CHE511

CHE 511
BIOCHEMICAL
ENGINEERING
CHE 511: Biochemical Engineering.
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Instructor/Author: Dr. A.O. Ayeni
MODULE 1
1.1 The Biochemical Engineer
• Biochemical Engineering involves the
application of chemical engineering principles
and approaches to biological based systems
and processes.
• It is central to the areas of environmental
engineering, and to biotechnology processes
that produce pharmaceuticals, fine chemicals
and genetically engineered products.

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• Study of the biological and biochemical
principles supports the field of biochemical
engineering.
• “A Biochemical Engineer is someone who
when with engineers, talks biology, when with
biologists, talks engineering and when with
biochemical engineers, talk politics”… V.
Hatzlmanikatis

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• Biochemical Engineering is a multidisciplinary
field:
Material science
Chemical
engineering
Biology:
microbiology,
molecular
biology Biochemical
Engineering
Pharmacology
Biochemistry
Chemistry Genetics
Medicine

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Related disciplines/areas of specialization
• Metabolic engineering including metabolic
modeling, bioprocess engineering, including
process control, bio-separation, bio-
informatics, bio-material engineering, tissue
engineering, manufacture engineer, reactor
design.
• Biochemical engineering covers research at
the scale of the biocatalyst development
(microbe, insect cell, mammalian cell, plant
cell, enzyme)
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Bio-processing fundamentals
• Biotechnology is the art and science of
converting reactants (substrates) into useful
products by the action of microorganism or
enzymes.
• Bio-processing simply means any process in
which microbes or living organisms plays a
vital role in getting transformation of the feed
into useful products.
• Examples: converting milk to curds, fruit juices
into wines, sugars into alcohols
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Traditional and modern application of
biotechnology
• They are in the areas of food, bakery products
and alcoholic beverages.
• Earliest application was in the production of
wine from fruit juices.
• Areas of health and hygiene for the
production of vaccines, enzymes, various
fermented foods, organic acids etc.

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Interdisciplinary approach to bio-processing
• Bio-processing or biotechnology operations
are interrelated with a large number of
faculties in science.
• Alcoholic fermentation was considered to
involve both chemical and microorganisms.
• The biochemical engineers were considered to
provide sterile environments in most bio-
processing.

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Outline of integrated bioprocess
• Integrated bio-processing consists of various
steps
Step 1: Isolation of the strain
Identification and isolation of the microorganism
which brings out the desired bioconversion. This
is generally the task of a microbiologist.
It involves some shake flask experimentation,
etc. The techno-economic viability of the
process will be taken up subsequently if the
results of this step 1 are encouraging.
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Step 2: Preservation of the strain
• The strain isolated in step 1 will be preserved
for future use.
• Its ability to perform and yield the products
should also be preserved. The strain can
therefore be stored ;
a) Refrigerated temperature (2 – 6 oC)
b) At frozen storage temperature (-18 to -80 oC)
c) By freeze drying (lyophilization)

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Step 3: Growth of inoculums
• Before using the strain in a
fermenter/bioreactor, the inoculum is cultured
for growth.
• The preserved strain/culture is revived by
growth in shake flasks or by solid-state
fermentation on the solid surfaces.
Solid state fermentation(SSF) is a method of
growing microorganisms in an environment of
limited moisture without having free flow of
water.

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In other words, microorganisms grow on a solid
surface which is moisten bed and which has
also got free access to air.
Step 4: Pre-fermentation culturing
Before the cells are admitted into the fermenter
for performing bioconversion, they are initially
grown separately with nutrients and the
substrate so that the cells can multiply and
proliferate. This will help increase the cell
density. The optimal cell concentrations in a
fermenter are as follows:
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1) Bacterial: 0.1 – 3.0%
2) Actinomycetes: 5 – 10%
3) Fungi: 5 – 10%
Step 5: Fermentation
• Major activity in industrial operation in the
whole bioprocess is FERMENTATION.
• Fermentation is the heart of the bio-
processing operation.
• Fermenter sizes vary from 1 to 450 m3,
depending upon the type of fermentation
process.

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A Fermenter
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• Based on capacity, tonnage and nature of the
fermentation product fermenters can be
classified;
1. Batch fermenter.
2. Fed-batch fermenter.
3. Continuous fermenter.
The conditions could be aerobic (with bubbling
of air) or anaerobic (in the absence of air).

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Step 6: Recovery and purification of the product
Recovery and purification sometimes decide the
economic viability of the process.
• The cost of recovery can vary anywhere
between 20 and 60% of the total
manufacturing costs.
The methodology depends on;
• Nature of the cells.
• How the hold the products : is it intracellular
(product is held within the cells) or
extracellular (the product is excreted from the
cells).
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A process for converting biomass to ethanol
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• Solids can be removed by simple filtration or
centrifugation techniques.
• The intracellular material is obtained by cell
disruption techniques for example
intracellular enzyme extraction
• Purification and isolation of the final products
are carried out using different unit operations.

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Step 7: Treatment of effluents
• Effluents are released during fermentation
processes.
• The effluents may be rich in organic matter
and may be a potential hazard for the
environment if released like that.
• Ability to economically be able to treat
effluents before release to the environment
makes or mars the viability of the product
manufacture.

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• Various effluent-treatment techniques can be
classified as:
-Physical
-Chemical
-Biological
The final choice depends upon the individual
cases and local circumstances.

