Chapter 3 Immobilized enzyme

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Chapter 3

Immobilized Enzyme

 Prepared By Jemal A (MSc)


 Learning objectives:

After the completion of this lesson you will be able to understand enzyme
immobilization techniques and the effect of mass-transfer resistance.
Outline

• Introduction to enzyme immobilization;

• Immobilization Techniques;

• Effect of mass-transfer resistance.


 Self-test:

1) What is immobilized enzyme.

2) How enzymes can be immobilized?

3) Discuss the merit & demerits of immobilized enzyme.


 Introduction

Enzyme immobilization may be defined as a process of confining the enzyme molecules to a


solid support over which a substrate passed and converted to products.

• The process whereby the movement of enzymes, cells, organelles, etc. in space is completely
or severely restricted usually resulting in a water- insoluble form of the enzyme.

• It is one whose movement in space has been restricted either completely or to a small
limited region.

• Since most enzymes are globular protein, they are soluble in water. Therefore, it is very
difficult or impractical to separate the enzyme for reuse in a batch process.
Enzymes can be immobilized on the surface of or inside of an insoluble matrix either by
chemical or physical methods.

• They can be also immobilized in their soluble forms by retaining them with a semipermeable
membrane.

 A main advantage of immobilized enzyme is that it can be

reused since it can be easily separated from the reaction solution and

easily retained in a continuous-flow reactor.

• Furthermore, immobilized enzyme may show selectively altered chemical or physical properties
and it may simulate the realistic natural environment where the enzyme came from, the cell.
• As enzymes are biological catalysts that promote the rate of reactions but are not
themselves consumed in the reactions; they may be used repeatedly for as long as they
remain active.

• However, in most of the processes, enzymes are mixed in a solution with substrates and
cannot be economically recovered after reaction and are generally wasted.

• Thus, there is an incentive to use enzymes in an immobilized or insolubilized form so that


they may be retained in a biochemical reactor for further analysis.
 Self-test:

Why immobilize enzyme?


Protection of enzymes from degradation and deactivation;

Re-use of enzymes for many reaction recycles, lowering the total production cost of
enzyme mediated reactions;

Ability to stop the reaction rapidly by removing the enzyme from the reaction
solution;

Enhanced stability;

Easy separation of the enzyme from the product;

Product is not contaminated by the enzyme;

Allows development of a multi-enzyme reaction system;

Reduces effluent disposal problems.


 Self-test :

What are the enzyme (or cell) immobilization techniques?


Classification of Enzyme Immobilization Techniques
>
 Chemical Method

Covalent Attachment:

• The covalent attachment of enzyme molecules via nonessential amino acid residues (i.e., amino
acids minus water) to water-insoluble, functionalized supports are the most widely used
method for immobilizing enzymes.

• Functional groups of the nonessential amino acid residues that are suitable for the
immobilization process are

free 𝛼-, 𝛽-, or 𝛾-carboxyl groups,

𝛼- or 𝛽- amino groups, and

phenyl, hydroxyl, sulfhydryl, or

imidazole groups.
• An other variation of immobilization by covalent attachment is the copolymerization of the
enzyme with a reactive monomer (M) such as
𝐧𝐌 + 𝐄 → 𝐌𝐧 𝐄

where Mn E may have the following structure:

• Commonly employed water-insoluble supports for the covalent attachment of enzymes include:

Synthetic supports: acrylamide-based polymers, maleic anhydride-based polymers,


methacrylic acid-based polymers, styrene-based polymers, and polypeptides, and

Natural supports: agarose (Sepharose), cellulose, dextran (Sephadex), glass, and starch.
• Already active polymers such as maleic anhydride copolymers will be simply mixed with
enzymes to produce immobilized enzymes.

Normally, natural or synthetic polymers need to be activated by treating them with reagents
before adding the enzyme.

• The activation involves the chemical conversion of a functional group of the polymer.

• The enzyme's active site should not be involved in the attachment, in which case the enzyme
would lose its activity upon immobilization.
 Cross-linking Using Multifunctional Reagents

• It involves intermolecular cross-linking of enzyme molecules in the presence/absence of solid


support.

• The method produces a 3D cross-linked enzyme aggregate (water – insoluble) by means of a


multifunctional reagents that links covalently to the enzyme molecules.
• Water-insoluble enzymes can be prepared by using multifunctional agents that are all
bifunctional in nature and have low molecular weight, such as glutaraldehyde.

• There are several different methods for producing immobilized enzymes with multifunctional
reagents.

Fig. 3.1 Several different methods for producing immobilized enzymes with multifunctional reagents: (a) enzymes
are adsorbed on the surface active support followed by intermolecular cross-linking, (b) functional groups are
introduced on the support to react covalently with enzymes, and (c) enzymes are cross-linked intermolecularly.
• Enzymes can be reacted with multifunctional reagent alone so that they are cross-linked
intermolecularly by the reagent to form a water insoluble derivative.

• Another method is to adsorb enzymes on a water-insoluble, surface-active support


followed by intermolecular cross-linking with multifunctional reagents to strengthen the
attachment.

