Immobilization of enzymes

What is immobilization?
Arresting the free movement of enzymes and fixing them on a insoluble solid support without affecting its catalytic behaviour

Advantages of immobilization
Stability of immobilized enzymes is more than the free enzymes. The turn over number of substrates and products is high As the enzymes are attached to the solid support, the re-useage is possible The recovery of enzymes from the reaction mixture is very easy and the purification cost is minimized. Long lasting reuseage

ontinuous use Less labour intensive Saving the capital cost !inimum reaction time Less chance of contamination in products !ore stability "mproved process control #igh enzyme $ substrate ratio

Classification of Immobilization
"mmobilization

%ntrapment "mmobilization

Surface "mmobilization

!atrix %ntrapment

!embrane %ntrapment

Adsorption

ovalent &onding

Methods of immobilization

'hysical adsorption ovalent lin(age ross lin(ing or co-polymerization %ntrapment !embrane confinement or encapsulation

Physical adsorption

&inding either external or internal surface of the carrier !ineral support

Aluminium oxide lay )rganic support-starch

#ydrogen bond, van der *aals forces arrier size is very small- +,, A to -mm
.o pore diffusion limitations 'rotected from abrasion, inhibitory bul( solution / microbial attac(

Procedures

Static process 0ynamic batch process 1eactor loading process %lectrodeposition process

Covalent binding

2ormed between the chemical groups of enzymes and chemical groups on the surface of the carrier
p# "onic strength )ther variable conditions

Steps followed
Attachment of coupling agent Activation process or attachment of functional group Attachment of the enzyme

Procedures

0iazoation
Aminobenzyle cellulose Aminosilanised porous glass Aminoderivatives of tyrosyl or histidyl group

2ormation of peptide bond

Amino or carboxyl group of the support or enzyme

3roup activation
4se of .&r to glycol group ellulose, syphadex, sepharose

'olyfunctional reagents
3lutaraldehyde 5amino group on both support and enzyme

Disadvantages
"nactivated due to conformational changes )vercomed by using activated polymers

elluloses, polyacrylamides -diazo, carbodimide or azide

Entrapment
'hysically entrapper inside a matrix !atrix - *ater soluble polymer

ellulose triacetate, agar, gelation, carragreenan, alginate

!atrix pore size can be ad6usted to prevent enzyme loss by diffusion

Cross lin ing

)r co-polymerization ovalent bond between enzyme and polyfunctional reagent 3lutaraldehyde, diaonium salt, hexamethylene disocyanate / ., .7- ethylene bismaleimide

0isadvantages
%nzyme denaturation .ot used for pure proteins 8ery little of enzyme immobilized

Applications
2ormation of an insoluble aggregates- papain with glutaraldehyde ross lin(ing by adsorption- trypsin on colloidal silica particles "mpregnation of porous materials 5 papain in collodion membrane

Membrane confinement or Encapsulation

%nclosing of enzyme droplet in a semipermeable membrane apsule- cellulose nitrate or nylon %nzymes effectively retained in capsule 4sed in medical sciences only Large 9uantity can be immobilized

0"SA08A.TA3%S

Small substrate molecule can be utilized

Applications
4sed as analytical agents in enzyme electrodes %nzyme electrodes are available to detect the following

0rugs 'esticides Toxins 3lucose levels in blood

In food industries
4sed to convert starch into glucose 2or the conversion of mil( whey lactose into simple sugars 'reparation of cottage cheese "mmobilized glucose isomerase- manufacturing of fructose syrup oagulation of mil( proteins and treating waste whey

Aminocylase used in fluidised bed reactors or ST1 in pharmaceutical industry. onversion of 0:L-amino acids %nzyme Therapy
Asparagenase covalently lin(ed with ;inc and used in leu(ameia treatment. ;inc used to treat insulin. "t increases the survivability and reaction potential of the insulin.

0etermine the characteristic of cellular metabolism in living cells under different physiological conditions.

!able "#eneralised comparison of different enzyme immobilisation techni$ues

Characteristics
Preparation Cost Bindingforce En#$melea"age Applicabilit$ 'unningProblems Matri(effects Largediffusional barriers Microbialprotection

Adsorption
Simple Low ariable %es !ide High %es &o &o

Covalent binding
Difficult High Strong &o Selective Low %es &o &o

Entrapment
Difficult Moderate !ea" %es !ide High %es %es %es

Membrane confinement
Simple High Strong &o er$wide High &o %es %es