2. INTRODUCTION
WHAT ARE STEROIDS?
A steroid is a biologically active organic compound with four rings arranged in a
specific molecular configuration.
also known as Gonane, sterane or
cyclopentaperhydrophenanthrene.
Steroids have two principal biological functions:
as important components of cell membranes which alter membrane fluidity;
as signaling molecules.
Steroids are found in plants, animals and fungi.
About more than 250 sterols and its derivatives exist in :
insects (e.g., ecdysteroids),
plants (e.g., phytosterols, diosgenin),
vertebrates (e.g., cholesterol; corticosteroids: glucocorticoids, mineralocorticoids;
sex hormones: androgens, estrogens; bile acids, vitamin D; neurosteroids),
fungi (e.g., ergosterol, ergosteroids).
3. TYPES OF STEROIDS
Corticosteroids:
Glucocorticoids: Cortisol - immunosuppression
Mineralocorticoids: Aldosterone -regulate blood pressure
Sex steroids:
Progestogens: - regulates cyclical changes in the endometrium of the uterus.
- maintains a pregnancy.
Androgens: Testosterone-male secondary sex characters.
Estrogens: Estradiol- female secondary sex characteristics
Bile salts or bile acid. These steroids are made in the liver. They don't function as hormones,
but are necessary for digestion and absorption of fats.
Sterols. The most commonly known of these is cholesterol.
Other sterols help your body to make vitamin D from sunlight and to build cell walls.
Cyclopentanoperhydrophenanthrene
Cholestane (C27): R1= R2= CH3,
R3=CH(CH3)(CH2)3CH(CH3)2
Pregnane (C21): R1= R2= CH3, R3=
CH2CH3
Androstane (C19): R1= R2= CH3, R3= H
Estrane (C18): R1: H, R2: CH3, R3: H
Gonane (C17): R1= R2= R3= H
4. BIOSYNTHESIS
By Mevalonate Pathway
which uses acetyl-CoA as building
blocks for dimethylallyl pyrophosphate
(DMAPP) and isopentenyl
pyrophosphate (IPP) .
By Steroidogenesis
Steroidogenesis is the biological process
by which steroids are generated from
cholesterol and changed into other
steroids.
5. FUNCTIONS OF STEROIDS
It act on target sites to regulate a cascade of physiological functions.
Steroid hormones and their derivatives have been used for a wide range of
therapeutic purposes.
Utilization as immunosuppressive, anti-inflammatory, anti-rheumatic,
progestational, diuretic, sedative, anabolic and contraceptive agents.
Recent applications of steroid compounds include the treatment of some forms
of cancer, osteoporosis, HIV infections and treatment of declared AIDS.
Nowadays, steroid manufacturing occupies a prominent place in the
pharmaceutical industry with an annual global market over $10
billion.
6. TRANSFORMATION OF STEROIDS
The manufacture of steroid drugs as well as steroid hormones
by the biotransformation process is an excellent example of
the outstanding use of microbial technology at an industrially
large scale.
The chemical synthesis and transformations of steroids
-requires multiple steps ,
-makes the use of reagents that have health risks
and -cause serious environmental disposal problems.
Alternative is FUNGAL TRANSFORMATION.
7. FUNGAL TRANSFORMATION
Several fungi, such as Rhizopus Curvularia lunata,
Aspergillus sp, Penicillium sp., Gliocladium sp.,
Fusarium solani and yeasts are some of the important
organisms of steroid biotransformation.
Steroid transformation is different from other
microbiological process (organic acids, solvents, antibiotics)
because these products are synthesized from the ingredients
in the medium which also serve as substrate for growth and
reproduction of microorganisms.
CONTENT
8. Fungal cells are ideal choice for biotransformation due to certain reasons like:
I. Surface-volume ratio: fungal washed mycelia(resting cells) and spores
suspension have high surface-volume ratio.
II. Growth Rate: Higher growth rate of fungal immobilized cells reduces the time
of biomass transformation.
III. Metabolism Rate: Higher rate of the metabolism in fungal cells due to specific
enzymes regulate efficient transformation of substrate.
IV. Sterility: It is easier to maintain sterile conditions when fungal cells are used.
V. FUNGAL transformations cleave the complex side chains of precursor steroids
in one single step and incorporate desirable modifications in steroid nucleus.
VI. Fungal transformations are regiospecific and stereospecific.
VII. Easy Purification and Isolation
WHY FUNGAL TRANSFORMATION?
9. HISTORY OF FUNGAL TRANSFORMATION
In 1934- 625 Kg ovaries from 50,000 cows were required to isolate 20 mg of
progesterone.
In 1946- chemical synthesis of cortisone from deoxycholic acid developed at
Merck and Company.
(615 Kb deoxycholic acid produced 1Kg of cortisone acetate through 37 steps.)
In 1949- anti- inflammatory property of cortisone in rheumatoid arthritis was
demonstrated by HENCH .
10. In 1953- Peterson and Murray –economically transformed progesterone by
Rhizopus nigricans.(85% more yield).
In 1954-Hanson reported transformation by Cunninghamella blakesleeana.
in 1955- Shull and Kita –transformation of cortisone by Curvularia lunata .(60%
effective specific yield.)
11. In 1959-Sebek and Spero - valuable corticoid do not cause sodium retention.
In 1961- Kondo and Masuo- psuedo-crystallofermentation- high yield (93%
more).
In 1962- Applezweig proved steroids as oral contraceptives.
in 1980 -With the introduction of biotransformation reactions, the number of
steps (microbial and chemical put together) were reduced and cost of the product
was reduced to just $1/g from $200/g (in1950).
