Steroids are little organic molecules that are synthesized in steroidogenic tissues and act on mark sites to modulate a cascade of physiological maps [ 1 ] . Examples of natural happening steroids include: steroid alcohols, steroidal saponins, cardioactive glycosides, bile acids, corticoids and mammalian sex endocrines [ 2 ] . They are based on the steran skeleton which is composed of three six-carbon pealing units and one five-carbon ring unit. The rings are labelled A, B, C, and D get downing from the far left ( see fig. 1 ) . In of course happening steroids, all four rings are in the chair conformation [ 3 ] with rings B, C, and D in trans- constellation with regard to each other. For rings A and B the place of the C-19 methyl group attached to C-10 and the H attached to C-5 determines the construction and their cis-/trans- constellation. Overall, neighbouring substituent are trans- if they are diaxial or diequatorial like in fig. 1a, and are cis- if they are axial-equatorial ( fig. 1b ) .
Fig. 1. An illustration of the steroid skeleton in the chair conformation demoing the difference in setereochemistry with regard to C5.
However, the two methyl groups attached to C-10 and C-13 are ever axial in comparative to peal B and D, with C-10 substituent being the conformational mention point [ 3 ] . Hence, the 5?- steroid skeleton ( see fig. 1a ) is in the ‘trans-trans-tans- ‘ constellation, and therefore is loosely two-dimensional. The cognition of the stereochemistry of steroid molecules is extremely important in understanding its biotransformation reactions which is the footing of this survey.
Steroids represent a category of natural merchandises with diverse curative belongingss. It has been observed that minor alterations in the molecular construction of steroids can impact their biological activity [ 4,5 ] . Hence legion research have been conducted to better the activity of bing steroid compounds and to synthesise fresh steroidal compounds with pharmacological activity, and therefore the most important country of these research is the transmutation of steroids utilizing biocatalysts.
Biotransformation could be defined as the alteration of an organic compound into a recoverable merchandise by chemical reactions catalysed by enzymes arising from a biological system [ 6 ] . It should be noted that the organic compound which is the substrate is non involved in the primary or secondary metamorphosis of the biological system concerned, and therefore distinguishes this procedure from biogenesis. The biotransformation of steroids is one of the most of import microbic procedures that are extremely regio- and stereospecific, affecting chemical alterations ( e.g. oxidization, decrease, hydrolysis, isomerization, epoxidation, etc. ) to the parent steroid which are catalysed by the microbic enzymes. In add-on, the characteristics which govern their regiospecificity differ from those commanding chemical specificity, and so it is possible to obtain biotransformation at Centres that are chemically unreactive [ 6 ] . For illustration, in the survey conducted by Peterson and Murray utilizing Rhizopus arrhizus, it was observed that Lipo-Lutin was hydroxylated at C-11 which is an ureactive site in this steroid molecule [ 7 ] . Therefore, these features alongside the rapid growing and high metabolic rates of micro-organisms give biotransformation reactions an advantage over conventional chemical procedures as a tool in the production of curative agents ( e.g. anti-inflammatory, water pills, anabolic, prophylactic, anti-cancer, anti-androgenic, postgestational etc. ) in the pharmaceutical industry. The of all time turning research into the survey of microbic transmutation of steroids have led to newer engineering in this country of scientific discipline such as: genetically alteration of micro-organisms to better their steroid transforming capablenesss, the immobilisation of whole cells or isolated enzymes in a suited matrix for insistent economic use of the enzymes, use of civilization media to better merchandise outputs by the usage of foils e.g. cyclodextrin, and the betterment of the solubility of substrates are indissoluble ( or meagerly soluble ) in H2O [ 8 ] . Furthermore, the progresss in microbic steroid biotransformation have led to the find of new microbic reactions and novel metabolites which may be of involvement within academe and clinical medical specialty.
