Enzyme and cell immobilization By Dr. Ahmed Z. Abdel Azeiz College of Biotechnology Misr university for Science and Technology Egypt E-mail: alrahman3@hotmail.com

introduction Immobilized enzyme is defined as: the enzyme that physically or chemically confined in defined materials with retention of its catalytic activity. The term "immobilization" is applicable also for cells, either microbial, plant or animal cells and even for cell organelles. The first discovery of this process was by Nelson and Griffin 1961, when they find that invertase is immobilized (adsorbed) on charcoal and able to hydrolyze sucrose.

After this publication, several researches were published about immobilization of several proteins by covalent bonding with saving its biological activity. It can be referred to this process as immobilization of biocatalysts (enzymes or cells). The biochemical reaction occur inside the living cell (microbial, plant or animal) are catalyzed by enzymes. These enzymes are usually water soluble proteins. These reactions take place at mild environmental conditions such as temperature, pH and pressure. The enzyme reactions have strict substrate specificity and stereospecificity.

These facts suggest that energy saving, resources saving, low pollution process and low waste products could be achieved by using immobilization of the biocatalysis agent. Furthermore, the recovery of the product from the reaction mixture is a problem when the biocatalysts are used in a free form. Disadvantages of the cell immobilization: 1- Some unwanted byproducts can be formed due to presence of other enzymes in the used cells. 2- The cell wall and cell membrane of the used cells prevent substrate, products or other reaction components to penetrate inside or outside the cell.

The immobilized cells can be used in growing state by continues supply of nutrients. The advantage of this process is that this technique serves as self-proliferating and self-regenerating biocatalyst. The disadvantage of this system is that the product can be contaminated by the cells leaking from the carrier. This is in addition to use of nutrients and energy sources to maintain the cell life.

Steps for enzyme and cell immobilization 1- Selection of the suitable enzyme or cell 2- Selection of the suitable carrier. 3- Selection of the suitable immobilization technique. Selection of the suitable enzyme or cell to perform a required reaction is carried out through several screening researches; while selection of the suitable carrier and immobilization technique depends on the type of the biocatalyst.

The immobilization methods A- Carrier-binding methods. B- Cross-linking methods. C- Entrapping methods. D- Combined method. Some of these methods are suitable for immobilization of both enzymes and cells; while other are suitable for either enzyme or cells only..

A- Carrier-binding methods This method is based on binding of the biocatalyst with a non-soluble carrier by a covalent bond, ionic bond, physical adsorption or bio-specific binding. Several materials can be used such as: 1- Polysaccharides: cellulose, dextran and agarose derivatives. 2- Proteins: gelatin, albumin. 3- synthetic polymers: Polystyrene derivatives, ion exchange resins, polyurethane. 4- Inorganic materials: glass, sand, ceramic and magnetite.

1- Covalent bonding methods One of the most common used techniques for enzyme immobilization. This is performed through the amino acid residue which is not included in the active site. For example: by using the ε- amino group of lysine, β-carboxyl group of aspartic, hydroxyl group of serine or threonine, mercapto group of systeine, imidazole group of histidine. Each of these groups can reacts with a suitable function group of the carrier such as diazonium salt, acid-azide, isocyanate, activated halide alkyle or aldehyde.

Advantages and disadvantages of the covalent binding method 1- The enzyme doesn't leak from the carrier. 2- The enzyme can be easily interacts with the substrate because it is in the surface of the carrier. 3- The enzyme stability is often increased due to its strong binding with the carrier. Disadvantages: 1- The yield activity of the enzyme is low due to using of toxic materials during immobilization. 2- The optimal immobilization conditions are difficult to be find. 3- Renewable of the carrier and recovery of the enzyme is impossible.

a) Cyanogen Bromide method (CNBr) In this method the cyanogen brimde reacts with hydroxyl groups of a carrier such as cellulose, glass beads or ceramic to yield the reactive imidocarbonate derivative (the activated carrier). The subsequent reaction is between this activated carrier and amine groups of the enzyme (figure 1). In some cases a "spacer" is needed to be added to prevent hindrance, which may be result from the stereo-configuration of either enzyme or substrate or both (figure 2). The ready made activated carriers are available commercially in the market. For example: CNBr-activated Sepharose 4B, that produced by Pharmacia Fine Chemicals.

Figure 2: The spacer effect in enzyme immobilization. Figure1: Steps of CNBr enzyme immobilization method Figure 2: The spacer effect in enzyme immobilization.

b) Acid-azide derivative method This method is also used for peptide synthesis. In this method the carboxyl group of the carrier is converted firstly to methyl ester. Then, it reacts with hydrazine to form the hydrazide. The hydrazide is reacted with nitrous acid to form azide derivative. This azide can be easily reacts with the amino group of the enzyme. Carboxymethylcellulose (CMC) is the best carrier in this process.

