Bio-chemical Engineering

Dr. Md Salatul Islam Mozumder

Professor
Department of Chemical Engineering and
Polymer Science
Shahjalal University of Science and Technology
Sylhet 3114
 Chapter 1
Biomass, biomass (bacterial/cells)
growth and kinetics
Biomass:

What is biomass?
Source of biomass

World resources plant biomass yields

Biomass conversion
- Thermal conversions
- Chemical conversion
- Biochemical conversion
- Electrochemical conversion
Environmental damage due to using biomass:

 Using biomass as a fuel produces air pollution in the form of

carbon monoxide, carbon dioxide, NOx, VOCs, particulates
and other pollutants
 the carbon storage capacity of the forest may be reduced
overall if destructive forestry techniques are employed
 The pros and cons of biomass usage regarding carbon
emissions may be quantified with the Indirect Land Use
Change (ILUC) factor.
What are Bacteria? What is Bacterial growth?

What are Bacteria?

 Bacteria are microscopic single-celled organisms

What is Bacterial growth?

 reproduction, or cell division

Growth curve:
A= Lag phase;
B= Exponential growth phase;
C= Stationary phase and
D= Death phase
Environmental conditions for bacterial growth:

Temperature
Psychrophiles:
cold-loving bacteria or archaea
maximal temperature for growth at 20 °C,
minimal temperature for growth at 0 °C or lower.

Mesophiles:
bacteria that thrive at moderate temperatures,
growing best between 20° and 45 °C.
These temperatures align with the natural body temperatures of
humans, which is why many human pathogens are mesophiles.

Thermophiles:
A thermophile is an organism—a type of extremophile—that thrives
relatively high temperatures, between 41 and 122 °C.
Environmental conditions for bacterial growth:

Acidity
Optimal acidity for bacteria = around pH 6.5 to 7.0
acidophiles grow between pH 3-5.5.
In an anaerobic digestion:
Acetogenesis worked well at low pH; 3-5
Methanogenesis worked well at neutral pH; 6.5-7.5.

Drastic variations in cytoplasmic pH that is affected by the pH

of the medium disrupt the plasma membrane or inhibit the
activity of enzymes and membrane transport proteins.
Environmental conditions for bacterial growth:

Water activity
 Water activity is the partial vapor pressure of water in a substance
divided by the standard state partial vapor pressure of water.
 As temperature increases, water activity typically increases, except
in some products with crystalline salt or sugar.
 Higher water activity substances tend to support more
microorganisms.
 Bacteria usually require at least 0.91, and fungi at least 0.7.
Environmental conditions for bacterial growth:

Oxygen
Aerobic organism
C6H12O6 + 6 O2 + 38 ADP + 38 phosphate → 6 CO2 + 6 H2O + 38 ATP

Anaerobic organism
 obligate anaerobes, which are harmed by the presence of oxygen
 aerotolerant organisms, which cannot use oxygen for growth but
tolerate its presence;
 facultative anaerobes, which can grow without oxygen but use
oxygen if it is present.

C6H12O6 + 2 ADP + 2 phosphate → 2 lactic acid (C3H6O3) + 2 ATP

C6H12O6 + 2 ADP + 2 phosphate → 2 C2H5OH + 2 CO2 + 2 ATP
Environmental conditions for bacterial growth:

Micronutrients
 Ammonium-N and Phosphate are required for synthesis of DNA and
ADP/ATP – essential for the production of microbial cells.
 NH4+ and PO4-3 are essential nutrients for the growth.
 For enzymatic functions and to produce the body fluid some tress
elements such as Fe, Cu, Mg, Na, K, Zn etc. are needed.

Osmotic pressure
 The optimal osmotic pressure of the mineral medium is the same
osmotic pressure of body liquid.
 High osmotic pressure inversely affects the microbial growth and
the production.
 Increasing salt concentration increase the osmotic pressure - effects
the process through the damaging the cell well.
Specific growth rate:

- steepness of a bacterial growth curve,

- the rate of increase of biomass per unit of biomass concentration.
This period of time occurs between the lag phase and stationary phases.
1 𝑋𝑜 − 𝑋
µ=
𝑋𝑜 𝑡

1 𝑑𝑋
µ=
𝑋 𝑑𝑡

1
𝑑𝑋 = µ 𝑑𝑡
𝑋

𝑙𝑛𝑋 = 𝑙𝑛𝑋𝑜 + µ𝑡

Where, XO is the initial biomass (or cell density)

X is the biomass (or cell density) at time t
µ can be calculated from the slope of the line between ln x and t.
Specific growth rate:

𝑋
𝑙𝑛 = µ𝑡
𝑋𝑜

𝑋 = 𝑋𝑜 𝑒 µ𝑡
While µ and XO are known the biomass concentration (X) can be calculated from the above
relationship.
Cell doubling time
2𝑋𝑜
𝑙𝑛 = µ𝑡𝑑
𝑋𝑜

𝑙𝑛2 0.692
𝑡𝑑 = =
𝜇 𝜇

And then the biomass concentration can be calculated by:

0.692
𝑡
𝑋 = 𝑋𝑜 𝑒 𝑡𝑑
 𝑑𝑋
For the batch culture = µ𝑋
𝑑𝑡
For the continuous culture, volume is not considered as a constant:

𝑑(𝑉𝑋)
= µ𝑋𝑉
𝑑𝑡

𝑑(𝑋) 𝑑(𝑉)
𝑉 +𝑋 = µ𝑋𝑉
𝑑𝑡 𝑑𝑡

𝑑𝑋 𝑑𝑉
𝑉 = µ𝑋𝑉 − 𝑋
𝑑𝑡 𝑑𝑡

𝑑𝑋 𝑑𝑉
= µ𝑋 − 𝑋
𝑑𝑡 𝑉𝑑𝑡

𝑑𝑋 𝑑𝑉 1
= µ𝑋 − 𝐷𝑋, 𝐷= = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 ( )
𝑑𝑡 𝑉𝑑𝑡 ℎ

𝑑𝑋
At steady state, = 0 and then µ = 𝐷 .
𝑑𝑡
 Monod growth kinetics:

The Monod equation is:

𝑆
µ = µ𝑚𝑎𝑥
𝐾𝑠 +𝑆

Linearizing the Monod equation:

1 𝐾𝑠 1 1
= +
µ µ𝑚𝑎𝑥 𝑆 µ𝑚𝑎𝑥
Specific growth rate for mixed substrate or
substrate and nutrien:
𝑆1
µ1 = µ𝑚𝑎𝑥1 = µ𝑚𝑎𝑥
0.5 𝐾𝑆1 + 𝑆1
S2= g/L
Specific growth rate, µ (1/h) 0.4

