Biochemical Engineering

Mohit Garg
Department of Chemical Engineering
BITS Pilani B.I.T.S-Pilani, Pilani Campus
Pilani Campus
Contact: mohit.garg@pilani.bits-pilani.ac.in
 Effect of pH

●
Certain enzymes have ionic groups on their active sites, and these ionic groups must be in a suitable
form (acid or base) to function.
●
Variations in the pH of the medium result in changes in the ionic form of the active site and changes in
the activity of the enzyme and hence the reaction rate.

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Effect of pH

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Effect of pH

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 Temperature effects
●
The rate of enzyme-catalyzed reactions increases with temperature up to a certain limit.
●
Above a certain temperature, enzyme activity decreases with temperature because of
enzyme denaturation.
●
The variation of reaction rate with temperature and the presence of an optimal
temperature.

The ascending part of Fig. is known as temperature activation.

The rate varies according to the Arrhenius equation in this region.

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Temperature effects
The descending part of Fig. is known as temperature inactivation or thermal
denaturation. The kinetics of thermal denaturation can be expressed as

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 Insoluble Substrates
●
Enzymes are often used to attack large, insoluble substrates such as wood chips (in bio-
pulping for paper manufacture) or cellulosic residues from agriculture (e.g., cornstalks).
In these cases access to the reaction site on these biopolymers by enzymes is often limited
by enzyme diffusion.
●
The number of potential reactive sites exceeds the number of enzyme molecules.
●
This situation is opposite that of the typical situation with soluble substrates, where access to the enzyme’s active
site limits reaction.
●
If we consider initial reaction rates and if the reaction is first order with respect to the concentration of enzyme
bound to substrate (i.e., [ES]), then we can derive a rate expression:

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Enzyme Immobilization


Immobilization is defined as the process of confining cell or enzyme in a support or

matrix.


Movement in the space is restricted.


The support or matrix on which the enzymes are immobilized allows the exchange

of medium containing substrate or inhibitor molecules.


Immobilization enhances the stability of the enzyme.

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Advantages of immobilized enzyme


Stability of the enzyme increases.


Can be reused again and again.


Products are enzyme free.


Can be used for continuous product formation.


Suitable for industrial and medical use.


Minimized effluent disposal problem.

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Applications of immobilized enzyme system

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Classification of solid matrices

1. Organic
a. Natural polymers:
Polysaccharides: cellulose, dextrans, agar, agarose, chitin, alginate
Protiens: collage, albumin
Carbon
b. Synthetic polymers
Polystyrene, PMMA, Polyacrylamide and Vinyl and allyl – polymers

2. Inorganic
a. Natural minerals: bentonite, silica
b. Processed minerals: glass, metals, porous metal oxides

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Carrier morphology and configuration


It can be porous and non-porous.


The porous carrier can have controlled pore, broad pore size distribution or can form a gel structure.


The main advantage of a non-porous carrier is that the diffusional effects are minimal and large

substrate in solution can be reacted with minor difficulties.


The disadvantage of non-porous carrier is the low surface area.


In porous carrier protects the enzyme from turbulent external environment due to internal surface bonding.


The disadvantage is that the large substrate molecules cannot penetrate the smaller pores.


Broad pore distribution can reduce the substrate-enzyme interaction

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Desirable characteristics of solid matrix

1. Chemical durability

2. High surface area for enzyme attachment

3. Mechanical strength and dimensional stability to avoid compaction and to protect enzyme structure.

4. Microbial resistance to avoid the destruction of both enzyme and carrier.

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Methods of Immobilization

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 Entrapment


Entrapment is the physical enclosure of enzymes in a small space.

Matrix entrapment and membrane entrapment, including microencapsulation, are the two major methods of
entrapment.

Matrices used for enzyme immobilization are usually polymeric materials such as Ca-alginate, agar, k-
carrageenin, polyacrylamide, and collagen.

Solid matrices such as activated carbon, porous ceramic, and diatomaceous earth can also be used.

The matrix can be a particle, a membrane, or a fiber.

Membrane entrapment: Membranes of nylon, cellulose, polysulfone, and polyacrylate are commonly used.

Microencapsulation: In this technique, microscopic hollow spheres are formed. The spheres contain the
enzyme solution, while the sphere is enclosed within a porous membrane.

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Disadvantages of Enzyme entrapment

Enzyme entrapment may have its inherent problems:


Enzyme leakage into solution: Enzyme leakage can be overcome by reducing the MW cutoff of

membranes or the pore size of solid matrices.


Significant diffusional limitations: Diffusion limitations can be eliminated by reducing the particle size of ma-

trices and/or capsules.


Reduced enzyme activity and stability, and


Lack of control of microenvironmental conditions.

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Surface immobilization

The two major types of immobilization of enzymes on the surfaces of support materials are adsorption
and covalent binding.

Adsorption is the attachment of enzymes on the surfaces of support particles by weak physical forces,
such as van der Waals or dispersion forces. The active site of the adsorbed enzyme is usually
unaffected, and nearly full activity is retained upon adsorption. However, desorption of enzymes is a
common problem, especially in the presence of strong hydrodynamic forces, since binding forces are
weak.

Covalent binding is the retention of enzymes on support surfaces by covalent bond formation. Enzyme
molecules bind to support material via certain functional groups, such as amino, carboxyl, hydroxyl,
and sulfhydryl groups. These functional groups must not be in the active site. One common trick is to
block the active site by flooding the enzyme solution with a competitive inhibitor prior to covalent
binding.
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