A Not On Biochemical Engineering

1
BIOCHEMICAL ENGINEERING I (CHE 461)

2 credits
Introduction – Preamble, Scope of Biochemical Engineering, Microbiology, Viruses.

Chemicals of Life. Kinetics of substrate utilization. Product yield and biomass production in
cell cultures. Kinetics of enzyme – catalyzed reactions. Applied enzyme catalysis. Metabolic
stoichiometry and energetic. Molecular genetics and control systems. Transport phenomena in
microbial systems.
30h (T) PR: CHM212 C.
CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Department of Chemical Engineering, University of Ilorin.
Engr. Dr E.O. Ajala - 2018
2
CHAPTER ONE
Introduction
Who is a biochemical engineer?
A biochemical engineer is someone who when with engineers, talks biology, when with
biologists, talks engineering, and when with biochemical engineers, talks politics. (V.
Hatzimanikatis) Biochemical engineering is a multidisciplinary field:
Related disciplines, and also areas of specialization of biochemical engineering:
1. Metabolic engineering, including metabolic modelling
2. Bioprocess engineering, including process control
3. Bio separations
4. Bioinformatics
5. Biomaterials engineering
6. Tissue engineering
7. Manufacturing engineer, reactor design
Areas of biochemical engineering cover research at the scale of the biocatalyst development
(microbe, insect cell, mammalian cell, plant cells, and enzyme) from the genetic level to the
culture development, including reactor design and manufacturing plant.

3
An example of what a biochemical engineer might do:
A biologist selects for a microbe to overproduce acetic acid from carbon monoxide, hydrogen
and carbon dioxide gas. In order to sell the product, a biochemical engineer can evaluate the
following:
What is the key constraint in the process? i.e. what are the problems to be solved before the
process can be profitable?
Profit = Selling price of product – (Cost of starting materials + Cost of production
Here is a scenario:
A microbiologist gives you a bacterial strain of bacteria, and he claims that it can produce 15
g/l (grams/litre) of a potential drug candidate for Parkinson's disease prevention. First, you
throw it in a laboratory reactor and after some media optimization using literature reports and
statistical experimental design, you achieve the following result: 30 g/l titre, 1.5 g/l/h rate and
a yield of 15% on your starting material. So you try it in larger reactors, keeping the
temperature, pH and starting material constant in each case, and put together the following
table of results:
Reactor stir speed titer (g/l) rate yield on

working (rpm) (g/l/h) starting
volume material
(liters)
1 1500 30 1.5 15%

10 1500 50 2.5 30%
1000 600 60 2.8 35%
10,000 300 20 0.8 10%
Why do you think the titre and rate increased on going from a 1L to a 10L reactor? From a 10L
to a 1000L reactor? Do you think the effect of scale up on the yield is related to the increase in
titre and rate? Why do you think the titre and rate decreased upon scale-up to a 10,000 litres
reactor?
Knowing that you must use a 10,000 litres reactor to produce sufficient drug to meet the market
demand and pay your bills, you might consider that you should optimize your reaction
conditions in the large reactor.
Why it is a bad idea and what could be an alternative approach?
As a B.Sc. Chemical Engineer (biochemical engineer) you could do the following:

Manufacturing , Fermentation development and optimization, Media optimization, Screening,
Separations, Analytical methods, Research, Development of all of the above at small scale with
4
guidance. An important aspect of manufacturing in pharmaceutical companies is Good

Manufacturing Practices (GMP) which involves:
• Methods
• Facilities
• Documentation
SCOPE OF BIOCHEMICAL ENGINEERING
Biochemical Engineering has a multiplicity of applicability and some of them are as follows:
Fermentation processes :- These involve both aerobic and anaerobic processes for the
production of ethanol, butanol, acetone, citric acid, ethanoic acid, oxalic acid, antibiotics,
sewage disposal, curing of hides, silage making, food products which are dependent on
microorganisms and enzymes systems for their characters and flavours, examples of which are
beer, cheese, wine and cocoa.
Operation of food processing :- This includes pasteurization (heating a material to 700C for
30 minutes to partly destroy the microbes to remove the harmful ones), sterilization (total
annihilation of microbes which is usually carried out using wet steam at 1210C) and
preservation using chemicals (addition of chemicals such as sodium benzoates and nitrite, in
ppm, to prevent growth of microbes), deep freezing, cooking and drying (since microbes need
water for survival, drying destroy microbes), salting (to increase the osmotic pressure outside
the microbes which eventually dehydrates the microbes). In the manufacture of sera (serum
(singular) is a liquid containing substances that fight infection) and vaccines. This involves the
development of methods of producing and preserving the products.
Extraction processes :- These are used for the production of insulin and other hormones, fats
from oil seeds and blubbers, essential oils and perfumes, infusions such as tea and coffee, bone
products such as glue and gelatin. Physical processing of clothing materials like cotton, wool,
leather and fibres. Processing of forests and crops products such as timbers as raw materials
for paper together with other products. Annexing of photosynthesis such as extraction of
protein from leaves.
Single cell production: - This involves the production of microbial proteins for animal feeds
and food supplement from renewable resources and wastes e.g. production of lucozade, glucose
D. etc.

5
CHAPTER TWO
MICROBIOLGY
Introduction
Microbiology has been defined as the study of living organisms, which are too small to be seen
clearly with the naked eyes. These organisms may be visualized as the expanding chemical
reactors, which take in chemical species called nutrients from the environment, grow,
reproduce and release products into the environment or surrounding. In sewage treatment, the
consumption of nutrients is the engineering objective. In this case, we are looking for an
organism which consumes a lot of the substrate but grow very little indicating low biomass
yield. As food sources, in SCP production, the mass of the microbial matter produced is the
object of importance. Here we look for an organism that consumes a little of the substrate but
grows a lot this implies a high biomass yield. The products formed and released during
biological activities are of major importance e.g penicillin and ethanol manufacturing.
Weight of cell produced

Biomass yield =
Weight of cell consumed
Classification of microbes
Most of microbes belong to the Protist Kingdom and the classifications as depicted in the figure
below:
Protist
Prokaryotes Eukaryotes
Bacteria Blue-green Protozoa Algae Fungi

algae
Yeast Mould
Protists
These are all living things with very simple biological organisation relative to plants and
animals. They are generally unicellular organisms or organisms containing multiple cells which
are all of the same type.

6
Prokaryotes
These are simplest organisms with very little internal cell differentiation. They usually exist
alone and can be spherical (cocci), rod-like (bacilli) or spirila (spiral shape). The volume is
usually about 10-12 ml/cell and they usually contain 50-80% water and with mass of
approximately 10-12 g/cell, hence they have a density approximately equal to that of water,
therefore they form a kind of suspension in water but in large quantity they can settle. They
grow rapidly and are very widespread. This relate to the omnipresence of microbes
(everywhere is full of microbes). They can accept a wide variety of nutrients but they select
the best from the available resource.
Characteristics
• They have a rigid cell wall which is about 2000A. They gives structural strength to the
organism. (0A=10-8 cm)
• They have cell membranes or plasma membrane which is about 700A in thickness. This
is a semi-permeable membrane which is used for osmotic control (regulation).
• They have a nuclear zone and not a nucleus. This is the control centre for all cell
operations and the genes are contained here.
• The ribosomes which are the sites for important biochemical reactions are scattered in
the cytoplasm which is the fluid occupying the rest of the cell.
• Examples of prokaryotes include bacteria and blue-green algae. In blue-green algae
which are usually found in the oceans, we have photosynthesis.
Eukaryotes
Unlike prokaryotes which lack internal cell differentiation, these cells have a substantial degree
of spatial organization and differentiation. They are about 1000 to 10,000 times larger than
prokaryotes. All the cells of higher animals are of this type. They have different forms. The
Eukaryotes have organelles. Eukaryotes cells exist in higher animal.
The structure is as follows:
• A cell wall or coat depending on the cell. The plants have rigid cell wall while animals
have soft cell coat.
• A plasma membrane.
• Endoplasmic reticular which are convoluted membrane systems from the cell
membrane into the cell.
• They have the nucleus with a porous membrane. They also have the ribosomes which
are attached to the endoplasmic reticular. They have the mitochondria which are
organelles involved in oxidative phosphorylation (ATP production). In photosynthetic
cells, we have chloroplasts. Eukaryotes can have vacuoles, nucleus, and ribosomes. All
are called organelles (organ-like).

7
Bacteria
These are prokaryotes that are typically unicellular, which could be spherical (cocci) with a
dimension of 0.5-4m in diameter. They can also be rodlike (bacilli) with dimension of 0.5-4m
in diameter and 0.5-20m in length. They can be in form of a spiral (spirila) which has the
dimension of 10m in length and 0.5m in diameter. They have a density slightly greater than
1.0g/cm3.
Classification of organisms
The classification may base on several factors, some of which are described below:
1. Staining characteristics: - Generally, most microbes or bacteria are transparent; hence

they cannot be seen distinctly under a microscope. To facilitate the observation they are
generally stained. When cells are stained with dye-crystal violet, a purple coloured cell
can be formed. When it is treated with iodine solution and washed in alcohol those ones
that retain the purple colour are said to be Gram positive while those that do not have
the purple colour are Gram negative. These are all functions of the cell wall.
2. Morphology: - This classification is based on the association between the cells during
colony formation. Those cells that grow only in a single plain and do not possess sticky
cell wall stay as single cells while those that possess sticky cell wall stay as strings and
are called Streps. The streps which are cocci are called Streptococcus while those that
are bacilli are called Streptobacillus. When the cells grow in the three plains and
possess sticky cell walls and stay in grape-like clumps called Staphs. They are called
Staphylococcus and Staphylobacillus when they are cocci and bacilli respectively.
Those organisms which are bean shaped and always paired are the Niseria.
3. Mode of respiration: - This considers the way by which the organism obtains its ATP
and they are classified as stated below:
I. An aerobe is an organism that respires in the presence of free oxygen. Such a
process where oxygen is present and used is called aerobic. Examples of such
are the production of (a) Vinegar (b) Antibiotic and single cell protein (SCP)
(like bacteria and yeast) which contain 50% protein in dry form.
II. Anaerobes: - These are organisms which respire in the absence of free oxygen.
Such organisms obtain their oxygen from sulphate and phosphate. Such
processes in which oxygen is not present are called anaerobic. Examples are
beer and ethanol production. The word fermentation was obtained from Latin
word fermentiar which means to be boiling was reserved initially to anaerobic
processes but presently it loosely used for every activity that takes place in a
fermentor, including the aerobic ones.
III. Obligate aerobes: - These are organisms that can grow only in the presence of
oxygen. Examples are Rhizobium species like the Nitrobacter which is found in
the roots of leguminous plants and the yeast Trichosporon cutaneum which is a
strict aerobs and is used as a model organism used to study oxygen transfer
efficiency in bioreactor systems.

8
IV. Obligate anaerobes: - These are organisms that cannot tolerate a slightest
amount of oxygen. An example is the Niseria gonorrhea or the tetanus causing
organism.
V. Facultative aerobes or facultative anaerobes: - These are organisms that
grow both in the presence and absence of oxygen. Example is the yeast
Saccharomyces cerevisiae. In fermentation systems, lack of oxygen leads to the
production of ethanol while with a lot of oxygen supply will produce more of
the microorganism though ethanol may be produced under aerobic conditions
due to Crabtree effect or glucose effect.
4. Sources of Carbon and Energy: - Organisms may be classified based on its source of
carbon and energy and this is summarized below.
Classification Source of Carbon Source of Energy

Chemoheterotroph Organic compound Organic compound
Chemoautotroph Carbon dioxide Organic compound
Photoheterotroph Organic compound Sunlight
Photoautotroph Carbon dioxide Sunlight
5. Mode of Existence
I. Vegetative cells: – This is the growing form of the organism which can be
easily destroyed by heat and chemicals.
II. Spore or endospore: - This is the dormant and protective form which can
survive the moderate heat and chemical environment. The spore formers are
usually difficult to destroy because it is the presence of heat that stimulates their
growth. So they cannot be destroyed by pasteurization. These can only be
destroyed by heat teasing and sterilization. Sterilization is performed usually by
subjecting the system to 15psi of wet (saturated) steam for about 20mins.
III. Temperature of growth: Microorganisms can also be classified according to
the temperature at which they grow best.
• Psycrophiles – grow best at less than 200C temperature.
• Mesophiles – grow best at 20 -350C temperature.
• Thermophiles – grow best at temperature greater than 400C e.g.
Streptococcus thermophillus.
6. Reproduction mode: - The mode of growth may be used in the classification of
microbes;
• Binary fission – When an initial surface area per unit volume is reached by
growth, the organism starts to reproduce by breaking.
• Budding – An out-growth takes place from the mature cell and this out growth
develops to the same size as the mother cell before breaking off. The number of
generation may be determined by the number of scars on the cell.
9
• Spore formation – when a cell is in distress due to environmental changes, it

forms spores which can develop into vegetative cell when the situation changes.
Yeast
These are eukaryotes which are elliptical in shape, having a length of about 8-15m and diameter
of 3-5m. They are facultative aerobes and they are the most industrially important group of
microbes being used in wine production, bear production, single cell protein for animal feed
and human diet supplementation because of the high content of Vitamin B complex. Also they
are used in bakery operation. They reproduce both by binary fission and budding.
Molds
These are highly branched myceliary eukaryotic organisms. Molds, like yeasts, do not contain
chlorophyll. Reproduction, which may be sexual or asexual, is typically accomplished by
means of spores. They are important in industrial microbiology such as the production of citric
acid and antibiotics. Examples are Aspergillus niger and Aspergillus flavus. Their shapes
influence the viscosity of the medium and create big rheological problems in the fermentor
because they become non-Newtonian at high concentration
Algae and protozoan
These are relatively large Eukaryotes and the examples of the algae are the Euglena and
Diatom. Examples of protozoan are amoeba.
Tutorials
1. With the aid of diagram, explain the kingdom of protists