0.3 S1= g/L

0.2 𝑆2 𝑆1 𝑆2
µ = µ𝑚𝑎𝑥1 = µ𝑚𝑎𝑥
𝐾𝑆2 + 𝑆2 𝐾𝑆1 + 𝑆1 𝐾𝑆2 + 𝑆2
0.1

0
0 5 10 15 20 25
Substrate (S1 or S2) (g/L)

For substrate (S), ammonium – nitrogen (N), phosphate :

𝑆 𝑁 𝑃
µ = µ𝑚𝑎𝑥
𝐾𝑠 + 𝑆 𝐾𝑁 + 𝑃 𝐾𝑃 + 𝑃
Types of bioreactor:

Batch culture:
The following parameters are monitored while the batch process continues:
• Cells and cell by-product
• Concentration of nutrients
• Desirable and undesirable products
• Inhibition
• pH, temperature, substrate concentration

Disadvantages exist in the batch system such as

 substrate depletion with limited nutrients
 product inhibition growth curve
 toxic by-products accumulate
 exponential growth is continuously changing
Types of bioreactor:

Continuous culture:
1. Chemostat
growth rate controlled by dilution rate, D, (h-1)
2. Turbidostat
constant cell density that is controlled by the fresh medium

Fed-batch culture:
Mass balance of biomass:

General mass balance equation:

Cell accumulation = Cell in – Cell out + Cell growth – Cell consumption

Fin, Sin

Mass balance on biomass:

𝑑(𝑋𝑉)
= 𝐹𝑖𝑛 𝑋𝑖𝑛 − 𝐹𝑜𝑢𝑡 𝑋𝑜𝑢𝑡 + µ𝑋𝑉 – 𝛼𝑋𝑉
𝑑𝑡
S => X
S => P

Fout, Sout,
Xout, Pout,
 Fin, Sin

In CSTR reactor, 𝑋𝑖𝑛 = 0 and 𝑋 = 𝑋𝑜𝑢𝑡 . So

𝑑𝑋 𝑑𝑉
𝑉 +𝑋 = 0 − 𝐹𝑜𝑢𝑡 𝑋 + µ𝑋𝑉 – 𝛼𝑋𝑉 S => X
𝑑𝑡 𝑑𝑡 S => P
𝑑𝑋
=> 𝑉 + 𝑋( 𝐹𝑖𝑛 − 𝐹𝑜𝑢𝑡 ) = − 𝐹𝑜𝑢𝑡 𝑋 + µ𝑋𝑉 – 𝛼𝑋𝑉 Fout, Sout,
𝑑𝑡
Xout, Pout,
𝑑𝑋 outflow rate (𝐹𝑜𝑢𝑡 ) do
=> 𝑉 = µ𝑋𝑉 − 𝐹𝑖𝑛 𝑋 − 𝛼𝑋𝑉
𝑑𝑡 not affect the biomass
𝑑𝑋 𝐹𝑖𝑛 concentration as well as
=> = µ𝑋 − 𝑋 − 𝛼𝑋 the reactor performance.
𝑑𝑡 𝑉
In fed-batch 𝐹𝑜𝑢𝑡 = 0 , as
𝑑𝑋
=> = µ𝑋 − 𝐷𝑋 − 𝛼𝑋 𝐹𝑜𝑢𝑡 do not affect the process
𝑑𝑡
performance, the performance
𝐹𝑖𝑛 of CSTR and fed-batch are
[ = 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒, 𝐷]
𝑉 the same.
 Fin, Sin
Mass balance of biomass:

𝑑𝑋 S => X
In steady state condition, = 0 , then S => P
𝑑𝑡
µ𝑋 − 𝐷𝑋 − 𝛼𝑋 = 0
If the decay (𝛼) is negligible then Fout, Sout,
Xout, Pout,
µ𝑋 − 𝐷𝑋 = 0 and µ = 𝐷

In steady state condition at CSTR indicate the same inflow and

𝒅(𝑽)
outflow that means 𝐹𝑖𝑛 = 𝐹𝑜𝑢𝑡 , and = 0. However, it does
𝒅𝒕
not affect the process performance as the outflow (𝐹𝑜𝑢𝑡 ) do not
affect it and the dilution only depends on inflow (𝐹𝑖𝑛 ).
 Fin, Sin
Mass balance of biomass:

In steady state condition, the outflow substrate S => X

concentration was calculated by: S => P

𝑆 Fout, Sout,
µ = 𝐷 = µ𝑚𝑎𝑥
𝐾𝑠 + 𝑆 Xout, Pout,
In CSTR type reactor S = Sout.
𝑆𝑜𝑢𝑡
µ = 𝐷 = µ𝑚𝑎𝑥
𝐾𝑠 + 𝑆𝑜𝑢𝑡

𝐾𝑠 𝐷
=> 𝑆𝑜𝑢𝑡 =
µ𝑚𝑎𝑥 − 𝐷
Mass balance of product : Fin, Sin

General mass balance equation:

S => X
Accumulation = In – Out + Production– Consumption S => P

Fout, Sout,
Xout, Pout,
Mass balance for product:
𝑑(𝑃𝑉)
= 𝐹𝑖𝑛 𝑃𝑖𝑛 − 𝐹𝑜𝑢𝑡 𝑃𝑜𝑢𝑡 + 𝑞𝑝 𝑋𝑉 – 𝛽𝑋𝑉
𝑑𝑡
Where, 𝑞𝑝 = Specific production rate (1/h)
𝛽 = Specific denaturation of product (1/h)
 Fin, Sin

In CSTR reactor, 𝑃𝑖𝑛 = 0 and 𝑃 = 𝑃𝑜𝑢𝑡 . So

𝑑𝑃 𝑑𝑉
𝑉 +𝑃 = 0 − 𝐹𝑜𝑢𝑡 𝑃𝑜𝑢𝑡 + 𝑞𝑝 𝑋𝑉 – 𝛽𝑋𝑉 S => X
𝑑𝑡 𝑑𝑡 S => P

𝑑𝑃 Fout, Sout,
=> 𝑉 + 𝑃( 𝐹𝑖𝑛 − 𝐹𝑜𝑢𝑡 ) = − 𝐹𝑜𝑢𝑡 𝑃 + 𝑞𝑝 𝑋𝑉 – 𝛽𝑋𝑉
𝑑𝑡 Xout, Pout,

𝑑𝑃 outflow rate (𝐹𝑜𝑢𝑡 ) do

=> 𝑉 = 𝑞𝑝 𝑋𝑉 − 𝐹𝑖𝑛 𝑃 − 𝛽𝑋𝑉 not affect the product
𝑑𝑡
concentration as well as
𝑑𝑃 𝐹𝑖𝑛 the reactor performance.
=> = 𝑞𝑝 𝑋 − 𝑃 − 𝛽𝑋
𝑑𝑡 𝑉 In fed-batch 𝐹𝑜𝑢𝑡 = 0 , as
𝐹𝑜𝑢𝑡 do not affect the process
𝑑𝑃
=> = 𝑞𝑝 𝑋 − 𝐷𝑃 − 𝛽𝑋 performance, the performance
𝑑𝑡
𝐹𝑖𝑛 of CSTR and fed-batch are
[ = 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒, 𝐷]
𝑉 the same.
 Fin, Sin
Mass balance of product:

𝑑𝑃 S => X
If, = 0 , then
𝑑𝑡 S => P
𝑞𝑝 𝑋 − 𝐷𝑃 − 𝛽𝑋 = 0
Fout, Sout,
If the product consumption (𝛽) is negligible then Xout, Pout,

𝑞𝑝 𝑋 − 𝐷𝑃 = 0 𝑎𝑛𝑑 𝑞𝑝 𝑋 = 𝐷𝑃

In steady state condition at CSTR indicate the same inflow and outflow
𝒅(𝑽)
that means 𝐹𝑖𝑛 = 𝐹𝑜𝑢𝑡 , and = 0. However, it does not affect the
𝒅𝒕
process performance as the outflow (𝐹𝑜𝑢𝑡 ) do not affect it and the
dilution only depends on inflow (𝐹𝑖𝑛 ).
Mass balance of substrate : Fin, Sin

General mass balance equation:

S => X
S => P
Accumulation = In – Out + Production– Consumption
Fout, Sout,
Xout, Pout,
Mass balance for substrate:
𝑑(𝑉𝑆) µ 𝑞𝑝
= 𝐹𝑖𝑛 𝑆𝑖𝑛 − 𝐹𝑜𝑢𝑡 𝑆𝑜𝑢𝑡 − 𝑋𝑉 − 𝑋𝑉 − 𝑚𝑋𝑉
𝑑𝑡 𝑌𝑋/𝑠 𝑌𝑝/𝑠

Where, 𝑆𝑖𝑛 = Substrate concentration in the inflow(g/L)

𝑆𝑜𝑢𝑡 = Substrate concentration in the outflow(g/L)
𝑚 = Specific maintenance rate (1/h)
In CSTR reactor, 𝑆 = 𝑆𝑜𝑢𝑡 . So Fin, Sin

𝑑𝑆 𝑑𝑉 µ 𝑞𝑝
𝑉 +𝑆 = 𝐹𝑖𝑛 𝑆𝑖𝑛 − 𝐹𝑜𝑢𝑡 𝑆 − 𝑋𝑉 − 𝑋𝑉 − 𝑚𝑋𝑉
𝑑𝑡 𝑑𝑡 𝑌𝑋/𝑠 𝑌𝑝/𝑠
S => X
𝑑𝑆 S => P
=> 𝑉 + 𝑆( 𝐹𝑖𝑛 − 𝐹𝑜𝑢𝑡 )
𝑑𝑡
µ 𝑞𝑝
= 𝐹𝑖𝑛 𝑆𝑖𝑛 − 𝐹𝑜𝑢𝑡 𝑆 − 𝑋𝑉 − 𝑋𝑉 − 𝑚𝑋𝑉 Fout, Sout,
𝑌𝑋/𝑠 𝑌𝑝/𝑠 Xout, Pout,

𝑑𝑆 µ 𝑞𝑝 outflow rate (𝐹𝑜𝑢𝑡 ) do

=> 𝑉 = 𝐹𝑖𝑛 𝑆𝑖𝑛 − 𝐹𝑖𝑛 𝑆 − 𝑋𝑉 − 𝑋𝑉
not−affect
𝑚𝑋𝑉the product
𝑑𝑡 𝑌𝑋/𝑠 𝑌𝑝/𝑠
concentration as well as
𝑑𝑆 𝐹𝑖𝑛 µ 𝑞𝑝 the reactor performance.
=> = (𝑆𝑖𝑛 − 𝑆) − 𝑋− 𝑋 − 𝑚𝑋
𝑑𝑡 𝑉 𝑌𝑋/𝑠 𝑌In 𝐹𝑜𝑢𝑡 = 0 , as
𝑝/𝑠 fed-batch

𝑑𝑆 µ 𝑞𝑝 𝐹𝑜𝑢𝑡 do not
𝐹𝑖𝑛affect the process
=> = 𝐷(𝑆𝑖𝑛 − 𝑆) − 𝑋− 𝑋− 𝑚𝑋 [
𝑑𝑡 𝑌𝑋 𝑌𝑝 performance,
𝑉 the performance
𝑠 𝑠 of CSTR and fed-batch are
= 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒, 𝐷]
the same.
 Fin, Sin
Mass balance of substrate:

𝑑𝑆 S => X
For a steady-state condition, = 0 , then S => P
𝑑𝑡
µ 𝑞𝑝
𝐷(𝑆𝑖𝑛 − 𝑆) − 𝑋− 𝑋 − 𝑚𝑋 = 0
𝑌𝑋/𝑆 𝑌𝑝/𝑆 Fout, Sout,
Xout, Pout,
If the substrate consumption for maintenance (𝑚) is negligible
then
µ 𝑞𝑝
𝐷(𝑆𝑖𝑛 − 𝑆) − 𝑋− 𝑋=0
𝑌𝑋/𝑆 𝑌𝑝/𝑆

µ 𝑞𝑝
=> 𝐷(𝑆𝑖𝑛 − 𝑆) = 𝑋+ 𝑋
𝑌𝑋/𝑆 𝑌𝑝/𝑆
The equation state that at steady state condition, the amount of
substrate freshly injected is fully consumed by growth, production
and maintenance.
Limitation and inhibition kinetics:

0.6

0.5
Specific growth rate (g/g/L)

0.4

0.3

0.2
Monod
0.1 Logistic
Modified Logistic
0
Haldane
-0.1
0 20 40 60 80
Substrate concentration (g/L)
 Limitation and inhibition kinetics:

Limitation Inhibition Overall kinetics

Monod, 𝑆
𝑆 µ = µ𝑚𝑎𝑥
𝐾𝑆 + 𝑆
𝐾𝑆 +𝑆
Monod, 𝑆 𝑆
𝑆
𝑆 Logistic, 1 − µ = µ𝑚𝑎𝑥 1−
𝑆𝑚 𝐾𝑆 + 𝑆 𝑆𝑚
𝐾𝑆 +𝑆
µ
Monod, Modified Logistic,
𝐶
𝑆 𝑆 𝐶 𝑆 𝑆
𝐾𝑆 +𝑆 1− = µ𝑚𝑎𝑥 1−
𝑆𝑚 𝐾𝑆 + 𝑆 𝑆𝑚
𝑆 𝑆
Haldane, µ = µ𝑚𝑎𝑥
𝐾𝑆 +𝑆+𝑆 2 /𝐾𝐼𝑆 𝐾𝑆 + 𝑆 + 𝑆 2 /𝐾𝐼𝑆
Monod, Non-competitive 𝑆 𝐾𝐼
𝑆 𝐾𝐼 µ = µ𝑚𝑎𝑥
inhibition, 𝐾𝑆 + 𝑆 𝐾𝐼 + 𝑆
𝐾𝑆 +𝑆 𝐾𝐼 +𝑆
 Limitation and inhibition kinetics:
𝑆
0.6 Monod, µ = µ𝑚𝑎𝑥
𝐾𝑆 +𝑆
0.5
Specific growth rate (g/g/L)

0.4 Monod+Logistic,
0.3 𝑆 𝑆
µ = µ𝑚𝑎𝑥 1−
𝐾𝑆 + 𝑆 𝑆𝑚
0.2
Monod
0.1 Logistic

0
Modified Logistic Haldane,
Haldane 𝑆
µ = µ𝑚𝑎𝑥 2 Τ𝐾
-0.1
0 20 40 60 80
𝐾𝑆 + 𝑆 + 𝑆 𝐼𝑆
Substrate concentration (g/L)
Monod+Modified Logistic
𝐶
𝑆 𝑆
µ = µ𝑚𝑎𝑥 1−
𝐾𝑆 + 𝑆 𝑆𝑚
 Chapter 2
Enzyme and enzyme kinetics
 Chapter 2
Enzyme and enzyme kinetics
 Enzymes are macromolecular biological catalysts.
 Enzymes are known to catalyze more than 5,000 biochemical reaction
types.
 Most enzymes are proteins, although a few are catalytic RNA
molecules.
 Enzymes' specificity comes from their unique three-dimensional
structures.

Enzyme activity can be affected by other molecules:

 inhibitors are molecules that decrease enzyme activity, and activators
are molecules that increase activity.
 Many therapeutic drugs and poisons are enzyme inhibitors.
 An enzyme's activity decreases markedly outside its optimal
temperature and pH.
 Mechanism of enzymatic reaction:
 very specific as to what substrates they bind
 binding pockets with complementary shape,
charge and hydrophilic/hydrophobic
characteristics
 distinguish between very similar substrate
molecules to be chemoselective,
regioselective and stereospecific.
 showing the highest specificity and
accuracy

"Lock and key"

model for
enzymatic reaction
 Classification of enzymes:

Classes Reaction type Examples

Oxidoreductases Oxidation reduction where oxygen Cytochrome oxidase,
and hydrogen are gained or lost lactate dehydrogenase
Transferases Transfer of functional groups, such Acetate kinase, alanine
as an amino group, acetyl group, or deaminase
phosphate group
Hydrolases Hydrolysis (addition of water) Lipase, sucrase
Lyases Removal of groups of atoms without Oxalate decarboxylase,
hydrolysis isocitrate lyase
Isomerases Rearrangement of atoms within a Glucose-phosphate
molecule isomerase, alanine
racemase
Ligases Joining of two molecules (using Acetyl-CoA synthetase,
energy usually derived from the DNA ligase
breakdown of ATP)
Michaelis-Mantens equation for enzymatic reaction:

Let us define the substrate [S], enzyme [E] and product is defined as [P]:
k1
E + S k−1 ⇌ [ES]
ks
ES + H2 O ՜ E + P
The rate of utilization of substrate -rs is stated as follows:
−𝑟𝑠 = 𝑘1 𝐸 𝑆 − 𝑘−1 𝐸𝑆
The rate of change of intermediate reached to zero is stated as:
−𝑟𝐸𝑆 = 0 = 𝑘1 𝐸 𝑆 − 𝑘−1 𝐸𝑆 − 𝑘𝑠 [𝐻2 𝑂] 𝐸𝑆

[Eo] =[E] + [ES], =>[E] = [Eo] - [ES];

𝑘1 𝐸0 − 𝐸𝑆 𝑆 = 𝐸𝑆 𝑘−1 + 𝑘𝑠 𝐻2 𝑂

𝑘1 𝐸0 [𝑆]
𝐸𝑆 =
𝑘−1 + 𝑘𝑠 𝐻2 𝑂 + 𝑘1 [𝑆]
Michaelis-Mantens equation for enzymatic reaction:

𝑘1 𝐸0 [𝑆]
𝐸𝑆 =
𝑘−1 + 𝑘𝑠 𝐻2 𝑂 + 𝑘1 [𝑆]
The rate of product formation (rate of reaction):

𝑟𝑝 = 𝑉0 = 𝑘𝑠 𝐸𝑆 𝐻2 𝑂
k0= ks[H2O] and then,
𝑟𝑝 = 𝑉0 = 𝑘0 𝐸𝑆

𝑘0 𝑘1 𝐸0 [𝑆]
𝑟𝑝 = 𝑉0 =
𝑘−1 + 𝑘0 + 𝑘1 [𝑆]

Michaelise Menten constant KM =k-1+ k0 and 𝑉𝑚𝑎𝑥 = 𝑘0 𝑘1 𝐸0

Then the Michaelis Menten rate equation, stated as follows:

𝑉𝑚𝑎𝑥 𝑆
𝑉0 =
𝐾𝑀 + 𝑆
 Michaelis-Mantens equation for enzymatic reaction:

𝑉𝑚𝑎𝑥 𝑆
𝑉0 =
𝐾𝑀 + 𝑆

Linearizing the Michaelis Menten

rate equation, we can find:
1 𝐾𝑀 1 1
= +
𝑉0 𝑉𝑚𝑎𝑥 𝑆 𝑉𝑚𝑎𝑥

Vmax at S=∞,
in reality: it is not possible
Vmax only theoretical value

Lineweaver–Burk plot
Eadie–Hofstee plot for the enzymatic reaction kinetics:

Disadvantage of Lineweaver–Burk plot:

- compressing the data points at high
substrate concentrations into a small
region
- emphasizing the points at lower
substrate concentrations, which are
often the least accurate.

