BIOTRANSFORMATION

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 transformationMicrobial 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.

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.

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)

• 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(20o -40oC) 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.

• 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.

– 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.

Types of microbial transformation/bioconversionChemically these transformations can be grouped

under the following categories:• Oxidation • Reduction• Hydrolysis• Condensation• Isomerization• Formation of new C=C double bond• Introduction of hetero functionsOxidation reactions are particularly useful in industrial

production. To a lesser extent, isomerization, reduction, hydrolysis and condensation also have industrial application.

• OXIDATION– Hydroxylation (100% conversion efficiency)

– Epoxidation ( approx. 25% conversion efficiency)

• 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)

• 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-CH3-demethylation, O-CH3-demethylation (100% conversion efficiency)

• REDUCTION– Reduction of carbonyl functions ( 50% conversion

efficiency)

– Reduction of hetero functions, particularly –NO2

– Hydrogenation of Carbon-Carbon double bonds

• HYDROLYTIC REACTIONS– Hydration of carbon- carbon double bonds

(55%conversion efficiency )

– Hydrolysis of carboxylic acid esters

• Hydrolysis of N- derivatives (conversion efficiency data unavailable )

• Amination

• Deamination

CH2 C

O

COOH

HO

HOCH2 C

HCOOH

HO

HO

NH2

L-DopaDihydroxy phenyl pyruvic acid

Corynbacterium aurantiacum

HN

CH2CH2NH2

HN

CH2CH2OH

TyrptamineTryptophol

Aspergillus niger

• CONDENSATION

– Phosphorylation

– N- Glycosidation (16% conversion efficiency)

O- Glycosidation (60% conversion efficiency)

Practical aspects of microbial conversions1. 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.

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.

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.

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.

Theoretical aspects of microbial transformations1. Conversion of uncommon substrates.2. Interference with metabolic pathways3. Interference with biotransformation4. Interference with biosynthesis.5. Rate of conversion depends upon the structure of substrate6. 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.

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 Ni2+ or Co2+ ions are added to the growth medium.

• Aspergillus orchraceus convert progesterone to 11--hydroxyprogesterone and this is further converted to 6--11--dihydroxyprogesterone in the presence of Zn2+. In the absence of Zn2+ only 11--hydroxyprogesterone is formed.

HO

CholesterolHO

O

Mycobacterium sp

Ni or Co

O

O

O

OHO

O

OHO

OH

Aspergillusorchraceus

Zn2+

2. Remove the product from the site were further transformation would occur.

• 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=3O

OCH2

HC C

CH2

n

2-demethylmenaquinones

O

OCH2

HC C

CH2

n

MicroorganismCH3

CH3 CH3

• Specificity of enzyme reactionOnly one specific reaction step is normally catalyzed by an enzyme. An enzyme from pseudomonas testosterone catalysis only the oxidation of 3 and 17 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

– Oxidation of alcohols to ketone: 3β-OH to 3-keto (with formation of Δ5 to Δ4)

– 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.

Hydroxylation• Hydroxylation involves the substitution of hydroxyl group directly for

the hydrogen at the position, be it or , 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.

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 1st 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

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.

CH3CH3

OCurvalaria lunata

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-17.

19-nortestosterone Estrone Estradoil-17

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

+ CH3-CH2-COOH

COOH

+ CH3-COOH

O

C27 C24 C22 C17

+ CH3-CH2-COOH

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

EstradiolFlavobacterium dehydrogenans

Esterification

• Usually involve acetylation

O

O

Androstenedione

OAc

O

Testosteron acetate

Sacromyces fragilis

• Steroid Ring Degradation

HO

O

O

O

O

O

O

OH

O

HO

O

Cholesterol Androstenedione androstadiendione

9 ydroxy-androstadiendione

androstatriendione

Degradation of cholesterol by mycobacteria

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 seriesn=3(digitoxose)

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

• N- Glycosidation (16% conversion efficiency)

• O- Glycosidation (60% conversion efficiency)

• 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.

Microbial transformation of Vitamins

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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