• Multifunctional reagents can be also used to introduce functional groups into water-
insoluble polymers, which then react covalently with water-soluble enzymes.
 Physical Method

Adsorption: It is the simplest way to immobilize enzymes.

• Enzymes can be adsorbed physically on a surface-active adsorbent by contacting an aqueous


solution of enzyme with an adsorbent.
Commonly employed adsorbents are:

 alumina,  collagen,

 anion- and cation-exchange resins,  colloid-ion,

 calcium carbonate,  conditioned metal,

 carbon,  glass plates,

 celluloses,  diatomaceous earth, and

 clays,  hydroxyapatite.
 Advantages of adsorption techniques are as follows:

1. The procedure of immobilization is simple.

2. It is possible to separate and purify the enzymes while being immobilized.

3. The enzymes are not usually deactivated by adsorption.

4. The adsorption is a reversible process.

 Disadvantages:

1. The bonding strength is weak.

2. The state of immobilization is very sensitive to solution pH, ionic strength, and
temperature.

3. The amount of enzymes loaded on a unit amount of support is usually low.


 Entrapment:

• Enzymes can be entrapped within cross-linked polymers by forming a highly cross-linked


network of polymer in the presence of an enzyme.
• In entrapment, the enzymes or cells are not directly attached to the support surface, but
simply trapped inside the polymer matrix.

• Enzymes are held or entrapped within the suitable gels or fibers.

• It is done in such a way as to retain protein while allowing penetration of substrate.

Inclusion in

gels: polyacrylamide gels, polyvinyl alcohol gels.

fibers: cellulose and polyacrylamide gels.

microcapsules: polyamide, polybasic-acid chloride monomers.


 Entrapment can be classified into lattice and microcapsule types.
Lattice – type entrapment:

• It involves entrapping enzymes within the interstitial spaces of a cross-linked water-insoluble


polymer.

• Some synthetic polymer such as polyacrylamide, polyvinyl alcohol, … etc. and natural polymer
(starch) have been used to immobilize enzymes.
Microcapsule –Type Entrapment/Encapsulation/Membrane Confinement

• Enzymes can be immobilized within semipermeable membrane microcapsules.

This can be done by the interfacial polymerization technique.

• Organic solvent containing one component of copolymer with surfactant is agitated in a


vessel and aqueous enzyme solution is introduced.

• The polymer membrane is formed at the liquid-liquid interface while the aqueous phase is
dispersed as small droplets.

E.g. polyamide nylon system, in which 1, 6-diaminohexane is the water soluble diamine
and 1,10-decanoyl chloride is the organic-soluble diacid halide.
• The organic solvent for this system is a chloroform cyclohexane mixture (1:4 v/v) containing
usually 1 percent (v /v) Span85 surfactant.

• This technique provides an extremely large surface area.

E.g. semipermeable colloid-ion or nylon membranes in the shape of spheres.


 Major advantage of entrapment:
 No chemical modification of the enzyme, i.e., the intrinsic properties of an enzyme are not altered.

 Relatively stable forms.

 Easy to handle and re-usage.

 Disadvantage:
 Enzyme may be deactivated during the gel formation.

 Enzyme leakage from the pore is also a problem.

• The most commonly employed cross linked polymer is the polyacrylamide gel system.

• This has been used to immobilize alcohol dehydrogenase, glucose oxidase, amino acid oxidase, hexokinase,
glucose isomerase, urease, and many other enzymes.
Effect Of Mass-transfer Resistance
Effect Of Mass-transfer Resistance

 The immobilization of enzymes may introduce a new problem which is absent in free soluble
enzymes.

• It is the mass-transfer resistance due to the

large particle size of immobilized enzyme or

inclusion of enzymes in polymeric matrix.

 If we follow the hypothetical path of a substrate from the liquid to the reaction site in an
immobilized enzyme, it can be divided into several steps.
 Steps:

1. Transfer of substrate from the bulk liquid to a relatively unmixed liquid layer surrounding the
immobilized enzyme;

2. Diffusion of substrate through the relatively unmixed liquid layer; and

3. Diffusion of the substrate from the surface of the particle to the active site of the enzyme in an inert
support.

Steps 1 and 2 are the external mass-transfer resistance.

Step 3 is the intra-particle mass-transfer resistance.


 External Mass-Transfer Resistance

• If an enzyme is immobilized on the surface of an insoluble particle, the path is only composed of the first
and second steps, external mass-transfer resistance.

• The rate of mass transfer is proportional to the driving force, the concentration difference, as

𝑁𝑆 = 𝑘𝑆 𝐴 𝐶𝑆𝑏 − 𝐶𝑆 (3.1)

where 𝐶𝑠𝑏 and 𝐶𝑠 are substrate concentration in the bulk of the solution and at the immobilized enzyme
surface, respectively.

The term 𝑘𝑠 is the mass-transfer coefficient (length/time) and A is the surface area of one immobilized
enzyme particle.
• During the enzymatic reaction of an immobilized enzyme, the rate of substrate transfer is equal to that
of substrate consumption.