Multiplying both sides of linearized

Michaelis Menten rate equation by 𝑉0 .
𝑉0 𝐾𝑀
𝑉𝑚𝑎𝑥 = 𝑉0 +
𝑆
𝑉0 𝐾𝑀 Eadie–Hofstee plot
=> 𝑉0 = 𝑉𝑚𝑎𝑥 −
𝑆
 Exercise 2.1:

Express the enzyme kinetics for the following

enzyme reaction:
(i) E+S = ES; equilibrium constant = K1,
ES=P+E; equilibrium constant = K2,
(ii) E+S = ES1; equilibrium constant = K1,
ES1=ES2; equilibrium constant = K2,
ES2=P+E; equilibrium constant = K3,
 𝐾1 𝐸 [𝑆] 𝐸 [𝑆]
(i) 𝐸 + 𝑆 ֞ 𝐸𝑆, 𝐾1 = ⇒ 𝐸𝑆 =
[𝐸𝑆] 𝐾1
𝐾2
𝐸𝑆 ֞ 𝑃 + 𝐸
 𝐾1 𝐸 [𝑆] 𝐸 [𝑆]
(i) 𝐸 + 𝑆 ֞ 𝐸𝑆, 𝐾1 = ⇒ 𝐸𝑆 =
[𝐸𝑆] 𝐾1
𝐾2
𝐸𝑆 ֞ 𝑃 + 𝐸
𝐸 𝑆
Total enzyme, 𝐸𝑜 = 𝐸𝑆 + [𝐸] = + 𝐸
𝐾1

𝐸𝑜
∴ 𝐸 =
[𝑆]
1+
𝐾1
𝑑𝑝
Now, the reaction rate: 𝑉= = 𝐾2 𝐸𝑆
𝑑𝑡
𝐸 [𝑆] 𝐾2 𝑆 [𝐸0 ]
= 𝐾2 =
𝐾1 𝐾1 +[𝑆]

Here, 𝐾2 𝐸0 = 𝑉𝑚𝑎𝑥 , then the rate equation is:

𝑉𝑚𝑎𝑥 𝑆
𝑉=
𝐾1 + [𝑆]
 𝐸 [𝑆] 𝐸 [𝑆]
(ii) 𝐸 + 𝑆 ⟺ 𝐸𝑆 ; 𝐾1 𝐾1 = ⇒ [𝐸𝑆1 ] =
[𝐸𝑆1 ] 𝐾1
[𝐸𝑆1 ] 𝐸𝑆1 𝐸 [𝑆]
𝐸𝑆1 ⟺ 𝐸𝑆2 ; 𝐾2 𝐾2 = ⇒ 𝐸𝑆2 = ⇒
[𝐸𝑆2 ] 𝐾2 𝐾1 𝐾2
𝐸𝑆2 ⟺ 𝑃 + 𝐸; 𝐾3
 𝐸 [𝑆] 𝐸 [𝑆]
(ii) 𝐸 + 𝑆 ⟺ 𝐸𝑆 ; 𝐾1 𝐾1 = ⇒ [𝐸𝑆1 ] =
[𝐸𝑆1 ] 𝐾1
[𝐸𝑆1 ] 𝐸𝑆1 𝐸 [𝑆]
𝐸𝑆1 ⟺ 𝐸𝑆2 ; 𝐾2 𝐾2 = ⇒ 𝐸𝑆2 = ⇒
[𝐸𝑆2 ] 𝐾2 𝐾1 𝐾2
𝐸𝑆2 ⟺ 𝑃 + 𝐸; 𝐾3

The total enzyme; 𝐸0 = 𝐸 + 𝐸𝑆1 + [𝐸𝑆2 ]

𝐸 [𝑆] 𝐸 [𝑆] 𝐸0
= 𝐸 + + ⇒ 𝐸 = [𝑆] [𝑆]
𝐾1 𝐾1 𝐾2 1+ +
𝐾1 𝐾1 𝐾2

𝑑𝑝 𝐸 [𝑆]
Now, the reaction rate: 𝑉= = 𝐾3 [𝐸𝑆2 ] = 𝐾3
𝑑𝑡 𝐾1 𝐾2

𝐾3 𝐸0 [𝑆] 𝐾3 𝐸0 [𝑆]
= 𝑆 𝑆 =
𝐾1 𝐾2 (1+𝐾 +𝐾 𝐾 ) 𝐾1 𝐾2 +𝐾2 𝑆 +[𝑆]
1 1 2

Here, 𝐾3 𝐸0 = 𝑉𝑚𝑎𝑥 , then the rate equation is:

𝑉𝑚𝑎𝑥 [𝑆]
𝑉=
𝐾1 𝐾2 + 𝐾2 𝑆 + [𝑆]
Enzymatic reaction rate using two substrates:

The rate of this enzymatic reaction depends on the reaction pathway.

Let consider S1 and S2 are two substrates produce product P in presence
of catalyst E. Several pathways are possible to conduct this reaction.
1st pathway E+S1 = ES1; K1,
ES1+S2 = ES1S2; K2,
ES1S2=P+E; K3,

2nd pathway E+S1 = ES1; K1,

E+S2 = ES2; K2,
ES1+ES2=P+2E; K3,

3rd pathway E+S1 = ES1; K1 ,

E+S2 = ES2; K2,
ES1+ES2= ES1ES2; K3,
ES1ES2=P+2E;
 1st pathway E+S1 = ES1; K1,
ES1+S2 = ES1S2; K2,
ES1S2=P+E; K3,
𝐸 [𝑆1 ] 𝐸 [𝑆1 ]
𝐾1 = ⇒ [𝐸𝑆1 ] =
[𝐸𝑆1 ] 𝐾1
[𝐸𝑆1 ][𝑆2 ] 𝐸𝑆1 [𝑆2 ] 𝐸 [𝑆1 ][𝑆2 ]
𝐾2 = ⇒ 𝐸𝑆1 𝑆2 = ⇒
[𝐸𝑆1 𝑆2 ] 𝐾2 𝐾1 𝐾2
The total enzyme;
𝐸0 = 𝐸 + 𝐸𝑆1 + [𝐸𝑆1 𝑆2 ]
𝐸 [𝑆1 ] 𝐸 [𝑆1 ][𝑆2 ] 𝐸0
= 𝐸 + + = [𝑆 ] [𝑆 ][𝑆 ]
𝐾1 𝐾1 𝐾2 1+ 𝐾1 + 𝐾1 𝐾 2
1 1 2
Now, the reaction rate:
𝑑𝑝
𝑉= = 𝐾3 [𝐸𝑆1 𝑆2 ]
𝑑𝑡
𝐸 [𝑆1 ][𝑆2 ] 𝐾3 𝐸0 [𝑆1 ][𝑆2 ] 𝐾3 𝐸0 [𝑆1 ][𝑆2 ]
= 𝐾3 = [𝑆 ] [𝑆 ][𝑆 ] =
𝐾1 𝐾2 𝐾1 𝐾2 (1+ 𝐾1 + 𝐾1 𝐾 2 ) 𝐾1 𝐾2 +𝐾2 𝑆1 +[𝑆1 ][𝑆2 ]
1 1 2
𝑉𝑚𝑎𝑥 [𝑆1 ][𝑆2 ]
Here, 𝐾3 𝐸0 = 𝑉𝑚𝑎𝑥 , then the rate equation is: 𝑉 =
𝐾1 𝐾2 +𝐾2 𝑆1 +[𝑆1 ][𝑆2 ]
2nd pathway E+S1 = ES1; K1 ,
E+S2 = ES2; K2 ,
ES1+ES2=P+2E; K3 ,

