Immobilised Enzymes 13573o

IMMOBILIZED ENZYMES AND THEIR APPLICATIONS Swathin S Naik Ist MSc Ist Semester Postgraduate Department of Biotechnology Alvas College, Moodabidri

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction. Definition. Purpose and Advantages of Immobilization. Methods of Immobilization. Properties of Carrier for Immobilization. Properties of Immobilized Enzymes. Applications of Immobilized Enzymes. Summary. References.

Introduction • Enzymes are normally tightly packed in cellular organelles or in enzyme cascades. • This enables catalytic process to take place precisely when and where they are needed. • Artificial application of such compartmentation or packing go back to 1950s.

Introdution continued…… • Since 1950s numerous efforts have been devoted to development of soluble immobilized enzymes for various applications. • Immobilized enzymes were first prepared intentionally by inclusion in polymeric matrix or binding onto carrier molecules.

• For Cross-linking of enzymes, either by protein crosslinking or by addition of inert materials, considerable effort was put.

Definition “An Immobilized enzyme is one, whose movement in space has been restricted; either completely or to small limited region by attachment to a solid structure.” OR “An Immobilized enzyme is one, which has been attached to or enclosed by an insoluble medium or one where the enzyme molecules have been cross-linked to each other, without loss of catalytic activity.”

Purpose and Advantages of Immobilization of Enzymes • Immobilization is natural phenomenon existing in environment. • Microorganisms in nature are distributed and often exits in biofilms • This natural phenomenon encouraged humans to utilize for his benefit. • Immobilized Enzymes have several advantages over soluble enzymes.

Biofilm

Purpose and Advantages of Immobilization of Enzymes continued…… • The Immobilized Enzymes can be easily removed from reaction making it easy to reuse it in the reaction • Since Immobilized enzymes can be removed from reaction mixture. The reaction mixture (solvent and Reaction product) does not get contaminated; thus lowering the cost thus making reaction product more economical. • Immobilized Enzyme have Greater thermal and operational stability. • Ability to stop the reaction by removing the enzyme from reaction solution.

Method of Enzyme immobilization • Adsorption – First among the immobilization methods. – Immobilization by adsorption is the simplest method and involves reversible surface interactions between enzyme and material – Procedure consists of • mixing together the biological components and a , under suitable conditions of pH and ionic strength, • for a period of incubation, • followed by collection of the immobilized material and • extensive washing to remove the unbound biological components.

Adsorption based Enzyme Immobilization

• Advantages of enzymes immobilized using the adsorption technique: – Reversibility: which enables not only the purification of proteins but also the reuse of the carriers. – Simplicity: which enables enzyme immobilization under mild conditions. – Possible high retention of activity because there is no chemical modification. – Cheap and quick method – No chemical changes to the or enzyme occurs.

• Disadvantages of enzymes immobilized using the adsorption technique – Enzymes tend to leak from the carriers, due to the relatively weak interaction between the enzyme and the carrier, which can be destroyed by desorption forces such as high ionic strength, pH, etc, –Contamination of product. –Non-specific binding. –Overloading on the . –Steric hindrance by the .

• Covalent Binding – involves formation of a covalent bond between the enzyme and material. – provide the strongest linkages between enzyme and carrier. – leakage of enzyme from the carrier used is often minimized. – bond is normally formed between functional groups present on the surface of the and functional groups belonging to amino acid residues on the surface of the enzyme.

Enzyme immobilization by covalent binding

• Advantages of enzymes immobilized using the covalent technique – No leakage of enzyme. – The enzyme can be easily in to substrate due to the localization of enzyme on materials. – Increase of the thermal stability.

• Disadvantages of enzymes immobilized using the covalent technique – The cost is quite high as the good s are very expensive. – Loss of enzyme activity.

