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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5472_Библиотеки_им_академика_М_И_Перельмана.pdf
Antibiotics
185
Microbial Production of Vitamin B
12
Vitamin B
12
was known to the scientific world in the early 1920s, when two American physicians,
Minot and Murphy, demonstrated to the world that they were able to cure pernicious anaemia, a
disorder first described in 1835, with a diet containing whole liver. Their initial discovery led to
further investigations into identifying the so-called extrinsic factor, giving rise to a whole new
scientific research field that has culminated into a number of Nobel Prizes, awarded most notably
to Minot and Murphy (together with Whipple) in 1934 for their discoveries concerning liver
therapy in cases of anaemia, and to Hodgkin in 1964 for her work on X-ray techniques for structure
elucidation of important biochemical substances.
After several years of intensive research, the full chemical synthesis of vitamin B
12
was
achieved by Woodward and Eschenmoser in 1973. This highly complicated synthesis—involving
about 70 synthetic steps—makes any industrial production of vitamin B
12
by chemical methods far
too technically challenging and expensive.
Today, vitamin B
12
is exclusively produced by biosynthetic fermentation processes, using
selected and genetically optimized microorganisms. The species capable of producing vitamin
B
12
belong to the genera of Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus,
Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia,
Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia,
Streptomyces, Streptococcus and Xanthomonas. For the industrial production of cobalamin, it is a
common strategy to use random mutagenesis in order to generate strains that produce vitamin B
12
in
high yields. This is achieved by treating the appropriate microorganisms with mutagenic agents like
UV light, ethylene amine, nitrosomethylurethane or N-methyl-Nv-nitro-N-nitrosoguanidine and
selecting the strains with practical advantages, such as productivity, genetic stability, reasonable
growth rates and resistance to high concentrations of toxic intermediates present in the medium.
Bioproduction
Vitamin B
12
is recovered as a by-product of streptomycin and aureomycin antibiotic fermentations.
A soluble cobalt salt is added to the fermentation reaction as a precursor to yield vitamin B
12
.
Relatively high amounts of this vitamin B
12
accumulate in the fermentation medium at concentrations
that are not toxic to Streptomyces species.
Vitamin B
12
is also produced on large scale by direct fermentation processes. The bacterial
strains of Propionibacterium shermanii or Pseudomonas denitrifi cans are used, nowadays, for
fermentation processes. P. shermanii is grown in anaerobic culture for 3 days at 30°C and in aerobic
culture for 4 days. The fermentation medium (growth medium) used for production contains
glucose, corn steep liquor (a waste product of starch manufacture), ammonia and cobalt chloride.
Ammonia is used in the form of ammonium hydroxide that maintains the pH of the medium at 7.
P. denitrifi cans is grown for 2 days in aerobic culture medium containing sucrose, betaine, glutamic
acid, cobalt chloride, 5,6-dimethylbenzimidazole and salts.
Textbook of Pharmaceutical Biotechnology
186
The vitamin resulted in the fermentation is retained within the cells. The cells are, therefore,
collected by high-speed centrifugation, and vitamin B
12
is recovered by releasing the vitamin from
the cells by treating with acid, heating and cyanide treatment. The vitamin thus released from the
cell is absorbed on ion-exchange resin IRC-50 or charcoal. It is then purified by phenol and water.
The vitamin is finally crystallized from aqueous acetone solutions. The normal yield is 23 mg/L
when Propionibacterium is used.
Tetracycline Production with Sweet Potato Residue by SSF
Tetracyclines are broad-spectrum antibiotics, and the hydrocarbon derivatives of octahydronaph-
thalene with four annelated six-membered rings. Submerged culture was usually used for tetracy-
cline production, and around 5500 different types of antibiotics were reported by 1982. However,
most of them had no economic value due to toxicity or ineffectiveness in clinical use. The biopro-
duction of therapeutically important antibiotic was successfully carried out in Taiwan using sweet
potato as raw material. Sweet potatoes and its residue are abundantly available in Taiwan. In 1985,
the cultivation area of sweet potato was 23,239 hectares and annual production was 3,69,461 tonnes.
