UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA

BIOCHEMISTRY LABORATORY (CBE 461)
NAME
GROUP
EXPERIMEN
T
DATE
PROG/CODE
SUBMIT TO

No
1
2
3
4
5
6
7
8
9
10
11
12
13

:
:
:
: 22ND MAY 2013
: EH 222
: MISS NUR SHAHIDAH AB AZIZ

Title
Abstract
Introduction
Objectives
Theory
Procedures/Methodology
Apparatus
Results
Calculation
Discussion
Conclusion
Recommendations
References
Appendices
TOTAL

Allocated Marks(%)
5
5
5
5
10
5
10
10
20
10
5
5
5
100

Marks

Remarks:

Checked by:

Rechecked by:

Date:

Date:

ABSTRACT

The experiment is to study the enzyme activity through different pH of buffer

solution. The enzyme used is beta amylase which is extracted from sweet potato through
centrifugation method. First maltose standard graph is by using three concentration of
maltose that are 0.8, 1.0 and 1.4 mg/ml. all the concentration is tested using
specthrophometer to get the absorbance value at 540 nm. After the graph plotted, the y=
mx +c equation is created and get the R2 value to compare with the theoretical value. The
enzyme is tested at different pH solution like 3, 5, 7, 9and 11 by adding maltose and
DNSA reagent. Then, get the absorbance value for each pH solution. Gather the results
for all pH and make comparison. From the result, the best pH for enzyme activity can be
determined.

INTRODUCTION

Enzyme is a catalyst which enhances the rate of reaction but is not permanently
altered. Catalyst work by decreasing the activation energy for a reaction. The best way to
interpret the characteristic of an enzyme is by its kinetics and specificity. Enzymes

selectively recognize proper substances over other molecules. The substances upon which
an enzyme acts are traditionally called substrates. Enzymes produce products in very high
yields which often much greater than 95%. The selective qualities of an enzyme are
recognized as its specificity. Specificity is controlled by structure of enzyme which the
unique fit of substrate with enzyme controls the selectivity for substrate and the product
yield. The specific site on the enzyme where substrate binds and catalysis occurs is called
the active site.

Kinetics is concerned with the rates of chemical reactions. Enzyme kinetics

addresses the biological roles of enzymatic catalyst and how they accomplish their
remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction
velocity that the enzyme can attain and its binding affinities for substrates and inhibitors.
This information can be exploited to control and manipulate the course of metabolic
events.

Any environmental factor that disturbs protein structure may change enzymatic
activity. Enzymes are especially sensitive to change in temperature and pH. Enzyme
catalyzed reaction also affected by different temperature. Each enzyme has own optimum
temperature at which it operates at maximum efficiency. If temperature is raised beyond
the optimum temperature, the activity of many enzymes will be affectless because the
enzyme has denatured. While the pH affects enzyme in several ways. Catalytic activity is
related to the ionic state of the active site. Change in pH can affect ionization of active
site group and tertiary structure of enzyme. Changes in pH may not only affect the shape
of an enzyme but it may also change the shape or charge properties of the substrate so
that either the substrate cannot bind to the active site or it cannot undergo catalysis.
OBJECTIVES
The experiment is to identify the ideas behind protein purification and to determine the
enzyme specific activity.

THEORY

In the experiment, the enzyme of beta amylase is extracted from the seewt potato
using the centrifugation method. Centrifugation is a basic separation technique. A
centrifuge is a device for separating particles in an applied centrifugal field in a solution.
The particles to be separated are suspended in a specific liquid media, held in tubes or
bottles which are located in rotor in centrifuge machine, positioned centrally to the drive
shaft. These particles are differing in size, shape and density. By increasing the effective
gravitational force on a test tube so the process will more rapid and completely cause the
precipitate (pellet) to settle on the bottom of the tube. The remaining solution is properly
called the (supernate or supernatant liquid) . The supernatant liquid is then either quickly
decanted from the tube without disturbing the pellet.

