Review of Microbiology and Biochemistry a.

Monosaccharides are simple sugars, the most common of which is

Biochemical Engineering is a branch of chemical and biological glucose. (mono- = “one”; sacchar- = “sugar”) are simple sugars, the
engineering that deals with the study of designing and developing the most common of which is glucose. Monosaccharides have a formula
units that come in direct contact with people. Involving molecules of (CH2O)n and they typically contain three to seven carbon atoms.
(bioreactors) and other biological organisms, these professionals are Most monosaccharide names end with the suffix -ose.
trained to use and implement their scientific knowledge and engineering Most of the oxygen atoms in monosaccharides are found in
principles to create products such as medicines, processed foods, oil, hydroxyl (OH) groups, but one of them is part of a carbonyl (C=O)
paints, cosmetics, paper, plastic amongst others. group. The position of the carbonyl (C=O) group can be used to
categorize the sugars:
Microbiology is the study of all living organisms that are too small to be ● If the sugar has an aldehyde group, meaning that the
visible with the naked eye. This includes bacteria, archaea, viruses, fungi, carbonyl C is the last one in the chain, it is known as an
prions, protozoa and algae, collectively known as 'microbes'. These aldose.
microbes play key roles in nutrient cycling, ● If the carbonyl C is internal to the chain, so that there
biodegradation/biodeterioration, climate change, food spoilage, the cause are other carbons on both sides of it, it forms a ketone
and control of disease, and biotechnology. Thanks to their versatility, group and the sugar is called a ketose.
microbes can be put to work in many ways: making life-saving drugs, the ● Depending on the number of carbon atoms in the sugar,
manufacture of biofuels, cleaning up pollution, and producing/processing they may be known as trioses (three carbon atoms),
food and drink. pentoses (five carbon atoms), and hexoses (six carbon
atoms).
MACROMOLECULES b. Disaccharides form when two monosaccharides undergo a
History and Introduction dehydration reaction (a reaction in which the removal of a
The term macromolecule (macro + molecule) was coined by Nobel water molecule occurs). During this process, the hydroxyl
laureate Hermann in the 1920’s. group (–OH) of one monosaccharide combines with a
The large molecules necessary for life that are built from smaller organic hydrogen atom of another monosaccharide, releasing a
molecules are called biological macromolecules. These macromolecules molecule of water (H2O) and forming a covalent bond between
may consist of thousands of covalently bonded atoms, some with mass atoms in the two sugar molecules. Common disaccharides
greater than 100,000 daltons. include lactose, maltose, and sucrose.
Four major classes of biological macromolecules c. A long chain of monosaccharides linked by covalent bonds is
● Carbohydrates known as a polysaccharide. The chain may be branched or
● Lipids unbranched, and it may contain different types of
● Proteins monosaccharides. Polysaccharides may be very large
● Nucleic acids molecules. The molecular weight of a polysaccharide can be
quite high, reaching 100,000 daltons or more if enough
and each is an important component of the cell and performs a wide monomers are joined. Starch, glycogen, cellulose, and chitin
array of functions. are some major examples of polysaccharides important in
living organisms.
Combined, these molecules make up the majority of a cell’s mass.
Biological macromolecules are organic, meaning that they contain Lipids
carbon. In addition, they may contain hydrogen, oxygen, nitrogen, Fats are just one type of lipid, a category of molecules united by their
phosphorus, sulfur, and additional minor elements. Biochemists have inability to mix well with water. Lipids tend to be hydrophobic, nonpolar,
determined the detailed structures of many macromolecules, which and made up mostly of hydrocarbon chains, though there are some
exhibit unique emergent properties arising from the orderly arrangement variations on this, which we'll explore below. The different varieties of
of their atoms. lipids have different structures, and correspondingly diverse roles in
Carbohydrates organisms. For instance, lipids store energy, provide insulation, make up
Macromolecules with which most consumers are somewhat familiar. cell membranes, form water-repellent layers on leaves, and provide
Carbohydrates provide energy to the body, particularly through glucose, building blocks for hormones like testosterone.
a simple sugar. Carbohydrates also have other important functions in
humans, animals, and plants. Fats and Oils
General names for carbohydrates include sugars, starches, saccharides,A fat molecule consists of two kinds of parts: a glycerol backbone and
and polysaccharides. The term saccharide is derived from the Latin three fatty acid tails. Glycerol is a small organic molecule with three
word “sacchararum” which means the sweet taste of sugars. The name hydroxyl (OH) groups, while a fatty acid consists of a long hydrocarbon
“carbohydrate” means a “hydrate of carbon” which was given to it due chain attached to a carboxyl group. A typical fatty acid contains 12–18
to its composition. carbons, though some may have as few as 4 or as many as 36.
Carbohydrates can be represented by the formula (CH2O)n, where n is Fat molecules are also called triacylglycerols, or, in bloodwork done by
the number of carbon atoms in the molecule. In other words, the ratio of
your doctor, triglycerides. In the human body, triglycerides are primarily
carbon to hydrogen to oxygen is 1:2:1 in carbohydrate molecules. stored in specialized fat cells, called adipocytes, which make up a tissue
The simplest carbohydrates are monosaccharides, or simple sugars. known as adipose tissue. While many fatty acids are found in fat
Carbohydrates are the most abundant class of organic compounds found molecules, some are also free in the body, and they are considered a type
in living organisms. They originate as products of photosynthesis, an of lipid in their own right.
endothermic reductive condensation of carbon dioxide requiring light Omega Fatty Acids
energy and the pigment chlorophyll. Another class of fatty acids that deserves mention includes the omega-3
n CO2 + nH2O + energy CnH2nOn + nO2 and omega-6 fatty acids. There are different types of omega-3 and
omega-6 fatty acids, but all of them are made from two basic precursor
Carbohydrates are classified into three subtypes: monosaccharides,
forms: alpha-linolenic acid (ALA) for omega-3s and linoleic acid (LA)
disaccharides, and polysaccharides.
for omega-6s.
The human body needs these molecules (and their derivatives), but can't
synthesize either ALA or LA itself. Accordingly, ALA and LA are
classified as essential fatty acids and must be obtained from a person’s
diet. Some fish, such as salmon, and some seeds, such as chia and flax,
are good sources of omega-3 fatty acids.
Omega-3 and omega-6 fatty acids have at least two cis-unsaturated
bonds, which gives them a curved shape.
Role of Fats
Fats have received a lot of bad publicity, and it’s true that eating large
amounts of fried foods and other “fatty” foods can lead to weight gain
and cause health problems. However, fats are essential to the body and
have a number of important functions.
For instance, many vitamins are fat-soluble, meaning that they must be
associated with fat molecules in order to be effectively absorbed by the
body. Fats also provide an efficient way to store energy over long time
periods, since they contain over twice as much energy per gram as
carbohydrates, and they additionally provide insulation for the body.
Like all the other large biological molecules, fats in the right amounts are
necessary to keep your body (and the bodies of other organisms)
functioning correctly.
Waxes
Waxes are another biologically important category of lipids. Wax covers
the feathers of some aquatic birds and the leaf surfaces of some plants,
where its hydrophobic (water-repelling) properties prevent water from
sticking to, or soaking into, the surface. This is why water beads up on
the leaves of many plants, and why birds don’t get soaked through when
it rains. These are just some role of wax besides the many industrial
products that are made out of it.
Phospholipids
Cells are surrounded by a structure called the plasma membrane, which
serves as a barrier between the inside of the cell and its surroundings.
Specialized lipids called phospholipids are major components of the
plasma membrane. Like fats, they are typically composed of fatty acid
chains attached to a backbone of glycerol. Instead having three fatty acid
tails, however, phospholipids generally have just two, and the third
carbon of the glycerol backbone is occupied by a modified phosphate
group. Different phospholipids have different modifiers on the phosphate
group, with choline (a nitrogen-containing compound) and serine (an
amino acid) being common examples. Different modifiers give
phospholipids different properties and roles in a cell.
A phospholipid is an amphipathic molecule, meaning it has a
hydrophobic part and a hydrophilic part. The fatty acid chains are
hydrophobic and do not interact with water, whereas the phosphate-
containing group is hydrophilic (because of its charge) and interacts
readily with water. In a membrane, phospholipids are arranged into a
structure called a bilayer, with their phosphate heads facing the water and
their tails pointing towards the inside (above). This organization prevents
the hydrophobic tails from coming into contact with the water, making it
a low-energy, stable arrangement.
