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

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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.

o competitive inhibitors are molecules that bind to the

same site as the substrate — preventing the substrate

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from binding as they do so — but are not changed by the

enzyme.

o 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

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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

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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):

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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:

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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.

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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

MaterialsDNS ReagentSoluble starchAmylase enzyme powderGlucose powderDistilled waterBuffers

ApparatusTest tubes Water bath Beaker Measuring cylinder Micro pipette Cuvette

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Vortex mixerSpectrophotometerHotplate

PROCEDURE

I. Glucose Standard Curve Preparation.

1. Different concentrations of glucose solution (ranging from 1

to 10 g/L)

were prepared.

2. 1 ml of glucose solution of each concentration were added

into individual

test tubes, and they were labeled according to their

concentrations.

3. 1 ml of DNS reagent was added into each test tube and mixed

for a few

seconds with a vortex mixer.

4. All the tubes were placed in a water bath ( at T= 100°C) for

10 min and

left to cool at room temperature.

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5. The absorbance of the sample was taken at a wavelength of

540 nm.

6. The standard curve of absorbance vs glucose concentration

was plotted.

II. Demonstration of Enzyme Activity

1. After 10 minutes of reaction time, 4ml of DNS reagent was

added to each

of the test tubes to stop the enzyme reaction.

2. The test tubes were boiled for 10 minutes and then left to

cool to room

temperature.

3. The absorbance value for each sample was measured at 540 nm

and recorded.

III. Preparation of Starch Solution

1. 4g of soluble starch was mixed with 200 ml of distilled

water.

2. The solution was stirred until well-mixed, then 100 ml of

boiling water

was added to the mixture.

3. 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

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1. 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.

2. 1 ml of each of the corresponding buffers were added into

the test tubes.

3. Another 5 test tubes were filled with 2 ml of amylase

solution.

4. All 10 test tubes were placed in a 37°C water bath for 5

minutes to allow

the temperature to equilibrate.

5. 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.

6. The tubes were placed in the water bath for another 10

minutes (for hydrolysis

reaction to occur).

7. Absorbance was measured using method stated in section II.

8. A graph of optical density vs pH was plotted.

V. Effect of temperature on the activity and stability of amylase

enzyme

1. 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.

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2. 2 ml of amylase solution was added to another test tube.

3. Both test tubes were placed into 30°C water bath for 5

minutes to allowing

the temperature to equilibrate.

4. 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.

5. The tubes were placed in the same temperature water bath

for another 10

minutes (for hydrolysis reaction to occur).

6. The OD was determined using the method stated in section

II.

7. Steps 1 to 6 were repeated for temperatures ranging from 40

– 70°C.

8. A graph of OD vs temperature was plotted.

VI. Effect of substrate concentration on the activity of amylase

enzyme

1. Starch solutions of concentrations 0.5, 1.5, 2.0 and 2.5 %

(w/v) were prepared

for use as the substrate.

2. Each test tube was labeled accordingly and 1 ml of starch

solution of the

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corresponding concentrations were added to the tubes

respectively.

3. 1 ml of pH 7 buffer was added to each of the tubes.

4. 2 ml of amylase solution was added into another 4

individual test tubes.

5. All test tubes were placed into a 37°C water bath for 5

minutes to allow

the temperature to equilibrate.

6. 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.

7. The tubes were placed in the water bath for another 10

minutes (for hydrolysis

reaction to occur).

8. The OD was determined using the method stated in section

II.

9. A graph of OD vs starch concentration is plotted.

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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) =

GlucoseConcentration (gL

)

Reactiontime(min)

Sample calculation for temperature 30 °C, with OD of 0.582 is as

follows:

Enzyme Activity ( gL.min) =

0.18g /L10min = 0.018 g

L.min

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Temperature

(°C)

Absorbance

Value (540 nm)

Glucose

Concentration

(g/L)

Enzyme Activity

(g/L. min)

30 0.582 0.18 0.01840 0.731 0.245 0.024550 0.279 0.165 0.016560 0.240 0.090 0.009070 0.180 0.055 0.0055Table of Manipulated Temperatures for Enzyme Reaction

2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 3.450246810

Enzyme Activity Based on Temperature

Series2

Temperature (°C)

OD

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.

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pH Absorbance

Value (540 nm)

Glucose

Concentration

(g/L)

Enzyme Activity

(g/L. min)

4 3.081 1027 102.75 3.300 1100 1106 3.375 1125 112.58 3.384 1128 112.89 3.014 1005 100.5

Manipulated pH Values for Enzyme Reaction.

4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.52.8

2.9

3

3.1

3.2

3.3

3.4

3.5

Enzyme Activity Based on pH

Series2

pH

OD

Effect of Substrate

Substrate

Concentration

Absorbance

Value (540 nm)

Glucose

Concentration

Enzyme Activity

(g/L. min)

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(g/L) (g/L)0.5 0.103 1027 102.71.5 0.130 1100 1102.0 0.160 1125 112.52.5 0.188 1128 112.8

Manipulated substrate concentrations for enzyme reaction.

0 2 4 6 8 10 120

5

10

15

Enzyme Activity based on Substrate Concentration

Series1

Substrate Concentration (g/L)

OD

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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

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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

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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.

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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.

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Appendix 1

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