Download Enzyme immobilization

Enzyme
“A biological catalyst that promotes & speeds
up a chemical reaction without itself being
altered in the process.”
Cut (degradation)
Build (synthesis)
Change (modification)
Structure of an enzyme
ENZYMES
NON-PROTEIN
COENZYMES
( VITAMIN)
COFACTOR
(MINERAL)
PROTEIN
Enzyme action
substrate
enzyme
products
Cells and enzymes as biocatalysts
enzyme
S
P
cells
cell based versus enzymatic
processes
glucose
glucose/fructose
glucose isomerase
glucose
ethanol
multi-enzymes acting sequentially
• whole cells preferred when multi-step
• enzymes preferred for 1 or 2 step transformations
• competing side reactions with whole cells
• sterility problems
• cell lysis
• other physiological requirements (nutrients, O2)
Properties of enzymes
• Control ripening.
• Cause food spoilage (rotting).
• Responsible for changes in flavor, color, texture and
nutritional properties.
• Can be inactivated by heat to extend storage
stability of foods.
• Control oxidation and spoilage (bioconservation)
• Increase nutritive values ( phytase , proteases etc.)
• Used for fermentation purposes in foods.
• Can be extracted and purified to a high degree.
Properties of enzymes
1. Biological catalysts (activity can be regulated)
2. Efficient, specific, stereospecific
3. Speeds up the rate of a reaction (activation energy) but does not
change the equilibrium
4. Speeds up both forward and reverse reactions
5. Product purity is 100% (due to reaction specificity - lack of formation
of wasteful by-products)
Properties of enzymes
1. Reaction specificity reduces energy demand in a cell
2. Couple reactions (energy gained from one reaction is used in second
reaction)
3. Some are control points in metabolic pathways
4. Have separate binding sites for substrates and effectors
5. (All enzymes) are proteins
Sources of enzymes
There are three major sources of enzymes :
Plants ( 4%)
(papain, bromilain)
Animals ( 8%)
(renet)
Microorganisms (>80%)
(yeast, fungi and bacteria)
Why important ?

Added or used to cause particular reaction
 Advantages
 Natural, Nontoxic
 Catalyze specific reactions
 Active under mild conditions
 Active at low concentrations
 control rate of reaction
 Can be inactivated
Enzyme Production/Isolation Methods
Uses of enzymes
1.
2.
3.
4.
5.
6.
1.
2.
3.
4.
5.
Analytical Applications of Enzymes
The Animal Feed Industry
The Meat and Fish Processing Industry
The Dairy Industry
The Leather Industry
cleaning of microfilters -- Detergents
The modification of Fats and Oils
The Pulp and Paper Industry
The Fruit Juice Processing Industry
The Production of Bulk and Fine Chemicals
Enzyme-Replacement Therapy
enzyme
Source
Action in food
Food application
Papain
Latex of unripe
papaya fruit
protein hydrolysis
Meat tenderisation
Bromelain
Pineapple juice
and stem
Muscle and connective
tissue protein hydrolysis
Meat tenderisation
Ficin
Fig fruit latex
Muscle and connective
tissue protein hydrolysis
As bromelain & papain but
not widely used due to cost
Chymosin
(rennet)
Calf abomasum
Kappa casein hydrolysis
Cheese making
Pepsin
Bovine
abomasum
casein
hydrolysis in cheese
Help for rennet action
Lysozyme
Hen egg white
Hydrolysis of bacterial Prevention of late blowing
cell wall
defects in cheese by
polysaccharides
spore-forming bacteria
Lactoperoxidase
Cheese whey:
Oxidation of
bovine colostrum thiocyanate
ion to bactericidal
Hypothiocyanate
Cold sterilisation of milk
Aminopeptidase
Lcictococcus
lactix
Axpergillux spp.
