Course Title: Microbial Biotechnology

Course No: 3104

According to the class lectures of our Honorable Teacher

Mostafa Jamal Happy Sir

[Ref: Molecular Biotechnology by Pasternak]

[It is prepared by Saad Bin Islam]
 Chapter name: Vaccine

Questions:
1. Definition of vaccine & vaccination. (2)
2. Principal of vaccination. (4)
3. What do you mean by viral infection? How will be the treatment strategy of viral infection? (5-6)
4. What is the mechanism of vaccination? Describe it. (5)
5. Classify the vaccine. Describe their advantages & disadvantages. (6-8)
6. What are the requirements of a successful vaccine? (4)
7. Distinguish between antibiotic & vaccine. (3)
8. Difference between killed vaccine and live attenuated vaccine. (3)
9. Describe the production of Herpes Simplex Virus-HSV. (6)
10. What is subunit vaccine? Mention its advantages and disadvantages. (3/4)
11. What is recombinant vaccine? Describe the general strategy of recombinant vaccine. (5)
12. Describe the Hepatitis-B recombinant vaccine. (5).
13. What is Tuberculosis? Describe the general strategy of tuberculosis. (4/5)
14. Describe the subunit vaccine production method of Foot & Mouth Disease. (5)
15. What is DNA vaccine? Describe the general strategy of DNA vaccine. (5)
16. What is the difference between DNA vaccine and recombinant vaccine? (2)
17. How do vaccine work? Do they work against virus and bacteria? (2)
18. Short Note- (4)
a. Bacterial Vaccine
b. Genetic immunization
1. Definition of vaccine & vaccination.
Vaccine: A vaccine is a biological preparation that improves immunity to a particular disease.
A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often
made from weakened or killed forms of the microbe, its toxins or one of its surface proteins.
The agent stimulates the body's immune system to recognize the agent as foreign, destroy it, and
"remember" it, so that the immune system can more easily recognize and destroy any of these
microorganisms that it later encounters.
Vaccination: Vaccination is the administration of antigenic material (a vaccine) to stimulate an
individual's immune system to develop adaptive immunity to a pathogen.
Vaccination is aimed at inducing active immunity in an individual, so that subsequent contact with the
microorganism following natural infection induces strong protective immune response.

2. Describe the principle of vaccination.

Principle of vaccination: Vaccination protects a recipient from pathogenic agents by establishing an
immunological resistance to infection. Vaccination process is developed by following principles-
 Identification of pathogenic agent.
 Convert the pathogenic agent to non-pathogenic agent.
 Administrate the agent into human body to develop immunity against the pathogen.
These are described below:
Identification of pathogenic agent: Not only whole microorganism but also some components can be
antigen, such as – protein, carbohydrate. We have to identify pathogenic agent.
Convert the pathogenic agent to non-pathogenic agent: To develop non-pathogenic antigen the
microorganisms can be killed or attenuated. Some components can be produced by recombinant DNA
technology.
Administrate the agent into human body to develop immunity against the pathogen: The
attenuated pathogen or components of pathogens are administrate through injection or orally. Then
human body produced immunity against the antigen. An injected or oral vaccine induces the host to
generate antibodies against the disease causing organism. Therefore, during future exposures, the
infectious agent is inactivated (neutralized, or killed), its proliferation is prevented, and the disease state
is not established. Over 200 years ago, in 1796, Edward Jenner had discovered the principle of
vaccination.

3. What do you mean by viral infection? How will be the treatment strategy of viral
infection?
Viral infection: Infection caused by the presence of a virus in the body. Depending on the virus and
the person's state of health, various viruses can infect almost any type of body tissue, from the brain to
the skin.
Viral infections cannot be treated with typical antibiotics (antibacterial antibiotics); in fact, in some
cases the use of antibacterial antibiotics may cause side-effects that complicate the viral infection.
Viral infection like-
Smallpox CMV Infection
COVID-19 Hemorrhagic Fever
HIV Infection Human Papilloma Virus
Influenza Rubella

Treatment strategy of viral infection: The vast majority of human viral infections can be effectively
fought by the body's own immune system, with a little help in the form of proper diet, hydration, and
rest. As for the rest, treatment depends on the type and location of the virus, and may include anti-viral
antibiotics or other drugs.
There have 6 steps to treat the viral infection:
A. Vaccination
B. Antiviral drugs
C. Natural sources
D. Nutrient
E. Management
F. Medication for minimizing symptoms

A. Vaccination: A vaccine helps the body’s immune system to recognize and fight pathogens like
viruses or bacteria, which then keeps us safe from the diseases they cause.
Most vaccines against viral infection are effective at preventing disease. However, they are not 100%
effective for a number of reasons, reactions can occur after vaccinations.
Bacterial vaccines contain killed or attenuated bacteria that activate the immune system. Antibodies are
built against that particular bacteria, and prevents bacterial infection later.
Some vaccines are-

 Mumps, measles and rubella/MMR Vaccines

 Typhus Vaccine
 Typhoid Vaccines
 HSV vaccines
 Rabies Vaccines
 Viral hepatitis
B. Antiviral Drug: Antiviral drugs are used for the treatment of viral infection. Antivirals are a class
of medications that are used to treat viral infections. Most viral infections resolve spontaneously in
immunocompetent individuals. The aim of antiviral therapy is to minimize symptoms and infectivity as
well as to shorten the duration of illness. These drugs act by arresting the viral replication cycle at
various stages.
Antiviral drugs do not deactivate or destroy the microbe (in this case, the virus) but act by inhibiting
replication. Some examples are-
Arbidol Balavir
Combivir Entecavir
Fusion inhibitor Ganciclovir
Indinavir Inosine

C. Natural Source: The scientific jury is still out concerning natural antibiotics. While people have
used remedies like these for hundreds of years, most treatments have not been thoroughly tested.
1. Garlic: Cultures across the world have long recognized garlic for its preventive and curative powers.
Research has found that garlic can be an effective treatment against many forms of bacteria, including
Salmonella and Escherichia coli (E. coli). Garlic has even been considered for use against multi-drug
resistant tuberculosis.
2. Honey: The antibacterial effects of honey are usually attributed to its hydrogen peroxide content.
However, honey fights off bacteria, though it has a lower hydrogen peroxide content. Antibacterial
properties aside, honey may help wounds to heal by providing a protective coating that fosters a moist
environment.
3. Ginger: Ginger as a natural antibiotic. Its ability to fight many strains of bacteria. Researchers are
also exploring ginger’s power to combat seasickness and nausea and to lower blood sugar levels.
4. Echinacea: Echinacea used for hundreds of years to treat infections and wounds. The extract of
Echinacea purpurea can kill many different kinds of bacteria.
5. Goldenseal: Goldenseal is usually consumed in tea or capsules to treat respiratory and digestive
problems. However, it may also combat bacterial diarrhea and urinary tract infections.
6. Clove: Clove has traditionally been used in dental procedures. Research is now finding that clove
water extract may be effective against many different kinds of bacteria, including E. coli.
7. Oregano: Some believe that oregano boosts the immune system and acts as an antioxidant. It may
have anti-inflammatory properties.

D. Nutrient: Micronutrients essential to fight infection include vitamins A, B, C, D, and E, and the
minerals iron, selenium, and zinc.
1. Vitamin A: Vitamin A maintains the structure of the cells in the skin, respiratory tract and gut. It is
found in oily fish, egg yolks, cheese, tofu, nuts, seeds, whole grains and legumes.
2. B vitamins: B vitamins, particularly B6, B9 and B12, contribute to body’s first response once it has
recognized a pathogen. They do this by influencing the production and activity of “natural killer” cells.
3. Vitamins C and E: Vitamin C and vitamin E help protect cells from oxidative stress. Vitamin C also
helps clean up this cellular mess by producing specialized cells to mount an immune response, including
neutrophils, lymphocytes and phagocytes.
Good sources of vitamin C include oranges, lemons, limes, berries, kiwifruit, broccoli, tomatoes and
capsicum. Vitamin E is found in nuts, green leafy vegetables and vegetables oils.
4. Vitamin D: Some immune cells need vitamin D to help destroy pathogens that cause infection.
Although sun exposure allows the body to produce vitamin D, food sources including eggs, fish and
some milks.
5. Iron, zinc, selenium: Iron helps kill pathogens by increasing the number of free radicals that can
destroy them. It also regulates enzyme reactions essential for immune cells to recognize and target
pathogens.
Zinc helps maintain the integrity of the skin and mucous membranes. Zinc and selenium also act as an
antioxidant, helping mop up some of the damage caused by oxidative stress.
E. Management: The following Eight steps can be done for management

1. Determine the severity of infection. 2. Evaluate host defenses.

3. Decide on the setting of care. 4. Treat surgically.
5. Support medically. 6. Choose and prescribe antibiotic therapy.
7. Administer the antibiotic properly. 8. Evaluate the patient frequently.

F. Medication for minimizing symptoms: An infection is caused by the over-growth of a

microorganism somewhere in the body. Micro-organisms include bacteria, viruses and fungi amongst
others. Bacterial infections are treated with antibiotics such as amoxicillin, erythromycin and
ciprofloxacin.
There are many different types of antibiotic, with different ways of working; the choice depends on the
type of infection you have. Fungi commonly cause skin infections such as athlete's foot and ringworm.
Antifungal medicines like miconazole and nystatin can be taken by mouth or applied directly to the
affected area. Aciclovir is an antiviral medicine used to treat viral infections like shingles and herpes.

4. What is the mechanism of vaccination? Describe it.

Mechanism of vaccination: A vaccine is a biological preparation that provides active acquired
immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing
microorganism and is often made from weakened or killed forms of the microbe, its toxins, or one of
its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat,
destroy it, and to further recognize and destroy any of the microorganisms associated with that agent
that it may encounter in the future.
The vaccine triggers our body's natural immune response into action to protect against the disease
without the risk of infection. The vaccine contains antigens: harmless substances (such as dead bacteria
or molecules) associated with the disease. Vaccines work by stimulating our immune system to produce
antibodies (substances produced by the body to fight disease) without actually infecting us with the
disease. They trigger the immune system to produce its own antibodies, as though the body has been
infected with a disease. This is called "active immunity". If the vaccinated person then comes into
contact with the disease itself, their immune system will recognize it and immediately produce the
antibodies they need to fight it.

FIGURE: Principal basis of vaccines

New born babies are already protected against several diseases, such as measles, mumps and rubella,
because antibodies have passed into them from their mothers via the placenta. This is called "passive
immunity". Passive immunity only lasts for a few weeks or months. In the case of measles, mumps and
rubella, it may last up to one year.
Vaccination provides protection to our body by following way:
1. Entry into body
2. Showed antigenicity
3. Primary immune response
4. Memory cell formation
5. Secondary response
6. Rapid and effective prevention of body
All these steps are described below:
Entry into body: Most vaccines are given by injection but some are given orally (by mouth) or nasally
(sprayed into the nose).
Showed antigenicity: After the inoculation into the body, vaccines show antigenicity and induce the
immune system of the body.
Primary immune response: The primary immune response occurs when a vaccine comes in contact
to the immune system for the first time. During this time the immune system has to learn to recognize
antigen and how to make antibody against it and eventually produce memory lymphocytes.
Memory cell formation: The goal of immunization is to produce memory of the vaccine antigen via a
large population of memory cells. If the real pathogen enters the body in the future, memory cells will
recognize it.
Secondary response: The secondary immune response occurs when the second time (3rd, 4th, etc.) the
person is exposed to the same antigen.
Rapid and effective prevention of body: At this point, the immune system can start making antibodies
immediately against the antigen because immunological memory has been established yet.

5. Classify the vaccine. Describe their advantages & disadvantages.

Vaccine Classification: Vaccines are dead or inactivated organisms or purified products derived from
them. There are several types of vaccines in use. These are given below:
i. Killed or inactivated vaccine
ii. Live attenuated vaccine
iii. Subunit vaccine
iv. Toxoid vaccine
v. Recombinant vaccine
vi. Vector vaccine vii. DNA vaccine

All types of vaccines are described below:

i. Killed or Inactivated Vaccine: Some vaccines contain inactivated, but previously virulent,
microorganisms that have been destroyed with chemicals (e.g., formaldehyde or formalin), heat,
radioactivity, or antibiotics. This destroys the pathogen’s ability to replicate, but keeps it “intact” so
that the immune system can still recognize it. Examples are influenza vaccine, cholera vaccine, polio
vaccine, rabies vaccine etc.

