2.Introduction to Enzyme biotechnology.pptx

ENZYME BIOTECHNOLOGY
Prepared by:
Ms. Harshada R. Bafna.
M. Pharm (Quality Assurances)
1

 Enzymes are soluble, amorphous, colloidal, proteinases, bioactive organic
catalyst products by living cell.
 Enzymes are mainly classified as extracellular enzymes (exoenzyme) and
intracellular enzymes (Endoenzyme).
 Exoenzymes are secreted outside the cell such as cellulose, polyglucturonase,
pectinmethylesterase etc.
 Endoenzymes are isolated by breaking the cells by means of a homogenizer or
a bead mill and extracting them through the biochemical processes.
2

 Enzymes- Biological catalysts- promote chemical reactions in living organisms
 Have the ability catalyze reactions under very mild conditions with high degree of
substrate specificity thus decreasing the formation of by products.
 “Immobilized enzyme” refers to “enzymes physically confined or localized in a
certain defined region of space with retention of their catalytic activities, and which
can be used repeatedly and continuously”
 Immobilization refers to restricting the mobility of an enzyme or protein and fixing
it into a state without disturbing its functional ability. An immobilized enzyme is
fixed to an inert, carrier that not only allows the exchange of substrate and product
outflow but also restricts the changes in enzyme conformation due to change in pH
or temperature.
3

Advantages of immobilizedenzymes:
 Increase functional efficiency of enzyme
 Enhanced reproducibility of the process
 Continuous use of enzyme
 Minimum reaction time
 More stability of product
 Less chance of contamination in product
 Less labor input in process
 improved process control
4

 The use of enzymes in industrial applications is limited because most of enzymes
are relatively unstable after immobilization.
 High cost of isolation, purification and recovery of active enzymes from the
reaction mixtures after the completion of catalytic process.
 Enzymes are inactivated by the heat generated in the system
Disadvantages
5

Carriermatrix:
The materials used for immobilization of enzymes are inert matrices and are polymer in
nature.
they should have following properties :
 Cost effective
 Inert
 Good physical strength
 Reduced product inhibition
 Reduction in non specific adsorption
 Reduction in microbial contamination
Carrier matrix:
 Enhancement in specificity of an enzyme
 Renewability of an immobilized enzyme
 Adequate stability
6

Methods of enzyme immobilization
Immobilization methods
Surface Immobilization Within Surface Immobilization
Adsorption Covalent
Bonding
Complexation Encapsulation Entrapment
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Adsorption: It is the simplest and oldest method. It was first used by Nelson and
Griffin in 1916 when they immobilized Invertase on an activated charcoal. Enzyme is
adsorbed on the physical outer surface of the support. Being a nonchemical method it
can affect the functional ability of enzyme by blocking its active / modulating site.
Weak bond or interactions (Hydrogen bond, Vander wall forces) play role in stabilizing
the enzyme to carrier.
Carriers used in adsorption can be
 Mineral based support : Aluminum oxide or clay, alginate beads
 Organic Bimolecular based support : Starch , Cellulose
 Modified ion exchange resin : Sepharose
1. Adsorption
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Enzymes: Invertase, Catalase, glucose oxidase, Alpha- amylase
Methods for adsorbing enzymes on carrier surface:
1. Static Method: In this method the carrier is just dipped and left in the enzyme
solution without any stirring.
2. Dynamic Method: In this method carrier is placed in the enzyme solution and
stirring is maintained to load the enzyme on carrier surface.
3. Reactor loading Method: In this method the carrier is placed in reactor and enzyme
solution is loaded with continuous stirring.
4. Electrode position Method: In this method carrier is put in vicinity of an electrode.
Now the enzymes migrate to carrier in presence of electric current.
9

ADVANTAGES
1. Simple and economical method
2. Limited loss of activity.
3. No reagent required
4. Can be Recycled, Regenerated & Reused.
DISADVANTAGES
1. Relatively low surface area for binding.
2. Exposure of enzyme to microbial attack.
3. Disturbance of the enzyme resulting from
changes in temperature, pH, and ionic
strength.
1. Adsorption
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Carriers used in Covalent Bonding are:
 Biomolecules: Carbohydrates are commonly used as a carrier
like Cellulose, DEAE Cellulose etc.
 Synthetic Molecules: Synthetic molecules like polyacrylamide
are used having good chemical reactivity to form bonds.
 Protein carriers: Protein carriers like Collagen, gelatin are also
used.
2. CovalentBonding:
Covalent Bonding: As the name suggests this method utilizes chemical groups present
on both enzyme and carrier for immobilization. A Covalent bond is formed in between
chemical groups of enzyme and carrier. This is one of the most widely used methods
for enzyme immobilization. Hydroxyl groups and amino group of support or enzyme
from covalent bonds more easily.
11

