Biotransformation

1. BIOTRANSFORMATION

2. Biotransformation (regiospecific and steriospecific bioconversion)
is a biological process whereby an organic compound is
modified into reversible product(s). These involves simple,
chemically defined reactions catalyzed by enzymes present in
the cell. Cells (i.e., microbial, plants and animal) provide the
enzymes to catalyze the transformation reactions.
Types of transformations/Bioconversion
•1) Microbial transformation
•2) Plant cell culture transformation
•3) Animal cell culture transformation
Microbial transformation
•When the transformation of the organic compounds is carried
out by microorganism then the process is called as microbial
transformation.
•The microorganisms have got the ability to chemically modify a
wide variety of organic compounds. These microbes during the
bioconversion provide enzymes which act upon and convert the
organic compound into other compounds or modify it.
E.g. production of Vinegar: the oldest and most established
transformation process.

3. Comparison of microbial transformation with others:
Microbial cells are preferred more as compared to
animal cells or plant cells due to the following
reasons:
•Surface-volume ratio: The microorganisms have high
surface-volume ratio as compared to the plant or
animal cell culture.
•Growth Rate: The microorganism have high growth
rate as compared to the plant or animal cell culture
thus the Transformation using the cell culture is a
less time consuming.
•Sterility: Sterility is the major factor that should be
taken care of. In case of plant or animal cell culture it
is difficult to maintain sterility as compared to the
transformation using microorganism.
•Metabolism Rate: The microorganisms possess high
rate of metabolism for the efficient transformation of
the substrate added as compared to the plant or
animal culture.

4. Comparison with chemical synthesis
•Substrate specificity: Only one specific reaction
step is normally catalyzed by an enzyme.
•Site specificity (regiospecificity): If several
functional groups of one type are present in the
molecule, only one specific position may be
affected. It is possible to obtain conversions at
centers that are chemically unreactive.
•Progesterone to 11alpha hydroxyprogesterone by
Rhizopus nigricans
•Stereoselectivity: If a racemic mixture is used as
starting material, only one specific enantiomer is
converted. They are well suited to obtain specific
conformation.
•Ibuprofen(R&S) --------------------Ibuprofen(S) 100%
•Pseudomonas antartica active at(100mg)

5. •Reaction conditions: Enzymatic reactions do not
cause destruction of sensitive substrates due to
the mild conditions of conversion.
•Several reactions can be combined, either in one
fermentation step using an organism with suitable
enzyme systems, or by step-wise conversions
using different microorganisms.
–Conditions are usually mild. The reaction conditions
cause less environmental hazard, as they take place
chiefly in water.
–These reactions proceed at ambient temperature(20
o

-40
o
C) and
•pressure normal in aqueous media.
–The number of process reaction steps are much less. The
total chemical transformation of one steroid to another
may require many steps, and the process may be costly
and provide only low yields because of certain difficult
steps in the process.

6. •Example: Biosynthesis of Androsterone by Chemical
Route.
•The biosynthesis of Androsterones via chemical
route involves several steps as compared to the
steroidal transformation (catalyzed by
microorganism) where the multi step reaction is
reduced to a single step reaction.

7. –Conversion gives mostly high yields
•Tryptophan to 5-hydroxy tryptophan by B.
subtilis (90% yield)
–Combination of two or more reaction can carried
out by the same organism.
–Microbial conversions are much cheaper than
chemical synthesis.
Disadvantages
–Chemical reactions are easier to handle and less
complicated equipment used.
–Tedious
–Specific organism required.
–Selection of organism is a laborious job.
–Some time process is not economical.
–The substrate concentration added bound by
certain limits.

8. Types of microbial transformation/bioconversion
Chemically these transformations can be grouped
under the following categories:
•Oxidation
•Reduction
•Hydrolysis
•Condensation
•Isomerization
•Formation of new C=C double bond
•Introduction of hetero functions
Oxidation reactions are particularly useful in industrial
production. To a lesser extent, isomerization,
reduction, hydrolysis and condensation also have
industrial application.

