Steroid transformation by fungi

fungal biotechnology,steroid transformation,fungi

1. STEROID TRANSFORMATION BY FUNGI Under the guidance of: Prof. G. R. Janardhan By: Himanshi Chauhan M.Sc.Botany First semester C2 -25/10/2018 University of Mysore

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: 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. I n 1959- Sebek and Spero - valuable corticoid do not cause sodium retention. In 1961- Kondo and Masuo- psuedo-crystallofermentation - high yield (93% more). I n 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: 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 .

17. 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.

18. MAJOR SITES OF FUNGAL REACTIONS

19. 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 : 3 β- 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 .

20. SOME IMPORTANT FUNGAL STEROID TRANSFORMATIONS

21. 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 I ndia then excessive import of steroidal drugs could be reduced to a greater extent.

22. 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

23. 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

24. THANK YOU