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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5648_Библиотеки_им_академика_М_И_Перельмана.pdf
242 Pharmaceutical Chemistry
solvent extraction may be performed using butyl, isobutyl and amyl acetate. In recent
times, highly automated and computerized technology has been used for the penicillin
fermentations. With this technology the level of sugar, NH
, CO2, O2, temperature and pH
3
(6.4-6.8) may be controlled for optimal antibiotic production. Previously, penicillin was
produced using the fungus
Penicillium notatum while nowadays it is produced by using
Penicillium chrysogenum with an improved yield using submerged culture technique,
however scientists are still looking for better processes.
Modern production methods
Modern techniques result in low cost and increased production of penicillin. The
commercial production of penicillin is carried out using
P. chrysogenum strains in submerged
culture with constant agitating and aerated stainless steel tanks having 50,000 gallon
capacity. Now, these industrial strains may produce 40 to 50 grams of penicillin/litre of
culture with a 90% recovery yield which was 50 times more efficient than the past.
10.3.2 Cephalosporin
Cephalosporins were developed to overcome the side effects of
penicillin such as allergic problems due to penicillin. Cephalosporins
are bactericidal agents belonging to the E-lactam antibiotics
(containing a dihydrothiazine ring with D-D-aminoadipic acid)
originally derived from the fungus Acremonium, which was previously
known as “Cephalosporium”. Cephalosporins basically kill the bacteria
by inhibiting the synthesis of peptidoglycan in the cell wall of
H
R
2
N
O
O
Cephalosporin
S
N
R
1
OHO
bacteria. Cephalosporins are used in the treatment of numerous bacterial infections, viz.,
skin infections, respiratory tract infections (tonsillitis, bronchitis, pneumonia and strep
throat), urinary tract infections and also effective against Gram-positive and Gramnegative bacteria. They are usually broad-spectrum antibiotics and exhibit less toxicity as
compared to ampicillin.
Cephalosporins can be divided into: cephalosporin-N (CPN), cephalosporin-C (CPC)
(CPN and CPC are related to penicillin) and cephalosporin-P (CPP) (steroid antibiotic
resembles fusidic acid). In 1955, Newton & Abraham isolated an antibacterial agent CPC
by aerobic fermentation of various strains such as Streptomyces clavuligerus, Cephalosporium
acremonium (or A. chrysogenum ATCC 36225). CPC exhibits weak antibacterial activity,
however semi-synthetic side chain modified analogues exhibit more antibacterial activity.
Organisms for Cephalosporin production
During the culture of C. acremonium (a fungus), an antibacterial agent cephalosporin-C was
discovered and this process is still a major choice for the production of CPC. Streptomyces
species, Emericeliopsis species and Paecilomyces species are also used for the production of
cephalosporin. Various C. acremonium mutants (resistance to the S-analogs) are also
developed to improve the production of cephalosporin.
Fermentation 243
Production process of cephalosporin
The fermentation process of the production of cephalosporin is almost similar to penicillin.
Mostly, corn-flour and soya-flour based culture media are used in a continuous feeding
system for the production of cephalosporin. Some other ingredients also used in the
medium include glucose, sucrose, methionine (source of sulphur) and ammonium salts.
This fermentation process is generally carried out between pH 6 and 7 and temperature
ranges between 25 and 28°C. During the production of cephalosporin-C, increased oxygen
consumption has been noted and thus good oxygen supply may have better microbial
growth. Column chromatography and ion exchange resins are mostly used to recover the
cephalosporin-C from the broth culture and it can be precipitated as pure or Zn/
Na/K-salt.
Production of 7-Aminocephalosporanic acid (7-ACA)
All cephalosporins consists of 7-aminocephalosporanic acid as a core structure. Various
typical reactions of fermentation lead to cephalosporin-C which on hydrolysis yield 7-ACA
with several drawbacks. Recently, the synthesis of 7-ACA has been explored using
enzymatic hydrolysis of cephalosporin-C. These conversions undergo enzymatic reactions
in the presence of enzymes, D-amino acid oxidase (isolated from Trigonopsis variabilis) and
glutaryl amidase (isolated from Pseudomonas species), respectively. These enzymes are
efficiently used nowadays for better results.
