NAME: Latiyah Timothy LAB PARTNER: Jasiel Mohammed

ID# 816012983 DATE: Session 1: 11th February, 2020

Session 2: 27 th February 2020

Course Code: Bioc 2169: Practical Skills in Biochemistry II

Title of Lab: Partial Purification of Yeast Invertase using the four methods: Centrifugation, Heat
extraction, Alcohol Solvation and Ion exchange Chromatography
Aim: To extract and purify yeast invertase from Saccharomyces cerevisiae using centrifugation, heat
extraction, alcohol solvation and ion-exchange chromatography.

Theory:
Yeast invertase also called β-fructofuranosidase or sucrase (EC: 3.2.1.26) is an enzyme that folds into
catalytic β-propeller and β-sandwich domains. There are two isozymes of invertase in yeasts,
extracellular yeast invertase and intracellular yeast invertase. Extracellular yeast invertase is found
between the plasma membrane and the outer cell wall. It is a glycoprotein that contains 3%
glucosamine, 5% mannose and 50% carbohydrate and has a molecular weight of 270 kDa.
Intracellular yeast invertase is found in the cytosol, has no carbohydrates and has a molecular weight
of 135 kDa. (Kulshrestha et al, 2013)

Invertase catalyses the hydrolysis of sucrose into an equimolar mixture of glucose and fructose. It
cleaves the terminal non-reducing β-fructofuranoside in sucrose. The resultant mixture of glucose and
fructose is called an invert sugar because sucrose rotates a plane of polarized light to the right while
the products rotate a plane of polarized light to the left. (Timerman, 2012)

Figure 1: Equation for the Hydrolysis of Sucrose via Invertase (Pito, 2012)

In this lab, invertase was extracted from Baker's Yeast. Mechanical homogenisation was first done
with sand and mortar and pestle so that the protein can be released into the extraction medium.
Toluene and cold deionized water were also used for cell lysis as well as aid in the extraction of the
invertase. To extract the yeast invertase extraction methods such as centrifugation, heat and alcohol
solvation.
Centrifugation was used to separate the biomolecules based on size, weight and density. The speed
used was 15000rpm which is used to pellet nucleic acids or proteins from solution. Heat extraction
was the next method used. It was adjusted with acetic acid to pH 5 as this is the optimum pH for
invertase. A temperature of 50oC was used to denature other proteins present that was not the protein
of interest.
Ethanol was used to precipitate the protein of interest. It decreases the solubility of the protein in
solution by forming hydrogen bonds with the water molecules, making fewer water molecules
available to hydrate the protein. Ethanol also has a lower dielectric constant which causes the protein
to aggregate with positive ions in solution, forming a solid precipitate at the bottom of the tube.
During the extraction process the conditions where maintained carefully such as using a Tris buffer of
pH 7.3 and grinding on ice to prevent protein degradation as well as inhibit enzymatic proteins that
may degrade the protein. Resuspension of the pellet in a Tris buffer was also done to maintain the pH
and prevent degradation.

Ion exchange chromatography was used as a purification technique. This type of chromatography is
based on the attraction between the oppositely charged stationary phase known as the ion exchanger
and the analyte. In this lab, an anion exchange was done. Invertase has a negative charge, therefore,
the resin chosen was Diethylaminoethyl cellulose. This is a positively charged resin commonly used
for the purification of proteins.
The loaded column is then washed with increasing concentrations of NaCl until the chloride ion
reaches a high concentration and displaces each protein from the column, this leads to eluation of
proteins in the order of increasing binding affinity to the resin. Ion exchange chromatography also
helps in increasing the concentration of the protein. (Timerman, 2012)

