IMMOBILISED ENZYMES

ARGUMENT FOR IMMOBILISATION

Enzymes are expensive, they should be utilized in an efficient manner As catalytic molecules, enzymes are not directly used up. After the reaction the enzymes cannot be economically recovered for re-use and are generally wasted This enzyme residue remains to contaminate the product and its removal may involve extra purification costs

Simple and economic methods must be used to separate the enzyme from the reaction product

Immobilization of enzymes refers to the technique of confining/anchoring the enzymes in or on inert support for their stability and functional reuse

Advantages of immobilized enzymes Stable and efficient in function Can be reused again and again Products are enzyme free Ideal for multiple enzyme reaction systems Control of enzyme function is easy Suitable for industrial and medical use Minimize effluent disposal problems Disadvantages: Loss of biological activity expensive

METHODS OF IMMOBILISATION Adsorption 2. Covalent binding 3. Entrapment 4. Membrane confinement

1.

METHODS OF IMMOBILISATION

MATRICES FOR ENZYME IMMOBILISATION

Inert. 2. Physically strong and stable. 3. Should be cheap enough to discard. 4. Better if it could be regenerated after the useful lifetime of the immobilised enzyme. 5. The surface available to the enzyme.
1.

ADSORPTION OF ENZYMES ONTO INSOLUBLE SUPPORTS

Physical binding of enzymes on the surface of an inert support Support materials- inorganic (alumina, silica gel, calcium phosphate gel, glass) Organic starch, carboxymethyl cellulose, DEAEcellulose, DEAE- sephadex Weak forces - vander waals force and hydrogen bonds Adsorbed enzymes- easily removed by changing pH,ionic strength or temperature

EXAMPLES OF SUITABLE ADSORBENTS

Ion-exchange matrices Porous carbon Clays Hydrous metal oxides Glasses Polymeric aromatic resins

ENTRAPMENT OF ENZYMES WITHIN GELS OR FIBRES

Purely

physical caging: size of the matrix pore is such that the enzyme is retained, substrate and pdt pass thru Tech commonly referred lattice entrapment Matrices used- polyacrylamide gel, collagen, gelatin, starch Entraped cells used- production of aminoacids (Lisoleucine,L-aspartic acid), L-malic acid and hydroquinone

Microencapsulation
It

is a type of entrapment refers to the process of spherical particle formation wherein a liquid or suspension is enclosed in a semipermeable membrane Membrane-polymeric,lipoidal,lipoprotein based or non-ionic in nature

Three distinct ways of microencapsulation Building of special membrane reactors Formation of emulsions Stabilization of emulsions to form microcapsules It is recently used for immobilization of enzymes and mammalian cells

Covalent binding
Covalent

bond b/w chemical groups of enzymes and the chemical groups of support It is often associated with loss of enzyme activity Inert support needs pretreatment before it binds enzymes

Common methods of covalent binding

Cyanogen

bromide activation: Inert support materialscellulose,sepharose,sephadex containing glycol groups activated by CNBr,which then binds to enzymes and immobilize them

Diazotation
Support

materials-aminobenzyl cellulose, amino derivatives of polystyrene, aminosilanized porous glass These are subjected to diazotation on treatment with NaNO2 and HCl In turn bind covalently to tyrosyl or histidyl groups of enzymes

Peptide bond formation

Peptide

bond b/w amino or carboxyl groups of the support and carboxy or amino group of enzyme Support material is chemically treated to form active functional groups

Activation by bi- or polyfunctional reagents

Some

of the reagents such as glutaraldehyde used to create bonds b/w amino groups of enzymes and amino groups of support Eg., aminoethylcellulose, albumin,aminoalkylated porous glass

Cross linking

The absence of solid support Enzymes immobilized by creating cross-links between them,thru polyfunctional reagents Reagents react with enzymes and create bridges which form the backbone to hold enzymes Crosslinking reagents: glutaraldehyde, diazobenzidine, hexamethylene diisocyanate and toluene diisothiocyanate

Choice of immobilization technique

Trial

and error method Factors to decide a technique Catalytic activity Stability Regenerability Cost factor

The most commonly used method for immobilizing enzymes on the research scale involves Sepharose (poly-{-1,3-D-galactose--1,4-(3,6anhydro)-L-galactose}), activated by Cyanogen bromide, because it is a commercially available beaded polymer which is highly hydrophilic and generally inert to microbiological attack

IMMOBILIZED ENZYME ACTIVITY DEPENDS ON CORRECT AND UNSTRAINED CONFORMATION

CONFINEMENT OF ENZYMES INSIDE A MEMBRANE

Semipermeable membrane: Hollow fibre

membrane Membrane-bound droplets: Nylon-6,6 Liposome: Phospholipid

GENERALISED COMPARISON OF DIFFERENT ENZYME IMMOBILISATION TECHNIQUES

Characteristics Preparation Cost Binding force Enzyme leakage Applicability Running Problems Matrix effects Large diffusional barriers Microbial protection Adsorption Simple Low Variable Yes Wide High Yes No No Covalent binding Difficult High Strong No Selective Low Yes No No Entrapment Difficult Moderate Weak Yes Wide High Yes Yes Yes Membrane confinement Simple High Strong No Very wide High No Yes Yes

Enzyme

EC number

Product

Aminoacylase Aspartate ammonia-lyase

3.5.1.14 4.3.1.1

L-Amino acids L-Aspartic acid

Aspartate 4-decarboxylase

4.1.1.12

L-Alanine

Cyanidase

3.5.5.x

Formic acid (from waste cyanide)

