Title: Specificity of enzyme: sugar metabolized by yeast

Introduction: Metabolism is the set of life-sustaining chemical tranformation within the cells of
living organism. The three main purposes of metabolism are the conversion of food to energy
to run cellular processes, the conversion of food to build blocks for protein, lipids, nucleic acid,
and some carbohydrates, and the elimination of nitrogen wasted. These enzyme-catalyzed
reactions allow organisms to grow and reproduce, maintain their structures, and respond to
their environments. The word metabolism can also refer to the sum of all chemical reactions
that occur in living organisms, including digestion and the transport of substances into and
between different cells, in which case the set of reactions within the cells is called intermediary
metabolism or intermediate metabolism.

Metabolism is usually divided into two categories which is catabolism, the breaking down of
organic matter for example, the breaking down of glucose to pyruvate, by cellular respiration,
and anabolism, the building up of components of cells such as proteins and nucleic acid form
simpler molecules. Usually, breaking down releases energy and building up consumes energy.

In this experiment we are focus on anaerobic energy production in yeast.In anaerobic

conditions, yeast cells will break down sugar into source of cellular energy. This process called
alcoholic fermantation or ethanol fermantation. Ethanol fermantation is a biological process
which converts sugars such as glucose, fructose, and sucrose into cellular energy and producing
ethanol and carbon dioxide as a side-effect. It also takes place in some species of fish
(including gold fish and carp) where (along with lactic acid fermentation) it provides energy
when oxygen is scarce. In this experiment we will determine which sugar is best metabolized by
yeast.

Ethanol fermentation also apllied on the production of ethanol fuel, alcoholic beverage
and bread cooking by using yeast. Yeast is able to metabolize some foods, but not others. In
order for an organism to make use of a potential source of food, it must be capable of
transporting the food into its cells. It must also have the proper enzymes capable of breaking
the foods chemical bonds in a useful way. Sugars are vital to all living organisms. Yeast is
capable of using some, but not all sugars as a food source. Yeast can metabolize sugar in two
ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen.

Although the aerobic fermentation of sugars is energetically much more efficient, in this
experiment we will set the conditions so that yeast carries out anaerobic respiration.
Aerobic respiration

The aerobic repiration of yeast occurs in three stages: glycolysis (involves soluble enzymes in
the cytoplasm), Krebs cycle (uses soluble enzymes in the matrix of mitochondria), and the
electron transport chain (a chain of proteins found on the inner membrane of the
mitochondria). Both alcoholic and lactic acid fermentation involve only glycolysis. Since both
the Krebs cycle and the electron transport chain require oxygen to function, neither process
can occur under anaerobic conditions. Fermentation and aerobic respiration involve oxidation-
reduction reactions. Redox reactions involve electron transfers: oxidation is the loss of
electrons from a substance, while reduction is the gain of electrons. In cellular respiration, two
hydrogen atoms at a time are enzymatically removed from glucose (oxidation) and transferred
to the co-enzyme NAD + (Nicotinamide Adenine Dinucleotide; Niacin, a common vitamin
supplement, is a component of NAD), reducing it to NADH + H + . Since each hydrogen atom
consists of one proton and one electron, think of this redox reaction involving the transfer of
two electrons, one from each hydrogen atom, and one proton to NAD+ , with the second
proton, H+ , released into the solution within the cell. Under aerobic conditions NADH transfers
the two electrons to the electron transport chain. The transfer of electrons from one E.T.C.
protein to another releases energy. Roughly 40% of this energy is used to produce ATP (usually
3 ATP per NADH), while the other 60% is released as heat. The final electron acceptor of the
E.T.C. is oxygen. One molecule of water is produced for every two electrons and two hydrogen
ions transferred to each oxygen atom by the electron transport chain.
e.t.c
2H++2e-+ 1/2 O2 H2O

Some animals such as the kangaroo rat of eastern Washington generate enough metabolic
water by aerobic respiration so that they can live a lifetime without drinking a single drop of
water. During glycolysis, the 6-carbon glucose molecule is converted via a series of enzyme-
controlled reactions into two molecules of pyruvic acid, a 3-carbon organic acid along with a net
gain of 2 ATP and 2 NADH + H+ . In the presence of oxygen (aerobic respiration), pyruvate
enters the mitochondria and another series of enzyme-mediated reactions called the Krebs
cycle. The Krebs cycle converts pyruvate molecules into carbon dioxide and water. This is
accomplished by a series of reactions that generate ATP, NADH + H+ , and FADH2 (Flavine
Adenine Dinucleotide; Vitamin B2 or riboflavin, common vitamin supplement, is a
component of FAD). FAD is a hydrogen carrier that generates 2 ATP for each pair of electrons it
transfers to the E.T.C.

