INSTITUTO POLITÉCNICO NACIONAL

Unidad Profesional Interdisciplinaria de

Ingeniería Campus Guanajuato.

Bioconversions Laboratory

Students
Cortes Cañamar Yordan
Chavez Centeno Samuel
Martinez Carrion Jessica Berenice
Rodríguez Becerra Paola de la Luz
Rojas Aguirre Kaled Alejandro

4BM1

Teachers
Anastasio Cortés Mendoza
Mario Josue Aguilar Mendez

Practice #3.

Separation and Purification of enzymes.

February 18th, 2019.

Objectives.
● Recover, precipitate and purify the enzyme (∝-amylase) using different
mechanisms for later use

Specific Objectives.
● Centrifuge the enzyme to separate it from cell debris
● Perform the salting-out method to precipitate the alpha-amylase enzyme.
● Purify the enzyme by the dialysis method

Introduction

Once the fermentation of our enzyme is done, it is processed to recover it and then
purify it. It is said that the first step for this is obtaining a Homogenate that we will
have it through a homogenization

Homogenization​: Here we will make a cellular break in which the fractions are the
most suitable for its purification, there are 5 types of homogenization
a) Mechanical homogenization: I​ n this method glass homogenizers are used and
the abrasives can be alumina or sand together with a suitable solvent and
likewise a good buffer
b) Sonic homogenization: Here it is based on the shock of waves as it will trigger
pressure gaps and consequently breaking the wall of the cell is commonly
used in bacteria and yeasts
c) Thermal disintegration: T ​ he structure of the cell will be destroyed by freezing
and thawing fast and several times
d) Chemical disintegration: Chemical agents are used for cell lysis such as
ethanol
e) Homogenization by dehydration:​ here the protein is precipitated
(Sanchez, 2013)

There are 3 principles to precipitate a protein which are:

● Decrease in solubility.
● Selective denaturation
● Affinity
We will use a solubility method using a precipitating agent: ammonium sulfate, which
is the most used for its easy use and low cost.
Proteins act as amphoteric molecules, this means that they can act either as a base
or as an acid since in their surface they have hydrophobic and hydrophilic regions.
Precipitation with salts is done by adding high concentrations of salts in the solution
where we have the protein for its precipitation.
(Tejada, 2011)

Figure 1. Solubility according to salt concentration.

Salting in:
In Figure 1 we can see how in this mechanism the solubility will increase according
to how the salt concentration is increasing, this means that the protein will have few
hydrophobic regions

Salting out:
Otherwise, we can also observe in figure 1 how when adding salt to our protein there
will be hydrophobic regions in freedom to be able to be combined so it will have a
low solubility.

Once the protein is precipitated as the densities are different it undergoes through
centrifugation to obtain our protein aggregates.

Centrifugation is used to yield particles with different densities and analyze the
component of interests is placed in a glass or plastic falcon tube and placed in a
centrifuge.

There are different types of centrifugation

According to its purpose
a) Analytical centrifugation:​ Consists in measuring the sedimentation coefficient
and the molecular mass of the particles that sedimented.
b) Preparative centrifugation:​ isolates the particles for analysis.

Depending on where you are going to centrifuge

a) Differential centrifugation: generally an angular rotor is used and the particles
are separated according to their sizes, the larger ones settle in the tube.
b) Zonal centrifugation: will separate the components with the highest
sedimentation coefficient will go to the bottom.
 c) Isopycnic centrifugation: join to separate macromolecules as DNA sediment at
different speeds.

(Bolivar,2018)

There are several factors that can denature an enzyme and damage its function or
properties, such as changes in:
a) pH:​ its acid and basic groups can cause variations in its structure.
b) Polarity of the solvent: it will not be possible to solvate and stabilize its loads
since other less polar substances will be added to it
c) Temperature: the enzymes are thermosensible that's why you can reach
temperatures close to 25 ° C
That is why Buffer is added and kept at low temperatures to avoid this denaturation
(Correa,2009)

Buffer:
To maintain a constant pH a buffer is used, which is a solution of a weak acid and a
conjugate base, the acid part of a buffer reacts with the OH ions and the acid part,
on the other hand, will react with the H, even though there are several we are
interested in the phosphate buffer
(Cabrera,2014)

Dialysis is the separation of molecules by their size using semi-permeable

membranes cellophane (cellulose acetate) its purpose is that the molecules find their
balance between the concentration outside and inside the bag, as the
macromolecules will be inside the bag.

