Immobilization

Dr.Aun MuhammadEjaz-ul-Hassan(BS-BT-27)

Faraz Ahmad(BS-BT-11)

Waseem Akram(BS-BT-09)

Institute of Molecular Biology & Biotechnology (IMBB)

B.Z.U. Multan

Immobilizati

on

“The permanent attachment or fixation of

enzymes, Bacteria or other cells (plant or animal) to a hard substrate or within

a stable matrix via any of a number of

physical and/or chemical means”

“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.”

Enzyme Immobilization

* Enzyme* Matrix* Mode of attachment* They are soluble in water. So it is very

difficult to separate the enzyme for reuse in a batch process.

* Enzymes can be immobilized on the surface or inside of an insoluble matrix either by chemical or physical methods.

Major Components

* They can also be immobilized in their soluble forms by retaining them with a semipermeable membrane.

* The enzymes can be attached to the support by interactions ranging from reversible physical adsorption and ionic linkages to stable covalent bonds.

* As a consequence of enzyme immobilization, some properties such as catalytic activity or thermal stability become altered.

“Cell immobilization is a technique to fix whole cells either on a

suitable matrix or on any support with

retention of its function”.

Cell Immobilization

* This method is implemented when the enzymes are difficult or expensive to extract. E.g intracellular enzymes

* If a series of enzymes are required in the reaction; whole cell immobilization may be used for convenience

* This is only done on a commercial basis when the need for the product is more justified.

* Cells are absorbed onto support materials

Properties

PhysicalChemical

Cross Linking

IonicCovalen

t

Carrier Binding

Entrapment

Micro

Encapsulation

Adsorption

Ionic Network Formation

Precipitation

Performed Structures

Polymerization

Methods

* It is intermolecular cross-linking of enzymes by means of multifunctional reagents(glutaraldehyde, etc)

* Performed by the formation of intermolecular cross-linkages between the enzyme molecules by reagents.

Cross Linking

Oldest Method Binding of enzymes to water-

insoluble carriers (polysaccharide derivatives)

Care is required in the selection of carriers as well as in binding techniques

Carrier Binding

* Carried out under severe conditions. * If we need high activity of immobilized

enzymes than immobilization of an enzyme by covalent binding is carried out under well controlled conditions.

* The binding forces between the enzyme and carrier are strong, and the enzyme cannot easily be lost from carriers even in the presence of substrates or salts at high concentrations.

* Covalent methods for immobilization are employed when there is a strict requirement for the absence of the enzyme in the product.

Covalent

* Carried out under mild conditions

* Weak Binding forces relative to others methods

* Leakage of the enzyme from the carrier may occur after changes in the ionic strength, pH of the substrate, or product solution

Ionic

In physical methods, there is physical immobilization of enzymes or cell. A physical connection is established between the support and the enzyme to immobilize it partly or completely. Physical methods is divided into following categories: Adsorption Microencapsulation Entrapment

Physical

* Simplest way to immobilize enzymes.

* Enzymes can be adsorbed physically on a surface-active adsorbent by contacting an aqueous solution of enzyme with an adsorbent.

* Commonly employed adsorbents are alumina, ammonium-exchange resins, calcium carbonate, carbon, cation exchange resins, celluloses, clays, collagen, colloid-ion.

Adsorption

Enzymes can be immobilized within semipermeable membrane microcapsules.

This can be done by the interfacial polymerization technique.

Organic solvent containing one component of copolymer with surfactant is agitated in a vessel and aqueous enzyme solution is introduced.

The polymer membrane is formed at the liquid-liquid interface while the aqueous phase is dispersed as small droplets.

Micro Encapsulation

“Entrapping is the incorporation of enzymes into the latticeof a semipermeable gel,

or enclosing of enzymes in a semipermeable polymer

membrane, such ascollagen, gelatin, cellulose, triacetate, polyacrylamide”.

Entrapment

It is the most widely used method, especially in the form of alginate beads.

Alginate is a polysaccharide that forms a stable gel in the presence of cations, with calcium the most frequently used. Beads of Alginate-containing cells, are formed by dipping a cell suspension-sodium alginate solution mixture into a stirred calcium chloride solution.

Κ-Carrageenan can also be used in similar manner instead of alginate, using either calcium or potassium.

Advantage of this method is that the gel can be reversible by adding EDTA.

Ionic Network

Formation

Preparations of agar and agarose can be used to trap plant cells by precipitation.

The polysaccharides form gel when a heated aqueous solution is cooled. The gel can be dispersed into particles in the warm liquid state by mixing in a hydrophobic phase, e. g. olive oil.

When particles of the desired size are obtained, the entire mixture is cooled and this results in solidification

Precipitation

Gel entrapment by polymerization is most commonly carried out using polyacrylamide.

The toxicity of the initiator and cross-linking agents used in the polymerization has caused a loss of cell viability.

Polymeriza

tion

Entrapment in preformed structures involves some form of open network through which nutrient medium may pass, but which entraps plant cells or cell aggregates.

Such structures can be facilitated by using cotton fiber, fiberglass mats, reticulate polyurethane foam, and in a cloth nonwoven polyester short fibers.