Anaerobic respiration
The anaerobic respiration of is similar to aerobic cellular respiration in that electrons extracted
from a fuel molecule are passed through an electron transport chain, driving ATP synthesis.
Some organisms use sulfate as the final electron acceptor at the end ot the transport chain,
while others use nitrate , sulfur, or one of a variety of other molecules.
For glycolysis to function there must be a continuous supply of NAD + . Under anaerobic
conditions NADH is unable to release its cargo of electrons to the E.T.C. since oxygen is not
available to be the final electron acceptor. To keep glycolysis operating under anaerobic
conditions, animal cells regenerate NAD+ by NADH transferring its cargo to pyruvate to produce
lactic acid.
On the other hand, yeast first decarboxylate pyruvate to produce acetaldehyde, a two carbon
compound. Acetaldehyde is then reduced by NADH to form ethanol.

The chemical equations below summarize the fermentation of sucrose (C12H22O11) into ethanol
(C2H5OH). Alcoholic fermentation converts one mole of glucose into two moles of ethanol and
two moles of carbon dioxide, producing two moles of ATP in the process.
The overall chemical formula for alcoholic fermentation is:
C6H12O6 2 C2H5OH + 2 CO2
Sucrose is a dimer of glucose and fructose molecules. In the first step of alcoholic fermentation,
the enzyme invertase cleaves the glycosidic linkage between the glucose and fructose
molecules.
C12H22O11 + H2O + invertase 2 C6H12O6
Next, each glucose molecule is broken down into two pyruvade molecules in a process known
as glycolysis. Glycolysis is summarized by the equation:
C6H12O6 + 2 ADP + 2 Pi + 2 NAD+ 2 CH3COCOO + 2 ATP + 2 NADH + 2 H2O + 2 H+
The chemical formula of pyruvate is CH3COCOO. Pi stands for the inorganic phosphate.
Finally, pyruvate is converted to ethanol and CO2 in two steps, regenerating oxidized NAD+
needed for glycolysis:
1. CH3COCOO + H+ CH3CHO + CO2
catalyzed by pyruvade decarboxylase.
2. CH3CHO + NADH+H+ C2H5OH + NAD+
This reaction is catalyzed by alcohol dehydrogenase (ADH1 in baker's yeast).
As shown by the reaction equation, glycolysis causes the reduction of two molecules of NAD+ to
NADH. Two ADP molecules are also converted to two ATP and two water molecules
via substate-level phosphorylation.

Objective:

Materials:

Prosedure:

1) All five femantation tubes has been prepared and labelled by student by writting down
the number.
2) Syringes were used to transfer 1cm3 distilled water, 1cm3 glucose, 1cm3 fructose, 1cm3
galactose, 1cm3 arbinose.
3) 1cm3 of yeast suspesion was added into each tube and top up with distilled
water.Before yeast suspesion added into each tube, the yeast suspesion was mixed by
swirling .
4)

Result

Type of solution Heigth of liquid (mm) Heigth of liquid after Different between
 (Time:0) 40 minutes(mm) heigth of liquid and
(Time:40 minutes) heigth of liquid after
40 minutes(mm)
(Volume of CO2)
Glucose 720 270 450
Fructose 720 320 400
Galactose 740 650 90
Distilled water 730 660 70
Arbinose 730 660 70
Question

If the tubes were put in a 10 degrees colder than the coldest Celsius solution how would
thataffect the results?

In the data we see that the lower temperature the water was, the slower the yeastfermented.
Because of this we can draw that if water 10 degrees cooler had been added to theexperiment,
then the results would show that the yeast would ferment even slower.

Explain why and how yeast is used in bread?

During the process of fermentation yeast creates two waste gases; CarbonDioxide andEthanol.
Since these gases are poisonous to the cell, the yeast gets rid of the gases. This processes what
causes bread to rise. The bubbles of gas that are expelled from the yeast molecules create air
pockets in the dough therefore creating light and fluffy bread. Without yeast in the bread, it
would remain flat and hard.

Explain why the maximum concentration of alcohol is 12%-20% in wine?