Advantages of Inactivated Vaccines:

 Safe to use and can be given to immune deficient and pregnant individuals.
 Cheaper than live attenuated vaccine.
 Storage not as critical as live vaccine.
Limitations of Inactivated Vaccines:
Totally inactivation is not possible because some microbes may form spore and can lead to vaccine-
associated disease.
  Inactivation or killing may alter antigenicity.
 Since the microorganisms cannot multiply, a large number are required to stimulate immunity.
 Periodic boosters must be given to maintain immunity.
 Only humoral immunity can be induced.
 Most killed vaccines have to be injected.
 Some vaccines may induce ill effects.
ii. Live Attenuated Vaccine: An attenuated vaccine is a vaccine created by reducing the virulence of
a pathogen, but still keeping it viable. Attenuation takes an infectious agent and alters it so that it
becomes harmless or less virulent. Examples are measles vaccine, mumps vaccine, typhoid vaccine etc.
Advantages of Live Vaccine: Being live, attenuated vaccine is the closest thing to a natural infection,
these vaccines are good “teachers” of the immune system. It is usually advantageous to develop a live
vaccine, because they are generally much more effective than killed or subunit vaccines.
 Infectious microbes can stimulate generation of memory cell as well as humoral immune
responses.
 Since these can multiply in the host, one or few doses required.
 A single administration of vaccine often has a high efficacy in producing long-lived immunity.
 Some live vaccines can be given orally; such vaccines induce mucosal immunity and IgA
synthesis, which gives more protection at the normal site of entry.
 Oral preparations are less expensive than giving injections and they can lead to elimination of
wild type virus from the community.
Limitations of Live Vaccine:

 Risk of reversion to its virulent form and may cause disease.

 There is possibility exists that an attenuated microbe in the vaccine could revert to a virulent
form and cause disease.
 Live attenuated vaccines is not applicable for anyone, people who have damaged or weakened
immune systems or immunocompromised people (i.e., having HIV or undergone
chemotherapy), cannot be given live vaccines.
 Since they are live and because their activity depends on their viability, proper storage is
critical.
 Controlled attenuation is required.

iii. Subunit Vaccine: Vaccines that use components of a pathogenic organism rather than the whole
organism are called “subunit” vaccines. Recombinant DNA technology is very well suited for
developing new subunit vaccines.
Advantages of Subunit Vaccines:

 Using a purified protein(s) as an immunogen ensures that the preparation is stable and safe.
 They can safely be given to immunosuppressed people.
 They are less likely to induce side effects.
Limitations of Subunit Vaccines:

 Purification of a specific protein can be costly.

 Antigens may not retain their native conformation, so that antibodies produced against the
subunit may not recognize the same protein on the pathogen surface.
  Isolated protein does not stimulate the immune system as well as a whole organism vaccine,
therefore, less effective.

iv. DNA Vaccines: DNA vaccines consist of DNA that is administered directly into the body. Like
recombinant vaccines, genes for the desired antigens are located and cloned. The direct injection of a
naked DNA plasmid into muscle as a vaccine system with the ability to induce an immune response
and protection after challenge is now well established. The idea behind the DNA vaccine system is that
the antigen can be expressed directly by the cells of the host in a way similar to that occurring during
viral infection.
Advantages of DNA Vaccines:
 DNA is very stable, it resists extreme temperature and hence storage and transport are easy.
 A DNA sequence can be changed easily in the laboratory.
 Because of the way the antigen is presented, there is a cell-mediated response that may be
directed against any antigen in the pathogen.
Limitations of DNA Vaccines:
Potential integration of DNA into host genome leading to insertional mutagenesis.
Induction of autoimmune responses: Anti-DNA antibodies may be produced against introduced
DNA. Induction of immunologic tolerance: The expression of the antigen in the host may lead to
specific non-responsiveness to that antigen.
v. Toxoid Vaccines: Toxoid vaccines are made from inactivated toxic compounds that cause illness
rather than the micro-organism. Examples of toxoid-based vaccines include tetanus and diphtheria. Not
all toxoids are for microorganisms; for example, Crotalus atrox toxoid is used to vaccinate dogs against
rattlesnake bites.
Advantages of Toxoid vaccine:
They are safe because there is no possibility of reversion to virulence.
 Highly immunogenic.
 They are usually stable and long lasting as they are less susceptible to changes in temperature,
humidity and light.
Limitations of Toxoid vaccine:

 Possible contamination by toxins.

 They usually need an adjuvant and require several doses for the reasons
 May occasionally cause allergic reactions or infiltration at the injection site.

vi. Recombinant Vaccine: A recombinant vaccine is a vaccine produced through recombinant DNA
technology. This involves inserting the DNA encoding an antigen (such as a bacterial surface protein)
that stimulates an immune response into bacterial or mammalian cells, expressing the antigen in these
cells and then purifying it from them.
Advantages of recombinant vaccine:
 No risk of pathogenicity.
 Defined composition.
  Various delivery systems available.
 Large scale production simplified.
 Further genetic engineering possible.
Limitations of recombinant vaccine:

 Multiple doses typically needed.

 Adjuvants needed.

vii. Vector Vaccine: A vector vaccine is a vaccine that uses a chemically weakened virus to transport
pieces of the pathogen in order to stimulate an immune response. The genes used in this vaccine are
usually antigen coding surface proteins from the pathogenic organism. They are then inserted into the
genome of a non-pathogenic organism such as adenovirus or vaccinia virus where they are expressed
on the cell's surface and can elicit an immune response.
Advantages of Vector Vaccine:
 Vector vaccine expresses high levels of the inserted gene product, which can then serve as a
potent immunogen in an inoculated host.
 Those vectors that are not only safe but also easy to grow and store can be chosen.
Limitations of vector vaccine:

 Since the genes for the desired antigens must be located, cloned, and expressed efficiently in
the new vector, the cost of production is high.
 When engineered vaccinia virus is used to vaccinate, care must be taken to spare
immunedeficient individuals.

6. What are the requirements of a successful vaccine?

Requirements of a Successful Vaccine: The overall requirements for a successful vaccine are the
following:
Safety: A vaccine should not cause disease, and side effects should be minimal. This is the most
important concern when developing a vaccine. The vaccine should be safe, even in
immunocompromised people.
Specificity: A vaccine should induce immune response against a specific virus, bacteria or any other
pathogenic organism.
Effectiveness: The third requirement is that the vaccine should be highly effective and optimally induce
‘sterilizing’ immunity. Vaccinated animals should be protected from illness due to the pathogen.
Ideally, appropriate innate, cellular and humoral responses should be evoked by the vaccine, and long
term immunity should be conferred.
Stability: The vaccine should be stable (for storage).
Practical Issues: The vaccine should be affordable and should have the practical implications.
Cheapness: To be included in the WHO Expanded Programme for Immunization, a vaccine should
cost less than 50 cents/dose.
7. Distinguish between antibiotic & vaccine.
Primary discussion about antibiotics and vaccine:
Antibiotics: Antibiotics are compounds that are effective in treating infections caused by organisms
such as bacteria, fungi and protozoa.
Vaccines: Vaccines are compounds that are used to provide immunity to a particular disease. Vaccines
are usually dead or inactivated organism or compounds purified from them.
o Vaccine is taken once and has permanent effect whereas antibiotics work during the time of
disease.
o Antibiotics are available in different forms like tablets, capsules, drops or ointments. Vaccines
can be given orally or through injection.
o Vaccines are preventive method that is taken before getting infected. Antibiotics are taken after
getting infected.
Difference between antibiotic and vaccine are given below:

Features Antibiotics Vaccines

Definition Antibiotics are small molecules Vaccines are dead or
that are effective in treating inactivated organisms or
infections caused by organisms compounds that are used to
such as bacteria, fungi and provide immunity to a
protozoa. particular infection or disease.
Source Antibiotics can be derived from Sources of vaccine include live
natural, semi-synthetic sources or inactivated microbes, toxins,
and synthetic sources antigens etc.
Types Antibiotics are classifiedVaccines are of different types-
according to their structure and killed, live attenuated, subunit,
mechanism of action into 3 toxoid, conjugate, DNA,
classes: cyclic lipopeptides, recombinant, vector vaccines
oxazolidinones and glycycline and other experimental
vaccines
Side effect Some antibiotics may have side Some vaccines may causes
effects like diarrhea nausea and allergic reaction
allergic reactions.
8. Difference between killed vaccine and live attenuated vaccine.
Live attenuated vaccine: An attenuated vaccine is a vaccine created by reducing the virulence of a
pathogen, but still keeping it viable. Attenuation takes an infectious agent and alters it so that it becomes
harmless or less virulent. Examples are measles vaccine, mumps vaccine, typhoid vaccine etc.
Killed vaccine: Some vaccines contain inactivated, but previously virulent, microorganisms that have
been destroyed with chemicals (e.g., formaldehyde or formalin), heat, radioactivity, or antibiotics. This
destroys the pathogen’s ability to replicate, but keeps it “intact” so that the immune system can still
recognize it. Examples are influenza vaccine, cholera vaccine, polio vaccine, rabies vaccine etc.

Killed Vaccine Live Attenuated Vaccine

Usually administered via injection Can be administered via injection or natural route
(oral, respiratory)
Produces humoral immunity (IgG) in Produces humoral immunity (IgG) in
bloodstream. bloodstream plus secretory IgA response if
administered via oral or respiratory route
Absence of living virus permits use in Immunodeficient hosts might become ill.
Immunodeficient hosts
Absence of living virus eliminates the danger of Vaccine virus may mutate (revert) towards
mutations that lead to increased virulence. increased virulence towards increased virulence.
May also recombine with other viruses in host
No spread of live virus to other animals Vaccine strain may spread to other animals and
may mutated to more virulent forms.
Variable induction of cellular immunity. Good induction of cellular immunity
Requires adjuvant. Does not require adjuvant.
Generally, repeated booster shots are required to Generally induces long-term immunity, due to
maintain long-term immunity full activation of both humoral and cellular
immunity.
Stable (not heat labile) Heat labile.
Less expensive. More expensive.

9. Describe the production of vaccine against Herpes Simplex Virus-HSV

Herpes Simplex Virus-HSV: Herpes simplex virus (HSV) has been implicated as a cancer-causing
(oncogenic) agent, in addition to its more common roles in causing sexually transmitted disease, severe
eye infections, and encephalitis, so prevention of HSV infection by vaccination with either killed or
attenuated virus may put the recipient at risk for cancer.
Thus, protection against HSV would be best achieved by a subunit vaccine, which would not be
oncogenic.
Production of Vaccine against HSV:
The primary requirement for creating any subunit vaccine is identification of the component(s) of the
infectious agent that elicits antibodies that react against the intact form of the infectious agent.
The steps of vaccine production against HSV are given below:
• The HSV type 1 (HSV-1) envelope glycoprotein D (gD) is a component, which after injection into
mice, elicits antibodies that neutralize intact HSV.
• The HSV-1 gD gene was isolated and then cloned into a mammalian expression vector and expressed
in Chinese hamster ovary (CHO) cells.
• The complete sequence of the gD gene encodes a protein that becomes bound to the mammalian host
cell membrane.
• Consequently, the gD gene was modified by removing the nucleotides encoding the C-terminal trans
membrane-binding domain.
• The modified gene was then transformed into CHO cells, where the product was glycosylated and
secreted into the external medium.
10. What is subunit vaccine? Mention its advantages and disadvantages.
Subunit vaccine: Vaccines that use components of a pathogenic organism rather than the whole
organism are called “subunit” vaccines. Recombinant DNA technology is very well suited for
developing new subunit vaccines.
Instead of the entire microbe, subunit vaccines include only the antigens that best stimulate the immune
system. Subunit vaccines can contain anywhere from 1 to 20 or more antigens. Of course, identifying
which antigens best stimulate the immune system is a tricky, time-consuming process.
Scientists can make subunit vaccines in one of two ways:
•They can grow the microbe in the laboratory and then use chemicals to break it apart and gather the
important antigens.
• They can manufacture the antigen molecules from the microbe using recombinant DNA technology.
Vaccines produced this way are called “recombinant subunit vaccines.”

There are advantages and disadvantages to the use of subunit vaccines.

Advantages: Using a purified protein(s) as an immunogen ensures that the preparation
-is stable and safe,
-is precisely defined chemically, and
-is free of extraneous proteins and nucleic acids that can initiate undesirable side effects in the host
organism.
Disadvantages: Purification of a specific protein can be costly, and in certain instances, an isolated
protein may not have the same conformation as it does in situ (within the viral capsid or envelope), with
the result that its antigenicity is decreased.