-Amylase is an enzyme that releases successive maltose units from the

nonreducing end of a polysaccharide chain by hydrolysis of 1,4--glucosidic linkages . Amylase is found primarily in the seeds of higher plants and sweet potatoes. It yields a
single product called maltose. The enzyme is useful in structural studies of starch and
glycogen. These -amylases are characterized by having optimum temperatures in the
range from 40 C. to 65 C, and optimum pH in the range from 4-5 and the pH stability
is about pH 3.5 to 6.
Here, is the formula used to calculate the activity of the enzyme:
Enzyme activity , U/mL = _________micromoles__________
Reaction time x 2.5 mL beta amylase

APPARATUS

1)

Pipette

2)

Spectrophotometer

3)

pH meter

4)

Centrifuge bottle

5)

Beaker

MATERIALS

1)

Sweet potato

2)

DNSA reagent

3)

Acetate/acetic acid buffer (pH 3, 5, 7, 9, 11)

4)

Ammonium sulphate

5)

Maltose

6)

Distilled water

PROCEDURE

Extraction of active enzyme

1)
the

20 ml of sample (sweet potato) is centrifuge for 15 minutes at 12,000 rpm. Then,

insoluble debris is removed.

2)
0.47g 0f ammonium sulphate is added in each 1ml supernatant. Then, it is
centrifuge at 12,000 rpm for 10 minutes.
3)
Pellets were suspended with 1ml of distilled water for each 8ml of original pulp
and are
centrifuge at 12,000 rpm for 10 minutes.

Analysis: Determination of amount of amylase activity

1)
7, 9,

0.1 ml of sample is mixed with 0.1 ml of 1% starch and 0.1ml of buffer at pH 3, 5,

and 11 (around 0.1 M acetate /acetic acid).

2)

The solution is incubated at 37C for 15 minutes.

3)

Next, 0.3ml of DNSA reagent is added. The solution is heated in boiling water for
10 minutes.

4)

Then, it is dilute with 4ml distilled water.

5)

The absorbance is determined at 540nm.

Standard curve
1)

Maltose is prepared with concentration from 0.1mg/ml until 1mg/ml.

2)
0.3ml of DNSA is added. Then, the solution is heated in boiling water for 10
minutes.
3)
The solution is dilute with 4ml of distilled water and the absorbance is read at
540nm.

RESULTS

Concentration of Maltose (mg/mL)

Absorbance (540 nm)

0.8

0.241

1.0

0.461

1.4

0.755
Table of standard curve of maltose

pH of buffer solution

Absorbance (540 nm),y

Concentration (mg/mL),x

0.824

1.470

0.992

1.670

0.713

1.337

0.861

1.514

11

0.887

1.545

Table of value obtained from equation y=mx+c

pH value of
buffer solution

Concentration
(mg/mL)

Micromoles of
maltose (moles)

Rate of reaction
of micromoles
maltose formed
per minute (u)

Enzyme activity
(u/mL)

1.470

4294.53

286.30

114.52

1.670

4878.82

325.25

130.10

1.337

3905.98

260.40

104.16

1.514

4423.07

294.87

117.95

11

1.545

4513.64

300.91

120.36

Table of value of micromoles,rate of reaction of micromoles maltose formed per minute

and enzyme activity.

Graph of standard curve for maltose

CALCULATION

Dilution of maltose

To carry out this experiment,maltose has to be diluted to different concentration of 0.8,1.0

and 1.4 g/mL. Blank sample also has been prepared and the amount of water needed is
determined by using this formula
Molarity = mass of maltose (g) x volume of water (mL)
Concentration of 0.8 g/mL
0.8 g/mL = ? g x 10 mL
= 8g maltose
Concentration of 1.0 g/mL

g/mL = ?g x 10 mL
= 10 g maltose

Concentration of 1.4 g/mL

1.4 g/mL = ?g x 10 mL
= 14 g maltose

Concentration of maltose using y=mx+c

Equation y = 0.839x - 0.409 is obtained when a linear is plotted on the standard curve of
maltose. When y is absorbance value, thus x is the value of concentration
y = 0.839x - 0.409
at pH 3, y = 0.824
0.824 = 0.839x - 0.409
x = 1.470 g/mL

Same step of calculation is repeated for pH 5,7,9 and 11.the results are tabulated.

Micromolecule of maltose

Molecular weight of maltose is 342.296 g/mol.1 mol of maltose is equivalent to 342.296

g of maltose
At pH 3, x = 1.470 g/mL
1.470 g

1 mole

1 moles

342.296 g

1 x 10-6 mole

= 4294.53 moles

Same step of calculation is repeated and the result is tabulated

Rate of reaction of micromoles maltose formed per minute

u=
at pH 3 =

= 286.302u
Same step of calculation is repeated and the result is tabulated.

