17
ABSTRACT
Parameters such as temperature, pH and substrate concentration, were put to test on the activity of Amylase enzyme to hydrolyze starch to produce glucose and the reaction time was also taken. 2% starch solution was added with amylase enzyme along with citrate-phosphate buffer (pH7). The solution was used as the basis for all experiments regarding different parameters to be tested. The Amylase activity is quantified by taking the absorbance value for each sample is taken at 540 nm and graph of amylase activity versus pH, temperature and substrate concentration were plotted. In order to obtain the glucose concentration in reference to a previously prepared glucose concentration calibration curve the reactions at different parameters were measured according to the optical density (OD) of the solution. It was found out that the functions optimally at temperature and pH of 40 and 9 respectively. Starch was used as the substrate medium and the enzyme was found to be increasingly active as the substrate concentration increased accordingly.
INTRODUCTION
Enzyme kinetics represents the study of the rate at which an enzyme works usually as a function of the enzyme parameter available to the enzyme. Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical reaction without being used up in the process. They achieve their effect by temporarily binding to the substrate and, in doing so, lowering the activation energy needed to convert it to a product (Campbell, 2008).
(Retrieved from www.biotek.com, June 4, 2014)
The rate at which an enzyme works is influenced by several factors; and for examples:
the concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in units of molarity.
the temperature. As the temperature rises, molecular motion — and hence collisions between enzyme and substrate — speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective.
the presence of inhibitors.
competitive inhibitors are molecules that bind to the same site as the substrate — preventing the substrate from binding as they do so — but are not changed by the enzyme.
noncompetitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power.
pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected.
Enzyme amylase according to Singh & Kayastha (2014), is a metalloenzyme belonging to the glycosyl hydrolase family and functions primarily in catalysing the endohydrolysis of starch molecules into a range of shorter chains of polymers or monomoers such as glucose, maltose and maltodextrins. Amylase occurs in mammalian saliva and small intestines and is found in the intestinal tract of most animals. It can also be found in plants where its presence acts as a defense mechanism which not only degrade stored starch and in some bacteria and fungi that attack plants (Adams and Kelly, 1995).
Amylase is very sensitive towards the changes in their environment giving rise to vulnerability and instability. They require a specific environment in order for them to function optimally. Slightly changes in pH or temperature can halt the enzyme functionality due to shape changing occurring within the enzyme molecule itself (Robert K. Scopes, 2002). Thus scientists are constantly trying to improve the functionality of the amylase through studies and research to optimize processes.
OBJECTIVES
To determine the effect of temperature on enzymatic activity and changes in enzyme concentration of an enzyme-catalyzed reaction.
To describe the relationship between substrate concentration and the maximum velocity of an enzyme.
To estimate the Michaelis-Menten parameters, effect of pH and temperature on enzyme activity and kinetics of inhibition.
THEORY
As we all know enzymes are protein molecules composed of small chains of amino acids and are manufactured by the living cell. These molecules provide energy for the organism by catalyzing various biochemical reactions. If enzymes were not present in cells, most of the chemical reactions would not be able to take place at measurable rates and at the temperatures of living systems. Each enzyme has at least a single active site which is the location where the enzyme binds to the substrate. In this way the substrate is held rigidly in the most favorable orientation. Within the active site there are various chemical groups that are involved in the reaction. It is important to remember that enzymatic reactions usually result in the addition or removal of some molecule or radical such as H2O, -OH, -H, -NH2 , etc.
Each enzyme possesses a pH and a temperature optimum for its activity. This optimum pH and temperature can be easily determined in the laboratory by carrying out the reaction in buffers over a wide range of pH or conducting tests at different temperatures. Enzymes demonstrate a rather high degree of specificity with respect to their substrates. The degree of specificity varies from enzyme to enzyme: some enzymes carry out a reaction in only one direction (e.g., dehydrogenation) but some will catalyze a reaction in both forward and reverse directions although usually at greatly different rates (e.g., hydrogenation in addition to dehydrogenation). Some enzymes will accept only one or two specific substrate molecules; others accept whole classes or subclasses of molecules as substrates. This specificity is the basis for enzyme nomenclature: according to the kind of reaction performed (e.g., hydrogenase) or, even more specifically, according to the substrate acted upon (e.g., succinic dehydrogenase).
The simplest possible case of an enzyme (E)-catalyzed reaction involves a single substrate (S) molecule giving rise to one product (P):
E + S ---> P + E
and we find that the amount of P formed increases with time until a plateau is reached as shown
in figure below:
Product concentration as a function of time for an enzyme catalyzed reaction.
We are usually concerned with the initial rate (or initial velocity) value (V0) which is the slope measured very near t=0; it is an important value for characterizing an enzymatic reaction. It is observed that the velocity depends on the concentration of E as depicted in figure below:
Product concentration as a function of time and enzyme concentration.
A more critical demonstration of the effect of enzyme concentration upon the reaction in question is given by varying [E] and holding time constant. This produces a curve similar to that of the previous one but with [E] replacing t at the abscissa.
