Instituto Politécnico Nacional

Professional Interdisciplinary Engineering Unit

campus Guanajuato

Biotechnological Engineering

Bioconversion Laboratory

Practice 5

Enzyme immobilization

4BV1

Teacher: ​Guadalupe Hortencia

Luevano

Team members:
 Marisol Estefanía Jasso Castro.
Genesis Abigail Martinez Medina.
Eduardo Moran Rangel Luis
Antonio Mendoza Soto.
General objective: ​immobilize the enzymes contained on the extracts of
amylases obtained previously from the solid and liquid state fermentation.

Specific objectives:
● learn how to immobilize the enzymes and determine the conditions that are
best for the method.

Introduction: ​Methods of immobilization: ​Sodium alginate Immobilization of Enzyme

defined, as “the imprisonment of an enzyme in a phase that allows interaction with substrate
effectors or inhibitor molecules but it is separate from them​‟​.

The imprisonment of an enzyme by covalent attaching it to a solid matrix (like sepharose) wich
allows the interactivo with the substrate.

Immobilized enzymes are used in organic syntheses to fully exploit the technical and
economical advantages of biocatalysts based on isolated enzymes. Immobilization enables the
separation of the enzyme catalyst easily from the reaction mixture, and can lower the costs of
enzymes dramatically. (​Tischer & Wedekind,1999)

There are many methods available for immobilization which span from binding on prefabricated
​ repared carriers.
carrier materials to incorporation into ​in situ p

Basically, there are four ways to immobilize enzymes: 1. Adsorption, in which enzymes are
adsorbed onto supporting matrix. 2. Covalent cross-linking. Enzyme or cell bounded covalently
with matrix. 3. Micro-encapsulation. Enzymes enclosed in a semipermeable membrane capsule.
4. Physical entrapment. Such as gelation in which enzyme is entrapped in Polymer matrix made
of cellulose triacetate, agar, gelatin or alginate.

Alginates are unbranched copolymers of (1→4)-linked β-D-mannuronic acid (M) and

α-L-guluronic acid (G) residues. If the uronic acid groups are in the acid form (–COOH), the
polysaccharide, called alginic acid, is water insoluble. The sodium salts of alginic acid
(–COONa), or sodium alginates, are water-soluble. The sequence of mannuronic and guluronic
residues significantly affects the physicochemical properties of alginates. (M.Masuelli,C.Illanes,
2014)

Alginate is located in the cell wall and in the matrix of the algae, cementing the cells together
and giving certain mechanical properties to the algae. In its native state, alginate exists as an
insoluble mixed salt of all the cations that are found in seawater, the main ones being sodium,
magnesium, and calcium. The three types of blocks in alginate have been
characterized by partial hydrolysis with HCl; ie mannuronic-guluronic (MG-block), mannuronic
(M-Block) and guluronic (G-Block). The material thus solubilized corresponds to the MG-block.
The resistant part was then fractionated at pH 2.9. The soluble fraction corresponds to the
M-block, the insoluble to the G-block. For commercial and scientific purposes, the most
important property of alginates is their ability to form viscous solutions in water, and thus
alginate samples can be characterized by intrinsic viscosity.
The ability of alginates to form gels in the presence of calcium ions is due to glycuronan as it
has one of its main biofunctional properties, and therefore is of great industrial interest. The
formation of gels depends mainly upon autocooperatively-formed functions between chain
regions enriched in GG-sequences [2]. These polysaccharides show interesting rheological
properties: that improve the viscosity of aqueous solutions to low concentrations, and to gels
form or thin films.
2 Na(Alginate) + Ca​++​→ Ca(Alginate)​2 +
​ 2 Na​
+

(Wang, 2017)

(Flores, 2011)
Methodology for the creation of beads

steps for creation of sodium alginate beads:

1.- Separate half of the volume obtained from the enzyme of the microorganism
corresponding to the beads to be made (S. cerevisiae, A. nigger, A. oryzae).