2. Compare and contrast the following;
I. Prokaryotes and Eukaryotes
II. Budding, Sexual fusion, fission and sporulation
III. Protozoa, algae and amoeba
3. Explain the following;
I. Sprilla, cocci, bacilli
II. Morphology

10
CHAPTER THREE
Viruses
These are complex set of entities which consists of two parts; nucleic acid and protein coat.
Viruses are infectious agent that are two small to be seen with a light microscope, they are not
microbial cells. They have no cell nucleus. When they in fade susceptible host cell, virus
displaces some properties of living organism and so appear to be on the border line between
living and non-living thing. They are not in actual sense a living organism but when they
attached with a host, they become living organism. Viruses can replicate or multiply, only
inside a living host cell as such they are called obligate intracellular parasites. A distinction
they share with Chlamydias and Rickettsias.
Viruses differ from cells in some important ways, whereas both Prokaryotic and Eukaryotic
cells contain both DNA and RNA. Individual virus particles contain only one kind of nucleic
acid, either DNA or RNA, but never both. Cells grow and divide but viruses do neither grow
nor divide. Viral replication requires that virus particles infect a cell and programme the host
cell machinery to synthesis the component required for the assembly of new virus particle. The
infected cells may produce one hundred to one thousands of new viruses and then dies.
Structure:
They are complex set of entities which consists of two parts:
(1) Nucleic acid which either contains DNA or RNA and not both together. However, about
99.9% are DNA viruses. DNA can exist either as single stranded or double stranded. It is also
true of RNA. However, there are relatively very few double stranded RNA. The double
stranded RNA stimulates the production of interferon which is a protein produced by virus
infection and it presents successive virus infection e.g. having measles and small pox injection
most likely immunes you against further infection.
(2) Protein coat - it may be simple or complex and it may be covered with membrane which
can be dissolved by acetone or liquid solvent - The membrane in our integral part of the
infection mechanism. If the virus has its nucleic acid enclosed by the protein coat and covered
with membrane it is called an enveloped virus.
Distinguishing Characteristics
(1) The Composition- which is as described above.
(2) They are obligate intercellular parasites.
(3) They replicate in a different way from a bacterium. This it does by replicating its
Nucleic acid and then produce protein coats which are then assembled and followed by
membrane addition. After this a burst size of about 200 viruses are released. Thus this is a
viral manufacturing.

11
- 1 Virus DNA injection
- Nucleic acid production
- Protein coat and tail production
- Assembling of parts
- Membrane addition
200 viruses
(4) Host specificity (extreme) - Viruses are extremely host specific. We have highly
contagious diseases in certain species which cannot infect other species e.g. rabies. This is as
a result of the receptor factor. Every cell in nature can be found to be a host of virus.
Shapes of Viruses:
Viruses have different shapes governed by the crystallographic principle
Consider the protein
Mixing them together allows the sides which have tremendous affinity for each other to
aggregate and form hexagonal polyhedral
When there is only triangle
Variability of Viruses

12
The protein coat is coded by the nucleic acid (NA) and a modification of the amino of the coat
can change the specification of the virus. There are various types of the virus and they vary
from season to season (e.g. running nose during the maize harvest period).
Viruses have 2 components - the protein coat and the nucleus
The T-4 replication cycle
The T-4 virus is a bacteria virus which infects Eschericia coli (E. coli)
Note: When the bacteria and virus form the bases i.e bacterial and virus DNA, we have
bacteriophage. This is because it involves both the viral DNA and bacterial DNA
Assignment
1. Discuss why some are carriers of HIV and not infected.

2. Differentiate between the bacteriophage and the cell.
Specificity between bacteriophage and cell:
Virus has an attachment site while the cell has a receptor site. The virus also has a coat, which
surrounds its DNA and a complicated mechanism for injecting the DNA.
Head DNA
Core
Fibres
Protein tail
fibre
Center plug
Plug
Structure of T4 bacteriophage
The T-4 bacteriophage has a tail structure with a hexagonal base plate. It consists of long
protein tail fibres which stick out. There is a hollow core in the tail, which is surrounded by a
chord wound around, with a plug at the bottom. The tail fibre consists of long protein
molecules with NH2 and COOH groups at the end which have positive and negative signs
respectively. When placed on E. coli, only the tail fibres touch the cell. On E. coli cell wall
we have lipopolysaccharides which are oriented in such a way that we have two charges equal
but opposite to the charges on the tail fibre. This allows the fibre to attach to the cell wall. As
soon as a tail fibre hits, it sticks to the cell, the others then stick because the bouncing due to
Brownian motion is reduced. This is the first level of fitness between the attachment and the
receptor sites. There is another level of fitness when the base plate makes contact with the cell
wall. The phage lysozyme makes its way into the cytoplasmic membrane and eats a hole into
the membrane. This causes the DNA to be injected from the core into the cytoplasmic
13
membrane. The bacteria phage DNA can attach both inside and outside. When the DNA gets
inside the cell, it circularizes. There is a tremendous specificity on the phage as cells have
charges and configuration. If the cell manipulates its sugar structure, the phage may not be
able to attach. This is phage resistance.
When the DNA gets into the cell, there are promoter regions on the DNA that are recognized
by the RNA polymerase, which transcribes message which then translates to protein and
produces phage nuclease protein. The phage also has hydroxyl methylcytosine consuming
enzymes and also phage sigma. These three enzymes are called the early enzymes which are
produced by the early genes. The phage nuclease feeds on the hydroxyl methyl cytosine and
glucose. It also destroys the cell genetic information by chewing out the cell DNA. The
bacteria DNA is full of promoter regions capable of transcription and translation but when the
virus attaches, it transplants its own DNA.
The early enzymes in the process of transcription enzymes appear only for a short time and
right at the start. The nuclease chops off whole cell DNA. Phage sigma which is a globular
protein interacts with RNA polymerase and changes it in such a way that it no longer recognizes
the promoters on the bacteria DNA but the new promoters on the viral DNA and it gets involved
in actual viral protein syntheses. At the same time, viral DNA syntheses also occur.
After this, the protein and the viral DNA are assembled.
Viral
DNA
Protein
During this time, lysozyme is also being produced which begins to eat away the bacteria cell
wall such that the viruses produced can come out after the osmotic pressure in the bacteria
ruptures it. Generally, we have approximately 200 viruses. However, if the bacteria can be
prevented from bursting, the size of the batch can be increased because we have longer time.
The key events which take place in this type of infection which is virulent, because it lyses the
host cell, can be summarized as follows.
(i) Attachment on the receptive side of the host (outside).
(ii) Injection of viral DNA.
(iii) Circularization of viral DNA and attachment to cell membrane.

14
Transcription of early genes
Production of viral DNA
Production of protein coats and tails
Destruction of bacteria DNA
(vii) Transcription of “late” genes
(viii) Lysing and bursting out of the new viruses
The Step Growth Curve
The time between the entrance of one virus and the point of appearance of outside virus is the
latent period. The virus kills the cell but they cannot be killed by antibiotic even when there is
one to kill the host cell.
Classification of viruses:
Virus may be classified into two main groups, which are:
(a) Virulent - they are the viruses that always go lytic and lyse the host cell.
(b) Temperate - there are viruses that have biochemical decision making ability to go either
lytic or lysogeny. The lysogeny is a phage when viral DNA integrates with the bacteria DNA
and actually replicates with it.
Virus DNA
Bacteria DNA
CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 The Cell

SESSION
15
This is called lysogenised bacteria. In this case, you do not have an intact virus but viral
information.
Why lytic or lysogeny?
The cell contains the phage repressor which stops the syntheses of early enzymes. If the
repressor is synthesized fast enough, and then we have lysogeny as it destroys the early
enzymes and no late genes are made. If the syntheses of the phage repressor is repressed, the
lytic cycle is again induced and each bacterium ruptures producing the burst size of the virus.
When the virus DNA is inside the cell of a bacterium, it is called a prophage. The prophage
can exist in two forms which are:
Autonomous prophage –
This is when the bacteria cell contains its DNA with the viral DNA somewhere near but not
touching; yet closes enough for regulatory activities. The virus DNA in this case exists
independently and replicates orderly with the bacteria DNA.
Viral DNA attached
to membrane
Cell DNA
Integrated prophage –
In this situation, the viral DNA which was originally membrane attached now comes in contact
with the cell DNA such that a particular base comes in contact with some particular bases which
are opposite in signs to those on the cell DNA. Then they stick and there is breakage and then
exchange and finally it becomes integrated.
When the bacteria lose its repressor level, it starts producing lytic enzymes. The first enzyme
produced is called exercises enzyme which cuts out the DNA of the viruses. When induced
prophage is carried out, the exercises enzyme makes mistake and cuts off viral DNA and 20%
more cell DNA. This contains chromosomes and so it can replicate. Now we can have a
defective viral DNA. The lytic cycle that results is a normal cycle. When the cell is lysed, the
DNA containing viral particle bursts out. If the viral particle injects its DNA into another
bacterium, it can no longer go to the lytic cycle but it has to go to the prophage and this leads
to genetic exchange in bacteria. Any phage mediated genetic exchange is called transduction.
If it occurs as a function of integrated prophage (improper excision), it is called restricted
transduction, because there are particular genes to be packaged.
Bacteria phages that remain autonomous can give rise to another form of transduction called
generalized transduction. In this case, the autonomous piece of viral DNA goes through the
16
lytic induction which chops off the cell DNA but leaves a small amount of cell DNA of the
same length as the viral DNA. This may then be packaged and we now have a virus particle
consisting of the bacteria DNA among the normal viral particles. When this virus containing
the bacteria DNA infects another bacterium, it does so in a form in which it can integrate. This
is called generalized transduction because there are no particular genes to be packaged.
Pseudo-Sexual Exchange:
This is a form of genetic exchange in bacteria. This mechanism in bacteria has certain things
in common.
(1) The exchange is completely unidirectional and is not towards the production of
embryo
(2) The donor cells either dies or does not benefit from the exchange.
(3) Very small amount of information are transferred.
The conditions for this to happen must be perfectly right which implies very far
occurrences.
Pseudo-sexual exchange is necessary since it allows a mean of genetic variability. Bacteria

however, have been able to get on without this phenomenon because they reproduce very fast
(they possess high rate of reproduction).
Forms of Genetic Exchange:
Genetic exchange takes place in the form of:
(a) Transduction - this is a phage-mediated exchange, which can either be generalized or

restricted.
(b) Transformation - this is a function of naked DNA, which floats around in the
environment. For transformation to occur, the recipient cell must be receptive to the DNA.
There must be holes on the cell wall of the receptive cell to allow the DNA in without leading
to their death. The receptivity is called competence
The competence may be categorized as genetic or physiological. In genetic competence, we

can have a bacterium, which will have holes in the cell wall without dying. In physiological
competence, it can only occur under certain conditions, which is usually at the end of the log
phase (exponential phase). At this point, the autolytic enzyme concentration is high and there
are more holes made than are being repaired. At this point, the cell is taking in a lot of DNA.
(c) Conjugation - this is a more complex form of genetic exchange with levels of
precondition since it is a pseudo-sexual transfer due to cell-cell contact.
1. Sex factor F

17
Female F Male: F+
2. HFX-----Integrated F
When F integrates with the DNA, sometimes it can be excised and becomes autonomous, at
times the cut is not accurate and some of the cell DNA is cut with it.
In this case, it is autonomous F+ Bacteria DNA. This is called F1.
3. R - Factor - Code for the sex pilli, and it codes also for antibiotic resistance. It is like
an F1 except that we do not know its history. When an F+ cell conjugates with an F- cell and
transfer the sex factor, then we have sexduction thus the transfer of R - factor is an example
of sexduction.
Practical Application
The practical application of microorganism is what is called biochemical engineering.

An example is in penicillin production, which involved:
• Discovery of penicillin by Fleming (surface fermentation)

• Realization of its use by Florrey
• Production in submerged systems - 1000 gallons which involves:
- sterilization of air by the application of heat or by filtration
- agitation involving the design of agitators or impellers
- supply of nutrients - e.g. corn steep
- chalk
- phosphates
- lactose
- air supply (through small holes known as sparger) by compression
- heat remover or addition (Heat exchanger)
- Product recovery (down-stream processing).
In the case of Penicillin Production the following were carried out:
• Development of the organism by say X-ray or UV radiation to obtain for the process
• Growth of the culture - in large fermentors e.g. 1000gal. This would include:
- sterilizing the medium
- maintaining sterility
- filtering the air
- maintaining a temp of 250C
- supplying O2 (sparging of air and agitation)
- foam controller
- mycelium is filtered off on a rotary filter
Extraction of small concentration of the delicate and complex molecules  this step involves
extraction by butanol, crystallization under vacuum after removing water by distillation.