While fermenting, yeast creates two waste products; Carbon dioxide and Ethanol. Ethanol is
another name for Alcohol and is therefore commonly drunk in wines and other alcoholic
beverages. As a yeast molecule ferments its waste products build up and will eventually be
released from the molecule. Since too much Ethanol in a yeast molecule will poison it, the
maximum concentration of alcohol is 12%-20% because it is the maximum the yeast molecule
can contain before dying

Explain why one needs controls in an experiment?

Controls are necessary in an experiment because they help guarantee that the results of an
experiment are valid. They are used to eliminate the possibility of other explanations for
your data. If something happens in the control that disrupts the experiment it can be concluded
that an outside factor, not being tested caused the disturbance because there were no factors
be tested in your control. Without controls its unknown whether results are valid or not.
Discussion:
The result have shown that all forms of sugar produced energy and among that glucose w a s
the most efficient. The carbon dioxide produced can be
d i r e c t l y r e l a t e d t o t h e e n e r g y produced through fermentation because carbon
dioxide is a by-product of ethanol fermentation. The control that contained no sugar
produced no energy because a source of sugar is required for glycolysis and fermentation
to occur. Glucose had the greatest rate of energy production because its rate of carbon
dioxide production was the largest. Fructose had the second highest rate of production while
galactose had the lower rate followed by arabinose. Glucoses rate of energy production was
more than three times that of fructose. Glucose was directly used i n t h e g l y c o l y s i s c y c l e
and did not require any extra energy to convert it into a usable
f o r m . T h i s s u p p o r t e d w h y g l u c o s e w a s t h e m o s t e f f i c i e n t . Fructose could
not be used immediately in the glycolysis chain but had to be altered to enter the chain as
one of the intermediates. These processes required to convert the non-glucose sugars
into a usable form reduced their efficiency when compared to glucose. However th
fermentation process of arabinose is combination of isomerase pathway and a fungal
reduction/oxidation D-xylose pathway. The enzymes involved in the reduction-oxidation steps
of fungal pentose sugar assimilation display different co-factor preferences. The fungal L-
arabinose pathway consists of two oxidations and two reductions, the conversion of L-
arabinose to D-xylulose is redox neutral. However, the reductions are NADPH-linked and the
oxidations NAD- linked so that there is an imbalance of redox cofactors. This imbalance of
redox cofactors could be solved by a transhydrogenase activity facilitating the equilibration
between NADH/NADP and NAD/NADPH. Yeasts are believed not to have such activity in that
enviroment.

The largest source of error for the experiment was the start time of
fermentation. The yeast was added to the fructose solution well after the glucose
and fructose yeast solutions began fermenting. Fermentation takes time to reach
its maximum rate of energy production so the time gap left glucose and sucrose further
ahead than fructose in t h e f e r m e n t a t i o n p r o c e s s . T h e d a t a o n r a t e o f c a r b o n
d i o x i d e p r o d u c t i o n w a s therefore skewed because the start of fermentation was
not controlled. Glucose and f r u c t o s e a p p e a r f a r m o r e
efficient than galactose and arabinose may because of this error. If this
e x p e r i m e n t w e r e t o b e repeated, extra care would be taken to ensure that
fermentation began at the same time. The measurements of sugars would be measured
in equal molarity and not by percent in a solution so that the sugar molecules are equal
across all of the tests. Other follow-up experiments may include testing other types of
yeasts to see how fermentation rates are impacted. The results of these experiments could
impact what sugars are the most efficient in alcohol fermentation. This could determine
what types of sugar brewers should use for the most efficient production of alcohol
Conclusion:
The different types of sugars used in fermentation had a significant impact on the amount of
carbon dioxide produced. Glucose produced the most with a gas bubble of 450mm
while galactose yielded 900mm of carbon dioxide.

The gas byproduct in fructose measured only 400mm.The gas produced distilled water and
arabinose resulted in 70mm of carbon dioxide and was the least productive of the tubes. The
slope of the line of best fit was analyzed to determine the average rate of carbon
dioxide production over the 40 minute time frame. Glucose was the most efficient,
producing 11.25mm of carbon dioxide per minute. Galactose 2.25yielded mm of carbon
dioxide per minute during fermentation while fructose functioned at a rate of 10mm of carbon
dioxide per minute.T h e d i s t i l l e d w a t e r a n d a r n i n o s e p r o d u c e d 1 . 7 5 m m o f
carbon dioxide per minute during fermentation.

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