𝐸 [𝑆1 ] 𝐸 [𝑆1 ]
𝐾1 = ⇒ [𝐸𝑆1 ] =
[𝐸𝑆1 ] 𝐾1
[𝐸][𝑆2 ] 𝐸 [𝑆2 ]
𝐾2 = ⇒ 𝐸𝑆2 =
[𝐸𝑆2 ] 𝐾2
The total enzyme; 𝐸0 = 𝐸 + 𝐸𝑆1 + [𝐸𝑆2 ]
𝐸 [𝑆1 ] 𝐸 [𝑆2 ] 𝐸0
= 𝐸 + + ⇒ 𝐸 = [𝑆 ] [𝑆 ]
𝐾1 𝐾2 1+ 𝐾1 + 𝐾2
1 2
𝑑𝑝
Now, the reaction rate: 𝑉= = 𝐾3 [𝐸𝑆1 ][𝐸𝑆2 ]
𝑑𝑡
𝐸 [𝑆1 ] 𝐸 [𝑆2 ] 𝐾3 𝐸0 2 [𝑆1 ][𝑆2 ]
= 𝐾3 = [𝑆1 ] [𝑆2 ] 2
𝐾1 𝐾2 𝐾1 𝐾2 1+ 𝐾 + 𝐾
1 2

2 𝑉𝑚𝑎𝑥 [𝑆1 ][𝑆2 ]

Here, 𝐾3 𝐸0 = 𝑉𝑚𝑎𝑥 , then the rate equation is: 𝑉 = [𝑆 ] [𝑆 ] 2
𝐾1 𝐾2 1+ 1 + 2
𝐾1 𝐾2
 Enzyme Inhibitor :

Any molecules that decrease the rate of velocity of an enzyme-catalyzed

reaction are known as enzyme inhibitor. The inhibitor may be organic or
inorganic in nature.
Example: Drugs, Poisons, antibiotics etc.

Three types of enzyme inhibition.

1. Competitive inhibition
2. Uncompetitive inhibition
3. Non-competitive inhibition
Competitive Inhibition:

- Reversible reaction
- Overcome by increasing substrate
concentration
Uncompetitive Inhibition:

- usually irreversible
- permanent damage
Non-competitive Inhibition:

- is usually reversible
- are not influenced by
concentration of the substrate
 Comparison among different inhibition:

Competitive inhibition Uncompetitive inhibition Non-competitive inhibition

Effect of competitive inhibitor on enzymatic kinetics:
E+S = ES; K S,
The sequence of reaction mechanisms is: E+I = EI; KI,
ES = P+E; K,
𝐸 [𝑆] 𝐸 [𝑆]
𝐾𝑆 = ⇒ [𝐸𝑆] =
[𝐸𝑆] 𝐾𝑆
[𝐸][𝐼] 𝐸 [𝐼]
𝐾𝐼 = ⇒ 𝐸𝐼 =
[𝐸𝐼] 𝐾𝐼

The total enzyme; 𝐸0 = 𝐸 + 𝐸𝑆 + [𝐸𝐼]

𝐸 [𝑆] 𝐸 [𝐼]
= 𝐸 + +
𝐾𝑆 𝐾𝐼
𝐸0
⇒ 𝐸 =
[𝑆] [𝐼]
1+ +
𝐾𝑆 𝐾𝐼
Effect of competitive inhibitor on enzymatic kinetics:

𝑑𝑝
Now, the reaction rate: 𝑉= = 𝐾[𝐸𝑆]
𝑑𝑡
𝐸 [𝑆] 𝐾[𝐸0 ][𝑆]
=𝐾 = [𝑆] [𝐼]
𝐾𝑆 𝐾𝑆 1+ +
𝐾𝑆 𝐾𝐼

𝐾[𝐸0 ][𝑆]
=
[𝐼]
[𝑆] + 𝐾𝑆 1 +
𝐾𝐼
Here, 𝐾[𝐸0 ] = 𝑉𝑚𝑎𝑥 , then the rate equation is:
𝑉𝑚𝑎𝑥 [𝑆]
𝑉=
[𝐼]
[𝑆] + 𝐾𝑆 1 +
𝐾𝐼
Michaelise Menten constant known as the apparent Michaelis constant.
𝑎𝑝𝑝 𝐼
𝐾𝑀 = 𝐾𝑠 1 +
𝐾𝐼
 Chapter 3
Immobilization of Enzyme
 Chapter 3
Immobilization of Enzyme

the imprisonment of cell or enzyme in a distinct support

or matrix
Advantages of immobilized enzymes:

• Increased functional efficiency of enzyme

• Enhanced reproducibility of the process
• Reuse of enzyme
• Continuous use of enzyme
• Less labour input in the processes
• Saving in capital cost and investment of the process
• Minimum reaction time
• Less chance of contamination in products
• More stability of products
Disadvantages of enzyme immobilization:

• High cost for the isolation, purification and recovery of

active enzyme (most important disadvantage)
• Industrial applications are limited
• Catalytic properties of some enzymes are reduced or
completely lost after their immobilization on support or
carrier.
• Some enzymes become unstable after immobilization.
• Enzymes are inactivated by the heat generated in the
system
Applications of enzyme immobilization:

(1) Industrial production: antibiotics, beverages, amino

acids etc.
(2) Biomedical applications:
(3) Food industry: Enzymes like pectinases and cellulases
immobilized on suitable carriers are successfully used
in the production of jams, jellies and syrups from
fruits and vegetables.
(4) Research:
(5) Production of bio-diesel from vegetable oils.
(6) Waste water management: treatment of sewage and
industrial effluents.
Applications of enzyme immobilization:

(7) Textile industry: scouring, bio-polishing and

desizing of fabrics.
(8) Detergent industry: immobilization of lipase
enzyme for effective dirt removal from cloths.
Supports or Matrix used in immobilization technology

(1) Natural polymers

(2) Synthetic polymers
(3) Inorganic materials
(1) Natural polymers

(a) Alginate: derived from the cell wall of some algae.