Glucoamylase Glucose isomerase Histidine ammonia-lyase Hydantoinasea Invertase Lactase Lipase Nitrile hydratase Penicillin amidases Raffinase Thermolysin

3.2.1.3 5.3.1.5 4.3.1.3 3.5.2.2 3.2.1.26 3.2.1.23 3.1.1.3 4.2.I.x 3.5.1.11 3.2.1.22 3.2.24.4

D-Glucose High -fructose corn syrup Urocanic acid D- and L-amino acids Invert sugar Lactose-free milk and whey Cocoa butter substitutes Acrylamide Penicillins Raffinose-free solutions Aspartame

Some of the more important industrial uses of immobilised enzymes

Immobilization of L-aminoacid acylase

First

enzyme to be immobilized 40 different methods attempted 3 methods found useful Covalent binding to iodoacetyl cellulose Ionic binding to iodoacetyl cellulose Ionic binding to DEAE-sephadex and entrapment within polyacrylamide

Stabilization of soluble enzymes

Some

enzymes- cannot be immobilized and have to be used in soluble form Such enzymes-stabilized by using additives or by chemical modification Stabilized enzymes - longer half - lives,

Methods of enzyme stabilization

Solvent

stabilization: solvents at low conc stabilize enzymes Substrate stabilization: active site can be stabilized by adding substrates Stabilization by polymers: enzymes stabilized against increased temperature by addition of polymers such as gelatin, albumin and polyethylene glycol

Stabilization by salts : Stability of metalloenzymes achieved by adding salts Ca,Fe,Mn,Cu and Zn Eg protease can stabilized by adding ca Stabilization by chemical modification : Addition of polyamino side chain Eg., poly tyrosine, poly glycine Acylation of enzymes by adding groups such as acetyl, propoinyl and succinyl Stabilization by rebuilding: Theoretically the stability of the enzymes due to hydrophobic interactions in the core of the enzyme. It is therefore proposed by enhancing hydrophobic interactions it can be stabilized the enzymes is first unfold and rebuilt

The enzymes is first unfold and rebuilt Enzyme is first chemically treated and then refolded
Refolding

can be done in presence of low mol wgt ligands certain enzymes, refolding at higher temp stabilizes them

For

Stabilization by site-directed mutagenesis

site-directed

mutagenesis used to produce more stable and functionally more efficient enzymes

Immobilization of cells
Immobilized

individual enzymes-used for single step reaction They are not suitable for multienzyme rxns requiring cofactors Whole cells or cellular organellesimmobilized to serve as multienzyme systems Sometimes cells are used for single step rxn due to cost factor in isolating enzymes

Immobilized viable cells

Viability

of the cells preserved by mild immobilization Immobilized cells used for fermentation Mammalian cells- used as immobilized viable cells

Immobilization non-viable cells

It

is prefered over the enzymes or even the viable cells Becoz of costly isolation and purification processes Eg., immobilization of cells containing glucose isomerase for the industrial production of fructose syrup

Limitations of immobilizing eukaryotic cells

Due

to the presence of cellular organelles the metabolism is slow It is used for the production of complex proteins such as immunoglobulins For the proteins that undergo post translational modification Usually prokaryotes fit well for industrial production of biochemicals

Effect of immobilization on enzyme properties

Enzyme

immobilization associated with alterations in enzyme properties Substantial decrease in enzyme specificity due to conformational change occur during immobilization Kinetic constants Km and Vmax of an native enzyme different from that of the immobilzed enzyme

Immobilized enzyme reactors

Immobilized

enzyme cells are utilized in the industrial processes in the form of enzyme reactors Batch reactors Continuous reactors Membrane reactors

Batch reactors

Immobilized enzymes and substrates placed Reaction is allowed to takes place under constant stirring As rxn completes the pdt is separated from enzyme Soluble enzymes commonly used in batch fermentation Difficult to separate enzyme and limitation of their reuse

Stirred tank reactors

Composed

of reactor with stirrer- allows good mixing and appropriate temperature and pH Chance of loss of enzyme activity Modification of stirrer tank is basket reactor Enzyme is retained in the impeller blades

Plug flow type reactors

The

flow rate of fluids controlled by a plug system It is in the form of packed bed or fluidized bed Useful for obtaining kinetic data on the reaction systems

Continuous reactors
Substrate

added continuously and product removed simultaneously Advantages Control over product formation Convenient operation of the system Easy automation of the entire process

Membrane reactors
Membrane

materials include polysulfone, polyamide and cellulose acetate Biocatalyst normally retained on the membranes of the reactor Recycle model membrane reactor, the contents, cofactors and substrates along with the freshly released product- recycled using a pump The product passes out can be recovered

Applications of immobilised enymes and cells

Manufacturing of commercial products Production of L-aminoacids are important for use in food, feed and medical purpose Chemical methods employed for their production result in racemic mixture of D and L-aminoacids They are then acylated to form D,L-acyl aminoacids Immobilised enzyme aminoacylase selectively hydrolyse D,L-acyl aminoacids to form Laminoacids

Production of high fructose syrup

High

fructose syrup contains approx equivalent amount of glucose and fructose HFS is a good substitute for sugar HFS can be produced from glucose by employing an immobilized enzyme glucose isomerase

Immobilized enzymes and cells analytical applications

Enzyme

electrode- glucometer Substance such as urea, cholesterol, lactate, alcohol can be assayed In affinity chromatography immobilized enzymes- used to purify several compounds, antigens and antibodies