temperature dependent according to the equation:

− E A ,d / RT
k d = kd0 e

List of Reagents and Instruments

A. Equipment: Flasks, Spectrophotometer, Sample tubes, Micropipette

B. Reagents

• Reagent A: A1. Dissolve 67. 5 gm of Sodium potassium tartrate in 100 ml of


water

A2: Dissolve 1.56 gms of Phenol in 10% Sodium Hydroxide (4.375 gm


of Sodium Hydroxide in 43.75 ml of water)

Mix A1 and A2 and keep in dark bottle

• Reagent B (DNS reagent): Dissolve 3, 5- DinitroSalicylic acid 1 gm in 100 ml


of Water

C. Enzyme and Chemicals Required

• Enzyme: α - amylase.

• Chemicals: Starch

Acetate Buffer composition

Chemical name Composition


Acetic Acid 1M
Sodium acetate 1M
Procedure:

Enzyme Assay:

• Chemicals/Reagents required: 0.25 ml starch 2 %; 0.10 ml enzyme;


• Incubate at 75oC for 10 min.
• Perform glucose analysis of the sample by DNS method
• Enzyme activity is defined as the amount of enzyme required for the formation of 1
µmol. of glucose per minute per ml of enzyme.

Enzyme Deactivation Kinetics:

For studying the deactivation kinetics of the enzyme, upon incubation under conditions
shown in the following table , the enzyme assay is performed as above.
Table: Plan for testing α-amylase activity
No. T (oC) Activity (a) after
H U
1 65 1
2 2
3 3
4 4
5 75 1
6 2
7 3
8 4
9 85 1
10 2
11 3
12 4
13 95 1
14 2
15 3
16 4

From the measurements of enzyme activity at various times and different temperatures
the order of the deactivation kinetic, the rate constant of deactivation and the activation
energy of the deactivation will be calculated.

A reaction is of the first order if the plot of ln (a/a0) against time (t) gives a straight line:

ln ( a / a0 ) = −kd t

where a0 is the activity of the native enzyme and kd is the slope of the straight line.
If logn of the various deactivation rate constants kd(T) is plotted against the reciprocal of
the absolute temperature, the slopes of the straight lines obtained give the negative
quotient of the deactivation energy (EA) and the gas constant (R = 8.314 J mol-1 K-1).

Task Required

Determine the activity of the enzyme at various temperatures and time of incubation

Calculate the order of reaction for the deactivation kinetics at a specific temperature

Calculate the rate constant for the deactivation reaction

Calculate the energy of deactivation.

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