Prepared by Leong Chean Ring (Modified by Nor Afifah Khalil)

CPB 30103 Biochemical Engineering

Experiment 2:
ENZYME ASSAYS AND AFCTORS AFFECTING ENZYME ACTIVITY

Overview

Enzymes

Enzymes are Biological molecules (protein) that accelerate the rates of biochemical reactions by lowering
the activation energy of the reactions. Thus, enzymes are also called biocatalyst and their catalytic action
results from their complex structure. The great diversity of protein structure allows enzyme action to be
highly specific. The action of an enzyme in speeding the biochemical conversion of a substance into
something else can be simply described as follows:

Absorption Photometry

Because of its simple technique and reliable, reasonably priced instruments, photometry is today one of
the preferred methods of enzyme assay. It can be carried out most quickly and conveniently when the
substrate or the product is colored or absorbs light in the ultraviolet region because the rate of
appearance or disappearance of a light-absorbing product or substrate can be followed with a
spectrophotometer.

Figure 1: Schematic diagram of UV- visible spectrophotometer

According to the Bouguer- Lambert-Beer law, which is valid for very dilute solutions, the following
relationship exists between absorbance A and concentration:

absorbance, A = = . .

A
and c= . d

Where c is the concentration, is the absorption coefficient and d is the path length.

Some bio-molecules have properties which allow direct measurement. For example, proteins and nucleic
acids can be measured at 280 nm and 260 nm respectively. Other molecules have chemical properties
which can be used in indirect measurement.

Standard Curve

Standard curve uses dilutions of a solution of known concentration to determine concentration of

unknown. We can assume that the unknown will respond in assay the same as the known.

A540

m = y/x
b
(may or may
not equal 0) 0
[glucose(red)]
0

Figure 2: example of a standard curve

Determination of Reducing sugars by DNS Method

Sugars with reducing property (arising out of the presence of a potential aldehyde or keto group) are
called reducing sugars. Some of the reducing sugars are glucose, galactose, lactose and maltose.

Figure 3: Reducing sugar have the aldehyde group that can be oxidized.
This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This
involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone
functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-
nitrosalicylic acid under alkaline conditions:

oxidation
aldehyde group -------------------> carboxyl group

reduction
3,5-dinitrosalicylic acid ---------------> 3-amino,5-nitrosalicylic acid

Because dissolved oxygen can interfere with glucose oxidation, sulfite, which itself is not necessary for the
color reaction, is added in the reagent to absorb the dissolved oxygen.

The above reaction scheme shows that one mole of sugar will react with one mole of 3,5-dinitrosalicylic
acid. However, it is suspected that there are many side reactions, and the actual reaction stoichiometry is
more complicated than that previously described. The type of side reaction depends on the exact nature
of the reducing sugars. Different reducing sugars generally yield different color intensities; thus, it is
necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups in the sugar,
other side reactions such as the decomposition of sugar also competes for the availability of 3,5-
dinitrosalicylic acid. As a consequence, carboxymethyl cellulose can affect the calibration curve by
enhancing the intensity of the developed color.

Introduction

Starch makes up a large proportion of our diet, and it needs to be made soluble before it can be absorbed
into our bodies. In order to carry out this chemical breakdown, various glands produce digestive juices
containing amylases to be mixed with the food. Saliva contains one amylase, and pancreatic juice contains
another. Different amylase enzymes are produced by other organisms, including fungi and bacteria, which
carry out external digestion. In fact, any organisms that grow on starch must produce an enzyme to break
it down. Plant seeds often contain a reserve of rather dry insoluble starch for the embryo plant, and when
a seed germinates it must produce an amylase to convert the starch into soluble sugars which can be
transported to the growing points at tips of root and shoot of the growing plant. Amylase is a general
name for an enzyme which breaks down starch. Amylase hydrolyzes starch (a polysaccharide) into maltose,
a sugar (dissacharide). In this investigation, this enzyme will be used to demonstrate the effect of
temperature, pH and enzyme concentration on enzyme activity.

OBJECTIVES

To comprehend physical characteristic and properties of enzymes

To study the factors affecting their activity.
LABORATORY PROCEDURE

Apparatus and Reagent

1. 15-20 test tube

2. UV-Vis spectrophotometer

3. Amylase enzyme (commercial)

4. Starch solution (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%; w/v)

5. Glucose standard (0, 200, 400, 600, 800, 1000 mg/L)

6. 3,5-dinitrosalicylic acid
7. Potassium sodium tartarate

8. Sodium hydroxide

9. Distilled water

10. Phosphate buffer pH 5.0, 7.0, 8.0, 9.0, 11.0

Procedure
Part 1
A. Preparation of the DNS reagent.

1. Prepare the DNS reagent by dissolving 5 g of DNS and 8g of NaOH in 100 ml of distilled water and top
the volume to 300ml.
2. Add 150g of potassium sodium tartarate slowly until all the DNS dissolve and top up the volume to
500 ml.

