Immobilization of enzymes

Immobilization of enzymes

Content

Ø Definition

Ø Advantages

Ø Support or matrix materials

Ø Methods of immobilization

Ø Application of immobilized enzymes

Session Objectives

At the end of the session, student will be able to

Ø Discuss advantages of immobilization

Ø Discuss the support or matrix materials used

Ø Explain the various methods of immobilization

Ø Discuss the application of immobilized techniques

Immobilization

Ø Enzymes – biocatalyst – promote the rate of reactions – but not themselves consumed in reactions

Ø They may be used repeatedly – as long as they remain active

Ø In most processes – enzymes are mixed in a solution with substrate – cannot be economically recovered after the reaction – generally get wasted

Ø Hence enzymes are used – immobilized or insolubilised form – so that they may be retained in a biochemical reactor for further catalysis

Ø Done by enzyme immobilization

Ø Immobilization - imprisonment of cell or enzyme in a distinct support or matrix

Ø The support or matrix on which the enzymes are immobilized allows the exchange of medium containing substrate

Ø The practice of immobilization of cells is very old –

Ø First immobilized enzyme -Nelson & Griffin - adsorption of invertase on charcoal - 1916

Advantages:

Ø Better stability

Ø Increased functional efficiency of enzymes - Better efficiency

Ø Enzyme can be recovered and can be reused repeatedly

Ø Enhanced reproducibility of the process

Ø Less chance of contamination in products

Ø Improved process control - ability to stop the reaction rapidly by removing the enzyme from the reaction solution

Supports or Matrix used in immobilization technology

• The matrix or support immobilizes the enzyme by holding it permanently or temporarily for a brief period of time

• Wide variety of matrixes or carriers or supports available for immobilization

• Matrix - cheap and easily available

• Their reaction with the components of the medium or with the enzyme should be minimum as possible

Supports or Matrix

• The matrixes or supports for immobilization of enzymes or whole cells are grouped into three major categories

(1) Natural polymers

(2) Synthetic polymers

(3) Inorganic materials

Natural Polymers

(a) Alginate:

• Derived from the cell wall of some algae

• Calcium or magnesium alginate - commonly used matrix

• They are inert and have good water holding capacity.

(b) Chitosan and chitin:

• Polysaccharides - cell wall of fungi - exoskeleton of Arthropods

• The various functional groups in enzymes can bind to the – OH group of chitin and can form covalent bonds

(c) Collagen:

• Main structural protein – animal connective tissue

• Proteinaceous support - good porosity and water holding capacity

• The side chains of the amino acids in the collagen + of enzyme can form covalent bonds to permanently hold the enzyme to the support

(d) Carrageenan:

• Sulfated polysaccharide - some red algae

• Good gelling properties together with its high protein holding capacity makes it good support for immobilizing enzymes

(e) Cellulose:

• Most abundant polymer of nature

• Cheapest support available as carrier of enzymes

• The hydroxyl group - monomer units (glucose) can form covalent bonds with that of the amino acids of enzyme

(f) Starch:

• A natural polymer of amylose and amylo-pectin

• It has good water holding capacity

(g) Pectin:

• Structural polysaccharide - plants - primary cell wall - they also acts as the inter-cellular cementing material in plant tissues

• Gelling agent with good water holding capacity

Synthetic polymers

• Ion exchange resins or polymers - insoluble supports with porous surface

• Their porous surface can trap and hold the enzymes or whole cells

• Example:

Diethylaminoethyl cellulose (DEAE cellulose)

Polyvinyl chloride (PVC)

UV activated Polyethylene glycol (PEG)

Inorganic Materials

(a) Zeolites:

They are microporous, alumino silicate minerals with good adsorbing properties and extensively used for immobilizing enzymes and whole cells

(b) Ceramics:

They are nonmetallic solids consisting of metal and nonmetal atoms held in ionic and covalent bonds

(c) Diatomaceous earth:

• Siliceous sedimentary rocks - formed - fossilized accumulations of the cell wall of diatoms