18
CHAPTER FOUR
CARBOHYDRATES
Carbohydrates constitute a major class of naturally occurring organic compounds, including

sugars, starches, and celluloses. They are essential to the maintenance of plant and animal life.
Carbohydrates are classified into three major groups: monosaccharides, oligosaccharides, and
polysaccharides. Monosaccharides are the simplest carbohydrate units. Oligosaccharides
contain two or more of these simple mono saccharide units, and polysaccharides contain
hundreds or thousands of them.
4. 1.1 Monosaccharides
The basic carbohydrate molecules are simple sugars, or monosaccharides, which are
polyhydroxy aldehyde, polyhydroxy ketone, and their derivatives. All simple monosaccharides
have the general empirical formula, (CH20)n, where n is the whole number ranging 3 to 8.
All monosaccharides can be grouped into two general classes as:
1. aldoses: contain a functional aldehyde grouping (-CHO), or
2. ketoses: contain a functional ketone grouping (> CO)
Oligo means few.
As indicated in Table 4.1, sugars can be further sub classified according to the number of
carbons: trioses, tetroses, pentoses, and hexoses. Monosaccharides have asymmetric carbon
atoms2
Table 4.1 Classification of Monosaccharides

19
The number of possible optical isomers for a compound can be determined by the formula 2n,
where n stands for the number of asymmetric carbons. As an example, aldohexose (see Table
4.1) has four asymmetric carbon atoms, second carbon through fifth carbon from the top.
Therefore, it has 16 possible isometric forms, with eight L forms and eight D forms~ The D
form has OH on the right side of the highest-numbered asymmetric carbon (fifth carbon for
aldohexos) and rotates polarized light in the +direction, while the L form has OH on the left
side and rotates polarized light in the - direction.
Glucose (or dextrose) is one of aldohexoses which has two isometric forms (Figure 4.1): D and
L. The D form predominates in the nature.
Glucose is the most common and most important hexose and is found in most sweet fruits and
in blood sugar.
D-glucose
Fig. 4.1 Two isomeric forms of glucose: fischer projection
In solution, very few sugar molecules exist with free aldehyde or ketone functional groups.
Aldehydes and hydroxyls in a sugar molecule can react in a solution so that the H from the OH
at the fifth carbon joins the aldehyde and the 0 from the same OH bonds to the first carbon, as
shown in Figure 4.2.
(X-D-glucose D-glucose P-D-glucose
Fig. 4.2 Two stereoisomeric forms of D-glucose in solution: Fischer projection formulas.

20
Haworth projection Shorthand haworth
Fisher projection
The resulting structure has a ring form which is known as cyclic hemiacetal. There is
equilibrium between the ring and open forms.
The open form allows the aldehyde or ketone group to react. The formation of a cyclic
hemiacetal generates an additional asymmetric center at the original carbonyl atom. The new
asymmetric center is known as the anorneric carbon (C* in Figure 4.2). In the linear Fischer
projection forrnulas, the structure with the anomeric hydroxyl group oriented to the right is
termed the a-form, and that to the left is {3-form. Pure fj-D-glucose equilibrates in water to
give a mixture of 64 percent f3- and 36 percent a-D-glucose. A more realistic representation
for the hemiacetal ring structure is the Haworth projection formulas. The formulas for a-D-
glucose are shown in Figure 4.3. The shorthand form of the Haworth projection eliminates the
Hs and indicates OHs by dashes. Five- and six membered cyclic sugars are called furanose and
pyranose, respectively.3 6CH20H CH20H
Fig. 4.3 Haworth projection formula of a-D-glucose (or a-D-glucopyranose)
Even though Haworth formulas give a sound representation of the ring structures of sugars, the
real structure conformation can be most accurately represented by the chair forms of
cyclohexane as shown in 3 the terms furanose and pyranose arise from attachment of
carbohydrate endings to the names of the cyclic compounds, furan and pyran.
Industrial Applications of Enzymes 73
Figure 4.4. However, despite the inaccuracy of the Haworth formulas, they are used more
frequently than the chair conformation, because they are easier to draw and interpret.

21
Fig. 4.4 Haworth and chair conformation of f3-D-glucose (or f3-D-glucopyranose).
Fructose is a keto sugar and is found in fruits and honey. Cyclization of fructose by formation
of a hemiketal between the carbonyl group at the second carbon and the hydroxyl group at the
fifth carbon gives a five-membered furanose ring, which can have two anomers, a- and {J-D-
fructose (or a- and fJ-D-fructofuranose), as shown in Figure 4.5. Fructose sweeter than other
natural sugar. If we take the relative sweetness of cane sugar as one, glucose is measured
to be 0.7 whereas fructose is 1.7. (Cheng and Lee, 1992)
Fig. 4.5 Fructose
4.1.2 Disaccharides
Two sugars can link to each other by losing water from OHs to form disaccharides. Figure 4.6
shows the Haworth projection formulas of four important disaccharides: sucrose, lactose,
maltose, and cellobiose, which all have the same molecular formulas, C12H22011' Sucrose and
lactose are the most abundant and most important disaccharides of natural origin. Maltose and
cellobiose are repeating units of polymeric starch and cellulose, respectively. Disaccharides
may hydrolyse to form two monosaccharide molecules.
Sucrose, known as table sugar, is comprised of a-D-glucose and fJ-D-fructose. The aldehyde
group (1' carbon) of glucose is linked with the ketone group (2' carbon) of fructose (1' - 2'), so
22
that no carbonyl group (-CO) from either monosaccharide portion is available as reducing
agent. For this reason, sucrose is termed a non-reducing sugar. Sucrose is the only non-reducing
sugar among the four disaccharides. Lactose, sugar present in milk, is a dimer of fi-D-galactose
bonded (1' - 4') with D-glucose. The aldehyde group of the left ring of lactose is used for
linkage. However, the right ring of the lactose can be opened to react because its aldehyde
group is not used for linkage. As a result, lactose is a reducing sugar.
Fig. 4.6 Important disaccharides
Maltose is repeating units of starch and can be obtained by the hydrolysis of starch using the
diastase enzyme. Further hydrolysis of maltose yields two molecules of glucose. Cellobiose, a
stereoisomer of maltose, is obtained by the partial hydrolysis of cellulose. Maltose and
cellobiose are both reducing sugars, since the right rings may open to react, as reducing agents.
4.1.3 POLYSACCHARIDES
Polysaccharides consist of many simple sugar units linked together. One of the most important
polysaccharides is starch, which is produced by plants for food storage. Animals produce a
related material called glycogen.
Starch comprises a large percentage of cereals, potatoes, corn, and rice. Complete hydrolysis
of starch yields glucose, but partial hydrolysis gives maltose as well. This shows that starch is
a polymer of glucose units, joined by a-glycosidic linkage. Starch can be separated into two
main fractions by treatment with hot water. The insoluble component (10 percent to 25 percent)
is amylose; the soluble component (75 per cent to 90 percent) is amylopectin. The glucosyl
residues (glucose minus water) of amylose are linked by a (1' - 4') glycosidic bonds in a single
chain that contains up to 4,000 glucose units (Figure 4.7). The long linear molecule of amylose
exists as a helix that contains six glucosyl residues per turn.

23
Fig.4.7 Structure of amylose and amylopectin.
Amylopectin is a highly branched amylose. Various length of the linear chains, a (1' - 4')
glucans containing 20 to 25 residues, are linked to a core chain by a (1' - 6') glycosidic bonds
(Figure 4.7).
Amylose and amylopectin are degraded by a- and f3-amylase, which are found in the pancreatic
juice and saliva of animals, a-amylase is an endoglycosidase which attacks the amylose and
amylopectin randomly along a (1' - 4') bonds. F3-amylase is an exoglycosidase which removes
maltose units successively from the non-reducing end of the chain. Neither enzyme can
hydrolyse a (1' - 6') branch points, which can be degraded by other enzymes, called debranching
enzymes. Another enzyme, amylogucosidase (also called glucoamylase), releases glucose units
from the non-reducing end of the chain. Enzymatic hydrolysis of starch by sequential treatment
with a-amylase and glucoamylase will produce glucose as the main final product.
Dextrins, products of the partial hydrolysis of starch, are polysacchandes of lower molecular
weight than starch. They are used in infant food because they are easier to digest than starches.
Dextrins are sticky when wet and are used as mucilage on postage stamps and envelopes.
Cellulose is one of the three major structural components of all plant cell walls with two other
components, hemicellulose and lignin.
Cellulose is the most abundant organic compound of natural origin on the face of the earth.
Complete hydrolysis of cellulose gives glucose. The cellulose molecule is comprised of long
chains of cellobiose molecules joined together by fi-I, 4-glucosidic bonds as shown in Figure
4.8. The molecular weight of cellulose ranges from 300,000 to 500,000 (1,800 to 3,000 glucose
units). The digestive systems of man and most other animals (except ruminants) do not contain
the necessary enzymes (cellulase) for hydrolyzmg p-glucosidic linkages. However, cellulases
are found in ruminants, various insects, fungi, algae, and bacteria.

24
Fig. 4.8 Structure of cellulose
4.2 STARCH CONVERSION
In recent years, the conversion of starch to fructose has become a very important commercial
process. High-fructose corn syrup (HFCS) is approximately twice as sweet as sucrose. It is
used in soft drinks, canned fruits, lactic acid beverages, juice, and bread, ice cream, frozen
candies, and so on. HFCS can be obtained from a variety of cereals and vegetables, such as
corn, wheat, rice, potatoes, and cassava. Corn is the most important source of HFCS because
of low costs and excellent utilities of its by-products, corn meal, oil, gluten, germ, and fibre.
Corn Wet Milling: The first step of the HFCS process is the corn wet milling (Joglekar et al.
1983). Corn is cleaned, shelled, and transferred to a large steep tank containing warm water
(54°C) with 0.1 to 0.2 percent sulfur dioxide (pH 3-4). The steeping lasts about 40 hours.
The sulfur dioxide inhibits fermentation and helps softening of the kernel.The steeped corn
kernels are torn apart in a degerminating mill to free the germ (containing corn oil) and to
loosen the hull. The germ is separated in a continuous liquid cyclone, washed, and dried for oil
recovery. Starch and hull are ground and screened to eliminate the hull. The resulting mill
starch contains 5 to 8 percent protein which is separated in a centrifuge. The separated-out
starch is further purified in a hydroclone to reduce the protein content to a minimum level of
0.3 percent.

25

26
Fig. 4.9 Corn refining process (D.E. means dextrose equivalent)
Corn Refining Process: The starch slurry from the wet milling process is broken down to
glucose and isomerized to fructose as shown in Figure 4.9. The starch slurry is gelatinized by
cooking at high temperature (104 - 107°C) for 5 to 8 minutes and liquefied by a-amylase into
low-molecular-weight dextrins and maltose. The syrup produced has D.E. (dextrose
equivalent) of 15%. The enzyme used for this step, a-amylase, is thermostable and splits the
starch at the interior of the molecule (Joglekar et al., 1983).
After liquefaction, the syrup is further hydrolysed to glucose by the action of fungal
glucoamylase, which acts on starch by splitting glucose units from the non-reducing end. It
takes about 40 to 80 hours at a temperature between 55-60°C and pH of 4-4.5. After
saccharification, the liquor (95 percent free glucose) is filtered, and passed through activated
carbon, diatomaceous earth' and ion exchange columns to remove impurities, colour, and salts.
It is then concentrated in an evaporator to 60 percent solids.
The glucose syrup obtained is isomerized to fructose by passing through an immobilized

isomerase colum. The glucose isomerase is immobilized by an inert carrier, such as glass beads
or DEAEcellulose. A typical residence time is 30 minutes. The isomerisation reaction is
reversible with an equilibrium constant of about 1.0 at 60°C. Therefore, the expected final
concentration of fructose will be less than half of the inlet glucose concentration. The finished
product leaving the reactor contains 42 percent fructose, 50 percent glucose, and other
saccharides. After isomerization, the syrup is purified by passing through a filter and ion-
exchanger and is concentrated in an evaporator.
4.3 Cellulose Conversion
Cellulosic wastes have great potential as a feedstock for producing fuels and chemicals.
Cellulose is a renewable resource that is inexpensive, widely available and present in ample
quantities. Large amounts of waste cellulose products are generated by commercial and
agricultural processes. In addition, municipal facilities must treat or dispose of tremendous
quantities of cellulosic solid waste.
4.3.1 LIGNOCELLULOSIC MATERIALS
Lignocellulosic materials have a common basic structure, but vary greatly in chemical
composition and physical structure.4 Typically, these materials contain 30 percent to 60
percent cellulose, 10 percent to 30 percent hemicellulose (polyoses), and 10 percent to 20
percent ligmn. Cellulose provides strength and flexibility, while lignin supports and protects
the cellulose from biological and chemical attack. Hemicellulose bonds lignin to cellulose.
Native cellulose is basically composed of microfibrils, which are bundles of lamellae
containing an indefinite number of fibrillar units.