Performed

Structures

Preparation & Characterization Of Immobilized EnzymesSr

. #

Charac-teristics

Physical Adsorp-tion

Ionic Binding

Covalent Binding

Cross-Linking Method

Entrapping Method

1. Preparation

Easy Easy Difficult Difficult Difficult

2. Enzyme Activity

Low High High Moderate High

3. Substrate Specificity

Unchangeable

Unchange-able

Unchange-able

Unchange-able

Unchange-able

4. Binding Force

Weak Moderate Strong Strong Strong

5. Regeneration

Possible Possible Impossible Impossible Impossible

6. General Applicability

Low Moderate Moderate Low High

7. Cost Low Low High Moderate Low

Factors Affecting

Immobilization

Cell Matrix Interaction

LightMass

Transfer

Aeration

When using reticulated polyurethane foam, in order for any immobilized cells to function well, the volumetric fraction of the foam has to be sufficient enough for all the cells and the reticulated pores of the foam is large enough to contain the cells.

The importance of initial interaction of cells with the surface of the polyurethane particles, intrusion of cells into the foam, simultaneous growth, coalescence of cells in the foam and retention of cells in the foam must occur.

Cell Matrix

Interaction

The transfer of compounds through immobilized cell matrix is usually assumed to be by molecular diffusion, as in gel entrapment.

The microstructure of immobilization matrix may bring other types into action such as capillary and active transports.

These types of transport mechanism are true for reticulated foam, membrane and fiber mats matrices.

Mass Transfer

The resistance towards mass transfer that results in a decrease in the transport of nutrients, can be an advantage in creating a stress factor for undesired secondary metabolite synthesis.

Mass transfer restriction within the biomass can be minimized by surface immobilization in a layer form where there is maximum contact between the surface of the immobilized cells and the liquid phase.

Relationship between metabolism and dissolved oxygen concentration is complex, a conclusion cannot be reached about the effect of reduced availability of oxygen in immobilized plant cell system on secondary metabolite production and growth.

Aeration

Metabolism can be affected by periodic exposure to light, and the quality and intensity of the light are significant.

Only the outer cell layers of the cultures in the immobilized matrix may receive some light.

This may be advantageous in the case where some precursors are form in light and some in dark condition.

The supply of light to the interior of the immobilized cell matrix may be possible by the use of optical fibers.

Light

The characteristics of the matrix are of paramount importance in determining the performance of the mobilized enzyme system. Ideal support properties include : Physical resistance to compression Hydrophilicity Inertness toward enzymes ease of derivation Biocompatibility Resistance to microbial attack Availability at low cost High stability against physical, chemical, and

microbial degradation Pore parameters Particle size determine the total surface area

Choice of

Support

Nonporous supports show few diffusional limitations and have a low loading capacity.

Porous supports are generally preferred because the high surface area allows for a higher enzyme loading and the immobilized enzyme receives greater protection from the environment.

Porous supports should have a controlled pore distribution in order to optimize capacity and flow properties.

The hydrophilic character is one of the most important factors determining the level of activity of an immobilized enzyme.

An excellent matrix that has been extensively used is agarose.

In addition to its high porosity, which leads to a high capacity for proteins, some other advantages of using agarose as a matrix are hydrophilic character, ease of derivation, absence of charged groups and commercial availability

Immobilized enzymes are widely used for variety of applications.

The immobilized enzymes are also widely used in food industry, pharmaceutical industry, bioremediation, detergent industry, textile industry, etc.

Several hundreds of enzymes are immobilized and used for various large scale industries.

Applications

In food industry, the purified enzymes are used but during the purification the enzymes will denature.

Hence the immobilization technique makes the enzyme stable.

The immobilized enzymes are used for the production of syrups.

Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production of baker’s yeast.

Food Industry

The detergent industry also employs enzymes for removal of stains.

The enzymes used in detergent industry are protease which is used to remove the stains of blood, egg, grass and human sweat.

Amylase used to remove the starch based stains like potatoes, gravies, chocolate.

Lipase used to remove the stains of oil and fats and also used to remove the stains in cuffs and collars.

Detergent

Industry

Cellulase is used for cotton based fabrics in order to improve softening, color brightening and to remove soil stains.

Nowadays Biotech cleaning agents are widely used in the detergent industries. When compared to synthetic detergents the bio based detergents have good cleaning property.

The increasing consumption of fresh water and water bodies are mixed up with polluted industrial waste water and the waste water treatments are necessary at present.

The sources of dye effluents are textile industry, paper industry, leather industry and the effluents are rich in dye colorants.

Nowadays enzymes are used to degrade the dye stuffs.

The enzymes used in the wastewater treatments are peroxidases, laccase, AZO reductases.

Waste Water

Managment

The Horse radish Peroxidases are entrapped in calcium alginate beads, this method is still in lab scale research.

The immobilized laccase enzyme has the ability to degrade dyes anthracinoid dye, Lancet blue and Ponceau red.

Adsorption method is widely used because of its easy regeneration.

During the covalent method of immobilization the conformational change in the enzyme occurs which will affect the activity of the enzyme.

The enzymes derived from microbial origin are of great interest in textile industry. The enzymes such as cellulase, amylase, laccase, pectinase and these are used for various textile applications such as scouring, bio polishing, designing, denim finishing, treating wools etc.

The processing of fabrics with enzymes requires high temperatures and increased pH, the free enzymes does not able to withstand the extreme conditions.

Textile Industry

Hence, enzyme immobilization for this process able to withstand at extreme and able to maintains its activity for more than 5-6 cycles.

Enzymes are used for diagnosis and treatment of diseases in the medical field.

The inborn metabolic deficiency can be overcome by replacing the encapsulated enzymes (i.e enzymes encapsulated by erythrocytes) instead of waste metabolites, the RBC acts as a carrier for the exogenous enzyme drugs

The enzyme encapsulation through the electroporation is an easiest way of immobilization in the biomedical field and it is a reversible process for which enzyme can be regenerated.

Biomedical

Industry

WE

CLASS