INSTITUTO POLITÉCNICO NACIONAL

Unidad Profesional Interdisciplinaria de

Ingeniería Campus Guanajuato

Bioconversion’s Laboratory

Practice 5: ​Immobilization of
enzymes

Teacher ​Guadalupe Hortensia Luévano

Colmenero

4BV1

Students:
• ​López Pardo Genaro Uriel
• ​Mireles
Zavala Elizabeth Guadalupe
• ​Ramírez Arzola Fátima Montserrat
• ​Roque Salinas María Guadalupe
 BIOCONVERSION’S LABORATORY
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OBJETIVE:
- Immobilize the enzymes obtained in the extracts of the previous practices by
Solid state fermentation and liquid fermentation.
PARTICULAR OBJECTIVES:
- Determining the optimal concentration of calcium alginate for immobilization. -
Realize the production of alginate beads for each enzyme extract, by means of
microencapsulation. - Prepare the necessary buffer solution for the total dissolution
of the alginate
beads - Determine the average final concentration of the alginate beads for each
enzyme extract using the Bradford method. ​INTRODUCTION: ​The processes
catalyzed by enzymes in the industry are increasingly numerous, since present a series
of advantages over conventional non-biological catalysts, it presents a great catalytic
activity; They show a high substrate specificity (including stereoselectivity and Regio
specificity); They are very active at room temperature and atmospheric pressure. The
immobilization of enzymes is a process in which the enzyme is confined or located in a
defined region of space, to give rise to insoluble forms that retain their catalytic activity
and can be reused repeatedly. Subsequently this definition has been extended to that
process by which the degrees of freedom of movement of enzymes, organelles, cells,
etc., are restricted, completely or partially, by their attachment to a support.

As advantages of the use of immobilized enzymes we can highlight:

1. Increase in the stability of the enzyme; 2. The possible reuse of the derivative,
which reduces the costs of the process. 3. The possibility of designing an enzymatic
reactor of easy handling and control, adapted to the application of the immobilized
enzyme. The different types of enzymatic reactors appear in Figure x. These
reactors with immobilized enzymes allow the use of high loads of enzyme, which will
maintain its activity for longer. These systems may include recycling, which allows
obtaining higher purity products.
 Figure 1.- Enzymatic Reactors employing immobilized enzymes

BIOCONVERSION’S LABORATORY
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The main drawbacks of the immobilization process are: 1. The alteration of the
conformation of the enzyme with respect to its native
state. 2. The great heterogeneity of the enzyme-support system where different
fractions of immobilized proteins can exist with a different number of bonds to the
support. 3. There is always a loss of activity of the enzyme during mobilization. 4.
The biocatalyst is more expensive than the native enzyme.

In general, immobilization methods are usually classified into two broad categories:
Physical retention and Chemical unión:

Figure 2. Methods of immobilization by physical retention.

Within the classification of inclusion of membrane is the technique of

 Microencapsulation: In this technique, the enzymes are surrounded by semipermeable
membranes that allow the passage of substrate and product molecules, but not of
enzyme. These semipermeable membranes can be permanent (originated by interfacial
polymerization) or non-permanent (generated by surfactants, also called "reverse
micelles"). The obtained microcapsules are spherical in shape, with sizes ranging from
1 to 100 μm in diameter. By means of this method, a large variety of enzymes, cells or
biomolecules can be encapsulated simultaneously, allowing certain reactions that occur
in multiple steps to be carried out. This physical process is the employed in the
methodology of encapsulation and immobilization of the work enzymes.
Another of the existing classifications is the chemical union, where its sections are
shown below:
PHYSICAL

RETENTION

Entrapment

In layers

In Fibers
Inclusion in
Membranes

Encapsulation

Reactors

BI
3
 CHEMICAL

UNION
eticulated

Union Supports
Pure Reticulated

Ionic
Absorption
Co-reticulated

Covalent Union

Figure 3.- Methods of immobilization by chemical

union.