27
Their schematic representation is shown in Figure 4.10. Cellulose molecules, hydrophilic linear
polymers, are linked together to form elenzentary fibrils (or photofibrils), about 40A wide, 30A
thick, and 100A long. The linear polymers in an elementary fibril are oriented in a parallel
alignment and are bounded by hydrogen bonds to form a crystalline region, which is
surrounded by a disordered layer of cellulose molecules, an amorphous region or
paracrystalline region (Ranby, 1969). Microfibrils in cell wall components are again
surrounded by hemicelluose layer and lignin.
Fig. 4.10 Schematic cross section of a lamellae of cellulose microbibrils
Although cellulose and lignin are both polymers and major components of woody biomass,
their chemical characteristics are totally different. Lignin is a complex aromatic biopolymer of
high molecular weight and is formed by the polymerization of oxidatively formed radicals of
p-hydroxycinnamyl alcohols (Hira et aI., 1978). It should be noted that the term lignin cannot
be regarded as one individually defined compound, but rather, as a collective term for a whole
series of similar, large polymeric molecules which are closely related structurally to one
another. The complexity of the chemical structure of lignin makes it very difficult to utilize
except as a fuel. Since isolated lignin is a by-product of the pulp industry, its economical
utilization has been actively sought. Because of its relatively high calorific value (12,700
BTUlib), lnost of waste lignin is being used as fuel in the chemical recovery processes of the
pulp plants. Only a small part of lignin is utilized in adhesives, structural polymers, coating,
dispersants, soil conditioner, pesticide carrier, and so on. Several processes for the conversion
of polymeric lignin to simple chemical feedstock have been developed. However,
0111yvanillin, dimethyl sulfide, and methyl mercaptan are produced in commercially
significant quantities (Drew et al., 1978). Hemicellulose (or polyose) is primarily composed of
xylan, a branched polymer composed of five-carbon sugar, xylose. Typical polymerization
degree of hemicellulose is 50 - 200, which is shorter than the cellulose molecules. The acid
28
hydrolysis of hemicellulose, (C6H100S)n, produces mainly xylose (C6H100S), which can be

converted to furfural, a chemical feedstock, or can be fermented to ethanol.
Cellulose Pre-treatment and Hydrolysis
Major obstacles in the hydrolysis of cellulose are the interference of lignin (which cements
cellulosic fibres together) and the highly ordered crystalline structure of cellulose. These
obstacles necessitate a costly pre-treatment step in which elementary cellulosic fibrils are
exposed and separated. Many pre-treatments have been employed to enhance the degradation
of lignocellulosic materials to glucose. The treatments fall into two general areas (Ryu and Lee,
1983):
1. physical pre-treatment milling, irradiation, heating, and heating with other pre-
treatment, and
2. Chemical pre-treatment alkali treatments, acid treatments, delignification, and
dissolving and re-precipitating.
Ball milling is the most commonly used pre-treatment. It reduces crystallinity and particle
sizes, while it increases surface area, bulk density, and the water soluble fraction. The major
drawbacks of the milling are cost and the fact that non-cellulosic substances are not removed.
Common chemical pre-treatments of alkali and acid contacting improve hydrolysis by breaking
down the lignin, hemicellulose, and cellulose. However, chemical hydrolysis is not specific
and a variety of products are formed. A balance must be met between the enhanced hydrolysis
and production. of undesirable by-products. Delignification treatment, such as Kraft and
sulphite pulping used in the pulp-and-paper industry, is too expensive to be considered as an
economical pre-treatment. The rate and extent of enzymatic hydrolysis was found to be
increased significantly by combining the pretreatment and reaction steps into one process
(Kelsey and Shafizadeh, 1980; Ryu and Lee, 1983; Deeble and Lee, 1985; Jones and Lee,
1988). The separate processes can be combined by using an attrition bioreactor, which is a
stirred reactor with stainless-steel balls (Ryu and Lee, 1983; Deeble and Lee, 1985; Jones and
Lee, 1988). By using this reactor, the amount of time required for the hydrolysis of newsprint
or sawdust can be reduced to hours as compared to days which are necessary in a regular stirred
reactor. Enhanced conversion of cellulose in the attrition bioreactor is due to a combination of
factors, including a reduction in crystallmity, an increase in pore volume and surface area, and
an increase in the accessibility of glucosidic bond sites to the cellulase complex. It was also
found that enzyme deactivation in the attrition bioreactor is not significant, since interfacial
forces, not shear forces, cause the most deactivation. Elimination of the air- liquid interface by
covering the reactor substantially increased the enzyme stability.

29
CHAPTER FIVE
PROTEINS
Introduction
Proteins are the most abundant biological macromolecules, occurring in all cells and all parts
of cells. Proteins also occur in great variety; thousands of different kinds, ranging in size from
relatively small peptides to huge polymers with molecular weights in the millions, may be
found in a single cell. What is most remarkable is that cells can produce proteins with strikingly
different properties and activities by joining the same 20 amino acids in many different
combinations and sequences. From these building blocks different organisms can make such
widely diverse products as enzymes, hormones, antibodies, transporters, muscle fibers, the lens
protein of the eye, feathers, spider webs, rhinoceros horn, milk proteins, antibiotics, mushroom
poisons, and myriad other substances having distinct biological activities. Among these protein
products, the enzymes are the most varied and specialized. Virtually all cellular reactions are
catalyzed by enzymes.
Proteins are polymers of amino acids, with each amino acid residue joined to its neighbor by
a specific type of covalent bond. (The term “residue” reflects the loss of the elements of water
when one amino acid is joined to another.) Proteins can be broken down (hydrolyzed) to their
constituent amino acids by a variety of methods, and the earliest studies of proteins naturally
focused on the free amino acids derived from them. Twenty different amino acids are
commonly found in proteins.
Amino acids and peptides
Proteins are a diverse and abundant class of biomolecules, constituting more than 50%
of the dry weight of cells. This diversity and abundance reflect the central role of
proteins in virtually all aspects of cell structure and function. An extraordinary diversity
of cellular activity is possible only because of the versatility inherent in proteins, each
of which is specifically tailored to its biological role. The pattern by which each is
tailored resides within the genetic information of cells, encoded in a specific sequence
of nucleotide bases in DNA. Each such segment of encoded information defines a gene,
and expression of the gene leads to synthesis of the specific protein encoded by it,
endowing the cell with the functions unique to that particular protein. Proteins are the
agents of biological function; they are also the expressions of genetic information.
Proteins Are Linear Polymers of Amino Acids

30
Chemically, proteins are unbranched polymers of amino acids linked head to tail, from
carboxyl group to amino group, through formation of covalent peptide bonds, a type of
amide linkage (Figure 5.1). Peptide bond formation results in the release of H2O. The
peptide “backbone” of a protein consists of the repeated sequence –N-Cα-C-, where the
N represents the amide nitrogen, the Cα is the α -carbon atom of an amino acid in the
polymer chain, and the final C is the carbonyl carbon of the amino acid, which in turn
is linked to the amide N of the next amino acid down the line. The geometry of the
peptide backbone is shown in Figure 5.2. Note that the carbonyl oxygen and the amide
hydrogen are trans to each other in this figure. This conformation is favoured
energetically because it results in less steric hindrance between non-bonded atoms in
neighbouring amino acids. Because the -carbon atom of the amino acid is a chiral centre
(in all amino acids except glycine), the polypeptide chain is inherently asymmetric.
Only L-amino acids are found in proteins.
Figure 5.1: Peptide formation is the creation of an amide bond between the carboxyl group of one amino
acid and the amino group of another amino acid. R1 and R2 represent the R groups of two different
amino acids.
Peptide Classification
Peptide is the name assigned to short polymers of amino acids. Peptides are classified
by the number of amino acid units in the chain. Each unit is called an amino acid residue,
the word residue denoting what is left after the release of H2O when an amino acid
forms a peptide link upon joining the peptide chain. Dipeptides have two amino acid
residues, tripeptides have three, tetra-peptides have four, and so on. After about 12
residues, this terminology becomes cumbersome, so peptide chains of more than 12 and
less than about 20 amino acid residues are usually referred to as oligopeptides, and,
when the chain exceeds several dozen amino acids in length, the term polypeptide is
used. The distinctions in this terminology are not precise.
Proteins Are Composed of One or More Polypeptide Chains
The terms polypeptide and protein are used interchangeably in discussing single
polypeptide chains. The term protein broadly defines molecules composed of one or
more polypeptide chains. Proteins having only one polypeptide chain are monomeric
proteins. Proteins composed of more than one polypeptide chain are multimeric
31
proteins. Multimeric proteins may contain only one kind of polypeptide, in which case
they are homomultimeric, or they may be composed of several different kinds of
polypeptide chains, in which instance they are heteromultimeric. Greek letters and
subscripts are used to denote the polypeptide composition of multimeric proteins. Thus,
an α2-type protein is a dimer of identical polypeptide subunits, or a homodimer.
Hemoglobin (Table 5.1) consists of four polypeptides of two different kinds; it is a α2β2
heteromultimer. Polypeptide chains of proteins range in length from about 100 amino
acids to 1800, the number found in each of the two polypeptide chains of myosin, the
contractile protein of muscle. However, titin, another muscle protein, has nearly 27,000
amino acid residues and a molecular weight of 2.8 X 106. The average molecular weight
of polypeptide chains in eukaryotic cells is about 31,700, corresponding to about 270
amino acid residues. Table 5.1 is a representative list of proteins according to size. The
molecular weights (Mw) of proteins can be estimated by a number of physicochemical
methods such as polyacrylamide gel electrophoresis or ultracentrifugation. Precise
determinations of protein molecular masses are best obtained by simple calculations
based on knowledge of their amino acid sequence. No simple generalizations correlate
the size of proteins with their functions. For instance, the same function may be fulfilled
in different cells by proteins of different molecular weight. The Escherichia coli enzyme
responsible for glutamine synthesis (a protein known as glutamine synthetase) has a
molecular weight of 600,000, whereas the analogous enzyme in brain tissue has a
molecular weight of just 380,000.

32

33
Acid Hydrolysis of Proteins
Peptide bonds of proteins are hydrolysed by either strong acid or strong base. Because
acid hydrolysis proceeds without racemization and with less destruction of certain
amino acids (Ser, Thr, Arg, and Cys) than alkaline treatment, it is the method of choice
in analysis of the amino acid composition of proteins and polypeptides. Typically,
samples of a protein are hydrolysed with 6 N HCl at 110°C for 24, 48, and 72 hr in
sealed glass vials. Tryptophan is destroyed by acid and must be estimated by other
means to determine its contribution to the total amino acid composition. The OH-
containing amino acids serine and threonine are slowly destroyed, but the data obtained
for the three time points (24, 48, and 72 hr) allow extrapolation to zero time to estimate
the original Ser and Thr content (Figure 5.5). In contrast, peptide bonds involving
hydrophobic residues such as valine and isoleucine are only slowly hydrolysed in acid.
Another complication arises because the β- and γ-amide linkages in asparagine (Asn)
and glutamine (Gln) are acid labile. The amino nitrogen is released as free ammonium,
and all of the Asn and Gln residues of the protein become aspartic acid (Asp) and
glutamic acid (Glu), respectively. The amount of ammonium released during acid
hydrolysis gives an estimate of the total number of Asn and Gln residues in the original
protein, but not the amounts of either. Accordingly, the concentrations of Asp and Glu
determined in amino acid analysis are expressed as Asx and Glx, respectively. Because
the relative contributions of [Asn + Asp] or [Gln + Glu] cannot be derived from the
data, this information must be obtained by alternative means.
FIGURE 5.5 ● (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course
of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero
allows an accurate estimation of the amount of these amino acids originally present in the protein
sample. (b) Peptide bonds involving hydrophobic amino acid residues such as valine and isoleucine
resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations
approach a limiting value that can be approximated with reliability.

34
Amino Acid Analysis of Proteins
The complex amino acid mixture in the hydrolysate obtained after digestion of a protein
in 6 N HCl can be separated into the component amino acids by either ion exchange
chromatography or by reversed-phase high-pressure liquid chromatography (HPLC).
The amount of each amino acid can then be determined. In ion exchange
chromatography, the amino acids are separated and then quantified following reaction
with ninhydrin (so-called postcolumn derivatization). In HPLC, the amino acids are
converted to phenylthiohydantoin (PTH) derivatives via reaction with Edman’s reagent
(see Figure 5.19) prior to chromatography (precolumn derivatization). Both of these
methods of separation and analysis are fully automated in instruments called amino acid
analyzers. Analysis of the amino acid composition of a 30-kD protein by these methods
requires less than 1 hour and only 6 g (0.2 nmol) of the protein. Table 5.2 gives the
amino acid composition of several selected proteins: ribonuclease A, alcohol
dehydrogenase, myoglobin, histone H3, and collagen. Each of the 20 naturally
occurring amino acids is usually represented at least once in a polypeptide chain.
However, some small proteins may not have a representative of every amino acid. Note
that ribonuclease (12.6 kD, 124 amino acid residues) does not contain any tryptophan.
Amino acids almost never occur in equimolar ratios in proteins, indicating that proteins
are not composed of repeating arrays of amino acids. There are a few exceptions to this
rule. Collagen, for example, contains large proportions of glycine and proline, and much
of its structure is composed of (Gly-x-Pro) repeating units, where x is any amino acid.
Other proteins show unusual abundances of various amino acids. For example, histones
are rich in positively charged amino acids such as arginine and lysine. Histones are a
class of proteins found associated with the anionic phosphate groups of eukaryotic
DNA. Amino acid analysis itself does not directly give the number of residues of each
amino acid in a polypeptide, but it does give amounts from which the percentages or
ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight
and the exact amount of the protein analyzed are known (or the number of amino acid
residues per molecule is known), the molar ratios of amino acids in the protein can be
calculated. Amino acid analysis provides no information on the order or sequence of
amino acid residues in the polypeptide chain. Because the polypeptide chain is
unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-
terminal or C-terminal end.
The Sequence of Amino Acids in Proteins
The unique characteristic of each protein is the distinctive sequence of amino acid
residues in its polypeptide chain(s). Indeed, it is the amino acid sequence of proteins
that is encoded by the nucleotide sequence of DNA. This amino acid sequence, then, is
a form of genetic information. By convention, the amino acid sequence is read from the
35
N-terminal end of the polypeptide chain through to the C-terminal end. As an example,
every molecule of ribonuclease A from bovine pancreas has the same amino acid
sequence, beginning with N-terminal lysine at position 1 and ending with C-terminal
valine at position 124 (Figure 5.6). Given the possibility of any of the 20 amino acids
at each position, the number of unique amino acid sequences is astronomically large.
The astounding sequence variation possible within polypeptide chains provides a key
insight into the incredible functional diversity of protein molecules in biological
systems, which is discussed shortly.

36
*Proteins are as follows:
RNase: Bovine ribonuclease A, an enzyme; 124 amino acid residues. Note that RNase lacks tryptophan.
ADH: Horse liver alcohol dehydrogenase, an enzyme; dimer of identical 374 amino acid polypeptide
chains. The amino acid composition of ADH is reasonably representative of the norm for water-soluble
proteins.
Mb: Sperm whale myoglobin, an oxygen-binding protein; 153 amino acid residues. Note that Mb lacks
cysteine.
Histone H3: Histones are DNA-binding proteins found in chromosomes; 135 amino acid residues. Note
the very basic nature of this protein due to its abundance of Arg and Lys residues. It also lacks
tryptophan.
Collagen: Collagen is an extracellular structural protein; 1052 amino acid residues. Collagen has an
unusual amino acid composition; it is about one-third glycine and is rich in proline.
Note that it also lacks Cys and Trp and is deficient in aromatic amino acid residues in general.

