Immobilization of Enzymes

Introduction

• Immobilization of enzymes can be defined as the confinement of an enzyme (bio-catalyst) in a distinct phase, separated from the bulk phase but allowing it to exchange with the latter.

• Bulk Phase consists of a substrate, an effecter or inhibitor.

• Immobilized enzyme is either physically entrapped or covalently bonded by chemical means to an inert insoluble matrix or carrier.

• In other words, it involves the restrictive localization of enzymes.

• Matrix is generally a high molecular weight polymer.

Ex : cellulose, polyacrlamide, alginate, etc. S

Introduction

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EnzymeIn

Solution Form

Substrate/Bulk

Phase/Inhibitor

Introduction

SSubstrate/Bulk

Phase/Inhibitor

EnzymeIn

Solution Form

Introduction

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EnzymeIn

Solution Form

EnzymeIn

Encapsulated Form

Substrate/Bulk

Phase/Inhibitor

Introduction

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EnzymeIn

Solution Form

EnzymeIn

Encapsulated Form

Substrate/Bulk

Phase/Inhibitor

Introduction

S

EnzymeIn

Solution Form

EnzymeIn

Encapsulated Form

Substrate/Bulk

Phase/Inhibitor

Need for Immobilization

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• Accelerates the chemical reaction.

• Specificity & un-modified enzyme.

• Cost effective.

• Not difficult to separate.

• Attachment to polymers/matrix, causes re-use.

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. S

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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Methods of Immobilization

• Physical Methods

AdsorptionEntrappingMembrane confinement

• Chemical Methods

Covalent BondingCross LinkingComplexation & Chelation

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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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• Types of Carriers :Naturally occuring

Structural proteins (Ex: ceratin, collagen)Globular proteins (Ex: albumin)Carbohydrates (Ex: dextran)

Synthetic organicEx: polyvinyls, epoxide,etc

InorganicEx: glass, silica gel, bentonite, titania,etc.

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Carrier for Immobilized Enzymes

A• Non-specific binding like electrostatic or

hydrophobic affinity binding to special ligand.• Mostly explained in following terms:

Static pores Dynamic pores Reactor Loading Electro Deposition

• NOTE: Adsorbent (mostly polymeric matrix)Ex: alumina, bentonite, CMC, Silica gel,

Titania, etc.

DSORPTION

A

ADSORPTION

• Advantages: Simple & Economical Limited Loss of activity Can be Recycled, Regenerated & Reused (R3)

• Disadvantages: Relatively Low surface area for binding 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.

E

E

E

E

ADSORPTION

MATRIX

ENTRAPPING

• Enzymes are held or entrapped within the suitable gels or fibres.

• In a gel it may causes: Matrix polymerization or Precipitation or Coagulation

• Entrapment in calcium alginate is the most widely used for entrapment for :

Microbial Animal & Plant enzymes/cells

Ex: Glucose oxidase + Polyacrlamide (gel entrapment)

• NOTE: Adsorbent (mostly commonly used)Ex: polyacrylamides, collagen, silica gel,

alginates, etc.

ENTRAPPING

• 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.

ENTRAPPING

Enzyme + Sod.alginate Mixture is added dropwise

CaCl2 Solution

Beads of Calcium alginates

CONFINEMENT

Membrane• Enzyme molecules (usually in aq. form) are

confined within semi-permeable :Reaction vesselo Partitioning into two chambers by a semi

permeable membraneo One chamber contains the enzyme while

the other have substrate & product.Hollow fiber membraneo Entrapment in semi permeable fibres

(cellulose, triacetate) or spheres (nylon, collodion).

o In which, the enzyme will be in the lumen/hollow space, while the fibres/spheres will be submerged in the substrate.

CONFINEMENT

MembraneMicro capsuleso Enzymes are packed in microcapsules

formed by polymerization (like phase separation or chemical polymerization).

Liposomeso Enzymes can be bounded in a concentric

spheres of lipoidal membrane formed by addition of phospholipid.

• Advantages: No enzyme leakage No change in enzyme activity

• Disadvantages: Diffusional barrier to the substrate &

product. Not cost effective.

CONFINEMENT

Membrane

E

EE

E E

E E

CONFINEMENT

Membrane

E

EE

E E

E E

BO

N

D

I

N

G

Chemical• Enzyme forms co-valent link with active group of

the matrix (like terminal -NH2, -COOH,etc,).

• Support with groups like :

-OH : support activation covalently by CNBr.

-COOH : supports (like CMC) activation covalently by azide derivatives.

-NH2 : support activation covalently by forming diazonium chlorides on treatment with NaNO2 + HCl.

NOTE: The functional group of enzyme which is involved in the linkage, should not affect the active properties of the said enzyme.

BO

N

D

I

N

G

Chemical• Advantages:

Not affected by pH Ionic Strength

• Disadvantages: Active site may be modified Not cost effective.

NOTE: Adsorbent

Ex: Agarose, Cellulose, sepharose, Polyacrlamide,etc.

BO

N

D

I

N

G

Chemical

E

E

E

E

E

E

LI

N

K

I

N

G

Cross• It involves cross linking of enzyme to a multi

functional reagent without use of any solid

support.

• Alternatively, chemical bridge of some other

molecule between & with the chemical support

(i.e., reaction of enzyme with reagent bridge or

chemical bridge).

• Activated carriers are used.Ex : Sepharose by CNBr (most commonly

used)Ex : Sepharose by ethyl chloroformate

LI

N

K

I

N

G

Cross• Advantages:• Strong linkage leads to low enzyme leakage

while use.• Higher stability (i.e., pH, ionic & substrate

concentration.

• Disadvantages:• Partially or wholly inactivation by active site

modification.• Not cost effetcive.

LI

N

K

I

N

G

Cross

E

E

E

E

E

E

E

E

E

E

E

E

Uses of Immobilized Enzymes

• Biotransformation• Secondary metabolite production• Biosensors• Enzyme-linked immunosorbent assays (ELISAs)• Biological washing Powders• Food Industry• Seed Germination

Glucose oxidaseGlucose Hydrogen peroxide

peroxidase

Dye: Blue---Green---Brown

Dye changes according to amount of glucose

Enzyme-linked immunosorbent assays (ELISAs) detect antibodies to infections.

Enzymes in Medicine

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• Proteases break down the coloured, insoluble proteins that

cause stains to smaller, colourless soluble polypeptides.

• Can wash at lower temperatures

Enzymes in biological washing Powders

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• 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.

Enzymes in Food Industry

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starch

embryo plant

amylasesecreted

maltose

absorbed

Enzymes in Seed Germination

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References

Pharmaceutical BiotechnologyBy Dr. S.P. Vyas & Dr. V.K. Dixit

Enymes & its Immobilization PresentationBy Dr. S. Khanam

Internet Resources :http://www.eplantscience.comhttp://www.clickbiology.comhttp://www.lsbu.ac.ukhttp://www.tech-ceramics.co.uk

Thank You !!!

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