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Unit Operations in Bioprocess
• The whole bio-processing operations can be
broadly classified into two classes/types of
operations.
1. Fermentation
2. Product recovery and effluent treatment.
• Fermentation is biochemical operation and can
be classified as unit process.
• Operations carried out on the fermentation
broth for product recovery and downstream
operations including effluent treatment may be
classified as unit operation.
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• Some operations carried out on the fermenter
such as fluid dynamics, mixing, air sparging
(introducing air or gas in order to agitate the
mixture), heat transfer to the fluids, etc may
also be categorize as unit operations.
• Product recovery and downstream processing
lines involve mixing and separations of various
phases, streams and fluids.
• We can broadly comprehend the systems
encountered as

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• Solid-Liquid, L-L, L-L-S, L-G, S-L-G, S-S.
The separation may be carried out with or
without phase change.
Mixing is also one of the most important unit
operations in bio-processing. It could be for
• G-L, L-L, L-S, G-L-S systems.

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Review questions
• What are the traditional and modern
applications of biotechnology?
• Distinguish between unit operation and unit
process.
• Biotechnology is an interdisciplinary subject.
Justify this statement.
• What are the various unit operations involved
in downs stream processing?

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1.1 Microbiology
• Microbiology is the study of microorganism
which are not only microscopic and exist as
single cells, but also ultramicroscopic
organism which are not cellular and hence
cannot exist independently e.g. viruses.
• It deals with study of functioning of cells, their
diversity and evolution, their interaction with
environment, other living organism and man.

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• Microbiology forms the basis for the
complimentary development in cell biology,
biochemistry, molecular biology and genetics.
• Microbiology is studied with respect to 2
major aspects:
1. As basic biological science.
2. As applied biological science.
As biological science it provides a system to
understand the nature of life processes, the
principle behind it, and the genetics which is
involved .

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Branches of basic microbiology
• Medical microbiology
• Agricultural ,,
• Environmental ,,
• Food & Dairy ,,
As applied biological science, microbiology deals

with the study of useful microorganisms as
well as that of pathogenic organisms.

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• Orientation of applied microbiology is in several
fields, and it uses the basic information and
techniques of different sub-divisions.
• Industrial microbiology is the study of
exploitation of the biochemical potentials of
microbes for the production of various products
like antibiotics, vaccines, steroids, solvents,
vitamins, leaching of minerals, etc.
• Microbiology also serves as a base for GENETIC
ENGINEERING which is an upcoming branch of
biotechnology and deals with improving the
performance of microorganisms.
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MICROSCOPY
• Microscopy is an important tool for unlocking
the secrets of the world of small creatures.
• Microorganisms and their structural components
are measured in smaller units such as
micrometers or microns (10-6 m), nanometers or
millimicrons (10-9 m), and angstroms (10-10 m).
• Microscopic resolutions are limited by the
size/type of the microbe. Some microbes are
unicellular, algae, fungi, protozoa, viruses,
proteins, lipids, small molecule.
• Operation ranges could vary from unaided eye, to
light microscope, and to electron microscope.
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MICROBIAL TAXONOMY
• Taxonomy is the subject of classification of
biological entities.
• Earlier, all living organisms were classified
under two main kingdoms, namely, Plantae
and Animalia by Swedish botanist Carolus
Linnaeus in the 18th century.
• Finally, in 1969, Robert H. Whittaker proposed
a 5-kingdom classification.

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The 5- Kingdoms;
• Monera: true bacteria and blue green algae.
• Prostita: eucaryotes such as protozoa which
are unicellular, contains chlorophyll (plant like)
and possess movement (animal like).
• Fungi: include non-green eucaryotic organisms
like slime moulds, bread moulds, sac fungi.
Composition of their cell wall differed from
that of plants, and had adjacent cells without
separation, hence are not considered as true
multicellular organism
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• Plantae: include algae, all mosses, ferns,
cornifers, and flowering plants.
• Animalia: Sponges (are primitive multicellular
animals, incapable of moving), worms, insects,
and vertebrates.
Microbial diversity
• Evolution roots of cell form a basis for
understanding the microbial diversity.
• Microbial diversity can be seen in terms of
variations in cell size, morphology,
metabolism, adaptation, growth etc.
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Microbial nomenclature
• Basic taxonomic unit is species which can be
defined as a collection of similar strains.
• Groups of species are collected as genera.
They share major properties.
• Groups are classified into families, families
into order, and orders into divisions.
• Following the binomial system of
nomenclature, microorganisms are given
genius and species name, which are either
Latin or Greek derivations.
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Example; Bacillus subtilus or Bacillus meaning
“rod shape”, which indicates its slender
nature;
B. cereus meaning “waxen”(shape can easily
change);
B. stearothermphilus meaning “heat loving”;
B. acidoceldarius meaning “acid-thermal”
They are written in italics with the genus name
stating with capital letter and the species
name with a small letter.

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• There are many ways of grouping
(classification) of procaryotes. The
characteristics of taxonomic approach include
morphology, Gram reaction (Gram reaction is
a classical technique to identify the basic
difference in the cell wall structure of the
bacteria. The cells are stained with crystal
violet dye, and then treated with iodine
solution. The cells are washed with alcohol.
Those which retain the blue crystal violet
colour are classified as gram positive, and
those which do not retain the colour are
called as gram negative species),
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nutrition, cell wall chemistry, pigment, storage
products, ability to use various substrates,
fermentation products, gaseous needs,
temperature, pH requirement, tolerances,
sensitivity.
• Molecular taxonomy includes guanine and
cytosine ratio (GC ratio) in organisms, the
extent of DNA: DNA hybridization, amino acid
sequencing and protein analysis.

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Chemical composition
• Cells are composed of small molecules as well
as macromolecules.
• They are made up of 4 basic elements, viz.
Carbon, Oxygen, Hydrogen and Nitrogen.
• Phosphorus, Iron, Sulphur, Zinc, Manganese,
Copper, Molybdenum, and Cobalt are also
present apart from the 4 basic elements.
• Water accounts for 90% of the weight of the
cells.

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• Of the macromolecules, proteins are most
abundant by weight (55%).
• Most procaryotes require an organic
compound of some sort as their source of
carbon. After carbon, the most abundant
element in cell is nitrogen (12% by dry
weight), which is a major constituent of
protein and nucleic acids. Nitrogen is found in
organic and inorganic forms, and is mostly
assimilated as ammonia NH3, nitrates NO3 or
N2.

