ENZYME PRODUCTION,

IMMOBILIZATION AND
APPLICATION
SCE 3204-INTRODUCTION TO BIOCHEMICAL
ENGINEERING
SESSION 1
DATE OF SUBMISSION: FRIDAY 27TH MARCH 2020 1
GROUP MEMBERS
NO NAME REGISTRATION NUMBER
1 KASULE HANNAH TALINDA 17/U/6870/CHD/PD
2 KIVUMBI ACHILEO 17/U/6879/CHD/PD
3 NDAHAYO PHILIPO 17/U/6932/CHD/PD
4 OUNDO FREDRICK 17/U/6950/CHD/PD
5 BARAKA ALEXIS 17/U/6839/CHD/PD
6 ECUNGO SAMSON 17/U/6847/CHD/PD
7 KAGENI JULIUS CEASER 17/U/127/CHD/GV
8 OCHOLA DAVID 17/U/6939/CHD/PD
9 NAMUGALA KIMERA ABDU NOOR 17/U/18163/CHD/PD
10 MPAATA HENRY 17/U/18159/CHD/PD
2
GROUP MEMBERS
NO NAME REGISTRATION NUMBER
11 OTIM JONATHAN 17/U/6949/CHD/PD
12 MUHKWANA ALEXANDER 17/U/6903/CHD/PD
13 MANGENI DEOGRACIOUS 17/U/6890/CHD/PD
14 NAMULONDO VIOLET 17/U/6923/CHD/PD
15 KYAMBADDE STEVEN 17/U/6883/CHD/PD
16 NDAULA VIAN 17/U/7027/CHD/PD
17 OLENGE JASPER 17/U/6945/CHD/PD
18 BALA FRANK 17/U/6838/CHD/PD
19 GYAZA ELTON 17/U/6855/CHD/PD
20 MAKANGA ROGER 17/U/6889/CHD/PD
3
INTRODUCTION
This presentation focuses on the use of microbial
enzymes as an alternative to conventional
chemical industrial processes in order to minimize
environmental pollution.
The following topics are discussed:
Production of enzymes
Methods of immobilizing enzymes
Applications of enzymes in industrial and textile
manufacturing processes. 4
ENZYME MANUFACTURING
PROCESS
Enzymes are manufactured by culturing micro
organisms in appropriate media using large
fermentation chambers (fermenters) under mild
conditions (Hema Anto et al., 2006)
Microorganisms either secrete enzymes outside their
cells (extra cellular) or within their cells (intracellular).
After fermentation, enzymes are purified using various
chemical, mechanical or thermal techniques e.g
precipitation, chromatography
Stabilizers, standardizing agents, preservatives and
salts are added to the pure concentrate to form the 5
final product.
ENZYME MANUFACTURING PROCESS
FLOW CHART

6
STAGES OF LARGE SCALE FERMENTATION

Selection of micro organisms: Microbes to be cultured

are selected according to the desired final enzyme e.g
B.coagulans is used in amylase production
Selection of media: This is the source of food to be
consumed by microbes. Should have sources for carbon,
nitrogen and some trace elements. Examples are sugar
sources like molasses, grape juice etc.
Sterilization: This process removes all unwanted
microbial life from raw materials and fermentation
vessels. High temperatures of 100°C are used.
7
STAGES OF LARGE SCALE FERMENTATION
Cont’d
Inoculation: This involves introducing microbes into the
culture medium. Microbes multiply exponentially on a
small scale and are later inoculated in large cultures
(Robinson et al., 2001).
Fermentation: In this stage, enzymes are produced by
the fermentation of substrate by microbes. Cultivation of
microbes can either be a batch/continuous process
(Mala et al., 2007). Batch process produces higher
enzyme concentration. Fermenter must have constant
oxygen supply.