Cephalosporin C
D-Amino acid oxidase
Glutaryl amidase
7-Aminocephalosporanic acid
Novel E-Lactam technology for the production of 7-ACA
Scientists also succeeded in the production of 7-ACA using fermentation of P. chrysogenum
by the possible genetic manipulations. Adipic acid in presence of P. chrysogenum leads to
adipyl-6-aminopenicillanic acid (structural similarity with penicillin-N) which on addition
with expandase and hydroxylase gene (cefEF), followed by the addition of acetyl-transferase
gene (cefG) results in the production of adipyl-7-ACA (Fig. 10.2). Then adipyl-7-ACA is
treated with the enzymes D-amino acid oxidase (isolated from Pseudomonas diminuta)
followed by the treatment with enzyme Glutaryl-amidase to produce 7-Aminocephalosporanic acid.
Fig. 10.2: Biosynthesis of 7-APA by P. chrysogenum transformants
244 Pharmaceutical Chemistry
10.3.3 Chloromycetin or Chloramphenicol
It is a broad spectrum aromatic compound which exhibits strong
antibiotic action especially against Gram-positive and Gramnegative bacteria. Many side effects are associated with the
administration of chloramphenicol, however major side effect is
bone marrow damage. Chloramphenicol is used as a reserve
antibiotic and selectively binds to 50S ribosomal subunit and
HO
Cl2HC
OH
NH
O
Chloramphenicol
NO
2
ultimately blocks the protein biosynthesis. Chloramphenicol can be produced by natural
sources such as Streptomyces omiyanesis and S. venezuelae.
Single-stage unit continuous fermentation is used for chloramphenicol production,
because the batch fermentation may lead to unwanted antibiotic product. Sometimes it
produces very less amount of amine after the reduction of nitro group at aromatic ring,
which can be improved calorimetrically by Ratton-Marshall diazotization procedure. The
culture of S. ienezuelae 05072 strain can be used to produce ~400 Pg/mL chloramphenicol.
Batch-fermentation of chloramphenicol
A modified chloramphenicol medium (with 1 mL of General-Electric No. 60 silicone
emulsion defoamer) is used in the fermenter after a series of batch fermentations with a
proper air supply (with rate of 2 volumes/min), however temperature was regulated at
28°C. After four days, the maximum yield is obtained without any contamination.
Continuous-fermentation of chloramphenicol
This process starts in the same manner as batch fermentation does and is then followed by
the culture progress for additional 24 hours with continuous feeding. Three confirmatory
experiments with the same culture dilution usually decreased the yield but later it was
maintained between 80-110 pg/mL. The yield fluctuation observed during continuous
fermentations might be possible due to the operational deviation. After 18 days of
experiment the appearance of contamination was observed which may affect the total
yield and thus, to save further contamination the process may be terminated after 20 days.
Drop in the yield of chloramphenicol production might be due to strain degeneration or
unwanted organisms. Isolation of unwanted organisms can be between 20 days at the start
of the continuous operation and batches.
10.3.4 Streptomycin
Streptomycin is a perfect example of aminoglycoside
antibiotics which is produced by Actinomyces sp. Some
hybrid aminoglycosides have been produced from the
organism by recombinant DNA techniques. They are
majorly responsible for increasing the fermentation
yield. Some examples are as follows (Table 10.1):
HO
HO
NHMe
O
HO
OH
H2N
HN
O
O
OH
H
N
O
O
Me
Streptomycin
HO
OH
H
N
HN
NH
2
Fermentation 245
Table 10.1: Examples of aminoglycosides and organisms responsible for their production
Aminoglycoside Organism
Streptomycin Streptomyces griseus
Neomycin B and C S. iradiae
Kanamycin A, B, and C S. kanamyceticus
Hygromycin B S. hygroscopicus
Gentamicin Micromonospora purpurea
Sisimicin M. inyoensis
Biosynthesis of streptomycin
Biosynthesis of streptomycin is carried by many enzymatic reactions which include >30
enzymatic steps. Glucose-6-phosphate (G6P) synthesized from glucose takes three different
routes to produce streptidine-6-phosphate-L-dehydrostreptose and N-methyl glucosamine,
respectively. Later streptidine-6-phosphate-L-dehydrostreptose and N-methyl glucosamine
are condensing to form an intermediate called dihydro-streptomycin-6-phosphate which
finally converts into streptomycin after a couple of reactions. The biosynthetic pathway of
streptomycin is mentioned in below (Fig. 10.3).
