TITLE

Super awesome Amylase Enzyme Experiment Presentation thing Jeff Blattner, Melody Lamis, Sabina
Muminovic, and Marcus Montuy

INTRODUCTION
Amylase is an enzyme that catalyzes the hydrolyses of the (-linked) polysaccharide starch into the
disaccharide sugar maltose. By breaking the long chain of glucose molecules at 1-4 glycosidic (6 carbons)
alpha bonds, amylase breaks dietary starches down into sugars to be converted into energy. Without amylase,
the process of digestion would lack fluency and proficiency. Polysaccharides would remain in long sequences
of glucose molecules and our bodies would use greater amounts of energy to breakdown these long
polysaccharide chains. Amylase is present in human saliva and produced by the pancreas, but humans are not
the only species that use amylase for digestion. There are 3 types of amylase: bacterial, fungal, and porcine
(pig/animal) amylase. Bacterial Amylase is a waterdispersible blend of the extracts of Bacillus licheniformis,
which includes high concentrations of alpha-amylase, -glucanase (gummase) and hemicellulose with moderate
levels of protease. Bacillus licheniformis is known for its high temperature stability and rapid reduction of
starch suspensions (The suspension feels like a solid when squeezed but acts like a liquid when no pressure is
applied). In adition, Bacillus Licheniformis optimal growth temperature is around 30C, while its optimal
temperature for enzyme secretion is 37oC. Bacillus licheniformis is cultured in order to obtain protease for use
in biological laundry detergent. The licheniiformis protease has an optimal pH level between 9-10. Bacillus
licheniformis has been found to degrade feathers of birds (especially parrots). However, Bacillus Licheniformis
decays white feathers much more optimally than colorful feathers that contain Psittacofulvin. Psittacofulvin
pigments cause the bright-red, orange, and yellow colours of parrots.Fungal amylase is produced by living cells
that are derived from Aspergillus oryzae, a fungus that is used for the production of many commercial and food
related products (baking industry). Fungal amylase is also found in mushrooms. This amylase is tolerant to wide
range of culture pH values (3 to 8) and temperature (25 to 35oC), with an optimum pH of 5 and temperature of
35oC. Lentinus edodes proved to be the most promising strain for amylase production. The amylolytic activity
of Lentinus edodes, a mushroom reached its maximum (621 U/ml) after seven days of incubation, the dry
mycelium yield being 75 g/l with a protein content of 34%. The third type of amylase observed was porcine
(pig) amalyase. Maltopentaose Porcine pancreatic alpha-amylase (PPA) is used in the baking industry to
remove starch and in the process of ripening tomatoes. -Amylase isolated from porcine pancreas is a
glycoprotein. It is a single polypeptide chain of ~475 residues containing two SH groups and four disulfide
bridges and a tightly bound Ca2+ necessary for stability. Chloride ions are necessary for activity and stability.
The pH range for activity is 5.5 to 8.0, with the pH optimum at 7.

OBJECTIVES
The purpose of the experiments was to 1) Establish a standard curve for Maltose to determine the absorbance of
light at 540nm. The equation generated will be used to measure the effects of pH and temperature on the
Amylase. The various results will allow us to determine which type of amylase we are sampling in these
experiments.

MATERIAL & METHODS

To measure the effect of pH on alpha-Amylase Activity we labeled one test tube as Blank and five other test
tubes as pH5, ph6, pH7, pH8, and pH9. Using Ph7 as our control, we added 1 ml of 1% starch solution to the
blank tube. The starch was the substrate in this experiment. Then we added 1 ml of the appropriate pH starch
solution to the corresponding assay tube careful to use a fresh pipette tip for each pH to avoid crosscontamination. Then we mixed 100 l of the concentrated enzyme stock solution with 9.9 ml of dH2O. Also,
we added 1 ml of dH2O to the blank tube only. To the other five tubes we set a timer to 12 mins and countdown
from the moment we added 1 ml of diluted -amylase to each of the five tubes. We allowed the tubes to
incubate at room temperature for 12 min and then, in the same order that the diluted enzyme was added, 1 ml of
Maltose Color Reagent was added to each tube including the blank tube. The Maltose Color Reagent is
handled while wearing gloves as it is harmful to the skin. After the tubes are thoroughly mixed they are
incubated at 100C for exactly 15 min. After the tubes are incubated and cooled to room temperature, 9 ml of
dH2O is added to each tube and then 4 ml of each tube is transferred to spec tubes to measure the absorption of
light at 540nm in a spectrophotometer.

To measure the effect of Temperature on -Amylase activity 6 test tubes were labelled: blank, 4C, 23C, 37C,
65C, and 100C. 1ml of 1% starch solution at pH7 was added to each tube. The tubes were set to the
appropriate temperatures for which they were labelled. Blank and 23C were left at room temperature; 4C
was set on ice, 37C, and 65C were placed in the corresponding water baths, while 100C was set in a heating
block. A fresh dilution of the -Amylase was made by mixing 100 l of concentrated enzyme stock solution
with 9.9 ml of dH2O and then placed on ice. All the tubes were allowed to incubate at the given temperatures
for 10 mins while 1 ml of dH2O was added to the blank tube only. After incubation the other 5 tubes received 1
ml of diluted -Amylase and returned to the appropriate temperatures to incubate for 12mins. After incubation 1
ml of Maltose Color Reagent was added to each tube, including the blank tube, while wearing gloves. The tubes
were then all placed at 100C for exactly 15mins to stop the enzymatic reaction. When the 15mins were
complete, all the tubes were cooled on ice to reach room temperature and then 9 ml of dH2O was added to each
tube and 4ml of that solution is then transferred to a spec tube to measure the absorption of light at 540nm in a
spectrophotometer.