Figure 2. Dialysis through a semi-permeable membrane

(Amoros, 2013)
Materials and Equipment
❖ Centrifugal
❖ Erlenmeyer flask (250mL)
❖ Eppendorf tubes
❖ Potentiometer
❖ Beaker (250mL)
❖ Spectrophotometer
❖ UV-VIS cells
❖ Microppipets of 2-20 μL and 100-1000 μL
❖ Racks for eppendorf tubes
❖ Ammonium Sulphate
❖ Phosphate Buffer pH=7 (amylase)
❖ Bradford Reagent

Experimental Development

Results:
Table 1.- Weight to falcon tubes of first centrifugation.

Tube Weight (g) Second Weight (g)

1 36.53 40.01
 2 38.14 40.06

3 36.50 40.00

4 35.58 40.02

5 33.22 40.04

6 32.12 40.02

Centrifugal condition: 7010 rpm, 4ºC, 7 minutes.

Six falcon tubes were placed inside the centrifuge with their respective counterweight
(Table 1) to maintain balance in the centrifuge. All tubes were filled with medium.

Table2. Weight to falcon tubes of second centrifugation

Tube Weight (g)

E1 45.03

E2 45.04

E3 45.02

E4 45.02

E5 39.12

E6 39.13

Water conductivity reading

After centrifugation, the supernatant was placed in a cellulose bag and place inside a
beaker with water. To prove dialysis, water conductivity was read:
Distilled water before dialysis: 1.31 µS at 24,4°C
Distilled water after dialysis: 146.6 µS at 11.7°C

Table 3: Determination of total protein concentration by spectrophotometry visible UV through

Bradford method at wavelength 595 nm to supernatant after second centrifugation.

Sample Absorbance Concentration (µg/mL)

White 0 0

1 0.50 186.6

2 0.096 36.94
 Average 111.8

Table 4. Determination of total protein concentration by spectrophotometry visible UV through

Bradford method at wavelength 595 nm after 16 hours approximately in dialisis for tube #1.

Sample Absorbance Concentration (µg/mL)

White 0 0

1 0.900 334.7

2 0.851 316.6

Average 325.7

Table 5. Determination of total protein concentration by spectrophotometry visible UV through

Bradford method at wavelength 595 nm after 16 hours approximately in dialisis for tube #2.

Sample Absorbance Concentration (µg/mL)

White 0 0

1 0.155 * 235.2

2 0.139 * 211.5

Average 223.4

● * We used a dilution factor to 1:4 because the absorbances exceeded the limit
to absorbance 1.
Discussion

Figure 3 . Test to check if we have a amilase in our sample a) cornstarch, b) cornstarch with purified
enzyme, c) cornstarch with saliva.

We realized a test to check if in our sample we had alfa amilase enzyme with a
simple test where in little tray put a low quantity of cornstarch, after in a first little tray
only added lugol solution (negative control), in the second little tray added purified
enzyme then lugol (sample) and the last tray added saliva then lugol solution
(positive control).
For our qualitative test for the identification of α-amylase (García,1998) mentions us,
in his work of identification of colonial morphologies of yeasts in CHROMagar
medium, which S. cerevisiae presents a lilac coloration, mates and with halo around.
This test is based on the behavior of the iodine with starch, when the test is negative
the iodine is bound in the chain of starch giving the blue coloration as shown in the
negative control. While reacting the enzyme ɑ-amylase, it hydrolyzes the starch,
returning a brown color. In order for the reaction to be carried out, Iodopovidona, a
complex iodine with potassium iodide where the complex I3-, since iodine has a low
solubility in water, but the I3 complex- is very soluble (Gary, 2003). And when
checking our results You may notice a small halo around purple with our test of the
medium in comparison to the positive test that is the saliva, even without having
been extracted and purified from the medium, you can confirm the presence of it by
this test.