37
Architecture of Protein Molecules
Protein Shape
As a first approximation, proteins can be assigned to one of three global classes on the
basis of shape and solubility: fibrous, globular, or membrane (Figure 5.7). Fibrous
proteins tend to have relatively simple, regular linear structures. These proteins often
serve structural roles in cells. Typically, they are insoluble in water or in dilute salt
solutions. In contrast, globular proteins are roughly spherical in shape. The polypeptide
chain is compactly folded so that hydrophobic amino acid side chains are in the interior
of the molecule and the hydrophilic side chains are on the outside exposed to the
solvent, water. Consequently, globular proteins are usually very soluble in aqueous
solutions. Most soluble proteins of the cell, such as the cytosolic enzymes, are globular
in shape. Membrane proteins are found in association with the various membrane
systems of cells. For interaction with the nonpolar phase within membranes, membrane
proteins have hydrophobic amino acid side chains oriented outward. As such,
membrane proteins are insoluble in aqueous solutions but can be solubilized in solutions
of detergents. Membrane proteins characteristically have fewer hydrophilic amino acids
than cytosolic proteins.
FIGURE 5.7 ● (a) Proteins having structural roles in cells are typically fibrous and often water
insoluble. Collagen is a good example. Collagen is composed of three polypeptide chains that
intertwine. (b) Soluble proteins serving metabolic functions can be characterized as compactly folded
globular molecules, such as myoglobin. The folding pattern puts hydrophilic amino acid side chains on
the outside and buries hydrophobic side chains in the interior, making the protein highly water soluble.
(c) Membrane proteins fold so that hydrophobic amino acid side chains are exposed in their membrane-
associated regions. The portions of membrane proteins extending into or exposed at the aqueous
environments are hydrophilic in character, like soluble proteins. Bacteriorhodopsin is a typical
membrane protein; it binds the light-absorbing pigment, cis-retinal, shown here in red.

38
The Levels of Protein Structure
The architecture of protein molecules is quite complex. Nevertheless, this complexity

can be resolved by defining various levels of structural organization.
Primary Structure
The amino acid sequence is the primary (1°) structure of a protein, such as that shown
in Figure 5.6, for example.
Secondary Structure
Through hydrogen bonding interactions between adjacent amino acid residues

(discussed in detail in Chapter 6), the polypeptide chain can arrange itself into
characteristic helical or pleated segments. These segments constitute structural
conformities, so-called regular structures that extend along one dimension, like the coils
of a spring. Such architectural features of a protein are designated secondary (2°)
structures (Figure 5.8). Secondary structures are just one of the higher levels of structure
that represent the three-dimensional arrangement of the polypeptide in space.
FIGURE 5.8 ● Two structural motifs that arrange the primary structure of proteins into a higher level
of organization predominate in
proteins: the _-helix and the _-pleated strand. Atomic representations of these secondary structures are
shown here, along with the symbols used by structural chemists to represent them: the flat, helical
39
ribbon for the _-helix and the flat, wide arrow for _-structures. Both of these structures owe their
stability to the formation of hydrogen bonds between NOH and OPC functions along the polypeptide
backbone
Chymotrypsin ribbon
FIGURE 5.9 ● Folding of the polypeptide chain into a compact, roughly spherical conformation creates the
tertiary level of protein structure. (a) The primary structure and (b) a representation of the tertiary structure of
chymotrypsin, a proteolytic enzyme, are shown here. The tertiary representation in (b) shows the course of the
chymotrypsin folding pattern by successive numbering of the amino acids in its sequence. (Residues 14 and 15
and 147 and 148 are missing because these residues are removed when chymotrypsin is formed from its larger
precursor, chymotrypsinogen.) The ribbon diagram depicts the three-dimensional track of the polypeptide in
space.
Tertiary Structure
When the polypeptide chains of protein molecules bend and fold in order to assume a
more compact three-dimensional shape, a tertiary (3°) level of structure is generated
(Figure 5.9). It is by virtue of their tertiary structure that proteins adopt a globular shape.
A globular conformation gives the lowest surface to- volume ratio, minimizing
interaction of the protein with the surrounding environment.
Quaternary Structure
Many proteins consist of two or more interacting polypeptide chains of characteristic

tertiary structure, each of which is commonly referred to as a subunit of the protein.
Subunit organization constitutes another level in the hierarchy of protein structure,
defined as the protein’s quaternary (4°) structure (Figure 5.10). Questions of quaternary
structure address the various kinds of subunits within a protein molecule, the number
of each, and the ways in which they interact with one another. Whereas the primary
structure of a protein is determined by the covalently linked amino acid residues in the
polypeptide backbone, secondary and higher orders of structure are determined
principally by noncovalent forces such as hydrogen bonds and ionic, van der Waals,
and hydrophobic interactions. It is important to emphasize that all the information
40
necessary for a protein molecule to achieve its intricate architecture is contained within
its 1° structure, that is, within the amino acid sequence of its polypeptide chain(s).
Chapter 6 presents a detailed discussion of the 2°, 3°, and 4° structure of protein
molecules.
Protein Conformation
The overall three-dimensional architecture of a protein is generally referred to as its

conformation. This term is not to be confused with configuration, which denotes the
geometric possibilities for a particular set of atoms (Figure 5.11). In going from one
configuration to another, covalent bonds must be broken and rearranged. In contrast,
the conformational possibilities of a molecule are achieved without breaking any
covalent bonds. In proteins, rotations about each of the single bonds along the peptide
backbone have the potential to alter the course of the polypeptide chain in three-
dimensional space. These rotational possibilities create many possible orientations for
the protein chain, referred to as its conformational possibilities. Of the great number of
theoretical conformations, a given protein might adopt, only a very few are favoured
energetically under physiological conditions. At this time, the rules that direct the
folding of protein chains into energetically favourable conformations are still not
entirely clear; accordingly, they are the subject of intensive contemporary research.
FIGURE 5.10 ● Haemoglobin, which consists of two _ and two _ polypeptide chains, is an example of
the quaternary level of protein structure. In this drawing, the _-chains are the two uppermost
polypeptides and the two _- chains are the lower half of the molecule. The two closest chains (darkest
colour) are the _2- chain (upper left) and the _1-chain (lower right). The heme groups of the four globin
chains are represented by rectangles with spheres (the he me iron atom). Note the symmetry of this
Macromolecular arrangement. (Irving Geis)

41
FIGURE 5.11 ● Configuration and conformation are not synonymous. (a) Rearrangements between configurational
alternatives of a molecule can be achieved only by breaking and remaking bonds, as in the transformation between the D- and
L-configurations of glyceraldehyde. No possible rotational reorientation of bonds linking the atoms of D-glyceraldehyde yields
geometric identity with L-glyceraldehyde, even though they are mirror images of each other. (b) The intrinsic free rotation
around single covalent bonds creates a great variety of three-dimensional conformations, even for relatively simple molecules.
Consider 1,2-dichloroethane. Viewed end-on in a Newman projection, three principal rotational orientations or conformations
predominate. Steric repulsion between eclipsed and partially eclipsed conformations keeps the possibilities at a reasonable
number. (c) Imagine the conformational possibilities for a protein in which two of every three bonds along its backbone are
freely rotating single bonds. Later we return to an analysis of the 1° structure of proteins and the methodology used in
determining the amino acid sequence of polypeptide chains, but let’s first consider the extraordinary variety and functional
diversity of these most interesting macromolecules.
The Many Biological Functions of Proteins
Proteins are the agents of biological function. Virtually every cellular activity is
dependent on one or more particular proteins. Thus, a convenient way to classify the
enormous number of proteins is by the biological roles they fill. Table 5.3 summarizes
the classification of proteins by function and gives examples of representative members
of each class.
Enzymes
By far the largest class of proteins is enzymes. More than 3000 different enzymes are
listed in Enzyme Nomenclature, the standard reference volume on enzyme
classification. Enzymes are catalysts that accelerate the rates of biological reactions.
Each enzyme is very specific in its function and acts only in a particular metabolic
reaction. Virtually every step in metabolism is catalyzed by an enzyme. The catalytic
power of enzymes far exceeds that of synthetic catalysts. Enzymes can enhance reaction
rates in cells as much as 1016 times the uncatalyzed rate. Enzymes are systematically
classified according to the nature of the reaction that they catalyze, such as the transfer
of a phosphate group (phosphotransferase) or an oxidation–reduction (oxidoreductase).
42
The formal names of enzymes come from the particular reaction within the class that
they catalyze, as in ATP:D-fructose-6-phosphate 1-phosphotransferase and
alcohol:NAD oxidoreductase. Often, enzymes have common names in addition to their
formal names. ATP:D-fructose-6-phosphate 1-phosphotransferase is more commonly
known as phosphofructokinase (kinase is a common name given to ATP-dependent
phosphotransferases). Similarly, alcohol:NAD oxidoreductase is casually referred to
as alcohol dehydrogenase. The reactions catalyzed by these two enzymes are shown in
Figure 5.12. Other enzymes are known by trivial names that have historical roots, such
as catalase (systematic name, hydrogen-peroxide:hydrogen- peroxide oxidoreductase),
and sometimes these trivial names have descriptive connotations as well, as in malic
enzyme (systematic name, L-malate:NADP oxidoreductase).
Regulatory Proteins
A number of proteins do not perform any obvious chemical transformation but

nevertheless can regulate the ability of other proteins to carry out their physiological
functions. Such proteins are referred to as regulatory proteins. A well-known example
is insulin, the hormone regulating glucose metabolism in animals. Insulin is a relatively
small protein (5.7 kD) and consists of two polypeptide chains held together by disulfide
cross-bridges. Other hormones that are also proteins include pituitary somatotropin (21
kD) and thyrotropin (28 kD), which stimulates the thyroid gland. Another group of
regulatory proteins is involved in the regulation of gene expression. These proteins
characteristically act by binding to DNA sequences that are adjacent to coding regions
of genes, either activating or inhibiting the transcription of genetic information into
RNA. Examples include repressors, which, because they block transcription, are
considered negative control elements. A prokaryotic representative is lac repressor (37
kD), which controls expression of the enzyme system responsible for the metabolism
of lactose (milk sugar); a mammalian example is NF1 (nuclear factor 1, 60 kD), which
inhibits transcription of the gene encoding the _-globin polypeptide chain of
hemoglobin. Positively acting control elements are also known. For example, the E. coli
catabolite gene activator protein (CAP) (44 kD), under appropriate metabolic
conditions, can bind to specific sites along the E. coli chromosome and increase the rate
of transcription of adjacent genes. The mammalian AP1 is a heterodimeric transcription
factor composed of one polypeptide from the Jun family of gene-regulatory proteins
and one polypeptide from the Fos family of gene-regulatory proteins. AP1 activates
expression of the _-globin gene. These various DNA-binding regulatory proteins often
possess characteristic structural features, such as helix-turn-helix, leucine zipper, and
zinc finger motifs.

43

44
Transport Proteins
A third class of proteins is the transport proteins. These proteins function to transport
specific substances from one place to another. One type of transport is exemplified by
the transport of oxygen from the lungs to the tissues by haemoglobin (Figure 5.13a) or
by the transport of fatty acids from adipose tissue to various organs by the blood protein
serum albumin. A very different type is the transport of metabolites across permeability
barriers such as cell membranes, as mediated by specific membrane proteins. These
membrane transport proteins take up metabolite molecules on one side of a membrane,
transport them across the membrane, and release them on the other side. Examples
include the transport proteins responsible for the uptake of essential nutrients into the
cell, such as glucose or amino acids (Figure 5.13b). All naturally occurring membrane
transport proteins studied thus far form channels in the membrane through which the
transported substances are passed.
Storage Proteins
Proteins whose biological function is to provide a reservoir of an essential nutrient are

called storage proteins. Because proteins are amino acid polymers and because nitrogen
is commonly a limiting nutrient for growth, organisms have exploited proteins as a
means to provide sufficient nitrogen in times of need. For example, ovalbumin, the
protein of egg white, provides the developing bird embryo with a source of nitrogen
during its isolation within the egg. Casein is the most abundant protein of milk and thus
the major nitrogen source for mammalian infants. The seeds of higher plants often
contain as much as 60% storage protein to make the germinating seed nitrogen-
sufficient during this crucial period of plant development. In corn (Zea mays or maize),
a family of low molecular weight proteins in the kernel called zeins serve this purpose;
peas (the seeds of Phaseolus vulgaris) contain a storage protein called phaseolin. The
use of proteins as a reservoir of nitrogen is more efficient than storing an equivalent
amount of amino acids. Not only is the osmotic pressure minimized, but the solvent
capacity of the cell is taxed less in solvating one molecule of a polypeptide than in
dissolving, for example, 100 molecules of free amino acids. Proteins can also serve to
store nutrients other than the more obvious elements composing amino acids (N, C, H,
O, and S). As an example, ferritin is a protein found in animal tissues that binds iron,
retaining this essential metal so that it is available for the synthesis of important iron-
containing proteins such as hemoglobin. One molecule of ferritin (460 kD) binds as
many as 4500 atoms of iron (35% by weight).

