7803 Biochem

Biochemical Engineering – Enzyme kinetics
Presence of a enzyme (biocatalyst) provides an alternative route for the reaction with
lower activation energy!
CH6705 BIOCHEMICAL ENGINEERING 1
Functions:
Inside the cell: In the cytoplasm, the protein is
involved in glycolysis and gluconeogenesis.
Outside the cell: It functions as a neurotrophic
factor for spinal and sensory neurons.
The same protein is also secreted
by cancer cells, where it is stimulates
metastasis.
For the glucose-6-phosphate isomerase reaction

Enzyme catalysis does NOT change Keq = + 2.0 kJ / mol

Mechanism:
• Lock and Key model
•Enzyme – substrate complex formation
•Proximity effect
•Orientation effect
•Conformation change

Consider the following single substrate – enzyme catalysed reaction:
S 
E
P
From the experiments, the rate equation is found to be:
vmax [ S ]
v
Km  [S ]
Elementary or non-elementary reaction?

Proposed mechanism for the single substrate – enzyme catalysed reaction:
k1

S  E  ES 
k2
P  E
k1
The rate equation for the above elementary equations are :
rS  k1  S  E   k1  ES  (1)

rE  k1  S  E   k1  ES   k2  ES  (2)
rES  k1  S  E   k1  ES   k2  ES  (3)
rP  k2  ES  (4)

Since the net amount of enzymes in the systems remain constant at any
given time:
[ E ]0  [ E ]  [ ES ] (5)
Assumption by Michaelis – Menton:

k1
S  E   ES is in equilibrium
k 1
which implies,
k1 S E   k 1  ES (6)

Substituting eqn.(5) into (6) and obtain an expression for [ES],
 
k1 S  E 0   ES  k 1  ES
k1 S E 0  k1 S ES  k 1  ES
S E 0
  ES (7)
 k 1 
  S 
 k1 

Substituting eqn.(7) into (4) and we obtain,

k 2  E 0 S
rp 
v max
(8)
k 1
 S
k1
KM

Assumption by Briggs - Haldane:
Quasi  steady state ,
d  ES
i.e., 0 (9)
dt
Substituting [ES] obtained using this assumption into (4) and we obtain,
k 2  E 0 S
rp 
vmax
(10)
k 1  k 2
 S
k1
KB

Enzyme kinetics: Evaluation of parameters
Lineweaver-Burk plot:

Eadie-Hofstee plot:

Hanes-Woolf (Langmuir) plot:

Enzyme kinetics: Inhibition
Lineweaver- Burk
plot

Significance of Km and Vmax
 Km is the parameter intrinsic to the type of substrate and the enzyme. It is a function
of rate parameters and varies upon changing the temperature or pH.
 Vmax is a function of rate parameter k2 and initial enzyme concentration [E]0.
For highly purified enzyme preparations, [E]0.can be presented in g/l or mol/l. For crude
preparations, it is measured in “unit”. The “unit” is the amount of enzyme that gives
predetermined amount of activity under specific conditions.
 For example, One U is defined as the amount of the enzyme that produces a certain
amount of enzymatic activity, that is, the amount that catalyzes the conversion of 1 micro
mole of substrate per minute.
Specific activity is the number of units of activity per amount of total protein. For
example: 10 units/mg protein.
Activation and inhibition
The binding of oxygen to the iron(II) heme pulls
the iron into the plane of the porphyrin ring,
causing a slight conformational shift. The shift
encourages oxygen to bind to the three
remaining heme units within hemoglobin (thus,
oxygen binding is cooperative).

Enzyme kinetics: Competitive Inhibition
 Competitive inhibitors compete for the substrate-binding site of the
enzyme with the substrate.
 Two equilibria exist in parallel, one between the enzyme and the
inhibitor and another between the enzyme and the substrate.
 Example: Malonic acid which competes with succinate for active sites
of succinic dehydrogenase
Mechanism: (competitive) vmax [ S ]
v
 I  
K m 1    [S ]
 Ki 
where, Ki 
 E  I 
 EI 
Enzyme kinetics: Uncompetitive Inhibition
 These inhibitors bind only to the ES complex without
binding to the free enzyme.
Example: Substrate inhibition is a type of uncompetitive
inhibition.
Mechanism: (Uncompetitive)
vmax [ S ]
v
 I  
K m  1  '  [ S ]
 Ki 
where, Ki 
'  ES  I 
 ESI 
Enzyme kinetics: Substrate Inhibition
This is a special case of uncompetitive inhibition where there subsutrate
itself acts as inhibitor at high concentrations.
When large amount of substrate is present, the enzyme catalysed

reaction is inhibited by the excess substrate.
At [ S ]  [ S ] v vmax , v  vmax .