11. What is recombinant vaccine? Describe the general strategy of recombinant vaccine.
Recombinant vaccine: A recombinant vaccine is a vaccine produced through recombinant DNA
technology. This involves inserting the DNA encoding an antigen (such as a bacterial surface protein)
that stimulates an immune response into bacterial or mammalian cells, expressing the antigen in these
cells and then purifying it from them.
Features of recombinant vaccine:
-Usually antigenic gene sequence is inserted into a plasmid vector.
-The recombinant plasmid is then inserted into host cell like E.coli or yeast cells.
-The cloned host cells are cultured for expression of Ag gene.
-Finally the expressed Ag particles are used as recombinant vaccine.
-No risk of pathogenicity.
-Defined composition.
-Various delivery systems available.
-Large scale production simplified.

12. Describe the Hepatitis-B recombinant vaccine

Vaccine against Hepatitis B virus: Hepatitis B virus (HBV) is wide spread in man and produces
several chronic liver disorders such as Fulminant chronic hepatitis, cirrhosis and primary liver cancer.
Hepatitis B vaccine is developed for the prevention of hepatitis B virus infection. The vaccine contains
one of the viral envelope proteins, hepatitis B surface antigen (HBsAg).
Recombinant vaccine for HBV is produced by cloning HBsAg gene of the virus in yeast cells.
Production of recombinant vaccine against HBV:
• The HBsAg gene contains 6 bp long sequence preceding the AUG that synthesizes N-terminal
methionine.
• This is joined to ADH promoter cloned in the yeast vector PMA-56.
• The recombinant plasmid is then inserted into yeast cells.
• The transformed yeast cells
are multiplied in tryptophan-
free medium.
• The transformed cells are
selected.
• The cloned yeast cells are
cultured for expression of
HBsAg gene.
• The expressed HBsAg
particles have similarity in
structure and
immunogenicity with those
isolated from HBV-infected
cells of patients.
Its high immunogenicity has
made it possible to market
the recombinant product as vaccine against HBV infection.

13. What is Tuberculosis? Describe the general strategy of tuberculosis.

Tuberculosis: Tuberculosis or TB is a common and often deadly infectious disease caused by various
strains of mycobacteria, usually Mycobacterium tuberculosis in humans.
A 30-kDa mycolytransferase also known as
αantigen, or antigen 85B is the major antigenic
part of M.tuberculosis which produces disease
in humans.
In some countries, bacillus Calmette-Guerin
(BCG), an attenuated strain of Mycobacterium
bovis, was used as a vaccine against
tuberculosis.
An E.coli–mycobacterium shuttle vector that
contained the gene for the 30-kDa protein
(αantigen) under the control of its own promoter was introduced into two different BCG strains.

Production of vaccine against tuberculosis: Recently, tuberculosis vaccine is developed by using

rDNA of bacterial strain and commercially synthesized from rBCG30.
rBCG30 is a live vaccine that is a recombinant form of BCG. The vaccine has been genetically
engineered to secrete large amounts of 30kDa protein of Mycobacterium tuberculosis.
A key feature of the rBCG30 vaccine is the use of a plasmid (pMTB30) to overexpress the M.
tuberculosis 30 kDa protein.

The basic procedure of rBCG30 vaccine is given below:

• rBCG30 is a recombinant BCG overexpressing the 30kDa major secretory protein of M.tuberculosis.
• This protein, also known as α-antigen or antigen 85B, is a mycolytransferase.
• rBCG30 is constructed by inserting the plasmid pMTB30 into BCG. This plasmid contains full-length
30kDa protein gene of M.tuberculosis.
• rBCG30 secretes approximately 5-fold more 30kDa protein than the parental BCG strain.
After purification, this 30kDa recombinant protein is used as vaccine against tuberculosis.

14. Describe the subunit vaccine production method of Foot & Mouth Disease.
Subunit Vaccine for Foot and Mouth Disease: Foot-and-mouth disease virus (FMDV) has a
devastating impact on cattle and swine and is extremely virulent, but for the most part, it has been
possible to keep the negative effects of the virus to a minimum by using formalin-killed FMDV
preparations as a vaccine.
Production of Subunit Vaccine against FMD:
Research on FMDV found that the major antigenic determinant that induces neutralizing antibodies is
capsid viral protein 1 (VP1).
Although purified VP1 is a much less potent antigen than intact viral particles, it can still elicit
neutralizing antibodies by itself and therefore can protect animals from infection by FMDV.
Thus, the gene for VP1 became a target for cloning. The genome of FMDV is composed of
singlestranded RNA.
The main steps of production of vaccine are given below:
• The entire viral RNA is made into cDNA, which is then digested with restriction enzymes.
• The DNA fragments are cloned into an expression vector in frame with the gene for the E. coli
bacteriophage MS2 replicative protein.
• The plasmid constructs are used to transform E. coli, and then the stable fusion protein is isolated and
used to inoculate animals.
The fusion protein containing the VP1 protein fragment is able to generate neutralizing antibodies to
FMDV.
15. What is DNA vaccine? Describe the general strategy of DNA vaccine
DNA vaccine: DNA vaccines consist of DNA that is administered directly into the body. These
vaccines are still in experimental stage.
A DNA vaccine uses a plasmid expression vector to generate a peptide within the cells.
DNA vaccine production against tuberculosis:
• Generally, peptide motifs are identified that are immunogenic and generate a strong host immune
response.
• For infectious agents, these may be coat or capsid proteins, extracellular receptors, or other peptides
that the host would ordinarily contact during the course of an infection.
• These peptides are analyzed, and cDNA sequences are generated.
• The coding DNA sequences are inserted into plasmid expression vectors, which are used to transform
bacteria.
• The DNA is subsequently purified and may be used as a vaccine.

• Immunogenic peptide motifs are identified in the genome of the organism.

• These motifs are inserted into a bacterial plasmid vector, which is grown, amplified, and purified in
vitro.
• The purified plasmid DNA is then used as a vaccine to protect against infection with the organism.

16. What is the difference between DNA vaccine and recombinant vaccine?
Difference between DNA Vaccine and Recombinant Vaccine: The key difference between DNA
vaccine and recombinant vaccine relies on the preparation of vaccines. DNA vaccine preparation takes
place using desired genes or DNA fragments, while recombinant vaccine preparation takes place using
a recombinant molecule or a vector containing the desired gene fragment.
 DNA Vaccine Recombinant Vaccine
Definition DNA Vaccines are vaccines Recombinant vaccines are
which use fragments of DNA vaccines that carry a
recombinant DNA molecule or
a vector.
Forms of vaccination DNA Recombinant vectors or
recombinant organism.
Recombinant DNA Technology Does not use recombinant DNA Use recombinant DNA
Technology technology

17. How do vaccine work? Do they work against virus and bacteria?
How vaccine works: A vaccine works by training the immune system to recognize and combat
pathogens, either viruses or bacteria. To do this, certain molecules from the pathogen must be
introduced into the body to trigger an immune response.
These molecules are called antigens, and they are present on all viruses and bacteria. By injecting these
antigens into the body, the immune system can safely learn to recognize them as hostile invaders,
produce antibodies, and remember them for the future. If the bacteria or virus reappears, the immune
system will recognize the antigens immediately and attack aggressively well before the pathogen can
spread and cause sickness.
Vaccines against Viral & Bacterial Diseases
Most vaccines against viral infection are effective at preventing disease. However, they are not 100%
effective for a number of reasons, reactions can occur after vaccinations.
It is difficult for many of us today to appreciate the dangers of childhood viral infections.
Most of the vaccines in use against viruses are very effective at preventing disease. However, for a
variety of reasons, they can fail:
The vaccine becomes inactive due to incorrect storage, if used past its expiry date, or if incorrectly
administered.
Individuals unpredictably fail to produce an adequate immune response to the vaccine.
Vaccine immunity “fades” over time.
The different vaccine combinations at each time point do not interfere with one another and there is no
increased risk of serious side-effects when they are given at the same time.
Bacterial vaccines contain killed or attenuated bacteria that activate the immune system. Antibodies are
built against that particular bacteria, and prevents bacterial infection later.
Most vaccines against bacterial infections are effective at preventing disease, reactions can occur after
vaccinations. Vaccines are available against tuberculosis, diphtheria, tetanus, pertussis, Haemophilus
influenzae type B, and cholera, typhoid, and Streptococcus pneumonia.
Mumps, measles and rubella/MMR Vaccines
o Live attenuated vaccines
o Typhus Vaccine
o Typhoid Vaccines
o HSV vaccines
 o Rabies Vaccines
o Viral hepatitis
o Shingles
o Inactivated vaccines

18. Short Note

i. Bacterial Vaccine: Bacterial vaccines contain killed or attenuated bacteria that activate the immune
system. Antibodies are built against that particular bacteria, and prevents bacterial infection later. An
example of a bacterial vaccine is the Tuberculosis vaccine.
Creative Bio-labs can offer high-quality Bacterial Vaccines for use in bacterial diseases. Bacterial
Vaccines contain attenuated or killed bacteria that activate the immune system. For Bacterial Vaccines,
a pathogen strain is cultured and inactivated to produce a “whole-cell” vaccine (e.g., Bordetella
pertussiss), an attenuated bacterium is used (BCG), or pathogen bacterial strains is cultured to produce
inactivated and purified toxins or virulence factors
Usage Bacterium Disease Antigen
Common Usage Clostridium tetani Tetanus Toxoid
Haemophilus Meningitis Capsular
influenzae polysaccharide
conjugated to carrier
protein
Neisseria meningitidis Meningitis Capsular
polysaccharide or
capsular
polysaccharide
conjugated to a carrier
protein
Streptococcus Pneumonia Capsular
pneumoniae polysaccharide or
capsular
polysaccharide
conjugated to carrier
protein

Special Situation Salmonella typhi Typhoid fever Live organisms or

capsular
polysaccharide
Vibrio cholerae Cholera Killed organisms
Bacillus anthracis Anthrax Killed organisms
Mycobacterium bovis Tuberculosis Live Organism
(BCG)

ii. Genetic immunization: Genetic immunization, also known as DNA or polynucleotide

immunization, is a novel strategy for vaccine development in which plasmid DNA encoding either
individual or a collection of antigens is directly administered to a host.
Direct injection of naked plasmid DNA induces strong immune responses to the antigen encoded by the
gene vaccine.
Once the plasmid DNA construct is injected the host cells take up the foreign DNA, expressing the viral
gene and producing the corresponding viral protein inside the cell.

Advantages of genetic immunization over conventional vaccines:

• Cultivation of dangerous agents is not required.
• Since genetic immunization does not utilize any viral or bacterial strains, there is no chance that an
attenuated strain will revert to virulence.
• Since no organisms are used, attenuated organisms that many cause disease in young or
immunocompromised animals are not a problem.
• Approach is independent of whether the microorganism is difficult to grow or attenuate.
• Production is inexpensive because protein does not need to be produced or purified.
• Storage is inexpensive because of the stability of DNA.
• One plasmid could encode several antigens/vaccines, or several plasmids could be mixed together and
administered at the same time.
 Chapter Name: Microbial Production of Therapeutic Agent

Questions:
1. What is therapeutic agent? Briefly describe some therapeutic agents which are produced by microbial
biotechnology. (4)
2. How do you isolate human interferon cDNA? (5)
3. Production of human interferon as therapeutic agent. (6)
4. Synthesization of the Human growth hormone. (6)
5. Why do you construct cDNA library? (3)
6. What is monoclonal antibody? Describe the role of monoclonal antibody as a therapeutic agent. (3)
7. Describe the therapeutic agents against HIV. (4)
8. Describe the production method of monoclonal antibody in E. coli. (5)
9. What is the role of DNase-1 against cystic fibrosis? Describe the production of recombinant DNase-
1. (5)
10. What is the role of Alginate Lyase against cystic fibrosis? Describe the production of recombinant
Alginate Lyase. (6)
11. Short Note (4)
i. HIV Inhibitor
ii. Interferon
1. What is therapeutic agent? Briefly describe some therapeutic agents which are
produced by microbial biotechnology.
Therapeutic Agent: Therapeutic Agent means a drug, protein, peptide, gene, compound or other
pharmaceutically active ingredient. In a word a clinical trial evaluating the safety & efficacy of an
angiogenic gene therapy is called therapeutic agent.
Some therapeutic agents: Some therapeutic agents are described below:
1) Interferon: Interferons (IFNs) are proteins and released by host cells in response to the presence of
pathogens (i.e., viruses, bacteria, parasites or tumor cells). Interferon was named for its ability to
interfere with viral proliferation. They are important modulator of immune response.

IFNs can be classified into three different groups: IFN-α, IFN-β, and IFN-γ. The proteins IFN-α and
IFN-β are synthesized in cells that have been exposed to viruses or viral RNA; IFN-γ is synthesized in
response to cell growth-stimulating agents.