Rhizopux oryzae
Releases free amino
acids from N-terminus
of proteins and
peptides
Releases free amino acids
from N-terminus
of proteins and peptides
Lipase/
esterase
Gullet of
goat&lamb: calf
abomasum:
pig pancreas
Triglyceride (fat)
hvdrolvsis
Flavour enhancement in
cheese products:
Enzymes
Uses
Amyloglucosidase
Starch breakdown in early season fruit
Cellulase
Liquefaction of fruit
Esterase
Aroma development
Lipoxygenase
Aroma development
Pectinesterase
Clarification of juice
Polygaclacturonase
Clarification of juice
Polyphenoloxidase
Color and flavor
Textile industry
• 'Amylases' isolated from bacteria, fungi, pancreas and malt are used
in textile industry as softening agents for starched clothes.
• Starch is often added to cotton fibres as a stiffening agent, before
weaving the fibre into cloth.
• Since, a starched cloth does not take good colour, the cloth is to be
destarched before dyeing it.
• This is done with an amylase preparation, which hydrolyses starch).
Leather industry
• Proteolytic enzymes from certain bacteria and fungi are used in the
manufacture of leather.
• These enzymes digest the collagen or connective tissue holding the
hairs to the hide in the skin and thus cause dehairing of the skin.
• These enzymes are also used for softening or plumping of dehaired
skin, a process popularly called bating.
Uses of Enzymes in the Food Industry
• Meat and beer processing
• The proteases 'papain' (from papaya) or 'bromelain'(from pineapple),
are used to tenderise meat by hydrolysing peptide bonds.
• It is also used to stabilise chill proof beer.
Manufacture of cheese
• 'Renin' obtained from the calf stomach is used in the manufacture of
cheese, since it converts calcium-casein of the milk to calciumparacaseinate, which is curd like in appearance.
• The curd is solidified and processed as cheese after inoculation with an
appropriate mixture of micro organisms.
• Another enzyme 'catalase' is also used for making cheese, since it breaks
down H2O2 produced during cold pasteurization of milk during cheese
making process.
• 'Lipase' is added during the processing of cheese, for flavour production.
• Beverage industry
Enzymes from yeast are used for alcoholic fermentation in beverage
industry, since they convert sugars to alcohol and CO2.
Juice and wine processing
'Pectinases' are often added in canned fruit juices and in wine, since
they hydrolyse the pectin making the juice or wine clear.
• Chocolate and candies
'Invertase' is used in the manufacture of chocolate covered berries
and other such candies.
Soft drinks industry
'Glucose isomerase' is used for the production of fructose and high
fructose syrups from hydrolysed maize starch, to be used in soft
drinks.
Ice-cream industry
'Lactase' is used for the prevention of lactose crystals in ice-cream
Enzymes for Detergent
• For most people, the most popular known application of enzymes is
in the manufacture of enzymatic washing agents (detergents).
• Since last 40 years, the use of enzymes in detergents has been the
largest of all enzyme applications.
• Consumers of detergents are actual users of an enzymatic product.
Proteases
• Proteases are the most widely used enzymes in the detergent industry.
• They remove protein stains such as grass, blood, egg and human sweat.
These organic stains have a tendency to adhere strongly to textile fibres.
• The proteins act as glues, preventing the waterborne detergent systems
from removing some of the other components of the soiling, such as
pigments and street dirt.
•
The inefficiency of nonenzymatic detergents at removing proteins can result
in permanent stains due to oxidation and denaturing caused by bleaching
and drying.
• Blood, for example, will leave a rustcoloured spot unless it is removed
before bleaching.
• Proteases hydrolyse proteins and break them down into more soluble
polypeptides or free amino acids. As a result of the combined effect of
surfactants and enzymes, stubborn stains can be removed from fibres.
Lipases
• Though enzymes can easily digest protein stains, oily and fatty stains
have always been troublesome to remove.
• The trend towards lower washing temperatures has made the
removal of grease spots an even bigger problem.
• This applies particularly to materials made up of a blend of cotton
and polyester.
• The lipase is capable of removing fatty stains such as fats, butter,
salad oil, sauces and the tough stains on collars and cuffs.
Amylases
• Amylases are used to remove residues of starch-based foods like
potatoes, spaghetti, custards, gravies and chocolate.
• This type of enzyme can be used in laundry detergents as well as in
dishwashing detergents.
Cellulases
• The development of detergent enzymes has mainly focused on enzymes capable of
removing stains.
• However, a cellulase enzyme has properties enabling it to modify the structure of
cellulose fibre on cotton and cotton blends.