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Nutritional requirements
• Organisms assimilate various organic
compounds, and use them to make new cell
wall materials, which may be amino acids,
fatty acids, organic acids, sugars, nitrogen
bases and aromatic compounds.
• Macronutrients required in nature and media
for organisms include Carbon, Hydrogen,
Oxygen, Nitrogen, phosphorus, Sulphur,
Potassium, Magnesium, Sodium, Calcium,
Iron.

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Micronutrients requirements by cells include;
• Chromium, Cobalt, Copper, Manganese,
Molybdenum, Nickel, Selenium, Tungsten,
Vanadium, Zinc, Iron.
GROWTH FACTORS
• They are the organic compounds required in
small amounts for growth and metabolism, and
these include vitamins, amino acids, purines,
pyrimidines. Vitamins are not only needed for
growth, but also play an important role as co-
enzymes. Most of the vitamins are synthesized by
organisms themselves, but some depends on
external sources.

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1.2 Metabolism
• Metabolism is a sum of biochemical reactions
in living organisms which include release of
energy by breakdown of complex organic
compounds into simpler forms (as
Catabolism) and release energy to form new
molecules like synthesis of proteins from
amino acids, sugar to polysaccharides (as
Anabolism).
• Life support activity of simple organisms
involves complex biochemical reactions.

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• Catabolism reactions furnish the energy
needed to drive anabolic reactions.
• The coupling of energy requiring reactions and
electron releasing reactions is made possible
through ATP (Adenosine tri-phosphate)
• ATP stores the energy released by catabolic
reactions and makes the energy available for
anabolic reactions and other cellular works.
• ATP is known as the currency of chemical
energy in cells.

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Assignments
Read more on catabolism especially the glycolic
pathway, Kreb’s cycle, Pentose phosphate
pathway. Anabolic reactions in living
organisms.

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1.3 Cell structure and function
Cells have evolved two architectural plans;
1. Procaryotes : cells without a nucleus.
2. Eucaryotes: cells with a nucleus.
THE PROCARYOTE
• In Greek means primitive nucleus. Includes
Bacteria and Archaea.
• Genetic material is not enclosed within
membrane.
• Lack membrane-bound organelles.
• Cell walls contain complex polysaccharide
peptidoglycon.
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Procaryotic cell
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• They have a simple method of reproduction.
• Procaryotes include a vast heterogeneous
group of very small unicellular organisms
which comprise eubacteria and
archaebacteria.
• Most range from 0.5 to 3 µm in diameter and
2-8 µm in length.
• They have a volume of 10-12 ml per cell and
may weigh approximately 10-12 g per cell.
• Variously shaped like cocci, spherical to oval,
streptococci, as tetrads, rod-shaped bacillus
etc.
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• They have external appendages (secondary
supports) as glycocalyx, flagella, procaryotic
flagellum, axial filaments, fimbriae, pili.
• Bacterial cell wall is complex, semi-rigid and is
responsible for characteristic shape.
• The cell wall protects cell organelles from
external environment and helps in anchorage.
• The cell wall is credited for classification of
major types of bacteria.
• Some primitive groups do not possess at all or
possess a very little cell wall material, e.g.
mycoplasm which are smallest bacteria.
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*Read on other features such as the cytoplasm
membrane, cytoplasm, nuclear area, ribosomes,
endospores etc.
THE EUCARYOTIC CELL
• The word means “true nucleus”.
• Consist of three main components: cell
membrane, cytoplasm, nucleus.
• The cells are larger and more complex than
the procaryotic cells.
• They have the generic material enclosed in a
specialized membrane.
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Eucaryotic cell
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• They contain distinct structures called cell
organelles within the cytoplasm (see
diagram).
• Typical eucaryotic cells range from 2 to 200
µm in diameter.
*Read up on the structure of eucaryotic cell
such as cell membrane, cytoplasm, nucleus.
Assignment: Draw and label the Eucaryptic Cell.
Highlight the different functions the cell
organelles,

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VIRUSES
• They are not classified as a part of any 5-
kingdom.
• There are no orders, divisions and kingdoms
established for viruses.
• They lack formal names, and most of them
acquired their names from source e.g.
measles virus derive its name after the
disease; Coxsackie virus after the place
Coxsackie, New Tork, where it was originally
isolated.
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• However, viruses are categorized into genera,
and each genus name ends with a suffix.
• Virus and genera have been organized into
families each ending with –viridae.
• Viruses are not composed of cells, and use
anabolic machinery within living host cells to
multiply.
• A virus genome (set of chromosomes) can
direct biosynthesis inside a host cell, and can
get incorporated into host genome.

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Virus
Fungi
Yeast

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Instructor/Author: Dr. A.O. Ayeni Algae 54
Fungi
• They are a diverse group of eucaryotes lacking
chlorophyll and are filamentous, with the cell
wall made up of chitin and coenocytic (i.e.
cytoplasm mingles between adjacent walls
through pores).
• They are typically aerobic, found in damp and
dark places.
• They are active producers of various hydrolytic
enzymes, and hence they can decompose
cellulose and lignin (like paper and wood
products).
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• They can also grow on the surface of electrical
insulators and make them transmit electricity.
• They are mostly saprophytic with complex life
cycles involve spore formation.
• They are mainly classified into;
1) moulds
2) Yeasts
• Yeasts are capable of anaerobic growth and
survive in different environments. If access is
given to oxygen, yeasts perform aerobic
respiration to metabolize carbohydrates to CO2
and H2O.