• Interferon stimulates the infected cells to prevent the virus from replicating within them.
• Interferons inhibit cell division, and prevent the differentiation of infected cells. Further production of
the virus is thereby inhibited.
• Interferons also have immune-regulatory functions—they inhibit B-cell activation, enhance T-cell
activity, and increase the cellular-destruction capability of natural killer cells.
• IFN-γ directly activates other immune cells, such as macrophages and natural killer cells.

2) Human growth hormone: Growth hormone (GH or HGH), also known as somatotropin, is a peptide
hormone that stimulates growth, cell reproduction and regeneration in humans.

HGH can be used as therapeutic agents in many purposes. Infants and children
-who lack sufficient endogenous levels of human growth hormone,
-patients with chronic renal insufficiency (defective kidneys), and
-individuals with Turner syndrome respond to treatment with growth hormone, which stimulates tissue
and bone growth, increases protein synthesis and mineral retention, and decreases body fat storage.

It also serve some other functions such as:

• Growth factor increases the total growth of human.
• It increases the synthesis of DNA and RNA synthesis in all cells.
• It enhances the oxidation of fats in liver and muscle.
• It promotes lipolysis and protein synthesis.
• It increases the growth of internal organs.
• It promotes gluconeogenesis in liver.
• It contribute to the function and maintenance of pancreatic cells
• It also stimulate the immune system.

3) Enzymes: Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in
nature (exception - RNA acting as ribozyme), colloidal and thermo-labile in character, and specific in
their action.

Some important therapeutic enzymes and their uses:

• DNase-I - used in cystic fibrosis treatment.
• Alginate lyase - used in cystic fibrosis treatment.
• Asparaginase - used in leukemia treatment.
• Collagenase - used in skin ulcers treatment.
• Hyaluronidase - used in heart attack treatment.
• Lysozyme - used as antibiotics.
• Ribonuclease - used as antiviral drug.
• Streptokinase - used to remove blood clots.
• Uricase - used in gout treatment.

4) Monoclonal antibody: ‘Mono’ means single and ‘clone’ means a group of cells all of which are
progeny of a single cell. So, monoclonal antibodies (mAb) are immunoglobulins produced from a single
specificity determining cell. They are made by identical immune cells that are all clones of a unique
parent cell.

Followings are important applications of monoclonal antibodies:

• Diagnostic Tests- Once mAbs for a given substance have been produced, they can be used to detect
the presence of this substance or purify it. mAb is used in the Western blot test to detect the specific
protein.
• Cancer Treatment- mAbs bind only to cancer cell-specific antigens and induce an immune response
against the target cancer cell.
• Autoimmune Diseases- mAbs are used for autoimmune diseases include infliximab and adalimumab,
which are effective in rheumatoid arthritis, Crohn's disease and ulcerative Colitis by their ability to bind
to and inhibit TNF-α.
• Analytical uses- mAbs can also be used to purify their target compounds from mixtures, using the
method of immunoprecipitation.

2. How do you isolate human interferon cDNA?

At first mRNA from human leucocyte cell will be collected.

After that by reverse transcription mRNA is converted to cDNA. Then it is cloned into plasmid PBR322.
Followed by it is transferred into E. coli and selected by marker Ampicillin resistance gene. Mean
plasmid has ampicillin resistance gene so ampicillin antibiotic is added in the media. So only that E.
coli will survive whose has cDNA of interferon.
Then the selected E. coli will be cultured to get man colonies. Followed by group of colony is made.
The approximately 6000 clones that were produced following transformation of E. coli were divided
into 12 pools of 512 clones. Each pool of clones rather than individual clones was tested to speed up
the identification process. Which means there are 12 groups and each group contain 512 colonies.
The plasmid DNA from each pool were hybridized to a crude IFN mRNA preparation. Which means
from every group cDNA hybridize with INF mRNA.
The input mRNA that hybridized to the plasmid DNA was separated from the cloned DNA-mRNA
hybrids and translated in a cell free protein synthesis system. Which means it is given in a media to
synthesize INF. Then INF is purified and its activity is tested.
Each translation mixture was then assayed for IFN antiviral activity. The pools that showed IFN activity
contained at least one clone with a cDNA that hybridized to IFN mRNA.
Positive pools were divvied into eight subgroups of 64 clones each and retested. This subgrouping
process was repeated until a clone with the complete cDNA for a human IFN was identified. Which has
interferon activity that cDNA will be selected and divide into subgroup and selected cDNA will be used
in the production of interferon.
Steps:
1. Size-fractionated mRNA was isolated from human leukocytes, reverse transcribed, and inserted into
the PstI site of plasmidpBR322.
2. The approximately 6,000 clones that were produced following transformation of E. coli were divided
into 12 pools of 512 clones each. Pools of clones, rather than individual clones, were tested to speed up
the identification process.
3. The plasmid DNA from each pool was hybridized to a crude IFN mRNA preparation.
4. The input mRNA that hybridized to the plasmid DNA was separated from the cloned DNA–mRNA
hybrids and translated in a cell-free protein synthesis system.
5. Each translation mixture was then assayed for IFN antiviral activity. The pools that showed IFN
activity contained at least one clone with a cDNA that hybridized to IFN mRNA.
6. Positive pools were divided into eight subgroups of 64 clones each and retested (i.e., steps 3 to 5 were
repeated). This subgrouping process was repeated until a clone with the complete cDNA for a human
IFN was identified.
Subsequently, whenever large quantities of the IFN were required, the
IFN cDNAs could be sub cloned into an E. coli expression vector and expressed at high levels.

3. Production of human interferon as therapeutic agent.

Answer: The Production of human interferon as therapeutic agent has two parts.
i) cDNA isolation (without figure)
ii) Gene shuffling

i) Isolation of Interferon cDNAs: A number of different strategies have been used to isolate either the
genes or cDNAs for human proteins. In some cases, the target protein is isolated and a portion of the
amino acid sequence is determined. From this information, a DNA coding sequence is deduced. The
appropriate oligonucleotide is synthesized and used as a DNA hybridization probe to isolate the gene
or cDNA from either a genomic or a cDNA library. Alternatively, antibodies are raised against the
purified protein and used to screen a gene expression library. For human proteins that are synthesized
primarily in a single tissue, a cDNA library from the messenger RNA (mRNA) of that tissue is enriched
for the target DNA sequence. For example, the major protein synthesized by the islets of Langerhans
of the pancreas is insulin; 70% of the mRNA fraction isolated from these cells encodes insulin.
Before the completion of sequencing of the human genome in 2001, it was often necessary to devise
innovative approaches to isolate human genes or cDNAs, especially when the proteins encoded were
found in very low concentrations or when the site of synthesis was not known. The human interferon
(IFN) proteins, which include IFN-α, IFN-β, and IFN-γ, are naturally occurring proteins, each one with
somewhat different biological activity. When the IFN cDNAs were initially isolated in the early 1980s,
very little was known about the encoded proteins (IFN was originally thought to be a single protein), so
a novel scheme had to be devised to overcome the scarcity of both the mRNAs and the proteins. Also,
at the time, Escherichia coli expression vectors for eukaryotic cDNAs were not readily available, so it
was necessary to devise an indirect scheme to isolate IFN cDNA.
The isolation of IFN cDNAs included the following steps:
1. Size-fractionated mRNA was isolated from human leukocytes, reverse transcribed, and inserted into
the PstI site of plasmid pBR322.
2. The approximately 6,000 clones that were produced following transformation of E. coli were divided
into 12 pools of 512 clones each. Pools of clones, rather than individual clones, were tested to speed up
the identification process.
3. The plasmid DNA from each pool was hybridized to a crude IFN mRNA preparation.
4. The input mRNA that hybridized to the plasmid DNA was separated from the cloned DNA–mRNA
hybrids and translated in a cell-free protein synthesis system.
5. Each translation mixture was then assayed for IFN antiviral activity. The pools that showed IFN
activity contained at least one clone with a cDNA that hybridized to IFN mRNA.
6. Positive pools were divided into eight subgroups of 64 clones each and retested (i.e., steps 3 to 5 were
repeated). This subgrouping process was repeated until a clone with the complete cDNA for a human
IFN was identified.
Subsequently, whenever large quantities of the IFN were required, the IFN cDNAs could
be sub cloned into an E. coli expression vector and expressed at high levels.
ii)Interferon gene shuffling: Several research groups have attempted to engineer IFNs with combined
properties based on different members of the IFN-α gene family that vary in the extents and specificities
of their antiviral activities.
Theoretically, this can be achieved by splicing a portion of one IFN-α gene with a DNA
sequence from a different IFN-α gene to create, after translation, a hybrid protein that exhibits novel
properties, i.e., properties different from either of the contributing genes. In one study, hybrid genes
from IFN-α2 and IFN-α3 were constructed in an effort to create proteins with novel IFN activities.
Comparison of the sequences of the two IFN-α cDNAs indicated that they had common restriction sites
at positions 60, 92, and 150. Digestion of both cDNAs at these sites and ligation of the DNA fragments
yielded a number of hybrid derivatives of the original genes. These hybrids were expressed in E. coli,
and the resultant proteins were purified and examined for various biological functions. When tested for
the extent of protection of mammalian cells in culture against viral infection, some of the hybrid IFNs
were found to have greater activity than the parental molecules. In addition, many of the hybrid IFNs
induced test cells to synthesize (2′-5′)-oligoisoadenylate synthetase. This enzyme generates (2′-5′)-
linked oligonucleotides, which in turn activate a latent cellular endoribonuclease that cleaves viral
mRNA. Other hybrid IFNs had an antiproliferative activity against various human cancers that was
greater than that of either of the parental molecules. More recently, additional hybrid IFN molecules
have been generated by a variation of the above-mentioned procedure. In this case, the entire IFN-α
cDNA family was PCR amplified and then digested with DNase into small DNA fragments (~50 to 60
nucleotides long) before the fragments were shuffled and amplified by PCR. This procedure works
because the PCR mixture contains many overlapping single-stranded DNAs that can act as PCR
primers. Following testing of the many shuffled IFN cDNAs, it is possible to select hybrid IFNs with
vastly improved antiviral or antiproliferative activities. In fact, some hybrid IFNs have recently
undergone successful clinical trials and have been approved for use as human therapeutic agents. The
strategy for creating hybrid IFNs can also be applied to other gene families whose products have
therapeutic potential.
Figure: Structure of the IFN-α2 and IFN-α3 genes and four hybrid genes. Comparison of the sequences
of the IFN-α2 and IFN-α3 genes shows shared restriction enzyme sites (RE1, RE2, and RE3). Digestion
of the genes at the indicated restriction sites and ligation of the resulting fragments generate a number
of different hybrid IFN genes, of which four possibilities are shown.

4. Synthesization of the Human growth hormone.

Production of human growth hormone: The strategy of designing human growth hormone by either
functional domain shuffling or directed mutagenesis can be used to augment or constrain its mode of
action.
For example, native human growth hormone binds to both growth hormone and prolactin receptors that
occur on a number of different cell types.
To avoid unwanted side effects during therapy, it is desirable that human growth hormone bind only to
growth hormone receptors.
Because the segment of the growth hormone molecule that binds to the growth hormone receptor
overlaps but is not identical to the portion of the molecule that binds to the prolactin receptor, it should
be possible to selectively decrease the binding to the prolactin receptor.
Site-specific mutagenesis of the cloned human growth hormone cDNA was used to change some of the
amino acid side chains that act as ligands for Zn2+ (i.e., His-18, His-21, and Glu-174), because the ion
is required for the high-affinity binding of human growth hormone to the prolactin receptor.

Figure: Schematic representation of native and modified human growth hormone. Oligonucleotide-
directed mutagenesis was used to alter human growth hormone so that it no longer bound to the prolactin
receptor but retained its specificity for the growth hormone receptor.
As a consequence of its relatively short half-life in plasma, human growth hormone therapy currently
requires subcutaneous injection once a day. This treatment is both inconvenient and expensive.
Therefore, it would be advantageous to have a long-lasting form of human growth hormone. To this
end, the extracellular domain of the human growth hormone receptor was fused to human growth
hormone using a 20-amino-acid-long linker peptide consisting of four repeats of the amino acids
Gly4Ser (Fig. 10.6). This construct has a very strong tendency to dimerize as the growth hormone
moiety from one molecule binds with the receptor portion of another molecule. When this growth
hormone construct was tested in rats, a single injection promoted growth for 10 days (compared to the
usual requirement in rats for daily injections). It is thought that the dimerization of the growth hormone
construct stabilizes human growth hormone in vivo so that it is cleared from plasma approximately 300
times more slowly than free human growth hormone. Under these conditions, the active monomeric
form (Fig.10.6A) is slowly released from the inactive dimeric growth hormone (Fig.10.6B), allowing it
to bind to the growth hormone receptor (Fig.10.6C). This experiment is certainly intriguing. It remains
to be determined whether humans respond in a similar manner to the dimerize complex.