• When it is added to a detergent, it results into the following effects:
• Colour brightening-When garments made of cotton or cotton blends have been
washed several times, they tend to get a 'fluffy' look and the colours become duller.
• This effect is due to the formation of microfibrils that become partly detached from
the main fibres.
• The light falling on the garment is reflected back to a greater extent giving the
impression that the colour is duller.
• These fibrils, however, can be degraded by the cellulase enzyme, restoring a smooth
surface to the fibre and restoring the garment to its original colour.
• Softening-The enzyme also has a significant softening effect on the fabric, probably
due
to
the
removal
of
the
micro
fibrils.
• Soil removal-Some dirt particles are trapped in the network of micro fibrils and are
released when the micro fibrils are removed by the cellulase enzyme.
What Is Enzyme Immobilization ?
Enzyme immobilization may be defined as a
process of confining the enzyme molecules to
a distinct phase from the one wherein the
substrates and the products are present.
What Is An Immobilized Enzyme?
An immobilized enzyme is one whose
movement in space has been restricted either
completely or to a small limited region.
Advantages of Immobilized Enzymes
• Recovered at the end of the reaction thereby can be reused.
• Economy of the reaction is improved.
• Easy separation of enzyme from the products occurs.
• Stability of immoblilised enzyme increases.
• Enhanced enzyme properties.
• Efficiency of the catalytic reaction is better in a few cases.
• Better control of reaction can be achieved.
• Catalytic process can be operated continuously.
• Multi enzyme reaction possible.
• Potential in industrial & medicinal use.
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Methods of Immobilization
• Parameters for Method Selection :Overall catalytic activity.
Effectiveness of the catalytic utilization.
Deactivation & Regeneration characteristics.
Cost effective.
Intended application of immobilized enzyme.
Toxicity of immobilized enzyme.
Waste disposal (of immobilization process).
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Carrier for Immobilized Enzymes
• Ideal Characteristics of the Carrier:Low Cost & of optimum quality
Inertness
Physical Strength
Stability
Regenerability
Enhancement of enzyme specificity
Reduction of product inhibition
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CLASSIFICATION OF CARRIERS
Inorganic
Carriers
•High pressure
stability.
• May undergo
abrasion
Examples:
1. Commercialy SiO2
available
materialso Porous glass.
o Silica.
2. Mineral materials (clays)
Celite ,Centonite
Organic Natural
Carriers
•Favourable
compatibility with
proteins.
Examples:
1. cellulose
derivativeso DEAE-cellulose
o CM-cellulose.
2. Dextran.
3. Polysacharides
Agarose, Starch
Pectine ,Chitosan
Organic
Synthetic
Carriers
•High chemical
and mechanical
stability.
Examples:
1. Polystyrene
2.Polyvinylacetate
3. Acrylic
polymers
IMMOBILIZATION
METHODS
SURFACE
IMMOBILIZATION
ADSORPTION
COVALENT
BONDING
WITHIN SURFACE
IMMOBILIZATION
COMPLEXATION
ENCAPSULATION
ENTRAPMENT
ADSORPTION
1.Surface Immobilization/Carrier Binding
According to the binding mode of the enzyme, this
method is further sub classified into:
1(a) Physical Adsorption:
• Enzyme molecules get adhered to the surface of
carrier matrix.
• Surface of carrier may be charged or neutral
• Driving force is hydrophobic intxn and salt bridge
Typical Adsorbents
• Cellulose derivatives
• Polystyrene resins
• Glass
• Alumina
• Silica gel
• Charcoal
• Starch
• Modified sepharose
Immobilization by Adsorption
•Dependant on PH, ionic strength,
temprature,
nature
of
solvent,
concentration
of
enzyme
and
adsorbent.
•Binding forces are ionic, hydrophobic,
hydrogen bonds, or Van der Waals’
interactions
•Binding is simple (stir together in a
beaker) but is reversible. Substrate
addition can cause desorption.
PROCEDURE
Enzyme mixed with adsorbent
Appropriate pH & desired
ionic strength
Incubation for a stipulated duration
Carrier matrix washed thoroughly to get
rid of unabsorbed enzyme molecules
Immobilized enzymes
D
• Advantages:
Simple & Economical
Limited Loss of activity
3
Can be Recycled, Regenerated & Reused (R )
Little damage to enzyme
No chemical change to support / enzyme
• Disadvantages:
Exposure of enzyme to microbial attack.