Mechanism: (Uncompetitive)

Modified Michaelis  Menton equation :
v max S
rp  (8)
 S
2
K M  S 
KS
At low substrate concentration,
S
2
1
KS
v max S
rp 
K M  S
Modified Michaelis  Menton equation :
v max S
rp  (8)
 S
2
K M  S 
KS
At high substrate concentration,
KM
1
S
v max
rp 
1
 S
KS
Enzyme kinetics: Noncompetitive Inhibition
 There are inhibitors that can bind both to the free enzyme as well as to the
ES complex.
Mechanism: (Noncompetitive)
vmax [ S ]
v
 I    I  
1   K m  1  '  [ S ]
 Ki   Ki 
where, Ki 
'  ES  I 
; Ki 
 E  I 
 ESI   EI 

CH6302 MECHANICS OF SOLID 23
Factors affecting enzyme activity
 pH
Temperature
Mechanical forces (hydrodynamics forces eg. Shear force)
Chemicals (such as alcohol, urea and hydrogen peroxide)
Irradiation (Light, sound)

Effect of pH
Optimal pH

Effect of pH
Mechanism
Mathematical model

Effect of Temperature
Ea
-
vmax  Ae RT
[Eqn. for enzyme denaturation]
The rate of the rxn increases
d[ E ]
with temperature at T<Topt   kd [ E ]
dt

Enzyme bioreactors – Ideal batch reactor
 What is a batch reactor?
 Mass balance equation for a batch reactor?
General mass balance equation:

dM
 M i  M O  RG  RC
dt
How the mass balance equation will change for a batch reactor?
Mi  ? MO  ? RG  ? RC  ?

Enzyme bioreactors – Ideal batch reactor
 Mass balance equation for a batch reactor
 v max S 
Mi  0 MO  0 RG  0 RC    V
 K  S 
 M 
d S  vmax  S 
  rp  for S 
E
P
dt KM  S 
Kinetic equation

Ideal Enzyme batch reactor - Problem

Enzyme bioreactors – Ideal CSTR
 What is a CSTR?
 Mass balance equation for a CSTR?
General mass balance equation:

dM
 M i  M O  RG  RC
dt
How the mass balance equation will change for a CSTR?

Mi  ? MO  ? RG  ? RC  ?

Enzyme bioreactors – Ideal CSTR
 Mass balance equation for a CSTR

 vmax  S  
M i  F  S 0 MO  F S  RG  0 RC   V
 KM   S  
 vmax  S  
F  S 0  F  S     V  0
 KM  S  
 Kinetic equation Residence time
vmax  S 
F
V
  S 0   S  
KM  S 
0
Dilution rate

Enzyme immobilisation

Enzyme immobilisation
- Easy separation from reaction mixture, providing the ability to control
reaction times and minimize the enzymes lost in the product.
- Re-use of enzymes for many reaction cycles, lowering the total

production cost of enzyme mediated reactions.
- Ability of enzymes to provide pure products.
- Possible provision of a better environment for enzyme activity
- Diffusional limitation

Enzyme immobilisation - Methods
Entrapment Immobilization is based on the localization of an enzyme
within the lattice of a polymer matrix or membrane.
- retain enzyme, - allow the penetration of substrate.
It can be classified into matrix and micro capsule types.

Matrix Materials:
Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides.
e.g. Ca-alginate, agar,
K-carrageenin, collagen
Immobilization procedures:
Enzyme + polymer solution → polymerization
→ extrusion/shape the particles
Inorganics: activated carbon, porous ceramic.
Shapes: particle, membrane, fiber

Entrapment
challenges:
- enzyme leakage into solution
- diffusional limitation
- reduced enzyme activity and stability
- lack of control micro-environmental conditions.
It could be improved by modifying matrix or membrane.

Surface immobilization
According to the binding mode of the enzyme, this method can be further
sub-classified into:
- Physical Adsorption: Van der Waals
Carriers: silica, carbon nanotube, cellulose, etc.
Easily desorbed, simple and cheap,
enzyme activity unaffected.
- Ionic Binding: ionic bonds

Similar to physical adsorption.
Carriers: polysaccharides and synthetic polymers
having ion-exchange centers.

Surface immobilization
- Covalent Binding: covalent bonds
Carriers: polymers contain amino, carboxyl,

sulfhydryl, hydroxyl, or phenolic groups.
- Loss of enzyme activity

- Strong binding of enzymes

Cross-linking is to cross link enzyme molecules

with each other using agents such as
glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.

Cross-linking is to cross link enzyme molecules

with each other using agents such as
glutaraldehyde.
Features: similar to covalent binding.
Several methods are combined.

