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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5586_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 6.1. A selected list of important biotransformation reactions.
Type of
reaction Example
Reduction Benzaldehyde → benzyl alcohol
nitropentachlorobenzol →
pentachloroaniline
Oxidation Tryptophan → 5 hydroxytryptophan
naphthalene → salicylic acid
Hydrolysis Anhydrotetracycline → tetracycline menthyl
laureate → menthol
Condensation Streptomycin → streptomycin-phosphate Streptomyces griseus
Commonly used microorganism(s)
Saccharomyces cerevisiae,
Streptomyces aureofaciens
Bacillus subtitis,
Corynebacterium spp.
Streptomyces aureofaciens,
Mycobacterium phlei
with the microorganisms involved are listed in table 6.1. The conversion time
required for biotransformation is related to the type of reaction, the substrate
concentration and the micro-organism used. Usually, oxidation, hydrolysis and
dehydration reactions are accomplished in a few hours.
6.3 Sources of biocatalysts and techniques for biotransformation
There are number of techniques available to enhance biotransformation and
bioconversion processes, which may include immobilization techniques, genetic
engineering and the use of biocatalysts that can tolerate organic solvents [1]. The
utilization of enzymes to conduct biotransformation reactions is often challenging,
as it faces difficulties such as:
• Reactant or product toxicity.
• Inhibition.
• High dilution.
• Conformational changes.
• Stability issues.
• pH and temperature dependence.
Nevertheless, biocatalysts are the only source for conducting biotransformation
reactions [2]. Another challenge is its dependence on the substrate; if the selected
substrate is toxic then it can kill the micro-organism and may further delay the
biotransformation reaction. The micro-organism also utilizes the substrate as a
nutrient and energy source, which can affect product recovery. Limitations related
to process time and the choice of biocatalysts makes biotransformation more
complicated in the manufacturing of a small molecule pharmaceutical [3].
Another challenge is our inadequate understanding of complex biological systems
and the need to increase the yield of the desirable product. However, the slow action
(in comparison to chemical transformation) and specific nature of enzymes makes
them more suitable for biotransformation. An extensive range of biological catalysts
is available for biotransformation reactions [4]. These include growing cells, resting
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
cells, killed cells, immobilized cells, cell-free extracts, enzymes and immobilized
enzymes. The most significant sources of biocatalysts and the protocol used for
biotransformation are briefly described below.
6.3.1 Growing cells
Suitable cells are cultured in a suitable medium. As the desired growth of the cells
occurs (6–24 h), a concentrated substrate is supplemented to the culture.
Occasionally, supplementation of emulsifiers (Tween, organic solvents) is required
to solubilize substrates and/or products, e.g. steroid biotransformation. The substrate transformation to the product can be examined by chromatographic procedures. Biotransformation can be stopped when the formation is optimum.
6.3.2 Non-growing cells
Non-growing cells are considered for biotransformation reactions for a number of
reasons, such as:
• A very high concentration of substrate can be used (with high substrate
concentration, growing cells stop their growth).
• The conversion efficiency of substrate to product is high.
• Cells can be washed and used and thus there will be no contaminating
substances.
• Biotransformation can be optimized by creating specific environmental
conditions (pH, temperature, etc). Product isolation and recovery are easy.
6.3.3 Immobilized cells
Biotransformations can be performed constantly by utilizing immobilized cells.
Further, the same cells can be used again and again. Several bioconversion reactions
with single or multistage reactions are in fact performed using immobilized cells, e.g.
large-scale production of l-alanine and malic acid.
6.3.4 Immobilized enzymes
Immobilized enzymes are frequently used in biotransformation, due to a number of
advantages, such as:
• Cell-free enzyme systems in the form of immobilized enzymes.
• There is no transport barrier across the cell membrane for the substrate or
product.
• The isolation and recovery of the product is simpler and easier.
• The desired products are not degraded.
• There is no occurrence of undesirable side reactions.
Several immobilized enzyme systems have been established for biotransformations,
such as glucose isomerase and penicillin acylase.