The mechanism of Hydroxylation
The hydroxylation of a compound is a really of import metabolic procedure, in worlds ; this procedure is catalysed by cytochrome P450 enzymes and consequences in merchandises with a higher mutual opposition than the parent compound, and therefore helping its elimination from the organic structure [ 1,3 ] . The procedure of hydroxylation, involves the transition of a carbon-hydrogen to a carbon-hydroxyl bond, and when catalysed by the enzyme hydroxylase, the reaction is more regio- and stereospecific in contrast to the conventional chemical procedure [ 8-12 ] . As a consequence, microbic hydroxylation is instead used for the synthesis of hydroxysteroid.
Fungal hydroxylation of steroids continues to be the focal point of attending at different degrees of research and merchandise development. In malice of its popularity this procedure is non to the full understood because few surveies have been conducted on the hydroxylase enzyme due to the trouble in insulating this enzyme [ 10,11 ] . However, most surveies have shown that the cytochrome P450 enzyme is besides responsible for steroid hydroxylation in filiform Fungis [ 9-11,13,22 ] .
Cytochrome P450 ( CYP 450 ) enzyme is an iron-haem system which carries out a broad scope of biocatalytical transmutation. These enzymes are besides known as monooxygenases because they transfer one atom of molecular O to an organic substrate. The catalytic mechanism for this reaction involves the binding of the substrate to the active site of the enzyme and so the supplanting of a H2O molecule ( see fig.2 ) . This is followed by a decrease of the Fe in the CYP 450-haem composite to its ferric province ( Iron II ) by an negatron transportation. The ferric province so binds to molecular O to organize a ferrous-dioxy ( Iron ( III ) -OOH ) species. This species so loses a hydroxyl anion to organize an Fe ( IV ) -oxygen extremist. This extremist may retreat a H atom from the substrate to bring forth a C group and an Fe ( IV ) -hydroxyl species. The C extremist so accepts a hydroxyl group from the Fe ( IV ) -hydroxyl species to organize a hydroxylated merchandise and Fe ( III ) . A simple general reaction equation for this procedure is summarised below: ( where R represents the substrate and NADPH is the negatron reassigning species ) .
RH + NADPH + H+ + O2 > ROH + NADP+ + H2O
Fig. 2. Hydroxylation by a cytochrome P450. [ 6 ] .
In other to to the full understand the mechanism of fungous hydroxylation of steroids, the relationship between the construction of the CYP 450 hydroxylase enzyme and its regio- and stereoselective feature has to be defined. However, as mentioned earlier non much surveies have been conducted on the structural characteristics of this enzyme, and so ‘active site theoretical accounts ‘ was developed to hold on the construct of the regio- and stereoselective result of microbic hydroxylation reactions.
The first theoretical account, postulated by Brannon et al suggested the possibility for a steroidal substrate to be bound by a individual steroid hydroxylase in more than one orientation due to two- sites adhering, which could ensue in hydroxylation taking topographic point at more than one place given the appropriate geometrical relationship between the active site of the enzyme and the C atom of the substrate undergoing the reaction [ 9,14 ] . These four orientations are represented as normal, contrary, inverted and change by reversal inverted ( see fig. 3 ) and has been observed in the metabolic handling of 3?-hydroxy-17a-oxa-D-homo-5?-androstan-17-one by a filiform fungus ; Aspergillus tamarii [ 15 ] .
Fig. 3. Possible orientations of a steroid as a consequence of two-site binding.
The other theoretical account, Jones ‘ theoretical account takes into history merely the normal and rearward binding orientations [ 6 ] . It requires the being of three active Centres on the steroid hydroxylase enzyme. These active Centres have double functions and could move both as a binding site or a hydroxylating site [ 16 ] . However, these functions are reciprocally sole, and so hydroxylation would happen at the closest atomic Centre to the steroid. Hence the enzyme-substrate interaction proposed by Jones would propose a triangular location with an approximative spacial correspondence to C-3, C-11 and C-16 atoms of the steroid karyon [ 6 ] ( fig. 4 ) .