To remove the microorganism six falcon tubes were centrifuged. These tubes
contained the production medium, in which ​Saccharomyces cerevisiae cells were
inoculated. The objective of this step was to obtain cell separation and protein, the
centrifugate condition was 7010 rpm at 4°C for 7 minutes.
In the first centrifugation we discard the precipitate, the precipitate contained the
cellular rest of ​Saccharomyces and our protein was contained in the supernatant.
According to Chung (1995) the supernatant is the phase where the protein is
contained and centrifugation at 7000 x g for 30 min works best. We differ from
Chung only in centrifugation time but we still performed a correct protein purification.

After the first centrifugation and separation of precipitate and supernatant ammonium
sulfate was slowly added to yield 45% saturation (the calculations are shown in the
annexes) and we let it precipitate at 4°C. Then, the sample was stored in a fridge for
48 h approximately in an Erlenmeyer flask. Gomez (2013) stated the parameters at
45% ammonium sulfate saturation for 15 h at 4°C and obtained remarkable results.
We can rely on that the conditions for prior precipitation were adequate.

(Bedon, M. 2013) also established that the enzyme precipitated during 15 hours in a
45% of saturation too at 4°C, but in this step, the desalination was realized with the
help of tubes and filters (Amicon Ultra 10K-NMWL).

Desalination process was made in two steps: in the first step the solution, the
supernatant of the first centrifugation and the salt added were applied to a second
centrifugation during 10 minutes at 4°C at 8000 rpm, the finality of this step was a
apart the major amount of protein of the rest the solution though in the supernatant
of second centrifugation exist a low amount of enzyme and the same form and the
bottom of tube exist a amount of salt. The second step to obtain desalination of
solution was a dialysis process, the precipitate of the tubes after second
centrifugation was emptied at a cellophane bags this bags was a closed with heat
and before suspended inside a precipitate glass with distilled water.

At the difference with (Bedon, M. 2013) the full desalinitatión and purification process
the supernatant obtained of the filtration process let in a second precipitatión with
ammonium sulphate at 60% during 15 hours at 4 °C and before the precipitate
separation they realized using polyacrylamide gel electrophoresis and before they
quantified the amount of amylase protein using the spectrophotometer method of
Gornal.
Figure 4. Summary of amylase purification of from germinated seeds of Chenopodium quinoa (Bedon,
M. 2013).

Compare the table 2 (Bedon, M. 2013) after first precipitate with 45% of saturation it
was obtained 1870 (µg/mL) and we was obtained with the same percent of
saturation only in the tube #1 325.7 (µg/mL) and the tube #2 223.4 (µg/mL) (table 5)
in average after the desalination process including the dialysis.

Including the process the amylase production process (practice 2) we can obtain a
graph of total amylase production during the process in where shown the
concentration of amylase during the process doing a average the final concentration
of protein (Tube #1 and tube #2).

Figure 5. Amylase concentration of the full process.

Conclusión
➔ By means of qualitative tests on cornstarch, the presence of
alpha-amylase could be determined, which concluded that from the S.
cerevisae in culture medium the production of the aforementioned
enzyme was achieved.
➔ A successful cell density count was achieved with the camara de
Neubauer.
➔ We managed to correctly prepare the pH 7 buffer which will help us
preserve the enzyme alpha-amylase
➔ Centrifugation was successfully achieved

Post-Laboratory Questionnaire

● Mentions three methods of enzyme precipitation

❖ ISOELECTRIC POINT: This technique is based on the decrease in
solubility at the isoelectric point. Proteins, due to their amphoteric
nature, present a negative net charge to high pH and a positive net
charge at low pH. There is a pH called pH isoelectric to which the net
charge of the protein is zero. At this pH the molecule is unable to move
in an electric field. (Huerta, 2008)
❖ PRECIPITATION WITH SOLVENTS: This technique is based on the
decrease of solubility by the addition of a slightly polar organic solvent.
Water is characterized by its high dielectric constant, which means that
the ions in aqueous solution present weaker interactions than with
other media. The addition of an organic solvent produces aggregates
of protein molecules that tend to precipitate, this occurs because there
is a dielectric constant less than that of water, which produces an
increase in the attractive forces between opposite charges and a
decrease in the degree of ionization of the protein radicals, and
consequently a decrease in the solubility of is.(Huerta, 2008)
❖ PRECIPITATION WITH NON-IONIC POLYMERS: Ogston's model is
based on thermodynamic theory and concludes that the systems can
be explained in terms of interaction coefficients protein-polymer, and
protein-protein of the species present. Laurent's model is based on a
mechanism of geometric exclusion of hydrated regions of the protein
by the polymer. (Huerta,2008)

● What are the centrifugation conditions used industrially?