If a drop of phospholipids is placed in water, it may spontaneously form
a sphere-shaped structure known as a micelle, in which the hydrophilic
phosphate heads face the outside and the fatty acids face the interior of
this structure. Formation of micelle is an energetically favored because it
sequesters the hydrophobic fatty acid tails, allowing the hydrophilic
phosphate head group to instead interact with the surrounding water
Steroids
Steroids are another class of lipid molecules, identifiable by their
structure of four fused rings. Although they do not resemble the other
lipids structurally, steroids are included in the lipid category because they
are also hydrophobic and insoluble in water. All steroids have four linked
carbon rings and several of them, like cholesterol, also have a short tail.
Many steroids also have an –OH functional group attached at a particular
site, as shown for cholesterol below; such steroids are also classified as
alcohols, and are thus called sterols.
Cholesterol, the most common steroid, is mainly synthesized in the liver
and is the precursor to many steroid hormones. These include the sex
hormones testosterone and estradiol, which are secreted by the gonads
(testes and ovaries). Cholesterol also serves as the starting material for
other important molecules in the body, including vitamin D and bile
acids, which aid in the digestion and absorption of fats from dietary
sources. It’s also a key component of cell membranes, altering their
fluidity and dynamics.
Of course, cholesterol is also found in the bloodstream, and blood levels
of cholesterol are what we often hear about at the doctor’s office or in
news reports. Cholesterol in the blood can have both protective effects
(in its high-density, or HDL, form) and negative effects (in its low-
density, or LDL, form) on cardiovascular health.
Proteins
Proteins are one of the most abundant organic molecules in living
systems and have the most diverse range of functions of all
macromolecules. Proteins may be structural, regulatory, contractile, or
protective; they may serve in transport, storage, or membranes; or they
may be toxins or enzymes.
Proteins can function as enzymes or hormones:
Enzymes - are produced by living cells, are catalysts in biochemical
reactions (like digestion) and are usually proteins. Each enzyme is
specific for the substrate (a reactant that binds to an enzyme) upon which
it acts.
Hormones - are chemical signaling molecules, usually proteins or
steroids, secreted by an endocrine gland or group of endocrine cells that
act to control or regulate specific physiological processes, including
growth, development, metabolism, and reproduction.
Amino acids are the monomers that make up proteins. Each amino acid
has the same fundamental structure, which consists of a central carbon
atom bonded to an amino group (–NH2), a carboxyl group (–COOH),
and a hydrogen atom.
Peptide Bonds
Each amino acid is attached to another amino acid by a covalent bond,
known as a peptide bond, which is formed by a dehydration reaction. The
carboxyl group of one amino acid and the amino group of a second
amino acid combine, releasing a water molecule. The resulting bond is
the peptide bond. The products formed by such a linkage are called
polypeptides.
Each protein has its own unique sequence and shape held together by
chemical interactions. If the protein is subject to changes in temperature,
pH, or exposure to chemicals, the protein structure may change, losing
its shape in what is known as denaturation.
Nucleic Acids
The amino acid sequence of a polypeptide is programmed by a unit of
inheritance known as a gene. A gene consists of DNA, a polymer known
as a nucleic acid. Nucleic acids are key macromolecules in the continuity
of life. They carry the genetic blueprint of a cell and carry instructions
for the functioning of the cell.
The two main types of nucleic acids are deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA). DNA is the genetic material found in all
living organisms, ranging from single-celled bacteria to multicellular
mammals. RNA is a molecule that is present in the majority of living
organisms and viruses. RNA and DNA are the molecules that enable
living organisms to reproduce their complex components from generation
to generation.
DNA and RNA are made up of monomers known as nucleotides. The
nucleotides combine with each other to form a polynucleotide, DNA or
RNA. Each nucleotide is made up of three components: a nitrogenous
base, a pentose (five-carbon) sugar, and a phosphate group . Each
nitrogenous base in a nucleotide is attached to a sugar molecule, which is
attached to a phosphate group.
What are the key differences between DNA and RNA?
We can identify five key categories where DNA and RNA differ:
● Function
● Sugar
● Bases
● Structure
● Location
What are the three types of RNA?
1. Messenger RNA (mRNA) copies portions of genetic code, a
process called transcription, and transports these copies to
ribosomes, which are the cellular factories that facilitate the
production of proteins from this code.
2. Transfer RNA (tRNA) is responsible for bringing amino acids,
basic protein building blocks, to these protein factories, in
response to the coded instructions introduced by the mRNA.
This protein-building process is called translation.
3. Finally, Ribosomal RNA (rRNA) is a component of the
ribosome factory itself without which protein production would
not occur
Cells
Characteristics Of Biochemical Process:
Industrial applications of biochemical processes are to use living cells or
cellular components to effect the desired physical or chemical changes.
Biological Agents
Biological agents include bacteria, viruses, fungi, other microorganisms
and their associated toxins. They have the ability to adversely affect
human health in a variety of ways, ranging from relatively mild, allergic
reactions to serious medical conditions—even death. Biotechnological
reactions are usually categorized as whole cell-mediated processes or
enzyme catalyzed reactions. The biological agents used for biochemical
processes are:
COMMON AGENTS:
● Microbial Cells (such as bacteria and fungi) - a pathogenic
bacterium, which falls under the category of microbial cells, is a
tiny living organism that cannot be observed without the aid of a
microscope. The term "microbial cells" is a broad one,
encompassing a wide variety of life forms such as bacteria, archaea,
fungi, and protists, each of which exhibits distinct sizes and
characteristics.
○ Enzymes (usually from a bacteria and fungi) - Enzymes are
proteins that help speed up metabolism, or the chemical
reactions in our bodies. They build some substances and break
others down. All living things have enzymes. Our bodies
naturally produce enzymes. But enzymes are also in
manufactured products and food.
● LIMITED APPLICABILITY:
○ Plant Cells (production of secondary metabolites) - Plants are
unique among the eukaryotes, organisms whose cells have
membrane-enclosed nuclei and organelles, because they can
manufacture their own food. Chlorophyll, which gives plants
their green color, enables them to use sunlight to convert
water and carbon dioxide into sugars and carbohydrates,
chemicals the cell uses for fuel.
○ Animal Cells (production of injectable polio vaccine) -
Animal cells are typical of the eukaryotic cell, enclosed by a
plasma membrane and containing a membrane-bound nucleus
and organelles. Unlike the eukaryotic cells of plants and
fungi, animal cells do not have a cell wall. This feature was
lost in the distant past by the single-celled organisms that
gave rise to the kingdom Animalia. Most cells, both animal
and plant, range in size between 1 and 100 micrometers and
are thus visible only with the aid of a microscope.
THE CELL STRUCTURE
Cells serve as the fundamental building blocks of all living
beings, ranging from enormous creatures like blue whales to tiny
archaebacteria residing in volcanoes. They come in various shapes and
sizes, with examples like giant squid nerve cells stretching up to 12
meters, while human eggs are a mere 0.1 millimeters in diameter. Plant
cells have cellulose-based protective walls, akin to celery strings, while
fungal cells resemble lobster shells in structure. Despite these
differences, all these cells share essential machinery.
There are two primary cell types: prokaryotic and eukaryotic.
Prokaryotes, including single-celled organisms in Bacteria and Archaea,
lack a true nucleus, while eukaryotes, encompassing animals, plants,
fungi, and protists, possess eukaryotic cells. Nonetheless, some
prokaryotes can coexist with eukaryotic cells, as seen in humans.
Prokaryotic Cells or Prokaryotes
Prokaryotes are simple, single-celled organisms lacking a nucleus and
membrane-bound organelles. They have no internal compartments
separated by membranes, consisting instead of an open space. These cells
are typically much smaller than eukaryotic cells, ranging from 0.1 to 5.0
micrometers in diameter.
Prokaryotes, including extremophiles, thrive in various extreme
environments such as hydrothermal vents, hot springs, swamps, and even
within the digestive tracts of humans and animals. They display
remarkable adaptability, existing virtually everywhere, including on
human skin, within the body, and on everyday objects in the
environment.