D-Glucose
D-Glucose-6-phosphate
myo-Inositol-1-phosphate D-Glucose-1-phosphate
myo-Inositol
Streptidine-6-phosphate
4-(O-Dihydrostreptosyl)-
streptidine-6-phosphate
Fig. 10.3: Streptomycin Biosynthesis
L-Dehydo-
streptose-1-dTDP
Dihydrostreptomycin-
6-phosphate
Streptomycin-6-phosphate
Streptomycin
D-Glucosamine-
6-phosphate
UDP- -methyl-D-N
glucosamine-6-
phosphate
-Methyl-L-glucosamine
N
Production process of streptomycin
Generally, soya flour or corn flour is used as a medium for the production of streptomycin
because they usually supply glucose at a slow rate (Streptomyces species have low amylase
246 Pharmaceutical Chemistry
activity) and should have optimal conditions (viz., pH between 6.5–7.5, temperature
between 27–30°C and low aeration rate) for fermentation. The duration of fermentation
process depends on the days (6-8) and the strain used in it. Soya meal also helps in producing
initial supply of nitrogen and phosphate in the medium. Thus, the high amount of production
of NH
and phosphates from glucose usually inhibit the streptomycin synthesis.
3
10.4 PRODUCTION OF LYSINE
It is an important essential amino acid or vital nutrient and majorly
isolated using microbial processes. Plants have very low
concentration of lysine proteins while the addition of lysine can
therefore increase the quality of plant foods. It can be produced by
H2N
Lysine
NH
O
O
3
a variety of microbial approaches as follows.
(a) Direct fermentation – production of strains: Glutamic acid producing mutants of
Brevibacterium and Corynebacterium (viz., homoserine auxotrophs or among
methionine-threonine double auxotrophs) are the major producers of lysine. The
most important lysine-secreting strains are Corynebacterium glutamicum,
Brevibacterium flavutn, B. lactofermentum, etc. Protoplast fusion between high-yielding
strains and wild strains of B. lactofermentum, Corynebacterium and Brevibacterium
mutants has led to strains with improved growth properties or higher efficiencies.
Cloning studies with
Escherichia coli, using plasmid pBR322 as a vector, have shown
that only in transformed strains that contain dap-A gene does a significant increase
in lysine production occurs. The enzyme dap-A, dihydrodipicolinate synthase
(DDPS) is therefore indicated as a rate-limiting step for lysine biosynthesis.
(b) Biosynthesis: For the synthesis of lysine there are two pathways, viz., Diaminopimelic
acid pathway or the aminoadipic acid pathway. However, in any single organism,
only one of the two alternatives is used: Bacteria, Actinomycetes, Cyanobacteria, some
Phycomycetes, all Ascomycetes and Basidiomycetes are used by aminoadipic pathway.
The two organisms may use the same pathway while the mechanism could be
different.
(i) In Escherichia coli, three distinct regulatory processes are involved.
7ZRLVRHQ]\PHVRIKRPRVHULQHGHK\GURJHQDVHH[LVW ZKLFK DUHUHSUHVVHG
by L-Methionine or L-Threonine.
7KUHH LVRHQ]\PHV RI DVSDUWLRNLQDVH H[LVW RQH VKRZLQJ UHSUHVVLRQ E\
L-methionine, the second showing multivalent repression by L-Isoleucine
and L-Threonine in addition to feedback inhibition by L-Threonine, and the
third showing feedback inhibition and repression by L-Lysine.
'LK\GURSLFROLQDWHV\QWKDVHWKHILUVWVSHFLILFHQ]\PHRIO\VLQHELRV\QWKHVLV
shows feedback inhibition due to L-Lysine.
For all three of these enzymatic reactions, regulatory mechanisms must be eliminated
to obtain the overproduction of L-lysine which is necessary for its commercial
Fermentation 247
preparation as well. In contrast to E. coli, the regulatory mechanism for lysine
producing strains, such as C. glutamicum or B. flavum is much simpler. There is only
one aspartokinase and one homoserine dehydrogenase. Aspartokinase is regulated
via multivalent feedback inhibition from L-Threonine and L-Lysine.
(ii) Other methods for the production of Lysine
/\VLQH SURGXFWLRQ YLD GLDPLQRSLPHOLF DFLG Lysine-histidine double
auxotrophic mutants of E. coli (ATCC 13002) produce diaminopimelic acid
(DAP) on a molasses medium with 19–24 g/L yield. The entire fermentation
solution, including the cell material, is subsequently incubated with Aerobacter
aerogenes (ATCC-12409 strain) at 35°C. After 20 hours, DAP has been
quantitatively decarboxylated to L-Lysine, the LL-DAP must be transformed
into the meso form by racemization before the decarboxylation step.