R—COOH + CH3OH R—COOCH3 R—COOCH3 + NH2—NH2 R—CONH—NH2 R—CONH—NH2 + HNO2 + EN-----NH2

c) Condensing reagent methods The carboxyl group of the carrier and amino group of the enzyme can be condensed together to form peptide linkage by using condensing reagents. These reagents such as carbodiimides. R—COOH + NH2—Enzyme R—CO-N--Enzyme

d) Diazo coupling methods Carriers having aromatic amino groups can be azotized by using nitrous acid to form diazonium derivative. This derivative is bind with the amino group, imidazole group, or phenolic hydroxyl group of a target enzyme. Carrier-Ar—NH2 +HNO2 Carrier—Ar—N=N Carrier—Ar—N=N + NH2—enzyme Carrier—Ar—NH—enzyme

e) Alkylation method Carriers having alkyl groups can react with phenolic hydroxyl groups or sulfhydryl (mercapto) groups of the enzyme. Halogenated acetyl derivatives carriers can be used in this method. X—CH3 + HO—Enzyme X—CH2---Enzyme + H2O Carrier X—CH3 + HS—Enzyme X—CH2---Enzyme + H2S X—C=O CH2Cl + HO—Enzyme X—C=O CH2—Enzyme Halo-acetyl carrier

2- Ionic binding method The renewable of the carrier and recovery of enzyme are available from this method. Binding with the ionic carrier are affected by buffer pH, ionic strength of the buffer and temperature. Several materials can be used such as polysaccharide derivatives such as Diethyl amino ethyl- cellulose (DEAE-cellulose) which is a weak anion exchanger, and other synthetic resins.

3- Physical adsorption This method depends on the physical interaction between the carrier and the biocatalyst (enzyme or cells). These forces such as hydrogen bonding, Van der Waals force and hydrophopic interaction. But the enzyme binding is weaker than the previous methods. So, it can be affected by the environmental conditions such as temperature and solute concentration. One of the most used carriers is derivatized tannin.

4- Bio-specific binding method The enzyme is bind with a specific compound such a co-enzyme, inhibitor, effector, lectin or antibody. Each of these compounds has been bind with a carrier. Use of antibody or inhibitors may lead to enzyme inhibition. One of the most used lectins is "concanvalin A" which is obtained from Jack bean. This compound bind well with the enzyme that are glycoproteins.

B- Cross-linking methods This method utilizes a bi-or multifunctional compound to bind with the functional groups of the enzyme to form an insoluble cross-linked complex. The most common used cross-linking reagent is glutaraldehyd. Other cross-linking reagents such as toluene diisocyanate and hexamethylene diioscyanate can be used. The glutaraldehyde bind with two molecules of enzyme (amino group of the enzyme with the carbonyl group of the glutaraldehyde). Several molecules of glutaraldehyde can bind together to form oligoglutaraldehyde. This polymer can bind with several enzyme molecules (cross-linking) to form the insoluble immobilized enzyme.

C- Entrapment methods The entrapment method of immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. It is done in such a way as to retain protein while allowing penetration of substrate. It can be classified into: lattice, microcapsule, membrane, and reversed micelle types. The advantage of this method is that it can be applicable for different types of enzymes, cell organelles and living cells.

1- Lattice-Type: entrapment involves entrapping enzymes within the spaces of a cross-linked water-insoluble polymer. Some synthetic polymers such as polyarylamide, calcium algenate polyvinylalcohol and natural polymers such as starch and agar have been used to immobilize enzymes using this technique.

a) Polyacrylamide: This polymer which is used in electrophoresis analysis. The disadvantage of this method is the toxicity pf acryl amide. b) Alginate gel method: Alginate is extracted from seaweeds, such as giant kelp (Macrocystis pyrifera). The chemical constituents of alginate are random sequences of chains of β-D-mannuronic and α-L-guluronic acids attached with 1→4 linkages. Alginates are insoluble in water, but absorb water readily. Alginate molecules are cross-linked by calcium ions to form the in-soluble polymer.

Procedure: A suspension of mixture of enzyme solution and alginate is passed through a narrow tube (1mm diameter) into a solution of calcium chloride. The insoluble polymer containing the enzyme is formed in a form of small beads. - Because of this, calcium alginate beads can be formed in extremely mild conditions, which ensure that enzyme activity yields of over 80% can be routinely achieved. The advantage of this method is that the enzymes or microbial cells can be easily recovered by dissolving the gel in a sodium solution.

2- Microcapsule-Type: entrapping involves enclosing the enzymes within semi permeable polymer membranes. The preparation of enzyme micro capsules requires extremely well-controlled conditions and the procedures for micro capsulation of enzymes can be classified as:

Interfacial Polymerization Method:  In this procedure, enzymes are enclosed in semi permeable membranes of polymers as follows: 1- An aqueous mixture of the enzyme and hydrophilic monomer are emulsified in a water-immiscible organic solvent. 2- Then the same hydrophilic monomer solution is added to the organic solvent by stirring. Polymerization of the monomers then occurs at the interface between the aqueous and organic solvent phases in the emulsion. 3- The result is that the enzyme in the aqueous phase is enclosed in a membrane of polymer.

Liquid Drying:  Procedure of this method: 1- a polymer is dissolved in a water-immiscible organic solvent which has a boiling point lower than that of water. 2- An aqueous solution of enzyme is dispersed in the organic phase to form an emulsion. 3- This emulsion containing aqueous micro droplets is then dispersed in an aqueous phase containing protective colloidal substances such as gelatin, and surfactants, and a secondary emulsion is prepared. 4- The organic solvent is then removed by warming in vacuum. A polymer membrane is thus produced to give enzyme micro capsules.

3- Membranes Enzyme membranes can be prepared by attaching enzymes to membrane-type carriers. The form of an immobilized enzyme can be classified into four types: particles, membranes, tubes, and filters. Most immobilized enzymes are in particle form for ease of handling and ease of application.

Difference between the general immobilization methods