B. Preparation of the glucose standard curve

1. Prepare glucose solution with the concentration of (0, 200, 400, 600, 800, 1000 mg/L).
2. Mix 0.5 mL glucose solution of each concentration with 0.5 mL, 0.2 M phosphate buffer (pH 7.0).
3. Incubate the mixture at room temperature for 30 min, mix every 10 minutes.
4. After 30 min, add the mixture in a test tube and subsequently add 1.0 ml of the DNS reagent.
5. Boil the reaction mixture for 5 min and allow to cool down. Add 10 ml of distilled water.
6. Determine the optical density of the mixture for each glucose concentration at 575nm using UV-Vis
spectrophotometer.
7. Plot the standard curve of the glucose concentration vs the absorbance at 575 nm. (Data are provided
in Table 1)

Concentration [g/L] Absorbance [/]

0 0
0.20 0.100
0.40 0.255
0.60 0.391
0.80 0.539
1.00 0.627
Table 1

C. Amylase Activity Assay

1. Prepare the enzyme solution by mixing 0.2 mL of Amylase with 0.3 mL, 0.2 M phosphate buffer (pH
7.0).
2. Add 0.5 mL of 1% (w/v) of starch solution into the enzyme solution in (a).
3. Incubate the mixture at room temperature for 30 min, mix every 10 minutes.
4. After 30 min, add the mixture in a test tube and subsequently add 1.0 ml of the DNS reagent.
5. Boil the reaction mixture for 5 min and allow to cool down. Add 10 ml of distilled water.
6. Determine the optical density of the mixture for each glucose concentration at 575nm using UV-Vis
spectrophotometer.
7. Calculate the concentration of the reducing sugar using the standard curve of glucose prepared in
Part 1 (B).
8. Determine the amylase activity. 1U (unit) is defined as the amount of enzyme required to release 1
g of reducing sugar per min under the conditions stated.

Part 2

A. Effect of Substrate Concentration on Enzyme Activity

1. Prepare starch solution with concentration of 0.5, 1.0, 1.5, 2.0 and 3.0% (w/v) as the substrate. Place
0.5 mL of the substrate of each concentrations into the test tubes.
2. Add 0.2 mL of enzyme solution and 0.3 mL of 0.2 M phosphate buffer (pH 7.0).
3. Incubate the mixture at room temperature for 30 min, mix every 10 minutes.
4. After 30 min, add the mixture in a test tube and subsequently add 1.0 ml of the DNS reagent.
5. Boil the reaction mixture for 5 min and allow to cool down. Add 10 ml of distilled water.
6. Determine the optical density of the mixture for each glucose concentration at 575nm using UV-Vis
spectrophotometer.
7. Calculate the concentration of the reducing sugar using the standard curve of glucose prepared in
Part 1 (B).
8. Determine the amylase activity. 1U (unit) is defined as the amount of enzyme required to release 1
g of reducing sugar per min under the conditions stated.
9. Plot the graph of product formed against substrate concentration.

B. Effect of pH on Enzyme Activity

1. Prepare the enzyme solution using buffer of various pH by mixing 0.2 mL of Amylase with 0.3 mL of
0.2 M phosphate buffer at pH 5.0, 7.0, 8.0, 9.0 or 11.0.
2. Add 0.5 mL of 1% (w/v) of starch solution into each enzyme solution in (a).
3. Incubate the mixture at room temperature for 30 min, mix every 10 minutes.
4. After 30 min, add the mixture in a test tube and subsequently add 1.0 ml of the DNS reagent.
5. Boil the reaction mixture for 5 min and allow to cool down. Add 10 ml of distilled water.
6. Determine the optical density of the mixture for each glucose concentration at 575nm using UV-Vis
spectrophotometer.
7. Calculate the concentration of the reducing sugar using the standard curve of glucose prepared in
Part 1 (B).
8. Determine the amylase activity. 1U (unit) is defined as the amount of enzyme required to release 1
g of reducing sugar per min under the conditions stated.
9. Plot the graph of product formed against different pH.
10. Determine the optimum pH for the enzyme.
Caution:

1. Proper labeling is very important for the experiments accuracy.

2. Make sure that you change the pipettes tip for substrate/glucose with different concentration.

Results/Discussion

1. Describe the effect of different temperature on the enzymes activity.

2. Determine and discuss the temperature for the optimum activity of amylase (for example the
environment where the enzyme catalyze its reaction).
3. Is the results obtained parallel to the results expected or previously reported? If not, make reasonable
suggestion of why you did not get them.
4. Describe what happens to the rate of enzyme reaction when you increase the concentration of the
substrate.
5. What is the effect of different pH on the rate of enzymatically catalyzed reactions? What is the
optimum pH for amylases activity?

Questions

1. Discuss about other applications using enzyme assay. For example; what does the portable
glucometers used by diabetic patients measure? How do they measure it?
2. Discuss about the importance of optimum pH and temperature in the industrial application of
enzymes.