Enzyme Carrier Matrix
Cellulase, Pectinase, Glucose Isomerase Polyurethane
Amino Group Alpha Carboxyl At C Terminal
Methodsof Covalent Bondingare:
1. Diazoation: this reaction occurs between amino group of the carrier and Tyrosil
and Histidyl group of the enzyme.
2. Peptide Bond: this is the most common bond formed between amino acids and
carrier. It occurs in amino and carboxyl groups of enzyme and carrier.
3. Poly functional Agent: In this method a multifunctional agent like Gluteraldehyde is
used to form bond between amino group of enzyme and carrier.
12

ADVANTAGES:
The strength of binding is very strong, so leakage of enzymes from the
support is absent or very little.
This is a simple, mild and often successful method of wide applicability
 Enzymes are chemically modified and so many are denatured during
immobilization.
 Only small amounts of enzymes may be immobilized (about 20 mg per gram of
matrix).
DISADVANTAGES:
13

Entrapment: In this method an enzyme is entrapped within a porous matrix. Bonds
involved can be covalent or non-covalent in nature. The matrix used is hydrophilic in
nature.
Type of polymer varies with the type of enzyme and its application.
Pore size is determined on the basis of the size of enzyme so that it does not leak out of
the matrix.
Matrices used in entrapment method are: Polyacrylamide gels, Agar, Gelatin, Alginate.
1. Inclusion in gels: In this method enzyme is trapped inside the gel. Gel is formed by
the polymer. Pore size of the gel is smaller than that of the enzyme so that there is no
leakage. Pore size depends on the concentration of polymer used.
Eg: Poly acrylamide gel, Poly vinyl alcohol gels.
3. Entrapment:
14

2. In fibers: In this method the enzymes are supported on the fibers of the supporting
material forming the matrix. The fibers are skeleton of the matrix in which enzyme is
trapped.
Eg: Cellulose and Poly -acryl amide gels.
 If the gel pore size is not proper then it may lead to leak of enzyme
 Low substrate accessibility of respective enzyme
DISADVANTAGES:
15

Encapsulation: In this method an enzyme is encapsulated within a capsule made up of
semi permeable membranes like nitro celluloses, nylon, hemi cellulosic structures etc. In
this method the effectiveness depends on the stability of the enzyme inside the
capsule...3 D conformation is maintained inside the capsule.
Advantages:
 Faster and cheaper method
 Large amount of enzymes can be encapsulated at a time.
Disadvantages:
 Pore size limitation
 Size of substrate and product a limitation in exchange through membrane.
4. Encapsulation:
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 This entrapment involves the formation of spherical particle called as “microcapsule”
in which a liquid or suspension of biocatalyst is enclosed within a semi permeable
polymeric membrane.
 Microcapsules: In this microcapsules are formed in which enzymes are entrapped.
Most commonly used polymers in microcapsules are polyamines and sodium alginate.
Microencapsulation:
17

Advantages:
 Limited or no conformational change of enzyme
 Mild conditions are required
 Easy at small scale application
Disadvantages:
 Leakage of enzyme
 Pore diffusion is a limitation both for substrate and product
 Microbial contamination
 Limited to small scale operations
18