8
DOWNSTREAM PROCESSING
This stage involves collection, concentration and
purification of the fermented media to obtain desired
enzyme.
For extra cellular enzymes, enzyme is concentrated,
separated and purified from spent media
Intra cellular enzymes are obtained by breaking the cell
and further separating and purifying it.
The purification process should not have too many
stages to avoid high production costs and loss of enzyme
activity
9
STAGES OF DOWNSTREAM PROCESSING

Cell Disruption: This is the use of mechanical or non

mechanical means to extract intra cellular enzymes from
microbes. Mechanical methods include shear forces and high
pressure homogenization. Non mechanical methods include
thermal and chemical lyses.
Separation of solid matter: Involves separation of extra and
intra cellular fragments from enzymes. Large cells are removed
by decantation. Other methods include centrifugation,
filtration and flocculation.
Concentration: this involves increasing the amount of enzyme
in specified volume without deactivating the enzyme. Mild
concentration procedures like thermal methods, precipitation
10
and membrane filtration are used.
STAGES OF DOWNSTREAM PROCESSING Cont’d

Purification: This stage is applied for enzymes which will be

used analytical applications. Procedures used include
crystallization, electrophoresis and chromatography
( molecules are separated according to their physical, chemical
or biological properties)
Formation of the final product: This involves the addition of
extra components to the purified enzyme to improve/maintain
its properties. These include stabilizers(ammonium sulphate)
which increase the ionic strength of enzyme proteins
(Kunanmnemi et al, 2005).
Final enzyme concentrate will contain the active enzyme and
various by products from fermentation.
11
ENZYME IMMOBILIZATION
• Is the process of attaching an enzyme to an inert, insoluble
material such as calcium alginate. This is done to provide
increased resistance to changes in conditions such as pH or
temperature.
• The enzyme is confined to a phase (matrix) different from the
one for substrates and products.
• Matrix must be affordable, inert, stable, physically strong and
able to reduce product inhibition (Singh, 2009).
• Immobilizing an enzyme can lead to high investment capacity
ratio and recovery of a product with greater purity.

12
IMMOBILIZATION TECHNIQUES
Adsorption: The enzyme is attached to the outside of an inert
material. This can be through bathing the support in
enzyme/drying the enzyme on electrode surfaces (Spahn,
2008). This method shields the enzymes from aggregation,
proteolysis and interaction with hydrophobic surfaces. Eco
friendly supports like coconut fibers, kaolin and silanized
molecular sieves are used.
Covalent binding: Enzyme molecules are covalently bound to
an insoluble support such as silica gel and macro porous
polymer beads. This improves the thermal stability and half
life of enzymes. It is also the strongest enzyme/support
interaction (Singh 2009).
13
IMMOBILIZATION TECHNIQUES CONT’D

Affinity immobilization: This method uses the specificity of an

enzyme to its support (Sardar et al., 2000). It involves joining
the support to an affinity ligand for the target
enzyme/conjugating the enzyme to a substance that can
develop affinity for the support. This method can also be used
to simultaneously purify enzymes.
Entrapment: Is the caging of an enzyme in a gel/fiber by use
of covalent or non covalent bonds. Carriers like alginate,
gelatin and calcium help to prevent enzyme leakage and
provide increased mechanical stability (Singh, 2009). However,
these gels can hinder the enzyme substrate interaction and
exit of the final product.
14
MATERIALS USED FOR IMMOBILIZATION