The activity of the enzyme was monitored using spectrophotometric analysis and the Clinistix test.
UV-Vis spectrophotometry was used as an indicator for the presence of invertase. Proteins can absorb
light at a maximum of 280nm due to their aromatic rings present. The detection limit is 0.1-100
ug/ml.
The disadvantage for this method as an indicator of the presence of invertase is that other
contaminants also absorbs light at 280nm, this can lead to inaccurate or fluctuating absorbance
readings.
The Clinistix Test was also used to test the presence of invertase. This test is used to detect the
presence of glucose. A portion of the uriscan strip contains glucose oxidase that has been dried. In the
presence of glucose, the glucose is oxidized to form gluconic acid and hydrogen peroxide. Therefore,
when a sample of the eluate is added to sucrose if there is invertase present, sucrose would be
hydrolysed to fructose and glucose and then result in a positive clinistix test. The disadvantage of this
test is that if it is read after 1 minute it can result in inaccurate results.
Apparatus and Materials:
DEAE: Diethylaminoethyl cellulose
0.05M Tris buffer at pH 7.3
0.05M Tris buffer with 200mM NaCl at pH 7.3
10g Baker's Yeast
4g Sand
30ml Deionized water
0.4 M acetic Acid
95% Cold Ethanol
Acetate buffer pH 4.5
0.5M Sucrose
Uriscan urine strip.

Method:

SESSION 1:

10g of yeast was weighed as well as 4g of sand. The samples were placed in a mortar in an ice bath
and it was ground to a very small powder. 10mL of toluene was added and the sample was ground
until a smooth paste was achieved (all the toluene was added at once). 30mL of cold deionized water
was added at a rate of 3mL every 3 minutes. Grinding was continued after each addition. The mixture
was transferred to two 50ml centrifuge tube and it was spun at 15000rpm for 10 minutes in a Sorvall
refrigerated centrifuge using the SS-34 rotor. A long tipped Pasteur pipette was used with a rubber
bulb and the murky cream aqueous layer was removed and transferred to a 25mL measuring cylinder.
The demonstrator was consulted for instructions on how to best remove the layer. The volume
transferred was recorded.

2mL was saved in two microcentrifuge tubes (1mL each tube) for invertase activity and protein
demonstrations. The tubes were closed and secured with parafilm. These tubes were labelled
fraction1- crude. The remaining solution was transferred to a 50ml centrifuge tube and the pH was
adjusted to 5 by the dropwise addition of 0.4M of acetic acid. After the addition of each acid, the
solution was mixed gently. The temperature of this extract was rapidly brought to 50 o C using a water
bath and the sample was incubated for 30 minutes at this temperature with occasional gentle mixing
by hand during the incubation. The next step taken was cooling the sample in an ice bath rapidly for
10 minutes and it was then centrifuged at 15000rpm for 15 minutes in a refrigerated centrifuge. The
supernatant was poured off and into a measuring cylinder and the exact volume was recorded.

A 1.5ml aliquot was saved for invertase activity and protein determinations- this sample was labelled
fraction 2. The remaining solution was poured into a 100mL beaker sitting in a larger 600ml beaker
with an ice-water jacket between the walls of the two beakers. A small magnetic stirring bar was
placed in the 100ml beaker and the larger beaker containing the smaller beaker was placed on a
magnetic stirrer. Over a period of 30 minutes, a volume of 95% EtOH was added which is equal to the
volume of the remaining extract from the previous step.

The mixture was stirred for an additional 15 minutes and it was then centrifuged at 15000rpm for 25
minutes in the refrigerated centrifuge. The supernatant was poured off into a clean test tube and it was
then tested for activity with the diagnostic clinistix strip. The tube containing the pellet was covered
and stored in a freeze until the next lab session.  
The Clinistix Test

In a spot plate, two drops of acetate buffer of pH 4.5 were mixed, 2 drops of sucrose and 3 drops of
supernatant. The sample was incubated for 5 minutes and the diagnostic strip (URISCAN urine strip)
was then dipped into the incubation mixture for 2 seconds. The glucose, protein and pH portions were
submerged in the incubation mixture. The strip was held up in a horizontal position and the test was
read after 60 seconds with the test area held near the appropriate colour chart on the bottle label.

SESSION 2:

The pellet was collected and it was allowed to thaw out at room temperature. The column was first
prepared. The exchange resin was prepared and equilibrated in 0.05M Tris buffer of pH 7.3. An 18cm
x 1cm column was packed in the same buffer with DEAE cellulose slurry slowly until the height was
achieved. The sample was washed 10mL of the 0.05M tris buffer of pH 7.3 while 1mL of the buffer
was left just above the surface of the resin. (The column never ran dry).