Cross linking: As the name suggests enzymes are
immobilized in the cross-linked matrix formed by
the polymer.
 There is no any carrier or surface molecule
ligand for attachment present in this method.
This method is also called as copolymerization.
 Covalent bonds are formed in between poly
functional agents and enzyme. Active site
should not be covered in cross linking
otherwise the activity will be reduced.
5. Cross linking:
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Various polymers used for cross linking enzymes are:
1. Gluteraldehye
2. Diazonium salts
Advantages:
 Simpler process
Disadvantages:
 Structural modification
 Risk of denaturation by poly functional agents
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 Industrial production: Widely used in the commercial production of antibiotics,
beverages, amino acids and secondary metabolites etc. of industrial grade.
 Biomedical applications: Immobilized enzymes are most commonly used in the fast
diagnostic kits like ELISA and treatment of many pathogenic diseases.
 Food industry: Enzymes like Pectinases and Celluloses, amylases are immobilized
on suitable carriers or matrices and are successfully used in the commercial
production of jams, jellies and syrups from fruits and vegetables. E.g. Lactase is
immobilized on cellulose fibers and produce lactose free milk from milk and whey.
 Large Scale Production of bio-diesel from vegetable oils using bioreactors.
Applications of EnzymeImmobilization:
21

 Waste water management: In Treatment of sewage and industrial effluents using
packed bed reactors .
 Research: A research activity extensively uses many enzymes. Eg: Horse radish
peroxidase in blotting experiments and different proteases for cell and organelle.
 Textile industry: Souring, polishing and desizing of fabric
 Detergent industry: immobilization of lipase enzyme for effective dirt removal
from cloths.
22

Enzyme kinetics specifically refers to the in-depth study of ‘enzymes in action’. The
abnormally high inherent rate of the induced enzyme-catalyzed reactions enormously
obviates and facilitates this study
Factors affecting on enzyme activity:
 Concentration of enzyme:
 Concentration of substrate:
 Effect of temperature:
 Effect of pH:
 Effect of product concentration:
 Effect of activators:
Factors affectingon enzyme activity:
23

Effect of activator: some enzyme need inorganic metallic catons like Mg 2+, Mn2+,
Zn2+,Ca2+, Co2+, ect. For their optimum activity. Anions are also needed for enzyme
activity rarely Eg.: Cloride ion, for amylase.
Plant cell immobilization:
Immobilization of cells refers to the technique of confining the cells in or on an inert
support for their stability and functional reuse. By employing this technique, cells are
made more efficient and cost-effective for their industrial use.
Immobilization of plant cells would be one method of increasing productivity and
hence reducing the costs.
Immobilization is the newest culture technology of plant cell, and considered as to be the
most “natural
27

NEED FOR IMMOBILIZATION:
• Protection from degradation and deactivation.
• Retention of cell.
• Cost efficiency.
• Enhanced stability.
• Allows development of multi-enzyme reaction
• Immobilization of plant cell would be one method of increasing productivity and
hence reducing the cost.
28

ADVANTAGESOF PLANT CELL IMMOBILIZATION:
• Retention of biomass enables its continuous reutilization as a production system.
• Separation of cells from medium:
• Immobilization allows a continuous process, which increase volumetric
productivity and allows the removal of metabolic inhibitors.
• The immobilization reduces some of the physical problems associated with the
cultivation of plant cells
29

DISADVANTAGEOF PLANTCELLIMMOBILIZATION:
• Secretion of secondary metabolites requires cellular transport or artificially altered
membrane permeability.
• The immobilization process may reduce biosynthetic capacity.
• Products must be released from the cell into medium. Release of single cells from cell
aggregate may make processing of the product more difficult.
• Immobilization is an expensive affair often requiring sophisticated equipment.
• The possibility of loss of biological activity of an enzyme during immobilization or
while it is in use.
30

METHODS OF IMMOBILIZATION:
• a) On surface immobilization:
• 1) Covalent coupling
• 2) Adsorption on inert support or ion exchange resins
• 3) Complexation and chelation
• b) Within support immobilization:
• 1) Cross-linking
• 2) Entrapment by Occlusion within cross-linked gel.
• 3) Encapsulation in microcapsules, hollow fibers, liposomes.
31

APPLICATIONS OF PLANT CELL IMMOBILIZATION
 Enhanced production of secondary metabolites:
• Eg: capsaicin, L- DOPA, methyl xanthines etc.
 Biotransformation: Bioconversion of ß methyl digitoxin has been achieved using
Digitalis lanata immobilized cell cultures upto 70 days.
 Other uses:
• 1. It is used in synthetic seed technology.
• 2. It can be used for transport of protoplasts.
• 3. Immobilized plant cells can be cultured as single cells for prolonged period.
32

2.Introduction to Enzyme biotechnology.pptx

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2.Introduction to Enzyme biotechnology.pptx