In terms of complexity, prokaryotic cells are less intricate than
eukaryotic cells, as they lack a true nucleus, with DNA coiled in a region
called the nucleoid. Prokaryotic organisms exhibit diverse cell shapes,
with spherical, rod-shaped, and spiral forms being the most common.
Taking bacteria as an example of prokaryotes, the following structures
and organelles are typically found in bacterial cells:
1. Capsule: Some bacterial cells possess this outer covering,
which offers additional protection when the cell is engulfed by
other organisms. It also helps in retaining moisture and aids the
cell in adhering to surfaces and nutrients.
2. Cell Wall: The cell wall acts as an outer shield, safeguarding
the bacterial cell and giving it its shape.
3. Cytoplasm: Cytoplasm is a gel-like substance primarily
composed of water. It also contains enzymes, salts, cellular
components, and various organic molecules.
4. Cell Membrane or Plasma Membrane: Surrounding the
cytoplasm, the cell membrane regulates the passage of
substances into and out of the cell.
5. Pili (Pilus singular): Hair-like structures on the cell surface that
facilitate attachment to other bacterial cells. Shorter versions
called fimbriae assist in bacterial adherence to surfaces.
6. Flagella: Flagella are elongated, whip-like projections that
contribute to cellular movement.
7. Ribosomes: These cell structures are responsible for
synthesizing proteins.
8. Plasmids: Plasmids are circular DNA structures carrying genes
but are not involved in reproduction.
9. Nucleoid Region: This area within the cytoplasm houses the
single bacterial DNA molecule.
Prokaryotic cells lack organelles commonly found in eukaryotic cells,
like mitochondria, endoplasmic reticula, and Golgi complexes. The
Endosymbiotic Theory suggests that eukaryotic organelles may have
originated from prokaryotic cells that formed mutualistic relationships.
Bacteria, similar to plant cells, have a cell wall, and some have an
additional polysaccharide capsule layer. This layer facilitates the
production of biofilm, a slimy substance that aids bacterial colonies in
adhering to surfaces and offers protection against antibiotics and
chemicals.
Furthermore, certain prokaryotes possess photosynthetic pigments, akin
to plants and algae, enabling them to derive nutrients from sunlight
through photosynthesis.
● Bacteria
Bacteria exhibit a diverse array of shapes and characteristics, and not all
bacteria possess every feature depicted in the diagram. Nevertheless,
most bacteria have a robust cell wall made of peptidoglycan, a polymer
comprising carbohydrates and small proteins. This cell wall offers
protection, maintains structural integrity, and prevents dehydration.
Many bacteria also possess an outer carbohydrate layer called a capsule,
which aids in surface adhesion.
Certain bacteria have specialized surface structures that assist in
mobility, adhesion, or genetic material exchange. Flagella act as whip-
like rotary motors for movement, while fimbriae are hair-like appendages
used for attaching to host cells and surfaces. Some bacteria also have
rod-like pili with various functions, such as DNA transfer or facilitating
bacterial motion.
Archaea may possess similar surface features, but their versions of these
characteristics typically differ from those in bacteria. For instance,
archaea's cell wall lacks peptidoglycan but contains carbohydrates and
proteins.
● Eukaryotic cell or Eukaryotes
Eukaryotic cells, featuring enclosed membrane-bound structures, form
the basis of all multicellular life, encompassing animals, plants, humans,
and even certain unicellular organisms like protozoa.
Within these cells, specialized subunits known as organelles perform
specific functions. The nucleus, enveloped by the nuclear membrane,
safeguards genetic material, with nuclear pores regulating substance
passage. The endoplasmic reticulum (ER), in two forms (rough and
smooth), plays roles in protein and lipid/steroid synthesis, respectively.
The Golgi apparatus, connected to the rough ER, modifies and packages
proteins, while mitochondria are pivotal for energy production. Various
eukaryotic cells may also possess additional organelles, varying in
proportion according to their unique functions.
● Plant Cell Parts, Function and Cell Cultivation
Plants possess a unique distinction among eukaryotic organisms due to
their ability to self-generate nourishment through chlorophyll, using
sunlight to convert water and carbon dioxide into essential sugars and
carbohydrates.
Similar to fungi, another eukaryotic group, plant cells have retained the
protective cell wall structure from their prokaryotic ancestors. Although
the fundamental structure of plant cells resembles typical eukaryotic
cells, they lack certain components like centrioles, lysosomes,
intermediate filaments, cilia, or flagella, which are present in animal
cells. Conversely, plant cells feature specialized structures including a
robust cell wall, central vacuole, plasmodesmata, and chloroplasts.
Although plants and their cells are generally immobile, certain species
produce gametes with flagella, enabling movement.
● Plant Cell Parts and its Function
1. Cell Wall - Like their prokaryotic ancestors, plant cells have a rigid
wall surrounding the plasma membrane. It is a far more complex
structure, however, and serves a variety of functions, from
protecting the cell to regulating the life cycle of the plant organism.
2. Chloroplasts - The most important characteristic of plants is their
ability to photosynthesize, in effect, to make their own food by
converting light energy into chemical energy. This process is carried
out in specialized organelles called chloroplasts.
3. Endoplasmic Reticulum - The endoplasmic reticulum is a network
of sacs that manufactures, processes, and transports chemical
compounds for use inside and outside of the cell. It is connected to
the double-layered nuclear envelope, providing a pipeline between
the nucleus and the cytoplasm. In plants, the endoplasmic reticulum
also connects between cells via the plasmodesmata.
4. Golgi Apparatus - The Golgi apparatus is the distribution and
shipping department for the cell's chemical products. It modifies
proteins and fats built in the endoplasmic reticulum and prepares
them for export as outside of the cell.
5. Microfilaments - Microfilaments are solid rods made of globular
proteins called actin. These filaments are primarily structural in
function and are an important component of the cytoskeleton.
6. Microtubules - These straight, hollow cylinders are found
throughout the cytoplasm of all eukaryotic cells (prokaryotes don't
have them) and carry out a variety of functions, ranging from
transport to structural support.
7. Mitochondria - Mitochondria are oblong shaped organelles found
in the cytoplasm of all eukaryotic cells. In plant cells, they break
down carbohydrate and sugar molecules to provide energy,
particularly when light isn't available for the chloroplasts to produce
energy.
8. Nucleus - The nucleus is a highly specialized organelle that serves
as the information processing and administrative center of the cell.
This organelle has two major functions: it stores the cell's hereditary
material, or DNA, and it coordinates the cell's activities, which
include growth, intermediary metabolism, protein synthesis, and
reproduction (cell division).
9. Peroxisomes - Microbodies are a diverse group of organelles that
are found in the cytoplasm, roughly spherical and bound by a single
membrane. There are several types of microbodies but peroxisomes
are the most common.
10. Plasmodesmata - Plasmodesmata are small tubes that connect plant
cells to each other, providing living bridges between cells.
11. Plasma Membrane - All living cells have a plasma membrane that
encloses their contents. In prokaryotes and plants, the membrane is
the inner layer of protection surrounded by a rigid cell wall. These
membranes also regulate the passage of molecules in and out of the
cells.
12. Ribosomes - All living cells contain ribosomes, tiny organelles
composed of approximately 60 percent RNA and 40 percent protein.
In eukaryotes, ribosomes are made of four strands of RNA. In
prokaryotes, they consist of three strands of RNA.
13. Vacuole - Each plant cell has a large, single vacuole that stores
compounds, helps in plant growth, and plays an important structural
role for the plant.
■ Plant Cell Cultivation
Plant Tissue Culture, or micropropagation, is a technique for cultivating
new plants under controlled conditions, allowing rapid production of
uniform specimens. It has diverse applications, including boosting crop
yields in developing regions, ensuring consistent quality for home
growers, and enabling businesses to profit from producing identical plant
copies.
Successful tissue culture hinges on factors like maintaining sterility and
using the right growing medium. Once plants are propagated, they can be
transferred to more natural environments like nurseries or greenhouses
for rapid growth. While it offers numerous benefits, tissue culture also
comes with its own set of advantages and disadvantages.
There are several benefits associated with the tissue culture method.