• Celite - trade name - diatomaceous earth

• It is a good adsorbent and are resistant to high pH and temperature

(d) Silica

(e) Glass

(f) Activated carbon

(g)Charcoal

Types of Immobilization

Based on support or matrix and types of bonds involved

Adsorption method

Covalent binding method

Cross linking method

Entrapment method

Microencapsulation

Adsorption Method of Immobilization

• Oldest and simplest method of enzyme immobilization

• Nelson & Griffin - charcoal to adsorb invertase for the first time in 1916

• Enzyme is adsorbed to external surface of the support

• No permanent bond formation between carrier and the enzyme in adsorption method

The weak bonds (low energy bonds) involved are mainly:

(a) Ionic interaction

(b) Hydrogen bonds

(c) Van der Waal forces

• For significant surface bonding the carrier particle size must be small (500 Å to 1 mm diameter)

• The greatest advantage of adsorption method is that there will not be “pore diffusion limitations” since enzymes are immobilized externally on the support or the carrier

Adsorbents :Alumina, Amberlite CG, Bentonite, CMC, Calcium phosphate , Porous carbon, Silica gel, clay, glass, collagen, cellulose, titania etc

Adsorbent pack

Water-jacketed column

Preconditioning solution

Enzyme solution circulated at desired temp for several hours

Enzyme solution drained the column wash with water

Immobilized enzyme column

Advantages:

Ø Simple process, economical (cheapest)

Ø Limited loss of activity

Ø Immobilized enzyme and matrix can be recycled, regenerated

Disadvantages:

Ø Desorption of enzymes from carrier

Ø Enzyme is exposed and can be prone to proteolytic and microbial attack

Covalent binding Method of Immobilization

• Formation of covalent between – chemical groups of carrier and enzymes

Chemical groups – Carrier

• Amino groups

• Imino groups

• Hydroxyl groups

• Carboxyl groups

• Thiol groups

• Imidazole groups

Chemical groups – enzyme

• Alpha carboxyl group at ‘C’ terminal of enzyme

• Alpha amino group at ‘N’ terminal of enzyme

• β and γ carboxyl groups of Aspartate and Glutamate

• Phenol ring of Tyrosine

• Thiol group of Cysteine

• Hydroxyl groups of Serine and Threonine

• Imidazole group of Histidine

• Indole ring of Tryptophan

• Carriers or supports commonly used for covalent bonding are:

Carbohydrates: Eg. Cellulose, cellulose, Agarose

Synthetic agents: Eg. Polyacrylamide

Protein carriers:Collagen, Gelatin

Amino group bearing carriers: Eg. Amino benzyl cellulose

Inorganic carriers: Porous glass, silica

Cyanogen bromide (CNBr) - agarose and CNBr Sepharose

Advantages:

Ø No enzyme leakage or desorption problem

Ø Higher stability

Ø A variety of support with different functional groups available

Ø Strong linkage

Disadvantages:

Ø Enzyme may be partially or wholly inactivated by active site modification - overcome through immobilization in the presence of a competitive inhibitor

Ø Chemical modification of enzyme – loss of functional conformation

Cross linking or Copolymerization method of Immobilization

Enzymes

â Cross link

Bi or multifunctional reagent

Eg: Glutaraldehyde, diazobenzidine, N, N hexamethyl di iso cyanate, dimethyl derivative (adipimate, suberimate) etc

Basic approaches

Cross- linking of enzyme with glutaraldehyde

Adsorption of enzyme onto surface followed by cross-linking

Eg. Cross linking trypsin – adsorbed to colloidal silica particles

Disasdv:

• Not suitable for macromolecules

• Hard to regenerate the immobilized enzyme for reuse

• Cross linking may denature or structurally modify the enzyme – loss of catalytic activity

Entrapment method of Immobilization

Enzymes

â Physically entrapped

Porous matrix

(Water soluble polymers)

(Calcium alginate, PAA, starch, Agar, Cellulose triacetate)