Smaller particles cause high Pressure drop in continuous packed
bed reactor.
Yield are often low due to inactivation & desorption.
 Leakage of enzyme
 Non-specific binding
 Over-loading on the support
EXAMPLES
Enzymes
Carrier matrix
Amylase
Calcium phosphate
Catalase
Charcoal
Invertase
Charcoal,DEAE-sephadex
Subtilisin
Cellulose
Aminoglycosidase
Agarose gel, DEAE-sephadex
Glucose oxidase
cellophane
COVALENT BINDING
COVALENT BINDING
The covalent binding method is based on the
binding of enzymes and water-insoluble carriers
by covalent bonds.
•Covalent bond is formed between the chemical
groups of enzymes and chemical groups on
surface of carrier.
•Formation of covalent bond take place with the
side chains of amino acids present in the
enzymes.
•
• The Protein Functional Groups used for the covalent
coupling
o NH2-lysine
o COOH-α and β Aspertic acid,Glutamic acid
o OH- Phenol ring on tyrosine
o SH- Cysteines
• Polymeric Supports which are widely used:
o Hydroxyl groups of polysaccharide, PVA,
Polymethylacetate
o Amino ethyl coated polysaccharides, silica gels
o Aldehyde and acetyl groups of polymers
o Amide groups of polypeptides
Different methods of covalent bonding
• Diazotation
• Formation of peptide bond
• Group activation
• Polyfunctional reagents
Diazotation:• In this reaction involves bonding between the amino group of the
support
Formation of peptide bond:• The reaction occurs between the amino and carboxyl group of the
support and the amino and carboxyl group of enzymes.
Group activation:• In this method , cyanogen bromide is applied to support containing glycol
groups eg. Cellulose, sephadex, sepharose
Chemistry of Covalent Immobilization
• Hydroxyl group containing polymers:
Polyfunctional reagents:• In this method, bifunctional or a multifunctional reagents such as
glutardehyde is used to create bonding between the amino groups of
the support and the amino groups of the enzymes.
Gluteraldehyde based protein coupling :
Bi functionality of gluteraldehyde can be used for the formation of covalent bond
Covalent coupling
Advantages
• Very little leakage , prevents
elution of proteins in to the
production stream
• Stable method (not reversed
by pH ionic strength,
substrate)
• The wide range of choices is
possible by selecting carrier
materials and binding method.
This allows flexibility in
designing an immobilized
enzyme with specific physical
and chemical properties
Disadvantages
• Relatively expensive and
complicated in procedures.
• Low enzyme activity due to
exposure of the enzymes to
harsh environments and toxic
reagents.
• Active site may be modified
through the chemical reactions
used to create covalent
bonding
Enzymes
Carrier matrix
Binding agent/reagent
α- Amylase
DEAE-cellulose
Direct coupling
Aminoglucosidase
DEAE-cellulose
Cyanuric chloride
Cellulose
Polyurethane
Isocyanate
Glucose isomerase
Polyurethane
Isocyanate
Glucose oxidase
Polyurethane
Isothiocyanate
Pectinase
Polyurethane
Isothiocyanate
Pronase
Carbodiimide activation
CM-sephadex
COMPLEXATION
COMPLEXATION
• Based on chelating properties of the transition metals employed to
couple enzyme.
• Transition metal compounds (titanium, zirconium metal salts) used
for activation of the surface of organic carriers or using the
corresponding hydrous metal oxides.
• Metals like Co,Cu, Mn, tin, Zn,chromium, zirchonium are converted
into metal oxides
• In presence of enzyme it gives metal oxide enzyme.
• Interaction of transition metal compounds with biopolymers as
follows:• Titanium metal + chloride ion = octahedral co-ordination(ligand)
• Ligand + cellulose = glycosidic linkage=titanium chloride-cellulose.
ENTRAPMENT
3. ENTRAPMENT
The entrapment method of immobilization is based on the
localization of an enzyme within the lattice of a polymer matrix
,gels or capsule(micro encapsulation) .