Enzyme - Nomenclature

•Stirred tank batch reactor (STR), which contains all of the enzyme and
substrates) until the conversion is complete.
•Batch membrane reactor (MR), where the enzyme is held within membrane
tubes which allow the substrate to diffuse in and the product to diffuse out. This
reactor may often be used in a semicontinuous manner, using the same enzyme
solution for several batches.
•Packed bed reactor (PBR), also called plug -flow reactor (PFR), containing a settled bed of
immobilised enzyme particles;
•Continuous flow stirred tank reactor (CSTR) which is a continuously operated version of (a);
•Continuous flow membrane reactor (CMR) which is a continuously operated version of (b);
•Fluidised bed reactor (FBR), where the flow of gas and/or substrate keeps the immobilised enzyme
particles in a fluidised state.
Effect of mass transfer resistance due to immobilization
(1) Transfer from the bulk liquid to a relatively unmixed liquid layer surrounding the immobilized enzyme;
(2) Diffusion through the relatively unmixed liquid layer; and
(3) Diffusion 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 intraparticle mass transfer resistance.

Effect of mass transfer external resistance
(Surface immobilization)




Effect of mass transfer internal resistance

Problems on enzyme kinetics and enzyme inhibition


7803 Biochem

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Biochemical Engineering – Enzyme kinetics

For the glucose-6-phosphate isomerase reaction

CH6705 BIOCHEMICAL ENGINEERING 3

CH6705 BIOCHEMICAL ENGINEERING 4

Elementary or non-elementary reaction?

CH6705 BIOCHEMICAL ENGINEERING 5

The rate equation for the above elementary equations are :

rS  k1  S  E   k1  ES  (1)

CH6705 BIOCHEMICAL ENGINEERING 6

Assumption by Michaelis – Menton:

CH6705 BIOCHEMICAL ENGINEERING 7

CH6705 BIOCHEMICAL ENGINEERING 8

Substituting eqn.(7) into (4) and we obtain,

CH6705 BIOCHEMICAL ENGINEERING 9

CH6705 BIOCHEMICAL ENGINEERING 10

CH6705 BIOCHEMICAL ENGINEERING 11

CH6705 BIOCHEMICAL ENGINEERING 12

CH6705 BIOCHEMICAL ENGINEERING 13

CH6705 BIOCHEMICAL ENGINEERING 14

 Vmax is a function of rate parameter k2 and initial enzyme concentration [E]0.

CH6705 BIOCHEMICAL ENGINEERING 16

When large amount of substrate is present, the enzyme catalysed

At [ S ]  [ S ] v vmax , v  vmax .

CH6705 BIOCHEMICAL ENGINEERING 19

CH6705 BIOCHEMICAL ENGINEERING 22

Mechanical forces (hydrodynamics forces eg. Shear force)

Chemicals (such as alcohol, urea and hydrogen peroxide)

Irradiation (Light, sound)

CH6705 BIOCHEMICAL ENGINEERING 24

CH6705 BIOCHEMICAL ENGINEERING 25

CH6705 BIOCHEMICAL ENGINEERING 26

CH6705 BIOCHEMICAL ENGINEERING 27

 What is a batch reactor?

 Mass balance equation for a batch reactor?

General mass balance equation:

CH6705 BIOCHEMICAL ENGINEERING 29

CH6705 BIOCHEMICAL ENGINEERING 30

CH6705 BIOCHEMICAL ENGINEERING 31

 Mass balance equation for a CSTR?

General mass balance equation:

How the mass balance equation will change for a CSTR?

CH6705 BIOCHEMICAL ENGINEERING 32

 Mass balance equation for a CSTR

CH6705 BIOCHEMICAL ENGINEERING 33

CH6705 BIOCHEMICAL ENGINEERING 34

- Re-use of enzymes for many reaction cycles, lowering the total

- Ability of enzymes to provide pure products.

- Possible provision of a better environment for enzyme activity

CH6705 BIOCHEMICAL ENGINEERING 36

CH6705 BIOCHEMICAL ENGINEERING 37

Inorganics: activated carbon, porous ceramic.

Shapes: particle, membrane, fiber

CH6705 BIOCHEMICAL ENGINEERING 38

CH6705 BIOCHEMICAL ENGINEERING 39

- Ionic Binding: ionic bonds

CH6705 BIOCHEMICAL ENGINEERING 40

- Covalent Binding: covalent bonds

Carriers: polymers contain amino, carboxyl,

- Loss of enzyme activity

CH6705 BIOCHEMICAL ENGINEERING 41

Cross-linking is to cross link enzyme molecules

Several methods are combined.

CH6705 BIOCHEMICAL ENGINEERING 42

Cross-linking is to cross link enzyme molecules