2.- dissolve 4% weight / final volume of sodium alginate in a volume of water similar to
the volume of the separated enzyme and then mix both volumes

3.- prepare 100 ml of Cacl2 0.3 M which will be placed the beads

4.- Mount the system with the 0.3 M CaCl2 where the alginate beads will be made

5.- place the sodium alginate mixture with the enzyme in a syringe, which will be used
to make the beads

6.- Drop each bead in the solution of CaCl2 0.3M mounted in the system

7.- let the beads stand for 2 hours once all are finished

8.- after 2 hours store the beads in acetates buffer PH 5.9

To make the beads of S. cerevisiae, we took the volume obtained from the
alpha-amylase enzyme from the fermentation practice with S. cerevisiae, which was
approximately 5 ml to a final volume of 8 ml, ​this for have enough volume to perform
the later practices. Besides, we decided to use a concentration of 4% w/v of sodium
alginate.

Table 1: ​Parameters used for the determination of concentrations used for the
elaboration of pearls
of sodium
alginate
Author Concentration tration of
 CaCl2 2003
Resting time Temperature
Resting time Temperature
used
Talekar and
used
chavare, 2012
S
Bolaños,

Borgio,2011 2%(w/v) 1%(w/v) 5 minutes DS

Gargi,Bhupi
nder and
DS: Does not show
pintu, 2003
w/v) 2 hours DS

According to the bibliography shown above the concentrations used where:

Table 2​: Parameters used to create the pearls

Concentration of alginate sodium ​4%(w/v)

Concentration of CaCl2 ​2%(w/v)

Time ​1-2 hours

Storage of alginate beads

Table 3​: Comparison of conditions used in the method.

No. Microorganism Bead wash Buffer Instrument

for drops
 1 ​A. niger S
​ aline solution DS Glass pipette
(2 mm
diameter)
pipette(1 mm of
2 ​DS water Acetic diameter)
acid-Sodium
4 ​Dacetate
S Distilled water Sodium citrate
0.05M
pH 4.8 0
Pipette Syringe
(0.25-0.5mm)

5 ​DS Saline solution Phosphates

3 ​Bacillus acidocaldarius D
​ istilled water ​Acetates 50 Syringe
mM pH 5.9 at
4°C
DS*: Does Not show
Pasteur

The buffer agreed to store the beads according to the bibliography shown above was
acetates at 50mM with pH of 5.9. This buffer was made to storage the beads made of
the commercial enzyme, ​Saccharomyces Serevisiae, a ​ nd ​Aspergillus niger. ​The pearls
were left in the fridge for 10 days.

Dissolved beads ​Spherical beads have been prepared by ionotropic gelation of

sodium alginate in the presence of CaCl​2 ​and BaCl​2 ​solutions. The barium
ion-crosslinked beads exhibit
almost minimum swelling of 40 ± 3% in PBS at pH 7.4 but possess greater stability
while calcium alginate beads exhibit nearly 160% of water uptake and subsequently
dissolve. The beads appear to swell through ion-exchange process which was
confirmed by monitoring the Ca​2+ ​release from the calcium alginate beads. To dissolve
the alginate beads we used the bibliography shown on table 4. we used citrate buffer at
50mM with pH of 7.4. To see if it dissolved the beads, we made different proves. for the
first prove we put 1 ml of buffer for four beads and it took 30 minutes for its complete
degradation, for the next prove we put 4 ml of buffer in 4 pearls and it took from 10 to 15
minutes for its complete degradation which was the one used to make the quantification
of proteins by the bradford method.
 Table 4​: Methods to dissolve alginate beads