• Therefore, if the enzyme reaction can be described by the Michaelis - Menten equation,

𝑟𝑚𝑎𝑥 𝐶𝑆
𝑟𝑃 = 𝑘𝑆 𝑎 𝐶𝑆𝑏 − 𝐶𝑆 = (3.2)
𝐾𝑀 + 𝐶𝑆
where 𝒂 is the total surface area per unit volume of reaction solution.

• This equation shows the relationship between the substrate concentration in the bulk of the solution
and that at the surface of an immobilized enzyme.
 Eq. (3.2) can be expressed as in dimensionless form as:

𝟏 − 𝒙𝑺 𝜷𝒙𝑺
= (3.3)
𝑵𝑫𝒂 𝟏 + 𝜷𝒙𝑺

where

; ; (3.4)

 𝑵𝑫𝒂 is known as Damkohler number, which is the ratio of the maximum reaction rate over the maximum mass-
transfer rate.
• Depending upon the magnitude of 𝑁𝐷𝑎 , Eq. (3.2) can be simplified as follows:

If 𝑵𝑫𝒂 ≪ 𝟏, the mass-transfer rate is much greater than the reaction rate and the overall reaction is
controlled by the enzyme reaction,

(3.5)

If 𝑵𝑫𝒂 ≫ 𝟏, the reaction rate is much greater than the mass transfer rate and the overall rate of reaction is
controlled by the rate of mass transfer i.e. a first-order reaction,

(3.6)
• To measure the extent which the reaction rate is lowered because of resistance to mass transfer, we can
define the effectiveness factor of an immobilized enzyme, , as

𝜼 = rate ifactual reaction rate


not slowed by diffusion
(3.7)

(3.8)

where the effectiveness factor is a function of 𝐱 𝐬 and 𝜷.

 If 𝑥𝑠 = 1, 𝐶𝑠𝑏 = 𝐶𝑠 .
𝜼 = 1, which indicates that there is no mass-transfer limitation. or
If 𝒙𝒔 approaches zero, 𝜼 also approaches zero, which is the case when the rate of mass transfer is very
slow compared to the reaction rate.
 Internal Mass-Transfer Resistance
• If enzymes are immobilized by copolymerization or microencapsulation, the intra-particle mass-transfer
resistance can affect the rate of enzyme reaction.

• In order to derive an equation that shows how the mass-transfer resistance affects the effectiveness of an
immobilized enzyme, let's make a series of assumptions as follows:
1) The reaction occurs at every position within the immobilized enzyme, and the kinetics of the
reaction are of the same form as observed for free enzyme.
2) Mass transfer through the immobilized enzyme occurs via molecular diffusion.
3) There is no mass-transfer limitation at the outside surface of the immobilized enzyme.
4) The immobilized enzyme is spherical.

• The model developed by these assumptions is known as the distributed model.


First we derive a differential equation which describes the relationship between the substrate concentration
and the radial distance in an immobilized enzyme.

• The material balance for the spherical shell with thickness dr,
Input – Output + Generation = Accumulation (3.9)

(10)

where Ds is diffusivity of the substrate in an immobilization matrix.


For a steady-state condition, dCs/dt = 0.

• After opening up the brackets and simplifying by eliminating all terms containing dr2 or dr3, we obtain the
second order differential equation:

𝒅𝟐 𝑪𝑺 𝟐 𝒅𝑪𝑺 (3.11)
𝑫𝑺 𝟐
+ + 𝒓𝑺 = 𝟎
𝒅𝒓 𝒓 𝒅𝒓

Eq. (3.11) can be solved by substituting a suitable expression for 𝑟𝑆 .

• Let's solve the equation first for the simple cases or zero-order and first-order reactions, and for the
Michaelis-Menten equation.
 Zero-order Kinetics:

• Let's assume that the rate of substrate consumption is constant (zero order) with respect to substrate
concentration as

rs = -k0 if Cs > 0 (3.12)

rs = 0 otherwise

• This is a good approximation when KM << Cs for Michaelis-Menten kinetics, in which case k0 = r max.

• By substituting Eq. (3.12) into Eq. (3.11), we obtain:

(3.13)
• The boundary conditions for the solution of the preceding equation are
(3.14)

• Eq.(3.13) becomes,

(3.15)

Integrating Eq. (3.15) twice with respect to r, we obtain


(3.16)

Therefore,
(3.17)
• Applying the boundary conditions (Eq. (3.14) on Eq. (3.17) yields

(3.18)

(3.19)
• Therefore, the solution of Eq. (3.13) is

(3.20)

Eq. (3.20) is only valid when Cs > 0


• The critical radius, below which Cs = 0, can be obtained by solving

(3.21)
• Therefore, the effectiveness factor, the ratio of the actual reaction rate to the rate if not slowed down
by diffusion, is
 Further Reading on Internal mass - transfer resistance:

Zero-order kinetics

First-order kinetics

Effective Diffusivities
End Of Chapter 3

Thank You!

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