Enzymes

 Enzymes are important catalysts in

living organisms.
 They reduce the amount of activation
energy required for a reaction to
proceed.
 Enzymes may be pure proteins or
proteins plus cofactors such as
metallic ions or coenzymes.
Enzyme Function

• An enzyme works by binding with its substrate, the molecule

whose reaction is catalyzed.
• The active site is the location on the enzyme where the substrate
fits.
• Enzyme + Substrate = ES complex.
Normal Enzyme Reaction
Main differences between free microorganism & free enzyme

• Microorganism is used for multipurpose: formation of product and by-

product.
• Free enzyme produces a single product. Free enzyme can not be re-used
after use. Free enzyme is expensive. Therefore, immobilization
technique should be applied for re-use.
• Enzyme is more sensitive to temperature than microorganism. The
enzyme structure begins to break down (denature) at higher
temperatures (up to 37 °C)
 Immobilization technique: confining/linking the cells/enzymes to a solid
support over which a substrate is passed and converted to a products. Chitin,
chitosan and alginate could be used as a solid support.

Advantages:
 Re-use of enzymes/cells for many reaction
cycles.
 Lowering the total production cost.
 Lowering the contamination risk.
 Ability to stop the reaction
rapidly by removing the
enzyme/cell from the reaction
solution.
 Enhanced stability.
 Easy separation of the enzyme/cell
from the product.
Immobilization techniques
Immobilization techniques
Each microorganism is a tool
to obtain final product
for bioprocess engineering
 Cellular Metabolism
• Cellular metabolism refers to all of the chemical processes that occur inside
living cells.

• Kinetic energy – energy of motion.

• Potential energy – stored energy
• Free energy – the energy available for doing work.
-Most chemical reactions release free energy – they are exergonic.
-Some reactions require the input of free energy – they are endergonic.

First law of thermodynamics – energy cannot be created or destroyed – only

transformed. The second law of thermodynamics states that the total entropy
of an isolated system can never decrease over time.
• Activation energy is the energy required for a chemical reaction to
occur.

• Anabolic reactions involve the joining of smaller molecules together

to form larger or more complex molecules. This occurs through
dehydration synthesis reactions.
• Catabolic reactions are a type of metabolic reaction that take place
within a cell. Larger molecules are separated to form smaller
molecules, as in the case of respiration where glucose is broken down
to form carbon dioxide and water. Often they are hydrolysis reactions
in which water molecules are used to break bonds.
 Oxidation – Reduction - Redox
• An atom that loses an electron
has been oxidized. Oxygen is
a common electron acceptor.
• An atom that gains an
electron has been reduced.
Higher energy.

• Redox reactions always occur in pairs.

• One atom loses the electron, the other gains the electron.
• Energy is transferred from one atom to another via redox reactions.
 Raw material Microorganism or Enzyme Biomass
and/or Final product
Substrate (food) By-product
Energy
 Cellular Respiration
• Cellular respiration is the oxidation of food molecules to obtain energy
• In aerobic respiration, ATP forms as the electrons are harvested, transferred
along the electron transport chain and eventually donated to O2 gas.
• Oxygen is required!
• Glucose is completely oxidized.

C6H12O6 + 6O2 6CO2 + 6H2O + energy

Glucose Oxygen Carbon Water Heat or ATP
Dioxide

• Anaerobic respiration occurs in the absence of oxygen.

• Different electron acceptors are used instead of oxygen (sulfur, or nitrate).
• Sugars are not completely oxidized, so it does not generate as much ATP

• Anaerobic metabolism can only use glucose and glycogen, while aerobic
metabolism can also break down fats and protein. In aerobic metabolism, smaller
quantity of food required to maintain the given rate of metabolism.
Cellular Respiration in 3 Stages
1. Food is digested to break it into smaller
pieces – no energy production here.
2. Glycolysis – coupled reactions used to
make ATP (fuel molecule).
• Occurs in cytoplasm
• Does not require O2
3. Oxidation – harvests electrons and uses
their energy to power ATP production. That
pathway is called Krebs cycle (TCA cycle).
• Only occurs in mitochondria
• More powerful
 • Glycolysis – the second
stage in cellular respiration.
• A series of enzyme catalysed
reactions.
• Glucose converted to pyruvic
acid.
• 2 moles of ATP made in the
absence of oxygen.
• All living organisms use
glycolysis.