Calcium or magnesium alginate is the most commonly
used matrix. They are inert and have good water holding
capacity.
(b) Chitosan and chitin: structural polysaccharides
produced from cell wall of fungi and exoskeleton of
Arthropods. Enzymes can bind to the – OH group of
chitin and can form covalent bonds.
(c) Collagen: protenaceous support with good porosity
and water holding capacity. The side chains of the amino
acids in the collagen and that of enzyme can form
covalent bonds to permanently hold the enzyme
(1) Natural polymers

(d) Carrageenan: sulfated polysaccharide obtained from some

red algae. Their good gelling properties together with its high
protein holding capacity makes it good support for
immobilizing enzymes.
(e) Gelatin: Gelatin is the partially hydrolyzed collagen (f)
Cellulose: cheapest support available as carrier of enzymes.
The –OH can form covalent bonds with amino acids of enzyme.
(g) Starch: A natural polymer of amylose and amylo-pectin. It
has good water holding capacity.
(h) Pectin: structural polysaccharide of plants found in their
primary cell wall and they also acts as the inter-cellular
cementing material in plant tissues. Pectin is a gelling agent
with good water holding capacity.
(2) Synthetic polymers

They are ion exchange resins or polymers and are

insoluble supports with porous surface. Their porous
surface can trap and hold the enzymes or whole cells.

Example:
Diethylaminoethyl cellulose (DEAE cellulose)
Polyvinyl chloride (PVC),
UV activated Polyethylene glycol (PEG)
(3) Inorganic materials

(a) Zeolites: They are microporous, aluminosilicate minerals

with good adsorbing properties.
(b) Ceramics: They are nonmetallic solids consisting of metal
and nonmetal atoms held in ionic and covalent bonds.
(c) Diatomaceous earth: silicious sedimentary rocks formed by
fossilized accumulations of the cell wall of diatoms. Celite is
the trade name of diatomaceous earth. A good adsorbent and are
resistant to high pH and temperature.
(d) Silica:
(e) Glass:
(f) Activated carbon
(g) Charcoal
Methods of Immobilization:

• Adsorption
• Covalent bonding
• Entrapment
• Copolymerization
• Encapsulation
Methods of Immobilization: Adsorption

The support or carrier used may be of different types such

as:
• Mineral support (Eg. aluminum oxide, clay)
• Organic support (Eg. starch)
• Modified sepharose and ion exchange resins
There is no permanent bond formation.
Only weak bonds stabilize the enzymes to the support or
carrier. The weak bonds (low energy bonds) involved are
mainly:
(a) Ionic interaction
(b) Hydrogen bonds
(c) Van der Waal forces
Methods of adsorption:

(1). Static process: Immobilization to carrier by allowing

the solution containing enzyme to contact the carrier
without stirring.
(2). Dynamic batch process: Carrier is placed in the
enzyme solution and mixed by stirring or agitation.
(3). Reactor loading process: Carrier is placed in the
reactor, and then the enzyme solution is transferred to the
reactor with continuous agitation.
(4). Electrode position process: Carrier is placed near to
an electrode in an enzyme bath and then the current is put
on, under the electric field the enzyme migrates to the
carrier and deposited on its surface.
Advantages of adsorption method:
a. No pore diffusion limitation
b. Easy to carry out
c. No reagents are required
d. Minimum activation steps involved
e. Comparatively cheap method of
immobilization
f. Less disruptive to enzyme than
chemical methods
Disadvantages of adsorption method:
a. Desorption of enzymes from the carrier
b. Efficiency is less
Methods of Immobilization: Covalent bonding

Important functional groups :

1. Alpha carboxyl group at ‘C’ terminal of
enzyme
2. Alpha amino group at ‘N’ terminal of enzyme
3. Epsilon amino groups of Lysine and Arginine
in the enzyme
4. β and γ carboxyl groups of Aspartate and
Glutamate
5. Phenol ring of Tyrosine
6. Thiol group of Cysteine
7. Hydroxyl groups of Serine and Threonine
8. Imidazole group of Histidine
9. Indole ring of Tryptophan
Carriers or supports used for covalent bonding:

(a) Carbohydrates: Eg. Cellulose, DEAE cellulose, Agarose

(b) Synthetic agents: Eg. Polyacrylamide
(c) Protein carriers: Collagen, Gelatin
(d) Amino group bearing carriers: Eg. amino benzyl cellulose
(e) Inorganic carriers: Porous glass, silica
(f) Cyanogen bromide (CNBr)-agarose and CNBr Sepharose
Methods of covalent bonding:

(1). Diazoation: Bonding between amino group of support and

tyrosil or histidyl group of enzymes.
(2). Peptide bond: Bonding between amino or carboxyl groups
of the support and that of the enzyme.
(3). Poly functional reagents: Use of a bi-functional or
multifunctional reagent (glutaraldehyde) which forms covalent
bonds between the amino group of the support and amino
group of the enzyme.
Advantages of covalent bonding:

(a) Strong linkage of enzyme to the support

(b) No leakage or desorption problem
(c) Comparatively simple method
(d) A variety of support with different functional groups
(e) Wide applicability
Disadvantages of covalent bonding
(a). Chemical modification of enzyme leading to the loss of
functional conformation of enzyme.
(b). Enzyme inactivation by changes in the conformation when
undergoes reactions at the active site. This can be overcome
through immobilization in the presence of enzyme’s substrate
or a competitive inhibitor.
Methods of Immobilization: Entrapment

1. Enzymes are physically entrapped inside a

porous matrix
2. may be covalent or non-covalent bond
Commonly used matrixes for entrapment:

• Polyacrylamide gels
• Cellulose triacetate
• Agar
• Gelatin
• Carrageenan
• Alginate
Methods of entrapment:

• Inclusion in the gels: enzymes trapped inside the gels.

• Inclusion in fibers: enzymes supported on fibers made of
matrix material.
• Inclusion in microcapsules: Enzymes entrapped in
microcapsules formed by monomer mixtures such as
polyamine and calcium alginate.
Advantages of entrapment:

• Fast method of immobilization

• Cheap (low cost matrixes available)
• Easy to practice at small scale
• Mild conditions are required
• Less chance of conformational changes in enzyme
• Can be used for sensing application
Disadvantages of entrapment:

• Leakage of enzyme
• Pore diffusion limitation
• Chance of microbial contamination
• Not much success in industrial process
Methods of Immobilization:
Cross linking (copolymerization)

1. there is no matrix or support involved

2. Commonly used polyfunctional reagents
are glutaraldehyde and diazonium salt
Methods of Immobilization:
Cross linking (copolymerization)

Advantages of copolymerization:
• This technique is cheap and simple but not often used
with pure enzymes. This method is widely used in
commercial preparations and industrial applications.

Disadvantages of copolymerization:
• The greatest disadvantage or demerit of this method is
that the polyfunctional reagents used for cross linking
the enzyme may denature or structurally modify the
enzyme leading to the loss of catalytic properties.
Methods of Immobilization: Encapsulation

• enclosing the enzymes in a membrane capsule.