Fig. 4. The Jones ‘ theoretical account of enzyme-substrate interaction
This theoretical account could non explicate the hydroxylation reactions by some micro-organisms. Therefore another theory was developed by McCrindle et Al utilizing both theoretical accounts above and taking into history the 3- D nature of the steroid compound and hydroxylase enzyme [ 17 ] . In this theoretical account, the steroid ring Acts of the Apostless as a planar mention point ( fig. 5 ) . Binding site A favor O atoms below the plane of the ring and hydroxylation is alpha. Binding site B is similar to A but can besides hyroxylate alpha ( axial or equatorial ) or beta ( equatorial ) atoms. Whereas, adhering site C binds preferentially to oxygen atoms above the plane of the steroid ring and hydroxylate with -beta orientation. Overall, this theoretical account tends to suit the hydroxylation form of most micro-organisms.
Fig. 5. The McCrindle ‘s theoretical account of enzyme-substrate interaction.
The hydroxylation result of some steroids can be predicted based on the O maps or ‘directing groups ‘ on the steroid skeleton. As a regulation of thumb mono- oxygenated substrates are dihydroxylated and their transmutation merchandises are frequently in low outputs [ 16 ] . This is as consequence of the presence of one O map on the steroid compound doing it less polar and therefore diminishing its solubility which hinders its pervasion into the microbic cell. In add-on to this, the presence of merely one O map allows the steroid to adhere to the enzyme at merely one Centre, thereby increasing its rotary motion and oscillation about the active site which makes it more likely to be dihydroxylated. Whereas, di- oxygenated substrates are monohydroxylated because the presence of two O maps reduces the opportunity of multiple hydroxylations due to the decrease in the possible figure of adhering orientations [ 16 ] . Furthermore, the presence of two adhering O groups increases the rate of responsiveness of microbiological transmutation as the increased substrate mutual opposition improves solubility and therefore pervasion into the cell membrane of the micro-organism is really likely. A broad assortment of beings have shown this form of hydroxylation with a broad scope of substrates [ 15,16 ] .
Hydroxylated steroids possess utile pharmacological activities, for illustration, C-11 hydroxylation is regarded as indispensable for anti- inflammatory action, and 16?- hydroxylated steroids have increased glucocorticoid activity [ 8,12 ] . Hence the steroid industry exploits the usage of 11?- , 11?- , 15?- and 16?- hydroxylation chiefly for the production of adrenal cerebral mantle endocrines and their parallels [ 8 ] . A scope of micro-organisms have been observed to impact this type of hydroxylations. For illustration, 11?- hydroxylation is performed utilizing Rhizopus sp. Or Aspergillus sp. , Cuvularia sp. or Cunninghamella sp. and Streptomyces sp. generates 11?- and 16?- hydroxylations severally [ 8,18 ] . Further research has shown other hydroxylations ( e.g. 7?- , 9?- and 14?- hydroxylations ) of holding the potency for industrial development [ 18 ] .
The mechanism of Baeyer- Villiger Oxidation
Baeyer- Villiger oxidization is the oxidative cleavage of a carbon-carbon bond adjacent to a carbonyl, which converts ketones to esters and cyclic ketones to lactones [ 19,20 ] . The mechanism of this chemical procedure was originally proposed by Criegee [ 19 ] . It involves a two measure procedure: a nucleophillic onslaught of a carbonyl by a peroxo species ensuing in the formation of a ‘Criegee ‘ intermediate, which so undergoes rearrangement to the corresponding ester. Normally used peracids or oxidizers include: m-chloroperoxybenzoic acid, H peroxide, peroxyacetic acid and trifluoroperoxy acetic acid. This chemical procedure is extremely important, because the merchandises generated are compounds which are intermediates in the synthesis of natural merchandises or bioactive compounds. However, the oxidizers used in chemical Baeyer- Villiger oxidization ( BVO ) are expensive and risky and besides the reaction generates a big sum of waste merchandises [ 4 ] . Hence biological ( or enzymatic ) BVO offers a ‘greener ‘ attack for the production of chiral lactones.
Biological Baeyer- Villiger oxidizations are mediated by flavin- dependent monooxygenase enzymes i.e. Baeyer- Villiger monooxygenases ( BVMOs ) [ 19,21,22 ] . As a consequence of the various nature of flavoproteins [ 19 ] , BVMOs have been shown to execute a assortment of catalytic reactions including BVO of steroidal systems.