Glucose + 2ADP + 2Pi + 2 NAD+ 2 Pyruvic acid + 2 NADH + 2ATP

Producing Acetyl-CoA
• The 3-carbon pyruvate loses a
carbon producing an acetyl
group.
• Electrons are transferred to NAD+
forming NADH.
• The acetyl group combines with
CoA forming Acetyl-CoA.
• Ready for use in Krebs cycle.
The Krebs Cycle
• The Krebs cycle is the next stage in oxidative respiration and
takes place in the mitochondria.
• The Krebs cycle is aimed to produce biomass, protein, amino acid, and
energy (fuel molecules: ATP and NADPH)

Acetyl unit + 3 NAD+ + FAD + ADP + Pi 2 CO2 + 3 NADH + FADH2 + ATP

Glucose

Phospoenol
Pyruvic acid
Fatty acid

Amino acid

• Acetyl-CoA joins cycle, binding to a 4-carbon molecule to form a 6-carbon molecule.

• 2 carbons removed as CO2, their electrons donated to NAD+, 4-carbon molecules left.
• More electrons are extracted and the original 4-carbon material is regenerated.
• 1 ATP, 3 NADH, and 1 FADH2 are produced.
 SUMMARY

36 moles of ATP is produced

in cellular respiration

Glucose + 2 ATP + 36 ADP + 36 Pi + 6 O2 6CO2 + 2 ADP + 36 ATP + 6 H2O

 Anaerobic respiration

• In the absence of oxygen, pyruvate is used.

Entner–Doudoroff pathway (ED pathway)

• Glycolysis involves the conversion of

glucose-6-phosphate to fructose-6-
phosphate (F6P). This reaction
occurs with the help of the enzyme
phosphoglucose isomerase (PI).
 Metabolic Regulation
Biyocatalyst (cell): The cell acts as a reactor

Metabolites:
Primary metabolites are
occurred in the lag phase of cell
Inhibition
DNA Induction Metabolite growth. They are produced under
Enzyme
Repression Activation (Product) optimum conditions.
Secondary metabolites are
occurred in the stationary phase
of cell growth. They are produced
under stress/extreme conditions

Control of enzyme synthesis Control of enzyme activity: 1. Inhibition: competitive, uncompetitive and non-competitive inhibition
2. Activation
 Control of enzyme synthesis

• Biocatalyst (cell) does not always synthesize enzyme. If glucose is present in the culture
medium, the cell will not synthesize enzyme. If there is no glucose and starch is present in the
culture medium, the cell will synthesize amylase enzyme to degrade starch to glucose.
• Structure of DNA:
Promoter synthesizes RNA polymerase
Regulator gene synthesizes repressor
DNA Template

RNA polymerase
 None-Induction mechanisms

• RNA polymerase (the enzyme responsible for the

transcription of genes) is unable to bind to the
Promoter region of the operon.
• If RNA polymerase does not bind to the Promoter
region, the 3 lac operon structural genes (lacZ, lac Y,
and lacA) are not transcribed into mRNA.
• Without the transcription of these genes, the
enzymes are not synthesized.

Transcription is blocked An Inducible Operon in the Absence of an Inducer

 Induction mechanisms
• The binding of a molecule called an inducer alters the shape of the repressor in a way that
now blocks its binding to the operator and thus permits transcription. This is referred to as an
inducible system.

Structural genes are

transcribed into mRNA and
enzyme synthesis

Transcription is permitted: enzyme synthesis

An Inducible Operon in the Presence of an Inducer
 Repression mechanisms
• Regulator gene synthesises apo-repressor. In the presence of co-
repressor, the active repressor is formed and it binds to operator.
• RNA polymerase does not bind to the Promoter region, the 3 lac
operon structural genes (lacZ, lac Y, and lacA) are not transcribed
into mRNA.

