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CHAPTER 5
MICROBIAL BIOTECHNOLOGY……
BTEC3301
Microbial Biotechnology
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INDUSTRIAL USE OF ENZYMES
 Several thermostable enzymes, like the Taq
polymerase have been identified and widely
used in PCR and other reactions.
 Cellulase is obtained from E.coli and degrades
cellulose, a polysaccharide in plant cells.
Microbial Biotechnology
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INDUSTRIAL USE OF ENZYMES
 The denim jean is treated with cellulase, from
fungi Trichoderma reesei and Aspergillus niger,
to give the faded look and texture.
 The protease subtilisin, from Bacillus subtilis,
forms component of Laundry detergent to
remove and degrade protein stains.
Microbial Biotechnology
MICROORGANISMS
 Enzymes
can rightly AS
be TOOLS
called the catalytic
INDUSTRIAL USE OF ENZYMES
machinery of living systems.
 Enzymes are responsible for fermentation of
sugar to ethanol by yeasts, a reaction that forms
the bases of beer and wine manufacturing.
 Enzymes oxidize ethanol to acetic acid. This
reaction has been used in vinegar production for
thousands of years.
Microbial Biotechnology
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AS TOOLS
 Similar
microbial enzyme
reactions of acid
INDUSTRIAL USE OF ENZYMES
forming bacteria and yeasts are responsible for
aroma forming activities in bread making.
 Presently more than 2000 different enzymes have
been isolated and characterized.
 More than 75% of industrial enzymes are
hydrolases.
 40% of all enzyme sales are Protein-degrading
enzymes .
Microbial Biotechnology
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Enzyme Production by Microbial
Fermentation
 Extracellular enzymes are secreted outside
the cell makes the recovery and purification
process much simpler compared to production
of intracellular enzymes .
Microbial Biotechnology
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Enzyme Production
by Microbial
Fermentation
 Intracellular
enzymes
must
be purified from
thousands of different cell proteins and other
components.
 The organism producing the enzymes should
have a GRAS-status, which means that it is
Generally Regarded as Safe. This is especially
important when the enzyme produced by the
organism is used in food processes .
Microbial Biotechnology
 The organism
should be
able to produce high
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AS TOOLS
Enzyme Production by Microbial Fermentation
amount
of the desired enzyme in a reasonable
time frame.
 The industrial strains typically produce over
50-g/l extracellular enzyme proteins.
 Most of the industrial enzymes are produced
by a relatively few microbial hosts like
Aspergillus and Trichoderma fungi,
Streptomyces and Bacillus .
Microbial Biotechnology
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Production
by Microbial Fermentation
 Most Enzyme
of the
industrially
used microorganisms
have been genetically modified to
overproduce the desired activity and not to
produce undesired side activities.
Microbial Biotechnology
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Protein engineering
 Often enzymes do not have the desired
properties for an industrial application. E.g
extreme thermo stability or overproduction of
the enzyme.
Microbial Biotechnology
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Protein Engineering
 Protein engineering
is used to improve
commercially available enzyme to be a better
industrial catalyst.
 Several enzymes have already been
engineered to function better in industrial
processes. These include proteinases, lipases,
cellulases and few amylases
Microbial Biotechnology
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Protein
Engineering
 Xylanase from
fungus
Trichoderma sps. is a good
example of an industrial enzyme, used in pulp
and paper industry and needs to be stable in high
temperature.
 Xylanases is a good example of engineered
enzyme from Trichoderma. Its xylanase has been
purified and crystallized. By designed
mutagenesis its thermal stability has been
increased about 2000 times at 70oC and its pHoptimum shifted towards alkaline region by one
pH-unit.
Microbial Biotechnology
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Enzyme Technology
 Enzyme technology involves how to use
enzymes.
 The simplest way to use enzymes is to add
them into a process stream where they
catalyse the desired reaction and are gradually
inactivated during the process .