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6.4 Product recovery in biotransformations
During most biotransformation reactions, the anticipated end-products are extracellular. The product may either be in a soluble or a suspended state. When whole
cells are utilized, they have to be isolated and frequently washed (with water or
organic solvent) as required. The extracted product can be recovered, using common
techniques such as precipitation by salts, extraction with solvents, adsorption to ionexchangers, etc. The volatile products can be recovered by direct distillation from
the medium. A number of biotransformations are reported in the literature. Of these,
only a selected few are significant for large-scale purposes. The major drawbacks
with a number of biotransformations are low yields, expensive processes or very
limited markets. Recently, an increase in the rate of biotransformations and product
recovery was observed in a membrane bioreactor by using direct electric current.
The amount of production of benzoic acid was found to be escalated by 42% when
the current was applied. This type of process has immense potential in increasing
productivity from biotransformations by means of a simultaneous enhancement in
metabolic activity and in situ product recovery [5]. It was also observed that recovery
of 3-methylcatechol produced in a biphasic system (aliphatic alcohol/water bioreactor) was 10–20 fold more than in an aqueous medium. This demonstrates the
effectiveness of double-phase systems for this particular biotransformation [6].
Similarly, the recovery and productivity of vicinal diol was increased by using a
biphasic system in a hollow fiber membrane bioreactor [7]. In 1998, a combined
bioreactor–separator system was developed for simultaneous biotransformation and
recovery of the product by crystallization in an immobilized l-aspartate betadecarboxylase reactor system. An increase in the biotransformation rate and
recovery of the isoflavones genistein and daidzein from industrial antibiotic
fermentations was observed in 2013 [8].
6.5 Application of biotransformation in the production of pharmaceutical products
6.5.1 Biotransformation of steroids
The structure cyclopentanoperhydrophenanthrene is present in all steroids. In
general, steroids are hormones that have an extensive range of therapeutic functions,
e.g. cortisone is extensively employed in the treatment of rheumatoid arthritis and
skin diseases; progesterone and estrogen derivatives are used as contraceptives.
Some derivatives of cortisone (e.g. prednisolone) are more effective in their
therapeutic action. Steroid production at the commercial level is an important
endeavor, which has required the attention of many researchers. Cortisone production once involved 37 reactions and the cost of this product was approximately
$200 g
procedure of 37 reactions was reduced to 11. This also decreased the cost of the
product to just $1 g
shortening of lengthy reactions which can ultimately impact the cost of the final
product.
−1
in 1950. With the help of biotransformation reactions, this lengthy
−1
in 1980. Thus developments in biotransformation allow the
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 6.1. Biotransformation of commercially important steroids.
There are several reactions involved in the production of steroids, microbial
transformation in particular mainly involves oxidation (introduction of hydroxyl
groups, splitting of side chains, production of epoxides, etc), reduction (conversion
of aldehydes or ketones to alcohols, hydration of double bonds), hydrolysis and ester
formation. Steroid production exclusively by biotransformation reactions is not
possible, thus in addition to microbial transformation, chemical reactions are
required. Key steps involved in the biotransformation of steroids are shown in
figure 6.1. There are a number of precursors available to initiate biotransformation
reactions, such as stigma sterol extracted from soybeans or diosgenin isolated from
the roots of the Mexican barbasco plant.
In steroid synthesis, stigma sterol can be chemically converted to progesterone,
which is further subjected to biotransformation to ultimately produce 11 αhydroxyprogesterone through microbial transformation (Rhizopus nigricans).