The mechanism of microbic Baeyer- Villiger ‘s oxidization ( fig. 6 ) is based on consequences obtained with cyclohexanone monooxygenase ( CHMO ) isolated from Acinetobacter calcoaceticus [ 19,22 ] . This enzyme was shown to possess flavin adenine dinucleotide ( FAD ) as a prosthetic group and was besides found to be dependent on NADPH and O. The enzymatic procedure is initiated by the decrease of the tightly bound FAD by NADPH followed by rapid oxidization by molecular O to bring forth flavin 4a- peroxide anion, which acts as the oxygenizing species. Nucleophillic onslaught of the substrate carbonyl group by the flavin 4a- peroxide anion consequences in the ‘Criegee ‘ intermediate. This intermediate so undergoes rearrangement to organize the merchandise lactone and 4a- hydroxy- flavin. The catalytic rhythm is terminated by riddance of H2O to organize FAD and the release of the merchandise and co-factor. It should be noted that the mechanism for microbic BVO based on CHMO serves as a theoretical account for other BVMOs. However, there are some differences such as the co-factor NADPH can be replaced by NADH and the prosthetic group FAD can be replaced by FMN [ 19 ] . Overall, there are no important alterations to the mechanism.
Fig. 6. Mechanism of flavin-dependent Baeyer- Villiger monooxygenases. [ 19 ]
Microbial Baeyer- Villiger ‘s oxidization is extremely regio- and stereoselective [ 4,19-22 ] and as consequence it is normally utilised for the biotransformation of steroidal compounds. It has besides been shown in assorted surveies, the ability of microbic BVMOs to assail the different pealing systems of the steroid skeleton. Glomerella fusarioides was observed to biotransform eburicoic acid through an onslaught on the ring- A system by manner of BVO to organize a lactone, followed by a ring- cleavage to bring forth carboxylic acid [ 19 ] . In add-on, 3-ketosteroids were observed to undergo Baeyer- Villiger ‘s oxidization with an stray Baeyer- Villiger monooxygenase enzyme from Pseudomonas sp. assailing the C-3 ketone group on ring- A [ 4 ] . Ring- B lactone formation has besides been observed in the steroid system utilizing tomato cell suspension civilizations to bring forth 24- epibassinolide [ 19 ] . Ring- D lactonization is really common and has been demonstrated by rather a few fungous species such as Pencillium sp. , Cylindrocarim sp. , Mucor sp. and Aspergillus sp. These Fungis were able to biotransform Lipo-Lutin to testololactone by manner of Baeyer- Villiger ‘s oxidization via the intermediate steroid androst-4-ene-3,17-dione [ 19 ] . So far, ring- C lactonization has non been observed, although surveies have been conducted to see this ring onslaught but none have proven its possibility [ 4 ] . Overall, several research have been undertaken and are still been conducted to research the catalytic repertory of Baeyer- Villiger monooxygenase enzymes, and these surveies have shown the ability of this enzyme to catalyze the oxidization of 3- keto and 17- keto steroids with full control of the regiochemistry of the produced lactone therefore leting its application as an option to the conventional chemical procedure.