 It is done in such a way as to retain protein while allowing
penetration of substrate.
It can be classified into lattice and micro capsule types.
 The support should have very small size pores which
facilitates the movement of substrate inside the compartment.
 Inclusion in gels: Poly acrylamide gel,Poly vinyl
alcohol gels
 Inclusion in fibers: Cellulose and Poly - acrylamide
gels.
 Inclusion in micro capsules: Polyamine,
Polybasic acid chloride monomers .
Lattice-Type Entrapment
• Entrapment involves entrapping enzymes within the
interstitial spaces of a cross-linked water-insoluble
polymer.
•Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have
been used to immobilize enzymes using this technique.
ENTRAPPED IN POLYMER
NETWORK
ENTRAPPED IN LATTICE
ENTRAPMENT
ChallengesEnzyme leakage into solution
Diffusional limitation
Reduced enzyme activity and stability
Lack of control micro-environmental conditions.
It could be improved by modifying matrix or
membrane.
ENCAPSULATION
ENCAPSULATION
• Enzymes are immobilised within microcapsules prepared from
organic polymers.
• The membrane encloses the enzyme and remain semipermiable to
the substrate and the products.
• Cheap and simple
• Provides large surface area to contact with the substrate and several
enzymes can be immobilised in single step.
ENCAPSULATION
• Not applicable for HMW substrates.
The methods used for encapsulation of enzymes:• Phase separation method
• Interfacial polymerization method
• Liquid drying method
• Liquid surfactant membrane method
Encapsulation of
enzyme
Immobilization Procedure
Enzyme blended with polymer solution
Polymerization
Extrusion/Shape the particles
Enzyme entrapped within the
microcapsules
• Advantages:
No chemical modification
Relatively stable forms.
Easy handling & reusage.
• Disadvantages:
May diffusion of substrate & product occurs.
Substrate accessibility may reduced due to free radical
polymerization of gel.
Enzyme in-activation.
Loss of enzyme content.
• NOTE: Sometimes covalent bonding may forms between the
entrapped enzyme & the matrix.
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LIMITATIONS OF ENZYME
IMMOBILIZATION
• Cost of carriers and immobilization.
• Changes in properties (selectivity).
• Mass transfer limitations.
• Problems with cofactor and regeneration.
• Problems with multienzymes systems.
• Activity loss during immobilisation.
Uses of Immobilized Enzymes
• Biotransformation
• Secondary metabolite production
• Biosensors
• Enzyme-linked immunosorbent assays (ELISAs)
• Biological washing Powders
• Food Industry
• Seed Germination
Applications of immobilized enzymes:
• Production of antibiotics- immobilized penicillin amidase
used for production of Penicillin G, Amoxicillin and
ampicillin
• Production of steroids- immobilized cells of
Cornybacterium simplex is used to convert
hydrocortisone and prednisolone from cortesolone
• Production of amino acids- β tyrosinase used for the
production of L-dopa
• Production of organic compoundsPropiniobacterium produce vit B 12
Catharanthus roseus produce ajmalcine
Digitalis lanata produce digitoxin
IMMUNOADSORPTION TECHNIQUES:• ELISA
• Radioallergosorbent test- to detection of Ig E antibody
THERAPEUTIC APPLICATIONS:• Immobilized enzymes such as streptokinase, urokinase, fibrinolysis in
microgranules of sephadex- for treatment of thromboses and
thromboemboli
• Artificial cells – enzyme is immobilized
• Artificial organs
• In replacement therapy needed in hereditary enzyme deficiency conditions.
Enzymes in biological washing Powders
• Proteases break down the coloured, insoluble proteins that
cause stains to smaller, colourless soluble polypeptides.
• Can wash at lower temperatures
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Enzymes in Food Industry
• Pectinase break down substances in
apple cell walls and enable greater
juice extraction.

Lactase breaks down lactose in milk
into glucose and galactose.
This makes milk drinkable for lactose
intolerant people.
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INDUSTRIAL
APPLICATIONS
Compounds
Microbial Cells
Enzymes
Matrix For Immobilisation
Ampicillin
Bacillus megaterium
Penicillin amidase
DEAE- cellulose
Penicillin G
Penicillium chrysogenum
Multi – enzymes
Polyacralamide,calcium
alginate
Cephalexin
Achromobacter sp.