Cortisol or hydrocortisone, derived from 11 α-hydroxyprogesterone by chemical
reactions, is exposed to a microbe (Corynebacterium simplex) to produce prednisolone. In addition, product derived from cortisol can be subjected to biotransformation by a micro-organism (C. simplex) to synthesize prednisone. Once diosgenin is
used as the precursor compound, substance S can be synthesized by chemical
reactions, which can be further converted to cortisol by biotransformation with the
help of Curvularia lunata. Biotransformation of steroids is generally achieved by
batch fermentation. In addition, several immobilization techniques are emerging in
this area, which may further encourage more efficient biotransformation reactions
by using immobilized cells or enzymes. This is more profitable as the biotransformation is more efficient with a high substrate concentration, short conversion time and
good product recovery. As not all steroids are water soluble, the microbial transformation reactions are always performed in an organic solvent (water-immiscible)
system. However, organic solvents are toxic to microorganisms or enzymes. It is best
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to use a two-phase aqueous system for the biotransformation of steroids. Mibolerone
is a synthetic anabolic and androgenic steroid utilized for the treatment of estrous in
female dogs. Based on a recent report, microbial transformations of mibolerone
with Cunninghamella blakesleeana, Cunninghamella echinulata and Macrophomina
phaseolina ledtoproductionof11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one of
potential use [10]. Similarly, microbial transformation of drospirenone, an oral
contraceptive drug, with Cunninghamella elegans was also reported recently, which
helps in understanding the metabolism of this important drug and provide new
analogs of drospirenone [9].
6.5.1.1 Biotransformation of cholesterol
Recently, many research activities have focussed on the importance of the lymphatic
route in reversing cholesterol transport, as well as the biliary and the non-biliary
pathways for removal of cholesterol from the body, which will help in understanding
and preventing more of the causes of ischemic coronary heart disease [10]. Studies
are also conducted to understand the role of cholesterol in multiple sclerosis
(a progressive, neurodegenerative disease of the central nervous system) by specific
modification in the mRNA and protein expression of key molecules involved in the
maintainance of cholesterol homeostasis in the rat spinal cord. It was observed that
modification in the regulation of cholesterol metabolism at onset can result in the
progression of disease, whereas during the course of the recovery period it may have
beneficial effects, contributing to the regeneration of myelin sheath and restoration
of neuronal function [11]. The importance of cholesterol for normal brain function
was also realized and it is understood that astrocytes produce most of the brain
cholesterol which is important for brain functioning. During this study it was
observed that sterol regulatory element-binding protein (a transcription factor
decreased during diabetes which can ultimately lead to decrease in brain cholesterol
synthesis) mediated cholesterol synthesis in astrocytes plays an important role in
brain function and development [12]. Alteration in the expression of this transcription factor can significantly affect brain cholesterol synthesis which draws a
relationship between diabetes and altered brain function. Similarly, the key
importance of cholesterol in brain physiology and function, changes in cholesterol
homeostasis and levels related to brain disorders and neurodegenerative diseases
have been studied recently. It was observed that intracellular cholesterol pools in the
compartments of mitochondria and endoplasmic reticulum play an important role in
understanding the importance of cholesterol homeostasis disruption in neurodegeneration [13]. The above mentioned reports help in understanding the importance of
cholesterol in our human body and its biotransformation. A number of commercially important steroids (e.g. androstendione, androstadiendione) can be synthesized directly from cholesterol by biotransformation (figure 6.2).
6.5.2 Biotransformation of antibiotics
One of the most important priorities of the pharmaceutical industry is the
production of new antibiotics or modifications of existing ones for more effective
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 6.2. Biotransformation of cholesterol by mycobacteria to commercial products.
treatment against particular diseases or infections. In addition, antibiotics with a
broad spectrum, minimum toxicity, minimal allergic reactions and decreased
resistance are highly preferred [14]. The significant contribution of biotransformation allows the production of various new antibiotics. Based on recent reports
it is well understood that modification in bacterial metabolism, e.g. those related to
dormancy (metabolically inactive spores to survive harsh conditions) or biofilm
formation, control bacterial susceptibility against antibiotics. This is called phenotypic resistance, presenting a connection between bacterial metabolism and antibiotic resistance (table 6.2)[15].