Figure: Derivatization of growth

hormone by coupling it to a portion
of the growth hormone receptor
using a 20-amino-acid peptide. (A)
Monomeric derivative; (B) dimeric
derivative; (C) monomeric
derivative bound to a growth
hormone receptor.

Another method that has been

devised to prolong the active lifetime of human growth hormone includes fusing the coding sequences
for the C-terminal end of human growth hormone (~22 kDa) with the N-terminal end of human serum
albumin (~67 kDa). This fusion protein is called Albutropin (Fig. 10.7); it has a molecular mass of ~89
kDa and is produced by a strain of yeast that has been genetically modified so that the proteins that it
produces have a minimal number of posttranslational modifications. The stabilization of the human
growth hormone portion of Albutropin reflects the stability of human serum albumin, which has a half-
life in serum of about 19 days.

Figure: Schematic representation of the fusion protein Albutropin,

which includes human serum albumin (red) at the N terminus and
human growth hormone (blue) at the C terminus

Albutropin has been shown to be effective in both rats and monkeys, in which high levels of the protein
in serum were observed 5 days after it was administered. Moreover, Albutropin has successfully
completed phase I clinical trials.
5. Why do you construct cDNA library?
cDNA library: A cDNA library is a combination of cloned cDNA (complementary DNA) fragments
inserted into a collection of host cells, which together constitute some portion of the transcriptome of
the organism and are stored as a "library".
cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the
expressed genes of an organism.

cDNA Library uses: cDNA libraries are commonly used when reproducing eukaryotic genomes, as
the amount of information is reduced to remove the large numbers of non-coding regions from the
library.
 cDNA libraries are used to express eukaryotic genes in prokaryotes.
 Prokaryotes do not have introns in their DNA and therefore do not possess any enzymes that
can cut it out during transcription process.
 cDNA does not have introns and therefore can be expressed in prokaryotic cells.
 cDNA libraries are most useful in reverse genetics where the additional genomic information
is of less use.
 Additionally, cDNA libraries are frequently used in functional cloning to identify genes based
on the encoded protein's function.

[Good to Know: cDNA Library vs. Genomic DNA Library: cDNA library lacks the non-coding and
regulatory elements found in genomic DNA.
Genomic DNA libraries provide more detailed information about the organism, but are more resource-
intensive to generate and maintain.]

6. What is monoclonal antibody? Describe the role of monoclonal antibody as a

therapeutic agent.
Monoclonal antibody: ‘Mono’ means single and ‘clone’ means a group of cells all of which are
progeny of a single cell. So, monoclonal antibodies (mAb) are immunoglobulins produced from a single
specificity determining cell. They are made by identical immune cells that are all clones of a unique
parent cell. Antibody that is made by identical immune cells that are all clones of a unique parent cell.
In other word Monoclonal antibodies are immune system proteins that are created in the lab and used
to treat cancer.

Role of mAb as a therapeutic agent: In an intact antibody molecule, the Fc portion elicits several
immunological responses after antigen–antibody binding occurs.
1. The complement is activated. The components of this system break down cell membranes,
activate phagocytes, and generate signals to mobilize other components of the immunological
response system.
2. Antibody-dependent cell-mediated cytotoxicity (ADCC), which is the result of the binding of
the Fc portion of the antibody to an Fc receptor of an ADCC effector cell, is produced.
3. The bound effector cell releases substances that lyse the foreign cell to which the Fab portion
of the antibody molecule is bound.
4. After the Fab region binds to a soluble antigen, the Fc portion of an antibody can be bound to
Fc receptors of phagocytic cells, which engulf and destroy the antibody–antigen complex.
5. Preventing Rejection of Transplanted Organs: Passive immunization is reconsidered as a way
of preventing immunological rejection of a transplanted organ.
6. Monoclonal Antibody can bind to certain lymphocytes and diminish the immune response
directed against the transplanted organ.

The mouse monoclonal antibody OKT3 was approved in 1986 by the FDA for use as an
immunosuppressive agent after organ transplantation in humans.
Various members of the T-cell population act as immunological helper and effector cells and are
responsible for organ rejection.
The OKT3 monoclonal antibody binds to a cell surface receptor called CD3, which is present on all T
cells.
As a result, a full immunological response is blocked, and the transplanted organ is not rejected.

7. Describe the therapeutic agents against HIV.

Introduction to antiretroviral therapy: Antiretrovirals (ARVs) are the cornerstone of HIV/AIDS
management, as there is currently no cure nor vaccine available for HIV.
If an individual with a non-resistant strain of HIV takes the appropriate antiretroviral treatment as
directed, the replication of HIV will be effectively suppressed in about 80% of cases.
ARVs have been consistently proven to reduce death due to HIV/AIDS and to reduce the development
of AIDS-defining conditions. These AIDS-defining conditions are a range of infections, cancers and
illnesses that can occur due to advanced stages of HIV infection.

Antiretroviral: Antiretroviral medications are a group of drugs that inhibit different steps in the HIV
replication process. In this way, they can suppress HIV infection but never entirely eliminate it from
the body. There are four categories of ARV medications:

1. Nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs);

2. Non-nucleoside reverse transcriptase inhibitors (NNRTIs);
3. Protease inhibitors (PIs) (often ritonavir-boosted); and
4. Drugs that interfere with viral entry, such as fusion inhibitors and CCR5 antagonists.

Working method: HIV is a RNA virus that uses a range of viral enzymes to incorporate itself into
human DNA within certain types of immune cells. Once present in the DNA strand, HIV can use the
cell’s own mechanisms to create more HIV viral particles, to infect more human immune cells.
HIV binds to receptors on the surface of human immune cells, primarily CD4+ T-helper immune cells
that fight infections.
 The HIV virus then gains entry into the cell cytoplasm, where an enzyme called viral reverse
transcriptase creates viral DNA from HIV RNA.
 This DNA moves into the cell nucleus, where it is incorporated into the human DNA strand by
way of viral integrase.
 As normal cellular DNA transcription takes place, the HIV DNA within the human strand is
also transcribed, producing HIV-derived mRNA.
 This mRNA is then translated into the proteins required to generate more HIV viral particles
by viral protease.
 If this process is left unchecked, the HIV infection will rapidly spread amongst all CD4+
immune cells, eventually exhausting their activity and destroying them.
 These cells are critical to the human immune defenses against a range of other pathogens – their
loss renders the human body vulnerable to a number of cancers, infections and diseases.
 ARV drugs work by inhibiting the various viral enzymes critical to the HIV replication cycle,
specifically reverse transcriptase, integrase and protease.

8. Describe the production method of monoclonal antibody in E. coli.

An elaborate series of manipulations makes it possible to select, as well as produce, functional
monoclonal antibodies in E. coli.
1. cDNA is synthesized from mRNA isolated from mouse antibody producing cells (B lymphocytes).
2. The H and L chain sequences in the cDNA preparation are amplified separately by PCR.
3. Each amplified cDNA preparation is treated with a specific set of restriction endonucleases and
cloned into a bacteriophage λ vector.
The cDNA sequences of the H and L chains each have distinctive restriction endonuclease recognition
sites, an arrangement that facilitates the directional cloning of each sequence into a separate
bacteriophage λ vector.
At this stage of the process, many different H and L chain sequences are cloned.
4. The cDNAs of one H and one L chain are cloned into a single “combinatorial” vector, thereby
enabling the bacteriophage to co-express both chains, thus forming an assembled antibody Fv fragment.
5. The H and L chains are expressed during the lytic cycle of bacteriophage λ, so that the library of
combinatorial bacteriophage clones can be screened for the presence of antigen-binding activity.

Figure: Procedure to create a combinatorial

library of the VL and VH regions of antibody
chains in E. coli. Note that the H and L chains
are amplified in separate PCRs.
Figure: Production of chemically linked mAB.
Schematic representation of a monoclonal antibody-based drug delivery
system. (A) The drug is coupled to a monoclonal antibody. (B) An enzyme
that converts an inactive prodrug to an active drug is attached to a monoclonal
antibody. The active drug is formed only in the immediate vicinity of the
target cells. In both cases, the monoclonal antibody binds to a specific protein
on the surface of the target cell

9. What is the role of DNase-1 against cystic fibrosis? Describe the production of
recombinant DNase-1.
Cystic fibrosis: Cystic fibrosis is one of the most common fatal hereditary diseases. Individuals with
cystic fibrosis are highly susceptible to bacterial infections in their lungs.
The presence of bacteria, some alive and some lysed, contributes to the accumulation of a thick mucus
in the lungs of these patients, making breathing very difficult and acting as a source for further infection.
The thick mucus in the lungs is the result of the combination of the alginate that is secreted by the living
bacteria, the DNA that is released from lysed bacterial cells, and degenerating leukocytes that
accumulate in response to the infection, as well as filamentous actin derived from the cytoskeletons of
damaged epithelial cells.

Production of recombinant DNase-1: To address this problem, scientists at the U.S. biotechnology
company Genentech isolated the gene for the human enzyme deoxyribonuclease I (DNase I) and
subsequently expressed the gene in Chinese hamster ovary (CHO) cells in culture.
DNase I can hydrolyze long polymeric DNA chains into much shorter oligonucleotides. The purified
enzyme was delivered in an aerosol mist to the lungs of patients with cystic fibrosis.
Role of DNase-1 against cystic fibrosis: The DNase I decreased the viscosity and adhesivity of the
mucus in the lungs and made it easier for these patients to breathe.
While this treatment is not a cure for cystic fibrosis, it nevertheless relieves the most severe symptom
of the disease in most patients

10. What is the role of Alginate Lyase against cystic fibrosis? Describe the production of
recombinant Alginate Lyase.
Alginate Lyase: Alginate is a polysaccharide polymer that is produced by a wide range of seaweeds
and both soil and marine bacteria.
Alginate is composed of chains of the sugars β-d-mannuronate and α-L-guluronate.
α-L-guluronate residues form both inter-chain and intra-chain cross-links by binding calcium ions and
the β-d-mannuronate residues bind other metal ions.
The cross-linked alginate polymer forms an elastic gel.
Cause of cystic fibrosis: The excretion of alginate by mucoid strains of Pseudomonas aeruginosa that
infect the lungs of patients with cystic fibrosis significantly contributes to the viscosity of the mucus in
the airways.
Once mucoid strains of P. aeruginosa have become established in the lungs of cystic fibrosis patients,
it is almost impossible to eliminate them by antibiotic treatment.
This is because the bacteria form biofilms in which the alginate prevents the antibiotics from coming
into contact with the bacterial cells.

Role of Alginate Lyase against cystic fibrosis: Alginate lyase efficiently liquefies alginates that are
produced by mucoid strains of P. aeruginosa.
In one experiment, it was shown that the addition of alginate lyase, which can liquefy bacterial alginate,
together with or prior to antibiotic treatment, significantly decreased the number of bacteria found in
biofilms.
This result suggests that, in addition to the DNase I treatment, depolymerization of the alginate would
help clear blocked airways of individuals with cystic fibrosis.

Figure: Schematic representation of the detection of an alginate

lyaseproducing clone from a clone bank of a Flavobacterium sp.
in E. coli. The alginate that is present in the growth medium is
digested by alginase secreted by an E. coli clone. The alginate in
the vicinity of such a colony is not cross-linked when calcium is
added and instead produces a clear zone (halo) surrounding the
colony

In the presence of calcium, all of the alginate in the medium becomes cross-linked and opaque.
Analysis of a cloned DNA fragment from one of the positive colonies revealed an open reading frame
encoding a polypeptide with a molecular mass of approximately 69kDa.
Detailed biochemical and genetic studies indicated that this polypeptide is a precursor of the three
different alginate lyases produced by the Flavobacterium sp.
 Figure: Processing of the
recombinant Flavobacterium
alginate lyase protein precursor
in E. coli. A 6-kDa peptide is
removed from the N terminus
of the 69-kDa precursor to yield
a 63-kDa protein that can
depolymerize alginate from
both seaweed and bacteria. A
second cleavage event converts
the 63-kDa protein into a 23-
kDa protein that is active against seaweed alginate and a 40-kDa protein that hydrolyzes bacterial
alginate.