• semi-permeable membrane like nitro cellulose or
nylon
Methods of Immobilization: Encapsulation

Advantages of encapsulation:
• Cheap and simple method
• Large quantity of enzymes can be immobilized by
encapsulation

Disadvantages of encapsulation:
• Pore size limitation
• Only small substrate molecule is able to cross the
membrane
 Generalized comparison of different enzyme
immobilization techniques
Characteristics Adsorpt Covalent Entrapment/ Encapsula
ion binding cross linking tion
Preparation Simple Difficult Difficult Simple
Cost Low High Moderate High
Binding force Variable Strong Weak Strong
Enzyme leakage Yes No Yes No
Applicability Wide Selective Wide Very wide
Running Problems High Low High High
Matrix effects Yes Yes Yes No
Large diffusional No No Yes Yes
barriers
Microbial protection No No Yes Yes
Other methods of Immobilization: Ionic Binding

• method is simple and reversible but, in general, it is

difficult to find conditions under which the enzyme
remains both strongly bound and fully active
Other methods of Immobilization:
Chelation or Metal Binding
• The metal salt or hydroxide is precipitated onto the
support
• The method is quite simple and the immobilized
specific activities obtained with enzymes in this way
have been relatively high
Immobilization of whole cells:

• It is a well-developed method for the utilization of

enzymes from microbes
• Immobilization of whole cells become particularly
effective when the individual enzymes become
inactive during direct immobilization
• Bacteria or yeast cells are immobilized by adsorbing it
on woodchips
Advantages of whole cell immobilization:

• Multiple enzymes can be introduced to a single step

• Extraction and purification of enzymes are not
required
• Enzymes are stable for long time
• Native conformation of enzyme is best maintained
• Cell organelles like mitochondria and chloroplasts can
be immobilized
• Cost effective method
Disadvantages of whole cell immobilization:

• Concentration of enzymes will be less

• Production of unwanted enzymes and unwanted
products
• Modification of end products by other enzymes
produced by immobilized cells
Methods of immobilization of whole cells:

Methods of immobilization of whole cells are same as

that described for enzyme immobilization and they
include:
1. Adsorption
2. Covalent bonding
3. Cell to cell cross linking
4. Encapsulation
5. Entrapment
 Immobilization of cells:
Methods, Support materials, Cells and Reaction
Method Support material Cells Reaction
Gelatin Lactobacilli Lactose => Lactic acid

Adsorption Porous glass Saccharomyces Glucose => ethanol

Cotton fibers Zymomonas Glucose => ethanol
DEAE Cellulose Nocardia Steroid conversion
Cellulose+
Covalent S. cerevisiae Glucose => ethanol
cyanuric Chloride
bonding
Titanium oxide Acetobacter Vinegar
Crosslinking Glutaraldehyde E. coli Fumaric acid
Alluminium
Candida tropicalis Phenol degradation
Entrapment alginate
Calcium alginate S. cervisiae Glucose => ethanol
Polyester Streptomyces sps. Glucose => fructose
Encapsulation Alginate
Properties to select a carrier-materials :

Physical Requirements-
• Pore-size and Available Surface
• Internal Structure
• Particle Size
• Hydrophilicity/hydrophobicity
• Insolubility
• Mechanical stability/rigidity
• Form and size of support
• Resistance to microbial attack
• Regenerability
Properties to select a carrier-materials :

Chemical Nature of the Carriers -

• Nature of Binding Functionality
• charge properties
• polarity of the functionality
• Density of functionality
• Distributing of charge
• hydrophobicity
• size
• The Role of the Spacer
• The Nature of the Backbone
Mass transfer effects of immobilized enzyme
on enzymatic reaction:
• Immobilization of enzymes means a deliberate
restriction of the mobility of the enzyme, which can
also affect mobility of the solutes.
• The various phenomena, referred to as mass transfer
effects, can lead to a reduced reaction rate, in other
words to a reduced efficiency as compared to the
soluble enzyme.

• Porous Diffusion
• Reaction (Dynamic) and Support-Generated (Static)
Proton Gradients
Porous Diffusion:

what are the reasons for restrictions caused by mass

transfer, and how can they be avoided if necessary?

For Michaelis–Menten based enzyme kinetics the extent

of mass transfer control is usually expressed by the
efficiency coefficient or effectiveness factor η, expressed
as:
𝑉 ′ 𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑖𝑚𝑚𝑜𝑏𝑖𝑙𝑖𝑧𝑒𝑑 𝑒𝑛𝑧𝑦𝑚𝑒
𝜂=
𝑉0 (𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 𝑜𝑓 𝑓𝑟𝑒𝑒 𝑒𝑛𝑧𝑦𝑚𝑒)
𝑉𝑚𝑎𝑥 𝑆
𝑉′ = 𝜂
𝐾𝑀 + 𝑆
Porous Diffusion:

In which the assumptions are considered as:

- Enzyme is uniformly distributed in a spherical
support particle.
- The reaction kinetics follows Michaelis-Menten
kinetics.
- There is no external diffusion limitation.
Porous Diffusion:

𝜂 = න(𝛷𝑅 , 𝛽)

𝑉𝑚
𝛷𝑅 = 𝑅
𝐾𝑀 + 𝐷𝑒𝑓𝑓
𝑆
𝛽=
𝐾𝑀
ΦR=Thiele modulus
β =dimensionless Michaelis constant
𝑉𝑚 = assuming maximum intrinsic activity per accessible
catalyst volume,
𝐷𝑒𝑓𝑓 = effective diffusion coefficients
which can be used to increase efficiency:

• No diffusional limitations for high substrate

concentration
• Decreasing the particle size of the carriers.
• Lowering of enzyme loading with high specific activity
which is easily achieved by common fixation methods.
• Preferential binding on the outer shell of carrier
materials will enable increased efficiencies.
Reaction (Dynamic) and Support-Generated
(Static) Proton Gradients:
The observed pH-shifts in activity may indeed also be
caused by:
1. Static proton or substrate gradients caused by partition
of charged groups near surfaces with stationary
charges.
2. Dynamic proton gradients due to hydrolytic reactions
with liberation of protons as has frequently been
observed for immobilized enzymes.
Minimize the effects of reaction-generated
dynamic pH-gradients:
• By reducing enzyme density and/or particle size as
indicated for substrate-mediated diffusional control.
• By using buffers with sufficient capacity (> 0.05 M)
that minimize the dynamic pH-gradients. It should not
be lower than the pK-value of the weak acid and not
lower than the optimum pH of the enzyme.
• By operating at an external pH higher than the
optimum pH of the enzyme.
• By co-immobilization of a proton-consuming enzyme.
Any question?