Although many immobilization techniques have been developed and applied to

numerous enzymes, it is recognized that there is no universal method valid for all
enzymes in all cases. However, thanks to all the information currently available,
generalizations can be made about each method of immobilization and thus, we can
select the most appropriate for each specific application. The choice must take into
account the conditions of the biocatalyzed reaction, the type of reactor to be used, the
type of substrate that has to be processed and other factors.

MINIMUM LOGICAL REQUIREMENTS FOR THE IMMOBILIZATION OF AN

ENZYME ​- The enzyme or cell must be stable under the necessary experimental
conditions. - If cross-linking agents are used, they should not affect the active center.
If they could interfere with it, a cross-linking agent should be used as large as
possible. - If possible, the active center should be protected:
If there are -SH groups, they must be reacted with cysteine or glutathione, to
reactivate later after immobilization.
Immobilization can be performed in the presence of saturating substrate
concentrations. - The washing process should not adversely affect the enzyme. -
Consistency with the subsequent biotransformation. - If support = polyanion, the
conversion of an anionic substrate will be very
 difficult (repulsion) - Enzyme trapped in a gel: if the substrate has a high molecular
weight, the result
will be bad. - Great importance of the mechanical properties (stability of the support,
physical form thereof)

BIOCONVERSION’S LABORATORY
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- The support must have a high specific surface, to minimize the problems of
material transfer. - The support must confer immobilized derivative a good
resistance to microbial
contamination. - The support must allow a high amount of biocatalyst (high
"loading") to be
immobilized. - The support must be commercially accessible: low price and good
availability The immobilization technique in sodium alginate is relatively simple. Dissolve
the sodium alginate in a little warm water, mix well, and when it is cold, add the yeasts.
The final mixture is dropped, drop by drop, into a calcium chloride solution.

With the exchange of calcium ions (of the medium) and sodium (of alginate) a matrix of
calcium alginate is formed that retains the yeasts.

Despite having solid consistency, said matrix allows the circulation of substances, so
that biological agents retain their metabolic activity. Once the expected chemical
reactions are completed, the alginate pellets and yeast recover easily.

Alginates are salts of alginic acid that can be formed with Na, K, Mg, Ca, among others,
forming salts with different degrees of solubility in water, which confers varying degrees
of viscosity (Yabur et al., 2007).

They are the structural components of the cell wall of algae, whose main function is to
give rigidity, elasticity, flexibility and water binding capacity (Hernández et al., 2005).

Alginates are a family of linear polysaccharides, containing variable amounts of β-D-

mannuronic acid (M: 1,4- β-D-mannopyranosyluronic acid 4C1) and α-L-guluronic acid
(G: 1,4- linkage α-L-gulopiranosilurónico acid 1C4). Its composition (given by the
characteristic manuronic / guluronic relationship M / G) and sequences vary depending
on the source from which the polysaccharide comes.