Beyond its effectiveness in assisting developing nations with food
production, here are some other advantages that may be of interest:
❖ Rapid Growth: New plantlets can be cultivated in a considerably
short time.
❖ Minimal Initial Material: Only a small amount of initial plant tissue
is needed.
❖ Reduced Risk of Diseases: The resulting plantlets and plants are
more likely to be free from viruses and diseases.
❖ Year-Round Availability: The process is not reliant on seasonal
conditions and can be conducted throughout the year.
❖ Space Efficiency: It requires relatively little space to perform the
process, allowing for the cultivation of ten times the number of
plants in just one-tenth of the area.
❖ Diverse Supply: On a larger scale, tissue culture aids in supplying
the consumer market with new subspecies and varieties.
❖ Success with Challenging Plants: Individuals attempting to grow
difficult plants, such as specific orchid breeds, often achieve greater
success with tissue culture compared to traditional soil-based
methods.
Disadvantages of Plant Tissue Culture
❖ Tissue culture can entail increased labor and higher costs associated
with constructing the facility and outfitting the laboratory with
necessary instruments and chemicals.
❖ There is a risk that the propagated plants may exhibit reduced
resilience to diseases when grown outdoors due to the controlled
environment in which they were cultivated.
❖ Before undergoing the culture process, it is crucial to thoroughly
screen the plant material; overlooking any abnormalities could result
in the new plants becoming infected.
❖ Success in tissue culture is not guaranteed, emphasizing the
importance of precise protocols, which can be labor-intensive to
develop independently.
❖ Contamination is a substantial concern, as plants can be vulnerable
to bacterial, fungal, and viral infections in tissue culture settings.
❖ Tissue culture is an advanced technique, requiring a solid
foundation of knowledge and practice for individuals looking to
enter this field.
★ TYPES OF PLANT CELL CULTIVATION
Tissue culture is a method that involves taking healthy tissues
from living organisms. In plant tissue culture, this can include leaves or
other plant parts, depending on the specific protocol.
The classification of tissue culture types is based on the
explant, which is the initial material or plant tissue used for growing
plants.
★ Callus Culture: Callus is a collection of undifferentiated plant cells
that possess the remarkable ability to give rise to various plant parts.
When stimulated in a laboratory environment, plant tissues from any
organ can develop into callus, which can further differentiate into
different plant organs like roots and shoots.
★ Seed Culture:
★ Protoplast Culture: Protoplasts are plant cells lacking a cell wall. In
this method, the plant cell wall is removed through mechanical or
enzymatic processes. The resulting protoplasts are purified and then,
under controlled conditions, the cell wall is regenerated before
transferring them to appropriate media for further development into
complete plants.
★ Meristem Culture: Meristem culture entails isolating the
meristematic region, such as shoot tips, from plants and placing
them in a growth medium containing nutrients, vitamins, and plant
hormones. This technique stimulates cell division and tissue
differentiation in the cultured cells. Meristem culture has various
applications, including disease-free plant production, whole plant
regeneration, generation of transgenic and haploid plants, crop
improvement, and germplasm preservation.
★ Embryo Culture: Embryo culture involves isolating and cultivating
either immature or mature plant embryos to support their growth
into complete plants. Instead of sterilizing individual embryos, this
method sterilizes the organ (such as ovule, seed, or fruit) from
which the embryos originate and uses it in the culture process.
★ Ovary Culture: Ovary culture is a technique where fertilized or
unfertilized plant ovaries are cultured in a suitable environment to
facilitate their development into complete plants. This method, also
known as gynogenesis, is primarily used to overcome pre- and post-
fertilization barriers and has been employed for interspecific
hybridization.
★ Anther/Pollen Culture: Pollen/anther culture is a plant
biotechnology technique that isolates and cultures pollen grains or
anthers (the male reproductive parts of flowers) in a nutrient-rich
medium. This method facilitates the development and regeneration
of haploid plants or callus tissues from the cultured pollen or anther
cells. It is widely applied in plant breeding and genetic research to
create new plant varieties and study plant cell behavior in controlled
environments.
Tissue culture is a valuable method with numerous practical uses. It can
significantly boost plant yields in a short time and allow genetic
modification for disease resistance and specific traits. This is
advantageous for businesses seeking profitability and individuals with
personal preferences. Additionally, tissue culture supports the
preservation of rare or endangered plants. The technique relies on the
plant's rapid cell rejuvenation, resulting in cloned copies.
● Animal Cell Parts, Function and Cell Cultivation
Animal cells, typical of eukaryotic cells, have a plasma membrane, a
nucleus enclosed by a membrane, and organelles. Unlike plant and fungal
cells, they lack a cell wall, a trait lost in the evolutionary history of
single-celled organisms that gave rise to the Animalia kingdom. These
cells vary in size but are generally microscopic.
The absence of a rigid cell wall in animal cells allows for diverse cell
types, tissues, and organs to develop, enabling mobility through
specialized nerve and muscle cells, a unique characteristic of the animal
kingdom. Some animals like sponges lack differentiated tissues, while
protozoans move through nonvascular means like cilia, flagella, and
pseudopodia.
What distinguishes the animal kingdom is the use of collagen, a triple
helix protein, to bind most tissues together in an extracellular matrix.
This sets animals apart from other eukaryotes, like plants and fungi,
which use different molecules for cell binding. The presence of collagen
suggests a common unicellular ancestor for all animals, and when the
collagen-containing matrix calcifies, it forms structures like bones,
shells, and spicules.
● Animal Cell Parts and its Function
1. Centrioles - Centrioles are self-replicating organelles comprising
nine microtubule bundles and are exclusive to animal cells. They
appear to assist in organizing cell division but are not crucial for the
process.
2. Cilia and Flagella - In single-celled eukaryotes, cilia and flagella are
essential for individual organism locomotion. In multicellular
organisms, cilia move fluid or materials past immobile cells and can
propel a cell or group of cells.
3. Endoplasmic Reticulum - The endoplasmic reticulum, a sac
network, manufactures, processes, and transports chemical
compounds within and outside the cell. It is connected to the nuclear
envelope, forming a link between the nucleus and the cytoplasm.
4. Endosomes and Endocytosis - Endosomes, membrane-bound
vesicles, form through a process called endocytosis, found in nearly
every animal cell's cytoplasm. Endocytosis involves the inward
folding of the cell's plasma membrane to encircle macromolecules
or extracellular matter.
5. Golgi Apparatus - The Golgi apparatus serves as the cell's
distribution and shipping department. It modifies proteins and fats
from the endoplasmic reticulum for export outside the cell.
6. Intermediate Filaments - Intermediate filaments are a diverse group
of fibrous proteins with structural and functional roles in the
cytoskeleton. They provide tension-bearing support to maintain cell
shape and rigidity.
7. Lysosomes - Lysosomes primarily function in cellular digestion,
breaking down waste and debris into simple compounds for reuse in
the cytoplasm.
8. Microfilaments - Microfilaments, composed of actin proteins, have
a primarily structural role and contribute to the cytoskeleton.
9. Microtubules - Straight, hollow cylinders found throughout
eukaryotic cell cytoplasm, microtubules serve various functions,
including transport and structural support.
10. Mitochondria - Mitochondria, oblong-shaped organelles in
eukaryotic cells, act as the primary power generators, converting
oxygen and nutrients into energy.
11. Nucleus - The nucleus is a specialized organelle serving as the cell's
information processing and administrative center. It stores DNA and
coordinates cell activities, including growth, metabolism, protein
synthesis, and reproduction.
12. Peroxisomes - Peroxisomes are a common type of microbody
organelle found in the cytoplasm, responsible for various metabolic
functions.
13. Plasma Membrane - All living cells possess a plasma membrane that
encloses their contents, regulating the passage of molecules in and
out of the cell. In prokaryotes, it is surrounded by a rigid cell wall.
14. Ribosomes - Ribosomes, present in all cells, are tiny organelles
composed mainly of RNA and protein, with variations in eukaryotes
and prokaryotes. They play a crucial role in protein synthesis.
● Animal Cell Cultivation
Depending on their source, animal cells can be cultured in two
ways: as adherent monolayers or in suspension.
