TOPIC 4IMMOBILIZED BIOCATALYST

Cell and Enzyme Immobilization

Immobilization means associating the biocatalysts with an insoluble matrix, so that it can be retained in a proper reactor geometry for its economic reuse under stabilized conditions. 

Advantages to immobilizing enzymes and cellsincreased stability,weeks or monthsstable to heat, pH extremes, storage, reactionfacilitates recovery for repeated or

continuous use (essential for soluble enzymes)

cellular activity is enzymatic activity (biotransformations)

Immobilized Enzymes• Immobilized enzymes have several advantages over the

soluble enzyme: – Convenience: Miniscule amounts of protein dissolve in

the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

– Economical: easily removed from the reaction reusage

– Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme

Immobilized Enzyme Systems- 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

Immobilized EnzymesAdvantages

Retention in reactor Separation from reaction

components is facilitated Usable in a wide range

of reactor configurations High catalytic loadings Enhanced stability

toward T, pH, solvent, etc.

Modified selectivities

Disadvantages Mass-transfer limitations Loss of activity upon

immobilization Impractical for solid

substrates

Immobilization criteriaThere are a number of requirements to achieve a

successful immobilization:The biological component must retain substantial

biological activity after attachment It must have a long-term stabilityThe sensitivity of the enzyme must be preserved after

attachment Overloading can block or inactivate the active site of the

immobilized biomaterial, therefore, must be avoided

Immobilization methods

a) adsorption b) covalent binding c) entrapment d) Encapsulation (membrane

confinement)

Immobilization Techniques

Methods of Enzyme Immobilization

• Three major Methods of Enzyme Immobilization are :

- Entrapment

- Surface Immobilization

- Cross-linking

Assignment:Form a group of 5-6 person. Pick any of the methods:

Construct a mind-map: Describe the properties/characteristic of method Identify whether the method is reversible or irreversible. Justify. Identify whether the method is suitable for cell or enzyme

immobilization. Justify. Determine the type of carrier or matrices that can be used.

Justify why are they suitable. The advantages and disadvantages of the method selected in

biocatalysis reaction. Application of the method

a) adsorption b) covalent binding c) entrapment d) Encapsulation (membrane

confinement)

Try to evaluate!

Which one is more complicated to be immobilized? Cells or enzymes?

Selecting an Immobilization TechniqueIt is well recognized that no one method can be regarded as the universal method for all applications or all enzymes. Consider,

widely different chemical characteristics of enzymes

different properties of substrates and productsrange of potential processes employed

Criteria to ensure the immobilization technique chosen will be effective

1)The procedure done to immobilize the cells must :

Have minimal adverse effects on the desired biocatalytic properties

Generate no by-products requiring special disposal procedure

The matrix used must not reduce the desired biocatalytic activity

Must be safe and simple to useOnly few steps and ingredients are requiredBe amenable to scale-upBe adaptable to automationBe economical

2) The matrix selected must:

Not reduce the desired biocatalytic activity of cells Not react with the substrates, nutrients or products Retain their physical integrity and insoluble under the bioprocess

reaction conditions Be permeable to reactants and products Have large specific surface Have high diffusion coefficients for substrates, nutrients and products Provide appropriate hydrophilic-hydrophobic balance for mutrietns,

reactants and products Be resistant to microbial degradation Retain chemical and thermal stability under bioprocess and storage

conditions Be elastic enough to accommodate growing cells Have functional groups for cross linking Be generally recognized as safe for food and pharmaceutical bioprocess

applications Be generally available in adequate quantities with consistent quality

and acceptance price Be easy and simple to handle in the immobilization procedure Be environmentally safe to dispose of and/or be capable of recycling

Criteria to ensure the immobilization technique chosen will be effective

Home work:Read review paper on “Parameters Affecting the Performance of Immobilized Enzyme”

Describe the parameters need to be considered in preparing the immobilization technique which might affect the performance of immobilized enzyme.

Parameters might affect the performance of immobilized enzyme

Immobilization methodsIn the entrapment method, the enzyme must be entrapped in the internal structure of polymer material, therefore does not results in large loss of enzyme activity. Moreover, lipase encapsulated will exhibited higher thermal stability and operational stability. In addition, the encapsulated lipases had higher catalytic conversion in the hydrolysis and higher enantioselectivity due to the encapsulation immobilization preserves the mobility of the enzyme and allows the increasing of its activity and enantioselectivity. This immobilization method favors the activity retention of immobilized enzyme and improves the performance of immobilized enzyme.

Parameters might affect the performance of immobilized enzyme

Immobilization carrier materialsCarrier material should be readily available, nontoxic, and should offer a good biological compatibility for enzyme. As a part of the immobilized enzyme, the structure and property of the carrier have important impacts on the enzymatic properties. Several natural polymer materials, synthetic polymer materials and inorganic particles were commonly used. The material must exhibit good mechanical rigidity, high specific surface area, easy to change their surface characteristics, and their potential for bringing specific functional group according to actual needs. The material also can have good biocompatibility, various surface functional groups which are suitable to couple enzymes and magnetic feature which enables it to achieve a rapidly easy separation from the reaction medium in a magnetic field.

Parameters might affect the performance of immobilized enzyme

Immobilization enzyme loading.The excessive enzyme loading always causes protein-protein interaction and inhibits the flexible stretching of enzyme conformation, which will result in the steric hindrance and thus the inactivation of an enzyme. That is, the enzyme molecule may be difficult to modulate its most suitable conformation for catching the substrate molecules and releasing product molecules under molecular crowding condition. The excessive enzyme loading may resulted in an intermolecular steric hindrance and greatly affected the lipase activity. In general, the amount of enzyme immobilized on carriers has obvious influence on the performance of the immobilized enzyme.