Reference Basis Method

amsbio,2015 Sodium citrate Method 1 (Method of recovery of cells from only

a part of the 24-well plate)
● (1) Remove the medium from the well
using an aspiration pipette
● (2) Transfer the alginate beads to 1.5 mL tubes using a sterile spatula.
● (3) ​Add 1 mL sodium citrate solution to each tube, and mix for 5–15 min at room temperature to
dissolve the alginate beads.
● (4) Centrifuge at approximately 300 Xg for
3 min.
● (5) Remove the supernatant, and harvest the
cells as a precipitated pellet.
ammonia buffer (90 g NH4Cl and 375 mL NH4OH
Fabrication of patterned (28–30%) diluted to 500 mL in water). The excess
calcium cross-linked of EDTA was back titrated using a standardized
alginate hydrogel films solution of calcium chloride, the equivalence
and coatings through determined in the presence of Eriochrome Black T
reductive cation (2 drops of a 1% alcoholic solution) (Ray Sarkar &
exchange Marion Chauhan, 1967).
Bruchet, Artem Melman After reductive exchange, calcium alginate in a
5 May 2015 quartz cuvette was dissolved using 1 mL of a
D 0.125 mM ethylenediaminetetraacetate (EDTA)
on of standardized solution (pH = 10). After the
on of calcium dissolution the volume of the solution was
he hydrogel adjusted to 10 mL using distilled water and the pH
tive cation was adjusted to 10 using 1 mL of concentrated
ammonia buffer (90 g NH4Cl and 375 mL NH4OH
aminetetraac (28–30%) diluted to 500 mL in water). The excess
A) standardized of EDTA was back titrated using a standardized
H = 10). solution of calcium chloride, the equivalence
After reductive exchange, calcium alginate in a determined in the presence of Eriochrome Black T
quartz cuvette was dissolved using 1 mL of a (2 drops of a 1% alcoholic solution) (Ray Sarkar &
0.125 mM ethylenediaminetetraacetate (EDTA) Chauhan, 1967).
standardized solution (pH = 10). After the
dissolution the volume of the solution was amsbio,2015 ​The cells can be
adjusted to 10 mL using distilled water and the pH recovered if necessary
was adjusted to 10 using 1 mL of concentrated Method 2 (Method of recovery of cells from the
entire 24-well plate)
be adding a sequestering Characterisation of the
agent for the calcium ions degrading properties of
(such as polyphosphate or alginate under influence
EDTA); once the calcium of citrate Thijs Grünhagen
ions are removed from the July 2002 BMTE 02.37
gel, its structure is lost and itIn this experiment, degradation of alginate is defined
changes to a liquid with the by the reduction of weight during exposure to citrate.
cells suspended in it. Alginate samples of two different concentrations (1%,
● (1) Remove the medium from the entire well 5% (m/v); see appendix) were exposed to five
using an aspiration pipette. different citrate concentrations (1 mM, 5 mM, 10 mM,
50 mM and 100 mM​). An alginate cylinder with a
● (2) Add 1 mL sodium citrate solution to each
radius of 15 mm and a height of 20 mm was weighed
well, and mix for 5-15 min at room
and placed in a beaker filled with 30 ml of one of the
temperature to dissolve the alginate beads.
citrate concentrations, which was gently shaken.
● (3) Transfer the alginate solution to 1.5 mL Every five minutes, the alginate samples were taken
tubes and centrifuge at approximately 300 ×g out of the citrate solution and weighed. This
for 3 min. procedure was followed until the samples were fully
● (4) Remove the supernatant, and harvest the degraded or the degradation course was sufficiently
cells as a precipitated pellet characterised.

50 mM sodium citrate solution with pH 7.4.

The experiment in which degradation of alginate gels

was characterised in terms of weight reduction, gave
insight into the effect of several citrate concentrations
on the degradation speed of two different
concentrations of alginate gels. It turned out that
citrate concentrations of 50 mM and 100 mM brought
about complete degradation of the alginate samples.
This led to the choice to use these two concentrations
in the later mechanical experiment to measure the
alteration of some mechanical parameters during
degradation.

we know that calcium ions can easily be exchanged for sodium ions, as well as
enzymes can be easily recovered by dissolving the gel in a sodium solution (Wang,
2016). Ionic exchange is a reversible chemical reaction that takes place when an ion of
a solution is exchanged for another ion of equal sign that is attached to an immobile
solid particle. For calcium alginate beads it is possible for calcium ions to be replaced
(Gónzalez, 2009).
Results:
● Number of pearls in each experiment

Sample Number of pearls

White 180

​ 20
Saccharomyces Serevisiae 3

Aspergillus Niger ​280

Commercial Enzyme ​270

Table 5 . ​Quantification of protein obtained in calcium chloride solution.

