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)

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

2)
at

0.47g 0f ammonium sulphate is added in each 1ml supernatant. Then, it is centrifuge

12,000 rpm for 10 minutes.

3)
are

Pellets were suspended with 1ml of distilled water for each 8ml of original pulp and
centrifuge at 12,000 rpm for 10 minutes.

Analysis: Determination of amount of amylase activity

1)
9,

0.1 ml of sample is mixed with 0.1 ml of 1% starch and 0.1ml of buffer at pH 3, 5, 7,

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)

0.8
1.0
1.4

Absorbance (540 nm)

0.241
0.461
0.755

Table of standard curve of maltose

pH of buffer solution
3
5
7
9
11

Absorbance (540 nm),y

0.824
0.992
0.713
0.861
0.887

Concentration (mg/mL),x
1.470
1.670
1.337
1.514
1.545

Table of value obtained from equation y=mx+c

pH value of

Concentration

Micromoles of

Rate of reaction

Enzyme activity

buffer solution

(mg/mL)

maltose (moles)

of micromoles

(u/mL)

maltose formed
3
5
7
9
11

1.470
1.670
1.337
1.514
1.545

4294.53
4878.82
3905.98
4423.07
4513.64

per minute (u)

286.30
325.25
260.40
294.87
300.91

114.52
130.10
104.16
117.95
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 3-amino,5-nitro salicylic acid which is orangered 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 -1,4glucan 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 R 2 = 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_betw
een_maltose_and_DNS

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

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

APPENDIX