It was found that the strength of alginate gels depends on the number of cross-links
formed, the type of ionic crosslinking, and the length and stiffness of the blocks between
the bonds (Mancini et al., 1999). The above is important, since alginates that have large
regions of G blocks form a gel of high strength and exhibit a high porosity. Those with
large M blocks form a gel of medium strength, but with a high resistance to syneresis
and exhibit smaller pores that make them softer (Hernández et al., 2005). In an acid
solution the alginate will form a gel, since at low pH the carboxylic groups of the uronic
acids accept protons, which causes that the formation of bonds of the G blocks is
favored. The hydration of the alginic acid at low pH leads to the formation of a high
viscosity gel. In an acidic medium, the viscosity is increased by decreasing the solubility
of the free alginic acid, precipitating in gel form at a pH in the range of 3 to 4 (Lupo et
al., 2012).
structure of the alginate. (Adapted from Reddy
and Reddy 2010).
BIOCONVERSION’S LABORATORY
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Fig. 4. Model "egg box" that describes the
Assemble the agitation system for the formation of pearls. ​Adjust the agitation of the system
​ at
a not too high speed, which will cause damage to the formation of the pearls.
Perform the bradford technique to know the Pour the 100mL of the calcium chloride solution
into the system.
average final concentration of each pearl obtained for each of the working enzymes.
Depositing the mixture in the syringe calcium alginate with each corresponding enzyme.
To dissolve the pearls, prepare a solution of 0.5 M sodium citrate and 4 N sodium hydroxide.
Add 1mL per pearl. Shake until completely dissolved.
Begin with the formation of pearls. Note: never leave the system free of
Carefully wash the pearls with distilled water agitation.
Once the entire volume of the enzyme is finished. Allow a rest time of 1 to 2 hours approximately.
METODOLOGY
Immobilization of alpha amylase enzyme
Separate of each enzyme, from the final volume obtained from the part of the fermentation, half
of this concentration.
BIOCONVERSION’S LABORATORY ​6
Prepare the calcium alginate at a concentration of 3M for each enzyme. Add the
enzyme
Prepare the calcium chloride solution at a concentration of 3M in 100mL of distilled
water
RESULTS AND DISCUSSION
A 3% (w/v) sodium alginate solution in distilled water was prepared by warming at 40°C.
After cooling down, using half volume of enzyme stock solution was mixed with the
same volume of sodium alginate solution (the relation of matrix and enzyme mixture
being 1:1). The mixture was taken into a syringe, and beads were formed by dropping
the solution into 0.3M calcium chloride solution with gentle stirring at 4°C for 2 h. The
formed beads were recovered by filtration and thoroughly washed with distilled water.
(Talekar, 2012) After enzymes were stored in distilled water for its preservation. In table
1 shows the enzyme concentration, that wasn’t retained for sodium alginate.
Table 1. Protein quantification by Bradford test for CaCl​2 ​Microorganism ​Sample
Concentration
(μg/ml)
The enzyme entrapment in beads depends on the concentration of sodium alginate and
calcium ions. Size of the beads also affects the immobilization efficiency. (Bharat, 2015)
Also (Prakash, 2011) mentions It was found that 1M calcium chloride retained highest
(89%) α-amylase and as the concentration of calcium chloride increased beyond 1M,
immobilization yield of α-amylase was decreased (Fig.5). While (Awnar, 2009) says
where it got more relative activity was with a concentration of 0.3M calcium chloride.
This excessive release of 26.9375 and 24.125 μ ​ g/ml ​was attributed for the low
concentrations of alginate, causing very large pores was obtained that allowed the
diffusion of substrate, products and cells (Sossa et al., 2008).
Another factor is effect of curing time of calcium alginate beads, hardness of the calcium
alginate beads depends upon time required for the gel to set. (Dey et al, 2003) It is an
important parameter in immobilization as it produces stable calcium alginate beads
which could reduce the enzyme leakage and increase the immobilization yield of
enzyme. So, the effect of curing time on the enzyme immobilization yield was evaluated.
The
1 ​ 40.375 26.9375
Average concentration (μg/ml) ​Blank ​0 0 ​A. Niger ​ 2 ​
1 ​ 26 13.5
13.5 ​S. Cerevisiae ​ 2 ​
1
A. oryzae (commercial)
1 39.125 24.125 2 9.125 ​Figure 5. Effect of concentration of calcium
chloride on immobilization yield
Figure 6. Effect of curing time on immobilization yield
BIOCONVERSION’S LABORATORY ​7
treatment of the beads in a calcium chloride bath for 120min gave 90% immobilization
yield (Fig.6).