• Entrapment – Entrapment differs from adsorption and covalent binding in that enzyme molecules are free in solution, but restricted in movement by the lattice structure of a gel. – The porosity of gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme or cells, yet at the same time allows free movement of substrate and product. – also acts as a barrier and can be advantageous as it protects the immobilized enzyme from microbial contamination by harmful cells, proteins, and enzymes in the microenvironment. – Entrapment can be achieved by mixing an enzyme with a polyionic polymer material, such as carrageenan, and by cross-linking the polymer with multivalent cations.

Enzyme immobilization by Entrapment

• Advantages of enzymes immobilized using the entrapment technique –Enzyme loading is very high.

• Disadvantages of enzymes immobilized using the entrapment technique –Enzyme leakage from the . –Diffusion of the substrate to the enzyme and of the product away from the enzyme.

• Encapsulation – Encapsulation of enzymes can be achieved by enveloping the biological components within various forms of semi-permeable membranes. – similar to entrapment in that the enzyme is free in solution, but restricted in space. – Large proteins or enzymes can not out or into the capsule, but small substrates and products can freely across the semi-permeable membrane. – Many materials have been used to construct microcapsules varying from 10-100µm in diameter.

Enzyme immobilization by Encapsulation

• Advantages of enzymes immobilized using the en-capsulation technique: – The enzymes could be encapsulated inside the cell. – Possibility of co-immobilization. Where cells and/or enzymes may be immobilized in any desired combination to suit particular applications.

• Disadvantages of enzymes immobilized using the entrapment technique: – The problems associated with diffusion are acute and may result in rupture of the membrane if products from a reaction accumulate rapidly.

• Crosslinking – This type of immobilization is -free as and involves ing enzyme molecules to each other to form a large, three-dimensional complex structure. – Chemical methods of cross-linking normally involve covalent bond formation between the enzymes by means of a bi- or multifunctional reagent, such as glutaraldehyde, dicarboxylic acid or toluene di-isocyanate. – Flocculating agents, such as polyamines, polyethyleneimine, polystyrene sulfonates, and various phosphates, have been used extensively to cross-link cells using physical bonds. – Crosslinking is rarely used as the only means of immobilization, because poor mechanical properties of the aggregates are severe limitations – Crosslinking is most often used to enhance the other methods of immobilization.

Enzyme immobilization by Cross linking

• Advantages of enzymes immobilized using the crosslinking technique – The immobilization is -free. – Cross-linking between the same enzyme molecules stabilises the enzymes by increasing the rigidity of the structure

• Disdvantages of enzymes immobilized using the crosslinking technique – Harshness of reagents of cross-linking is a limiting factor. – The enzyme may partially lose activity or become totally inactivated in case the crosslinking reagent reacted across the active site.

Properties of Carrier for Immobilization • Availability from a reliable commercial sources. • Abundance of easily available functional group. • Good mechanical and chemical stability. • Carrier material has good capacity for the enzyme. • Carrier material should be friendly.

Properties of Immobilized Enzymes • The properties of an immobilized enzymes may be different from those of the enzyme in free solution. • Depends on method of immobilization and nature of carrier. • Reduction in specific activity may occur. Denaturation may take place if chemical process is involved. • The new micro-environment could influence the enzyme in various ways – like the characteristic of enzyme might be altered if active site undergo conformational changes due to the chemical or physical interaction between enzyme and carrier.

Properties of Immobilized Enzymes continued…..

• Stability of enzymes on heating and storing may increase or decrease or remain same depending on how the new micro-environment affects its tendency to denature. • pH optimum can change by as much as by 2 pH units owing to effect of new micro-environment. • The apparent Km may be effected similarly by the electrostatic field of the carrier and diffusion factors.

Applications of Immobilized Enzymes • Biomedical Application – Used for diagnostic treatment of diseases.

– The inborn metabolic deficiency can be overcome by encapsulated enzymes instead of waste metabolites. – Enzyme encapsulation through electroporation in the biomedical field and its is reversible and enzyme can be regenerated.