Microbial Biotechnology
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Enzyme
Technology
 Example liquefaction
of starch
with amylases, bleaching
of cellulose pulp with xylanases or use of enzymes in
animal feed.
 An alternative way to use enzymes is to immobilize them
so that they can be reused.
 One method of immobilization is to use ultrafiltration
membranes in the reactor system. The large enzyme
molecules cannot pass the membrane but the small
molecular reaction products can. Therefore enzymes are
retained in a reaction system and the products leave the
system continuously .
Reading Assignment for
Quiz:Detergent, Food &
BeveragesAnimal feed,personal
care etc slide 15-40
Large scale enzyme applications
Detergents
 Detergents were the first large scale application
for microbial enzymes.
 Bacterial proteinases are still the most important
detergent enzymes. Some products have been
genetically engineered to be more stable in the
hostile environment of washing machines with
several different chemicals present.
Microbial Biotechnology
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AS TOOLS
 Lipid degrading
enzymes,
lipase, were used in
Large scale enzyme applicationsDetergents
powder and liquid detergents to decompose
fats.
 Lipase is produced in large scale by Aspergillus
oryzae host after cloning the Humicola gene
into this organism.
 Amylases are used in detergents to remove
starch based stains.
Microbial Biotechnology
MICROORGANISMS
TOOLS
 Cellulases
have beenAS
part
of detergents since
Large scale enzyme applicationsDetergents
early 90s. Cellulase is actually an enzyme
complex capable of degrading crystalline
cellulose to glucose.
 In textile cellulases remove cellulose
microfibrils, which are formed during washing.
 Alkaline cellulases are produced by Bacillus
strains and neutral and acidic cellulases by
Trichoderma and Humicola fungi.
Microbial Biotechnology
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Large scale enzyme applications
Foods/Beverages produced by Microbial Activity
 Yogurt, cheese, chocolate, butter, pickles,
sauerkraut, soy sauce, food supplements (such as
vitamins and amino acids), food thickeners
(produced from microbial polysaccharides),
alcohol (beer, whiskeys, wines) and silage for
animals are all products of microbial activity.
Microbial Biotechnology
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Large scale enzyme applications Foods/Beverages produced by Microbial Activity
 The industrial microbiologist/ biotechnologist
may be involved in producing concentrated
microbial inocula for fermentations or the
maintenance of fermentation systems utilized
in production facilities.
 The use of starch degrading enzymes,
amylase, was the first large-scale application
of microbial enzymes in food industry.
Microbial Biotechnology
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Large scale enzyme applications Foods/Beverages produced by Microbial Activity
 Enzymes have many applications in drink
industry. The use of chymosin in cheese
making to coagulate milk protein.
 Another enzyme used in milk industry is beta-
galactosidase or lactase, which splits milksugar lactose into glucose and galactose. This
process is used for milk products that are
consumed by lactose intolerant consumers.
Microbial Biotechnology
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Large scale enzyme applications Foods/Beverages produced by Microbial Activity
 Enzymes are used also in fruit juice
manufacturing. Pectins are substances in fruit
lamella and cell walls. The cell wall contains
also hemicelluloses and cellulose. Pectinase,
xylanase and cellulase improve the liberation
of the juice from the pulp.
 Pectinases and amylases are used in juice
clarification.
Microbial Biotechnology
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Large scale enzyme applications Foods/Beverages produced by Microbial Activity
 Brewing is an enzymatic process. Malting is a
process, which increases the enzyme levels in
the grain. In the mashing process the
enzymes, amylase, are liberated and they
hydrolyse (Break down) the starch into soluble
fermentable sugars like maltose, which is a
glucose disaccharide.
 Similarly enzymes are widely used in wine
production to obtain a better extraction of the
necessary components and thus improving the
yield.