As already mentioned, the sodium alginate concentration also affects the enzyme
activity. Ertan ​et al. ​(2007) reported that when alginate concentration was increases
from 1 to 4% (w/v), the highest α-amylase activity was found to be at 3%(w/v). Figure 7.
 immobilization yield was found to be highest (90%)
for a final sodium alginate concentration of 3%
(w/v). Figure 8. The authors also mentioned that
the decreasing immobilization yield with increase in
sodium alginate concentration is due to the
decrease in the porosity of the gel beads, which
cause diffusion limitation of the substrate. While,
the lower immobilization yield in case of lower
concentration of sodium alginate solutions might
be due to larger pore size and consequently
greater leakage of the enzyme from matrix.
Figure 8. Effect of alginate concentration
on immobilization yield

In the same way, as Talekar (2012) reported, the

Also, in the article published by Wen-Taoy et al., (2005), it is observed that they
obtained higher concentration of enzyme because they worked a mixture of alginate-
chitosan -alginate as polymers to form beads with greater internal fluidity without having
an excessive release of cells, which favored the formation of cell aggregates that
compete for the available substrate (Sossa et al., 2008).

One the most important processes is gelation, alginates can be cross-linked by external
or internal gelation method using polyvalent cations, such as Ca​2+​.(Chan,
Figure 7. Effect of alginate concentration on enzyme
activity
 BIOCONVERSION’S LABORATORY
8
2005) The main difference between the mechanisms of external and internal gelation is
the kinetics of the process. If what is intended is the control of the solution-gel transition,
in the external gelation process the factors to be manipulated are the calcium
concentration and the composition of the polymer. While for the internal gelation
process, the solubility and concentration of the calcium salt and the concentration of the
sequestering agent and the organic acid used must be considered (Draget et al., 2002).
Taking this information, the process that was used is external gelation, then (Codd,
1991) mentions some comparisons between externally and internally gelled immobilized
this comparison of the traditional externally gelled beads and the internally immobilized
particles, showed that the externally gelled beads were superior. The internally gelled
particles produced were soft "crumbly” and were difficult to mould into the desired form.
The storage of the obtained pearls was made in distilled water, however, (Erickson,
1992) mentions that the Ca​+2 ​ion required in ​Aspergillus ​Species, for example, seems to
be so closely united that it will not be lost even if it is dissolved or stored in distilled
water. But the same author suggests for the Conservation treatment of pearls (without a
side effect of causing the precipitation of calcium ions) to use acetate buffers.
For protein quantification, the capsules were dissolved in 0.4N NaOH containing 0.5M
sodium citrate. The combination of alkaline pH and calcium chelator (citrate) ensures
the rapid and reliable dissolution of the capsules. (Pierre, 1993) Alpha amylase activity
increased from pH 3.0 and peaked at pH 6.0-8.0 beyond which it gradually decreased
until pH 10.0. (Tizon, 2012). pH can have an effect of the state of ionization of acidic or
basic amino acids. Acidic amino acids have carboxyl functional groups in their side
chains. Basic amino acids have amine functional groups in their side chains. If the state
of ionization of amino acids in a protein is altered, then the ionic bonds that help to
determine the 3-D shape of the protein can be altered. This can lead to altered protein
recognition or an enzyme might become inactive. (Frankenberger, 1982). In the next
table shows the results obtained the Bradford method.
Table 2 . Protein quantification by Bradford test for immobilized enzyme
Microorganism ​Sample Concentration
(μg/ml)
The immobilization efficiency was calculated as Sharma ​et al. (​ 2014) mentioned:
Average concentration in 3 beads (μg/ml)
Immobilization
efficiency %
Blank 0 0 -
A. Niger 1 ​6.625 28.1875 81.88
2 ​49.75 ​S. Cerevisiae 1 ​60.38 60.37 95.71
2 ​60.36 ​A. oryzae (commercial)
1 ​182.75 184.75 89.27 ​2 ​186.74
BIOCONVERSION’S LABORATORY ​9
Teorical enzyme loaded − amount of enzyme leached in CaCl​ solution and
Immobiliztion efficiency (%) = (​ 2​