9. •OXIDATION
–Hydroxylation (100% conversion efficiency)
–Epoxidation ( approx. 25% conversion
efficiency)

10. •Dehydrogenation ( 60% conversion efficiency)
•Oxidation of aliphatic side chains with the formation
of aldehydes, ketones or carboxyl functions ( 80%
conversion efficiency)
•Oxidative splitting of aromatic rings (70% conversion
efficiency)

11. •Oxidation of heterofunctions i.e. amino groups to
nitro groups; formation of N-oxides and sulfoxides
•Oxidative splitting of substituents i.e. oxidative
deamination, N-CH
3
-demethylation, O-CH
3
-
demethylation (100% conversion efficiency)

12. •REDUCTION
–Reduction of carbonyl functions ( 50% conversion
efficiency)
–Reduction of hetero functions, particularly –NO
2-
–Hydrogenation of Carbon-Carbon double bonds

13. •HYDROLYTIC REACTIONS
–Hydration of carbon- carbon double bonds
(55%conversion efficiency )
–Hydrolysis of carboxylic acid esters

14. •Hydrolysis of N- derivatives (conversion efficiency
data unavailable )
•Amination
•Deamination
CH
2C
O
COOH
HO
HO
CH
2C
H
COOH
HO
HO
NH
2
L-Dopa
Dihydroxy phenyl pyruvic acid
Corynbacterium aurantiacum
H
N
CH
2CH
2NH
2
H
N
CH
2CH
2OH
Tyrptamine
Tryptophol
Aspergillus niger

15. •CONDENSATION
–Phosphorylation
–N- Glycosidation (16% conversion efficiency)
O- Glycosidation (60% conversion efficiency)

16. Practical aspects of microbial conversions
1. Organism:
Two requirements should be fulfilled by the MO
•To obtain a particular product the MO should have the
enzyme which brings about the desired transformation.
•Should able to use the starting material as the substrate.
Organism which capable of utilizing substrate grow in
the medium.
Selection of Organism:
Random screening
•The method consists of addition of the substrate to a culture of
a large number of MO and after a given time, the medium is
analyzed for presence of the product.
•If a particular organism brings about the transformation the
organism is selected for further investigation.
Parallel system
•A better chance to find a suitable organism is by searching the
literature for similar type of conversions and testing the
organism or closely ones in the system under study.

17. Enrichment procedure
•Large amount of substrate is added to the soil samples together
with water and additional nutrients. Organism which capable of
utilizing substrate grows in the medium, these organisms are
isolated and used for transformation.
•Mixed cultures
•In some cases it may be advantageous to use a mixture of two
or more MO for conversion of a particular substrate.
Tropine Bacillus alvei & Enterococcus cerevisae Pseudotropine

•Not possible by individual bacteria
2. Sterilization
•Sterility is necessary because contamination can suppress the
desired reaction , induce the formation of faulty conversion
products or cause total substrate breakdown.
•Fermentation media is sterilized by
•Boiling
•Passing steam.
•Autoclaving.
•Synthetic media require shorter sterilization time
•Greater the viscosity of media longer the sterilization time.

18. 3. Aeration and stirring
•For efficient microbial transformation oxygen should
be placed in intimate contact with cellular structure.
Sterile air obtained by passing through sterile filter
under pressure. Aeration is always a problem with
filamentous organisms growth creates Non-
Newtonian broth ( Viscosity) therefore impellers are
used to facilitate aeration Newer methods like Airlift
fermentors, surface attached microorganism
(biofilms) in fixed fluidized bed reactor are now a
days used.
4. Diffusion
•Transformation takes place with in the cell. Hence to
reach the cell solubility of the substrate in medium
and its rate of diffusion are rate limiting step.
Emulsifiers such a Tween or water miscible solvents
with low toxicity (ethanol, acetone, DMF, DMSO) may
help to solublize poorly soluble compounds.

19. 5. Elimination of side reactions
•Side reactions can produce unwanted products and
complicate the process of isolation and can be
prevented by controlling heat or pH of the medium.
6. Product isolation
•The end product of transformation reactions are
usually extra cellular and may occur in either
dissolved form or suspended form. For further
purification, bacteria and yeast are not separated,
were as fungal mycelium is usually removed by
filtration. In all cases the separated cell must be
washed repeatedly with water or organic solvents
since significant amounts of the reaction product can
be adsorbed on to the cells. Depending upon the
solubility of the product recover is performed by
precipitation as calcium salt, by adsorption or ion
exchangers, by extraction with appropriate solvents,
or for volatile substances, by direct distillation from
medium.