● Adherent cells are reliant on a surface for attachment and grow as a
single layer connected to the culture vessel. This attachment is
crucial for their growth. Many adherent cell cultures will stop
growing when they cover the entire surface (reach confluence), and
some may die if left in this state for too long. Most tissue-derived
cells fall into this category.
● Suspension cells, on the other hand, can thrive and multiply without
attaching to a substrate. Hematopoietic cells (derived from blood,
spleen, or bone marrow), certain transformed cell lines, and cells
from malignant tumors can be cultured in suspension.
Primary cells, finite cultures, and continuous cell lines exhibit varying
proliferative capacities. Different cell types have diverse growth
characteristics and nutrient requirements. To ensure cells are healthy and
suitable for downstream applications, it's essential to optimize cell
culture conditions.
❖ Primary cell culture
Primary cell cultures originate from cells migrating from tissue
fragments or through disaggregation using enzymes, chemicals, or
mechanical methods. These cultures consist of surviving cells that adhere
to the culture vessel and begin to multiply.
Primary cells closely resemble the parent tissue in morphology but have
limited cell division capacity, eventually entering a non-proliferative
stage called senescence. Adherent primary cells are sensitive to contact
inhibition, ceasing growth when confluent but maintaining normal
characteristics at lower densities. Primary cell culture is generally more
challenging than working with continuous cell lines.
Researchers prefer primary cell cultures over continuous cell lines
because they closely mimic in vivo cell physiology. Continuous cell lines
can undergo changes over time, leading to inconsistencies in data across
different laboratories. Additionally, certain cell types are not available as
continuous cell lines.
❖ Finite cell culture
Finite cell cultures are established through the first subculturing of a
primary cell culture. These cultures can replicate for a limited number of
cell divisions before entering a senescent state. Some human finite cell
cultures can extend their proliferative potential by introducing viral
transforming genes like the SV40 transforming-antigen genes. These
cultures have an intermediate phenotype, between finite and continuous
cultures. They can proliferate for an extended period but eventually stop
dividing, akin to senescent primary cells. Using these cells can be more
convenient than primary cell cultures, particularly for creating stably
transfected clones.
❖ Continuous cell culture
Finite cell cultures will either decline with time or undergo a stable,
heritable mutation that transforms them into continuous cell lines with
unlimited growth potential, often linked to tumorigenicity. Rodent
primary cell cultures can easily transition into continuous cell lines,
sometimes spontaneously or after exposure to mutagenic agents. In
contrast, human primary cell cultures rarely become immortal in this
manner and often require additional genetic manipulation. However, cell
cultures from human tumors tend to exhibit immortality. Continuous cell
lines are more convenient to work with than primary or finite cell
cultures, but it's important to remember that they have genetic alterations,
and their behavior in vitro may not fully represent the in vivo context.
ANIMAL CELL COMMONLY USED IN CULTURE
❖ Lymphocytes - A type of white blood cell in the vertebrate immune
system. Play an important role in the body's defenses.
❖ Epithelial cell - Cell composing the epithelium. Epithelium is the
tissue that lines the cavities and surfaces of the structures throughout
the body.
❖ Fibroblast cell - Most common cells of connective tissue. Play an
important role in wound healing.
I. Enzyme & Enzyme Kinetics
Enzymes
An enzyme is a protein that serves as a natural catalyst within living
organisms. Catalysts are substances that quicken chemical reactions
without undergoing any change or depletion in the process. Enzymes
play a pivotal role in numerous metabolic processes, facilitating these
processes to occur at a significantly accelerated pace compared to their
natural rate in the absence of enzymes.
Enzymes possess several distinctive attributes:
Specificity: Enzymes are highly specialized in their function, typically
catalyzing a specific type of chemical reaction or a limited range of
closely related reactions. This unique specialization is commonly
referred to as the enzyme's "substrate specificity."
Efficiency: Enzymes have the remarkable ability to greatly hasten the
rate of a chemical reaction, rendering many vital biological processes
feasible under the relatively mild conditions found in living organisms.
Without enzymes, numerous reactions would occur too slowly to sustain
life.
Reusability: Enzymes are not depleted or altered during the reactions
they facilitate. Once an enzyme has facilitated a reaction, it remains
available to catalyze many more similar reactions.
Regulation: Enzyme activity can be controlled through various
mechanisms, including the presence of specific molecules that either
enhance or inhibit their function. This regulatory capability enables cells
to manage and coordinate metabolic pathways.
Enzymes participate in a wide array of biological processes,
encompassing digestion (e.g., the digestive enzymes in the stomach and
small intestine), energy production (e.g., enzymes in cellular respiration),
DNA replication and repair (e.g., DNA polymerases), and numerous
other vital functions in living organisms. Enzymes are indispensable for
life and are often likened to the diligent "workers" of biochemical
reactions.
Composition of Enzyme
Enzymes consist primarily of proteins, which are extensive molecules
comprising chains of amino acids. Enzyme composition primarily
involves proteins, but it can also encompass non-protein constituents
referred to as cofactors or coenzymes. These supplementary elements can
be essential for the enzyme's operational effectiveness and purpose.
The fundamental makeup of enzymes can be summarized as follows:
Protein: Enzymes are primarily constructed from proteins, which are
macromolecules structured with amino acid sequences. The precise
sequence and organization of amino acids within the protein establish the
enzyme's three-dimensional structure, influencing its purpose and
selectivity.
Cofactors: Certain enzymes necessitate non-protein substances or ions,
known as cofactors, for complete functionality. Cofactors can comprise
either inorganic ions (like metallic ions such as zinc or magnesium) or
organic compounds recognized as coenzymes. Coenzymes often perform
a crucial role in expediting the enzyme's catalytic function by conveying
or transferring chemical groups during the reaction.
Active Site: Inside the enzyme's three-dimensional configuration, there
usually exists a specialized area known as the active site. This is where
the enzyme interacts with its substrates, the molecules it acts upon in a
chemical reaction. The shape and chemical properties of the active site
are pivotal for the enzyme's specificity and catalytic operation.
Allosteric Sites: Some enzymes incorporate allosteric sites, which are
distinct from the active site. Binding at these sites by particular
molecules can either enhance or hinder the enzyme's operation, providing
a means of regulation.
Substrate Binding Site: Enzymes possess a distinct region designated
for the binding of their substrates. This binding site is compatible in
terms of shape and chemical characteristics with the substrate, enabling
the enzyme to recognize and attach to the substrates with a high level of
specificity.
Quaternary Structure (in some cases): In specific instances, enzymes
may be composed of multiple protein subunits, forming a quaternary
structure. The arrangement of these subunits can influence the enzyme's
functionality and regulatory mechanisms.
In summary, the composition of enzymes is principally rooted
in proteins, but their activity can be influenced by supplementary
constituents like cofactors and coenzymes. The specific amalgamation of
protein structure, active site properties, and any associated cofactors or
coenzymes determines the enzyme's capacity to expedite particular
chemical reactions within living organisms.
B. Classification
Enzymes can be categorized using various criteria, which
encompass their role, the specific reactions they catalyze, and the way
they are named. The primary methods for classifying enzymes include:
Categorization by Function:
a. Oxidoreductases: These enzymes facilitate oxidation-reduction
reactions, where electrons are transferred between molecules. Examples
include dehydrogenases and oxidases.
b. Transferases: Transferases assist in the movement of functional
groups (like methyl, phosphate, or amino groups) from one molecule to
another. Kinases, methyltransferases, and transaminases are instances.
c. Hydrolases: Hydrolases expedite the breaking of chemical bonds
through the addition of water molecules. Lipases, proteases, and
nucleases are illustrative.
d. Lyases: Lyases initiate reactions that either add or remove functional
groups from substrates without involving hydrolysis or oxidation.
Decarboxylases and synthases are examples.
e. Isomerases: Isomerases cause rearrangements of atoms within a
molecule, transforming it into its isomeric form. Cis-trans isomerases and
racemases fall into this category.
f. Ligases or Synthetases: Ligases bond two molecules together by
forming covalent bonds, often utilizing energy from ATP. DNA ligase is
an example.