washings

) × 100
teorical enzyme loading ​
Immobilization techniques are applied to cells as well as to microorganisms and
enzymes. Its main advantages are to facilitate the separation of the product and allow
the recovery of the biological agent.
In one variation, adjusting the pH by adding NaOH to the reagent improves the
sensitivity of the assay and greatly reduces the variation observed with different proteins
(Stoscheck,1990). This is presumably caused by an increase the proportion of free dye
in the blue form, the ionic species that reacts with protein. However, the optimum pH is
critically dependent on the source and concentration of the dye. Pretreatment of the
samples with membrane-disrupting agents such as NaOH or detergents may reduce
this problem, but the results should be treated with caution. (Kirazov et al, 1993) Also
The addition of 1 M NaOH was suggested by Stoscheck (1990) to allow the
solubilization of membrane proteins and reduce the protein-to- protein variation in color
yield. With this information we know that NaOH doesn’t interfere in this process.
After an immobilization, the activity of the enzyme can decrease and even be lost by
varios reasons If you completely lose enzymatic activity, it may be because Ertan, F.,
Yagar, H., Balkan, B. (2007):
1. The connection to the support is produced in such a way that the passage from
the substrate to the active center is prevented, 2. The reactive groups of the support
react with any amino acid that forms part of
the center active or essential for the catalytic activity of the enzyme, 3. Immobilization
can lead to a conformational change that results in a inactive 4. The experimental
conditions of the process cause the denaturation or
deactivation of the enzyme.
If the loss of activity is not total after immobilization, the changes (decrease or increase
in enzymatic activity) will be mainly due to diffusional, electrostatic, steric and / or
microenvironment effects.
CONCLUSION
In accordance with the stated objectives, an external gelation was carried out. In
addition, important factors are identified that influence the efficiency of the
immobilization such as concentration of calcium chloride, the extension of the curing
time with the purpose of obtaining pearls with greater hardness and union with the
alginate as well as the size of the obtained pearls.
Effectively, the beads were dissolved in 0.4 N NaOH containing 0.5M sodium citrate to
perform quantification by Bradford method obtaining an average concentration of
28.1875, 60.37 and 184.75 μg / ml immobilized enzyme from Aspergillus Niger, S.
Cerevisiae Y commercial enzyme respectively, results uninfluenced by the factors
mentioned above​.
BIOCONVERSION’S LABORATORY ​10
ANEXXES
1. ​CALCULATIONS TO PREPARE THE CALCIUM CHLORIDE SOLUTION
CaCl​2 ​molecular weight: 110.98 g/mol Final concentration: 0.3M Final volume: 100 mL
mol
g​CaCl​2 =
​ (0.3​

)(0.1L)(110.98 ​ g) = 3.3204 g
L​ mol​
2. ​DETERMINATION OF SODIUM ALGINATE FOR EACH ENZYME
SOLUTION - For ​S. cerevisiae
Volume of enzyme extract: 14 ml
w⁄​ =​ grams of ​ solute
%​ v​ mililiters ​ of ​
 × 100
solution ​
3)(14) = 3.3710 g
g​solute =
​ ( 100​
- For ​A. niger
Volume of enzyme extract: 7 ml
3)(7) = 0.21 g
g​solute ​= ( 100​
- For commercial enzyme (​A. oryzae​) Volume of enzyme extract: 10 ml
3)(10) = 0.3 g
g​solute ​= ( 100​
3. ​GLOBAL BALANCE A-AMYLASE FROM ​S. CEREVISIAE
Destiled water Suspended
Pure enzyme 7 mL
MIXED ​enzyme in
Sodium alginate 14 mL
IMMOBILIZATION ​50mL ​WASHING
Sodium alginate 3% w/v 7mL Immobilized enzyme
in alginate beads

Calcium chloride 0.3 M ​100mL

Immobilized enzyme 630 beads
Suspended enzyme in CaCl​2
___ (μg/ml 13.5 μg/mL
suspensión)/3 beads60.
BIOCONVERSION’S LABORATORY ​11
μg) ∗ (7ml) = 31526.25μg ​
μg α − amylase = (4503.73 ml​ μg α − amylase in CaCl​2 =
​ (13.5