Transcription is blocked

In the Presence of co-repressor

Control of Enzyme Activity
Competitive Inhibition
• Competitive inhibition involves a molecule (inhibitor)
• The molecule (inhibitor) is structurally and chemically similar to the substrate (hence
able to bind to the active site). Inhibitor binds to the enzyme’s active site
• The competitive inhibitor blocks the active site and thus prevents substrate
binding
• As the inhibitor is in competition with the substrate, its effects can be reduced by
increasing substrate concentration. In other words, if an inhibitor is competitive, it
will decrease reaction rate when there's not much substrate.
Non-competitive Inhibition
• Non-competitive inhibition involves a molecule (inhibitor) binding to an allosteric
site.
• The binding of the inhibitor to the allosteric site causes a conformational change
to the enzyme’s active site.
• As a result of this change, the substrate cannot bind the active site.
• As the inhibitor is not in direct competition with the substrate, increasing
substrate levels cannot decrease the inhibitor’s effect.
Uncompetitive inhibition

An uncompetitive inhibitor is an inhibitor that only binds to the enzyme-substrate

complex. The uncompetitive inhibition does not work when additional substrates
are trying to be involved.
 Enzyme Activation
Some enzymes require a molecule known as an activator to attach to their
allosteric site in order for the active site to be receptive to substrate.
As a result, the enzyme catalyzes a reaction.
Engineering Calculations
 Unit Conversions
• 1 lb = 453.6 g
• 1 ft = 0.3048 m
• 1 inch = 2.54 cm
• 1 hp (horse power)= 42.41 Btu min-1
• 1 Btu=0.2520 kcal
• 1 K = °C + 273
• 1 mmHg = 1.316 x 10-3 atm
• 1 cm3 = 1 mL
• 1 N = 1 kgms-2
• 1 Ibm ft-1 h-1 = 4.134 x 10-4 kgm-1s-1
• 1 Pas = 1 kgm-1s-1
• 1 dyn cm-1= 1 gs-2
• 1 cP = 10-3 kgm-1s-1
• 1 ppm = (g solute/g solution)×106
 Dimensionless variables
• Reynolds number , Schmidt number, Prandtl number,
Sherwood number, Peclet number, Nusselt number,
Grashof number, power number and many others.
 Recall

• 5% NaOH = 5 g NaOH in 100 g solution

• 30% (v/v) H2SO4 solution = 30 ml H2SO4 in 100
ml water
• 0.15% w/v cell solution = 0.15 gr cell in 100 ml
solution
• Molecular weight = Dimensionless
Molecular weight of CO2 is 44
Q1)The volumetric flow rate of carbon tetrachloride (CCI 4) in a pipe
is 50 cm3 min-1. The density of CCI4 is 1.6 g cm-3. The molecular weight
of carbon tetrachloride (CCI 4) is 153.8 (1 gmol CCl4=153.8 g).
 What is the mass flow rate of CCl4?
 What is the molar flow rate of CCl4?
The volumetric flow rate of carbon tetrachloride
(CCI 4) in a pipe is 50 cm3 min-1. The density of CCI4 is
1.6 g cm-3. The molecular weight of carbon
tetrachloride (CCI 4) is 153.8 (1 gmol CCl4=153.8 g)

A1)
 Q2
Determine the amount of (NH4)2SO4 to be supplied in a
fermentation medium where the final cell concentration is
30 g/L in a 103 L culture medium. Assume that the cells are
12% nitrogen by weight and (NH4)2SO4 is the only nitrogen
source.
MW of (NH4)2SO4 = 132

Nitrogen content of cells: 0.12 g nitrogen/g cell

Total nitrogen in cells =?

 A2

Total nitrogen in cells =?

 A2

Total nitrogen in cells =

(Nitrogen content of cells)x(Final cell concent.)x(Cult.volume) =
(0.12 g nitrogen/g cells) x (30 g cells/L) x (1000 liters)
=3.6 kg nitrogen
 A2

Amount of (NH4)2SO4 needed = ?

 A2

Amount of (NH4)2SO4 needed = ?

3.6 kg nitrogen x132 kg (NH4)2SO4 / 28 kg nitrogen
= 17 kg (NH4)2SO4
Q3) What mass of oxygen required to produce 15 g glutamic acid ?
A3)