INTRODUCTION

An experiment was conducted on Thursday, 30th November 2017 with several objectives; to determine
the effect of temperature on the 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 and estimation of Michaelis-Menten parameters, effect of pH and temperature on
enzyme activity and kinetics of inhibition.

Enzyme kinetic by mean is the study of chemical reactions that are catalyzed by enzymes where the
reaction rate is measured.The enzyme used was Alpha Amylase enzyme and starch was used as the
substrate. The 2% starch solution was prepared by mixing soluble starch in cold water, then stirred until
it turn into slurry. The enzyme and starch reacted in a test tube and was placed in water bath according
to parameter set. After 10 minutes, the mixture were added with dinitrosalicylic acid (DNS reagent) which
strongly absorbed light at 540 nm. Enzyme perform the task of lowering a reactions activation energy
that the amount of energy that must be put in for the reaction to begin. The difference between the
transition state (unstable part of the reaction) and the reactants is the Gibbs free energy of activation,
commonly known as activation energy. Enzymes change the formation of the transition state by lowering
the energy and stabilizing the highly energetic unstable transition state. This allows the reaction rate to
increase, but also the back reaction occurs more easily.
Arrhenius equation is a description of the relationship between the activation energy and the reaction
rate.

k = Ae(-Ea/RT)

where: k = chemical reaction, T = temperature in Kelvin, Ea = activation energy, A = the pre-exponential

factor, R = the gas constant.

According to this equation, it is observed that at a higher temperature, the probability that the two
molecules will collide is higher, resulting in a higher kinetic energy, which leads to the lower requirement
on the activation energy.

Enzymatic activity changes according to temperature due to increase in velocity and kinetic energy
between molecules. The faster velocity, the less time between collisions. Since the molecules are also
moving faster, collision between enzymes and substrate also increase. Changes in pH may not only affect
the shape of an enzyme but it also change the shape or change properties of the substrate either the
substrate cannot bind to the active site or unable to undergo catalysis. The most favorable pH value
(where the enzyme is the most active) is known as the optimum pH.

The application of enzyme mostly in food processing where plant enzymes are important because the
enzymes are capable to digest food before the bodys own digestive process begins. In other words, the
enzymes can enhance the digestion of food and delivery of nutrients to the blood. Digestive enzymes
break down protein, carbohydrates and fats into progressively smaller components.

Furthermore, there are importance different enzymes with a special focus on amylase and lipase.
Generally, enzymes increase the reaction rates by several million times than normal chemical reactions.
Lipase play an important role in the food, detergent, chemical and pharmaceutical industries. The major
advantage of enzymes is being economical and easy to manipulate.
THEORY
This experiment was conducted to investigate on enzyme activity and kinetics. Enzymes are proteins that
speed up the rate of a chemical reaction without being consumed. Enzymes usually specific to particular
substrates. The function of substrates in the reaction is binding to active sites on the surface of the
enzyme. The enzyme-substrate complex undergoes a reaction to form a product. Enzymes are considered
as a potential biocatalyst for various reactions. The enzymes are also more active and stable than plant
and animal enzymes. In addition, microorganisms offer an alternative source of biochemical diversity and
susceptibility to gene manipulation.

The rate of a chemical reaction is affected by the total number of enzymes as well as the concentration of
substrates. The relationship between substrate concentration and reaction in enzyme-substrate complex
is of considerable theoretical importance. Consider reaction AB, single molecular (A) catalyzed by an
enzyme E. Supposed on molecule of A to combine reversibly with one molecule of E forming an enzyme-
substrate complex which then decomposes irreversibly into free enzyme and product of reaction (B). The
reaction may be represented:

+ +

In the experiment, amylase was used as the enzymes that degrade starch (polymer of glucose) into smaller
disaccharides (maltose). A molecule of water split during reaction and the OH- and H+ ions bind to the
exposed ends of the broken starch polymer. This reaction called as hydrolysis (water splitting) where it is
common for enzymes to break chemical bonds.

The reaction of hydrolysis can be measured through the use of an enzyme test or assay. An enzyme assay
will test for the enzyme activity that present but also can be used to measure the reaction rate of an
enzyme-catalyzed reaction. The assay can measure either the appearance of product or the
disappearance of the substrates over time.
Enzyme activity (reaction rate) is dependent upon temperature, pH and etc. This is because these
conditions can be alter the amino acid chains in protein, affecting protein structure and folding and
sometimes the enzymes active site.

Effect of changes pH on enzyme activity.

Changes in pH can alter an enzymes shape. Different enzymes work best at different pH values. Most
enzymes have a characteristic optimum pH at which the velocity of catalyzed reaction is maximal and
above and below which the velocity declines. As the pH changes, the ionization group at the enzymes
active site and on the substrate can alter, influencing the rate of binding of the substrate to the active
site. At the certain pH, the conformation of the protein will be optimal for its function and this is where it
will be have maximum activity (the curve can be steeper). As pH deviates to either side of the optimum,
the conformation changes and the structure will no longer be correct for proper function.

Effect of changes temperature on enzyme activity.

Changes in temperature give several effect on enzyme activity. Increasing temperature increases kinetic
energy that molecules possess. Since enzyme catalyse reactions by randomly colliding with Substrate
molecules, increasing temperature increases the rate of reaction, forming more product. Furthermore,
increasing temperature also increase the vibrational energy that molecules have that could put strain on
the bonds that hold them together. More bonds especially the weaker hydrogen and ionic bond will
break as a result of this strain. Breaking bonds within the enzyme will cause the active site to change
shape where it will be les complementary for the shape of Substrate. Due to that, catalyse reaction can
no longer happen and enzyme will become denatured. In conclusion, as the temperature increases,
initially the rate of reaction will increase because of increased in kinetic energy. However, the effect of
bond breaking will become greater that effect the rate of reaction will begin to decrease.
Effect of substrate concentration on enzyme activity.

During enzyme-substrate reaction, the initial velocity will gradually increase with increasing concentration
of the substrate. When a point is reached, beyond which the increase of initial will not depend on the
concentration of substrate. The velocity increases until it reached a certain point where afterwards the
velocity remains constant without any further increase. The maximum velocity of an enzyme catalyzed
reaction under substrate concentration is called Vmax, Maximum velocity.

Part A - at lower concentration of substrate, steeper slope show an increases in the rate of reaction with
increasing substrate concentration. The active site of the enzyme is empty, waiting for substrate to bind
for time and the rate at which product can be formed is limited by the concentration of substrate which
is available.
Part B - As the concentration of the substrate increases, the enzyme becomes saturated with substrate.
As soon as the active site is empty, more substrate is available to bind and undergo reaction. The rate of
formation of product now depends on the activity of the enzyme itself, and adding more substrate will
not affect the rate or reaction to any significant effect.

The relationship between rate of reaction and concentration of substrate depends on the affinity of the
enzyme for its substrate. Km (Michaelis constant) of the enzyme is an inverse measure of affinity. Km is
the concentration of substrate which permits the enzyme to achieve half Vmax. Enzyme with a high Km
has a low affinity for its substrate and requires a greater concentration of substrate to achieve Vmax.

The Lineweaver-Burk (double reciprocal) plot that rearranges from the Michaelis-Menten was used.

1 1
= +

Plotting using this equation give a straight line.

1
Intercept -

Gradient -

1
X-intercept -