RESULTS
y = 0.8097x - 0.0139

Absorbance at 540 nm
(AU)

Maltose Standard Curve

R = 0.9972

0.9
0.8
0.7
0.6
0.5

Series1

0.4
0.3

Linear (Series1)

0.2

Linear (Series1)

0.1
0
-0.1 0

0.2

0.4

0.6

0.8

1.2

Concentration of Maltose (mg/mL)

The Maltose Standard Curve graph shows how the increase of Maltose, increases the amount of light
absorbed at 540 nm. The concentration of Maltose at 0.0 mg/mL was used to tare the spectrophotometer as
having no Maltose in the test tube should have relatively no light absorbance value. At 0.2 mg/mL of Maltose,
0.145 AUs of light were absorbed. At 0.4 mg/mL of Maltose, 0.298 AUs of light were absorbed. At 0.4 mg/mL
of Maltose, 0.298 AUs of light were absorbed. Our sample of 0.6 mg/mL of Maltose was spilled during the
experiment and has been excluded from our data as it would thwart the accuracy of our standard curve and of
the equation needed for the remainder of the experiments. At 0.8 mg/mL of Maltose, 0.613 AUs of light were
absorbed. Lastly, at 1.0 mg/mL of Maltose, 0.818 AUs of light were absorbed. Once we graphed this data, we
were able to use the graph to create an equation y=0.8097x - 0.0139 where y= the absorbance of light at 540
nm. 0.8097= the slope of the line. x= the concentration of Maltose in mg/mL and -0.0139 = the y-intercept
which is the best fit of where the line crosses the y-axis. This equation was used in the following Amylase labs
to determine how pH and temperature would affect the amount of Maltose (x) produced.

Absorbance at 540 nm (AU)

Effect of pH on -amylase activity

1.60
pH 6

1.40

pH 8
pH 7

1.20
1.00

pH 9
pH 5

0.80

Series1
Linear (Series1)

0.60

Linear (Series1)

0.40
0.20
0.00
0

0.5

1.5

Concentration of Maltose (mg/mL)

During the pH lab, we discovered that our Amylase sample (B) produced the most amount of Maltose at
a pH of 6 where 1.838 mg/mL of Maltose was produced. The next closest optimal pH was 8 where 1.726
mg/mL of Maltose was produced. Then at a pH of 7, 1.672 mg/mL of Maltose was produced. At a pH of 9,
1.210 mg/mL of Maltose was produced and at a pH of 5, 1.084 mg/mL of Maltose was produced. We obtained
the Maltose (x) values by inputting our Absorbance Unit (y) values into our equation: y=0.8097x - 0.0139 and
then we balanced the equation. For example, to obtain the amount of Maltose produced at a pH of 6, we made
the following calculation and rounded the x value to 3 decimal places:
( )
( )
( )

Absorbance at 540
nm (AU)

Effect of temperature on -amylase activity

2

37C (Body Temp.)

1.5
1

Series1

22C (Room Temp.)

Linear (Series1)

4C

0.5

Linear (Series1)

0
0

0.5

1.5

2.5

Concentration of Maltose (mg/mL)

During the Temperature lab, we discovered that our Amylase sample (B) produced the most amount of
Maltose at a Temp. of 37C where 2.262 mg/mL of Maltose were produced. The next closest optimal

Temperature was 22C (Room Temp.) where 0.964 mg/mL of Maltose was produced. Lastly, at 4C, 0.670
mg/mL of Maltose was produced. At 65C and 100C, the spectrophotometer was blinking when it gave us the
Absorbance Units. We were advised to exclude these values from the data. We again obtained the Maltose (x)
values by inputting our Absorbance Unit (y) values into our equation: y=0.8097x - 0.0139 and then we balanced
the equation. For example, to obtain the amount of Maltose produced at a Temp. of 37C, we made the
following calculation and rounded the x value to 3 decimal places:
( )
( )
( )

CONCLUSION
Identify the source of the -amylase enzyme. Provide supporting evidence.
Established on information we provided in the introduction, about -amylases Bacillus licheniformis,
Aspergillus oryzae and Porcine pancreatic, the best choice for our amylase B, would be -amylase Aspergillus
oryzae. The activity of amylase produced by Aspergillus oryzae was measured at various temperatures and
pHs, with soluble starch as a substrate. We found that the activity of produced amylase from Aspergillus
oryzae gradually increased with the increase of the reaction temperature up to 37C and the pH from 6 to 8, by
which the maximal activity of the enzyme was documented. However, further rising in the reaction temperature
over this degree, and the pH below 6 and above 8, resulted in a gradual reduction in the enzyme activity. The
maximum produced Maltose of 1.838 mg/mL at pH 6, and 2.262 mg/mL at Temp. 37C, states that amylase
Aspergillus oryzae is amylase we worked with.