(Covalent or non-covalent bond)

Pore size is adjusted to prevent loss of enzyme - Done – adjusting the concentration of polymer used

Advantages:

Ø Enzyme activity is not damaged

Ø Fast method of immobilization

Ø Cheap (low cost matrixes available)

Ø Easy to practice at small scale

Ø Mild conditions are required

Ø Less chance of conformational changes in enzyme

Ø Minimize enzyme leaching

Disadvantages:

Ø Loss of enzyme activity due to free radicals produced during polymerization

Ø Chance of microbial contamination

Applications of Immobilized Enzymes

Ø Various immobilized enzymes are frequently in use in several large industrial processes

Ø Some of the immobilized enzymes are utilized in drug manufacture and drug analysis

Drug Manufacture:

Ø Production of antibiotics

Ø Production of amino acids

Ø Other medicinal compounds

i. Production of antibiotics

Antibiotics:

Ø Production of semi-synthetic 6 – amino penicillinic acid (6-APA) is an important intermediate for the production of semi-synthetic penicillins like ampicillin, amoxycilin

Ø This intermediate is derived from penicillin G or penicillin V by acylation with enzymes penicillin amidase

Ø Immobilized penicillin amidase is now being used in place of native or soluble enzyme

Immobilized enzyme (source)	Method of immobilization	Precursor	Product
Penicillin amidase (E.coli)	Trapping into cellulose triacetate fibres or covalent bonding to CN Br-activated sephadex	Penicillin G	6-APA
Penicillin amidase (E.coli)	Absorption on Bentonite or covalent bonding to amberite XDA-7 with glutaraldeyde	Penicillin V	6-APA
Penicillin amidase (E.coli)	Trapping into cellulose triacetate fibres	6-APA	Ampicillin
Penicillin amidase (E.coli)	Trapping into cellulose triacetate fibres	D-phenyl glycine methyl ester	Amoxycillin

Other antibiotics:

Antibiotics	Immobilized enzyme or cells
Cephalosporin derivates	Cephalosporin amidase
Bacitracin	Cells of Bacillus species
Tylosin and nikkomycin	Cells of Streptomyces species

iii. Production of amino acids

Ø Amino acids are in great demand for their nutritional and medical applications

Ø Chemical synthesis produces only racemic mixtures.

Ø d-isomer of the racemic mixture generally devoid of nutritional value

Thus, it is desirable to obtain l-amino acids

Ø Immobilized enzymes are helpful to get these physiologically active isomers

Ø For example, immobilized amino acylase can be used for the production of l-amino acids

iv. Other medicinal compounds

Ø A few notable examples are given below, which are produced by immobilized enzymes

Compounds	Immobilized enzyme	Used in
Dopamine	B-Tyrosinase	Parkinsonism
Vitamin. B₁₂	Propioni bacterium species	As. Vitamin
High fructose corn syrup	Glucose isomerase	Commercial application
Proinsulin	Bacillus substilis cells	Diabetes

Summary

Ø Enzymes are biocatalysts, high m.w, Proteinaceous and water soluble compounds

Ø Activity is affected by temp, pH and heavy metals, specific in their action

Ø Classification according to IUMAB and site of action

Ø Enzymes have medicinal, food and industrial applications

Ø Main source is plant, animal and micro organisms

Ø Isolation involves extraction, preparation of crude enzyme, precipitation and purification

Ø Imprisonment of enzyme or cell in / on a distinct phase that allows exchange with but it is separated from bulk phase

Ø Stable, economical and reusable

Ø Adsorption method

Ø Imprisonment of enzyme or cell in / on a distinct phase that allows exchange with but it is separated from bulk phase

Ø Stable, economical and reusable

Ø Covalent binding, cross linking, entrapment and microencapsulation method

Ø Immobilized enzymes are utilized in drug manufacture and drug analysis

Ø Production of antibiotics, steroids, amino acids and other medicinal compounds

Immobilization of enzymes

Prakash surya

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