Applications of Immobilized Enzymes continued…… – Examples of potential medical use of immobilized enzymes. • • • • • • • •

Asperginase for Leukemia. Urate oxidase for Hyperglacemia. Urease for artificial kidney, uraemic disorder. Glucose oxidase for artificial lungs. Carbomate hydratase and catalase for artificial lungs Glucomylase for glycose storage disease. Xanthine oxidase for Lysch-Nyhan disease. Phenylalanine ammonia lyase for phenylketouria.

Applications of Immobilized Enzymes continued……

• Food Industry – In food industry, the purified enzymes are used but during the purification, the enzymes will denature. – immobilization technique makes the enzymes stable. – The immobilized enzymes are used for the production of syrups.

– Immobilized beta-galactosidase used for lactose hydrolysis in whey for the production of bakers yeast. – The enzyme galactosidase was linked to resin (food grade) through cross linking. This method was used for the various purposes such as confectionaries and icecreams.

Applications of Immobilized Enzymes in Food Industry

Applications of Immobilized Enzymes continued……

• Biodiesel Production – Biodiesel is produced through triglycerides with esterification of alcohol in the presence of the catalyst.

– The production of catalyst is a drawback of high energy requirements, recovery of glycerol and side reaction which may affect the pollution. – Biological production of Biodiesel with lipases nowadays has a great consideration.

Applications of Immobilized Enzymes continued……

• Biodiesel Production continued…… –Lipase catalyses the reaction with less energy requirements and mild conditions required. –Production of lipase is of high cost, hence the immobilization of lipase which results in repeated use and stability. –In the biological production of biodiesel the methanol inactivates the lipase, hence the immobilization method is an advantage for the biodiesel production.

Applications of Immobilized Enzymes continued……

• Textile Industry – The enzymes derived from microbial origin are of great interest in textile industry.

– Textile industries now turned to enzyme process instead of using harsh chemical which affects the pollution and cause damage to the fabrics. – The enzymes such as cellulase, amylase, liccase, pectinase, cutinase and are used for various textile applications such ascouring, biopolishing, desizing, denim finishing, treating wool.

Applications of Immobilized Enzymes in textile industry

Applications of Immobilized Enzymes continued……

• Textile Industry continued…… – Among these enzymes cellulase has been widely used. – The processing of fabrics with enzymes requires high temperatures and increased pH, the free enzymes does not able to withstand the extreme conditions. – Immobilized Enzyme able to withstand at extreme and able to maintains its activity for more than 5-6 cycles.

Summary • Enzymes can be immobilized by binding to an insoluble carrier, by entrapping in gel or membrane or by cross-linking.

• Immobilization effect stability, pH optimum, apparent Km, apparent Vmax of enzyme, depending on method of immobilization and nature of carrier. • Immobilized enzymes can be removed from a reaction mixture; thereby stopping the reaction and making enzyme available for reuse.

Summary continued…… • Immobilized enzymes have technological advances and is useful tool to meet cost targets by the repeated use of enzymes. • Immobilized enzymes make laborious separation steps unnecessary by being easily separated from reaction mixture.

• Other benefit include stability against harsh reaction condition.

References 1. Trevor Palmer, Philip Bonner (2008) Enzymes Biochemistry, Biotechnology, Clinical Chemistry, 2nd Edition New Delhi: EastWest Press Private Limited. 2. Magdy M Elnshar (2010) Immobilized Molecules using Biomaterials and Nanobiotechnology: Review article, Journal of Biomaterials and nanotechnology. 3. Dr. Linqic Cao (2005) Carrier bound Immobilized enzymes principles application and designs, wiley-VCH verlag GmbH and Co 4. Hemalatha V et. al. (2016) Methods, Applications of Immobilized enzymes-A Mini Review, International Journal of Engineering sciences and research technology. 5. Wilhelm Tischer, Volker Kasche (1999) Immobilized Enzymes: crystals or carriers?: Review Elsevier Science, volume 7