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Full Report Enzyme 1
Full Report Enzyme 1
ABSTRACT................................................................................................................................................ 2
1.0INTRODUCTION .................................................................................................................................. 2
2.0 OBJECTIVES ....................................................................................................................................... 3
3.0 THEORY ............................................................................................................................................. 4
4.0 APPARATUS AND MATERIALS ........................................................................................................... 6
5.0 PROCEDURES..................................................................................................................................... 7
6.0 RESULT ............................................................................................................................................ 11
7.0SAMPLE CALCULATION .................................................................................................................... 17
8.0 DISCUSSION..................................................................................................................................... 20
9.0 CONCLUSIONS ................................................................................................................................. 22
10.0 RECOMMENDATIONS.................................................................................................................... 23
11.0 REFERENCES .................................................................................................................................. 23
11.0 APPENDIX ...................................................................................................................................... 24
ABSTRACT
The experiment is done to determine the effects of temperature, substrate concentrations, and
pH on the enzyme activity. Other than that, the experiment is done to calculate the maximum
velocity based on the result obtained whether it is same as in the theory or not. There are 4
experiments being done simultaneously. The enzyme used in this experiment is alpha
amylase. Amylase were being tested with starch by using different parameters; temperature,
pH and substrate concentration. Based on the results that has been plotted and calculated, it
can be said that the graph is successfully constructed. Each effect and parameters are all been
discuss. The objective of the experiment is achieved and hence the experiment is completed
successfully.
1.0 INTRODUCTION
Enzymes are often described as organic catalysts which increase the rate of reaction.
[1]
One of them is amylase. Amylase generally known as the enzyme which catalyses the
hydrolysis of starch and glycogen is among the most important industrial enzymes. Also, the
enzyme has been long studied and it may be said that the history of study amylase is as long
as that of enzymes. Hydrolysis is a common mechanism used by enzymes to break chemical
bonds. The hydrolysis of starch can be measured through the use of an enzyme test or assay.
An enzyme assay will test for the simple presence of enzyme activity but can also be used to
measure the reaction rate of an enzyme-catalysed reaction. The assay can measure either the
appearance of one of the products or the disappearance of one of the substrates over time.
The aims for this experiment is to determine the effect of temperature on the
enzymatic activity and changes in enzyme concentration of an enzyme-catalyzed reaction,
describe the relationship between substrate concentration and the maximum velocity of an
enzyme and estimation of Michaelis-Menten parameters, effect of pH and temperature on
enzyme activity and kinetics of inhibition. Enzyme activity is dependent upon the
environmental conditions either in nature or in the laboratory. This is because these
conditions can alter the amino acid side chains in protein, affecting protein structure and
folding and sometimes the enzyme’s active site. The effect of some conditions will be
explored in this experiment.
2.0 OBJECTIVES
Enzymes are proteins produced in living cells that helps to accelerate or catalyze the
metabolic processes of an organism. It is very selective in the molecules that they act upon,
called substrates which often reacting with only a single substrate. An active site located at an
enzyme is a placed for the substrate to bind before the reaction takes place. Enzymes can
speed up the chemical reactions but only function within a narrow temperature and pH range,
otherwise they can lose their structure and become denatured. It also involved in the
processes of breaking down of large protein, starch and fat molecules in food into smaller
molecules during digestion [2].
The second step’s rate (k2) is the enzyme’s turnover for enzyme-substrate converting
to product. The Michaelis constant, Km, obtain from the ratio of the reactions affecting the
enzyme-substrate complex: its formation (k1), dissociation (k-1), and progress towards
product (k2). A lower Km value indicates that an enzyme has a higher affinity for a substrate.
APPARATUS MATERIALS
Beaker Alpha amylase enzyme
Measuring cylinder Starch
Cuvette pH buffer solution (pH 4-9)
Falcon tube rack DNSA reagent
Falcon tube Water bath
Micropipette and tips
Label sticker
Schott bottle
Vortex mixer
Spectrophotometer
Hot plate
5.0 PROCEDURES
B. EFFECT OF TEMPERATURE
1) One test tube was labeled with 30°C and placed with 1ml of 2% starch solution and
1ml of pH=7 buffer.
3) Both tubes were placed in the 30°C water bath for 5 minutes until the temperature
equilibrated.
4) The content of amylase test tube poured onto the starch test tube and mixed on vortex
meter.