Microbial Biotechnology
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Large scale enzyme applications Foods/Beverages produced by Microbial Activity
 Brewing is an enzymatic process. Malting is a
process, which increases the enzyme levels in the
grain. In the mashing process the enzymes,
amylase, are liberated and they hydrolyse (Break
down) the starch into soluble fermentable sugars
like maltose, which is a glucose disaccharide.
 Similarly enzymes are widely used in wine
production to obtain a better extraction of the
necessary components and thus improving the
yield.
Microbial Biotechnology
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Large scale enzyme applications
Foods/Beverages cured or improved by microbial
activity
 Production of coffee, tea, cocoa, summer sausage,
vanilla, cheese, olives and tobacco all require
microbial activity.
Microbial Biotechnology
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Large scale enzyme applications
Food flavoring agents and preservatives
 Organic acids such as citric, malic and ascorbic
acids and monosodium glutamate are microbial
products commonly used in foods.
Microbial Biotechnology
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Large scale enzyme applications
Foods
 Mushrooms, truffles and some red and green algae
are consumed directly. Yeasts are used as food
supplements for humans and animals.
Microbial Biotechnology
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Large scale enzyme applications
Oil recovery/mining
 Oil recovery may be facilitated by the
development of unique bacteria which produce a
surfactant that forces trapped oil out of rocks.
 Extraction of minerals from low-grade ores is
enhanced by some bacteria (microbial leaching).
Microbial Biotechnology
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Large scale enzyme applications
Waste and Wastewater Management
 Isolating or developing microbial strains capable of
degrading and detoxifying hydrocarbon and
halogenated hydrocarbon waste (for example
organophosphates, acetylcholinesterase
inhibitors) of industrial, agricultural or military
origin is essential in waste management.
Microbial Biotechnology
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Large scale enzyme applications
Textiles
 The use of enzymes in textile industry is one of the
most rapidly growing fields in industrial
enzymology.
 Starch has for a long time been used as a
protective glue of fibers in weaving of fabrics. This
is called sizing.
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Large scale enzyme applications
Textiles
 Enzymes are used to remove the starch in a
process called desizing. Amylases are used in this
process since they do not harm the textile fibers.
 Laccase – a polyphenol oxidase from fungi is used
to degrade lignin the aromatic polymer found in all
plant materials .
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Large scale enzyme applications
Animal Feed
 The net effect of enzyme usage in feed has been
increased animal weight.
 The first commercial success was addition of betaglucanase into barley based feed diets. Barley
contains beta-glucan, which causes high viscosity
in the chicken gut.
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Large scale enzyme applications
Animal Feed
 Xylanase, from Trichoderma, are added to wheat-
based broiler feed and are nowadays routinely
used in feed formulations and animals gain
weight.
 Enzymes have become an important aspect of
animal feed industry. In addition to poultry,
enzymes are used in pig feeds and turkey feeds.
Microbial Biotechnology
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Large scale enzyme applications
Baking
 Alpha-amylases have been most widely studied in
connection with improved bread quality and
increased shelf life.
 Both fungal and bacterial amylases are used in
bread making and excess may lead to sticky
dough.
Microbial Biotechnology
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Large scale enzyme applications
Pulp and Paper
 The major application is the use of
xylanases in pulp bleaching for paper.
Microbial Biotechnology
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Large scale enzyme applications
Leather
 Leather industry uses proteolytic and
lipolytic enzymes in leather processing.
 Enzymes are used to remove animal skin,
hair, and any unwanted parts.
Microbial Biotechnology
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Large scale enzyme applications
Leather
 The used enzymes are typically alkaline
bacterial proteases.
 Lipases are used in this phase or in bating
phase to specifically remove grease.
Microbial Biotechnology
Large scale enzyme applications
Enzymes in Personal Care products
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Personal care products are a relatively
new area for enzymes and the amounts
used are small but worth to mention as a
future growth area.