Classification Based on Reaction Type:
Enzymes are often sorted according to the specific chemical
reactions they catalyze, including:
a. Nomenclature-Based Classification:
Enzymes are frequently named systematically, reflecting the
reactions they catalyze, followed by "–ase." For instance, lactase
catalyzes the hydrolysis of lactose, while DNA polymerase is involved in
DNA polymerization.
b. Enzyme Commission (EC) Number-Based Classification:
The Enzyme Commission (EC) numbers offer a structured classification
system through a hierarchical numbering scheme. Each enzyme receives
a unique EC number that signifies the type of reaction it catalyzes. The
EC number is composed of four levels, representing the enzyme's class,
subclass, sub-subclass, and a specific identifier. For instance, the enzyme
catalase bears the EC number 1.11.1.6, where:
Class 1 signifies oxidoreductases.
Subclass 11 indicates activity involving peroxide as an acceptor.
Sub-subclass 1 denotes catalase activity.
The specific identifier is 6.
EC 1. Oxidoreductases
EC 2. Transferases
EC 3. Hydrolases
EC 4. Lyases
EC 5. Isomerases
EC 6. Ligases
These classification methods are essential for researchers and scientists
to comprehend the diverse roles of enzymes and their contributions to
various biochemical processes within living organisms.
Enzyme Inhibition
Enzyme kinetics also explores the effects of inhibitors on enzymatic
reactions. Inhibitors can be competitive (compete with substrates for the
active site), non-competitive (bind to an allosteric site and inhibit enzyme
activity), or uncompetitive (bind to the enzyme-substrate complex).
Enzyme inhibitor - compounds that bind to enzyme and reduce their
activity
Types of Reversible Inhibition
a. Competitive Inhibition - Inhibitor usually a substrate analog which
competes for the active site of the enzyme.
b. Non-Competitive Inhibition - inhibitor is not a substrate analog
but bind on sites other than the active site which reduces affinity of
enzyme to substrate
c. Uncompetitive Inhibition - inhibitor binds to the ES complex only
and has no affinity for the enzyme itself .
Allosteric Inhibition - is when a molecule stops the activity of an
enzyme by entering a secondary site (an allosteric site) which changes
the shape of the active site blocking access for the substrate.
C. Enzyme Kinetics
Enzyme kinetics is the study of the rates at which enzymes
catalyze chemical reactions. It involves the investigation of several key
parameters and principles that help scientists understand how enzymes
function and how their activity is regulated. Enzyme kinetics provides
valuable insights into the mechanisms of enzyme-catalyzed reactions.
Here are some fundamental aspects of enzyme kinetics:
Theories on Enzyme-Substrate Complex
1. Emil Fischer’s Lock and Key Model
● This model assumes that the active site of the enzyme and the
substrate fit perfectly into one another.
● The lock is the enzyme and the key is the substrate.
● Enzymes catalyze the chemical reaction to take place, which can
either be a synthesis reaction (favors bond formation) or a
decomposition reaction (favors bond breakage).
2. Koshland’s Induced Fit Hypothesis
● The catalytic site of the enzyme is not complementary to substrate.
● This model propose that enzyme undergoes conformational change
to accommodate the substrate and form a stronger bond.
Michaelis-Menten Kinetics
The Michaelis-Menten equation is a fundamental concept in
enzyme kinetics. It describes the relationship between the initial reaction
rate (V₀), substrate concentration ([S]), and enzyme parameters (Km and
Vmax). The equation is given as:
Vmax [S]
Vo=
Km +[ S]
V₀: Initial reaction rate.
Vmax: Maximum reaction rate (when all enzyme active sites
are saturated with substrate).
[S]: Substrate concentration.
Km (Michaelis constant): A measure of the substrate
concentration at which the reaction rate is half of Vmax. It
reflects the affinity of the enzyme for its substrate.
Organic matrices used for enzyme immobilization can be categorized
into natural and synthetic polymers.
● Natural Polymers: These materials exhibit excellent
compatibility with proteins. Examples include polysaccharides
such as cellulose, dextran, agar, agarose, chitin, and alginate, as
well as proteins like collagen and albumin, along with carbon-
based materials.
● Turnover number ( Kcat ) - maximum number of molecules
of substrate that an enzyme can convert to product per catalytic
site per unit time and can be calculated as follows:
Vmax
Kcat =
ET
Enzyme Kinetics Applications
● Enzyme kinetics is crucial in various fields, including biochemistry,
pharmacology, and medicine. It is used to study drug interactions,
enzyme inhibition by pharmaceuticals, and the kinetics of metabolic
pathways.
● Understanding enzyme kinetics is essential for comprehending the
intricacies of biochemical reactions and for designing experiments,
optimizing enzymatic processes, and developing therapies in various
scientific and medical disciplines.
II. Immobilized Enzyme
ENZYME IMMOBILIZATION
- restricts the mobility of an enzyme or protein and fixes the enzyme
into a state without disturbing its functional ability. It can also
reduce the sensitivity of a native enzyme hence increasing the
functional efficiency of the enzyme.
- Confining the enzyme molecules to a distinct phase from the one in
which the substrates and the products are present.
- It is the process of attachment of an enzyme to a solid matrix so that
it cannot escape but can still act on its substrate.
-
The history of enzyme immobilization can be divided into three
developmental stages regarding immobilized enzyme technology.
❖ In the initial stage, around 1815, immobilized enzymes found
empirical applications in industrial processes, such as the
manufacture of acetic acid and wastewater purification.
❖ In the second stage, which unfolded during the 1960s, a pioneering
single enzyme immobilization system emerged. This system found
application in various processes, including the production of L-
amino acids and glucose isomerization.
❖ In the third stage, spanning from 1970 to 1995, a range of enzyme
immobilization techniques emerged, encompassing co-factor
regeneration and cell immobilization methods.
COMPONENTS OF ENZYME IMMOBILIZATION
The fundamental elements of enzyme immobilization
encompass the enzyme itself, a supporting matrix, and the method
employed to affix the catalyst to the carrier.
A. Enzyme - can be defined as biomolecules that function as
biocatalysts, facilitating numerous biochemical reactions by
mediating the conversion of substrates into products, without
being consumed or depleted in the process.
B. Support Matrix - a substance designed to enable the
immobilization of an enzyme
● Synthetic Polymers: These matrices offer high chemical and
mechanical stability. Some examples of synthetic polymers
used for enzyme immobilization are polystyrene, polyacrylate,
polyacrylamide, and polyamides.
Inorganic matrices for enzyme immobilization can be further classified
into two categories: natural minerals and processed materials.
 ● Natural Minerals: Examples of natural minerals used as
matrices include bentonite, celite, centolite, silica, and
charcoal.
● Processed Materials: Processed materials used for enzyme
immobilization include porous glass, metals, and metal oxides.
C. Mode of Attachment - Various modes of attachment between the
enzyme and the support matrix are elucidated in the methods of enzyme
immobilization, which typically encompass both physical and chemical
approaches.
METHODS OF ENZYME IMMOBILIZATION
Enzyme immobilization is classified into physical and
chemical processes based on their binding properties.
1. ADSORPTION
- the enzyme attaches to the surface of a water-insoluble carrier
matrix
- the bond between enzymes and the carrier matrix is typically strong
but can be weakened by factors such as the addition of a substrate or
changes in pH or ionic strength.
- The binding is nonspecific and can involve interactions such as
electrostatic or hydrophobic affinities with various ligands.
- the bonding is non-permanent and accomplished by the weak bonds,
mainly like hydrogen bond and Vander Waal forces.
- the matrix employed should have small particle sizes within the
range of 500Å to 1mm in diameter.
- various carrier materials are utilized in this type, including mineral
supports like aluminum oxide and clay, organic supports such as
starch, as well as modified sepharose and ion exchange resins.
Methods of Immobilization by Adsorption:
a. Static Method: This method is efficient but time-consuming. It
immobilizes the enzyme and carrier molecule without agitation.
b. Dynamic Process: In this approach, the enzyme is mixed with the
carrier under constant agitation, facilitating immobilization.
c. Reactor Loading: This method involves transferring both the
enzyme and carrier into a reactor with continuous agitation. It is
commonly employed in the commercial production of immobilized
enzymes.
d. Electro-Deposition: In this technique, a carrier is placed near an
electrode within an enzyme bath, and an electric current is passed
through it. This results in the enzyme moving towards the carrier
and ultimately depositing on its surface.