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• In the absence of oxygen, they ferment
carbohydrates to ethanol and CO2; hence they are
used in brewing, wine making and baking
industries.
ALGAE
• They are eucaryotic, photosynthetic,
multicellular, non-motile.
• More similar to plants, i.e. have branching
structures (leaf).
• Are attached to substrates by root-like hold fast.
• Some contain pigments (photosynthetic)
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• Cell organisation, process of photosynthesis and
storage materials link them strongly to higher
plants.
• Their mode of nutrition in some cases is non-
photosynthetic.
• Reproduction have resemblance close to animals.
• They are considered as primary food producers as
they convert chemically simple nutrients to
complex organic matter.
• Their dead cells contribute to the formation of a
complex organic material called “humus” which is
provided as food to other microbes.
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Protozoa
• It means “first animals”
• Except the flagellate algae, the phylum
protozoa are the only animal group in the
entire kingdom of Protista.
• They are heterogeneous group with highly
specialized cell structure, mode of life and
reproduction.
• They produce asexually by cell fission or by
genetic combination and multiplication. This
results in rejuvenation of an asexual process
called “Conjugation”

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Paramecium
Euglena
Amoeba
PROTOZOANS
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• All protozoa are chemoorganotrophic, capable
of taking solid particles into cell.
• They respond to stimuli such as heat,
chemicals, gravity and electricity.
• Certain organisms even have eye spot which
can distinguish light of different wavelength.

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Summary
• Cell structure, metabolism and function of the
cell constitute the core of microbiology.
• Biochemical Engineering is the application of
these core microbiology area to make
economically useful metabolic products.
• The understanding of these subjects and
ability to interweave them with process
engineering skills can lead to design of
bioreactors which make the cells to produce
useful metabolic products from organic
substrates.
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• Successful production and economization of a
fermentation process requires both industrial
microbiologist as well as a biochemical
engineer along with constant experimental
work and patience.
REVIEW QUESTIONS
• Describe the taxonomical classification of 5
kingdoms as proposed by Whittaker.
• Describe Kreb’s cycle
• Describe mitochondria. What is the function
of mitochondria in a cell
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Biochemistry
• It is the study of life cyclic processes in terms
of chemicals.
• It is not the study of chemistry of living
organisms.
• It obtains information from the basic
microorganisms viz., bacteria, viruses, algae
and fungi.
• It draws information on energetics from basic
thermodynamics.

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• Some of the chemical/biochemical reactions
in the living organisms are facilitated by
another type of compounds known as
enzymes.
• This facilitation is known as catalysis.
• Enzymes are therefore known as biocatalysts
or biological catalysts.
• Cells themselves contain some of the
enzymes.
• Cells regulate the type of enzyme and its
number by which it regulates both the type of
biochemical reaction and its rate.
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• Genetic engineering deals with the
information flow of the living organisms.
• Biochemistry, coupled with genetic
engineering principles play a vital role in
devising techniques for manipulating most of
the biochemical transformations.
• Living organisms contain various biomolecules
which are the building blocks of the cell and
also help in storing and releasing energy for
biotransformations.
• Biomolecules include carbohydrates, lipids,
proteins, nucleic acids, vitamins, etc.
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LIPIDS
• Classified as compounds which are fatty/oily
in nature and present in cells and tissues.
• Are insoluble in water
• Are soluble in non-polar solvents like n-
hexane, chloroform, benzene, etc., hence can
be extracted from the organisms using these
solvents.

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• They release a lot of energy on break down
and hence are considered as the energy
storage media.
• They contain a large proportion of C-H bonds.
• Upon saponification, they release fatty acids
and glycerol, and hence glycerol is considered
as a binding agent for fatty acids.
• They are synthesized by the cells from sugars.
• Lipids such as vitamins and hormones have
intense biological activity.
• They are grouped based on their chemical
composition.
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• They can be combined with other classes of
compounds and are known as; lipoproteins,
proteolipids, phosphatidopeptides,
lipoaminoacids, lipopolysaccharides.
• Some lipids contain glycerol while some do
not contain glycerol.
• As biomolecules, they are a constituent of cell
walls and form a protective coating to the cell.
• They are also energy carriers, and release the
energy as and when the cells require it.

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Fatty Acids
• Fatty acids are generally available in the
esterified form in cells and tissues, and are
rarely available in free form.
• They are named on the basis of the
hydrocarbon from which they are derived.
• The hydrocarbons may be saturated or
unsaturated.
• In saturated fatty acids, the carbon atoms hold
as many hydrogen atoms as possible in
addition to the carboxylic grouping.
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• In the unsaturated fatty acid, the carbon
atoms hold double and triple bonds.
• Fatty acids differ from each other primarily in
chain length and in the number and position
of their unsaturated bonds.
Place some structures of lipids biomolecules
here.
• Fatty acid esters of glycerol are called acyl-
glycerols or glycerides

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• Dependeing upon the number of hydroxyl
groups of the glycerol is esterified, it is known
as monoacyl-, diacyl-, or tracyl-glycerol.
• ESTER is an organic compound formed in a
reaction between an acid and alcohol with the
elimination of water.
(Place structures here).

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• Phospholipids consists of a glycerol connected
to a phosphate group.
• Some lipids do not contain glycerols. Examples
include Sphingolipids, Terpenes, steroids.
• NUCLEIC ACIDS, AMINO ACIDS & PROTEINS

(ARE TO BE TREATED IN MODULE 6)

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Carbohydrates
• Are also referred to as saccharides.
• Are large group of compounds which are poly-
hydroxyaldehydes or poly-hydroxyketones and
their derivatives.
• They contain carbon, hydrogen and oxygen,
with the ratio of hydrogen and oxygen being
2:1.
• Have general chemical formula Cx(H2O)y

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• May be classified as monosaccharides,
oligosaccharides (di- and tri-saccharides),
polysaccharides, high molecular weight poly-
saccharides.
(Place structures here)
• Monosaccharides are named based on the
numbers of carbon atoms; 3-trioses, e.g
glyceraldehyde; 4-tetroses, e.g erythrose; 5-
pentoses, e.g arabinose, xylose, ribose,
rhamnose; 6-hexoses, e.g. glucose, galactose,
fructose, mannose;
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• Disaccaharides are anhydrides of two
monosaccharides, which include sucrose,
maltose, lactose, and cellobiose.
• Oligosaccharides are anhydrides of several
monosaccharides residues. Compounds
containing ten or less monosaccharides units
are called oligosaccharides while those
containing more than ten are called
polysaccharides.
• Polysaccharides include starches, cellulose.
They are also known as glycans.