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Large scale enzyme applications
Enzymes in Personal Care products
 One application is contact lens cleaning. Proteinase
and lipase containing enzyme solutions are used for
this purpose. Hydrogen peroxide is used in
disinfections of contact lenses. The residual hydrogen
peroxide after disinfections can be removed by a
heme containing catalase enzyme, which degrades
hydrogen peroxide.
Microbial Biotechnology
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Large scale enzyme applications
Enzymes in Personal Care products
 Some toothpaste contains glucoamylase and
glucose oxidase.
 Dentures can be cleaned with protein degrading
enzyme solutions.
 Enzymes are also used for applications in skin and
hair care products.
Microbial Biotechnology
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Large scale enzyme applications
Enzymes in DNA-technology
 DNA-technology has revolutionized both
traditional biotechnology and opened totally new
fields for scientific study.
 Recombinant DNA-technology allows one to
produce new enzymes in traditional overproducing
and safe organisms .
Microbial Biotechnology
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Large scale enzyme applications
Enzymes in DNA-technology
 Protein engineering is used to modify and improve
existing enzymes.
 DNA is basically a long chain of deoxyribose sugars
linked together by phosphodiester bonds. Organic
bases, adenine, thymine, guanine and cytosine are
linked to the sugars and form the alphabet of genes.
The specific order of the organic bases in the chain
constitutes the genetic language.
Microbial Biotechnology
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Large scale enzyme applications
Enzymes in DNA-technology
 Genetic engineering means reading and modifying
this language. Enzymes are crucial tools in this
process. E.g.:
Microbial Biotechnology
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Large scale enzyme applications
Enzymes in DNA-technology

Genetic engineering means reading and modifying this language. Enzymes are crucial tools in this process.
E.g.:
1. Restriction enzymes recognise specific DNA sequences
and cut the chain at these recognition sites.
2. DNA modifying enzymes synthesize nucleic acids,
degrade them, join pieces together and remove parts
of the DNA.
3. DNA-polymerases synthesize new DNA-chains. Many
of them need a model template, which they copy.
4. Ligases join adjacent nucleotides together.
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Therapeutic Proteins by Gene
Transfer
 Recombinant DNA technology led to the rapid
development and production of Therapeutic
protein.
 There are many proteins essential to good
health that some people cannot produce
because of genetic defects.
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Therapeutic Proteins by Gene Transfer
 These proteins include various blood-clotting
factors causing hemophilia, insulin (resulting in
diabetes), growth hormone (resulting in lack of
proper growth), and other proteins, the
administration of which corrects pathological
conditions or results in other therapeutic
benefits.
 Plasmids are used to transfer human genes to
bacterial cells.
Microbial Biotechnology
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Therapeutic Proteins by Gene Transfer
 If the gene inserted into the plasmid of bacteria is
the human gene for insulin, for example, the
bacteria into which this gene is inserted produces
human insulin.
 Bacteria as such do not produce insulin, but the
recombinant bacterial cells do produce insulin, it
was an outstanding example of microbial
biotechnology.
Courtesy © John J. Cardamone, Jr.
Insertion of a DNA section into a plasmid
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Therapeutic Proteins by Gene Transfer
cDNA:
 Human genes composed of coding and non-
coding sequences. The copy of the coding
sequences is called cDNA.
 The synthesis of the insulin cDNA will allow
the production of a functional insulin
molecule.
Transfer of the Insulin gene into a plasmid vector
(schematic)
Microbial Biotechnology
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Therapeutic Proteins by Gene Transfer
Cloning the Insulin gene
(Mechanism):
 Insulin was first synthesized in 1979 in E. coli
cells through the use of recombinant DNA
techniques.
 Insulin is produced by beta cells in pancreas in
humans.
Biotechnology
 Microbial
Human
insulin
has
two
polypeptides
subunits
MICROORGANISMS AS TOOLS
called the A (21 amino acids) and the B (30
amino acids) which are bonded by disulphide
bond to create the active insulin.
