Enzyme immobilization techniques

              Enzyme immobilization techniques 

What is enzyme immobilization?

• imprisonment of cell or enzyme in a distinct support(matrix).
• The support/matrix allows change of medium.
• The medium contain substrate or inhibitor molecule.
• First immobilization technology : amino acylases by aspergillus oryzae for the production of L-amino acid japan.
• The main advantages of enzyme immobilization:    

       1.increase functional efficiency 

       2. enhanced reproducibility 

  Advantages-

• Reuse
• Continuous use 
• minimum reaction time 
• less chance of contamination 
• more stability 
• improve in process control 
• high enzyme substrate ratio

Disadvantages-

• loss of catalytic properties in sometime 
• some enzyme become unstable 
• some enzyme are inactivated by heat generation in system 
• high cost for isolation, purification and recovery of active enzyme 

Application-

• Industrial production: Eg. Antibiotics, beverage ,amino acid etc 
• Biomedical application: treatment, diagnosis and drug delivery .
• Food industry: Production of jams, jellies, and syrup.
• Research: blotting experiment, proteases for cell lysis 
• Waste water treatment: treatment of sewage & industrial effluents

    Support/matrix-

• The enzyme hold in support/matrix 
• The should be cheap and easy available 
• Their reaction with medium and enzyme should be minimums as possible 
• A wide range of matrix are used in immobilization of enzyme/ cell 
• Matrix categories into 3 grouped 

1.  Natural polymers
2. Synthetic polymer 
3. Inorganic polymer 

Types of immobilization-

1. Adsorption 
2. Covalent bonding 
3. Entrapment 
4. Copolymerization 
5. Encapsulation 

       (1).  Adsorption-

• Oldest method of enzyme immobilization 
• Simplest method of enzyme immobilization 
• Nelson and Griffin used charcoal to adsorb invertase 
• Enzyme are adsorbed to external surface of support 
• Support/ carrier may be ;

1. Mineral support (aluminum oxide, clay)
2. Organic support (starch)
3. Modified sephareose and ion exchange  resins               

     

Methods of adsorption ;

1.static process-

immobilization to be perform by the using solution which containing enzyme to contact with the carrier (without stirring)

2.dynamic batch process-

carrier is placed in a enzyme solution and mixed by using agitation 

3.Reactor loading process-

when enzyme solution is transfer to reactor the carrier will already present in the reactor 

4.Electrode position process-

 carrier is placed proximal to an electrode in enzyme bath and the current is put on , the enzyme migrate to the carrier and deposition on the surface 

Advantages-

• Easy to carry out 
• no reagent are required 
• minimum activation step is involve 
• comparatively cheap method 
• less disruptive to protein than chemical method 

Disadvantages-

• desoption of enzyme from the carrier 
• efficiency is less 

2.Covalent bonding;

• In this process involve the formation of covalent bond between the enzyme and support/ matrix
• widely used method for enzyme immobilization 
• chemical group in enzyme that form covalent bonds with support are;

1. Amino acid 
2. Hydroxyl group 
3. carboxyl group 
4. Thiol group and methyl group 
5. Guanidyl group and imidazole group 
6. phenol rings 

Matrix used for covalent bonding-

• Carbohydrates -cellulose, DEDE cellulose , agarose 
• synthetic agent - polyacrylamide 
• protein carrier 
• amino group bearing carrier -amino benzyl cellulose 
• inorganic carrier- porous glass , silica 
• cyanogen bromide (CNBr) - agarose and CNBr sepharose 
• hydroxyl and amino group form covalent bond more easily 

Methods of covalent bonding 

1. Diazoation ;bonding between amino groups of support and thyrosil or histidyl groups of enzyme 

2. Peptide bond;bonding between carboxyl /amino groups of support and enzyme 

3. Poly functional reagent ;use of bi-functional or multifunctional reagent (glutaradehyde) which form bonding between the amino group of the support and amino group of the enzyme

3. Entrapment-

• Enzyme are physically entrapped inside the support 
• Bonds are involve in entrapment such as covalent and non-covalent bond
• matrix used will be water soluble polymer like-

1. polyacrylamide gels 
2. cellulose triacetate 
3. agar 
4. gelatin
5. alginate 

• Form and nature of matrix varies 
• pore size of matrix is adjusted to prevent loss of enzyme 
• agar have larger pore sizes 
• easy to practice at small scale 

Method of entrapment-

1.Inclusion in the gels- enzyme are trapped in gels 

2.Inclusion of fibers- enzyme support in fiber format

3.Inclusion in microcapsules - enzyme are trapped in microcapsules formed by the using monomer mixture such as polyamine, calcium alginate .

Advantages-

• This is fast method and cheap compare to another method of immobilization 
• Mild condition are required 
• No conformation change in this method 

Disadvantages-

• chance of microbial contamination 
• leakage of enzyme 

4. Cross linking or co-polymerization ;

• In this method involve covalent bonding between various groups of enzyme via poly-functional reagent 
• there are not involve matrix or support
• Commonly used poly-functional reagents are -Glutaradehyde , Diazonium salt 

 5. Encapsulation 

• enzyme are enclosed in semi-permeable membrane capsule 
• capsule is made up of nitro cellulose or nylon 
• effectiveness depends on the stability of enzymes 

Advantages-

• Simple and cheap method , large quantity of enzyme can immobilized by the using this method 

Disadvantages-

• Pore size is limitation 
• only small size of substrate is able to pass through the membrane