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• Polysaccharides are anhydrides of one or
more monosaccharides in which a large
number of units are combined.
• Starches are nutritional reservoir in plants
corresponding alpha(1,4) linkages.
• The repeating disaccharide unit in starch is
maltose.
• Native starches are a mixture of two
compounds, viz, amylose and amylopeptin.
• Cellulose is the compound which gives
strength to plant tissues.

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• Acid hydrolysis of cellulose yields glucose of
about 95-96%.
• Glucose units are joined in beta(1,4)linkages.
• The purest form being cotton containing at
least 90% pure cellulose.
VITAMINS
• They are group of organic compounds
required in trace amounts to stimulate body
growth.
• They also keep away certain types of diseases.

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• They are also present in certain enzymes to
assist them in their catalytic activity. Hence
they are also known as co-enzymes.
• They are present in living cells in addition to
lipids, proteins, carbohydrates, nucleic acids,
etc.
• Most vitamins contain amines, some do not
contain amine but the name Vitamin is
retained.
• Vitamins are classified into two groups as
water soluble and fat soluble.

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• Examples include Ascorbic acid (Vit-C), Folic
acid, Biotin, Riboflavin (Vit-B2), Thiamin (B1),
Nicotinic acid (niacin), Vitamin A, D, E, K,
Inositol.
(Check the structures of some of these vitamins)
REVIEW QUESTIONS
• Describe water soluble and fat-soluble
vitamins.
• Write short notes on biomolecules.
• What are steroids, terpenes, saturated and
unsaturated fatty acids.
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MODULE 3: ENZYMES
• Enzymes are natural catalyst that make
biochemical reactions occur rapidly.
• A catalyst is a substance that accelerates a
chemical reaction but not consumed in the
overall process.
• They are biological catalysts used to increase
the rate of biochemical reactions taking place
in living systems, without undergoing any
overall change.
• All enzymes are important class of conjugated
proteins.

1/12/2021 81
TERMS IN ENZYMOLOGY
Cofactor: the non-protein component of an
enzyme.
Coenzyme: an enzyme with organic molecule as
cofactor.
Holoenzyme: an active enzyme including
cofactor.
Apoenzyme: the inactive portion of protein.
In some enzymes, metal may be conjugated to
the protein part.

1/12/2021 82
• A large number of enzymes has been isolated
from time to time.
• By 1985, as many as 2500 enzymes were
known out of which 250 were marketable.
Classification of enzymes
• They are usually termed in the reactions they
catalysed.
• -ase is customarily added at the end (suffix)
e.g. enzyme that attack urea is urease, argenine
is arginase.

1/12/2021 83
• They are also classified according to the
groups that catalyze similar chemical reactions
such as proteases, lipases, oxidases, etc. The
only major exception is trypsin.
• All the enzyme groups are essentially
classified into 2 groups, viz.
1. Hydrolyzing enzymes or hydrolases
2. Other enzymes or desmolases.
READ ON ENZYME COMMISSION’S
CLASSIFICATION

1/12/2021 84
Enzyme as biological catalysts
• Enzymes make biological reactions proceed.
Two possible important theories are
responsible for this;
1. Collision theory: proposes that reactions take
place by the collision of the reactant
molecules. That is, the more is the
concentration of the reactants, more are the
chances for the reactants to collide, and hence
more will be the rate of reaction. However, all
collisions may not necessarily results in the
reaction to proceed to produce products.
1/12/2021 85
2. Transition state theory: proposes that the
collision of certain molecules, which have
crossed certain potential energy barrier, alone
will result in the reaction to take place. This
potential energy barrier is known as activation
energy.

1/12/2021 86
Biocatalysis of enzymatic reactions
• This can be explained by the concept that the
enzyme form with the substrate an
intermediate complex which further
decomposes to form the bioproducts
(place enzymatic process of conversion of
substrate to product here)

1/12/2021 87
S P
S = Substrate, P = Product, the transition state
proposes a halfway point and unstable
intermediate complex where the bonds of S
are distorted sufficiently such that conversion
to P becomes possible.
• The rate of reaction obviously depends on the
number of molecules of S that enter the
transition state per unit time.
• The rate of reaction can be increased by
raising the temperature or lowering the
activation energy.
1/12/2021 88
• The enzyme complexes lower the energies of
activation of vital biochemical reactions.
• Thus enzymes can carry out the reactions at
ambient temperatures.
• This makes an enzyme catalyzed reaction at 25
oC to proceed at 106 – 1015 times faster as
compared to an uncatalyzed reaction.

1/12/2021 89
1/12/2021 90
Enzyme specificity
• They are specific in action.
• They usually catalyse only one particular type
of reaction by selectively lowering the
activation energy of only one of the several
possible chemical/biochemical reactions.
• Depending upon the reaction conditions and
the specific nature of the enzymes, the
catalysed process exhibit;
Absolute specificity, group, stereochemical,
product, substrate specificities, etc.

1/12/2021 91
Stereochemical specificity
• The concept of active site is useful. An
enzyme is conceived to possess two different
kinds of site;
1. Binding sites
2. Catalytic sites
(Place diagram here)
• The substrate-enzyme interaction can be at
the binding sites or catalytic sites.

