By : Radwa Ahmed Taha

Definition of microbial biotransformation

Examples of industrially relevant biotransformation andbiocatalytic processes

Advantages of microbial biotransformation of bioactive compounds

It is the specific modification of a definite compound to a distinct product with structural similarity, by the use of biological catalysts including microorganisms like fungi or bacteria.

The biological catalyst can be described as an enzyme, or a whole, dead microorganism that contains an enzyme or several enzymes produced in it.

Biotransformation is also known to comply with the green chemistry strategy today.

Green chemistry is a term used for sustainable chemical industrial manufacturing processes towards achieving minimal waste production and energy consumption

year process

5000 BC Vinegar production

800 BC Casein hydrolysis with chymosin for cheese production

1670 Orlean process for the industrial bio-oxidation of ethanol to acetic acid

1934 Regioselective biooxidation of sorbite to sorbose for Reichstein Vitamin Csynthesis

1950 Bioconversion of steroids

1970 Hydrolysis of penicillin to 6-aminopenicillanic acid

1974 Glucose to fructose isomerisation with immobilized glucose isomerase

1990 Hydrolysis by protease (trypsin) of porcine insuline to human insulin

1995 biotransformation of nicotinonitrile to nicotinamide

1. The process of microbial transformation can operate at near neutral pH, ambient temperatures and atmospheric pressures, while chemistry often requires extremes of these conditions which are not exactly environmentally friendly and industrially undesired

2. extreme pH, temperature and pressure may provide harmful effects towards personnel operating the harsh procedures and may also affect community surrounding the areas.

3. Regiospecificity and stereospecificity of the microbial biotransformation process which allows the production of chiral products of racemic mixtures.

4. the ability of microorganisms like fungi and bacteria produce large amounts of biomass and a great variety of different enzymes in a short time. Their small size has by far the largest surface-to-volume ratio ever living on the earth

5. Concerns regarding transmission of animal based-diseases such as bovine spongiform encephalopathy (BSE), scrapie , Kuru and Creutzfeld-Jacob syndrome

ex : growth hormone purified from cadaver pituitaries for dwarfism is now produced by a recombinant Escherichia coli

6. Several individual reactions can be combined by single microbial reaction

7. Sometimes it is cheaper to use a microorganism to prepare an organic compound than to synthesize it chemically

8. Production of novel metabolites

Importance of mammalian models

General strategy for use of microbial models

Biochemical basics for microorganisms as models for drug metabolism studies

Modes of bioconversion

Advantages of microbial systems as models for drug metabolism

Microbial model of steroid metabolism

Microbial oxidative metabolism of diclofenac

Biotransformation of celecoxib using microbial cultures

Microbial biotransformation systems can be used to complement mammalian drug metabolism studies

There is now increased availability of genetically engineered microorganisms expressing human drug-metabolising enzymes

The use of microorganisms as metabolite factories is a useful approach for biosynthesis of regioselective and stereospecific products instead of difficult chemical reactions

1. There are difficulties in carrying out metabolic studies in mammals because of small amount of metabolites obtained by these systems.

2. Such small amounts of mammalian metabolites do not allow full structural elucidation or biological evaluation.

3. it is not possible detect highly nascent intermediate

General strategy for use of microbial models-Suitable microorganism and culture conditions are determined. -A two-stage fermentation protocol used for study

1. Cytochrome P-450 monooxygenases in mammals:

Oxidative biotransformation in mammals are mostly achieved by Cyt-P 450 linked monooxygenases.

It mediates aromatic and aliphatic hydroxylations, N,O,S, dealkylations, S, N, oxidations ….. etc.

Monoxygenase enzymes of fungi are similar to those of mammals.

2. Several fungi displayed NIH shift during aromatic hydroxylation similar to hepatic microsomes with the following differences:

a-Ortho in fungi ------- Vs para in hepatic microsomes

b-Polar substrates are better utilized by fungi while non polar substrates are better utilized by hepatic microsomes.