After the 69kDa precursor is produced, a proteolytic enzyme cleaves off an N-terminal peptide of about
6kDa.
The 63kDa protein can lyse both bacterial and seaweed alginates. Cleavage of the 63kDa protein yields
a 23kDa enzyme that depolymerizes seaweed alginate and a 40kDa enzyme that is effective against
bacterial alginate.
To produce large amounts of the 40kDa enzyme, the DNA corresponding to the enzyme was amplified
by the polymerase chain reaction (PCR) and then inserted into a Bacillus subtilis plasmid vector fused
to a B. subtilis α-amylase leader peptide to direct the secretion of the protein and placed under the
transcriptional control of a penicillinase gene promoter.

Figure: DNA construct encoding the 40,000-Da alginate lyase. The leader peptide from a B. subtilis α-
amylase gene is fused to the N terminus of the alginate lyase coding sequence. The construct is under
the transcriptional control of a B. subtilis penicillinase gene expression system.

Transformation of B. subtilis cells with this construct yielded colonies with large halos on solid medium
containing alginate after calcium was added.
When these transformants were grown in liquid medium, the recombinant alginate lyase was secreted
into the culture broth.

11. Short Note

i. HIV Inhibitor
HIV inhibitor: HIV inhibitors are a group of drugs that inhibit different steps in the HIV replication
process. In this way, they can suppress HIV infection but never entirely eliminate it from the body.
Protease inhibitors are Anti-retroviral (ARV) HIV drug class that block protease (an HIV enzyme). By
blocking protease, these inhibitors prevent HIV from becoming a mature virus that can infect other CD4
cells. The licensed protease inhibitors are:
•amprenavir (Agenerase)
•atazanavir (Reyataz)
•darunavir (Prezista) etc.
Functions: Worldwide, the predominant mode of HIV transmission is by heterosexual contact. One
possible way to protect women, who currently comprise about half of all new cases of HIV/AIDS,
against HIV infection is a topical microbicide, delivered by a live vaginal Lactobacillus strain, that
prevents HIV infection directly at mucosal surfaces.

This strategy seems reasonable because naturally occurring vaginal Lactobacillus strains play a
protective role in preventing urogenital infections.
The compound cyanovirin N, isolated from the cyanobacterium Nostoc ellipsosporum, blocks several
steps of HIV infection, preventing virus entry into human cells.
Consequently, cyanovirin N is a candidate for a topical microbiocide to prevent HIV infections.

Production: To ensure that cyanovirin N would be expressed at a sufficiently high level in a vaginal
strain of Lactobacillus jensenii, the gene was chemically synthesized to reflect the codon usage found
in the bacterium.

Figure: Flowchart of the scheme used to develop a Lactobacillus strain that produces and secretes
cyanovirin N (CV-N).

Typically, the GC content of lactobacilli is about 36%. In addition, during the synthesis of the gene,
proline 51 was replaced by a glycine residue to stabilize the cyanovirin N, and four amino acids were
added to the N terminus to ensure proper cleavage of the signal sequence.

The modified cyanovirin N gene was fused to a strong and constitutive Lactobacillus promoter.
The final construct was integrated into the chromosomal DNA of a strain of L. jensenii and it was found
to be highly effective at preventing HIV infections in mice.
Under these conditions, about 4 μg of cyanovirin N per mL was released into the culture medium.

ii) Interferon
Interferon: Interferons (IFNs) are proteins and released by host cells in response to the presence of
pathogens (i.e., viruses, bacteria, parasites or tumor cells). Interferon was named for its ability to
interfere with viral proliferation. They are important modulator of immune response.
Structurally, they are part of the helical cytokine family which are characterized by an amino acid chain
that is 145-166 amino acids long.
Mechanism of Action: Interferons are small proteins released by macrophages, lymphocytes, and
tissue cells infected with a virus. When a tissue cell is infected by a virus, it releases interferon.
Interferon will diffuse to the surrounding cells. When it binds to receptors on the surface of those
adjacent cells, they begin the production of a protein that prevents the synthesis of viral proteins. This
prevents the spread of the virus throughout the body.
Types: IFNs can be classified into three different groups: IFN-α, IFN-β, and IFN-γ.

 The proteins IFN-α and IFN-β are synthesized in cells that have been exposed to viruses or viral
RNA.
 IFN-γ is synthesized in response to cell growth-stimulating agents.
 Functions:
 Interferon stimulates the infected cells to prevent the virus from replicating within them.
 Interferons inhibit cell division, and prevent the differentiation of infected cells. Further
production of the virus is thereby inhibited.
 Interferons also have immune-regulatory functions—they inhibit B-cell activation, enhance T-
cell activity, and increase the cellular-destruction capability of natural killer cells.
 IFN-γ directly activates other immune cells, such as macrophages and natural killer cells.
 Chapter Name: Single Cell Protein

Question:
1. What is Single cell Protein? Mention the criteria. Give some example. (3)
2. Mention some importance of Single cell protein. (3)
3. What are the disadvantages of single cell protein? (2)
4. Mention some sources of Single Cell protein. (4)
5. What are the substrates are used for production of Single Cell protein. (3)
6. Describe the method for food & feed grade Single cell protein. (3)
7. Describe the process of single cell protein production from high energy sources. (5)
8. Write down the SCP production from carbohydrate sources. (4)
1. What is Single cell Protein? Mention the criteria. Give some example.
Single Cell Protein: The term Single Cell Protein (SCP) refers to the dried microbial cells or total
protein extracted from pure microbial cell culture such as algae, bacteria, filamentous fungi, yeasts etc.,
which can be used as food supplement to humans (Food Grade) or animals (Feed grade).
If SCP is to be used successfully, there are five main criteria to be satisfied:
a) The SCP must be safe to eat.
b) The nutritional value dependent on the amino acid composition must be high.
c) It must be acceptable to the general public.
d) It must have the functionality, i.e. characteristics, which are found in common staple foods.
e) The economic viability of the SCP process.
A list of micro-organisms are used as single cell protein. Some are mentioned below:

Microorganism Example
Algae Spirulina sps.
Chlorella pyrenoidosa
Chondrus crispus
Bacteria Bacillus subtilis
Lactobacillus
Pseudomonas fluroescens

Fungi Aspergillus fumigatus

Aspergillus niger
Rhizopus cyclopium

Yeast Saccharomyces cerevisae

Candida tropicalis
Candida utilis

2. Mention some importance of Single cell protein.

Importance of Single cell protein: Single-cell proteins are the dried cells of microorganism, which are
used as protein supplement in human foods or animal feeds. Microorganisms like algae, fungi, yeast
and bacteria, utilize inexpensive feedstock and wastes as sources of carbon and energy for growth to
produce biomass, protein concentrate or amino acids.
The advantages of single cell protein is given below:
 Microorganisms have a high rate of multiplication to hence rapid succession of generation
(algae: 2-6hours, yeast: 1-3 hours, bacteria: 0.5-2 hours)
 They can be easily genetically modified for varying the amino acid composition.
 A very high protein content 43-85 % in the dry mass.
 They can utilize a broad spectrum of raw materials as carbon sources, which include even waste
products. Thus they help in the removal of pollutants also.
 Strains with high yield and good composition can be selected or produce relatively easily.
Microbial biomass production occurs in continuous cultures and the quality is consistent since
the growth is independent of seasonal and climatic variations.
  Land requirements is low and is ecologically beneficial.
 It is not dependent on climate
 Yeasts grown in this process possess high vitamin content.
 All essential amino acids are contained by single cell proteins.
 Utilization of solar energy.
 Production in continuous culture.

3. What are the disadvantages of single cell protein?

Disadvantages of single cell protein: Despite of the interesting benefits, there are some problems
regarding the Single Cell Protein. As mentioned before, the main problem of SCP production is that
this product may cost more than conventional food product.
There are also some technical problems, such as:

 High content of nucleic acids leading to elevated levels of uric acid.

 Development of kidney stone & gout if consumed in high quality.
 Poor Digestibility.
 Sometimes the microbial biomass when taken as diet supplement may lead to indigestion or
allergic reactions in humans.
 The high nucleic acid content of many types of microbial biomass products is also undesirable
for human consumption as single cell protein. Sometimes this high level of nucleic acid content
in microbial biomass will lead to kidney stone formation or gout.
 The high nucleic acid content of many types of microbial biomass may lead to poor
digestibility, gastrointestinal problem and also some skin reactions in humans.
 The possibility of presence of toxins or carcinogenic compounds may lead to some serious
health problems in humans as well as in animal stock.
 Single cell protein production is a very expensive procedure as it needs high level of sterility
control in the production unit or in the laboratory.
 Hypersensitivity skin reactions.

4. Mention some sources of Single Cell protein.

Sources of Single cell protein: Single cell protein is a protein extracted from cultured algae, yeasts or
bacteria and used as a substitute for protein rich foods, especially in animal feeds or as dietary
supplements. Many types of animal feeds contain single cell proteins. It is convenient to use
microorganisms for production of SCP as they have rapid growth and high protein content.
The main sources are:-
Bacteria as SCP: Purple photosynthetic bacteria e.g. Rhodopseudomonas capsulate, R. palustris are
frequently among the predominant populations of microorganisms in ponds, ditches and other water
sources polluted by sewage or other types of organic matter. The cultivation of photosynthetic bacteria
in municipal and agricultural wastes and attempts have been made to grow photosynthetic bacteria on
effluents of biogas plants. The biomass of photosynthetic bacteria is not only rich in high quality protein
but also contains significantly large amounts of carotenoid pigments, biological cofactors and vitamins.
Fungi as SCP: Many fungal species are good sources of protein, e.g. Candida, Hansenula, and
Saccharomyces. The most widespread and commonly used substrates for SCP production have been
those where the carbon and energy source is derived from carbohydrates. This is due to the fact that
their building blocks (mono and disaccharides) are natural microbial substrates and that carbohydrates
are a renewable resource which is widely distributed. Molasses is a by-product of the sugar
manufacturing process. Besides its high sugar content, molasses contains minerals, organic compounds
and vitamins which are valuable nutrients in the fermentation processes. In fact, about 9% of the dry
matter in yeast grown on molasses has been estimated to originate from substances other than sucrose.
Nevertheless, biomass production from molasses requires supplementation with a suitable nitrogen
source, as well as phosphorus. The traditional nitrogen sources used are ammonia or ammonium salts
and phosphorus can be added in the form of salts.
Algae as SCP: Algae are a source of high value biological molecules such as proteins, polyunsaturated
fatty acids, and pigments. For protein production, there are three species most commonly used with a
higher commercial value: Chlorella, Spirulina (Arthrospira) and Dunaliella, with 55%, 65% and 57%
protein content, respectively. Agricultural facilities and agro-industries have encountered a serious
problem due to the co-products and by-products generated in their operation. The recovery of these
products to produce single-cell protein (SCP) from algae may pose a solution to this problem, because
the composition of agricultural by-products is similar to the ideal medium for the growth of
microorganisms, e.g. the vinasse from ethanol distillation (from beet and cane molasses fermentation)
is a brown liquid, which contains mostly organic matter and a high amount of inorganic salts. Its
composition is similar to that of the culture medium for Spirulina, hence the interest in using this
corrected medium for the production of this algae.