1) Five test tubes were labeled with pH 5, 6, 7, 8 and 9 and placed with 1ml of 2% starch
solution and 1 mL of the appropriate buffer to each tube.
3) All the ten test tubes placed in the 37°C water bath for 5 minutes until the temperature
equilibrated.
4) The content of each amylase test tube was added onto each starch test tube and were
mixed on vortex meter.
1) A starch solutions varying concentration (0.5, 1.5, 2.0, 2.5 and 3.0% w/v) was
prepared as the substrate.
2) Each tube with starch concentration labeled and placed each of them with 1ml starch
solution.
4) 2ml of amylase solution was added onto five additional clean test tubes.
5) The content of each amylase test tube poured onto the starch test tube and were mixed
on vortex meter.
1) After the 10 minutes hydrolysis reaction, the reaction stopped by adding 4ml of DNS
agent.
4) The absorbance value was compared with the glucose standard curve that prepared to
obtain the glucose concentration.
3) 1 ml of DNS reagent was added in each test tube and mixed for few seconds on vortex
meter.
4) The test tubes were placed in water bath (T=100°C) for 10 minutes and left cooled at
room temperature.
5
ABSORBANCE OPTICAL DENSITY (OD)
0
0 500 1000 1500 2000 2500
STANDARD GLUCOSE CONCENTRATION
OD vs Temperature
2.35
Absorbance Optical Density (OD)
2.3
2.25
2.2
2.15
2.1
2.05
2
58 60 62 64 66 68 70 72
Temperature (oC)
9.00E-04
Enzyme activity, V (mol/min)
8.80E-04
8.60E-04
8.40E-04
8.20E-04
8.00E-04
7.80E-04
7.60E-04
58 60 62 64 66 68 70 72
Temperature (oC)
OD vs pH value
7
6
5
OD (nm)
4
3
2
1
0
0 2 4 6 8 10
pH
OD vs Substrate Concentration
4.6
4.5
4.4
OD (nm)
4.3
4.2
4.1
4
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration (%)
Figure 8: The effect of absorbance optical density (OD) reading on different substrate concentration
Table 7: The values of enzyme activity at different substrate concentrations and the data for michaelis-menten
2.10E-06
2.05E-06
2.00E-06
1.95E-06
1.90E-06
1.85E-06
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration (%)
1/V vs 1/S
540000
530000
520000
510000
1/V
500000
490000
480000 y = -91941x + 566550
R² = 1
470000
0 0.2 0.4 0.6 0.8 1 1.2
1/S
From the standard curve of glucose plotted in figure 3, the linear equation of the curve is presented
as:
y = 0.0024x
Where,
x: glucose concentration
y: absorbance reading
i) Effect of temperature
x=1.746x10-3 mg/L
i) Effect on Temperature
9.625x10−4 g/mL
Mol of glucose released (mol) = x 2mL
180 g/mol
= 1.069x10-5mol
1.896x10−3 mg/L
Mol of glucose released (mol) = x 2mL
180 g/mol
iii) Effect on pH
1.746x10−3 mg/L
Mol of glucose released (mol) = x 2 mL
180 g/mol
i) Effect on temperature
𝑚𝑜𝑙 1.069𝑥10−5 𝑚𝑜𝑙
Enzyme activity ( )=
𝑚𝑖𝑛 10 𝑚𝑖𝑛
iii) Effect on pH
In this experiment, we want to study the effect of pH, temperature and substrate concentration
toward the enzymes kinetic. In order to determine the glucose concentration for every sample
in this experiment, a standard glucose curve have to be plotted by using a glucose
concentration with a range concentration of 0 to 2000 g/L by serial dilution. In order to obtain
the value of the glucose standard curve, a number of glucose solution with different
concentration and after several step of procedure, the absorbance optical density (OD) value
was measured for each of the samples. The value then was use to plotted the standard glucose
curve and this graph was use to obtain the glucose concentration for each of the experiment
by interpolating the value of OD that was obtain in the experiment in this graph.