Advantages of Adsorption:
● It has no pore diffusion limitation.
● It is a simple and economical method to conduct.
● No reagents are required in this method.
● There is a limited loss of enzyme activity.
● It causes less disruption to an enzyme.
● It requires minimum activation steps.
● The adsorped enzyme can be recycled, regenerated and reused.
● It has a high loading efficiency of an enzyme.
Disadvantages of Adsorption:
● It provides low surface area for the enzyme binding.
● Desorption of an enzyme from the carrier usually occurs.
● Also, the yield is low.
2. ENTRAPMENT
- enzymes are confined within the pores of a porous polymer or gel
matrix, which is also referred to as lattice entrapment
- The interaction between the enzyme and the matrix can involve
either covalent or non-covalent bonding.
- The matrix used in this method is water soluble, and nature varies
with different enzymes. It can be polyacrylamide gels, cellulose
triacetate, agar, gelatine, alginate etc.
Methods of enzyme entrapment encompass the incorporation of enzymes
into various matrices, which can include:
a. Gels - This method entails entrapping the enzyme within a gel
matrix, where the enzyme is held in place within the gel structure.
b. Fibers - Enzyme entrapment can also occur within a fiber matrix,
where the enzyme is trapped and distributed within the
fibers.
c. Microoapsules - In this approach, enzymes are entrapped within
microcapsules, which are small, encapsulated structures that provide
a controlled environment for the enzymes.
Advantages of Entrapment Method:
● Its enzyme loading capacity is high.
● It’s a rapid method.
● The enzyme distortion is low.
● It is easy to practice.
Disadvantages of Entrapment Method:
● The diffusion of substrate and product create difficulties.
● It causes leakage of low molecular weight enzymes.
● There might some chances of microbial contamination.
● It also causes enzyme inactivation and sometimes loss of enzyme
activity.
● It has limited industrial use.
3. ENCAPSULATION
- This method involves membrane confinement, where an enzyme is
enclosed within the semipermeable membrane of a capsule
submerged in an aqueous solution.
- This arrangement permits the exchange of the surrounding medium,
including substrates and products, while retaining the enzyme
within the capsule.
- The matrix used here is a capsule which is made of a semi-
permeable membrane, and it can be polymeric, lipoid, non-ionic etc.
in nature. It includes nitrocellulose, nylon semi-permeable matrix
etc.
Encapsulation methods can be achieved through various approaches:
a. Encapsulation in a Reaction Vessel: This method divides a
chamber using a semipermeable membrane, with one chamber
containing enzymes and the other containing substrates and
products.
b. Encapsulation by Hollow Fiber Membrane: In this technique,
enzymes are entrapped within a semipermeable matrix, such as
cellulose or triacetate, within the matrix's confined space.
c. Microencapsulation: Enzyme molecules are enclosed within
microcapsules through chemical polymerization, typically utilizing
1-6-diaminohexane.
d. Encapsulation by Liposomes: Here, enzymes are bound to the
concentric lipoidal membrane of liposomes using phospholipids as
the binding agent.
Advantages of Encapsulation:
● No enzyme leakage
● Preservation of Enzyme Activity
● Simplicity
● High Loading Efficiency
Disadvantages of Encapsulation:
● Pore Size Limitation
● Cost- Effectiveness
4. COVALENT BONDING
- a widely employed enzyme immobilization method
- an enzyme molecule forms a robust covalent bond with the carrier,
resulting in a stable and complex interaction
- there is no enzyme loss in this process
- it occurs between the enzyme’s active site, represented byt its
functional group and the carrier molecule.
- The reactivity of functional groups in the binding process is influenced
by their charged status. In general, the order of reactivity, from highest to
lowest, is as follows:
● -S– (Thiolate)
● -SH (Thiol)
● -O– (Alkoxide)
● -NH2 (Amine)
● -COO– (Carboxylate)
● -OH (Hydroxyl)
● -NH3+ (Ammonium)
- The more negatively charged or electron-rich functional groups tend to
exhibit higher reactivity in covalent binding interactions with the carrier.
- Examples of polymeric carriers commonly used in covalent binding for
enzyme immobilization includes :Carboxylic Acid and Related Groups of
Polyglutamic Acid, Amide Groups of Polypeptides, Amino and Related
Groups of Polysaccharides
Some of the most frequently used polymers in this context are
polysaccharides like celluloses, agarose, and sepharose, as well as
polyvinyl alcohol, silica, and porous glasses. These polymers provide
functional groups that enable the formation of stable covalent bonds with
enzymes during the immobilization process.
Methods Used for Covalent Bonding:
a. Diazotation - involves bonding between the amino group of the
matrix and either the tyrosyl or histidyl group of an enzyme. This
bonding occurs through a reaction with sodium nitrite (NaNO2) and
hydrochloric acid (HCl). This process results in the formation of
covalent bonds between the enzyme and the matrix, facilitating
enzyme immobilization.
b. By Peptide Bond - entails the formation of bonds between either
the amino or carboxyl group of the matrix and the corresponding
carboxyl or amino group of an enzyme. In this process, the matrix is
chemically treated to facilitate the binding with the enzyme's active
functional group, resulting in covalent bonds that enable enzyme
immobilization.
c. Cyanogen Bromide Activation - involves the binding of glycol
groups present on a matrix to an enzyme. This binding is achieved
by activating the matrix with cyanogen bromide (CNBr), which
allows for the formation of covalent bonds between the glycol
groups and the enzyme, thereby facilitating enzyme immobilization.
d. By Polyfunctional Reagents - entails the formation of bonds
between the amino group of the matrix and the amino group of an
enzyme. An example of a reagent commonly used for this purpose is
glutaraldehyde, which is a bi-functional reagent. Glutaraldehyde
facilitates the cross-linking of amino groups from both the matrix
and the enzyme, resulting in covalent bonds that enable enzyme
immobilization.
Advantages of Covalent Bonding
● High Bonding Strength
● No Enzyme Leakage
● Simplicilty and Widely Used
● pH and Ionic Strength Insensitivity
Disadvantages of Covalent Bonding
● Enzyme Denaturation
● Limited Immobilization Capacity
● Cost-Effectiveness
5. CROSS LINKING
- often referred to as "Copolymerization" or "Cross-Linking." In this
approach, immobilized enzymes form covalent links with various
functional groups of the enzyme itself using polyfunctional reagents.
Notably, it does not require a support matrix.
- This cross-linking process results in the formation of three-dimensional
crosslinked enzyme aggregates
- Commonly used polyfunctional agents for this purpose include
glutaraldehyde and diazonium salts, among others
Advantages of the Cross-Linking Method
● Minimal Enzyme Leakage
● High Enzyme Stability
● Simplicity and Cost-Efficiency
● Wide Applicability
Disadvantages of the Cross-Linking Method
● Enzyme Inactivation
● Enzyme Denaturation
● Cost-Effectiveness
Advantages of Enzyme Immobilization:
● Enzymes can undergo multiple uses.
● Immobilization reduces labor requirements.
● It enhances the enzyme-to-substrate ratio.
● Minimum activation time is needed.
Disadvantages of Enzyme Immobilization:
● Limited industrial applicability.
● Enzymes may experience diminished catalytic activity and
stability.
● Heat generation can lead to enzyme inactivation.
● The method can be costly to implement.
Applications of Enzyme Immobilization:
● Industrial Applications: Such as the manufacturing of antibiotics,
amino acids, and other industrial processes.
● Biomedical Applications: Including enzyme usage for treatment,
diagnosis, and drug delivery in the field of medicine.
● Food Industry: For instance, in the production of jams, jellies,
syrups, and other food products.
● Sewage and Wastewater Treatment: Utilized in the treatment of
sewage and industrial effluents to manage and purify wastewater.
● Detergent Industry: Enzyme immobilization, particularly for
lipases, aids in breaking down lipids found in stains and dirt in
detergents.
● Textile Industry: Encompassing processes like scouring and bio-
polishing for fabric treatment and improvement.
The immobilization process is extensively applied across various
domains, allowing enzymes to be securely attached to specific phases,
thus enabling their reuse and stabilization for multiple reactions.
DISADVANTAGE OF IMMOBILIZED ENZYME:
● Adds to the cost.