&RQYHUVLRQ RI '/D-amino caprolactam: DL-Amino caprolactam solution
(10% and pH 8.0) is added to acetone dried cells of Achromobacter obae and
Cryptococcus laurentii (0.1% w/v). After 24 hours, DL-amino caprolactam is
converted into L-Lysine at 40°C (yield 99.8%).
Hydrolysis with
Cryptococcus laurentii
L-Amino caprolactum
hydrolase
L-Lysine
D- -Aminoa
caprolactum
Racemization with
Achromobacter abae
D-Aminocaprolactum
racemase
L- -Aminoa
caprolactum
10.5 PRODUCTION OF GLUTAMIC ACID
L-glutamic acid is found abundantly in nature and fermentation
technique is widely used in the production of amino acid. Glutamic
acid was discovered by KHL Ritthausen (1866). Hlasiwetz &
Habermann (1873) used HCl instead of H
as hydrolyzing agent
2SO4
for proteins to obtain HCl salt of L-glutamic acid but solubility was
the issue. Ikeda (1908) reported that monosodium glutamate (glutamic acid derivative) is
responsible for enhancing the taste of sea weed soup, thus it can be used as flavour
enhancing agent. Kinoshita (1963) & Asai (1959) also isolated starin Micrococcus glutamicus
from soil which was able to accumulate glutamic acid. During fermentation organism
consumes glucose (30%) and converts glucose-ammonium medium to glutamic acid.
Before 1957, glutamic acid was generated by hydrolysis of plant proteins (viz., gluten) but
later microbial fermentation was accepted by all and the search began for new and effective
stimulator microorganisms. Kinoshita et al. (1957) discovered glutamic acid producing
bacterium M. glutamicus or Corynebacterium glutamicum and they produced L-glutamic
acid (30 g/L) in glucose medium. Other organisms to produce glutamic acid are C. callunae,
C. lilium, Brevibacterium devaricatum, Br. Lactofermentum, Br. immariophilium, Br.
saccharolyticum, Br. flayum, Br. Roseum and Bacillus megatherium.
O
HO
Glutamic acid
NH
O
OH
2
248 Pharmaceutical Chemistry
Hydrolysis of protein constituents
(a) Acid hydrolysis of protein-rich constituents: Glutamic acid containing protein
raw materials (viz., gluten, soyabean cake or casein) are mostly used in this process.
In this process, raw material is hydrolysed using con. HCl for 5-6 hours at 110°C
using corrosion-resistant kettle. After completion of hydrolysis, humus (developed
during reaction) is removed by filtration followed by the evaporation of filtrate
under vacuo resulting in crude liquid (cooled at –5°C for several hours). After
cooling the liquid, a solid precipitate of HCl salt of L-glutamic acid was obtained
and finally crystallized using hot water. This process is performed in iron machines
thus iron may be present in trace amount and it can be removed using hot water
recrystallization technique. The colour due to excess amount of iron can be
decolourized using Na
S and activated charcoal treatment. Thus, crystalline
2
glutamic acid may be obtained by adjusting the pH of the solution to its isoelectric
point (pH 3.2) and finally separates from the solution (Fig. 10.4).
Fig. 10.4: Hydrolysis of raw protein material
(b) Alkaline hydrolysis of protein-rich components: Usually, Steffen’s molasses
(isolated from beet sugar plant) is used as the starting material for alkaline
hydrolysis. The Steffen’s molasses is first saturated with CO
, and thus various
2
solid impurities can be removed by filtration, followed by the evaporation of filtrate
Fermentation 249
thus resulting in crude precipitate. Some remaining impurities of the precipitates
can be removed by sedimentation and filtration followed by evaporation. The
resulting crude liquid was then hydrolyzed by alkali, maintaining the pH at 5.8 and
then the mixture was cooled down below 0°C resulting in crude crystalline glutamic
acid. Later this crude product was further purified by decolourization and filtration
(Fig. 10.5).
Fig. 10.5: Alkaline hydrolysis of Steffen’s molasses
Usually, glucose, molasses, starch or a mixture of these substances can be used as a
carbon source for the culture medium of fermentation for the production of glutamic
acid. Some other chemicals are also present in the culture for nitrogen source, viz.,
urea, etc. Thus, the prepared culture medium is sterilized by steam in a fermenter.