5. What are the substrates are used for production of Single Cell protein.
Substrates used for production of SCP: A variety of substrates are used for SCP production.
Availability of necessary substrates is of considerable biological and economic importance for the
production of SCP.
a) Algae which contain chlorophylls, do not require organic wastes. They use free energy from sunlight
and carbon dioxide from air.
b) Bacteria (except photoautotrophs) and fungi require organic wastes, as they do not contain
chlorophylls.
The major components of substrates are the raw materials which contain:
 sugars (found in sugarcane, sugar beet and their processed products),
 starch (found in grains, tapioca, potato, and their by-products),
 lignocelluloses from woody plants and herbs having residues with nitrogen and phosphorous
contents,
 Other raw materials (whey and refuses from processed food).
c) Organic wastes are also rich in aromatic compounds or hydrocarbons and their derivative products
such as methanol and ethanol can be used as substrates for production of SCP. But recent price-increase
in petroleum and refined petroleum products has made hydrocarbons and their derivative products less
attractive as raw materials for SCP production than renewable sources such as agricultural wastes or
by-products.
6. Describe the method for food & feed grade Single cell protein.
Method for food & feed grade Single cell protein: The name of the raw materials used in SCP
processes represents the main safety hazard. The acceptability of SCP when presented as a human food
does not depend only on its safety and nutritional value.
The effective use of microbial protein for human food requires:

 Liberation of cell proteins by destruction of indigestible cell walls

 Reduction of nucleic acid content
Methods of cell wall destruction: The use of microbes for refined SCP requires not only an adequate
amount of specific organism but also an efficient means of disrupting the cell wall. Mechanical
integration of cell wall can be carried out either by crushing, crumbling, grinding, pressure
homogenization or ultra sonification. Various enzymes or combination of enzymes can be used to digest
and disrupt cell wall, either partially or completely. Enzymatic hydrolysis of cell wall is attractive in
terms of its delicacy and specificity for only the cell wall structure. It may be used as an alternative to
the mechanical disruption, especially for materials that can be inactivated during the mechanical
process. Here are the methods for cell wall disruption:
Non mechanical methods:
1. Chemical treatment: acid, base, solvent, detergent
2. Enzyme analysis: lytic enzymes, phage Infection, autolysis
3. Physical treatment: freeze thaw, osmotic shock, heating and drying
Mechanical methods:
1. High pressure homogenization
2. Wet milling
3. Sonification
4. Pressure extrusion: French press, freeze pressing
5. Decompression (pressure chamber)
6. Treatment with grinding particles
Removal of nucleic acids: Several methods have been proposed to reduce nucleic acid levels in SCP.
These methods involve chemical and enzymatic treatments. Each has disadvantages both in terms of
cost and potential nutritional concern. In 1977, the extraction of nucleic acid by acidified alcohol, salt,
acid and alkalis has been proposed. Alkaline extraction of microbial biomass at elevated temperature
was also used in 1970.However, alkaline hydrolysis of nucleic acid at high temperature causes the
formation of toxic compounds such as lysinoalanine. It has been shown to reduce digestion and induce
kidneys changes in rats. It also implicated in skin allergy in some persons. However chemical
modification of yeast nucleoproteins with anhydrides has been used to reduce the nucleic acid levels.
Alternatively, nuclease has been added exogenously to reduce the nucleic acid content of SCP.
Pancreatic ribonuclease and a fungal ribonuclease of Aspergillus candidus strain M16 has been used as
the source of exogenous nuclease for the reduction of nucleic acid in the cells of yeast species.
Hydrolysis of NA has also been performed by using immobilized enzymes.

7. Describe the process of single cell protein production from high energy sources.
Single cell protein production from high energy sources: SCP production from High Energy
Sources: Materials possessing great commercial significance as sources of energy or derivatives of these
chemicals such as:
  methane,
 methanol
 N-alkanes
These have brought into being broad commercial interest. The microorganisms used for this purpose
are typically bacteria and yeast.
Methane: Methane as a source of SCP has been greatly investigated however is currently believed to
exhibit various technological obstacles to guarantee exploitation. On the contrary, methanol presents
vast SCP economic concern. Fermentation plant on large-scale for production of the methanol-
consuming bacterium which is Methylophilus methtlotrophus was built by ICI, UK.
Methanol: Methanol is used as a carbon source for SCP production as it has numerous inherited
benefits above n-paraffins, methane gas and even carbohydrates composition is not dependent upon
seasonal variation. Potential toxicity sources are not present in methanol as it solubilizes easily in the
aqueous solutions in all quantities.
The price of SCP resulting from methanol is two to five folds the price of fishmeal in the USA. In the
Middle East, methanol low price and higher prices of fishmeal together with a requirement to make
supplementary animal products could bring about SCP a striking suggestion.
N-alkanes: The N-alkanes as source of SCP substrate have been widely examined in several countries
and characterize an exceedingly complicated biotechnological practice. Nevertheless, the majority of
these procedures have currently stopped the operation as a consequence of expected health
vulnerabilities due to the occurrence of cancer causing agents (carcinogens) in the SCP. The immense
technology expanded in this discipline in Japan and other Eastern countries have been directed to the
study of alcohol-derived SCP and organic wastes-derived SCP.
Fermentation Process: A fermentation process is required for producing single cell protein. This is
achieved by multiplying desired strains of microbes on raw materials suitable for growth in industrial
cultivation process heading for the growth of microbial culture, as well as the isolation of resulting cell
mass is achieved by separation techniques.
i. The fermentation methods have need of the following steps as shown:
ii. Purified cultures of the selected organism which should be in the proper physiological state.
iii. Medium for growth is sterilized and is utilized for the microorganism culturing in that medium.
iv. A production fermentation tank, which is the major instrument, employed for managing the
medium used for culturing in stable condition.
v. Isolation of microbial cells from fermented culture medium is done.
vi. Cell free supernatant is collected.
vii. Product is then purified by using purification strategies.
viii. Effluent is treated
 Figure: Flow chart of production of single cell protein by fermentation process

8. Write down the SCP production from carbohydrate sources.

SCP production from carbohydrate sources: There are many agricultural and industrial carbohydrate
used for SCP production. Cellulose from agriculture and forestry sources constitutes the most abundant
renewable resource in the as potential substance for SCP production.
Cellulose in nature: It is usually found with lignin, hemicellulose, starch etc in a complex form. For
utilization of lignocellulose, a pretreatment is usually necessary. Well known cultivated mushroom
Agaricus bisporus are used SCP production from cellulose which are contained lignocellulytic enzymes.
Other microorganism are Vovariella spp., Lentinus edodes and Pleurotus spp. used for SCP production
from cellulose.
A cheaper and more amenable SCP of carbohydrate origin is starch. This very abundant carbohydrate
may be obtained from rice, maize and cereals. In tropical countries, cassava has been proposed as a
good source of starch for SCP proceeds.
1. Milk and Curd: Milk is a completely food but it can also transmit diseases. It is an excellent nutritive
medium for the growth of certain micro-organisms like bacteria, yeast and moulds. These microbes
alter the physical and chemical properties of milk.
2. Butter: Butter is composed of about 80% fat and 10-16% water. At first, cream is separated from
milk and it is then pasteurized to destroy the micro-organisms present in it. Starter of the bacterial
culture is the added which consists of lactic acid bacteria like Streptococcus lactis.
3. Cheese: It is another important item of dairy product. It is concentrated constituent of milk,
principally fat, casein and insoluble salts. To retain these constituents in concentrated form, milk is
coagulated either by fermenting bacteria which produce lactic acid or by the addition of rennet.
Fermentation makes the casein spongy, soft and tasty.
4. Ice Cream: It is made of cream & sugar and contains about 8-14% butter fat. Ice cream also contains
some non-pathogenic bacteria.
[Ref- A Textbook of Biotechnology by Rehana Khan, page-323]
 Chapter Name: Microbial production of enzyme & organic acid

Questions:
1. Why microbial productions become important for enzyme & organic acid industry?-4
2. What is melanin? Describe the production method of melanin.-5
3. What is adhesive protein? Why it is important? Briefly describe the microbial production method of
adhesive protein.-6
4. Why amino acid is commercially important? How tryptophan is produced from microorganisms?-5
5. What is indigo? How do you produce indigo from microorganism?-6
6. Describe the production method of L-ascorbic acid.
7. What is lipase? Write down its application.
8. Write down the production method of lipase.
1. Why microbial productions become important for enzyme & organic acid industry?
Importance of microbial production in enzyme and organic acid industry:
Microbes play a key role in maintaining life on earth, fixing gases and breaking down dead plant &
animal matter into simpler substances that are used at the beginning of the food chain. Microbes are
also economically important because they are used by humans for various purposes. Biotechnology
makes it possible to use microbes such as bacteria and fungi in the manufacturing and services industry.
These include chemical manufacturing such as ethanol, acetone, organic acid, enzymes etc.
Enzymes are commercially used in a variety of industries such as pharmaceuticals, chemical production,
biofuels, food & beverage, consumer products etc. Enzyme industries extensively used microbes for the
production of enzymes. Enzymes produced by microbes are- protease, keratinase, glucose isomerase,
lipase, amylase, xylanase, ligninase, cellulose etc.
Organic acids are chemical compounds widely distributed in nature as normal constituents of plant and
animal tissues. Organic acids constitute a key group among the building block chemicals that can be
produced by microbial process. Major types of organic acid produced by microbial activity are citric
acid, succinic acid, lactic acid, gluconic acid, acetic acid etc.
Before using microbial production process enzyme & organic acid industries used chemical or synthetic
processes to produce organic acids and enzymes. But now microbial productions become more
important in enzyme and organic acid industry day by day. Because microbial production process is the
complete solution for large scale production at a time. Microbial productions have the capacity to
produce desired products from grams to tons. Microbial production is less time consuming, convenient
and less expensive also. That’s why microbial productions become important in enzyme & organic acid
industry.

2. What is melanin? Describe the production method of melanin.

Melanin: Melanin is a large, diverse family of light absorbing biopolymers that are synthesized by
animals, plants, bacteria and fungi.
Biochemically melanins are composed of indoles, benzothiazole and various amino acid. Currently
melanins are obtained in small quantities either by extraction from natural source or by chemical
synthesis.
Use of melanin: Melanin pigment might be used as follows:
a) Tropical sunscreen.
b) Sunlight protective coatings for plastics.
c) Additives for cosmetic products.
Production method of melanin: Melanin can be produced by genetic engineering method.
Recombinant DNA technology has made it possible to produce a range of melanins with different
physical properties, inexpensively & on a large scale.

 The genes involved in melanin biosynthesis in the bacterium Streptomyces antibiotics have
been isolated and analyzed. They are consist of two open reading frame (ORF), one encoding
tyrosinase (MW 30,600) and one namely ORF t438 encoding a protein of unknown function
with a molecular weight of approximately 14,800 MW.
  However, melanin biosynthesis requires the products of both genes. The protein encoded by
ORF 438 may act as a copper donor to apotyrosinase, the inactive precursor from tyrosinase.
Apotyrosinase are activated by acquiring copper ions.
 Both of these genes were subcloned onto an E. coli expression vector pBGC620.3.
Transcription of the cloned gene is under the control of the E. coli bacteriophage T7 promoter
(pT7). RBS1 and RBS2 denote two different ribosome binding sites.
 Under natural conditions after dihydroxy phenylalanine quinone is produced by tyrosinase, a
variety of low molecular weight compounds (non-quinones) can be incorporated into the final
polymer, melanin.

3. What is adhesive protein? Why it is important? Briefly describe the microbial

production method of adhesive protein.
Adhesive protein: Adhesive proteins are biopolymer originally isolated from the blue mussel Mytilus
edulis.
Importance of adhesive protein: Adhesive proteins are exceptionally strong, waterproof that enable
the mussel to attach very tightly to a variety of surfaces.
Microbial production of adhesive protein:
• An intracellular precursor form of the adhesive protein, called the 130-kDa precursor protein is
isolated and analyzed biochemically.
It is found that the 130-kDa precursor protein is rich in serine, threonine, lysine, proline, and tyrosine
and 60 to 70% of the amino acids contain a hydroxyl group.
Most of the proline residues are hydroxylated to either 3- or 4-hydroxyproline (Hyp) and the majority
of the tyrosines are hydroxylated to 3,4-dihydroxyphenylalanine (DOPA).
• The cDNA for the 130-kDa precursor adhesive protein was isolated from a cDNA library that was
constructed with mRNA isolated from the gland that actively secretes the byssal adhesive.
• When either complete or partial cDNAs for the adhesive protein are cloned onto yeast expression
vectors and introduced into yeast cells, active novel forms of the adhesive protein ranging from 20 to
100 kDa are synthesized and represented a significant fraction (2 to 5%) of the total cell protein.
• Considerably higher expression levels are attained when a chemically synthesized adhesive protein
gene sequence is expressed in E. coli.
• In this case, repeating DNA units that encode the consensus decapeptide repeat of the adhesive protein
are used to construct a 600-bp synthetic gene that encoded a protein with a molecular mass of
approximately 25 kDa.
• The synthetic gene is expressed at very high levels by using the T7 promoter.
Thus, high levels of adhesive proteins are produced by microorganism.

4. Why amino acid is commercially important? How tryptophan is produced from

microorganisms?
Commercial importance of amino acids: Amino acids are organic compounds containing amine (-
NH2) and carboxyl (-COOH) functional groups, along with side chain (R group) specific to each amino
acid. Amino acids are used extensively in the food industry as flavor enhancers, antioxidants, and
nutritional supplements; in agriculture as feed additives; in medicine in infusion solutions for
postoperative treatment; and in the chemical industry as starting materials for the manufacture of
polymers and cosmetics.

It is estimated that more than 2.5 million tons of amino acids, worth more than $9 billion, were produced
worldwide in 2008.
L-Glutamic acid, which is used in the manufacture of the flavor enhancer monosodium glutamate,
makes up around half of the total volume. So, amino acids are commercially important.
Production of tryptophan: The important amino acid tryptophan can be produced in Corynebacterium
glutamicum but the rate of production is low, about 0.48 mg/ml. Because in their biosynthesis pathway,
the enzyme anthranilate synthase act as rate limiting enzyme. By inhibition of this enzyme microbe can
inhibit the overproduction of the amino acid tryptophan that is called feedback inhibition process.
To avoid this feedback inhibition for the production of high amount of tryptophan, scientists have
developed a genetic engineering technique.