μg) ∗ (100ml) = 1350 μg

ml​
μg Immobilized Enzyme= 31526.25μg - 1350 μg = 30176.25 μg/630 beads =47.89μg/beads
Volume per bead: 7mL/630 beads= 0.011 mL
4. ​GLOBAL BALANCE A-AMYLASE FROM ​A. NIGER
μg) ∗ (3.5ml) = 7434μg ​
μg α − amylase = (2124 ml​ μg α − amylase in CaCl​2 =
​ (26.9375

μg) ∗ (100ml) = 2693.73 μg

ml​
μg Immobilized Enzyme= 7434μg - 2693.73 μg = 4740.27 μg/380 beads =12.47 μg/bead
Volume per bead: 3.5 mL/380 beads= 0.009 mL
Destilled
Suspended
water 50mL
Enzyme pure 3.5 mL
MIXED ​enzyme in sodium alginate 7 mL
IMMOBILIZATION ​Immobilized
enzyme 380 beads
WASHING
Sodium alginate 3% w/v 3.5 mL
Immobilized
Calcium chloride 0.3M 100mL
Suspended enzyme in CaCl​2 ​26.9375 μg/mL
enzyme in alginate beads 28.1875 (μg/ml suspension)/3 beads
BIOCONVERSION’S LABORATORY ​12
5. ​GLOBAL BALANCE Α-AMYLASE FROM ​A. ORYZAE COMMERCIAL
μg) ∗ (5ml) = 22500μg ​
μg α − amylase = (4500 ml​ μg α − amylase in CaCl​2 ​= (24.125
μg) ∗ (100ml) = 2412.5 μg
ml​
μg Immobilized Enzyme= 22500μg - 2412.5 μg = 20087.5 μg/354 beads =56.74μg/bead
Volume per bead: 5mL/354beads= 0.014mL
BIBLIOGRAPHIES
• Anwar A., S. A. Ul Qader, A. Raiz, S. Iqbal and A. Azhar. (2009). Calcium Alginate: A
Support Material for Immobilization of Proteases from Newly Isolated Strain of Bacillus
subtilis KIBGE-HAS. World Appl. Sci. J. 7 (10): 1281-128
• Arroyo, M,. (2010) Inmovilización de enzimas. Fundamentos, métodos y aplicaciones
Departamento de Bioquímica y Biología Molecular I Facultad de Ciencias Biológicas
Universidad Complutense de Madrid 28040 Madrid
• Bharat, B. (2015). Improved Enzyme Catalytic Characteristics upon Glutaraldehyde
Cross-Linking of Alginate Entrapped Xylanase Isolated from Aspergillus flavus MTCC
9390. Enzyme Res. DOI: 10.1155/2015/210784.
• Chan, T. (2005). Mechanisms of external and internal gelation and their impact on the
functions of alginate as a coat and delivery system. Department of Pharmacy, Faculty of
Science, National University of Singapore, 18 Science Drive 4.
• Codd, G. (1991). Extracellular amylase production by calcium alginate immobilized
bacteria. MARDI Res. J. I9(2) : 265-272.
Destilled Suspended
water
Enzyme pure 5 mL
enzyme in sodium alginate 10 mL
Immobilized
IMMOBILIZED ​
50mL enzyme 354 ​MIXED ​ beads