Cephalosporin amidase
DEAE- cellulose
Bacitracin
Bacillus sp.
Multi- enzymes
Polyacralamide
tylosin
Streptomyces sp.
Multi-enzymes
calcium alginate
prednisolone
Arthrobacter simplex
Complete cell
Photo crosslinkable resin,
calcium alginate
hydrocortisone
Curvularia lunata
Complete cell
Photo crosslinkable resin,
polyacrylamide
1. Antibiotics
2. steroids
Compounds
Microbial Cells
Enzymes
Matrix For Immobilisation
L-alanine
Pseudomonas dacunhae
L-aspartate 4-decarboxylase
carrageenam
L-arginine
serratia marcescens
Multi-enzymes
carrageenam
L-glutamic acid
Brevibacterium flavum
Multi- enzymes
collagen
D-phenylglycine
Bacillus sp.
Hydantoinase
polyacrylamide
L-tryptophan
E. Coli
Tryptophan synthase
polyacrylamide
L-tyrosine
Erwinia herbicola
Β - tyrosinase
Collagen and glutaraldehyde
3. Amino acids
Compounds
Microbial Cells
Enzymes
Matrix For Immobilisation
Acetic acid
Acetobacter aceti
Multi- enzymes
Porous ceramic
Citric acid
Aspergillus niger
Multi- enzymes
calcium alginate
gluconic acid
Aspergillus niger
Glucose oxidase
calcium alginate
lactic acid
Lactobacillus casei
Multi- enzymes
polyacrylamide
2-ketogluconic acid
Serratia marcescens
Multi- enzymes
collagen
L-malic acid
Brevibacterium flavum
fumarase
carrageenam
4. Organic acid
Electochemical Devices
Sensor
Enzymes
Immobilisation
Oxygen electrode
Glucose
Glucose oxidase
Covalent
Ethanol
Alcohol oxidase
Crosslinked
Uric acid
uricase
Crosslinked
Inosine
Nucleoside phosphorylase
Covalent
Monoamine
Monoamine oxidase
Entrapement
L-alaginine
Alginine decarboxylase
Crosslinked
L-amino acid
L- amino acid oxidase
Covalent
L-aspargine
Asparginase
Entrapment
Urea
Urease
Crosslinked
Nitrite
Nitrite reductase
Crosslinked
L-methionine
Methionone ammonia lyase
Crosslinked
Ammonia gas electrode
ANALYTICAL APPLICATIONS
Electochemical Devices
Sensor
Enzymes
Immobilisation
CO2 gas electrode
L- Tyrosine
L- Tyrosine decarboxylase
Adsorption
PH electrode
Penicillin
Penicillinase
Entrapment
Nuetral lipid
Lipase
Covalent
Cholesterol
Cholesterol esterase
Covalent
Phospholipid
Phospholipase
Covalent
Platinum electrode
Immobilized cells and their applications
Immobilized micro organism
Applications
Anthrobacter simplex
Synthesis of prednisolone from hydrocortisone
Bacteria and yeast species
In biosensors
E. Coli
Production of L- tryptophan from indole and serine
Humicola species
Conversion of rifamycin B to rifamycin S
Pseudomonas chloraphis
Production of acrylamide from acrylonitrile
Saccharomyces cerevisiae
Hydrolysis of sucrose
Zymomonas mobilis
Production of sorbitol and gluconic acid from glucose
and fructose
IMMOBILIZED
PLANT
CELLS
Plant species
Cell type
Immobilized matrix
Cannabia sativa
cells
Calcium alginate
Catharanthus roseus
cells
Calcium alginate
Capsium frutescens
cells
Reticulate polyurethane
Digitalis lanata
Cells
Calcium alginate
Datura innoxia
cells
Calcium alginate
Daucus carota
Protoplast
Agarose + lectins
Mucuna pruriens
Cells
Calcium alginate
Papaver somniferum
Cells
Calcium alginate
Spinacia oleracea
Chloroplast
Calcium alginate
THANK YOU
-PHARMA STREET