 The most broad host range plasmid vectors replicate only in gram negative organisms. It is
necessary to construct expression vectors that are specifically suited for gram positive bacteria
Corynebacterium and Brevibacterium spp. Such cloning vehicles might take the form of (E.
coli- Corynebacterium) shuttle vectors.
 The E. coli portion of the plasmid could encode resistance to the antibiotic tetracycline,
chloramphenicol or kanamycin. Because both E. coli & Corynebacterium are susceptible to
these antibiotics. They could be used as selectable marker in both organisms.
 A library of tryptophan producing Brevibacterium flavum chromosomal DNA was cloned into
a Corynebacterium glutamicum that produced no active anthranilic acid synthetase.
 The mutant strain was unable to grow on minimal medium unless anthranilic acid was added.
Therefore transformants were selected by their ability to grow in the absence of anthranilic acid.
 The vector carrying the anthranilic acid synthetase gene was then transferred to a wild type
strain of C. glutamicum.
 Moreover, the effect of adding this gene to wild type Corynebacterium glutamicum was much
more dramatic with the synthesis of tryptophan being increased by approximately 130%. These
levels of over production refer more efficient utilization of available precursor material.

Figure: Simplified pathway and regulation of tryptophan biosynthesis in C. glutamicum.

5. What is indigo? How do you produce indigo from microorganism?
Indigo: Indigo is an organic compound with a distinctive blue color. Historically indigo was a natural
dye extracted from plants Indigofera tinetoria but currently it synthesized chemically. It replaces the
natural process because of cheapness and purity.
Importance of indigo:

 Indigo is a commercially important blue pigment that is used to dye both cotton and wool.
 Indigo, the coloring agent in blue jeans is the largest selling dye in the world.
 Indigo is used very extensively in printing inks.
 Using henna and indigo in hair coloring can results in black, raven black or blue black hair.
 Indigo has been used to reduce inflammation.
 Indigo is used to treat nematode infection and malignancies of the ovaries or stomach.
 Synthetic indigo has been commonly used intravenously or intramuscularly to test kidney.
 Microbial production of indigo: By using genetic engineering process scientists are able to
produce about 13*106 kg of indigo every year by using the bacteria Pseudomonas spp.
The genetic engineering process is given below:

 Pseudomonas spp. contains plasmid NAH7 that encode the enzyme for the degradation of
organic compounds, such as Naphthalene. This enzyme called Naphthalene dioxygenase, which
can convert indole into cis indole 2, 3dihydrodiol.
 NAH7 plasmid can be used in the genetic engineering for indigo biosynthesis. To do so, NAH7
plasmid DNA was digested with HindIII and the fragments (approximately 50-2000 kb) were
ligated with linear HindIII digested plasmid (pBR322).
 This recombinant vector was introduced into E. coli and transformant were selected on the basis
of their resistance to ampicillin and sensitivity to tetracycline.
 The transformants containing the recombinant NAH7 (10.5 kb) insert would convert tryptophan
existing in the minimal growth medium into indigo which is revealed by blue color.
This synthesis was achieved in four steps:
1. Conversion of tryptophan in the growth medium to indole by the enzyme tryptophanase, which
is produced by the E. coli host cell.
2. Oxidation of indole to cis-indole-2, 3-dihydrodiol by naphthalene dioxygenase which is
encoded by the DNA that was cloned from the NAH7 plasmid.
3. Spontaneous elimination of water.
4. Air oxidation to form indigo.
6. Describe the production method of L-ascorbic acid.
Synthesis of L-Ascorbic Acid: L-Ascorbic acid (vitamin C) is currently synthesized commercially
by a process starting with D-glucose that includes one microbial fermentation step and a number of
chemical steps.
The last step in this process is the acid-catalyzed conversion of 2-keto-L-gulonic acid (2KLG) to L-
ascorbic acid.
Biochemical studies of the metabolic pathways of a number of different microorganisms have shown
that it may be possible to synthesize 2-KLG by a different pathway.
For example, some bacteria (Acetobacter, Erwinia) can convert glucose to2, 5-di-keto-Dgluconic acid
(2, 5-DKG) and others (Corynebacterium, Arthrobacter) have the enzyme 2,5-DKG reductase, which
converts 2,5-DKG to 2-KLG.
Microbial production method: The current procedure for synthesizing ascorbic acid could be
improved by producing 2-KLG from glucose by co-fermentation with suitable organisms.
It is done by utilizing a tandem fermentation process in which the two organisms are cultivated in
succession.
The conversion of D-glucose to 2, 5-DKG by Erwinia herbicola includes several enzymatic steps,
whereas the transformation of 2, 5-DKG to 2-KLG by a Corynebacterium sp. requires only one.
Consequently, the simplest strategy for constructing a single organism that is able to convert D-glucose
to 2-KLG is to isolate the 2, 5-DKG reductase gene from the Corynebacterium sp. and express it in E.
herbicola.
The first step in cloning the 2, 5-DKG reductase gene from the Corynebacterium sp. involved purifying
the enzyme and determining the sequence of the first 40 amino acids from the N-terminal end of the
molecule.
On the basis of the known amino acid sequence, two 43-nucleotide DNA hybridization probes were
synthesized.
A Corynebacterium DNA clone bank was screened with these two probes. Any clones that hybridized
with only one of the probes were discarded.
A clone that hybridized with both probes was isolated and then sequenced since it contained the 2, 5-
DKG reductase gene.
Then, the regulatory gene sequences were deleted and replaced with transcriptional and translational
signals that function in E. coli because the regulatory sequences from Corynebacterium spp. are not
efficiently utilized by E. coli.
This construct expressed 2, 5-DKG reductase activity in E. coli and subsequently was sub-cloned onto
a broad-host-range vector which was used to transform E. herbicola since it is able to use E. coli
transcriptional and translational signals.
The transformed Erwinia cells were able to convert D-glucose directly to 2-KLG. The endogenous
Erwinia enzymes converted glucose to 2, 5-DKG and the cloned 2, 5-DKG reductase catalyzed the
conversion of 2,5-DKG to 2-KLG.
Thus, this recombinant organism should be useful as a source of 2-KLG for the production of L-ascorbic
acid.

7. What is lipase? Write down its application.

Lipase: A lipase is any enzyme that catalyzes the hydrolysis of fats (lipids).Lipases are a subclass of
the esterases. Lipases perform essential roles in digestion, transport and processing of dietary lipids
(e.g. triglycerides, fats, oils) in most living organisms. For example, human pancreatic lipase (HPL) is
the main enzyme that breaks down dietary fats in the human digestive system. It converts triglyceride
substrates found in ingested oils to monoglycerides and two fatty acids. Several other types of lipase
activities exist in nature, such as phospholipases and sphingomyelinases.
In other word, Lipase is a type of protein made by your pancreas, an organ located near your stomach.
Lipase helps your body digest fats. It's normal to have a small amount of lipase in your blood. But, a
high level of lipase can mean you have pancreatitis, an inflammation of the pancreas, or another type
of pancreas disease.
Application of lipase: Dairy Industry: Lipases are extensively used in the dairy industry for ---

 The hydrolysis of milk fat.

 The flavor enhancement of cheeses.
 The acceleration of cheese ripening.
 The manufacturing of cheese like products.
 The lipolysis of butterfat and cream.
Detergents: Lipase can be used in the laundry detergents and automatic dish washing machines
detergents. Lipase can reduce the environmental load of detergent products, since they save energy by
enabling a lower wash temperature to be used; allow the content of other, often less desirable, chemicals
in detergents to be reduced; are biodegradable, leaving no harmful residues; have no negative impact
on sewage treatment processes; and, do not present a risk to aquatic life.
Oleochemical Industry: The scope for the application of lipases in the oleochemical industry is
enormous as it saves energy and minimizes thermal degradation during hydrolysis, glycerolysis, and
alcoholysis.
Pharmaceutical Industry:

 The vast variety of synthetic pharmaceuticals and agrochemicals containing one or more chiral
centres, are still being sold as racemates. This is despite the fact that the desired biological
activity resides in one particular enantiomer. A single isomer is preferable to a racemate, but
there are severe technical and/or economic problems with the production of single isomers. The
usefulness of lipases in the preparation of chiral synthons is well recognised.
 Lipases have been found useful as industrial catalysts for the resolution of racemic alcohols.
 Lipases are currently being used by many pharmaceutical companies world-wide for the
preparation of optically active intermediates on a kilo-gramme scale. A number of relatively
small biotechnological companies, such as Enzymatix in the U.K, specialise in bio-
transformations and offer a whole variety of intermediates prepared via lipase mediated
resolution.
Cosmetic Industry:

 Lipases in Cosmetics and perfumery: Lipases have potential application in cosmetics and
perfumeries because it shows activities in surfactants and in aroma41 production. Retinoids
(Vitamin A and derivatives) are of great commercial potential in cosmetics and pharmaceuticals
such as skin care products. Water-soluble retinol derivatives were prepared by catalytic reaction
of immobilized lipase.
 Isopropyl myristate, isopropyl palmitate and 2-ethylhexylpalmitate are used as an emollient in
personal care products such as skin and sun-tan creams, bath oils etc. Immobilised Rhizomucor
meihei lipase can be used as a biocatalyst in the solvent free esterification to produce these. The
use of lipase in place of the conventional acid catalyst gives products of much higher quality,
requiring minimum downstream refining.
 Wax esters (esters of fatty acids and fatty alcohols) have similar applications in personal care
products and are also being manufactured enzymically by using Candida cylindracea lipase in
a batch bioreactor.
Medical applications: Possible medical applications of lipases are under consideration, for example
inhibition of the human enzyme as a method of reducing fatty acid absorption is being investigated as
a possible treatment for obesity.
Lipases as Diagnostic Tools: Lipases are also important drug targets or marker enzymes in the medical
sector. They can be used as diagnostic tools and their presence or increasing levels can indicate certain
infection or disease.
Lipases in Environmental Management: Employment of lipases in bioremediation processes is a new
aspect in lipase biotechnology. The wastes of lipid- processing factories and restaurants can be cleaned
by the help of lipases from different origins.
Lipases in Tea Processing: The quality of black tea is dependent largely on the dehydration,
mechanical breaking, and enzymatic fermentation to which tea shoots are subjected. During
manufacture of black tea, enzymatic breakdown of membrane lipids initiate the formation of volatile
products with characteristic flavor properties, emphasize the importance of lipid in flavor development.
Lipase produced by Rhizomucor miehei enhanced the level of polyunsaturated fatty acid observed by
reduction in total lipid content.
Lipases as Biosensors: A promising new field is the use of microbial lipase as biosensors. Biosensors
can be chemical or electronic in nature. An important analytical use of lipases is determination of lipids
for clinical purpose.

8. Write down the production method of lipase.

Production method of lipase: Lipase is produced by Pseudomonas alcaligenes at such low levels that
it is expensive to use in the cleaning of laundry.
Moreover, it is extremely difficult to overproduce the enzyme in a variety of heterologous hosts
including Bacillus licheniformis, E. coli, Aspergillus niger etc.
This difficulty reflects the requirement for the simultaneous expression of another gene product that is
involved in either the secretion or the stabilization of the bacterial lipase.
To isolate the lipase gene from P. alcaligenes, as well as any other gene whose expression was linked
to lipase gene expression, the enzyme was first purified.
The amino acid sequence of the N terminus was determined, and an oligonucleotide probe that
corresponded to 11 amino acids from this region of the protein was synthesized.
The oligonucleotide probe was used to screen a clone bank of P. alcaligenes DNA.
A clone bank of P. alcaligenes DNA contained the entire lipase gene, from which a portion of the
additional gene was obtained.
The DNA fragment encoding the lipase gene and a portion of the additional gene was used as a
hybridization probe to screen another clone bank, from which the rest of the second gene was isolated.
The two fragments were spliced together, cloned into a broad-host-range expression vector, and
used to transform P. alcaligenes.

The lipase structural gene is called lipA, and the second (helper) gene is called lipB.
When the vector was derived from a low-copy-number plasmid, the lipase activities of the transformants
were four- to five folds greater than that of the wild type, regardless of the presence or absence of the
second gene (lipB).
However, with a high-copy-number plasmid, the lipase activities of the transformants were about 20-
fold greater than that of the wild type in the absence of the lipB gene and approximately 35-fold greater
than that of the wild type in the presence of the lipB gene.
Since the lipase is secreted into the growth medium, very little purification should be necessary before
using it in laundry detergent.