Sample Average absorbance Concentration

(μg/mL)

A0 (White) ​0 0

A1 (S. cerevisiae) ​0.063 38.44

A2 (A. niger) ​0.116 61.47

Table 6. ​Quantification of protein in beads dissolved in citrate buffer

Sample Average absorbance Concentration (μg/mL)

A0 (White) ​0 0
60.60
A1 (S. cerevisiae) 5
beads

A2 (A. niger) 3 beads ​0.286 135.39

 A3 (A. oryzae) 3 beads ​0.05 32.78

Table 7. ​Quantification of protein in acetate buffer

Sample Average absorbance Concentration (μg/mL)

A0 (White) ​0 0

A1 (A. oryzae) ​0.0235 21.26

Figure 1. ​Diagram of the immobilization process of de enzyme alpha-amylase from S.

cerevisiae.

Global enzyme balance for α- amylase from S. cerevisiae

Where: A= Enzyme suspended in phosphate

buffer F= Suspended enzyme in calcium
chloride I= Immobilized enzyme in alginate
beads

Figure 2. ​Diagram of the immobilization process of de enzyme alpha-amylase from A.

niger.

Global enzyme balance for α- amylase from A. niger

Where: A= Enzyme suspended in phosphate

buffer F= Suspended enzyme in calcium
chloride I= Immobilized enzyme in alginate
beads
Figure 3. ​Diagram of the immobilization process of de enzyme alpha-amylase from A.
oryzae

Global enzyme balance for α- amylase from A. oryzae

Where: A= Enzyme suspended in phosphate

buffer H= Suspended enzyme in acetate buffer I=

Immobilized enzyme in alginate beads ​A-H-I=0

A 96.64% immobilized enzyme was obtained by A. oryzae. Having a loss of 3.36%

Immobilization efficiency

Immobilization efficiency was defined as follows:

Discussion:
● Effect of Sodium Alginate Concentration ​To have pearls with
minimal or no enzyme loss we resorted to the articles cited and found that most of these
cited 3% (w / v) and 4% (w / v) concentration of sodium alginate, for which It was
decided in the classroom that two teams would work with 3 % and another two with 4%,
as well as we also find in most of the articles that these are deposited in 2M calcium
chloride. the concentration of enzyme with the bradford test in the calcium chloride used
for the elaboration of the pearls was relatively low 38.65 ​Mg/ml ​for saccharomyces and
61.59 ​Mg/ml ​for aspergillus, that is, compared with the initial concentration of enzyme
that are of 194.3 Mg/mL for S. Cerevisiae and 6321.7 Mg/mL for A.Niger so we can say
that there was no loss of enzyme in this part and that although there were only some
remains of it.According to A.Anwar, S.Qader, S.A.Azhard
● Immobilization efficiency for α- amylase from ​A. oryzae
The percent entrapped activity was found maximal 45% ± 2 at 2% (w / v) sodium
alginate concentration. Maximum leakage of enzyme from beads occurred at 1% (w / v)
sodium alginate concentration owing to the larger pore size of the less tightly crossed
linked fragile Ca-alginate beads while at 3% and 4% (w / v) sodium alginate
concentration the entrapped activity of the enzyme was found comparatively very low
which might be due to the high viscosity of enzyme entrapped beads, which decreased
the pores size and thus hindered the penetration substrate in to the beads.To analyze
this part we need to perform future tests for example of enzymatic activity to be able to
quantify the enzyme that is still active after this method of immobilization and thus
determine the efficacy of it since with this concentration of alginate that.
● Effect of Calcium Chloride Concentration on Immobilization ​The
concentration of calcium chloride was chosen on the basis of some articles of which the
concentration of 2% (w / v) was chosen since according to A.Anwar, S.Qader,
S.A.Azhard,. Concentration of calcium chloride (0.05- 0.3 M) was also varied in order to
acquire stable beads capable to secure maximum enzyme and it was found that CaCl2
(0.2 M) retained highest activity of entrapped enzyme and as calcium chloride
concentration increased beyond 0.2 M the activity decreased. Roig et al. also reported a
decrease in the relative enzyme activity of alkaline amylase when they increased the
concentration of CaCl2 used to form capsule . They observed that the pH of the CaCl
solution changes with its concentration which might be a factor to affects the activity of
entrapped enzyme.
● Effect of temperature and PH ​In this part we find that the temperature at
which the active center as well as the cmom such enzyme does not suffer damage is 4c
since this will prevent the unfolding of the hydrogen bridges that make up the unions
between primary structures to shape the tertiary and secondary. When the temperature
is high, the kinetic energy of the molecules increases, which disorganizes the aqueous
envelope of the proteins, causing irreversible damage or denaturation. As well as an
optimum ph of 5.9 since according to R ​ oyhaila, Haziqah, Aziah, Huyop & Abdul,2015 ​At
lower pH solution, there is a high presence of protons that favors the formation of the
complex enzyme- calcium alginate At stable pH, the enzyme does not denaturalize and
the hydrophobic regions keep their natural position, this one allows the reaction enzyme
with alginate sodium

