Chapter 3 Immobilized enzyme
Chapter 3 Immobilized enzyme
Chapter 3 Immobilized enzyme
Immobilized Enzyme
After the completion of this lesson you will be able to understand enzyme
immobilization techniques and the effect of mass-transfer resistance.
Outline
• Immobilization Techniques;
• 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.
reused since it can be easily separated from the reaction solution and
• 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.
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;
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
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
• Commonly employed water-insoluble supports for the covalent attachment of enzymes include:
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
• 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.
• Multifunctional reagents can be also used to introduce functional groups into water-
insoluble polymers, which then react covalently with water-soluble enzymes.
Physical Method
alumina, collagen,
clays, hydroxyapatite.
Advantages of adsorption techniques are as follows:
Disadvantages:
2. The state of immobilization is very sensitive to solution pH, ionic strength, and
temperature.
Inclusion in
• 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
• 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.
Disadvantage:
Enzyme may be deactivated during the gel formation.
• 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.
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;
3. Diffusion of the substrate from the surface of the particle to the active site of the enzyme in an inert
support.
• 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
(3.8)
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 material balance for the spherical shell with thickness dr,
Input – Output + Generation = Accumulation (3.9)
(10)
• 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
• 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 = 0 otherwise
• This is a good approximation when KM << Cs for Michaelis-Menten kinetics, in which case k0 = r max.
(3.13)
• The boundary conditions for the solution of the preceding equation are
(3.14)
• Eq.(3.13) becomes,
(3.15)
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)
(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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