1/12/2021 92
1/12/2021 93
• The active site or active centre of the enzyme
is the region which contains the binding and
catalytic sites which are only a small fraction
of the total volume of the enzyme.
• It is usually situated at or near the surface of
the enzyme and thus easily accessible for
substrate molecules to get attached.
• The function of an enzyme depends upon the
arrangement of binding and catalytic sites.

1/12/2021 94
Enzyme specificity hypotheses
• The specificity in catalytic activity and ability
of enzyme to interact with the substrates have
been proposed by several hypotheses.
• The few prominent ones are;
1. Fisher lock-and-key hypothesis
2. Koshland induced fit hypothesis
3. Hypothesis involving strain or transition-state
stabilization.
(Please read about these hypotheses)

1/12/2021 95
ENZYME KINETICS
• The study of kinetics tells us how fast does a
reaction proceed.
• Reactions can take place in two phases;
1. Homogeneous phase (reactant and catalyst
in the same phase).
2. Heterogeneous phase (reactant and catalyst
in different phases).
• Enzymatic reactions are considered
analogous to catalytic reactions.
1/12/2021 96
• An enzyme is a protein or protein-like
substance with catalytic properties.
• A substrate, S is equivalent to the chemical
reactant whose reaction rate is accelerated
due to the action of enzymes.
• Three possible enzymatic reactions can be
established
Type 1: Soluble enzyme-Insoluble substrate
Type 2: Insoluble enzyme-Soluble substrate
Type 3: Soluble enzyme-Soluble substrate.

1/12/2021 97
• We are focusing on Type 3, which are known
as homogenous reactions. Note that
equations developed here are equally
applicable to the other types.
(Place the mechanism of the enzymatic reaction
here)

1/12/2021 98
Immobilization of enzymes
• This is the restriction of the movement of
enzymes in a fixed location.
• Immobilized enzymes are attached to an
insoluble support medium or enclosed by the
support medium which is also known as a
CARRIER.
• Their movement is restricted but their
catalytic activities are retained.
• The restriction could also be movement
completely or to some regions.
1/12/2021 99
• Since enzymes are proteins and are soluble in
water, it is difficult to separate them once the
reaction is over for reuse in a batch process.
• To overcome this difficulty, enzymes are
immobilized on the carrier materials or inside
an insoluble matrix by various physical or
chemical methods.
• Immobilization techniques can be divided into
four main groups based on whether the
enzymes are entrapped in a limited space or
bound to a carrier material.
1/12/2021 100
• Entrapment; Matrix entrapment and Micro
encapsulation.
• Binding; Adsorption (using adsorbents such as
alumina, clay, silica, anion-exchange resins,
CaCO3, C, cation exchange resins, collagen and
glass plates) and Covalent bonding

1/12/2021 101
ENZYME INHIBITION
• Apart from temperature and solution pH, one
other factor that greatly influence the rates of
enzyme-catalysed reactions is the presence of
an inhibitor.
• Inhibitor could be the substrate, or the
product. Other compound in the system could
also act as an inhibitor.
• Inhibitors are species that interact with
enzymes and render the enzyme ineffective to
catalyze its specific reaction.
1/12/2021 102
• The three most common types of reversible
(an assumption) inhibition occurring in
enzyme reactions are competitive,
uncompetitive, and non-competitive. The case
of irreversible inhibition leads to toxicity of
the medium.
• Each kind of inhibition leads to a different
form of the rate equation.
• The rate equations are altered from the
standard M-M form.

1/12/2021 103
Competitive inhibition
• Here, another substance, I, competes with the
substrate for the enzyme molecules to form
an inhibitor-enzyme complex.
• The inhibitor binds to the active site and
prevents binding of the substrate.
(Place equations here)

1/12/2021 104
• The final equation,
Vp = -Vs = Vmax[S]
KM {1+([I]/KI)} + [S]
• The M-M expression is altered.
• The enzyme appears to have a lower affinity
for the substrate (higher KM = lower affinity).
• It means more substrate is needed to achieve
a higher reaction rate, since the substrate can
compete for the binding site.

1/12/2021 105
Vmax and KM are defined as before when no
inhibition is present, that is,
Vmax = K3 (K2) Et and KM = (k2 + k3)/k1
and the inhibition constant, KI (mol/dm3) is
KI = k5/k4
(Rearrange to generate the L-B plot)

1/12/2021 106
Uncompetitive inhibition
• In this case, the inhibitor binds to the E.S
complex and prevents conversion to products.
• The inhibitor has no affinity for the enzyme
and does not compete with the substrate for
the enzyme, rather ties with ES to form
inactive [I.E.S.] complex.
• We assume that this binding is in equilibrium
and can be represented with their dissociation
constants k’s.
• Two additional steps are also added here.
1/12/2021 107
(place the equations here)
Non Competitive
• The inhibitor can bind to either free enzyme
or enzyme substrate complex. Likewise, the
substrate can bind to free enzyme or the
enzyme-inhibitor complex.
• Binding of one does not prohibit binding of
the other, however the E-I and E-S-I complexes
are both dead end( product cannot be formed
if I is bound)
(place the equations here)

1/12/2021 108
• It is also called mixed inhibition.
• Whenever the inhibitor is attached to the
enzyme it is inactive and cannot form product.
• The deactivating complex, E-S-I can be formed
by two reversible reaction paths.