It was reported that the surface of the beads in which the enzyme is localized has a
cationic or anionic nature. This charged surface of beads and entrapped enzyme
produces a charged microenvironment, which ultimately affects the nature of the active
enzyme protein and alters the pH of the entrapped enzyme
● Disolving the calcium alginate ​We know that calcium ions can easily be
exchanged for sodium ions, as well as enzymes can be easily recovered by dissolving
the gel in a sodium solution (Wang, 2016). Ionic exchange is a reversible chemical
reaction that takes place when an ion of a solution is exchanged for another ion of equal
sign that is attached to an immobile solid particle. For calcium alginate beads it is
possible for calcium ions to be replaced (Gónzalez, 2009). According to this statement
the buffer used contains sodium acetate which had a good result in the degradation of
the pearls.
 ● ​Results of the energy balance ​The results of the balance of the enzyme
obtained from the microorganisms were contradictory in the case of S. cerevisiae, since
the final balance did not coincide coherently with the established one, the amount of
initial enzyme differed from the sum of the readings obtained for the pearls, the buffer
where they were stored could contain an enzyme that was not retained inside the
beads, which would result in loss after immobilization. It should be taken into account
the possibility that the equipment used for alpha amylase measurements from s.
Cerevisiae y A Níger would perform an erroneous measurement due to its poor
calibration or to the poor performance of the Bradford technique.

Conclusion
● ​It was possible to imóbilize the enzymes ​contained on the extracts of amylases
correctly therefore the results given of the ​Immobilization efficiency for α- amylase
from ​A. oryzae ​was of 93.28%. it was only possible to determine one efficiency
because there were problems in the balance of the concentrations explained in the
discussion.
● it was also possible to determine the conditions that were best for the method
which were: 1. Sodium alginate solution concentration: 4% (w/v) 2. Calcium chloride
solution concentration: 2% (w/v) 3. Beads curing time: 1 h 4. Buffer for beads
suspension: Acetate 50 mM, pH 5.9

Post-Laboratory Questions ​1. Which were the parameters that affected the yield
of immobilization according to your practice and literature?

How the concentration of sodium alginate affects the yield of immobilization?

In case of having low concentrations it might be due to larger pore size and
consequently greater leakage of the enzyme from the matrix (Singh 2003). Since
concentration increases the pore size decreases.