45
Contractile and Motile Proteins
Certain proteins endow cells with unique capabilities for movement. Cell division,
muscle contraction, and cell motility represent some of the ways in which cells execute
motion. The contractile and motile proteins underlying these motions share a common
property: they are filamentous or polymerize to form filaments. Examples include actin
and myosin, the filamentous proteins forming the contractile systems of cells, and
tubulin, the major component of microtubules (the filaments involved in the mitotic
spindle of cell division as well as in flagella and cilia). Another class of proteins
involved in movement includes dynein and kinesin, so-called motor proteins that drive
the movement of vesicles, granules, and organelles along microtubules serving as
established cytoskeletal “tracks.”
Structural Proteins
An apparently passive but very important role of proteins is their function in creating
and maintaining biological structures. Structural proteins provide strength and
protection to cells and tissues. Monomeric units of structural proteins typically
polymerize to generate long fibers (as in hair) or protective sheets of fibrous arrays, as
in cowhide (leather). _-Keratins are insoluble fibrous proteins making up hair, horns,
and fingernails. Collagen, another insoluble fibrous protein, is found in bone,
connective tissue, tendons, cartilage, and hide, where it forms inelastic fibrils of great
strength. One-third of the total protein in a vertebrate animal is collagen. A structural
protein having elastic properties is, appropriately, elastin, an important component of
ligaments. Because of the way elastin monomers are cross-linked in forming polymers,
elastin can stretch in two dimensions. Certain insects make a structurally useful protein,
fibroin (a _-keratin), the major constituent of cocoons (silk) and spider webs. An
important protective barrier for animal cells is the extracellular matrix containing
collagen and proteoglycans, covalent protein–polysaccharide complexes that cushion
and lubricate.
Scaffold Proteins (Adapter Proteins)
Some proteins play a recently discovered role in the complex pathways of cellular
response to hormones and growth factors. These proteins, the scaffold or adapter
proteins, have a modular organization in which specific parts (modules) of the protein’s
structure recognize and bind certain structural elements in other proteins through
protein–protein interactions. For example, SH2 modules bind to proteins in which a
tyrosine residue has become phosphorylated on its phenolic OOH, and SH3 modules
bind to proteins having a characteristic grouping of proline residues. Others include PH
modules, which bind to membranes, and PDZ-containing proteins, which bind
specifically to the C-terminal amino acid of certain proteins.
46
Because scaffold proteins typically possess several of these different kinds of modules,
they can act as a scaffold onto which a set of different proteins is assembled into a
multiprotein complex. Such assemblages are typically involved in coordinating and
communicating the many intracellular responses to hormones or other signalling
molecules (Figure 5.14). Anchoring (or targeting) proteins are proteins that bind other
proteins, causing them to associate with other structures in the cell. A family of
anchoring proteins, known as AKAP or A kinase anchoring proteins, exists in which
specific AKAP members bind the regulatory enzyme protein kinase A (PKA) to
particular subcellular compartments. For example, AKAP100 targets PKA to the
endoplasmic reticulum, whereas AKAP79 targets PKA to the plasma membrane.
Protective and Exploitive Proteins
In contrast to the passive protective nature of some structural proteins, another group
can be more aptly classified as protective or exploitive proteins because of their
biologically active role in cell defense, protection, or exploitation. Prominent among
the protective proteins are the immunoglobulins or antibodies produced by the
lymphocytes of vertebrates.
47
Antibodies have the remarkable ability to “ignore” molecules that are an intrinsic part
of the host organism, yet they can specifically recognize and neutralize “foreign”
molecules resulting from the invasion of the organism by bacteria, viruses, or other
infectious agents. Another group of protective proteins is the blood-clotting proteins,
thrombin and fibrinogen, which prevent the loss of blood when the circulatory system
is damaged. Arctic and Antarctic fishes have antifreeze proteins to protect their blood
against freezing in the below-zero temperatures of high-latitude seas. In addition,
various proteins serve defensive or exploitive roles for organisms, including the lytic
and neurotoxic proteins of snake and bee venoms and toxic plant proteins, such as ricin,
whose apparent purpose is to thwart predation by herbivores. Another class of exploitive
proteins includes the toxins produced by bacteria, such as diphtheria toxin and cholera
toxin.
Exotic Proteins
Some proteins display rather exotic functions that do not quite fit the previous
classifications. Monellin, a protein found in an African plant, has a very sweet taste and
is being considered as an artificial sweetener for human consumption. Resilin, a protein
having exceptional elastic properties, is found in the hinges of insect wings. Certain
marine organisms such as mussels secrete glue proteins, allowing them to attach firmly
to hard surfaces. It is worth repeating that the great diversity of function in proteins, as
reflected in this survey, is attained using just 20 amino acids.

48
Some Proteins Have Chemical Groups
Other than Amino Acids Many proteins consist of only amino acids and contain no
other chemical groups. The enzyme ribonuclease and the contractile protein actin are
two such examples. Such proteins are called simple proteins. However, many other
proteins contain various chemical constituents as an integral part of their structure.
These proteins are termed conjugated proteins (Table 5.4). If the nonprotein part is
crucial to the protein’s function, it is referred to as a prosthetic group. If the nonprotein
moiety is not covalently linked to the protein, it can usually be removed by denaturing
the protein structure. However, if the conjugate is covalently joined to the protein, it
may be necessary to carry out acid hydrolysis of the protein into its component amino
acids in order to release it. Conjugated proteins are typically classified according to the
chemical nature of their nonamino acid component; a representative selection of them
is given here and in Table 5.4. (Note that comparisons of Tables 5.3 and 5.4 reveal two
distinctly different ways of considering the nature of proteins—function versus
chemistry.)
GLYCOPROTEINS.
Glycoproteins are proteins that contain carbohydrate. Proteins destined for an

extracellular location are characteristically glycoproteins. For example, fibronectin and
proteoglycans are important components of the extracellular matrix that surrounds the
cells of most tissues in animals. Immunoglobulin G molecules are the principal antibody
species found circulating free in the blood plasma. Many membrane proteins are
glycosylated on their extracellular segments.
LIPOPROTEINS.
Blood plasma lipoproteins are prominent examples of the class of proteins conjugated
with lipid. The plasma lipoproteins function primarily in the transport of lipids to sites
of active membrane synthesis. Serum levels of low density lipoproteins (LDLs) are
often used as a clinical index of susceptibility to vascular disease.
NUCLEOPROTEINS.
Nucleoprotein conjugates have many roles in the storage and transmission of genetic
information. Ribosomes are the sites of protein synthesis. Virus particles and even
chromosomes are protein–nucleic acid complexes.

49

50
PHOSPHOPROTEINS.
These proteins have phosphate groups esterified to the hydroxyls of serine, threonine,
or tyrosine residues. Casein, the major protein of milk, contains many phosphates and
serves to bring essential phosphorus to the growing infant. Many key steps in
metabolism are regulated between states of activity or inactivity, depending on the
presence or absence of phosphate groups on proteins, as we shall see in Chapter 15.
Glycogen phosphorylase a is one well-studied example.
METALLOPROTEINS.
Metalloproteins are either metal storage forms, as in the case of ferritin, or enzymes in
which the metal atom participates in a catalytically important manner. We encounter
many examples throughout this book of the vital metabolic functions served by
metalloenzymes.
HEMOPROTEINS.
These proteins are actually a subclass of metalloproteins because their prosthetic group
is heme, the name given to iron protoporphyrin IX (Figure 5.15). Because heme-
containing proteins enjoy so many prominent biological functions, they are considered
a class by themselves.
FLAVOPROTEINS.
Flavin is an essential substance for the activity of a number of important

oxidoreductases. We discuss the chemistry of flavin and its derivatives, FMN and FAD,
in the chapter on electron transport and oxidative phosphorylation
CELL KINETICS AND FERMENTER DESIGN

Understanding the growth kinetics of microbial, animal, or plant cells is
important for the design and operation of fermentation systems employing them. Cell
kinetics deals with the rate of cell growth and how it is affected by various chemical
and physical conditions. Unlike enzyme kinetics discussed in Chapter 2, cell kinetics is
the result of numerous complicated networks of biochemical and chemical reactions
and transport phenomena, which involves multiple phases and multicomponent
systems. During the course of growth, the heterogeneous mixture of young and old cells
is continuously changing and adapting itself in the media environment which is also
continuously changing in physical and chemical conditions. As a result, accurate
mathematical modeling of growth kinetics is impossible to achieve. Even with such a
realistic model, this approach is usually useless because the model may contain many
parameters which are impossible to determine. Therefore, we must make assumptions
51
to be able to arrive at simple models which are useful for fermenter design and
performance predictions. Various models can be developed based on the assumptions
concerning cell 'components and population as shown in Table 6.1 (Tsuchiya et al.,
1966). The simplest model is the unstructured, distributed rnodel which is based on the
following two assumptions:
1. Cells can be represented by a single component, such as cell mass, cell number, or
the concentration of protein, DNA, or RNA. This is true for balanced growth, since a
doubling of cell mass for balanced growth is accompanied by a doubling of all other
measurable properties of the cell population.
2. The population of cellular mass is distributed uniformly throughout the culture. The
cell suspension can be regarded as a homogeneous solution. The heterogeneous nature
of cells can be ignored. The cell concentration can be expressed as dry weight per unit
volume.
Besides the assumptions for the cells, the medium is formulated so that only one
component may be limiting the reaction rate. All other components are present at
sufficiently high concentrations, so that minor changes do not significantly affect the
reaction rate. Fermenters are also controlled so that environmental parameters such as
pH, temperature, and dissolved oxygen concentration are maintained at a constant level.
In this chapter, cell kinetic equations are derived from the unstructured, distributed
model, and those equations are applied for the analysis and design of ideal fermenters.
More realistic models which consider the multiplicity of cell components, structured
model, are introduced at the end of the chapter.
DEFINITIONS
First, let us define the terminologies we use for microbial growth. If we mention the cell
concentration without any specification, it can have many different meanings. It can be
the number of cells, the wet cell weight, or the dry cell weight per unit volume. In this
text, the following nomenclature is adopted:
𝑑𝐶𝑥
It appears that and rx are always the same, but this is not true. The former is the
𝑑𝑥
change of the cell concentration in a fermenter, which may include the effect of the
input and_ output flow rates, cell recycling, and other operating conditions of a

52
fermenter. The latter is the actual growth rate of the cells. The two quantities are the
same only for batch operation. The growth rate based on the number of cells and that
based on cell weight are not necessarily the same because the average size of the cells
may vary considerably from one phase to another. When the mass of an individual cell
increases without division, the' growth rate based on cell weight increases, while that
based on the number cells stays the same. However, during the exponential growth
period, which is the phase that we are most interested in from an engineer's point of
view, the growth rate based on the cell number and that based on cell weight can be
assumed to be proportional to each other. Sometimes, the growth rate can be confused
with the division which is defined as the rate of cell division per unit time. If all of the
cell in the vessel at time t = 0 ( CN = CN0 ) have divided once after a certain period of
time, the cell population will have increased to CNo × 2 If cells are divided n times after
the time t, the total number cells will be 𝐶𝑛 = 𝐶𝑛𝑜 × 2N and the average division rate
is;
𝑛
𝛿 = ……………………...(1)
𝑙
Since 𝑁 = log 2 𝐶𝑛 − log 2 𝐶𝑛 𝑜 according to (1), the average division rate is

1
𝛿= (log 2 𝐶𝑛 − log 2 𝐶𝑛 𝑜) ……………………………. (2)
𝑡
And the division rate at time t is

𝑑 log2 𝐶𝑛
𝛿= ………………………………………………. (3)
𝑑𝑡
Therefore, the growth rate defined as the change of cell number with time is the slope
of the CN versus t curve, while the division rate is the slope of the log2CN versus t curve.
As explained later, the division rate is constant during the exponential growth period,
while the growth rate is not. Therefore, these two terms should not be confused with
each other.
GROWTH CYCLE FOR BATCH CULTIVATION
If you inoculate unicellular microorganisms into a fresh sterilized medium and measure
the cell number density with respect to time and plot it, you may find that there are six
phases of growth and death, as shown in Figure 6.1. They are:
1. Lag phase: A period of time when the change of cell number is zero.
2. Accelerated growth phase: The cell number starts to increase and the division
rate increases to reach a maximum.
3. Exponential growth phase: The cell number increases exponentially as the
cells start to divide. The growth rate is increasing during this phase, but the
division rate which is proportional to dlnC/dt, is constant at its maximum value,
as illustrated in Figure 6.1.
53
4. Decelerated growth phase: After the growth rate reaches a maximum, it is

followed by the deceleration of both growth rate and the division rate.
5. Stationary phase: The cell population will reach a maximum value and will not
increase any further.
6. Death phase: After nutrients available for the cells are depleted, cells will start
to die and the number of viable cells will decrease.
Lag Phase
The lag phase (or initial stationary, or latent) is an initial period of cultivation during
which the change of cell is zero or negligible. Even though the cell number does not
increase, the cells may grow in size during this period. The length of this lag period
depends on many factors such as the type and age of the microorganisms, the size of
the inoculum, and culture conditions. The lag usually occurs because the cells must
adjust to the new medium before growth can begin. If microorganisms are inoculated
from a medium with a low nutrient concentration to a medium with a high
concentration, the length of the lag period is usually long. This is because the cells must
produce the enzymes necessary for the metabolization of the available nutrients. If they
are moved from a high to a low nutrient concentration, there is usually no lag phase.
Another important factor affecting the length of the lag phase is the size of the inoculum.
If a small amount of cells are inoculated into a large volume, they will have a long lag
phase. For large-scale operation of the cell culture, it is our objective to make this lag
phase as short as possible. Therefore, to inoculate a large fermenter, we need to have a
series of progressively larger seed tanks to minimize the effect of the lag phase. At the
end of the lag phase, when growth begins, the division rate increases gradually and
reaches a maximum value in the exponential growth period, as shown by the rising
inflection at B in Figure 6.1. His transitional period is commonly called the accelerated
growth phase and is often included as a part of the lag phase.
Exponential Growth Phase
In unicellular organisms, the progressive doubling of cell number results in a

continually increasing rate of growth in the population. A bacterial culture undergoing
balanced growth mimics a first-order autocatalytic chemical reaction (Carberry, 1976;
Levenspiel, 1972). Therefore, the rate of the cell population increase at any particular
time is proportional to the number density (CN) of bacteria present at that time:
𝐶𝑛
𝑅𝑛 = = 𝜇𝐶𝑛 ………………………………. (1)
𝑑𝑡