Adoption of new techniques such as miniaturization, nanotechnology, microdosing, chemometrics and high-throughput analysis helps in the exploration of new
molecules. Alternatively, it can be produced by ‘green chemistry’, a novel synthetic
approach resulting in reduced production of waste and the smallest environmental
impact [16]. Since the discovery of penicillin by Sir Alexander Fleming, more than
20 000 types of antibiotic molecules have been produced using microorganisms,
although only few are clinically useful. Reports also confirm a steep decline in the
discovery of new molecules, in particular antibiotics [17]. During the last three
decades, among the 28 new molecules discovered, only three semisynthetic molecules that have experienced chemical modifications have been approved for clinical
use [28]. Even after advancement in synthetic chemistry, biotransformation is
considered as the most cost-effective track to discover new molecules [18]. This
requires a focus towards developing more approaches to produce a substantial
number of molecules from microorganisms. This gives scope for considerable further
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 6.2. Production of new antibiotics.
Organism Antibiotics Reference
White rot fungi (Irpex lacteus, Panus
tigrinus, Dichomitus squalens,
Trametes versicolor and
Pleurotus ostreatus)
Streptomyces davawensis and
Streptomyces cinnabarinus
Ligninolytic fungi (Irpex lacteus,
Dichomitus squalens)
Marine-derived fungus
Paecilomyces spp.
Cystobacter fuscus Myxobacterial antibiotic (cystothiazole A) [20]
Streptomyces venezuelae strain Rosamicin antibiotic into 10,11-
Xylaria longipes Danofloxacin [22]
Spores of Streptomyces griseus Acylation of chloramphenicol [23]
Deoxystreptamine-negative mutant
of Micromonospora purpurea
Biodegradation of fluoroquinolones
(norfloxacin, ofloxacin and ciprofloxacin)
Antibiotics (riboflavin analogs) roseoflavin
and 8-demethyl-8-aminoriboflavin
Flumequine (fluoroquinolone antibiotic) [18]
Tetracycline, minocycline, chlortetracycline,
oxytetracycline, doxycycline
dihydrorosamicin
2,4,6/3,5-pentahydroxycyclohexanone and
2,4/3,5-tetrahydroxycyclohexanone
[16]
[17]
[19]
[21]
[24]
development biotransformation processes in the future [19 ]. However, there are still
various challenges that need to be addressed, such as:
• Determining the relationship between structural changes and time and cost
efficiency.
• Determining the relationship between the biological activity of products and
the optimization of pharmacokinetic/pharmacodynamic properties and
safety.
• The limited number of suppliers involved in large-scale drug production with
good quality practices [30].
6.5.2.1 Direct biotransformation
Various reactions such as acylation and de-acylation, phosphorylation, adenylation
and hydrolysis are involved in direct biotransformation, in particular during
microbial transformation of antibiotics.
6.5.2.1.1 Biotransformation of penicillin G
The earliest extraction of penicillin G from Penicillium chrysogenum and its
conversion into 6-aminopenicillanic acid and phenyl acetic acid was reported in
1992. In this study 6-aminopenicillanic acid was converted into ampicillin when a
liquid membrane carrier system was electro coalescence [20]. Recent study related to
the production of 6-aminopenicillanic acid has revealed production of penicillin G
acylase from immobilized whole cells of Escherichia coli. Later, E. coli was utilized
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 6.3. Biotransformation of penicillin G.
for the biotransformation of penicillin V to 6-aminopenicillanic acid to express a
highly active penicillin V acylase [21]. In association with chemical synthesis,
microbial transformation is usually used for the commercial synthesis of semisynthetic cephalosporins and penicillins. During this conversion, 6-amino-penicillanic acids derived from the enzymatic cleavage of penicillin by penicillin acylase is a
very important reaction (figure 6.3). With the help of enzyme penicillinase
(β-lactamase), penicillin G becomes inactivated by its conversion to benzylpenicilloic
acid.
6.5.2.1.2 Biotransformation of narbomycin
During 1998, hydroxylation of narbomycin was achieved by the pikC (P450
hydroxylase) encoded cytochrome P450 in Streptomyces venezuelae. In this process
hydroxylation of narbomycin to picromycin (by Streptomyces spp.) is performed
with the help of P450 hydroxylase [22].