20. Theoretical aspects of microbial transformations
2.Conversion of uncommon substrates.
3.Interference with metabolic pathways
4.Interference with biotransformation
5.Interference with biosynthesis.
6.Rate of conversion depends upon the structure of substrate
7.Specificity of enzyme reaction.
Conversion of uncommon substrates.
•A number of theories have been put forward to explain why an
organism should have enzyme system to achieve a
transformation of uncommon substrate.
•The ability to convert certain substrate may be due to presence
of enzyme that commonly functioning by catalyzing the
conversion of structurally related substances.
•An alternative hypothesis, according to which a mutation takes
place somewhere in the ancestry of the organism which
enables offspring to carry out the transformation, As long as
the compound (substrate) is not available the enzyme has no
function and it becomes apparent only in the presence of the
substrate.

21. Interference with metabolic pathways.
•Every substances formed inside the cell can
theoretically becomes a product if further
degradative pathway is inhibited.
•Majority of degradation of organic compounds occur
by oxidation and reduction. This give energy to the
cell needed for biosynthesis of cell constituents. The
right balance between biosynthesis and degradation
is kept by regulation of mechanism.
Interference with biotransformation
•Several methods are used to prevent certain
transformation.
1. The enzyme that is responsible for the conversion is
inhibited selectively.
Eg. Degradation of cholesterol by mycobacterium
species is restricted to side chain only when Ni
2+
or
Co
2+
ions are added to the growth medium.

22. •Aspergillus orchraceus convert progesterone to 11- a-
hydroxyprogesterone and this is further converted to 6-b-11-a-
dihydroxyprogesterone in the presence of Zn
2+
. In the absence of
Zn
2+
only 11-a-hydroxyprogesterone is formed.
HO
Cholesterol
HO
O
Mycobacterium sp
Ni or Co
O
O
O
OHO
O
OHO
OH
Aspergillus
orchraceus
Zn2+
2. Remove the product from the site were further
transformation would occur.

23. •Interference with biosynthesis
The biosynthesis of complex molecules involves enzyme catalyzed
combination of distinct moieties. The addition of one of the
subunits may result in a considerable improvement in the yield
where production of this substance is the limiting factor in the
synthesis.
Eg. Phenyl acetate is added during the production of benzylpenicillin
to improve the yield.
•Rate of conversion depends upon the structure of substrate.
•Methylation rate of 2-demethylmenaquinones depends upon the
length of side chain in the position 3.
• A optimum rate is obtained with n=3
O
O
C
H
2
H
C
C
CH
2
n
2-demethylmenaquinones
O
O
C
H
2
H
C
C
CH
2
n
Microorganism
CH
3
CH
3 CH
3

24. •Specificity of enzyme reaction
Only one specific reaction step is normally catalyzed by an enzyme. An
enzyme from pseudomonas testosterone catalysis only the oxidation
of 3b and 17a hydroxysteroid
Microbial transformation of steroids
•Naturally occurring steroids possess remarkable hormonal
properties which are of therapeutic importance to human well-
being, such as hormones of adrenal cortex (cortisone, cortisol,
corticosterone), the progestational hormone (progesterone), the
androgens or male sex hormones(testosterone,
dihydrotestosterone) and the estrogens or female sex
hormones (estradiol, estrone, etc.).
•Types of steroidal transformation
•Oxidation
–Hydroxylation
–Dehydrogenation.
–Epoxidations
–Oxidation to ketone through hydroxylation
–Ring A Aromatization
–Degradation of steroid nucleus

25. –Oxidation of alcohols to ketone: 3β-OH to 3-keto
(with formation of Δ
5
to Δ
4
)
–Side chain cleavage of steroids
–Decarboxylation of acids
•Reduction
–Double bond
–aldehyde and ketone to alcohol
•Hydrolysis
•Isomerization
•Resolution of racemic mixture
•Other reactions
–Aminations
–Enolization of carbonyl compounds
–Esterification.

26. Hydroxylation
• Hydroxylation involves the substitution of hydroxyl group directly for
the hydrogen at the position, be it a or b, in the steroid with a retention
of configuration. The oxygen atom in the hydroxyl group is derived
form molecular oxygen (gaseous), not from water, and the hydroxyl
group thus formed always retains the stereochemical configuration of
the hydrogen atom that has been replaced.
Example 2: Certain microorganisms can introduce hydroxyl groups at any
of several of the carbon atoms of the steroid molecule.
•Fungi are the most active hydroxylating microorganisms, but
some bacteria particularly the Bacilli, Nocardia and
Streptomyces show fair to good activity.
•The hydroxylation at the 11-position of progesterone was one of
the first hydroxylation described.