3. These similarities are considered as support for microbial models of mammalian metabolism which can be defined as use of microorganisms to facilitate study of drug metabolism by mammals.

1. Prospective: carried out in microorganisms first and then extrapolated to mammals.

2. Retrospective: carried out in animals first and then in microorganisms.

1. Maintenance of stock cultures is simple and cheap

2. Screening requires large number of strains to metabolize the drug and is a simple repetitive process, requiring only a periodical sampling of incubation media.

3. metabolic capabilities of microorganisms can be high, requiring the use of higher concentrations of the drug. This allows easier detection, isolation and structural identification

4. Novel metabolites can be isolated with new or different activities.

5. There is a possibility of predicting the most favored metabolic reactions.

6. The models can be scaled up easily for the preparation of metabolites for pharmacological and toxicological evaluation.

7. These models can be utilized in the synthetic reactions where tedious steps are involved.

8. The models can be useful in cases where regio- and stereo-specificity is involved.

9. In most cases relatively mild incubation conditions are used.

production

of 4’-hydroxydiclofenac using Epiccocum nigrum IMI354292

The study aimed to use microbial fermentatios to generate the 4’-hydroxylated metabolite of diclofenac, a major metabolite of this drug in man

An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselectiveconversion of diclofenac to its 4’-hydroxylated metabolite.

the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism. 2 g diclofenac was added to 30 L fermenter

Mammalian phase I metabolism of diclofenac

4’- hydroxydiclofenac was found to be the predominant metabolite produced by the organism investigated as well as in man according to in vivo studies.

Celecoxib , a non-steroidal anti-inflammatory drug (NSAID), is the first

specific cyclooxygenase-2 (COX-2) inhibitor approved by FDA for the treatment of osteoarthritis, rheumatoid arthritis, and familial adenomatouspolyposis.

Celecoxib is extensively metabolized by the CYP2C9 isozyme in humans and rats to produce the major metabolites by methyl hydroxylation and its further conversion to carboxylic acid.

The metabolic pathway involves oxidation of methyl group to produce hydroxymethyl metabolite, which is further converted to carboxylic acid.

Since the drug celecoxib is lipophilic in nature, it should be eliminated predominantly by metabolism, and hence, the study of metabolic pathways is important.

Bacterial, fungal, and yeast cultures were employed in the present study to elucidate the metabolism of celecoxib

HPLC analysis of biotransformed products indicated that majority of the metabolites are more polar than the substrate celecoxib.

The major metabolite was found to be hydroxymethyl metabolite of celecoxib, while the remaining metabolites were produced by carboxylation, methylation, acetylation, or combination of these reactions.

The methyl hydroxylation and further conversion to carboxylic acid was known to occur in metabolism by mammals.

The results further support the use of microorganisms for simulating mammalian metabolism of drugs.

The media used for biotransformation studies were:

1. dextrose broth for fungi

2. nutrient broth for bacteria

3. MGYP (Malt Glucose Yeast extract broth) for yeast

The first stage culture was initiated in 50-ml culture flasks containing 10 ml of sterile liquid medium and inoculated with a loop of culture scratched from freshly grown agar slant. The culture flasks were orbitally shaken then placed at 30°C in refrigerated shaker incubator

The second stage cultures were added with 2 mg each of celecoxib (in 100 μl methanol) to obtain a final drug concentration of 0.2 g/l.

after 10 days of incubation with celecoxib, were extracted with ethyl acetate. The combined ethyl acetate layers were evaporated, and the dried samples were reconstituted with HPLC grade methanol.

The samples were centrifuged , the supernatants were used for HPLC diode array detection (HPLC-DAD) and liquid chromatography tandem mass spectrometry (LC–MS/MS) analyses.

HPLC analysis of the extracts of the cultures showed that 23 out of 39 cultures were able to metabolize celecoxib to produce one or more metabolites