250 Pharmaceutical Chemistry
Later the mixture was cooled down up to 30°C and then microorganism (viz.,
Micrococcus glutamicus) was added and incubated for additional 36-48 hours. During
incubation the temperature, pH and aeration rate were carefully controlled. After
completion of fermentation, the fermentation broth was hydrolyzed using HCl. The
hydrolysed mixture also contains impurities which can be removed and purified
using crystallization, recrystallization, decolourization, filtration techniques and
finally neutralization resulting in the pure crystalline glutamic acid (Fig. 10.6).
Microbiological fermentation
HCl
Starch Molasses
Air
Culture seed Chemicals Urea
Hydrolysis Filtration
Sedimentation
Neutralization
Sterilization
Sterilization
Treated main
culture medium
Humus
fermentation
Sterilization
Fermenter
Fermentation
Broth
Concentration (vacuo)
Acid hydrolysis
Filtration
Concentration
Cooling & crystallization
Seed
Sterilization
pH control
Sterilization
Separation
Glutamic acid .HCl
Removal of Iron
Decolourization
Neutralization pH 3.2
Fig. 10.6: Fermentative method of glutamic acid production
Glutamic acid .HCl
mother liquor
Separation
Glutamic acid
10.6 PRODUCTION OF VITAMIN B2 (RIBOFLAVIN)
Fermentation 251
HN
O
N
Me
In 1920, riboflavin was discovered and in 1933 it was first isolated
from the egg albumen, however in 1935 its structure was
identified. It consists of flavin and ribose sugar units known as
lumichrome. It is an essential constituent of the human nutrition
and animal feed additive. The deficiency symptoms are hair loss,
vision deterioration, growth failure, and inflammation of skin in
humans. Recent studies revealed that it can be used in the
treatment of malaria and migraine. The commercial production
of riboflavin can be done using fermentation or by a combination
of fermentation.
O
N
N
H
CH2OH
Vitamin B2
Me
H
OHH
OHH
OHH
There are two Ascomycete fungi which are the major source of riboflavin named,
Eremothecium ashbyii and Ashbya gossypii, infect yeasts (viz., Candida sp.) and bacteria (viz.,
Clostridium sp.) are also good producers of riboflavin. A multiple times (~40,000 times) of
riboflavin can be produced by Ashbya gossypii. Candida and Clostridia can also produce less
amount of riboflavin. Ferrous ion usually responsible to inhibit riboflavin production or
inhibit enzymatic pathway (especially guanosine 5’-triphosphate (GTP) cyclohydrolase II)
results in less amount of riboflavin.
Production of riboflavin via fermentation
Submerged culture is used for the production of riboflavin via fermentation. There are
many factors which can affect the fermentative production of riboflavin such as carbon
source, minerals, microbial strain, temperature and pH. Now scientists are able to optimize
these factors to obtain riboflavin with a maximum yield.
Fermentative production of riboflavin from Ashbya gossypii
Various carbon sources such as glucose, corn steep liquor, palm oil, whey and molasses
have been tried for the fermentative production of riboflavin. Stahmann et al. used culture
broth of A. gossypii with an excess of carbon sources (molasses or oil containing plants) in
a sterile aerobic submerged fermentation for the production of riboflavin that led to a
15 g/L yield. Ertrk et al. reported that the production of riboflavin from culture broth of
A. gossypii in whey and the overall yield varies with the whey contents used in culture
broth, such as soybean oil (17.5 mg/L), peptone (23.2 mg/L), yeast extract (68.4 mg/L),
Glycine (78.3 mg/L), sucrose (87.5 mg/L), glycine + peptone (120 mg/L) and bran (389.5
mg/L). Agro-industrial by-products such as beet molasses, grape must and peanut seed
cake are also used for the fermentative production of riboflavin. The fermentation
production of riboflavin from a culture broth of A. gossypii with 40% palm oil (from earth
waste) for 10 days results in 2.1 g/L which is ~1.5 times higher yield than the pure palm
oil culture broth. Organic wastes broth culture of fish, beef extract, blood meal and hog
casings with E. ashbyii NRRL-1363 strain is also used for the production of Riboflavin.
Thus, overall optimizations for optimum yield depend on the culture broth that must
contain C-source at 50 g/L (dextrose equivalents) of molasses and N-source at 50 g/L of
peanut seed cake.