NATURAL POLYMERS INORGANIC MATERIALS

Alginate Zeolites

Chitosan Ceramics

Collagen Celite

Carrageenan Silica

Gelatin Glass

Cellulose Activated carbon

Starch Charcoal

15
APPLICATIONS OF ENZYMES
Detergents: Lipases, amylases and proteases efficiently
remove dirt and other stains on clothes at low temperatures.
Hydrolases remove soils formed from proteins, lipids and
polysaccharides (Pujii et al., 1986)
Paper: Amylases are used in the modification of starch
coatings and xylanases reduce consumption of bleaching
chemicals (Bernfield 1955). Estereases are used for stickiness
removal etc.. They also open up the pulp matrix for better
penetration of bleaching agents.
Leather: Enzymes assist in removing hair from hides, replacing
conventional chemicals like lime and sodium sulfide. Bacterial
proteases and trypsin also make leather more pliable (Sharma
16
et al, 2001).
APPLICATIONS OF ENZYMES
Cont’d
Fruit juice: Enzymes like pectinase act on pectin found in most
ripe fruits to change appearance of juice from cloudy to crystal
clear (Martin et al., 2004). It also improves the stability, taste
and texture of the final product.
Medicinal products: Pancreatic enzymes like trypsin can be
used for curing pancreatic insufficiency. Proteases remove
fibroin layers from wounds to aid in healing (Hooper, 2002).
Proteases also improve blood fluidity and cure peripheral
arterial diseases.
Scientific purposes: Enzymes can be used to determine the
concentrations of substrates, measure catalytic activity of
enzymes in biological samples and determine the
concentrations of enzymatically inert substances (Winkler et al., 17
1990).
APPLICATIONS OF ENZYMES IN
TEXTILE PROCESSING
Bio singeing: Enzymes with powerful cellulase compositions
can be used to treat fabric in order to improve absorbency and
softness without affecting fabric color (Koo et al., 1994).
Bio de sizing: Amylases can be used to effectively remove
starch based sizing agents without using harsh chemicals
(caustic soda) and degrading the cotton fibre.
Bio scouring: Enzymes are fairly effective in removing waxes,
pectins, sizes and other impurities on the surface of fabric
(Emilla et al., 1998). They are also a better alternative to
chemicals like caustic soda, which pollute the environment.
Bio bleaching: Some enzymes like lipase and laccase can be
used in place of toxic chemicals like chlorine to bleach fabrics
18
without affecting the cellulose within the fabric.
APPLICATIONS OF ENZYMES IN TEXTILE
PROCESSING Cont’d
Peroxide killers: Peroxidase or catalase are used to neutralize
residual peroxide from the bleaching process without reacting
with the dyes in the fabric. One molecule of catalyst destroys 5
million molecules of peroxide.
Bio polishing: Enzymes (cellulases like Aspergillus Niger) are
used in bio polishing to upgrade the quality of the fabric by
removing the protruded fibres from the surface and
modification of the surface structure of the fibre, making it
soft, smooth and long lasting.
Bio carbonizing: Cellulose based enzyme formulation is used
to dissolve cellulosic components from fabric in order to make
it soft and fluffy. This method is preferred to using sulphuric
19
acid, which is highly corrosive.
APPLICATIONS OF ENZYMES IN TEXTILE
PROCESSING Cont’d
Degumming of silk: Sericin specific proteins are used to degum
silk without causing damage, impart softness, and increase dye
uptake by about 30%. This is more efficient than using alkaline
treatment, which could damage the fibroin protein.
De colorization of dye water effluent: Enzymes can be used
along with other bio technological processes to remove
harmful chemicals during waste water treatment from textile
factories
Bio denim washing: Cellulase enzyme can be used to fade
denims through exposure of the un dyed core of the fibres
which gives a faded look to the denim fabric. This replaced the
used of pumice stones, which would generate a lot of sludge in
the effluent tank. 20
CASE STUDIES
1. EXTRACTION, PURIFICATION AND
CHARACTERIZATION OF PROTEASE FROM
ASPERGILLUS NIGER ISOLATED FROM YAM PEELS

2. PURIFICATION OF XYLANASES
SCE 3204-INTRODUCTION TO BIOCHEMICAL
ENGINEERING
SESSION 2
21
INTRODUCTION
• Waste is defined as any unwanted or unusable material that is
discarded after primary use. Some waste however, can be
turned into valuable resources once they are removed from
the waste stream.
• An example is Protease enzyme, which can be extracted from
Aspergillus Niger, a fungus isolated from waste yam peels and
used for various purposes.
• Protease enzyme can be used in various industries like
detergent for stain removal, leather for bating hides,
pharmaceuticals, food and waste water treatment.

