chapter 3 immobilized enzymes

Immobilization

• Immobilized biocatalysts can be made from killed cells, cell fragments, purified enzymes, and respiring cells

• Definition: Enzymes or cells which are physically confined to a defined region in space while retaining their catalytic activity and have the ability to be repeatedly and continuously used

Immobilized Enzyme Systems

Enzyme Immobilization:

- Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product.

- Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.

- Ability of enzymes to provide pure products.

- Possible provision of a better environment for enzyme activity

- Diffusional limitation

• Higher dilution rates can be used in a CSTR.

• Easier product recovery. • Lowered viscosity.• Easier enzyme recovery.

Why….

Advantages of Immobilization

• Mass transfer is inhibited

• Reduced activity of enzymes

• Bursting of immobilization medium due to cell growth

• Cells/enzymes can leak out of immobilization medium

Disadvantages of Immobilization

Immobilization Methods

Membranes Matrix entrapment Adsorption Covalent bonding

Immobilization

OrganicPolysaccharidesProtein chains

Synthetic Polymers

InorganicCeramic

Glass, SilicaAlumina

Supports

PhysicalChemical

Membranes

Membrane MicroencapsulationMembrane polymerized around aqueous enzyme solution in colloidal suspension (particle sizes on the order of 100-10 µm)

Organic solvent

Aqueous enzyme solution

mixing Add polymer

Microencapsulation

• Advantages

–High surface to volume ratio

–Thin membrane

–Relatively benign attachment method

• Problems

–More easily damaged than gel beads

Other Membrane Immobilization Methods- HFM

Hollow fiber reactors use micro- or ultra-filtration membranes to retain high MW enzymes, but pass low MW compounds

Enzymes or cells

Substrate and products outside the membrane

Substrate diffuses through membrane

Product diffuses out of the membrane

Close-up view of fiber

Hollow Fiber ReactorEnzymes or cells in

Substrate inProduct out

Two Types of Cartridges

Entrapment

Entrapment Immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane.

- retain enzyme - allow the penetration of substrate.

It can be classified into matrix and micro capsule types.

Immobilized Enzyme Systems

Immobilized Enzyme Systems

Entrapment - Matrix Entrapment - Membrane Entrapment

(microencapsulation)

Entrapment

• Advantages

– Relatively benign attachment method

– Easy to perform

• Problems

– Mass transfer limitations

– Cell/enzyme leakage

– Some cell/enzyme deactivation

• Alginate (polymer) common entrapment matrix

Immobilized Enzyme SystemsMatrix Materials:

Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar,

K-carrageenin, collagen

Immobilization procedures:Enzyme + polymer solution → polymerization

→ extrusion/shape the particles

Inorganics: activated carbon, porous ceramic.

Shapes: particle, membrane, fiber

Immobilized Enzyme Systems

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.

Covalent Bonding

Immobilized Enzyme SystemsSurface immobilization

According to the binding mode of the enzyme, this method can be further sub-classified into:

- Physical Adsorption: Van der WaalsCarriers: silica, carbon nanotube, cellulose, etc. Easily desorbed, simple and cheap, enzyme activity unaffected.

- Ionic Binding: ionic bonds Similar to physical adsorption.Carriers: polysaccharides and synthetic polymers

having ion-exchange centers.

Immobilized Enzyme Systems

Surface immobilization

- Covalent Binding: covalent bonds

Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups.

- Loss of enzyme activity - Strong binding of enzymes

Covalent Bonding• Most extensively used method of

immobilization due to high bond strength

• Three elements- structural polymer, enzyme molecule and bridge molecule–Activity of an immobilized enzyme

is a strong function of the hydrophilicity of the structural polymer

Covalent Bonding To SupportBridging molecule needs to be small, have two reactive groups, and not bond at the active site

Immobilized Enzyme Systems

Cross-linking is to cross link enzyme molecules with each other using agents such as glutaraldehyde.

Features: similar to covalent binding.

Several methods are combined.

Copolymerization of Enzymes• Copolymerization is performed with

multifunctional bridge molecules to yield and insoluble product

• Usually an inert protein is also included

Chemistry of Covalent Immobilization

Adsorption

Immobilization by Adsorption

• 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.

Typical Adsorbents

• Cellulose

• Polystyrene resins

• Kaolinite

• Glass

• Alumina

• Silica gel