27. Dehydrogenation
•Dehydrogenation with the concomitant introduction of a double
bond has been reported for all four rings of the steroid nucleus,
although the introduction of unsaturated bonds in Ring A is the
only reactions of commercial importance.
Example 1:
•In 1955, Charney and co-worker observed that they could
greatly enhance the anti-inflammatory properties of cortisol by
causing the compound to be dehydrogenated at 1
st
position by
Corynebacterium simplex. The resultant product, prednisolone,
was 3-5 times more active than the parent compound and
produced fewer side effects.
cortisol
prednisolone
Corynebacterium simplex

28. Epoxidation
The epoxidation of steroidal double bonds is a rare
example of biological epoxidation. The 9,11-
epoxidation of 9(11)-dehydro-compounds , and the
14, 15-epoxidation of 14(15)-dehydrocompounds ,
using Curvalaria lunata.
CH3
CH3
OCurvalaria lunata

29. Ring A Aromatization
•The microbial aromatization of suitable steroid substrates can
lead to ring A aromatic compounds, particularly the estrogens
which constitutes an important ingredient in oral contraceptives
drugs and play important role in replacement therapy for
menopause treatment
•Cell free extracts of Pseudomonas testosteroni could transform
19-nor-testosterone into estrone with small quantities of
estradiol-17b.
19-nortestosterone Estrone Estradoil-17b

30. Degradation of steroid nucleus
•Side chain degradation of steroids
Selectively removal of the aliphatic side chain with out
further breakdown of the steroidal nucleus. The
breakdown of the side chain to yield C-17 keto
steroids can be done by several organisms as given
below. (Nocardia species)
COOH
+ CH
3-CH
2-COOH
COOH
+ CH
3-COOH
O
C27 C24
C22
C17
+ CH
3-CH
2-COOH

31. Reduction
•Reduction of aldehydes and ketones to alcohols
Hydrolysis
•Hydrolysis of esters- Flavobacterium dehydrogenans
contain a specific enzyme acetolase which
hydrolyses the steroidal acetates
OH
Estradiol
Streptimyces
OAc
OH
Estradiol
Flavobacterium dehydrogenans

32. Esterification
•Usually involve acetylation
O
O
Androstenedione
OAc
O
Testosteron acetate
Sacromyces fragilis

33. •Steroid Ring Degradation
HO
O
O
O
O
O
O
OH
O
HO
O
Cholesterol
Androstenedione
androstadiendione
9a-Hydroxy-
androstadiendione
androstatriendione
Degradationofcholesterolbymycobacteria

34. Microbial transformation of Glycosides
•Reductive Inactivation of Digitoxin by Eubacterium lentum
Cultures
•The anaerobe Eubacterium lentum inactivated the cardiac
glycoside digitoxin by reducing the double bond in the lactone
ring. This conversion was quantitative when the substrate was
incubated at a concentration of 10 mcg/ml.
R= OH Digoxin series
n=3(digitoxose)

35. •On the biotransformation of phenol and
monofluorophenols by the cultured cells of
Eucalyptus perriniana, phenyl and fluorophenyl b -D-
glucosides were isolated after a 1-h incubation.

36. •N- Glycosidation (16% conversion efficiency)
•O- Glycosidation (60% conversion efficiency)

37. •Erythromycin can be inactivated by microorganisms
through phosphorylation. Erythromycin is
glycosylated and inactivated by Streptomyces
vendargensis and that the product of glycosylation is
2'-(0-[D-glucopyranosyl]) erythromycin A (Fig. 1;
structure 2). Such a glycosylation system could
protect macrolide-producing microorganisms during
antibiotic biosynthesis or be a mechanism of
macrolide resistance in pathogens.

38. Microbial transformation of Vitamins

39. Synthesis of Ascorbic Acid
•The starting material: D- glucose (which is converted into D-
sorbitol by electrolytic reduction)
•Next step: conversion of D- sorbitol into L- sorbose (carried out
using strains of Gluconobacter suboxydans or Acetobacter
suboxydans).

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