2. Which were the disadvantages of the protocol that you proposed?

The concentration of Calcium chloride:we used a concentration of 0.2 M which is a low

concentration according to the usual reported by authors as Lahiri (2015) who used
concentrations of 0.5 M to 3M

3. What are the advantages of using immobilized enzymes? As advantages of the use
of immobilized enzymes such as: 1. Increased stability of the enzyme; 2. The possible
reuse of the derivative, thus reducing the costs of the process. 3. The possibility of
designing an enzymatic reactor of easy handling and control, adapted to the application
of immobilized enzyme (Dey G.,2003).

4. What are the applications of enzymes immobilized in the industry?

-In protein hydrolysis: Proteolytic enzymes are used in the modification of the protein
content of foods. Whey protein hydrolysates have been achieved by the use of pepsin
and protease co-immobilized on chitosan. Other immobilized proteases have been used
in the reduction of lactoglobulin content in milk, in the dairy industry (Taylor,1991). -In
improving the organoleptic characteristics of certain foods: The use of Arthobacter
globilis cells trapped in polyacrylamide eliminates the bitter taste of citrus juice. On the
other hand, Leuconostoc oeno cells immobilized in alginate have been used in
deacidification of wine. -Waste water treatment: A method has been developed for the
reduction of nitrates to nitrites in wastewater using immobilized enzymes (Taylor,1991).

5. What is the mechanism of formation of calcium alginate gel?

The salts of the alginic acid are formed by three blocks, blocks M, G and GM. When two
G-block chains are aligned, coordination sites are formed due to the loop shape of
these chains, the cavities remain between them and these are sized to accommodate
the calcium ion, are coated with carboxylic groups and other oxygen atoms
electronegatives which are favorable ligands and allow a high degree of coordination of
the calcium ions. For this reason this model is called "egg box model" (Oliveira,2003).

References:
● A. Anwar, S. Qader, A. Raiz, S. Azhar Calcium Alginate: A Support Material for
Immobilization of Proteases from Newly Isolated Strain of Bacillus subtilis KIBGE-HAS,
2009
https://pdfs.semanticscholar.org/d9e4/52030ad73695a28d0c8b75394b9538359c31. pdf

● Martin Alberto Masuelli, Cristian Omar Illanes. Review of the Characterization of

​
Sodium Alginate by Intrinsic Viscosity Measurements. Comparative Analysis between
Conventional and Single Point Methods, International Journal of BioMaterials Science
and Engineering. Vol. 1, No. 1, 2014, pp. 1-11.
https://www.researchgate.net/profile/Cristian_Illanes/publication/272785338_Review
_of_the_characterization_of_sodium_alginate_by_intrinsic_viscosity_measurements
_Comparative_analysis_between_conventional_and_single_point_methods/links/54
edcc540cf25da9f7f233dc/Review-of-the-characterization-of-sodium-alginate-by-intrin
sic-viscosity-measurements-Comparative-analysis-between-conventional-and-single-
point-methods.pdf
​ . Flores, Efecto de un proceso de inmovilización por gelación iónica sobre la
● G
actividad proteolítica de mexicaína. Instituto Politécnico Nacional. México. (2011)
● Royhaila, Nur., Haziqah, N., Aziah, N., Huyop, F., & Abdul, R. (2015). An overview
of technologies for immobilization of enzymes and surface analysis techniques for
immobilized enzymes. Biotechnological equipment.
● ​Lahiri, P. (2015). Development of the Optimal Conditions for Alpha-Amylase Immobilization.
International Journal Of Scientific Research, [online] 4 (Diciembre 2015). Retrieved from
https://www.worldwidejournals.com/international-journal-of-scientific-research-
(IJSR)/file.php?val=December_2015_1450703201__150.pdf ​[Accesado 13 Mayo 2018]
● Taylor, R.F. (1991) Protein Immobilization: fundamentals and applications, Marcel Dekker,
New York.
● Dey G., B. Singh and R. Banerjee. (2003). Immobilization of α- amylaseProduced by Bacillus
circulans GRS 313. Braz. Arch. Biol.Techn. 46(2):167-176.