54
Where the constant is known as the specific growth rate [hr-1]. The specific growth rate
should not be confused with the growth rate, which has different units and meaning.
The growth rate is the change
of the cell number density with time, while the specific growth rate is
1 𝑑𝐶𝑛 𝑑𝐼𝑛𝐶𝑛
𝜇= = ……………. (2)
𝐶𝑛 𝑑𝑡 𝑑𝑡
This is the change of the natural log of the cell number density with time. Comparing
eqn 1 and 2 shows that:
𝑑𝐼𝑛𝐶𝑛 𝑑𝑙𝑜𝑔𝐶𝑛
𝜇= = 𝐼𝑛2 ( ) = 𝛿𝐼𝑛2 …………….. (3)
𝑑𝑡 𝑑𝑡
Therefore, the specific growth rate 𝜇 is equal to In2 times of the division rate, 𝛿.
If 𝜇 is constant with time during the exponential growth period, eqn 1 can be integrated
from time t1 to t as
𝐶𝑛 𝑑𝐶𝑛 𝑡
∫𝐶𝑛𝑜 = ∫𝑡𝑜 𝜇𝑑𝑡 ……………………….. (4)
𝐶𝑛
To give 𝐶𝑛 = 𝐶𝑛𝑜𝑒𝑥𝑝{𝜇(𝑡−𝑡𝑜)} …………………… (5)
Where Cno is the cell number concentration at t1 when the exponential growth
starts. Eqn 4 shows the increase of the number of cells exponentially with respect to
time. The time required to double the population, called the doubling time (td), can be
estimated from equation 4 by setting 𝐶𝑛 = 2𝐶𝑛0 and t1 = 0 and solving for t:
𝐼𝑛2 1
𝑡𝑑 = = …………… (6)
𝜇 𝛿
The doubling time is inversely proportional to the specific growth rate and is equal to
the reciprocal of the division rate.
This equation has the same form as the rate equation for an enzyme catalyzed reaction,
the Michaelis Menten equation:

55

56
Stationary and death phase
The growth of microbial populations is normally limited either by the exhaustion of

available nutrients or by the accumulation of toxic products of metabolism. As a
consequence, the rate of growth declines and growth eventually stops. At this point a
culture is said to be in the stationary phase. The transition between the exponential
phase and the stationary phase involves a period of unbalanced growth during which
the various cellular components are synthesized at unequal rates. Consequently, cells in
57
the stationary phase have a chemical composition different from that of cells in the
exponential phase. The stationary phase is usually followed by a death phase in which
the organisms in the population die. Death occurs either because of the depletion of the
cellular reserves of energy, or the accumulation of toxic products. Like growth, death
is an exponential function. In some cases, the organisms not only die but also
disintegrate, a process called lysis.

58
ENZYMES
Enzymes are biological catalysts that are protein molecules in nature.

They are produced by living cells (animal, plant, and microorganism)
and are absolutely essential as catalysts in biochemical reactions.
Almost every reaction in a cell requires the presence of a specific
enzyme. A major function of enzymes in a living system is to catalyze
the making and breaking of chemical bonds. Therefore, like any other
catalysts, they increase the rate of reaction without themselves
undergoing permanent chemical changes.
The catalytic ability of enzymes is due to its particular protein
structure. A specific chemical reaction is catalyzed at a small portion
of the surface of an enzyme, which is known as the active site. Some
physical and chemical interactions occur at this site to catalyze a
certain chemical reaction for a certain enzyme.
Enzyme reactions are different from chemical reactions, as follows:
1. An enzyme catalyst is highly specific, and catalyzes only one or
a small number of chemical reactions. A great variety of
enzymes exist, which can catalyze a very wide range of
reactions.
2. The rate of an enzyme-catalyzed reaction is usually much faster
than that of the same reaction when directed by nonbiological
catalysts. Only a small amount of enzyme is required to
produce a desired effect.
3. The reaction conditions (temperature, pressure, pH, and so on)
for the enzyme reactions are very mild.
4. Enzymes are comparatively sensitive or unstable molecules
and require care in their use.
Nomenclature of Enzymes
Originally enzymes were given nondescriptive names such as:

rennin curding of milk to start cheese-making processcr
pepsin hydrolyzes proteins at acidic pH
trypsin hydrolyzes proteins at mild alkaline pH

59
The nomenclature was later improved by adding the suffix -ase to the
name of the substrate with which the enzyme functions, or to the
reaction that is catalyzed.myfootnote1 For example:
Simple Enzyme Kinetics
Enzyme kinetics deals with the rate of enzyme reaction and how it is
affected by various chemical and physical conditions. Kinetic studies
of enzymatic reactions provide information about the basic
mechanism of the enzyme reaction and other parameters that
characterize the properties of the enzyme. The rate equations
developed from the kinetic studies can be applied in calculating
reaction time, yields, and optimum economic condition, which are
important in the design of an effective bioreactor.
Assume that a substrate (5) is converted to a product (P) with the
help of an enzyme (E) in a reactor as
If you measure the concentrations of substrate and product with

respect to time, the product concentration will increase and reach a
maximum value, whereas the substrate concentration will decrease
as shown in Figure 2.1

60
The rate of reaction can be expressed in terms of either the change

of the substrate Cs or the product concentrations C as follows:
In order to understand the effectiveness and characteristics of an

enzyme reaction, it is important to know how the reaction rate is
influenced by reaction conditions such as substrate, product, and
enzyme concentrations. If we measure the initial reaction rate at
different levels of substrate and enzyme concentrations, we obtain a
series of curves like the one shown in Figure 2.2. From these curves
we can conclude the following:
1. The reaction rate is proportional to the substrate concentration

61
(that is, first-order reaction) when the substrate concentration is

in the low range.
2. The reaction rate does not depend on the substrate
concentration when the substrate concentration is high, since
the reaction rate changes gradually from first order to zero
order as the substrate concentration is increased.
3. The maximum reaction rate rmax is proportional to the enzyme
concentration within the range of the enzyme tested.
Henri observed this behavior in 1902 (Bailey and Ollis, p. 100, 1986)
and proposed the rate equation
where rmax and KM are kinetic parameters which need to be

experimentally determined. Eq. (2.4) expresses the three preceding
observations fairly well. The rate is proportional to Cs (first order) for
low values of Cs, but with higher values of Cs, the rate becomes
constant (zero order) and equal to rmax. Since Eq. (2.4) describes the
experimental results well, we need to find the kinetic mechanisms
which support this equation.
Brown (1902) proposed that an enzyme forms a complex with its

substrate. The complex then breaks down to the products and
regenerates the free enzyme. 'Jhe mechanism of one substrateenzyme
reaction can be expressed as
Brown's kinetic ~nference of the existence of the enzyme-substrate

complex was made long before the chemical nature of enzymes was
known, 40 years before the spectrophotometric detection of such

62
complexes.
One of the original theories to account for the formation of the
enzyme-substrate complex is the "lock and key" theory. The main
concept of this hypothesis is that there is a topographical, structural
compatibility between an enzyme and a substrate which optimally
favors the recognition of the substrate as shown in Figure 2.3.
The reaction rate equation can be derived from the preceding

mechanism based on the following assumptions:
1. The total enzyme concentration stays constant during the
reaction, that is, CEO C ES + C

2. The amount of an enzyme is very small compared to the
amount of substrate.2 Therefore, the formation of the enzymesubstrate
complex does not significantly deplete the substrate.
3. The product concentration is so low that product inhibition
may be considered negligible.
In addition to the preceding assumptions, there are three different
approaches to derive the rate equation:
1. Michaelis-Menten approach (Michaelis and Menten, 1913): It
is assumed that the product-releasing step, Eq. (2.6), is much
slower than the reversible reaction, Eq. (2.5), and the slow ~tep
determihes the rate, while the other is at equilibrium. This i~ an
assumption which is often employed in heterogeneous catalytic
reactions in chemical kinetics. 3 Even though the enzyme is soluble in water, the enzyme
molecules have large and
complicated three-dimensional structures. Therefore, enzymes
can be analogous to solid catalysts in chemical reactions.
Furthermore, the first step for an enzyme reaction also involves
the formation of an enzyme-substrate complex, which is based

63
on a very weak interaction. Therefore, it is reasonable to assume

that the enzyme-substrate complex formation step is much
faster than the product releasing step which involves chemical
changes.
2. Briggs-Haldane approach (Briggs and Haldane, 1925): The
change of the intermediate concentration with respect to time is
assumed to be negligible, that is, d(CES)/dt = O. This is also

known as the pseudo-steady-state (or quasi-steady-state)
assumption in chemical kinetics and is often used in developing
rate expressions in homogeneous catalytic reactions.
3. Numerical solution: Solution of the simultaneous differential
equations developed from Eqs. (2.5) and (2.6) without
simplification.
Michaelis-Menten Approach
If the slower reaction, Eq. (2.6), determines the overall rate of reaction,
the rate of product formation and substrate consumption is
proportional to the concentration of the enzyme-substrate complex
as:4
Unless otherwise specified, the concentration is expressed as

molar unit, such as kmol/m3 or mol/L. The concentration of the
enzyme-substrate complex ES in Eq. (2.7), can be related to the
substrate concentration Cs and the free-enzyme concentration C
from the assumption that the first reversible reaction Eq. (2.5) is in

64
equilibrium. Then, the forward reaction is equal to the reverse

reaction so that
By substituting Eq. (2.8) into Eq. (2.7), the rate of reaction can be
expressed as a function of Cs and CE, of which CE cannot be easily
determined. If we assume that the total enzyme contents are
conserved, the free-enzyme concentration C can be related to the
initial enzyme concentration CEO
So, now we have three equation"s from which we can eliminate C

and CES to express the rate expression as the function of substrate
concentration and the initial enzyme concentration. By substituting
Eq. (2.8) into Eq. (2.9) for CE and rearranging for CES' we obtain
Substitution of Eq. (2.10) into Eq. (2.7) results in the final rate
Equation

65
which is known as Michaelis-Menten equation and is identical to the

empirical expression Eq. (2.4). KM in Eq. (2.11) is known as the
Michaelis constant. In the Michaelis-Menten approach, KM is equal to
the dissociation constant K or the reciprocal of equilibrium constant
Keg as
The unit of M is the same as s. When K is equal to s, r is equal to

one half of r according to Eq. (2.11). Therefore, the value of KM is
equal to the substrate concentration when the reaction rate is half of
the maximum rate rmax (see Figure 2.2). M is an important kinetic
parameter because it characterizes the interaction of an enzyme with
a given substrate.
Another kinetic parameter in Eq. (2.11) is the maximum reaction
rate r which is proportional to the initial enzyme concentration.
The main reason for combining two constants k3 and CEO' into one
lumped parameter rmax is due to the difficulty of expressing the
enzyme concentration in molar unit. To express the enzyme
concentration in molar unit, we need to know the molecular weight

of enzyme and the exact amount of pure enzyme added, both of
which are very difficult to determine. Since we often use enzymes
which are not in pure form, the actual amount of enzyme is not
known.
Enzyme concentration may be expressed in mass unit instead of
molar unit. However, the amount of enzyme is not well quantified in
mass unit because actual contents of an enzyme can differ widely
depending on its purity. Therefore, it is common to express enzyme
concentration as an arbitrarily defined unit based on its catalytic
ability. For example, one unit of an enzyme, cellobiose, can be
defined as the amount of enzyme required to hydrolyze cellobiose to
produce 1 ilmol of glucose per minute. Whatever unit is adopted for
CEO' the unit for 3CEO should be the same as r, that is, kmole/m3s.
Care should be taken for the consistency of unit when enzyme
concentration is not expressed in molar unit.
The Michaelis-Menten equation is analogous to the Langmuir
isotherm equation

66
where θ is the fraction of the solid surface covered by gas molecules

and K is the reciprocal of the adsorption equilibrium constant.
Briggs-Haldane Approach
Again, from the mechanism described by Eqs. (2.5) Eq. (2.6), the rates
of product formation and of substrate consumption are
2.14
Assume that the change of CES with time, dCES/dt, is negligible

compared to that of C or Cs.
Substitution of Eq. (2.16) into Eq. (2.15) confirms that the rate of
product formation and that of the substrate consumption are the
same, that is,
Again, if we assume that the total enzyme contents are conserved,
Substituting Eq. (2.9) into Eq. (2.16) for CE and rearranging for CES

67
Substitution of Eq. (2.17) into Eq. (2.14) results
which is the same as the Michaelis-Menten equation, Eq. (2.11),

except that the meaning of KM is different. In the Michaelis-Menten
approach, KM is equal to the dissociation constant k2/k1 while in the
Bnggs-Haldane approach, it is equal to K2 + K3)/k1. Eq. (2.18) can be
simplified to Eq. (2.11) if k2 » k3 which means that the productreleasing

step is much slower than the enzyme-substrate complex
dissociation step. This is true with many enzyme reactions. Since the
formation of the complex involves only weak interactions, it is likely
that the rate of dissociation of the complex will be rapid. The
breakdown of the complex to yield products will involve the makillg
and breaking of chemical bonds, which is much slower than the
enzyme-substrate complex dissociation step.
Evaluation of Kinetic Parameters
In order to estimate the values of the kinetic parameters, we need to

make a series of batch runs with different levels of substrate
concentration. Then the initial reaction rate can be calculated as a
function of initial substrate concentrations. The results can be plotted
graphically so that the validity of the kinetic model can be tested and
the values of the kinetic parameters can be estimated.
The most straightforward way is to plot r against Cs as shown in
Figure 2.2. The asymptote for r will be 'max and KM is equal to Cs
when r 0.5 'max. However, this is an unsatisfactory plot in
estimating rmax and KM because it is difficult to estimate asymptotes
accurately and also difficult to test the validity of the kinetic model.
Therefore, the Michaelis-Menten equation is usually rearranged so
68
that the results can be plotted as a straight line. Some of the better
known methods are presented here. The Michaelis-Menten equation,
Eq. (2.11), can be rearranged to be expressed in linear form. This can
be achieved in three ways:
An equation of the form of Eq. (2.32) was given by Langmuir

(Carberry, 1976) for the treatment of data from the adsorption of gas
on a solid surface. If the Michaelis-Menten equation is applicable, the
Langmuir plot will result in a straight line, and the slope will be equal
to 1lrMAX The intercept will be KMlrMAX as shown in Figure 2.5.