One of the variable that we use to determine the effect on the enzymes kinetics is
temperature. In the figure shows that that graph of OD versus temperature shows that the
graph become decreasing as the temperature increase. From the OD value that was recorded,
the enzymes activity was calculated and a graph of enzymes activity versus temperature was
plotted. Based from the graph that have been plotted, the graph become deceasing as the
temperature increasing. Based from this observation, the temperature of the solution can
affect the enzymes activity where the increasing of temperature will cause the enzymes
activity become lower and after a certain high temperature the enzymes will denatured. As
we already know, the temperature has a general effect on the chemical reaction where the
increasing of temperature can typically cause the increasing of reaction rate. As for the
enzymes, the increasing of temperature causes the kinetic motion of amino acid chain in the
enzymes increase. From the range of 0oC to 40oC, the rate of reaction in the enzyme
increasing by double for every 10oC increasing in temperature. Above 40oC, the increasing of
temperature will cause the reducing of the increase in enzymes activity. Most enzymes reach
its peak in activity between 40oC to 50oC and then it became steep at 55oC and start to drop at
60oC. Thus, it is recommended that to operate the process in the optimum temperature of the
most enzymes which is 40oC to 50oC, but different enzymes have different optimum
temperature in which it can reach a peak performance of enzymes kinetic and high
temperature can cause the enzyme kinetics become decreasing and eventually become zero.
For different pH values, the graph of absorbance optical density (OD) against
different pH values was plotted. The graphs decrease gradually as the pH value increases.
The graphs that the highest OD reading is at pH 5 with 6.567 nm and the lowest OD reading
is at pH 9 with only 3.663 nm. Then the results followed with a graph of enzyme activity
(mol/min) against different pH values. The graph shows a same pattern as in graph in figure.
There will be a pH, characteristic of each enzyme, at which the net charge on the
molecule is zero. This is called the isoelectric point (pI), at which the enzyme generally has
minimum solubility in aqueous solutions. In a similar manner to the effect on enzymes, the
charge and charge distribution on the substrate(s), product(s) and coenzymes (where
applicable) will also be affected by pH changes. Increasing hydrogen ion concentration will,
additionally, increase the successful competition of hydrogen ions for any metal cationic
binding sites on the enzyme, reducing the bound metal cation concentration. Decreasing
hydrogen ion concentration, on the other hand, leads to increasing hydroxyl ion concentration
which competes against the enzymes' ligands for divalent and trivalent cations causing their
conversion to hydroxides and, at high hydroxyl concentrations, their complete removal from
the enzyme. [4]
Based on the graph plotted in figure, the result shows that when the concentration of
substrate (starch) increases, the absorbance optical reading (OD) decreases. The graph started
at 1% starch concentration with 4.55 nm OD reading and then decreases to 4.03 nm OD
reading at 3% starch concentration. Next, a graph of enzyme activity (mol/min) against
substrate concentration (%) is also plotted. The graph shows an identical pattern such in the
graph plotted in figure. The graph shows decreasing value of enzyme activity from 2.107E-06
mol/min at 1% starch concentration to 1.866E-06 mol/min at 3% starch concentration. The
same pattern shows that the OD reading is equal to the enzyme activity against the substrate
where the enzyme activity is defined as the amount of glucose formed in reaction
mixture per unit time.
The graph results in such state because the reaction is already at its maximum reaction
rate at the beginning of the experiment. The enzyme and substrate molecules collide
vigorously at this stage and continuous addition of substrate concentration only caused the
reaction rate to slow down. This is because the enzyme slowly becomes ineffective as it is
being used to the maximum where there is no more free enzyme to bind with the substrate
and all the active sites of enzyme are already bound to the substrate.[3]
9.0 CONCLUSIONS
There are few recommendations that need to be taken to increase the Km and Vmax value
during conducting the experiment:
1) Increase the temperature because temperature effects enzymatic reactions as an
increase in reaction rate until it reaches a peak where enzyme can function well.
2) Use another enzyme as different enzymes has different characteristics.
3) Increase in enzyme concentration as it increase the reaction rate by allowing the
active sites in which substrate may bind to perform reactions.
11.0 REFERENCES
1. The Amylase Research Society (2014). Handbook of Amylases and Related
Enzymes: Their sources, isolation methods, properties and applications.
Retrieved from:
https://books.google.com.my/books?id=2lKeBQAAQBAJ&dq=amylase&source=gbs_
navlinks_s
2. The American Heritage® Science Dictionary Copyright © 2011. Published by
Houghton Mifflin Harcourt Publishing Company.
3. Peter J. Russell et al, Biology: The Dynamic Science 3rd Edition Volume 1, 2014,
page 81.
4. Enzyme Technology Effect of pH and ionic strength. Martin Chaplin (6 August, 2014 )
Retrieved from, www1.lsbu.ac.uk/water/enztech/ph.html
11.0 APPENDIX