6.5.2.1.3 Biotransformation of macrolides
The macrolides are prodrugs and semisynthetic derivatives that are used in a variety
of infections. The most important feature of macrolides is that they are frequently
administered with other drugs, thus this may increase chances for pharmacokinetic
interactions. The potential of macrolides to participate in the biotransformation of
some other drugs has been extensively documented, typically with erythromycin and
troleandomycin. Macrolides can induce their own hepatic biotransformation into
nitrosoalkanes. Less active products are derived from de-acylation of macrolide
antibiotics. These products can be used for the further synthesis of more efficient
semisynthetic macrolides [23].
6.5.2.2 Indirect biotransformation
During antibiotic production, biotransformation reactions can often be controlled
by the addition of certain inhibitors or modified substrates to the medium, or in
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
other words the biosynthetic processes of antibiotics occur in a controlled fashion in
indirect biotransformation.
6.5.2.2.1 Biotransformation of actinomycins
A good example of indirect biotransformation is biotransformation of actinomycins
which involves the production of new actinomycins in the presence of 4-methylproline (a proline analog) in the medium by using the micro-organism Streptomyces
parvulus. These actinomycins are more effective in their function as they have 4methylproline in place of proline [24].
6.5.2.2.2 Biotransformation of ribostamycin
Ribostamycin, an intermediate during biosynthesis of neomycin, can be synthesized
in large quantities by employing mutant strains of Streptomyces fradiae. Numerous
other mutant strains of microorganisms have been produced by rDNA technology
for the synthesis of modified antibiotics of aminoglycosides and rifamycins [25].
6.5.3 Biotransformation of arachidonic acid to prostaglandins
Prostaglandins are significant in pharmaceutical and therapeutic application, e.g.
PGE
as a contraceptive, PGG1in the treatment of CHF and PGG2for relieving
1
labor pains. Arachidonic acid (unsaturated fatty acid) is the starting compound for
the biosynthesis of prostaglandins. Several reports based on biotransformation of
arachidonic acid to PGE
, PGE2, PGF1and PGF2by using fungi area are available.
1
It is assumed that prostaglandins with better efficacy will be synthesized by
biotransformations in upcoming years [26].
6.5.4 Biotransformation for the production of ascorbic acid
Another common example of biotransformation includes the combination of
chemical and microbial transformation processes for the commercial production
of ascorbic acid (vitamin C).
6.5.5 Biotransformation of glycerol to dihydroxyacetone
Dihydroxyacetone is often employed in cosmetics and suntan lotions. By the process
of biotransformation certain acetic acid bacteria can convert glycerol to dihydroxyacetone (figure 6.4). Sufficient oxygen supply, a temperature of 26 °C–28 °C and
pH 6.0 are ideal for optimal biotransformation [27].
Figure 6.4. Biotransformation of glycerol to dihydroxyacetone.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
6.5.6 Biotransformation for the production of indigo
By means of microbial transformation, indigo can be produced. This can be
achieved by cloning a single Pseudomonas gene that encodes naphthalene dioxygenase in the creation of E. coli. The related reactions of biotransformation
for the synthesis of indigo are represented in figure 6.5.
6.6 Mechanisms of enzyme action in biotransformation
Biotransformation is primarily a metabolic process occurring in the liver, aiding the
excretion of both exogenous (outside the body) and endogenous (within the body)
substances. Enzymes catalyze reactions altering these substances’ chemical structures, potentially rendering the substrate inactive, active, or even toxic [28]. The
action of enzymes in biotransformation can be broadly categorized into Phase I
(functionalization reactions) and Phase II (conjugation reactions) as per the traditional understanding. This systematic categorization helps understand enzymes’
different roles in modifying substances to facilitate their elimination or utilization
within the body [29].
6.6.1 Enzyme kinetics and biotransformation
Enzyme kinetics is pivotal in understanding how enzymes interact with substrates
and how their catalytic activities are modulated during biotransformation processes.
Figure 6.5. Microbial production of indigo.
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