Therapeutic Proteins by Gene Transfer
Cloning the Insulin gene (Mechanism):
 When a human gene for insulin is cloned, gene
for each of the subunit is inserted into plasmid
vector separately (Fig 5.9).
Biotechnology
 Microbial
The
vector
has the Lac z gene encoding for the
MICROORGANISMS AS TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
enzyme β-galactosidase (β-gal).
 The genes that code for the two insulin chains in
human are fused to the E. coli gene (Lac z)
encoding for beta-galactosidase.
 The plasmid is then transformed into E.coli .
 Plasmids enter the bacteria in a process called
transfection .
Biotechnology
 Microbial
With
the recombinant
DNA molecule successfully
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AS TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
inserted into the bacterial host, another property of
plasmids can be exploited - their capacity to
replicate.
 Once inside a bacterium, the plasmid containing
the human cDNA can multiply to yield several
dozen copies.
 Because the insulin genes are connected to the lac z
gene, when bacteria synthesizes proteins from
these plasmids, they produce a protein containing
β-gal attached to human insulin protein and this is
called a fusion protein .
Biotechnology
 Microbial
The
fusion
protein here is called β-gal-insulin
MICROORGANISMS AS TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
fusion protein.
 When the bacteria divide, the plasmids are
divided between the two daughter cells and the
plasmids continue to reproduce.
 With cells dividing rapidly (every 20 minutes), a
bacterium containing human cDNA) will shortly
produce many millions of similar cells (clones)
containing the same human gene.
Biotechnology
 Microbial
After
theASchains
are synthesized, the bonds that
MICROORGANISMS
TOOLS
Therapeutic Proteins by Gene Transfer Cloning the Insulin gene (Mechanism):
hold the insulin molecule to the betagalactosidase are cleaved with cyanogen
bromide.
 Affinity column is used to separate the two
proteins.
 The two chains are then purified to give native
insulin.
 This form of insulin is an exact match to that
which is made in the body.
Fig 5.9: Using bacteria to produce Human insulin
NEW & VIEWS
Type 1 or insulin-dependent diabetes
mellitus & study steps in Fig.5.9.
Microbial Biotechnology
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Therapeutic Proteins by Gene Transfer
Microbes against microbes
 Antibiotics are antimicrobial drugs used
against microbes.
 An antibiotic is a substance, usually produced
by a microorganism which, in very small
quantities, inhibits or kills other
microorganisms .
Microbial Biotechnology
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Therapeutic Proteins by Gene Transfer Microbes against microbes
 Both natural and chemically enhanced microbial
products can be used to control human, animal
and plant diseases.
 Using traditional genetics or recombinant DNA
techniques, the microorganism can be modified
to improve the yield or action of antibiotics and
other antimicrobial agents.
Microbial Biotechnology
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Therapeutic Proteins by Gene Transfer Microbes against microbes
 New research directions are aimed at
discovering microbial metabolites with
pharmacological activities useful in the
treatment of hypertension, obesity, coronary
heart disease, cancer and inflammation.
Microbial Biotechnology
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Vaccines
Introduction
 A number of diseases are caused by microorganisms
 Vaccines are essential to protect humans and
animals from microbial diseases. Recombinant
DNA technology has allowed the production of
novel vaccines that offer protection without the
risk of infection (e.g. hepatitis B vaccine).
Microbial Biotechnology
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Vaccines: Introduction
 For many bacterial diseases, and some fungal
diseases, there are antibiotics, produced by
other micro-organisms.
 There are very few means of fighting viral
diseases.
 The production of vaccines against the microbial
pathogens, and more particularly the
pathogenic viruses, in order to immunise the
susceptible populations, is a safe and more
certain recourse.
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Vaccines: Introduction
 Biotechnology has made it now possible to
produce immunological agents to afford
protection from diseases to large numbers of
people.
 This area is immunotechnology, an arm of
biotechnology.