The two buffers provided (0.05M Tris pH 7.3 and 0.05M tris containing 200mM NaCl pH7.3) were
used to prepare eight buffers containing the 2.0mM, 10mM, 30mM, 50mM, 75mM, 100mM,150mM
and 200mM NaCl (only 10 ml of each buffer was needed). The pellets were resuspended and thawed
in 5mL of 0.05M Tris pH 7.3. A Pasteur pipette was used to dissolve the pellet solution. The
dissolved enzyme was centrifuged at 15000 rpm for 20 minutes in the refrigerated centrifuge and the
supernatant was collected in a measuring cylinder on ice. The volume of the supernatant was
recorded. 3.5mL was kept for the column and the remainder was saved for invertase and protein
determinations. This sample was labelled as fraction 3.

3.5mL of the supernatant was added to the column slowly without disturbing the surface of the
column. The supernatant was allowed to flow into the column without allowing the column to run dry.
Collection of 5ml fractions immediately began in an ice bath. After the sample ran into the column the
sides of the column were carefully washed with 6.0 mL of 0.05 M Tris buffer pH7.3 using a Pasteur
pipette. After the buffer has been added and the buffer level is 1cm above the top of the resin, stop the
flow through the column. 10mL of the first elution buffer (containing 2.0mM NaCl)  was carefully
collected as well as two 5mL aliquots on ice. The last elution was continued until the buffer has been
added and the fractions collected.  Between buffers, The flow was stopped before adding the next
buffer of higher salt concentration. The absorbance of EACH collected fraction was read at 280nm
and each fraction was tested for invertase activity with “Clinistix”. The two tubes were combined with
the highest level of activity and THE VOLUME WAS NOTED. This volume was divided among 10
microcentrifuge tubes (1ml per tube). One of these tubes was used later used in protein determination.
The tubes were covered and secured with parafilm and it was stored in the freezer until the next
session. These tubes were labelled Fraction 4 – column fraction. Also, the tube was immediately
combined and saved preceding the peak activity and immediately after the peak activity.
 Results:
SESSION 1:

Table 1: Volumes of the Crude and Heat fractions obtained.

FRACTION VOLUME (ml)
Crude 24.5
Heat 13.9

Table 2: Results of Clinistix Test of the Supernatant.

Fraction Glucose Approximate Protein pH
Concentration Concentration
(mg/dl) (mg/dl)
Supernatent 0 0 5

SESSION 2:

Table 3: Results obtained from Clinistix Test and UV Spectrophotometry of the Ion-exchange
chromatography eluates.
Fraction 6ml 0.05M 0.05M Tris 0.05M Tris 0.05M Tris 0.05M Tris 0.05M Tris 0.05M
Tris + 3.5ml and 2.0mM and 10mM and 30mM and 50mM and 75mM Tris
sample NaCl NaCl NaCl NaCl NaCl and
100m
M
NaCl
1 2 3 4 5 6 7 8 9 10 11 12 13
Absorbance 0.02 0.13 0.98 0.58 0.57 0.48 0.21 0.32 0.18 0.58 0.60 0.47 0.268
(280nm) 1 3 7 6 9 6 7 8 5 0 9 8
Glucose 0 0 0 0 0 0 0 0 0 14 21 28 14
Concentratio
n (mg/dl)
Approximate 0 0 0 0 0 0 0 0 0 0.1 0.2 0.3 0.1
Protein
Concentratio
n (mg/dl)
pH 5 5 5 5 5 5 5 5 5 5 5 5 5

The tubes with the highest activity where tubes 11 and 12.

Table 4: Volumes obtained for the Alcohol and Column Fractions.