Starchy materials have a high productivity per hectare and an excellent rate of fermentation by a
great number of fast growing microorganisms.
Materials and Methods
1. Sweet potato residue: Sweet potato residue purchased from the local market in Taiwan was
screened with 4–16 mesh to remove the dust and large aggregates. It contains 14.0–16.1%
moisture, 2.32% crude protein, 3.6% ash, 18.1% crude fibre and 65.4% carbohydrate.
2. Tested organisms: S. viridifaciens ATCC 11989 is used for tetracycline and chlortetracycline
production, and B. subtilis ATCC 6633 is used for bioassay of antimicrobial activity.
3. Culture media and culture conditions: The tested organism was cultivated in a slant of
inorganic salt starch agar at 26°C. The medium is made up of the soluble starch 10 g,
ammonium sulphate 2.0 g, calcium carbonate 2.0 g, sodium chloride 1 g, potassium hydrogen
phosphate 1.0 g, magnesium sulphate 1.0 g, trace element solution 1 mL (contained ferrous
sulphate 1.0 g, copper sulphate 0.5 g, zinc sulphate 1.0 g and manganese sulphate 0.1 g/L) and
agar 20 g/L. The basal solid medium contains sweet potato residue 100 g, nitrogen source
2.4 g, rice bran or wheat bran or soybean meal 20 g, CaCO
3
1.0 g and NaCl 0.2 g. The medium
is mixed thoroughly with conidia and distilled water, and incubated statically in flask (the
thickness of medium is about 2 cm) at 26°C for 5–7 days by sting once a day.
4. Extraction of antibiotics: After fermentation, the antibiotic is extracted by shaking with
5 times volume of distilled water at room temperature for 5 min.
5. Observation under scanning electron microscope: Morphogenesis of tested organism on the
solid substrate with different moisture contents or different incubation periods is observed
under Hitachi S-550 scanning electron microscope (Hitachi Co., Japan) at 20 KV with gold
metal shadowing.
6. Moisture content: The samples were dried at 60°C under vacuum for 8–12 h, until their weight
remained constant.
Antibiotics
187
7. pH: Initial pH of substrate is determined directly with pH meter, whereas final pH is determined
after diluting with 5 times the volume of distilled water.
8. Water activity: Samples with different moisture contents are placed in a sealed container at
25°C, and water activity is determined by a hygrometer or modified Conway method.
9. Bulk density: The dry weight or wet weight of samples per unit volume of 1 mL is the bulk
density in dry weight or wet weight, respectively.
10. Extraction of tetracycline: The solid substrate is extracted with 5 times the volume of distilled
water. It is noted that 5 min of shaking was enough for tetracycline extraction. Prolonged
shaking treatment could not improve the efficiency of extraction, but made the filtration or
centrifugation troublesome. The effect of extraction volume on the recovery of tetracycline
has been exclusively studied. It is reported that 4–5 times the volume of distilled water is
efficient for tetracycline recovery.
The optimum conditions for tetracycline production from sweet potato residue by SSF using
Streptomyces viridifaciens are moisture content 68–72%, initial pH 5.8–6.0, supplement with
(NH
4
)
2
SO
4
0.5%, CaCO
3
1.8%, NaCl 0.6%, K
2
HPO
4
0.4%, MgCl
2
0.8%, methionine 0.2%, sodium
glutamate 0.4% and incubation at 26°C for 5 days. Each gram of dry substrate produce 2129 pg of
tetracycline.
In case of peanut meal as the nitrogen source, the optimal conditions for tetracycline production
are sweet potato residue supplement with peanut meal 20%, (NH
4
)
2
SO
4
0.5%, CaCO
3
1.8%, NaCl
0.6%, K
2
HPO
4
0.4%, MgCl
2
0.8%, soluble starch 10%, methionine 0.2%, histidine 0.8%, sodium
glutamate 1.6%. Each gram of dry substrate produced 4720 pg of tetracycline.