The latest graph shows what happens when [E] is held constant and [S] is varied. Here we will obtain a curve. In this graph, the rate of product formation is called the velocity of the reaction. Notice how the velocity ( O.D. measured by a spectrophotometer) reaches a maximum as substrate concentration reaches a saturation level. Doubling [E], under certain conditions, doubles Vmax, but the reaction rate always reaches a plateau at high [S].
MATERIALS AND APPARATUS
Materials
DNS Reagent
Soluble starch
Amylase enzyme powder
Glucose powder
Distilled water
Buffers
Apparatus
Test tubes
Water bath
Beaker
Measuring cylinder
Micro pipette
Cuvette
Vortex mixer
Spectrophotometer
Hotplate
PROCEDURE
I. Glucose Standard Curve Preparation.
Different concentrations of glucose solution (ranging from 1 to 10 g/L)
were prepared.
1 ml of glucose solution of each concentration were added into individual
test tubes, and they were labeled according to their concentrations.
1 ml of DNS reagent was added into each test tube and mixed for a few
seconds with a vortex mixer.
All the tubes were placed in a water bath ( at T= 100°C) for 10 min and
left to cool at room temperature.
The absorbance of the sample was taken at a wavelength of 540 nm.
The standard curve of absorbance vs glucose concentration was plotted.
II. Demonstration of Enzyme Activity
After 10 minutes of reaction time, 4ml of DNS reagent was added to each
of the test tubes to stop the enzyme reaction.
The test tubes were boiled for 10 minutes and then left to cool to room
temperature.
The absorbance value for each sample was measured at 540 nm and recorded.
III. Preparation of Starch Solution
4g of soluble starch was mixed with 200 ml of distilled water.
The solution was stirred until well-mixed, then 100 ml of boiling water
was added to the mixture.
The solution was filled with distilled water up to the 200 ml mark, and
mixed well.
IV. Effect of pH on the activity and stability of amylase enzyme
5 test tubes were labeled with pH 4, 5, 6, 8 and 9. 1 ml of 2% starch solution
was added to each test tube.
1 ml of each of the corresponding buffers were added into the test tubes.
Another 5 test tubes were filled with 2 ml of amylase solution.
All 10 test tubes were placed in a 37°C water bath for 5 minutes to allow
the temperature to equilibrate.
The contents in the test tube containing the amylase solution were poured
into the test tubes containing the pH solution. It was then mixed with a
vortex mixer.
The tubes were placed in the water bath for another 10 minutes (for hydrolysis
reaction to occur).
Absorbance was measured using method stated in section II.
A graph of optical density vs pH was plotted.
V. Effect of temperature on the activity and stability of amylase enzyme
1 test tube was labeled with 30°C and 1 ml of a mixture of 2% starch solution
and pH 7 buffer was added to the test tube.
2 ml of amylase solution was added to another test tube.
Both test tubes were placed into 30°C water bath for 5 minutes to allowing
the temperature to equilibrate.
The contents in the test tube containing amylase solution was poured into
the test tube containing the buffer and starch solution. It was mixed with
the vortex mixer.
The tubes were placed in the same temperature water bath for another 10
minutes (for hydrolysis reaction to occur).
The OD was determined using the method stated in section II.
Steps 1 to 6 were repeated for temperatures ranging from 40 – 70°C.
A graph of OD vs temperature was plotted.
VI. Effect of substrate concentration on the activity of amylase enzyme
Starch solutions of concentrations 0.5, 1.5, 2.0 and 2.5 % (w/v) were prepared
for use as the substrate.
Each test tube was labeled accordingly and 1 ml of starch solution of the
corresponding concentrations were added to the tubes respectively.
1 ml of pH 7 buffer was added to each of the tubes.
2 ml of amylase solution was added into another 4 individual test tubes.
All test tubes were placed into a 37°C water bath for 5 minutes to allow
the temperature to equilibrate.
The contents in the test tubes containing amylase solution were poured
into the test tubes containing buffer and starch solutions. They were mixed
with the vortex mixer for a few seconds.
The tubes were placed in the water bath for another 10 minutes (for hydrolysis
reaction to occur).
The OD was determined using the method stated in section II.
A graph of OD vs starch concentration is plotted.
