Biochemical Engineering

8CH01

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT III
ENZYME CATALYZED REACTIONS
Mechanism & Michealis-Menten kinetics model
& estimation of kinetic parameters

Batch & continuous enzyme reactor kinetics

Multi-substrate enzyme reaction kinetics & estimation of

kinetic parameters

Kinetics of enzyme inhibition & estimation of kinetic

parameters

Effects of temperature, pH & shear

Immobilized enzymes & industrial applications

Immobilization of Enzymes
Restriction of Enzymes in a fixed place is Immobilization

Advantages
Enzyme reutilization
Elimination of E recovery & purification
Improved product purity
Minimized effluent handling
Longer lasting E activity
Unique environment (well-defined, pre-determined space)

Methods of Immobilization
Entrapment (Physical method)
Surface Immobilization (Chemical method)

How to select the immobilization strategy???

Overall catalytic activity & effectiveness of catalyst activity
Deactivation
Regeneration characteristics and COST
Toxicity of immobilization reagents/waste disposal
Immobilization of Enzymes
Physical Methods (Entrapment)

Physical enclosure of Enzymes in a small space

Enzymes in solution are entrapped either in

Matrix-entrapped (polymer network)
Membrane-entrapped (semipermeable membrane)

Porous for substrates and product

Impermeable to Enzymes and large molecules

 Matrix Immobilization
Minimal perturbation to enzyme’s native structure & function
Polymers (natural or synthetic) used as matrices
Calcium cross-linked alginate
k-carrageenin
Polyacrylamide
Collagen
Solid matrices: Activated carbon, porous ceramic, diatomaceous earth
Fibers, microparticles, etc (pore size: 100-400 nm & size: 0.3-2 µm)
E + monomer solution → polymerize and then extrude
E + polymer solution → crosslink polymer and entrap E

http://cerebralenhancementzone.wikispaces.com/
Membrane Immobilization
Polymers (both natural & synthetic) used as membranes
Nylon
Cellulose
Polysulfone
Polyacrylate
Copolymerization at the interface b/w organic phase and aqueous phase
containing enzyme
Membrane can be made substrate specific
Hollow fiber membranes, microcapsules, etc are examples

http://image.slidesharecdn.com/
Disadvantages of Matrix Immobilization
Enzyme leakage

Significant diffusional limitations (especially for crosslinked matrix)

Reduced enzyme activity and stability

Lack of control of micro-environmental conditions

Substrate needs to be significantly smaller than E

Surface Immobilization
Attaching E to the surface of the support material
Physical adsorption
Covalent binding
Crosslinking

Adsorption Immobilization
Surface attachment of E by weak physical forces
(van der Waals, ionic)
Insoluble support materials:
Inorganic (Alumina, Silica, Porous Glass,
Ceramics, etc)
Organic (Cellulose, Starch, Activated Carbon,
Resins, etc)
Pretreatment of surfaces is necessary
Enzyme activity is unaffected upon adsorption
Easy removal of E for regeneration and addition of
new E
Disadvantage: Strong hydrodynamic forces can
desorb E
Covalent Immobilization
Retention of E on support surfaces by covalent bond formation
Functional groups on E such as amine, carboxyl, hydroxyl, sulfhydroxyl, etc
bind to surfaces
Need to block active sites before covalent bonding
Surface modification of supports (Silanization)
Coating the surface with organic functional groups using an organo-
functional Silane reagent.
Attaching flexible spacer arm moieties (eg. n-propyl amine) to the
support and then the E.

Diazotization:
Amide bond formation:
Alkylation:
Schiff’s base formation:
Amidation reaction:
Thiol-Disulfide interchange:
Glutaraldehyde:
Support---N=N---Enzyme
Support---CO-NH---Enzyme
Support---CH2-NH---Enzyme
Support---CH2-S---Enzyme
Support---CH=N---Enzyme
Support---CNH-NH---Enzyme
Support---S-S---Enzyme
Support---CH-N---Enzyme
Covalent Immobilization
Crosslinking
Variation of direct covalent attachment

Crosslinking E by multifunctional reagents

Glutaraldehyde, bis-diazobenzidine, 2,2-disulfonic acid are used

Gives extensive crosslinking (with or without support)

Polyenzyme network

Very strong binding (minimal enzyme detachment)

Disadvantages
Regeneration is not possible
Severe diffusion limitations
Covalent attachment at multiple sites
reduces enzyme activity
Increased cost
Criteria for the Selection of Support Material
Support material & immobilization method depends on
Enzyme
Application

Criteria for selection

Interaction b/w support material and reaction mixture (Substrate)

Binding capacity of the support material

Charge density, functional groups, hydrophobicity

Stability & retention of enzymatic activity

Functional groups on support & micro-environmental conditions

Physical & mechanical properties of support material

Porosity, mechanical strength, etc.
Immobilized Enzymes - Video
Diffusional Path in Immobilized Enzyme Systems

Bulk liquid
containing the Immobilized
substrate enzyme

2
1 3 ["]
Reaction site

["# ]

1 - Transfer of substrate from bulk liquid to a relatively unmixed liquid film

surrounding the immobilized enzyme

2 - Diffusion of substrate through the relatively unmixed liquid film

3 - Diffusion from the surface of the particle to the active site of the enzyme in
an inert support.
Diffusional Limitations in Immobilized Enzyme Systems
Resistance to diffusion in immobilized enzyme systems depends on
Nature of the support material (porous, nonporous)
Hydrodynamic conditions surrounding the support material
Distribution of the enzyme inside or on the surface of the support material

To understand if diffusion resistance has a significant effect on the rate of

enzymatic reaction, we use Damkohler number (Da).

Da is defined as the ratio of maximum rate of reaction to the maximum rate of

diffusion.
$%
!" =
&' [)* ]

where, [)* ] is the substrate concentration in bulk liquid (g/cm3) and &' is the
mass-transfer coefficient (cm/s).

If Da >> 1, the diffusion rate is limiting.

If Da << 1, the reaction rate is limiting.
If Da = 1, the diffusion and reaction resistances are comparable.
Diffusion Effects in Surface-bound Enzymes on Non-porous
Support Materials
Assume a situation when enzymes are bound and evenly distributed on the
surface of a nonporous support material.

All enzymes are equally active, and substrates diffuse through a thin liquid film
surrounding the support surface to reach the reactive substrates.
Assume that the process of immobilization has
not altered the protein structure, and the kinetic
parameters (!" , #" ) are unaltered.

In surface-bound enzymes, the path is only

composed of steps 1 and 2, which is external
mass transfer resistance.

At steady state, the reaction rate is equal to the

the rate of mass transfer, and is given by:

, [) ]
$% = '( )* − )% = 0 -1[)% ….. (1)
- %]
Diffusion Effects in Surface-bound Enzymes on Non-porous
Support Materials
)* [,- ]
!" = $% &' − &" = ……. (1)
/* 0[,- ]

Where,
12 is the maximum reaction rate per unit of external surface area
$% is the liquid mass transfer coefficient
&' is the substrate concentration in the liquid bulk
&" is the substrate concentration at the surface

When the system is strongly mass transfer limited ie., &" = 0, since the
reaction is rapid compared to mass transfer, and hence:

4 = 56[78 ] for Da >> 1 and the system behaves as pseudo first order.

When the system is reaction limited (Da << 1), the reaction rate is often
expressed with appropriate assumptions as

9: [78 ] )*
4= …… (2) where, ?2,@AA = ?2 1 + D
;:,=>> 0[78 ] E ( ,G 0/* )
Thank you