Enzyme activity (u/mL)

Enzyme activity, u/mL =

micromoles
Reaction time x 2.5 mL beta amylase

At pH 3

= 286.302u
2.5 mL
=114.52 u/mL

Same step of calculation is repeated and the result is tabulated.

DISCUSSION

In this experiment, sweet potato is used to get its beta amylase enzyme. The

enzyme is next being tested with maltose to study the ideas behind protein purification
and determination of enzyme specific activity. The reaction of enzyme activity is tested in
different pH buffer solution as 3, 5, 7, 9 and 11. First, standard curve of maltose is
plotted by using different concentration of maltose is which is in range of 0.8, 1.0 and
1.8 mg/mL and added with DNSA reagent and heated with boiling water bath for 10
minutes. Then, get the absorbance value of each concentration based on the
specthrophometer method at 540 nm. DNSA or dinitrosalicylic acid has a nitro group in
its 3 and 5 positions each. Maltose being a reducing sugar reduces the amino group at 3
rd carbon to amino group and itself gets oxidized. The reduced product thus formed is 3amino,5-nitro salicylic acid which is orange-red in color.

Next the maltose is reacted with the enzyme beta amylase in different condition
of pH like 3, 5, 7, 9 and 11. Based on the result, it shows that the rate of reaction of
micromoles maltose formed per minute is highest at pH 5 and 11. Hence it shows that the
enzyme activity is best at acidic and basic condition. -Amylase is an exoenzyme that
releases successive maltose units from the nonreducing end of a polysaccharide chain by
hydrolysis of -1,4-glucan linkages. The standard curve plotted is used as reference for
enzyme activity at different pH. the equation of standard curve obtained is y = 0.839x 0.409 with R2 = 0.988 is valid At pH 5 the concentration of maltose obtained is 1.670
mg/mL and the enzyme activity is 130.10 U/mL. While pH 11 shows the maltose
concentration is 1.545 mg/mL with enzyme activity of 120.36 U/mL..

Based on the result obtained it can be said that beta amylase can be react in both
acidic and basic condition. This can be proven that reduction of starch to maltose
catalysed by beta amylase occur in mouth and stomach as the pH of the mouth is slightly
alkali and acidic for stomach.
CONCLUSION

In this experiment, the characterization of beta amylase from sweet potato in the term of
pH is analysed. The analysis is done by using the uv-vis spectroscopy method and taken
at an absorbance of 540nm at different concentration. To quantify the kinetics of beta
amylase, the concentration used is from 0.8, 1.0 AND 1.4. The result obtained shows the

activity of this enzyme at two points of pH which is pH 5 and 9. At this point the enzyme
activity is 130.10 U/mL and 120.36 U/mL respectively. Thus, showing the amount of
maltose reduced from starch 1.670 mg/mL for pH 5 and 1.545 mg/mL at pH 11. It
follows the official optimum pH is 4-5. Hence the results are valid by the calculation of
the R2 from the graph which is 0.9886 .According to all results and compared to official
standard curve, it can be concluded that beta amylase is presence in salivary amylase as
well as in stomach. Thus, based on the experimental result, it is possible to conclude that
beta amylase able to reduce starch to maltose in acidic and slightly alkaline condition.

RECOMMENDATION

1)

Careful while conducting the DNSA reagent because DNSA assay is corrosive.

2)
To obtain the pH value, the acid or alkaline solution must be added drop by drop
because
the small drop of the acid or alkaline solution can change the pH value.
3)
When using the spectrophotometer, the cuvette must be clean because it may
affect the
absorbance obtained.

4)

Goggles and gloves must be wearing while conducted the experiment.

5)
The apparatus must be clean first to ensure the apparatus is clean from the
chemical
reaction.

REFERENCE

http://wiki.answers.com/Q/What_is_the_oxidation_reduction_reaction_b
etween_maltose_and_DNS

http://www.worthington-biochem.com/ba/default.html

http://upendrats.blogspot.com/2011/12/centrifugation-separatingtechnique.html

APPENDIX