Escolar Documentos
Profissional Documentos
Cultura Documentos
Enzymes are proteins that catalyze chemical reactions. In these reactions, the molecules at
the beginning of the process are called substrates, and the enzyme converts them into different
molecules, the products. Almost all processes in a biological cell need enzymes in order to occur
at significant rates. Since enzymes are extremely selective for their substrates and speed up only a
few reactions from among many possibilities, the set of enzymes made in a cell determines which
metabolic pathways occur in that cell. Besides, the microbial amylases meet industrial demands
and a large number of them are available commercially although many microorganism produce
this enzyme. Amylases have attracted the world’s enzyme market because of their wide application
in starch based industries especially food, textile, paper, detergent and baking industries. Most of
the commercially produced amylases are of microbial origin. Therefore enzyme stability and its
The physical characteristic and properties of enzyme are first, enzyme generally work very
rapidly. The action and speed of an enzyme is expressed as its turnover number. The turnover
number is the number of substrate molecules which one mole of the enzyme turns into products
per minute. Then, enzymes can work in either direction. This means that the reaction is either to
the left or to the right until an equilibrium is reached between the substrates and the products
formed. Besides that, enzymes are not destroyed or altered by the reaction they catalyzed. Enzymes
can be reused because they are not destroyed by the reactions they catalyze. However, enzymes
cannot be used indefinitely because they are quite unstable as they can be inactivated by certain
parameters. Enzymes are also sensitive to pH changes because they operated at specific pH range
and any alterations can affect their activity and efficiency. Plus, different enzymes have different
specific pH range. Other than that enzymes are specific in their reaction and enzymes cannot
resistant to excessive heat. They are inactivated to excessive heat causing denaturing when
exposed to high temperatures and this explain that only few cell can tolerate with temperature that
Furthermore, the activity and stability of enzymes can be affected by some factors
including the concentration of substrate molecule and temperature. As the enzyme concentration
increase, the rate of enzyme activity increases up to a level where it becomes constant. This is
because the more the enzymes are available, the more substrates are broken in less time. It then
becomes constant as the substrate acts as a limiting factor which means that there are not enough
substrate to be broken down compared to the number of enzymes. For temperature, as the
temperature rises, molecular motion causing the collisions between enzyme and substrate speed
up. But as enzyme are proteins, there is a limit beyond which enzyme becomes denatured and
The objectives of this experiment is to study the correlation between enzyme concentration
and activity. Besides, this experiment was also to identify the effect of substrate concentration on
amylase activity.
METHODOLOGY
The different concentration of starch solution (0, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0) were
prepared as the substrate for this experiment. Then, about 0.5 ml of the substrate of each
concentration were added into each test tubes. Besides, an amylase solution is prepared by
dissolving 0.2 ml of amylase, 0.5 ml of starch solution, and 0.3 ml of 0.2M phosphate buffer (pH
7) into every test tubes. The solution then incubated in room temperature for approximately 30
minutes and shaken for every 10 minutes time intervals. Then, about 1 ml of DNS reagent was
added into every solutions. These test tubes then were put into the water bath and boiled for 10
minutes and then cooled down before added in 10 ml of distilled water. The absorbance for these
solution were read at the wavelength of 575 nm. A glucose solution were also prepared for the
standard curve by preparing the pure glucose solution in the range of 0 – 1000 mg/L by repeating
the steps used for the amylase solution preparation. The standard then were used in determining
the unknown glucose concentration for the amylase solution samples. The graph of product formed
0.06
0.05
Absorbance
0.04
0.03
y = 0.0122x - 0.0166
0.02
R² = 0.9482
0.01
0
0 61.5 125 250 500 1000
-0.01
Standard concentration of glucose (mg/L)
2.5
1.5
0.5
0
0 5 10 15 20 25 30 35 40
Product formed (mg/L)
This experiment involved the correlation between the percentages of substrate towards the
concentration of glucose produced. The substrate used in this experiment was starch while the
enzyme used to digest the substrate was amylase. Basically, break down or hydrolyzes of starch
by amylase will produce maltose which is disaccharide that made up of two glucose molecules and
therefore, glucose standard curve was constructed first by checking the absorbance of six different
known concentrations of glucoses. Two-fold series of dilution was applied in order to obtain all
these concentrations ranging from 0 mg/mL to 1000mg/mL and by plotting the standard curve.
In order to study the effect of substrate percentage which is substrate concentration towards
the production of glucoses, six solutions were used with different percentage of substrates starting
from 0%, 0.5%, 1.0%, 1.5%, 2.0% and 3.0%. DNS colorimetric method was applied to determine
the concentration of reducing sugar by adding the DNS reagent before boiled the solution. Based
on the principal, this method will identify the presence of free carbonyl group (C=O) which
represents reducing sugar (J Gou, 2004). Then, the absorbance of every solution was determined
by using spectrophotometer with wavelength of 575nm and the absorbance value obtained can be
used to identify the glucose concentration on the solutions by using glucose standard curve.
According to the results obtained, the concentrations of glucose in the solution were
increased when substrate percentage increased. According to C. J. Fieldeg (1972), in the presence
of a given amount of enzyme, the rate of enzymatic reaction increases as the substrate
concentration increases until a limiting rate is reached. In this case, when starch concentration
increased, there were high amount of starch molecules that bound to the amylase and hydrolyzed
to form glucoses thus increased the rate of reaction. This figure below proved the relationship
between the effects of substrate concentrations towards reaction rate that is catalyzed by fixed
amount of enzyme.
Figure 1.1, the product formed was increasing as the concentration of substrate increased. The
product formed was glucose concentration. As stated by I. Keyser (2011), after a certain
concentration, there will be no effect on the enzyme activity because the enzyme will effectively
become saturated and will be working at their maximum possible even though the substrate
concentrations keep on increasing. From the results obtained, it shows that the amylase were still
active bound to the starch molecules even at 3% concentration because the amount of glucose
concentration was still increased and not yet reached the limiting rate.
CONCLUSION
The main intention of doing this experiment was to study the correlation between
concentration of enzyme and its activity and also the effect of substrate concentration on the
amylase activity. In this experiment, the enzyme used was amylase while the substrate was starch.
Besides, based on the graph plotted, the product formed increasing as the concentration of the
substrate increased. Hence, by referring to the theory, increasing enzyme concentration will
increased the rate of reaction due to high collision occur between the enzymes and substrates
molecules.
APPENDIX
https://www.academia.edu/7347283/Enzyme_Kinetics_Haidhar
enzymes/
St. Rosemary. (2010). Factors Affecting The Activity of Catalase and Amylase Lab Answer.
http://schoolworkhelper.net/factors-affecting-the-activity-of-catalase-and-amylase-lab-
answers/
APPENDIXES
To get the concentration of substrate (shown in Table 3) from standard curve of glucose
y = mx + c
y = 0.0122x – 0.0166
y = 0.0122x – 0.0166
x = 13.0819