The mechanism of intoxicant oxidization
Alcohol oxidization is an of import reaction in organic chemical science. It leads to the production of aldehydes or carboxylic acids from primary intoxicant and ketones from secondary intoxicant. Third intoxicants are immune to oxidization because it is impossible to take a H ion or add an O atom to the compound without interrupting the C-C bond. The normally used reagents for the oxidization of intoxicant are Jones ‘ reagent, K permanganate and chromium- based reagents. However, the oxidization of primary intoxicants to aldehydes creates a job for the organic chemist because aldehydes are non stable when produced in the conventional chemical oxidization procedure therefore the usage of microbic cells is preferred to get the better of this job [ 22 ] . The enzymes used in the oxidization of intoxicant by micro-organisms are alcohol dehydrogenases ( ADH ) which are dependent on the co-factors NAD+ or NADP+ . The mechanism of this reaction consists of a series of equilibrium where the hydride from the intoxicant substrate is transferred to NAD ( P ) + in the treble complex ‘enzyme- NAD+- intoxicant ‘ complex [ 22 ] . In worlds, this procedure is carried in the same manner and is highly of import for several endogenous every bit good as drug metamorphosis. Therefore, micro-organisms could function as theoretical accounts for human metamorphosis utilizing this procedure. An unprecedented degree of regioselctivity of microbic oxidization of the alcoholic group in gall acids has been observed [ 23 ] . Some fungous species are known to hold the ability to oxidize the C-3 and C-17 hydroxyl groups of steroidal compounds. Aspergillus tamarii has been shown to possess the enzyme 3?- hydroxy- steroid- dehydrogenases which catalyses the 3?- hydroxyl group to a C-3 ketone [ 5 ] . Oxidation of the 17?- hydroxyl group has besides been observed in a figure of fungous species e.g. Penicillium sp. , Aspergillus sp. and Mucor sp [ 24,25 ] . In general, a figure of micro-organisms have shown the ability to oxidize the intoxicant groups on a steroid compound to bring forth the ketone parallel, which could function as an intermediate in the synthesis of lactones.
The mechanism of carbonyl decrease
The rearward reaction of oxidization is decrease. It involves the transportation of one hydride ion to the carbonyl group. In conventional chemical reaction, the accelerators normally used are sodium borohydride ( NaBH4 ) and Lithium aluminum hydride ( LiAlH4 ) , aldehydes are easy reduced to primary intoxicants utilizing these accelerators. However, the high stereoselective decrease of ketones to chiral secondary intoxicants is better performed with microbic enzymes [ 20,22 ] . This procedure is catalyzed by intoxicant dehydrogenases ( ADHs ) , necessitating the co-enzymes NADH or NADPH which transfers the hydride ion to the Si- or Re- face of the carbonyl group ensuing in the formation of the corresponding ( S ) – or ( R ) – intoxicant [ 22,25 ] . Microbial decrease of ketones to secondary intoxicants usually returns in conformity with Prelog ‘s regulation to give secondary intoxicants in the chief ( S ) – enantiomorph [ 25,26 ] . However, merely a really limited figure of microbic enzyme ( ADHs ) is available to let anti- Prelog activity and have been demonstrated in the fungus Myceliophthora thermophila [ 27 ] .
The ability of micro-organisms to cut down the carbonyl groups on steroid compounds was reported in 1937 by Mamoli and Vercelloni who described the decrease of the 17- keto group in androst-4-ene-3,17-dione to testosterone by Saccharomyces cerevisiae [ 25 ] . Since so this procedure has been demonstrated for a broad assortment of substrates and micro-organisms of different species. Carbonyl decrease frequently accompanies other reactions in steroid biotransformation, and therefore Acts of the Apostless as one of the procedures in the production of hydroxysteroids.
The micro-organism: Myceliophthora thermophila
Thermophilic Fungis are among the few fungous species of eucaryotic being that are able to last at temperatures every bit high as 60 – 62oC [ 28 ] . However, Cooney and Emerson ‘s definition of thermophilic Fungi is: Fungi that have a growing temperature lower limit at or above 20oC and a growing temperature upper limit at or above 50oC [ 29 ] . These Fungis have a widespread distribution in both tropical and temperate parts, populating assorted types of dirt and topographic points where decomposition of works stuff and organic affair occur therefore supplying the warm, humid and aerophilic environment which are the basic conditions for their development [ 28,29 ] . The enzymes of thermophilic Fungis have been studied to research their part in biotechnology, and these surveies have identified a singular scope of extracellular enzymes ( e.g. peptidases, lipases, ?-amylases, glucoamylases, cellulases, cellobiose dehydrogenases, xylanases, ?- D-glucuronidase, polygalacturonase, laccases, phytase and D-glucosyltransferase ) and intracellular enzymes ( e.g. trehalases, saccharases, ?-glycosidases, lipoamide dehydrogenases, ATP sulfurylases and protein disulfide isomerases ) [ 28 ] . The bulk of these enzymes are appreciably thermostable which have resulted in its application in sugar and paper industries [ 30 ] .
