THEORY

Amylase enzymes play a crucial role in biotechnological industries and has large potential
applications in food, fermentation, textile and paper industries. Amylases can be extracted many
sources like plant, animal, microbes. Based on Michaelis-Menten equation, Kinetics also can be
defined as velocity. Michaelis-Menten model was proposed in 20th century by Leonor Michaelis and
also Maud Leonora Menten to explain the mechanism of the enzyme work on the substrates.
Enzymes are found in every single living cell and controlling the metabolic procedures in which
they changed over supplements into energy and new cells.

Michaelis-Menten mechanism of enzyme kinetics shows as:

E: enzymes
S: substrate
ES: combination of enzymes and substrate
The rate of product then equals to

By applying pseudo steady state hypothesis (PSSH), The transient species that is enzyme-
substrate complex ES is set up for an equation for its rate of change. Rate of change ES equals to
zero.

Then [ES] as a subject

Substituting equation (3) into (2) to generate new equation

Rearrange all equation Michaelis-Menten constant KM is introduced

Km value, equation (6) t

Since the value of [E] that is the concentration of non-complexed enzyme (enzyme that is
not bind to the substrate) is difficult to find, therefore we insert the value of [E]0 that is known total
enzyme concentration. The following equation shows how [E] and [E]0 relates:

Equation (9) then is inserted into equation (7) to become velocity of reaction

Then Vmaxcan be determined by

The equation of v then is changed into lineweaver-burk equation to linearize the equation.

According to equation 14, graph 1/v vs 1/[S], a straight line in which the gradient is KM /
VMAX and the y-intercept that is 1/vmax. The enzyme -Amylase can catalyze the hydrolysis of inward
- 1, 4-glycosidic bond present in starch with the creation of lessening sugars. In the investigation
of substrate concentration on enzyme kinetics, the enzyme is kept steady whereas the concentration
of starch is taken in increasing order. As the substrate concentration increases, the measure products
produced in each progressive tube increases. Clarification by Michaelis and others that an enzyme
catalyzed reaction at different substrate concentrations is diphasic, that means, at low substrate
concentration the active sites on particles (enzyme) are not possessed by substrate and the enzyme
rate changes with substrate atoms concentration. As the quantity of substrate atoms builds, the
enzyme accomplishes the immersion level, subsequent to there is no more reaction sites staying for
binding. So the enzyme can work with full limit and its reaction rate is free of substrate
concentration .
B. subtilis is the bacteria that has been applied in industry to produce amylase. Once the
amylase is being produced, the solution that consist of enzyme and bacteria needed to be separated
by using centrifuged. So the centrifuging technique can separate supernantant and slurry under high
gravitational force exerted on it. Supernantant consist of amylase while slurry consist of the
bacteria. So supernantant is extracted and slurry is deposed. Then, the enzyme activity is
determined in the experiment enzyme-substrate. Using the spectrophotometer can be determined
concentration of starch converted over time. So high of optical density show high concentration of
enzymes is being produced by bacteria.

Reaction of enzyme-substrate in different temperature shows different result. Optimal

temperature for the amylase is 37C. so below the optimal temperature the reactivity is decreasing
while above the optimal temperature, the reactivity will also decrease and denaturation begins to
occur.

CONCLUSIONS

The experiment was initially aimed to extract the amylase enzyme produced, to quantify the
enzyme activity and to calculate glucose calculation by using a calibration curve. Based on the
result obtained, at low temperature the enzyme activity is high (0.3105) while at high temperature,
the enzyme activity is low(0119). In addition, microorganisms at low temperature produce a high
efficiency enzymes. By using a calibration curve, the concentration of the first sample is 152.75
mg/L and the second sample is 57 mg/L. This prove that high temperature killed the eszyme and not
suitable for microbial growth.

References
1. N/A http://cbc.arizona.edu/~salzmanr/480a/480ants/enzymekn/enzymekn.html
2. http://www.biology-pages.info/E/EnzymeKinetics.html
3.