22
MATERIALS AND METHODS
• Sourcing Material: Yam peels were obtained from a yam seller
in Nigeria, collected in clean cellophane and allowed to
biodegrade naturally
• Inoculation: Potato dextrose agar mixed with choramphenicol
was melted and poured over diluted samples of A. Niger.
Plates were then incubated at room temperature for 72 hours.
Pure culture was maintained at 4°C on potato agar slants
• Determination of optimum growth conditions. 100 ml of
sterile fermentation medium was inoculated with pure culture
and incubated for 3 days at 28°C, then centrifuged at 4°C. The
supernatant was then precipitated, dialysed and analysed to
estimate protease activity and protein content.
23
MATERIALS AND METHODS Cont’d
• Assay of protein content: Proteolytic activity was determined
by mixing 1% casein with phosphate buffer and culture
supernatant. This mixture was incubated at 40°C for 30
minutes and filtered. Its activity was then determined using a
PC spectrophotometer at 660 nm.
• Ion exchange chromatography: The dialyzed culture sample
was purified by application to a column, were it was washed
and eluted with increasing concentrations of NaCl. The eluted
fraction was then analyzed by a UV spectrophotometer.
• Gel filtration chromatography: The enzyme solution was then
collected, dissolved in phosphate buffer and eluted using gel
to obtain fractions which were used as the purified enzyme.
24
MATERIALS AND METHODS Cont’d
• Gel electrophoresis: The purified enzyme solution was then
electrophoresed to determine the purity and molecular
weight of the enzyme.
• Effect of pH on enzyme activity: Optimum enzyme pH was
investigated at 40°C Using buffers ranging from pH 3-12 for 30
min
• Effect of temperature on enzyme activity: Optimum enzyme
temperature was determined by estimating protease activity
at pH 7 and temperatures ranging from 30-90°C for 30 mins.
• Stability of pure enzyme toward pH: This was done by
measuring the activity of enzyme during 5 hour incubation
period at pH ranging from 3-12 in presence of 1% casein
25
substrate.
MATERIALS AND METHODS
• Enzyme stability towards temperature: Enzyme solution was
then subjected to temperatures ranging from 30 to 90°C for 30
minutes at pH 7. Residual enzyme activity was then assayed.
• Determination of kinetic parameters: the Michaelis Menten
Constant (Km) and maximum reaction velocity (Vmax) of
protease for casein was then determined at different substrate
concentrations using a Lineweaver Burk plot.
• Effect of metal ions/enzyme inhibitors on enzyme activity:
The purified enzyme was incubated with ions e.g. Cu, Fe, Mg,
Ca and Na for 30 minutes at 40°C
• Substrate specificity of enzyme: Finally, enzyme activity on
various substrates e.g. casein , yeast extract, malt extract, beef
extract, urea and peptone was determined. 26
 RESULTS

Growth pattern of pure culture Effect of metallic ions on enzyme

• The exponential phase was observed at 24 activity
hours. The rate of growth was between 24 • Iron (ii) sulphate, magnesium sulphate and
to 48 hours reaching peak growth at 42 copper (ii) sulphate inhibited protease
hours of incubation. Growth declined at 48 activity
hours and the culture entered the death • EDTA, sodium chloride and calcium chloride
phase.
promoted protease activity.
• There was a prolonged lag phase from 6
hours to 24 hours and a sharp growth phase
from 24 hours to 42hours. 27
• There was a gradual death phase from 48
hours to 72 hours.
 RESULTS Cont’d