RESULTS AND CALCULATIONS
Effect of Temperature
Based on the Glucose Calibration Curve (Appendix A1), the glucose concentration values obtained are as stated in the table shown below. From these values, Equation (2) is used to determine the amount of glucose produced (g/L) over the reaction time which was 10 minutes:
Enzyme Activity (gL.min) = Glucose Concentration (gL)Reaction time (min)
Sample calculation for temperature 30 °C, with OD of 0.582 is as follows:
Enzyme Activity (gL.min) = 0.18 g/L10 min = 0.018 gL.min
Temperature (°C)
Absorbance Value (540 nm)
Glucose Concentration (g/L)
Enzyme Activity (g/L. min)
30
0.582
0.18
0.018
40
0.731
0.245
0.0245
50
0.279
0.165
0.0165
60
0.240
0.090
0.0090
70
0.180
0.055
0.0055
Table of Manipulated Temperatures for Enzyme Reaction
Effect of pH
Based on the Glucose Calibration Curve (Appendix A1), the glucose concentration values obtained are as stated in Table 5.1. From these values, Equation (1) is used to determine the amount of glucose produced (g/L) over the reaction time which was 10 minutes.
pH
Absorbance Value (540 nm)
Glucose Concentration (g/L)
Enzyme Activity (g/L. min)
4
3.081
1027
102.7
5
3.300
1100
110
6
3.375
1125
112.5
8
3.384
1128
112.8
9
3.014
1005
100.5
Manipulated pH Values for Enzyme Reaction.
Effect of Substrate
Substrate Concentration (g/L)
Absorbance Value (540 nm)
Glucose Concentration (g/L)
Enzyme Activity (g/L. min)
0.5
0.103
1027
102.7
1.5
0.130
1100
110
2.0
0.160
1125
112.5
2.5
0.188
1128
112.8
Manipulated substrate concentrations for enzyme reaction.
DISCUSSION
It can be seen from the results that the enzyme activity was at its peak at 40 , where at temperatures beyond this, the activity of the enzyme decreased according to the absorbance reading. This shows that amylase has an optimum temperature of 40 . The obtained graph of the effect of pH on enzyme kinetics, the optimum pH for amylase from B. subtilis is around 8. Each enzyme has its own pH value where it functions the best. Any value above or below, as suggested in the theory, results in inefficient reaction between the enzyme and substrate, producing a low concentration of glucose as a result. This is proven through correspondence with the OD value obtained from the samples. There can be many factors as to why the enzyme activity decreased on either side of the optimum such as the affect of pH instability. Various physical, chemical and genetic approaches have been applied in order to enhance enzyme stability and activity. The major drawback to the widespread usage of many enzymes compared to chemical catalysts is their relatively low stability in their native state (Senyay-Oncel & Yesil-Celiktas, 2011). This gives it the tendency to be irreversibly inactivated. Other than that the kinetics of the reaction could also be affected by the pH as the stability of the enzyme-substrate complex. The optimum value is when the best reaction occurs and it indicates that the arrangement of an active sites of an enzyme is perfectly fixed by the hydrogen and ionic bond between the substrate and the enzyme. Theoretically, the more substrate is present or available for the enzyme to hydrolyze, the higher the concentration of the product should be however only up to a certain point which is known as Vmax. Beyond this point, the velocity doesn't change as all of the available enzymes have been converted into enzyme-substrate complexes. Although, in the graph plotted obtained from the experiment, no Vmax value is observed. This is probably due to the fact that the substrate concentration was still not saturated so as to cause all enzyme active sites to be occupied. This resulted in a graph that was still struggling and climbing to reach the Vmax. Without the value of Vmax, the optimum substrate concentration is not known, and calculations of Km using the Michaelis-Menten equations are not possible.
CONCLUSION
The experiment was conducted successfully and all the objectives were achieved; however not conclusive in relevance to the optimum substrate concentration. The optimum temperature and pH for efficient functionality of Amylase enzyme obtained from the experiment is about 8 and 40 respectively. The substrate concentration in correspondence to Vmax could not be obtained as no plateau of the graph was observed.
RECOMMENDATION
Every effects of the parameters involved should be handled carefully according to the laboratory manual and guidelines.
The Michaelis-Menten parameters can be estimated more appropriately when the experiment for effect of substrate concentration is extended for higher starch concentration maybe until 5.0 and 6.0 g/L of starch.
With higher concentration of starch, the reaction can proceed until it reaches constant level and the Vmax and Km of the reaction can be determined.
Starch analysis method can be used to enhance the reaction process. It can be accomplished by mixing the starch solution with the buffer.
To measure the starch consumed in the reaction carried out, the starch-iodine assay can also be used.
REFERENCES
Neil A. Campbell, Jane B. Reece, 2008 Biology, 8th Edition, Pearson Publication.
Kritika Singh, Arvind M. Kayastha, α-Amylase from wheat (Triticum aestivum) seeds: Its purification, biochemical attributes and active site studies, Food Chemistry, Volume 162, 1 November 2014, Pages 1-9.
A.Rogers & Y.Gibon.2009.Chapter 4.Enzyme Kinetics: Thoery & Practice.
Adams M. W. W, and Kelly, R. M. (1995). Enzymes from microorganisms in extreme environments. Chemical and Engineering News 73. Pp: 32-42.
Robert K. Scopes (2002). Enzyme Activity and Assays. Encyclopedia of Life Sciences. Retrieved 14 May 2014.
Appendix 1
Enzyme Activity based on Substrate Concentration
Substrate Concentration (g/L)
OD
Enzyme Activity Based on Temperature
Temperature (°C)
OD
Enzyme Activity Based on pH
pH
OD