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Vaccines
What are Vaccines
 A vaccine is an agent, sourced from the pathogen,
and is deliberately introduced into the
mammalian system in order to impart a ‘memory’
of the pathogen or its pathogenic component
 The memory is imparted on the first contact of
the vaccine with the mammalian immune system.
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Vaccines: What are Vaccines
 Vaccines contain antigens (that elicit the
production of antibodies), or immunogens (that
trigger the cellular component of immune
response)
 In the event of an encounter with the
corresponding antibodies, only the antigens can
bind with the antibodies, and form an antigenantibody complex that neutralises the harmful
effects of the antigens or the organisms that
produce them.
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Vaccines
Vaccination/Immunisation
 The process of the deliberate introduction of a
vaccine into the organism is vaccination, for
which the term inoculation is also often
used. Since vaccination immunises the
organism, the process is also called immunisation
 When an organism is vaccinated, the immune
system is readied to show an immune response
by way producing antibodies against the
pathogen.
Microbial Biotechnology
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Vaccines
Composition of vaccines
 Vaccines are suspensions, in saline , of
weakened pathogenic organisms or the
proteins they secrete, which have the
potential to cause a disease.
Microbial Biotechnology
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Vaccines
Types of vaccines:
Inactivated vaccines:
 The pathogen is killed using heat or formalin,
as for example, typhoid vaccines .
Microbial Biotechnology
Attenuated
vaccines:
Vaccines: Types of vaccines:
 The pathogen is weakened (attenuated) by
aging or altering growth conditions, but is alive,
as in the case of measles, mumps and rubella
vaccines .
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Avirulent organisms:
 A non-pathogenic strain of a pathogenic
organism is used as a vaccine, as in BCG (Bacillus
Calmette Guerin) vaccine against Mycobacterium
tuberculosis, the tuberculosis bacterium.
Microbial Biotechnology
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Vaccines: Types of vaccines:
Toxoids:
 The toxin from the pathogen is used as an
antigen to produce the vaccine .
Acellular vaccines:
 Only the antigenic component of the organism
is used instead of the whole organism, as in
Haemophilus influenza B vaccine .
Microbial Biotechnology
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Vaccines: Types of vaccines:
Subunit vaccines:
 Genetic engineering techniques have now made
it possible to use as a vaccine only a part of an
organism that is adequate to stimulate the
immune response e.g in Hepatitis B vaccine
a segment of genetic material is isolated from
the pathogens and introduced into bacteria or
yeasts .
Microbial Biotechnology
DNA
Vaccines: vaccines:
Types of vaccines:
MICROORGANISMS AS TOOLS
DNA vaccines are an offshoot of gene therapy
 Selected segments of DNA, when introduced into
the patients system synthesise and deliver proteins
that are needed to replace the defective enzyme
system or tag a cell for destruction.
 Viruses or lipid vehicles are used to deliver the DNA
into the cells.
 This recent technology is being tried to produce
vaccines against HIV, by a direct injection of
plasmid borne DNA
2-WAY- LEARNING
 Hepatitis B vaccine?
 Immunisation by DNA injection?
Bioterrorism
(Chapter 9)
REFERENCES:

Introduction to Biotechnology by W.J. Thieman and M.A. Palladino. Pearson &
Benjamin Cummings 2nd edition.

http://en.wikipedia.org

Matti Leisola, Jouni Jokela, Ossi Pastinen, Ossi Turunen

Laboratory of Bioprocess Engineering, Helsinki University of Technology, Finland

Hans Schoemaker, DSM Research, MD Geleen, The Netherlands

http://www.accessexcellence.org/RC/VL/GG/transfer_and.html

PRODUCTION OF THERAPEUTIC PROTEINS BY GENETIC ENGINEERING - IMPACT No. 299 May
1998 Duane T. G

http://www.fbae.org/Channels/biotech_in_medicine/vaccines.htmish