1/12/2021 109
Substrate inhibition
• In a number of cases, the substrate itself can
act as a inhibitor.
• In most cases are uncompetitive inhibition.
S + E-S S-E-S (Inactive)
(Place the rate law here)

1/12/2021 110
Toxicity
• Earlier, we assumed that the binding of the
inhibitor is reversible.
• We also assumed that a once bound inhibitor
has been released from the enzyme, the
enzyme has not been negatively affected by
the experience.
• A case of toxicity and not inhibition is
explained if the enzyme has been indeed
rendered inactive by the binding or if the
bound inhibitor does not release.
1/12/2021 111
• The rate expressions have a complex
mathematics.
For competitive inhibitor
[E] + [S] [E-S] [E] + [P]
For Toxicity,
[E] + [I] [E-I] [E-I]dead

1/12/2021 112
MODULE 4: KINETICS OF MICROBIAL GROWTH
• Microbial growth is an orderly increase in
cellular component.
• The increase leads to cell enlargement and
eventual cell division (sometimes cell division
does not occur, but conc. of biomass increases
i.e. increases in size).
• Growth kinetics can be modelled using
differential equations in a continuum model.

1/12/2021 113
• In general chemical reactions kinetics, the rate
of reaction is dependent upon the
concentration of reactant(s).
• However, for biochemical reactions, there are
other complications added to the kinetics.
• The reaction proceeds with the intervention of
living organisms or cells in the presence of
nutrients of the medium under specified
conditions of pH, temperature, etc.

1/12/2021 114
In a biochemical reaction;
• All microorganisms are not added in desired
quantities (unlike in the case of chemical
reaction)
• The cells consume part of the substrate
(reactant) and essential nutrients from the
medium of fermentation.
• The cells initially multiply and grow.
• Depending if cells are unicellular or multi-
cellular, the growth pattern varies.

1/12/2021 115
• Cells can divide when they grow, increasing
the number of cell or biomass.
• more and more of substrate is consumed as
cell number increases.
• Moulds increase in number, also increase in
size and hence density of the cells, which also
results in increase in the viscosity of the broth.
• All the changes manifest in the form of
changes in various other physico-chemical and
thermal parameters of the broth.

1/12/2021 116
Associated with cell growth are other processes
as;
• Uptake of some material from the cell’s
environment.
• The materials are converted into some
metabolic end products.
- Associated cell growth starts destabilizing the
cell growth and the availability of substrate for
competitive processes, namely: cell growth
and bioconversion of the substrate into
metabolic products.

1/12/2021 117
• To formulate a simple kinetic model involving
all the complexities mentioned is almost
impossible.
• A simple equation as -rA = k(CA)n is almost
never possible.

1/12/2021 118
PHASES OF CELL GROWTH
Growth Curve of cells in a batch process

1/12/2021 119
LAG PHASE
• In the lag phase, virtually no growth occurs
and the microbial population remains
relatively constant.
• The lag phase is a period of intense metabolic
activity as the microbial inoculum
adapts/adjusts to the new environment.
• The chemical composition of the fermentation
media influences the length of the lag phase.
• It is usually longer if the inoculum was grown
up using a carbon source different from that in
the fresh medium.
1/12/2021 120
LOG or EXPONENTIAL or GROWTH PHASE
• The cells start consuming nutrients in the
medium and multiply.
• Once growth starts, the cells multiply in an
exponential order.
• The growth is very fast.
• The phase is very important and all the kinetic
growth parameters are studied here.
• Since the growth is exponential, when the cell
conc. is plotted versus time on a semi-log plot,
the log phase results in a straight line.

1/12/2021 121
• At a maximum point the cells stop growing.
Hence growth becomes stagnant
STATIONARY PHASE
• Here, the growth rate decreases and
eventually stops.
• This can be due to depletion of essential
nutrients (carbon source, essential amino
acids, etc.)
• It could be a build up of toxic metabolites such
as ethanol and lactic acid

1/12/2021 122
• It could also be a combination of nutrient
depletion and toxin accumulation.
• It is probably in the stationary phase that cells
start converting the substrate into metabolic
products.
DEATH or DECAY PHASE
• The cells start decaying at the beginning of
this phase.
• Probably the cells face shortage of substrate
for their consumption. Hence they start
decaying.

1/12/2021 123
Batch reactor data analysis
• A biochemical reactor is a vessel in which
substrate (reactant) undergoes a biochemical
transformation by the action of living cells,
microorganisms or cell-free enzyme system.
• We can use terms like, biochemical reactor,
biological reactor, bioreactor, fermenter,
microbial reactor. All these have the same
meaning.
Thus, a bioreactor is for carrying out microbial
fermentation, enzyme (cell-free) reactions.
1/12/2021 124
• Depending on requirements, different types of
bioreactors are used, and are operated as
aerobic, anaerobic, solid state, enzymatic
(immobilized or soluble), well-
mixed/stratified.
• Fermentations give rise to a variety of
products based on the type of cell and its
growth pattern. They are: primary metabolites
such as alcohol, citric acid; biomass such as
Baker’s yeast, single cell protein (SCP);
transformed substrates such as steroids;
purified solvents as in the case of water
treatment.
1/12/2021 125
• Batch reactor is frequently used to collect the
kinetic data in view of its ease of construction,
and versatility i.e. same batch bioreactor can
be used for various reactions.
• Data are collected on concentration versus
time, and processed to evaluate the reaction
rate constant as given before.
(Place the procedure to find out the reaction
rate constants)

1/12/2021 126
KINETIC MODELS FOR CELL GROWTH
• Since the growth of cells in the log phase follows
an exponential path, we can describe the cell
growth in the form of mathematical equation.
• It is generally believed that the growth rate of the
cells at any time in the growth phase is
proportional to the number of the cells present at
that time (Malthus law).
dx/dt = µx M4-1
X = mass of cells per unit volume
µ = proportionality constant known as ‘Specific
growth rate’ in (h)-1 and t is the time in hours.
1/12/2021 127
• Equ. M4-1 is similar to –rA = k1CA, except with
a difference of –ve sign. This means as t
increases, x also will be increasing.
Integrating M4-1,
ln (x/x0) = µt
or ln x = ln x0 + µt …M4-2
The plot of ln x versus t from M4-2 gives a
straight line with ln x0 as its intercept and as
the slope µ.
Equation M4-2 enables us to calculate the
SPECIFIC GROWTH RATE µ with the
knowledge of x versus t data.
1/12/2021 128
• The biomass produced from the growth of the
cells is always due to a change in concentration of
the substrate.
Mathematically,
X = Yx/s (SR – s) …M4-3
X = biomass conc. which is produced by the
depletion of the substrate with its original
concentration SR , and s is the residual
concentration of the substrate.
Yx/s ,is some yield parameter which relates the
conversion of substrate to biomass and is
dimensionless.

