BIO 462

EXPERIMENT 3
ENZYMOLOGY
PART 1: DETERMINATION OF OPTIMUM
PARAMETERS

NAME: NURFADHILAH BINTI JAAFAR

STUDENT ID: 2016675256

PROGRAM: AS 246

GROUP: 4B

DATE OF THE EXPERIMENT: 5TH APRIL 2017

DATE OF SUBMISSION: 12TH APRIL 2017

LECTURER: MADAM AZANI BINTI SALEH

PROCEDURE

- Refer to Biochemistry Laboratory Manual page 15 until 23 -

RESULTS
1. Enzyme Concentration

Cuvette OD OD OD Mean SD
(replicate 1) (replicate 2) (replicate 3)
1 0.080 0.079 0.078 0.079 0.001
2 0.144 0.143 0.143 0.143 0.0006
3 0.213 0.214 0.214 0.214 0.0006
4 0.287 0.287 0.288 0.288 0.0006

2. Substrate Concentration

Cuvette OD OD OD Mean SD
(replicate 1) (replicate 2) (replicate 3)
1 0.096 0.099 0.100 0.098 0.002
2 0.103 0.105 0.107 0.105 0.002
3 0.094 0.096 0.098 0.096 0.002
4 0.094 0.094 0.095 0.094 0.0006
5 0.068 0.070 0.071 0.0700 0.002
6 0.051 0.053 0.053 0.0520 0.002
7 0.041 0.042 0.044 0.0420 0.002
3. pH

Cuvette OD OD OD Mean SD
(replicate 1) (replicate 2) (replicate 3)
1 0.092 0.087 0.090 0.090 0.003
2 0.072 0.073 0.072 0.072 0.0006
3 0.077 0.080 0.070 0.078 0.005
4 0.072 0.072 0.072 0.072 0
5 0.070 0.073 0.075 0.075 0.003
6 0.230 0.232 0.230 0.231 0.001
DISCUSSIONS

This experiment conducted with the use of spectrophotometer to identify the effect of
enzyme concentration, substrate concentration on the rate of reaction and to determine the
effect of pH on the enzyme activity. The spectrophotometer is a vital machine for researchers
and scientist to in studying various substances and elements. The spectrophotometer was
calibrated and set to 410 nm. The absorbance values and the transmittance of light for the
substance were measured by the photometer at the end of the spectrophotometer machine
which can be plotted on a calibration curve.

Enzymes are the vital part of life because enzyme controls all metabolisms and
metabolic process that occur. The substrate used in this experiment is starch solution while the
enzyme used in this experiment is amylase which is specialized to break down one specific type
of compound. Enzymes amylase can be found in human saliva and responsible to break down
the starches into simple sugars. The result for the effect of enzyme concentration on the rate of
reaction and it shows that as the enzyme concentration increases, the rate of reaction also
increases and the result of this reaction resulted with a linear graph which continuously
increases. In this experiment, the effect of substrate concentration on the rate of reaction also
can be observed with the reading from the spectrophotometer.

The graph of the absorbance against the starch concentration that we constructed
experimentally was not same theoretically. It shows that the reading of the absorbance become
lower as the substrate concentration been added which is opposite from theory. Supposedly
when the substrate concentration increases, the solution become more saturated, the rate of
reaction also increases, but once all the active enzymes are fully occupied by substrates, the
reaction reach the maximum rate and would not increases since all the active sites are all
occupied. The reaction initiates at faster rate and become slower as there is not enough
enzymes to convert the substrates and this can be solved by addition of more enzymes.

The relationship between the effects of pH on the enzyme activity is presented by the
graph constructed absorbance against the pH buffer. pH buffer used on this experiment are
pH3, pH5, pH7, pH9, pH11 and distilled water. The graph experimentally is differing with
theoretically as the enzyme activity become slower when the pH buffer increases. When the pH
was 3 or 5 the reaction yielded minimum result. Theoretically, if there is an increase in pH, the
rate of reaction also increases, until it reaches the peak value and starts to denature. Any small
changes in pH can drastically alter the productivity of the enzymes.
 Overall, we could only get the accepted result for the graph of the effect of enzyme
concentration on the rate of the reaction only. The possible transmission of errors in this
experiment might be due to material handling and personal error which contributed to
inaccuracy of result data. In this experiment the measurement used was by drops of the
concentration is inaccurate measures compared to unit volume such as millimeter which can
contribute to either predictable or accurate results. Other random error also happen when we
used the unclean cuvette, thus the interferences inside the unclean cuvette may react with the
enzyme or substrate concentration added. The systematic error may occur when the
spectrophotometer was not calibrated properly. Other possible ways to see improvements for
this result would have been more replicate to provide much more representation how the curve
on the graph actually is.

CONCLUSIONS

In conclusions, the objective of the experiment to calibrate the spectrophotometer,

determine the enzyme concentration, substrate concentration on the rate of reaction and the
effect of pH on the enzyme activity was achieved. It shows that the graph become linear when
as the amount of the enzyme concentration increases, the rate of reaction also increases. For
both graph of the effect of substrate concentration on the rate of reaction and the effect of the
pH on the enzyme activity, we could not get the desirable curve as theoretical graph curve
which may due to some error.

The pH on the enzyme amylase should have an effect and major influence on the
enzyme activity as the pH and if the substrates are unlimited in availability, then the rate of
reaction of the enzyme also decreases. Overall, it was apparent that change in enzyme
concentration, substrate concentration and pH level does have an effect on the enzyme activity
and the rate of the reaction.