QUESTIONS
1. Describe screening of soil for organisms producing antibiotics.
2. Draw a diagram of typical fementers.
3. Write a short note on bioreactors, vitamin B
12
antibiotics and antimicrobial spectrum.
4. How will you carry out the screening of soil for organisms producing antibiotics?
5. Write about the production of penicillin.
6. Write about the screening of soil for organisms producing antibiotics.
7. What do you know about antimicrobials spectrum?
8. What are antibiotics? Explain about the various methods of their standardization in brief.
9. Name the organisms and fermentations used in the production of streptomycin.
10. Write about the production of tetracyclines.
11. What is a fermenter? Write the factors involved in the designing of a fermenter.
12. Write about a typical batch fermenter.
13. Write a note on primary screening methods of soil for antibiotic-producing organisms.
14. Given an account of historical developments in discovery of antibiotics.
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INTRODUCTION
Since early 1990s, scientists have attempted to establish the transfer of genes to human somatic
cells as a valid therapy. The importance of gene transfer was highlighted by three trials, involving
participants with haemophilia B and two types of severe combined immunodeficiency X-linked and
adenosine deaminase-deficient participants. The most successful trials of gene transfer have been
unfortunately questioned about their prospects. In the haemophilia B trial, the detection of vector (the
agent that carries genes to cells in participants’ semen) raised concerns about modifications of the
germline. The X-linked severe combined immunodeficiency trial received setback by unexpected
vector-induced leukaemia in two participants. Despite these developments, number of gene transfer
trials approved worldwide has increased since 1989, of which 77% have been conducted in the
United States and UK. Most trials have used virus-based vectors (70%). Several hazards associated
with gene transfer have been verified by clinical experience, and others are predicted on theoretical
grounds. It is, therefore, worth considering whether the risks observed in studies related to human
gene transfer pose any unusual ethical and social challenges, and, if so, what is required to be done
to tackle the same.
Gene transfer or DNA uptake refers to the process that moves specific pieces of deoxyribonucleic
acid (DNA) into cells. DNA carries within its structure the hereditary information that determines
the structures of protein, which is a prime molecule of life. The most important contribution to the
fields of biotechnology and molecular biology has come from genetic engineering. Exogenous
DNA into organisms and their expression have been achieved successfully. Transgenic animals
Genetic Recombination
13
CHAPTER
Introduction
Methods of Gene Transfer in Plants
Horizontal Gene Transfer Technology
Protoplast Fusion
Gene Cloning
Development of Hybridoma by
Monoclonal Antibodies
Some Important Medicines Produced by
Biotechnology
Gene Transfer in Humans
Applications of Gene Transfer
Questions
Textbook of Pharmaceutical Biotechnology
190
and plants can be obtained by the introduction of exogenous DNA into targeted animals and plants
respectively accompanied by the stable expression.
The techniques for the transfer of DNA into organisms differ from organism to organism. Generally,
there are two approaches for DNA transfer. In the first case, the transfer of DNA takes place by natural
method, and in the second case the transfer is by artificial method. In a gene transfer, therapeutic DNA
is combined with a vector (often of viral origin). Vectors can be injected into recipient’s tissue directly
or used to modify cells ex vivo for transplantation to the recipient. The choice of methods of DNA
transfer depends on the target cells in which transformation is to be performed.
It also depends on the objectives of gene manipulation. The transfection may be either stable or
transient. Although, choice of DNA transfer method is very important, the other important steps involved
are: (1) selection of gene, (2) its isolation, (3) preparation of recombinant DNA and (4) selection of
transformed cells. The regeneration of organism with new characteristics is equally important.
METHODS OF GENE TRANSFER IN PLANTS
Gene transfer is a standard technique in biological research, and it has recently gained clinical
significance in the development of human gene therapy and gene marking protocol. The development
of recombinant DNA technology and techniques for transferring recombinant genes into plants
have revolutionized the field of plant science.