69
Similarly, the plot of 11r versus 1/Cs will result in a straight line
according to Eq. (2.33), and the slope will be equal to KMlrMAX The
intercept will be 1/ rmax' as shown in Figure 2.6. This plot is known as
Lineweaver-Burk plot (Lineweaver and Burk, 1934).
The plot of r versus r/Cs will result in a straight line with a slope of
M and an intercept of max' as shown in Figure 2.7. This plot is
known as the Eadie-Hofstee plot (Eadie, 1942; Hofstee, 1952).
The Lineweaver-Burk plot is more often employed than the other
two plots because it shows the relationship between the independent
variable Cs and the dependent variable r. However, l/r approaches

70
infinity as Cs decreases, which gives undue weight to inaccurate

measurements made at low substrate concentrations, and insufficient
weight to the more accurate measurements at high substrate concentrations. This is
illustrated in Figure 2.6. The points on the line
in the figure represent seven equally spaced substrate concentrations.
The space between the points in Figure 2.6 increases with the
decrease of Cs.
On the other hand, the Eadie-Hofstee plot gives slightly better

weighting of the data than the Lineweaver-Burk plot (see Figure
A disadvantage of this plot is that the rate of reaction r appears in
both coordinates while it is usually regarded as a dependent variable.
Based on the data distribution, the Langmuir plot (Cs/r versus Cs) is
the most satisfactory of the three, since the points are equally spaced
(see Figure 2.5).
The values of kinetic parameters can be estimated by drawing a
least-squares line roughly after plotting the data in a suitable format.
The linear regression can be also carried out accurately by using a
calclllator with statistical functions, spreadsheet programs such as
Excel (Microsoft., Redmond, WA) or other software packages stIch as
MathCad (MathSoft, Inc., Cambridge, MA). However, it is important
to examine the plot visually to ensure the validity of the parameters
71
values obtained when these numerical techniques are used.

Another approach for the determination of the kinetic parameters
is to use the SAS NLIN (NonLINear regression) procedure (SAS,
1985) which produces weighted least-squares estimates of the
parameters of nonlinear models. The advantages of this technique
are that: (1) it does not require linearization of the Michaelis-Menten
equ.ation, (2) it can be used for complicated multiparameter models,
and (3) the estimated parameter values are reliable because it
produces weighted least-squares estimates.
In conclusion, the values of the Michaelis-Menten kinetic

parameters, rmax and KM, can be estimated, as follows:
1. Make a series of batch runs with different levels of substrate
concentration at a constant initial enzyme concentration and
measure the change of product or substrate concentration with
respect to time.
2. Estimate the initial rate of reaction from the Cs or C versus
time curves for different initial substrate concentrations.
3. Estimate the kinetic parameters by plotting one of the three
plots explained in this section or a nonlinear regression
technique. It is important to examine the data points so that you
may not include the points which deviate systematically from
the kinetic model as illustrated in the following problem.
INHIBITION OF ENZYMES REACTION
A modulator (or effector) is a substance which can combine with

enzymes to alter their catalytic activities. An inhibitor is a modulator
which decreases enzyme activity. It can decrease the rate of reaction
either competitively, noncompetitively, or partially competitively.
Competitive Inhibition
Since a competitive inhibitor has a strong structural resemblance to

the substrate, both the inhibitor and substrate compete for the active
site of an enzyme. The formation of an enzyme-inhibitor complex

72
reduces the amount of enzyme available for interaction with the

substrate and, as a result, the rate of reaction decreases. A
competitive inhibitor normally combines reversibly with enzyme.
Therefore, the effect of the inhibitor can be minimized by increasing
the substrate concentration, unless the substrate concentration is
greater than the concentration at which the substrate itself inhibits
the reaction. The mechanism of competitive inhibition can be
expressed as follows:
If the slower reaction, the prodllct formation step, determines the

rate of reaction according to the Michaelis-Menten assunlption, the
rate can be expressed as:
The enzyme balance gives
From the two equilibrium reactions,

73
Therefore, since KM1 is larger than KS, the reaction rate decreases
due to the presence of inhibitor according to Egn Eg. (2.48). rt is
interesting to note that the maximum reaction rate is not affected by
the presence of a competitive inhibitor. However, a larger amount of
substrate is required to reach the maximum rate. The graphical
consequences of competitive in.hibition are shown in Figure 2.12.
Noncompetitive Inhibition

74

75
Therefore, the maximum reaction rate will be decreased by the

presence of a noncompetitive inhibitor, while the Michaelis constant
Ks will not be affected by the inhibitor. The graphical consequences of
noncompetitive inhibition are shown in Figure 2.12. Note that
making these plots enables us to distinguish between competitive
and noncompetitive inhibition.
Several variations of the mechanism for noncompetitive inhibition
are possible. One case is when the enzyme-inhibitor-substrate
complex can be decomposed to produce a product and the enzymeinhibitor
complex. This mechanism can be described by adding the
following slow reaction to Eq. (2.49)
This case is known as partially competitive inhibition. The derivation

of the rate equation is left as an exercise problem.

76
Influences of Enzyme activity
The rate of an enzyme reaction is influenced by various chemical and

physical conditions. Some of the important factors are the concentration of various
components (substrate, product, enzyme, cofactor, and so on), pH, temperature, and shear.
pH
The rate of an enzyme reaction is strongly influenced by the pH of the
}reaction solution both in vivo and in vitro The typical relationship
between the reaction velocity and pH shows a bell-shaped curve
Figure 2.13. The optimum pH is different for each enzyme. For
example, pepsin from the stomach has an optimum pH between 2 and
3.3, while the optimum pH of amylase, from saliva, is 6.8.
Chymotrypsin, from the pancreas, has an optimum pH in the mildly
alkaline region between 7 and 8.
The reason that the rate of enzyme reaction is influenced by pH can
be explained as follows:
1. Enzyme is a protein which consists of ammo acid residues (that
is, amino acids minus water).
2. The amino acid residues possess basic, neutral, or acid side
groups which can be positively or negatively charged at any
given pH. As an example (Wiseman and Gould, 1970), let's
consider one acidic amino acid, glutamic acid, which is acidic in
the lower pH range. As the pH is increased, glutamic acid is ionized
3. An enzyme is catalytically active when the amino acid
residues at the active site each possess a particular charge.
Therefore, the fraction of the catalytically active enzyme
depends on the pH.
Temperature

77
Shear
Enzymes had been believed to be susceptible to mechanical force,
which disturbs the elaborate shape of an enzyme molecule to such a
degree that denaturation occurs. The mechanical force that an
enzyme solution normally encounters is fluid shear, generated either
by flowing fluid, the shaking of a vessel, or stirring with an agitator.
The effect of shear on the stability of an enzyme is important for the
consideration of enzyme reactor design, because the contents of the
reactor need to be agitated or shook in order to minimize masstransfer
resistance.
However, conflicting results have been reported concerning the
effect of shear on the activity of enzymes. Charm and Wong (1970)
showed that the enzymes catalase, rennet, and carboxypeptidase
were partially inactivated when subjected to shear in a coaxial
cylinder viscometer. The remaining activity could be correlated with
a dimensionless group gammatheta, where gamma and theta are the
shear rate and the time of exposure to shear, respectively.IO In the case

78
of catalase, about 50 percent of the activity was lost when γθ was

0.5 x 107
However, Thomas and Dimnill (1979) studied the effect of shear on
catalase and urease activities by using a coaxial cylindrical
viscometer that was sealed to prevent any air-liquid contact. They
found that there was no significant loss of enzyme activity due to
shear force alone at shear rates up to 106 sec-I. They reasoned that the
deactivation observed by Charm and Wong (1970) was the result of a
combination of shear, air-liquid interface, and some other effects
which are not fully understood. Charm and Wong did not seal their
shear apparatus. This was further confirmed, as cellulase
deactivation due to the interfacial effect combined with the shear
effect was found to be far more severe and extensive than that due to
the shear effect alone (Jones and Lee, 1988).
The use of enzymes in detergents:
At present only proteases and amylases are commonly used. The -amylase supplied for
detergent use is Termamyl, the enzyme from B. licheniformis which is also used in the
production of glucose syrups. -Amylase is particularly useful in dish-washing and
destarching detergents.
Applications of proteases in the food Industry: Certain proteases have been used in food
processing for centuries and any record of the discovery of their activity has been lost in the
mists of time. Rennet (mainly chymosin), obtained from the fourth stomach (abomasum) of
unweaned calves has been used traditionally in the production of cheese. Similarly, papain
from the leaves and unripe fruit of the pawpaw (Carica papaya) has been used to tenderise
meats.

79
The use of proteases in the leather and wool industries: The leather industry consumes a
significant proportion of the world's enzyme production. Alkaline proteases are used to remove
hair from hides. This process is far safer and more pleasant than the traditional methods
involving
sodium sulfide. Relatively large amounts of enzyme are required (0.1-1.0 % (w/w)) and the
process must be closely controlled to avoid reducing the quality of the leather. After dehairing,
hides which are to be used for producing soft leather clothing and goods are bated, a process,
often involving pancreatic enzymes, that increases their suppleness and improves the softness
of their appearance.
The use of enzymes in starch Hydrolysis
Starch is the commonest storage carbohydrate in plants. It is used by the plants themselves, by
microbes and by higher organisms so there is a great diversity of enzymes able to catalyse its
hydrolysis.
The use of lactases in the dairy industry
Lactose is present at concentrations of about 4.7% (w/v) in milk and the whey (supernatant)
left after the coagulation stage of cheese-making.
Enzymes in the fruit juice, wine, brewing and distilling industries
One of the major problems in the preparation of fruit juices and wine is cloudiness due
primarily to the presence of pectins. These consist primarily of -1,4-anhydrogalacturonic acid
polymers, with varying degrees of methyl esterification. They are associated with other plant
polymers and, after homogenisation, with the cell debris. The cloudiness that they cause is
80
difficult to remove except by enzymic hydrolysis. Such treatment also has the additional
benefits of reducing the solution viscosity, increasing the volume of juice produced (e.g. the
yield of juice from white grapes can be raised by 15%), subtle but generally beneficial changes
in the flavour and, in the
case of wine-making, shorter fermentation times.
Glucose oxidase and catalase in the Food industry
Glucose oxidase is a highly specific enzyme (for D-glucose, but see Chapter 8), from the fungi
Aspergillus niger and Penicillium, which catalyses the oxidation of -glucose to glucono-1,5-
lactone (which spontaneously hydrolyses non-enzymically to gluconic acid) using molecular
oxygen and releasing hydrogen peroxide (see reaction scheme [1.1]). It finds uses in the
removal of either glucose or oxygen from foodstuffs in order to improve their storage
capability. Hydrogen peroxide is an effective bacteriocide and may be removed, after use, by
treatment with catalase (derived from the same fungal fermentations as the glucose oxidase)
which converts it to
water and molecular oxygen:
Medical applications of enzymes
Development of medical applications for enzymes have been at least as extensive as those for
industrial applications, reflecting the magnitude of the potential rewards: for example,
pancreatic enzymes have been in use since the nineteenth century for the treatment of digestive
disorders. The variety of enzymes and their potential therapeutic applications are considerable.
A selection of those enzymes which have realised this potential to become important
therapeutic agents is shown in Table 1

81
Table 1: Some important therapeutic enzymes


A Not On Biochemical Engineering

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

A Not On Biochemical Engineering

Uploaded by

Copyright:

Available Formats

1

BIOCHEMICAL ENGINEERING I (CHE 461)

Introduction – Preamble, Scope of Biochemical Engineering, Microbiology, Viruses.

30h (T) PR: CHM212 C.

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Who is a biochemical engineer?

Related disciplines, and also areas of specialization of biochemical engineering:

1. Metabolic engineering, including metabolic modelling

2. Bioprocess engineering, including process control

7. Manufacturing engineer, reactor design

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

An example of what a biochemical engineer might do:

Profit = Selling price of product – (Cost of starting materials + Cost of production

Reactor stir speed titer (g/l) rate yield on

1 1500 30 1.5 15%

Why it is a bad idea and what could be an alternative approach?

As a B.Sc. Chemical Engineer (biochemical engineer) you could do the following:

guidance. An important aspect of manufacturing in pharmaceutical companies is Good

SCOPE OF BIOCHEMICAL ENGINEERING

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Weight of cell produced

Bacteria Blue-green Protozoa Algae Fungi

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

The structure is as follows:

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

1. Staining characteristics: - Generally, most microbes or bacteria are transparent; hence

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Classification Source of Carbon Source of Energy

• Spore formation – when a cell is in distress due to environmental changes, it

Algae and protozoan

1. With the aid of diagram, explain the kingdom of protists

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

They are complex set of entities which consists of two parts:

(1) The Composition- which is as described above.

(2) They are obligate intercellular parasites.

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

- 1 Virus DNA injection

- Nucleic acid production

- Protein coat and tail production

Viruses have different shapes governed by the crystallographic principle

Consider the protein

When there is only triangle

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Viruses have 2 components - the protein coat and the nucleus

The T-4 replication cycle

1. Discuss why some are carriers of HIV and not infected.

Specificity between bacteriophage and cell:

(i) Attachment on the receptive side of the host (outside).

(ii) Injection of viral DNA.

(iii) Circularization of viral DNA and attachment to cell membrane.

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 SESSION

Transcription of early genes

Production of viral DNA

Production of protein coats and tails

Destruction of bacteria DNA

(vii) Transcription of “late” genes

(viii) Lysing and bursting out of the new viruses

The Step Growth Curve

Virus may be classified into two main groups, which are:

CHE 461 – BIOCHEMICAL ENGINEERING 1 2017/2018 The Cell

Why lytic or lysogeny?

(3) Very small amount of information are transferred.

Pseudo-sexual exchange is necessary since it allows a mean of genetic variability. Bacteria