WASHING
Sodium alginate 3% w/v 5 mL
Immobilized
Calcium chloride 0.3M 100mL
Suspended enzyme in CaCl​2 ​24.125 μg/mL
enzyme in alginate beads 184.75 (μg/ml suspension)/3 beads
BIOCONVERSION’S LABORATORY ​13
• Dey G., B. Singh and R. Banerjee. (2003). Immobilization of α-amylas e
Produced by Bacillus circulans GRS 313. Braz. Arch. Biol.Techn. 46(2):167- 176.
• Draget, K., I. (2000). Alginates. In Handbook of Hydrocolloids. Cambridge,
Inglaterra: Woodhead Publishing Limited - Boca Raton, FL, EE.UU. CRC Press
LLC. pp. 379-395.
• Draget, K.I., Smidsrød, O. y Skjå k-Bræk, G. (20029 Alginates from algae. En:
Steinb ̈uchel, A., De Daets, S., Vandame, E.J. y Springer, J. Appl Phycol 2007.
19:43–53 (eds) Biopolymers. vol 6: Polysacharides II. Wiley- VCH, Weinheim,
Alemania, pp 215–240
• Erickson,, H. (1992). Usage Recommendations for a-Amylases: Maximizing:
Enzyme Activity while Minimizing Enzyme-Artifact Binding Residues. The Book
And Paper Group Annual, 11, 24-33. Retrieved from
http://www.cool.conservation-us.org/byauth/erickson/enzymes/
• Ertan, F., Yagar, H., Balkan, B. (2007) Optimization of a-Amylase
Immobilization in Calcium Alginate Beads. Preparative Biochemistry &
Biotechnology. 37: 195–204.
• Frankerberger, W. (1989). Effect pH on enzyme stability. Soil Biology and
Biochemistry. Volume 14, Issue 5.Pages 433-437. DOI:
https://doi.org/10.1016/0038-0717(82)90101-8
• Kirazov, L. P., Venkov, L. G. and Kirazov, E. P. (1993) Comparison of the
Lowry and the Bradford protein assays as applied for protein estimation of
membrane-containing fractions. Analyt. Biochem. 208, 44–48.
• Lupo P. B., González A.C. y Maestro G. A. (2012). Microencapsulación con
alginato en alimentos. Técnicas y aplicaciones. Revista Venezolana de Ciencia
y Tecnología de Alimentos. 3 (1): 130-151.
• McDowell, R. H. (1977). Properties of alginates. London, Alginate Industries
Ltd., 67 p.
• Pierre, J. (1993), Method for the quantification of alginate in microcapsules. Cell
Transplantation, Vol. 2, pp. 429-436.
• Prakash O. and Jaiswal N. (2011). Immobilization of a Thermostable "Amylase
on Agarose and Agar Matrices and its Application in Starch Stain Removal.
World Appl. Sci. J. 13 (3): 572-577.
• Sharma, M., Sharma, V., Majumdar, D. (2014) Entrapment of α-Amylase in
Agar Beads for Biocatalysis of Macromolecular Substrate. Hindawi Publishing
Corporation International Scholarly Research Notices. Volume 2014, Article ID
936129, 8 pages http://dx.doi.org/10.1155/2014/936129
• Sossa, D., Navarro, M., Matiz, A., Mercado, M., Quevedo, B., & Pedroza, A.
(2008). Immovilization of Bacillus licheniformis and Saccharomyces cerevisiae
for ethanol production from potato starch. Revistas Javeriana, 3(2), 43-82.
Recuperado de:
http://revistas.javeriana.edu.co/index.php/scientarium/article/view/1419/44 40

BIOCONVERSION’S LABORATORY
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• Stoscheck, C. M. (1990) Increased uniformity in the response of the Coomassie
Blue protein assay to different proteins. Analyt. Biochem. 184, 111–116
• Talekar, S., Chavare, S. (2012) Optimization of immobilization of α-amylase in
alginate gel and its comparative biochemical studies with free α-amylase. Recent
Research in Science and Technology. 4(2): 01-05.
• Tizon, U. (2012). Effects of pH on amylase, cellulase and protease of the
Angelwing clam, Pholas orientalis. Northern Iloilo Polytechnic State College,
Estancia, Iloilo, Philippines 2 Institute of Aquaculture, College of Fisheries and
Ocean Sciences, University of the Philippines.
• Wen-Tao, Q.; Wei-Ting, Y.; Yu-Bing, X. y Xiaojun, M. Optimization of
Saccharomyces cerevisiae culture in alginate-chitosan-alginate microcapsule.
Biochemical Engineering Journal, 2005, 25, 151-157.
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