Some important features of gene transfer are as follows:
1. Gene transfer technology has facilitated the molecular cloning by tagging genomic sequence of
important genes whose gene products control the normal pattern of growth and differentiation
of plants.
2. It has allowed investigations using reporter genes in the transcription regulation of plant genes.
3. Gene transfer technology has applications in the improvement of plant agriculture productivity.
4. The success story of gene transfer demonstrates the potential use of plant as biotechnological
and chemical factories.
Gene transfer of plants is basically carried out by two methods (Fig. 13. 1).
Vector-Mediated Gene Transfer
This technique uses two types of methods: (1) using Agrobacterium rhizogenes and (2) virus-mediated.
Vector-Free DNA-Mediated (Direct) Gene Transfer
Physical Method
These are the techniques of electroporation, microinjection, macroinjections, biolistics, microprojectile
bombardment, liposome-mediated gene transfers and ultrasonication employed for effective gene
transfer in living cells.
Genetic Recombination
191
Chemical Method
This method employs the following:
1. Using polyethylene glycol (PEG)
2. Calcium phosphate-mediated DNA transfer
3. Transfer of DNA by polycation-dimethyl sulphoxide (DMSO)
4. DNA transfer by DEAE-dextran method
5. DNA transfer by polybrene
6. Gene transfer through peptide
Figure 13.1 Gene transfer in plants.
Gene transfer
Vector-mediated
Vector-free DNA-mediated (direct)
Viruses-mediated
Physical methods
Chemical methods
Polyethylene glycol-mediated
Calcium phosphate-mediated
By polycation-DMSO
By DEAE-D
By polybrene
Through peptide
Electroporation
Microinjection
Macroinjection
Biolistics
Liposome-mediated
Ultrasonification
Using Agrobacterium rhizogenes
Textbook of Pharmaceutical Biotechnology
192
Vector-Mediated Gene Transfer
The term plant gene vector refers to potential vector both for the transfer of genetic information
between plants and the transfer of genetic information from other organisms to plants. Plant gene
vectors being used for transfer of genes are plasmid of Agrobacterium viruses.
Using Vector of Agrobacterium
For many species of family of Leguminosae, the combination of in vitro techniques for transformation
and regeneration has proven difficult. The transgenic plants have been obtained with a selected
group of species and varieties. The A. rhizogenes approach generating composite plants with a
transgenic root system and untransformed wild-type shoot is especially useful for the study of root
nodule formation on legumes.
Classifi cation of Agrobacterium
1. A. tumefaciens-induced (Ti) crown gall disease
2. A. rhizogenes-induced hairy root disease
3. A. radiobacter—an avirulent strain
A. tumefaciens is a gram-negative bacterium. It commonly occurs in soil and causes crown gall disease
in higher plants. It enters the plant through the mechanically wounded tissue site. This infection causes
local proliferation of undifferentiated tissue. The transfer of DNA or xDNA of this plasmid encodes
substance that acts as plant hormone, causing proliferation of tissue seen in the infected plant.
A. tumefaciens has an extraordinary ability to transfer its DNA segment into the plant genome.
This DNA segment is known as T-DNA (Fig 13.2).
Transform into E. coli
Mate ith Agrobacterium
E. coli
T-DNA
T-plasmid
Agrobacterium
Agrobacterium
T-plasmid integrates into Ti plasmid
ecombinant Agrobacterium
nfected plant cell
Figure 13.2 Mechanism of gene transfer by using A. tumefaciens.
Genetic Recombination
193
Ti plasmid has four regions as follows:
1. Region A: comprises of T-DNA and responsible for tumour induction
2. Region B: responsible for replication
3. Region C: responsible for conjugation
4. Region D: responsible for virulence, mutation in the region
When A. tumefaciens is mixed with cells, it inserts the DNA containing the new gene into the plant
chromosomes. Genetically engineered plant cells with the outline resistance gene are able to grow
on antibiotic medium. Whole plants are generated from the single cell. Regenerated whole plant
and its progeny carry the antibiotic resistance trail.