❖ 20 minutes, 7000 rpm, 4ºC ( Quintero,2009)

● What are the methods for lysis to release intracellular enzymes?

 ❖ As an agitator, homogenizers and mills that produce vibration and
movement between cells causing shocks that result in breakage.
❖ The homogenizer at high pressure where, by pressure difference, the
cell wall exceeds its capacity
❖ ultrasound
❖ electrophoresis, by means of an electric charge can quantitatively
separate molecular specie

● Investigate a chromatographic method to separate enzymes

❖ CHROMATOGRAPHY BY AFFINITY: This type of chromatography
uses highly specific interactions between one type of molecule of
solute and a second molecule covalently bound (immobilized) to the
stationary phase. By example, the immobilized molecule could be an
antibody specific for a particular protein. When a crude mixture
containing thousands of proteins is passed through a column, only a
protein reacts with the antibody that is bound to the column. After
washing all the other solutes from the column, the desired protein is
displaced from the antibody by changing the pH, the ionic strength or
the polarity. The interactions between the ligand and target molecules
can be the result of hydrophobic interactions or electrostatic
interactions, van der Waals forces and / or bridges of hydrogen. Affinity
purification requires a biospecific ligand that can be linked covalently to
a chromatographic matrix. The ligand coupled must retain its affinity
specific binding to the target molecules, and after washing the unbound
material, the binding between the target molecule and the ligand must
be reversible and allow the target molecules to be removed in an active
way.

● Investigate the use of membranes in the purification of enzymes.

❖ Filtration processes in enzyme purification and dialysis methods
Bibliography

● Armando Tejeda. (2011). 9.2.1 Precipitación por disminución de la solubilidad

. En BIOSEPARACIONES(495-504). Mexico: PEARSON.
● Gabriel Bolívar. (2018). Centrifugación: en qué consiste, tipos, importancia,
ejemplos. 14/febrero/2019, de Lifeder Sitio web:
https://www.lifeder.com/centrifugacion/
● Dra. Sobeida Sánchez Nieto. (2013). Purificación de la enzima lactato
deshidrogenasa de músculo esquelético de pollo.. 14/febrero/2019, de UNAM
Sitio web:
http://depa.fquim.unam.mx/amyd/archivero/MATERIALAPOYOANTECEDENT
ES_22427.pdf
 ● Tania V. Correa . (2009). DESNATURALIZACION. 16/Febrero/2019, de - Sitio
web: ​http://bqmenmh.blogspot.com/2009/04/desnaturalizacion.html
● Dra. Bustamante Cabrera Gladys Cordón Patino Catherine. (2014).
AMORTIGUADORES ( BUFFERS). 16/Febrero/2019, de Revistar bolivianas
Sitio web:
http://www.revistasbolivianas.org.bo/scielo.php?pid=s2304-376820140001000
03&script=sci_arttext
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http://ufq.unq.edu.ar/Docencia-Virtual/BQblog/Dialisis%20y%20ultrafiltracion.
pdf
● Bendon, M., Nolasco, O., Santa Cruz, C., Guitiérrez, A. (2013). Purificación
parcial y caracterización de alfa amilasa de granos germinados de
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● http://www.scielo.br/scielo.php?pid=S1517-83822010000400004&script=sci_a
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Annexes

Previous calculations for buffer solution pH 7

pKa=7.2
pH= 7.0

(base)
pH= pKa + log (acid)
base + ácido = 0.1 M
(base)
7 = 7.2 + log (acid)
(base)
7 - 7.2 = log (0.1−base)
100.2 = base
0.1−acid
1.5849(0.1-base) = (base)
0.15849 = 2.5849 (base)
base = 0.15849
2.5849
= 0.0613

base = 0.0613 mol/L (1 L)(174.2 g/mol)= 10.68 g K 2 HP O4

acid = 0.03869 mol/L (1 L)(136.086 g/mol)= 5.265 g K H 2 P 04

Ammonium sulphate calculation to 45% to saturation.

g (N H 4 )2 SO4
( 258 L
)(0.158 L supernatant) = 40.76 g (N H 4 )2 SO4