Purification results for protease enzyme

• Specific activities for crude extract, ammonium sulphate precipitation, ion exchange
chromatography and gel filtration were 0.51, 0.67, 5.11 and 8.51 respectively.
• Yield for gel filtration was 10.96, an indication that purification increased with each
purification step while percentage enzyme yield reduced with each purification
step.
• The Line Weaver Burk plot of protease activity indicated that the Km and Vmax
values for casein hydrolysis are 401.3mg/ml and 7.8U respectively.
28
 RESULTS Cont’d
Effect of pH on enzyme activity
• The results revealed optimum pH at
10. At pH 11 and 12, the enzyme was
relatively stable and at lower pH it was
observed that activity was low at pH
3-7.
• At pH 8 there was a sharp and sudden
increase in activity, which means that
the enzyme is more stable at alkaline
pH.
Effect of temperature on enzyme
activity
• At temperatures ranging between 30-
50°C, the activity was high, while the
optimum temperature was at 50°C.
• There was a sharp and steady
decrease in activity from 60-90°C.
• It was observed that the higher the
temperature, the lower the activity. 29
 RESULTS Cont’d
Effect of pH on enzyme stability
• At pH 10-12 over a time of 5 hours, the
enzyme was relatively stable and had
residual activity of 70-55%
• At pH lower than 10 the enzyme was not
stable with residual activity ranging from
55-20%.
• The enzyme was relatively stable at pH 9-12
as and less stable at pH of 3 to 8 for a time
of 5 hours.
Effect of temperature on enzyme
stability
• The enzyme was more stable from 30-60°C
after an hour of incubation.
• At higher temperature, there was a decline
in residual activity.
• At incubation time of 20 minutes, activity
was high at temperatures between 30-50°C
• At temperatures between 60-90°C, the
enzymatic activity was moderate. 30
 RESULTS Cont’d

Effect of different protein substrates on protease activity

• Different protein substrates at 1% concentration were used including yeast
extract, malt, casein, peptone and beef.
• Maximum protease activity was observed in casein and yeast extract while
minimum protease activity was observed with beef and peptone. Malt 31
exhibited moderate enzymatic activity.
DISCUSSION
• Protease enzyme had 94% activity at 50°C, showing that it is stable
at lower temperatures. The optimum temperature was similar to
protease of A terreus at 50°C (Bushra et al., 2010)
• Protease has wide range thermo stability even at low temperatures,
making it suitable for use in food industries which utilize
temperatures of 50 to 60°C.
• Protease also displayed a broad range of activity in acidic, alkaline,
and neutral levels, which is consistent which literature reports which
state that fungi produces alkaline, acidic and neutral proteases.
(Siala et al., 2009 and Samantha et al., 2005)
• The promotion of protease activity by EDTA suggests that the
enzyme is not a metallo-protease, making it useful as a detergent
additive.
32
DISCUSSION Cont’d
• The strong activation of protease enzyme in presence of Mg ions
indicates that protease can be protected from thermal denaturation,
and this is consistent with scientific reports.
• Purified protease demonstrated a wide range of hydrolytic activity
on various protein substrates, with the highest activity performed on
casein substrate. This is consistent with the report of (Shankar et al.,
2011) and shows potential for use in biotechnological applications.
• The Vmax and Km values, 7.8 and 40.13 showed that protease had
high affinity and degradability. However, other reports (Shankar et
al., 2011) reported Km value of 5.1mg/ml.
• During gel filtration, protease migrated as a single band with a
molecular weight of 49kDa, which shows that the enzyme is
homogenous and monomeric. However, bands of contaminant
proteins were also observed. 33
CONCLUSION
• The publication described the isolation, purification and
characterization of protease enzyme obtained from Aspergillus
Niger.
• The enzyme was purified 16.6 fold and final yield was 10.96%
• After comparison of characteristics, it was determined that
protease obtained from this fungi can be a viable alternative in
industrial processes such as food, pharmaceuticals, cosmetics
etc.
• Protease produced from yam peels also has enormous
potential in reducing enzyme production costs, due to high
availability of cheap agricultural residue (yam peels).
34
 PURIFICATION OF XYLANASES

• Xylanase enzymes are purified in order to acquire information about their

structural and functional properties, and well as discovering their
applications.
• The ultimate objective of purification is to obtain the greatest possible yield
of a desired enzyme, with highest possible purity and catalytic activity.
• Purification of xylanases is usually based on a series of many techniques
(Wong and Saddler, 1992). Fewer steps generally have higher recovery
yields, with lower purity and vice versa.
• Examples of purification steps discussed include: ultra filtration, gel 35
filtration, chromatography and precipitation.
 ULTRA FILTRATION