Vector-Free DNA-Mediated (Direct) Gene Transfer
The species and genotype-independent transformation techniques—wherein no natural vector is
involved, but which are linked to the direct delivery of naked DNA to the plant cell—are covered
under this category. This is also referred to as DNA-mediated gene transfer. A general scheme for
the production of transgenic organism using direct DNA delivery method is explained below.
Physical Methods
Electroporation
It is a new technique that transfers genes into plants or animals by using electrical pulses to produce
transient pores in the plasma membrane. The microscopic pores induced in biological membrane by the
application of electric field are known as electropores. These electropores allow the molecules, ions and
water to pass from one side of the membrane to the other. The formation of electropores depends on the
type of cells used and the amplitude and duration of the electric pulse applied to them.
The electric currents may lead to dramatic heating of the cells that can result in the death of
cells. The heating effects are minimized by using relatively high amplitude for a short duration pulse
or by using two very short-duration pulses. The membranes reseal after a short-period. If properly
treated, the cells can then regenerate cell walls, divide to form a callus and finally regenerate
new plants. In mammalian transgenesis, electroporation is an effective technique of introducing
exogenous DNA into embryonic stem (ES) cells.
Advantages
1. It can be applied for a number of cell types.
2. Simultaneously a large number of cells can be treated.
3. The technique is fast and less.
5. High percentage of stable transformants may be produced.
Applications
1. It increases efficiency of transformation or transfection of bacterial cells.
2. It is useful in introduction of exogenous DNA into animal cell lines, plant protoplast, yeast
protoplast and bacterial protoplast.
3. It is possible to study the transient expression of molecular constructs.
Textbook of Pharmaceutical Biotechnology
194
4. Genes encoding selectable marker may be used to introduce genes using electroporation.
5. Electroporation of early embryo may result in the production of transgenic animals.
6. Wheat, maize, tobacco and rice have been stably transformed with frequency up to 1% by this
method.
7. Naked DNA may be used for gene therapy by applying electroporation device on animal cells.
8. Hepatocytes, haematopoietic stem cells, epidermal cells, fibroblast, mouse T and B
lymphocytes can be transformed by this technique.
Microinjection
In microinjection, DNA can be introduced into cells or protoplast with the help of very fine needles
or glass micropipettes with a diameter of 0.5–10 m. Some of the DNA-injected materials may be
taken up by the nucleus. The computerized handling of operations, such as holding pipette, needle,
microscope stage and video technology have improved the efficiency of this technique.
Advantages
1. Mere precise integration of recombinant gene in limited copy number can be obtained.
2. The method is effective in transforming primary cells, as well as, cells in established cultures.
3. The DNA injected is subjected to less-extensive modifications.
4. Frequency of stable integration of DNA is far better as compared to other methods.
Applications
1. The technique is ideally useful for producing transgenic animal quickly.
2. The process is applicable for plant cells, as well as animal cells but more common for animal
cells.
3. This can be applied to inject DNA into plant nuclei.
4. The procedure is important for gene transfer to embryonic cells.
5. The method has been successfully used with cells and protoplast of tobacco, alfalfa, etc.
Limitations
1. Skilled personnel are required.
2. Embryonic cells are preferred for manipulation.
3. This method is useful for protoplasts and not for the walled cells.
4. Knowledge of mating timing and acolyte recovery is essential.
5. The process causes random integration.
6. Rearrangement or deletion of host DNA adjacent to site of integration is common.
7. It is more useful for animal cells.
8. It is a costly technique.
Macroinjection
This method is tried for artificial DNA transfer to cereal plants that show inability to regenerate and
develop into whole plants from cultured cells. Needles used for injecting DNA have their diameter
greater than cell diameter. DNA is injected with conventional syringe into region of plant that