● Adversely affects stability and/or activity of the enzyme.
● Substrate cannot diffuse efficiently to reach the enzyme.
Microbial Growth
While growth for multicellular organisms is typically measured
in terms of the increase in size of a single organism, microbial growth is
measured by the increase in population, either by measuring the increase
in cell number or the increase in overall mass.
Two Types of Asexual Reproduction in Microbes:
● Binary Fission
Bacterial reproduction occurs through fission, a primitive form of cell
division that does not employ a spindle fiber apparatus. [A spindle fiber
apparatus made of protein filaments is responsible for moving the
chromosomes around during cell division (mitosis & meiosis) in most
eukaryotic cells. Bacteria do not have these structures.] The bacterial
cell doubles in size and replicates its chromosome. Following DNA
replication, the two chromosomes attach to separate sites on the plasma
membrane, and the cell wall is laid down between them, producing two
daughter cells.
● Budding
A few bacteria and some eukaryotes (including yeasts) may also replicate
by budding, forming a bubble-like growth that enlarges and separates
from the parent cell.
Growth Factors - Microbes can exist in a great many environments
because they are small, easily dispersed, need only small quantities of
nutrients, and are diverse in their nutritional requirements.
A. Physical Factors
● pH – bacteria can classified as:
➔ acidophiles (acid-loving) – grow best at a pH of 1 to 5.4; Ex.
Lactobacillus (ferments milk)
➔ neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause
human disease are in this category.
➔ alkaliphiles (base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio
cholerae (causes cholera)
● Temperature – bacteria can be classified as:
➔ psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC.
➔ mesophiles - grow best between 25oC and 40 C; human body temp
is 37oC.
➔ hermophiles (heat-loving) – 50oC to 60oC; found in compost heaps
and in boiling hot springs.
● Moisture – only the spores of sport-forming bacteria can exist in a
dormant state in a dry environment.
B. Oxygen Requirements
● Strict or obligate anaerobes – oxygen kills the bacteria; ex.
Clostridium tetani
● Strict or obligate aerobes – lack of oxygen kills the bacteria; ex.
Pserdomonas
● Facultative anaerobes – can shift their metabolism (anaerobic if
oxygen is absent or aerobic if oxygen is present); ex. E. coli,
Staphylococcus
● Aerotolerant – the bacteria don’t use oxygen, but oxygen doesn’t
harm them; ex. Lactobacillus
● Microaerophiles – like low oxygen concentrations and higher
carbon dioxide concentrations; ex. Campylobacter
C. Nutritional (Biochemical) Factors
● Carbon – carbon containing compounds are needed as an energy
source (ex. glucose) and for building blocks.
● Nitrogen - needed for amino acids and nucleotides; some can
synthesize all 20 amino acids; others have to have some provided in
their medium.
● Sulfur – needed for amino acids, coenzymes,
● Phosphorus – needed for ATP, phospholipids, and nucleotides
● Vitamins – a vitamin is an organic substance that an organism
requires in small amounts and that is typically used as a coenzyme;
many bacteria make their own, but some are required in the
medium; microbes living in the human intestine manufacture
vitamin K, needed for blood clotting, and some of the B vitamins,
thus benefiting their host.
● Certain trace elements – ex. copper, iron, zinc, sodium, chloride,
potassium, calcium, etc.; often serve as cofactors in enzymatic
reactions.
Growth Curve - refers to a graphical representation of changes in
comparable situations. Since bacteria are easy to grow in the lab, their
growth has been studied extensively. It has been determined that in a
closed system or batch culture (no food added, no wastes removed)
bacteria will grow in a predictable pattern, resulting in a growth curve
composed of four distinct phases of growth: the lag phase, the
exponential or log phase, the stationary phase, and the death or decline
phase. Additionally, this growth curve can yield generation time for a
particular organism – the amount of time it takes for the population to
double.
1. Lag Phase
➔ Period of adjustment to new conditions
➔ Little or no cell division occurs, population size doesn’t increase
➔ Phase of intense metabolic activity, in which individual organisms
grow in size
➔ May last from one hour to several days.
2. Log Phase (exponential growth)
➔ Cells begin to divide and generation time reaches a constant
minimum
➔ Period of most rapid growth. Number of cells produced > Number
of cells dying
➔ Cells are at highest metabolic activity
➔ Cells are most susceptible to adverse environmental factors at this
stage.
3. Stationary Phase
➔ Population size begins to stabilize. Number if cells produced =
Number of cells dying
➔ Overall cell number does not increase
➔ Cell division begins to slow down
4. Death Phase
➔ Population size begins to decrease. Number of cells dying >
Number of cells produced
➔ Cell number decreases at a logarithmic rate
➔ Cells lose their ability to divide
➔ A few cells may remain alive for a long period of time
Measuring Microbial Growth
Direct Method - can be done, visually, and by various types of
electronic particle counters.
1. Plate Count
➔ Most frequently used method of measuring bacterial populations
➔ Inoculate plate with a sample and count number of colonies
The sample is like water, food or soil that contains millions of bacteria
and hence it is practically impossible to count them. For such samples,
we first perform dilution methods. To dilute the bacterial number, the
sample is serially diluted 10 times and it is plated in appropriate medium.
2. Filtration
➔ Known for sterilization of heat labile or sensitive material.
➔ The filter used are made of inert material like acetate, cellulose
nitrate, polyamide, polycarbonate, polypropylene or
polytetrafluoroethylene.
➔ This method is employed for measuring bacteria of broth
culture. Under sterile conditions, the liquid culture is poured
through the filter, the pore size of the filter is 0.2 to 0.45
micrometer.
➔ The bacteria that are larger than the pore size will retain in the
filter.
3. Most Probable Number (MPN)
➔ It is used to estimate the concentration of viable microorganisms in
a sample by means of replicating liquid broth growth in ten-fold
dilutions.
➔ Commonly used in estimating microbial populations in soils, waters,
and agricultural products.
MPN is most commonly applied for quality testing of water i.e to ensure
whether the water is safe or not in terms of bacteria present in it. A group
of bacteria commonly referred to as fecal coliforms act as an indicator of
fecal contamination of water. The presence of very few fecal coliform
bacteria would indicate that water probably contains no disease-causing
organisms, while the presence of large numbers of fecal coliform bacteria
would indicate a very high probability that the water could contain
disease-producing organisms making the water unsafe for consumption.
4. Direct Microscopic Count
➔ Require the use of a specialized slide called the Petroff-Hausser
counting chamber, in which an aliquot of a eukaryotic cell
suspension is counted and the total number of cells is determined
mathematically.
➔ The Petroff-Hausser counting chamber is a thick glass microscope
slide with a chamber 0.02 mm (1/50 mm) deep in the center.

Indirect Method - can be performed by using a spectrophotometer and

measuring the turbidity (absorbance or Optical Density) of the sample.
1. Turbidity
➔ Turbidity can be measured by Calorimeter or spectrophotometer
which works on the principle of Beer Lambert’s Law which states
the concentration of solutes or cells is directly proportional to the
absorbance. It means higher the absorbance, higher is the cell
count.
2. Metabolic Activity
➔ The metabolic activity of living cells means anabolic (synthesis) and
catabolic activity occurring inside the cell.
➔ During metabolic activity, there could be oxygen consumption, acid
and gas production, nutrient utilization, change in pH and waste
production and accumulation.
➔ As bacteria multiply in media, they produce certain products:
carbon dioxide and acids
3. Dry Weight
➔ Cells growing in liquid medium are collected by centrifugation,
washed, dried in an oven, and weighed.
➔ The bacterial suspension must be free from extraneous matter for
accurate results.
➔ The drawback of this method is that we may not be sure if the dry
weight is only of bacterial cells.
➔ It also cannot distinguish between live and dead cells.

Characteristic of Matrix
1. Low cost
2. Inertness
3. Physical strength
4. Mechanical Strength
5. Stability
6. Regenerability after the useful lifetime of the immobilized enzyme,
7. Enhancement of enzyme specificity
8. Maintain optimum pH
9. Reduction in product inhibition
10. A shift in the pH optimum for enzyme action to a desired value for
the process
11.Reduction in microbial contamination and non specific adsorption
12. Hydrophilic Character
13. Mean Particle Size
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