• Is a variety of membrane filtration in which forces like concentration gradients lead

to separation through a semi permeable membrane.
• Used to separate xylanases from other proteins due to their low molecular weight.
• Can be used in conjunction with solvent exchange through ion exchangers to
produce high yields of cellulase-free xylanase from culture filtrate of T, harzianum.
• Can be an alternative to precipitation during maximum purification, by using
membranes with a 5-30 kDa molecular weight cut off value (Sa-Pereira et al.,
2003). 36
 GEL FILTRATION

• Separates proteins based on differences in molecular size.

• Utilizes adsorptive interactions between xylanases and gel filtration resin
matrices to elute the enzymes as proteins smaller than 12 kDa.
• Gel filtration has been used in combination with other chromatography
for maximum purification of xylanase (Lee et al., 1993).
• A highly thermo stable alkaline xylanase was purified to homogeneity
from a culture of Bacillus sp. Using gel filtration, with a 43.5% recovery 37
(Shrinivas et al., 2010).
 PRECIPITATION

• Used in many purification processes as an initial step

• Examples of re agents used for this method include ammonium sulphate,
ethanol, ionic polymers
• It removes the low molecular weight proteins (Nakamura et al., 1993)
Examples:
• (Gupta et al., 1994) purified xylanase by precipitation with an ionic polymer
Eudragit S 100, with a yield of 89%. Binding was by electrostatic interaction.
• (Kiddinamoorthy et al., 2008) purified xylanase from Bacillus sp. Using 38
ammonium sulphate (40-80% precipitation) resulting in a 71% yield.
 CHROMATOGRAPHY

• This method involves separating a mixture based on differential

partitioning between a mobile phase and a stationary phase.
• Examples include ion exchange, gel filtration, gel permeation, affinity,
hydroxyaphite , fast protein liquid chromatography and HPLC
• These methods are used in 2 to 5 purification steps, providing 39
recovery yields from 0.2% to 78%.
CHROMATOGRAPHY Cont’d
• Affinity chromatography, however, is rarely used due to high expenses and
short life of matrices.
• These techniques are usually combined with HPLC (High Performance Liquid
Chromatography) for better resolution.
Examples:
• (Breccia et al., 1999) isolated xylanase by adsorption on a cation exchanger,
leading to 82% yield.
• (Dhillon et al., 2000) used anion exchange chromatography to purify
xylanase from B. circulans.
• (Bronnenmeier et al., 1996) purified cellulose binding enzymes including
xylanase from Clostridium stercoranium using cellulose affinity
chromatography
• Use of cation exchange chromatography is also reported using
carboxymethyl sepharose for purification of xylanase from Fibrobacter
succinogens and Streptomyces rosei (Matte and Forsberg, 1992; 40
Lappalainen et al., 2000)
CLARIFICATION
• Is an important step in the purification of extra cellular
xylanases.
• It is mainly used to remove poly phenols, pigments and
nucleic acids, among others, which may be contaminants
during further purification steps.
• Clarification is done by precipitation, centrifugation or
filtration.
Example:
• (Dhillon et al., 2000) and (Nakamura et al., 1993) clarified
xylanase by centrifugation at high and moderate speed,
respectively.
41
WORKS CITED
Bajpal, P. (2014). Purification of Xylanases. In B. Pratima, Xylanolytic
Enzymes (pp. 53-61). Academic Press.
Brahmachari, G. (2016). Industrial Enzymes. In G. Brahmachan,
Biotechnology of Microbial Enzymes (pp. 23-47). Academic Press.
Enujiugha, V. N. (2015). Extraction, purification and characterization of
protease from Aspergillus Niger isolated from yam peels. International
Journal of Food Sciences and Nutrition, 125-131.
Sumitra Datta, R. C. (2012). Enzyme immobilization: an overview on
techniques and support materials. Springerlink.com.
 

42