12. PRESENT
The microbial steroid transformations were made cost effective by substituting
expensive precursor by cheaper
Plant sterols like stigmasterol, sitosterol (byproducts of soyabean oil production),
campesterol (by product of paper industry)
and diosgenin (Maxican yam roots- Dioscorea composite or Testudinaria sylvatica)
or animal sterol (cholesterol)
as the precursors used in steroid transformation reactions catalyzed by microbes.
These microbial transformations are accomplished at 37ºC in aqueous medium at
atmospheric pressure.
The market demand for four major steroids (cortisone, aldosterone, prednisone
and prednisolone) is about 700,000 kg/year (Table 7).
13. Fig:An overview of steroid production, from raw materials to finished products.
Full lines indicate bioconversion whereas dashed lines indicate chemical transformation
14. INDIAN SCENARIO OF FUNGAL STEROID TRANSFORMATION
In India inspite of the abundant availability of raw materials only few licensed firms
are involved in the actual production of steroidal drugs such as :
Glaxo India limited,
Ciba,Wyeth india Limited,
Cipla Ltd
and Merind Ltd.
thus there is excessive import is practiced .
the estimated demand of corticosteroids
in 1993-1994 was 4 to 5 times greater
than actual production.
Some Indian research institutions such as
Central Drug research institute,
Lucknow, Bose institute,
Indian institute of science Bangalore,
National Chemical laboratory, Pune etc
have continuously contributed to the
development of fungal steroid transformation.
15. Production process of steroids:
Production of steroids can be carried out by
following steps:
A. CULTIVATION OF FUNGI :
-culture of fungi in fermentation tanks
with aeration and agitation for 1or 2 days.
-glucose/sucrose as carbon source
-Corn steep liquor as nitrogen source.
B. INCORPORATION OF STEROIDS AND
INHIBITORS INTO THE MEDIUM:
- suitable amount of steroid (0.25 to 1.0 g/ lit )
is added in suitable solvent
(dimethylformamide, acetone, propylene glycol)
at the end of growth phase.
-inhibitors to avoid undesired enzyme activity.
16. C.TRANSFORMATION OF
STEROIDS:
left undisturbed for
maximum transformation
for 1 or 5 days .
D.SEPERARTION AND
PURIFICATION:
--sampled analyzed by
chromatographically using
TLC followed by
spectrophotometrically.
--transformed product is
extracted with methylene
chloride, chloroform, ethyl
acetate .
---separation by
crystallization or column
chromatography .
18. METHODS FOR TRANSFORMATION OF STEROIDS
In Aqueous Media And Facilitators
The major limitations on fungal steroid transformation are low water solubility, dispersibility, and
powder aggregation (Goetschel and Bar 1992).
Increasing the permeability of viable cells has been considered as a remedy using substrate micron
ization, fed-batch systems, permeabilizing the cell wall using antibiotics, surfactants, or cyclo dextrins.
Using Immobilized Biocatalysts
Whole-cell immobilization has been widely applied to minimize loss of enzyme activity and maintain
longer half-life(Ahmad et al. 1992)
Using Free And Immobilized Enzymes
Some efforts have been devoted to isolate and characterize enzymes from fungal sources for industria
land research purposes (Petri č et al. 2010).
In Two-phase Systems
Aqueous organic two-phase systems are often applied to improve the yield of fermentations in which
lipophilic substrates and products are present (Leon et al. 1998).
In A Cloud Point System
Cloud point systems (CPS) prepared by using non ionic surfactants, provide a micro-aqueous
environment to ensure the viability of cells and their enzymatic activity.
20. TYPES OF FUNGAL STREOID 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 3keto
– 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.
29. CONCLUSION
In India inspite of the plenty availability of
raw materials, very few Indian concerns have
entered in the actual production of steroidal
drugs using indigenous raw materials.
If few more concerns can divert their
attention to carry out microbiological steps in
India then excessive import of steroidal drugs
could be reduced to a greater extent.
30. REFERENCES
MICROBIAL BIOTECHNOLOGY--1997—S.B.Chincholkar—jodhpur scientific publisher—
ch: Steroid transformation with free and immobilized cells ---page no :7-11.
MICROBIAL TECHNOLOGY: microbial process---2009--H.J.Peppler and D.Perlman—
second edition—Elsevier press—ch 14 :Microbial transformation of steroids ---
page no.:483-495.
A TEXTBOOK OF MICROBIOLOGY—R.C.DUBEY and D.K.Maheshwari---2000—S Chand
company—ch 17:industrial microbiology—page no:403-407.
HANDBOOL OF SECONDARY METABOLITES—R.J.Cole and M.A.Schweikert—Academic
press New York—2003—page no :5-18 and 25-39.
FERMENTATION TECHNOLOGY---M.L.Srivastava—Narosa publishing home—2008—
page no 69-73.
MICROBIAL BOITECHNOLOGY---A.N.Glazer and H.Nikaido—Cambridge press—2008--
second edition—page no:400-404.
BOOKS
31. REFERENCES
WEBSITES
Microbial steroid transformations: Current state and prospects
https://www.researchgate.net/.../224914691_
Biotransformation of Steroids
https://www.tandfonline.com/doi/pdf/10.3109/07388558809146602
"Spore Plate Method" for Transformation of Steroids - NCBI
https://www.ncbi.nlm.nih.gov/pmc/articles/.../pdf/applmicro00115-0029.pdf
Biotransformation of steroids - SlideShare
https://www.slideshare.net/sudharajput/biotransformation-of-steroid