FRACTION VOLUME (ml)
Alcohol 5
Column 10
Discussion:
In this lab, yeast extract was extracted and partially purified using centrifugation, heat extraction,
alcohol solvation and ion-exchange chromatography. The crude extract was obtained from mechanical
homogenization using sand, mortar and pestle and toluene. The grinding of the yeast cells with sand
disrupted the cell membrane while the toluene was used in cell lysis. Centrifugation at 15000rpm
separated the protein of interest from other biomolecules. The volume of the crude extraction obtained
was 24.5ml.

Heat extraction was the next method used. The extract was adjusted to pH 5 and was rapidly brought
to the temperature of 500C and was incubated. The high temperature would degrade other proteins
present in the crude extract while leaving the protein of interest. The volume of the extract obtained
after the heat extraction was 13.9 ml.

Ethanol was used to precipitate the protein. This occurs because ethanol has a lower dielectric
constant which causes it to aggregate and forms a precipitate, it also lowers the solubility of the
protein in solution. The pellet formed after centrifugation was resuspended in a Tris buffer to prevent
degradation and maintain the pH. A clinistix test was performed on the supernatant. The results
obtained stated that there was no protein or glucose present but the pH was 5. This was a good
indicator that there was no invertase in the supernatant as the sucrose was not hydrolysed to form
glucose and fructose.

In the second session, the pellet resuspended in the buffer was centrifuged and the volume of the
alcohol fraction obtained was 5 ml. Ion exchange chromatography was done using 3.5 ml of the
alcohol fraction. Ion exchange chromatography is a technique used to purify proteins based on their
charges. The type of ion exchange occurred was an anion exchange. Invertase has a negative charge
and thus the resin used had a positive charge. Diethylaminoethyl cellulose was the resin chosen. The
loaded sample was washed using increasing concentrations of NaCl. The increasing salt concentration
would displace then displace the ions attached to the positive column. As a result, the ions would
eluate in the order of increasing binding affinity.

UV- Spectrophotometry was done on the eluates from the column chromatography. The results
obtained were 0.021, 0.133, 0.987, 0.586, 0.579, 0.486, 0.217, 0.328, 0.185, 0.580, 0.609, 0.478,
0.268 for 1,2 3,4,5,6,7,8,9,10, 11, 12 and 13 respectively. The readings were fluctuating and there is
no trend observed. These fluctuations may be as a result of other impurities absorbing at 280nm.
Therefore, the spectrophotometric analysis is not an accurate indicator of the presence of invertase.

The clinistix test was also done on the 13 eluates, the pH remained at 5 for each eluate, however, only
tubes 10-13 showed the presence of invertase. The concentration of glucose present were 14mg/dl,
21mg/dl, 28mg/dl and14mg/dl for tubes 10, 11, 12 and 13 respectively. The amount of protein present
was 0.1mg/dl, 0.2mg/dl, 0.3mg/dl and 0.1mg/dl for tubes 10,11,12 and 13 respectively. This test was
a more accurate indicator of the presence of invertase. This is because when samples of each eluate
were added to a sucrose solution if invertase was present it would hydrolyse the sucrose into fructose
and glucose and in turn, give a positive glucose result on the uriscan strip. The disadvantage of this
test is that if the strip is read after 1 minute it can give inaccurate results.

Tubes 11 and 12 which showed the highest activity were combined to create the column fraction. The
resultant volume obtained was 10ml.
Sources of Error and Precautions:
- The Clinistix test must be read after 1 minute any later would result in a false positive.
-There may be enzyme lost during the mechanical homogenization.
-Ensure that most of the supernatant is in the column before adding the buffer.
References:
Kulshrestha, Samarth, Prasidhi Tyagi, Vinita Sindhi, and Kameshwar Sharma Yadavilli.
"Invertase and its applications–a brief review." journal of pharmacy research 7, no. 9 (2013):
792-797.

Pito, D. S., I. M. Fonseca, A. M. Ramos, J. Vital, and J. E. Castanheiro. "Hydrolysis of

sucrose over composite catalysts." Chemical Engineering Journal 184 (2012): 347-351.

Timerman, Anthony P. "The isolation of invertase from baker’s yeast—an introduction to

protein purification strategies." Protein Purif (2012): 29-52.