So far merely two surveies to day of the month hold been conducted to look into the steroid biotransformation abilities of thermophilic Fungis. The first survey used the thermophilic filiform fungus, Rhizomucor tauricus and it was observed that all transmutations were oxidative bring forthing mono- and dihydroxylated merchandises with allylic hydroxylation been the prevailing path of onslaught on the steroid compounds [ 30 ] . The 2nd survey was conducted utilizing Myceliophthora thermophila [ 27 ] on which this nowadays survey is based.
Myceliophthora thermophla is a thermophilic filiform fungus classed as an ascomycetous fungus within the phyla of fungi [ 28 ] . It has another name which is sometimes used, Sporotrichum ( Chrysosporium ) thermophile [ 28,29 ] . However, M. thermophila is the sexual ( telomorph ) phase of the Fungis, while Sporotrichum ( Chrysosporium ) thermophile is the nonsexual ( anamorph ) phase [ 28 ] . Its chief home ground is in the dirt and it is found in the undermentioned states: USA, Canada, India, UK, Japan and Australia [ 29 ] . But this fungus can turn on simple media incorporating C, N and indispensable mineral salts such as Czapek- dox agar ( CDA ) . The optimal growing temperature for M. thermophila is within the scope 45 – 50oC [ 28 ] . It grows quickly on CDA at 45oC, bring forthing settlements that vary in surface texture from cottony to granular and its coloring material alterations from white to cinnamon brown [ 29 ] . This fungus has besides been observed to bring forth extracellular enzymes such as laccases, xylanases, cellulases and phytase which have been exploited for usage in the nutrient industry and as biocatalyst in biotechnological procedures [ 27 ] .
This present survey is a continuance of the research into steroid biotransformation by M. thermophila. Previously, a series of steroids ( Lipo-Lutin, testosterone ethanoate, 17?-acetoxy-5?-androstan-3-one, testosterone and androst-4-ene-3,17-dione ) were incubated with this fungus, and a broad scope of biocatalytical activity was observed with enzymatic onslaught at all four rings of the steroid karyon and the C-17? side- concatenation. This fungus demonstrated an unusual ring- A gap following incubation of the steroid 17?-acetoxy-5?-androstan-3-one, and therefore bring forthing 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid. It was besides identified to be the first thermophilic fungus to split the side- concatenation of Lipo-Lutin. M. thermophila besides demonstrated reversible acetylation and oxidization of the 17?- intoxicant of testosterone [ 27 ] ( fig. 8 ) .
Fig.7. The gap if pealing an A- gay steroid following incubation of 17?-acetoxy-5?-androstan-3-one by Myceliophthora thermophila.
Fig.8. Putative metabolic tracts present in Myceliophthora thermophila.
Further probe into the diverse biocatalytical activity of this being has led to the incubation of six concentrated steroids viz. : 17?-hydroxy-5?-androstan-3-one, 5?-prgnane-3,20-dione, 3?-hydroxy-5?-androstan-17-one, 3?-hydroxy-5?-androstan-17-one, 5?-androstan-3,6,17-trione and 5?-androstan-3,17-dione with M. thermophila
The proposed hypothesis from old survey is outlined as follows:
Presumed lactonohydrolase activity evident from the isolation of an unfastened lactone ring.
Enzymes responsible for the decrease of C3 ketone to a 3?- intoxicant and hydrogenation of the C-4-C-5 olefine are induced by Lipo-Lutin.
Organism ‘s ability for contrary metamorphosis, which is apparent from the acetylation of testosterone to bring forth testosterone ethanoate and the decrease of the C-17 ketone of androst-4-ene-3,17-dione to bring forth testosterone which farther undergoes acetylation.
Preference for stereochemistry of hydroxylation with onslaught at axial protons ( 6? , 7? , 11? , 14? ) .
Therefore, the chief purpose of this survey is to detect the consequence of concentrated steroids on the biocatalytical activity of Myceliophthora thermophila CBS 117.65 and to turn out the hypothesis from the old survey.