1/12/2021 129
• The stationary phase and death phase of the
cells are caused by the depletion of one of the
components in the medium, which we call as
the “limiting substrate”.
• Limiting substrate is that in the medium, the
concentration of which affects the cell growth
and changes the growth phase from the log
phase to the stationary phase and finally to
the death phase.
• The residual limiting substrate concentration
also affects the specific growth rate.
1/12/2021 130
• If s is more, the µ also increases up to a certain
extent and ultimately reaches a plateau as shown
in the graph .
(place the graph of specific growth rate on the

concentration of the limiting substrate)
• The graph indicates that more the substrate,

more is the specific growth rate up to a certain
extent, beyond which it does not have any effect.
• This plateau of µ is called MAXIMUM SPECIFIC
GROWTH RATE CONSTANT (µm).
1/12/2021 131
• Introducing the concept of “doubling time”
(i.e. the time required to double the number
of cells x = 2xo) for cell growth rates, we have
ln (x/xo) = ln 2 = µtd …M4-4
td = ln 2/µ = 0.693/µ
µ = specific growth for cells, which is defined as
the rate of increase in cell concentration per
unit of cell concentration (h-1), we can write
1 dx/dt = µ …M4-5
X
This equation is frequently referred to as the
growth equation.
1/12/2021 132
THE MONOD MODEL
• As shown earlier that the growth rate passes
through various phases, viz., high growth
phase, low growth phase and finally cessation.
• In other words, the specific growth rate varies
with the residual concentration of the limiting
substrate.
• This relationship is well explained by an
empirical equation proposed by Monod
(1949), which is for exponential growth.

1/12/2021 133
(Place the steps in calculating Monod constants
here)
• Monod equation is a expression which
reasonably predicts the kinetics of the cell
growth.
• The Monod equation depicts the general
variation of µ with s except when s>>Ks. In
such cases, the equation predicts the
percentage increase in µm as compared to µ
• In 1958, Moser included an additional
empirical feature to overcome some
limitations of the Monod model.
1/12/2021
134
GROWTH OF FILAMENTOUS ORGANISM
• Filamentous organisms like moulds, fungi etc.,
does not necessarily increase the number of
cells (i.e. biomass), but increase them in
length, hence physical properties like density
of the cell mass and viscosity of the broth
change.
• Therefore, product formation by the organism
may be classified as growth associated and
non-growth associated.

1/12/2021 135
Growth Associated product formation
• The rate of production of the product parallels
the rate of growth of cell population. Example
is the production of alcohol by the anaerobic
fermentation of glucose by yeast.
(Place the graph here)
Non-growth Associated product formation
• Products such as antibiotics and vitamins are
produced in batch fermentations at the end of
the log phase or only after entering into the
stationary phase.
1/12/2021 136
• Secondary metabolites as these are known as
non-growth associated products.
• In non-growth associated, the rate of product
formation is proportional to the cell
concentration rather than the growth rate.

1/12/2021 137
Substrate and product inhibition on cell growth
• In microbial processes, some higher
concentrations of the substrate or the product
inhibit the growth of the microorganism,
which make the growth rate to fall.
• In 1970, Edward used an equation originally
proposed for substrate-inhibited enzyme
reactions to describe the growth of
microorganisms, and how the substrate
concentrations affect the growth rate of the
cells.
1/12/2021 138
• The specific growth rate can be represented as
(a modified version of the Monod equation)
µ = µms/ (Ks + s + {s2/KI}) …M4-6

KI is the inhibition constant
As the substrate concentration increases, µ will
increase up to a certain extent. A maximum
value µm is attained. Subsequently, further
increase in substrate concentration results in
decreasing µ.

1/12/2021 139
µmax can be obtained by differentiation the last
equation with respect to s and equation to
zero.
Therefore, the value of s corresponding to µmax
is given as
s = (KsKI)1/2 …M4-7
(refer to the graph of influence of substrate
concentration on the specific growth rate)
substituting M4-7 into M4-6 results in

1/12/2021 140
µmax = µm /{2(Ks/KI)1/2 +1} …M4-8
The growth of microbes on acetate is an
example of the use of M4-6.
It shows the inhibitory effect of the substrate
concentration on the specific growth rate of
microorganisms.
• In certain cases, the product also inhibits the
growth of microorganisms, which is called
“product inhibition” cell growth. Example is
the build up of ethanol concentartion in
fermentation of glucose by yeast.
1/12/2021 141
• As the alcohol concentration builds up to 8-
9%, it will have an inhibitory effect on the
growth of the organisms.
• The yeast cell will not be able to survive any
more build up of alcohol concentration.
• In such cases, Monod equation does not give
an adequate estimate of the specific growth
rate.
• Mathematical representation of the situation
is to incorporate a term into the Monod
equation which will depress the value of µ with
increase in product concentration.
1/12/2021 142
µ = {µ s/ (Ks +s)} {Kp/(Kp + P)} …M4-8
Kp = constant representing the inhibitory effect
due to the product.
P = the concentration of the product.
As P increases, µ will decrease.
If P =0, equation reduces to Monod equation.

1/12/2021 143

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CHE 511

CHE 511: Biochemical Engineering.

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As applied biological science, microbiology deals

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(Place structures here).

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• NUCLEIC ACIDS, AMINO ACIDS & PROTEINS

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