Biotransformation of pregnenolone and dehydroepiandrosterone compounds by the mold Penicillium hirsutum

Natural products are substances that do not contribute to the growth and reproduction of live creatures. Because of their impacts on other organisms, natural substances benefit living things and attract more attention. Groups such as alkaloids, terpenoids, peptides, steroids, polyketides, special amino acids, phenylpropanoids, fatty acids and derivatives and special carbohydrates are commonly used to categorize natural compounds. Biotransformations are chemical changes that biological systems can perform on xenobiotic substances. Biotransformations are carried out by enzyme-containing biological systems and free or immobilized enzymes. Tissue cultures, cell cultures, microsomes, organ cultures, microsomes, microbe spores and microorganisms are often used as standard biological systems for biotransformations. In living organisms, enzymes carry out nearly every reaction by reducing the activation energy (EA). Enzymes reduce the time required for the reaction to achieve equilibrium, but they are neither consumed nor affected by the processes nor alter the ΔG or the state of equilibrium of the process. The International Union of Biochemistry has reported about 3200 enzymes, with an estimated 25,000 enzymes in nature. Because they are highly efficient catalysts, enzymes have certain benefits for their users. For example, the reaction rates of enzymatic reactions can be increased 108-1010 times, and in rare cases, this can even exceed 1012. Because they are highly efficient catalysts, enzymes have certain benefits for their users. For example, the reaction rates of enzymatic reactions can be increased 108-1010 times, and in rare cases, this can even exceed 1012. Since enzymes are completely biodegradable and are composed of amino acids, they are acceptable for the environment. While most other chemical reagents harm the environment, enzymes generally work in moderate environments (pH 7, 30 °C and 1 atm). As a result, certain problems such as isomerization, racemization, rearrangements and fragmentation are reduced. Since enzymes are compatible with each other, they typically work in the same or similar environments. As a result, many reactions can be performed in a single flask using multienzyme systems. While most enzymes have a high substrate tolerance, some are not limited to their intended function. These enzymes can accept a wide variety of natural and artificial substances. Enzymes can catalyze many different reactions, and practically any known reaction can be catalyzed by an enzyme. Enzymes are molecules with chemoselectivity, regioselectivity and enantioselectivity. Because they are chemoselective, enzymes usually only affect a certain type of functional group, leaving other functions unchanged. Enzymatic reactions, therefore, typically have a cleaner quality. Thanks to their regioselectivity, enzymes can discriminate between functional groups that occupy different chemical positions within the same substrate molecule. The complex three-dimensional architecture of enzymes may enable them to achieve this. As chiral catalysts derived from L-amino acids, enzymes are enantioselective. Therefore, enzymes can recognize any chirality on a substrate molecule. Both enantiomers in a racemic substrate can usually react at different rates, resulting in a kinetic reaction, and a prochiral substrate can be transformed into a chiral product. However, there are also disadvantages to using enzymes. One type of enantiomer found in nature is an enzyme. When the other kind of enantiomeric product is needed, an enzyme with the opposite stereochemical selectivity is required. But this is often not possible. Enzymes require special working conditions. Working in temperate environments can be problematic, as high temperatures and acidic pH can inhibit some enzymes. Water is the least optimal solvent for most organic processes due to its high boiling point and evaporation heat; however, enzymes demonstrate the highest catalytic activity in this solvent. In addition, most organic molecules are hardly soluble in aqueous solutions. Therefore, switching an enzymatic process from an aqueous to an organic medium is highly desirable. However, due to enzyme denaturation, this can lead to a decrease in catalytic activity. The natural cofactors on which enzymes rely are extremely important. Despite their extraordinary flexibility in accepting non-natural substrates, enzymes rely almost entirely on their natural cofactors such as NADH and NADPH. Unfortunately, because these molecules are relatively unstable, too costly to be used at stoichiometric levels, and both are not feasible, they cannot be substituted with more affordable synthetic counterparts. Enzymes are sensitive to inhibition events. Substrate or product inhibition, which causes enzymes to stop operating at increased substrate and/or product concentrations, can impact a wide range of enzyme functions. In addition, some enzymes can trigger allergies. Enzymes can cause allergic reactions, but this danger can be minimized by considering them as chemicals and exercising caution when utilizing them. Typically, biotransformations are carried out by whole, intact microorganisms or isolated enzyme systems. It is estimated that about 300 isolated enzyme systems are available for sale. Biotransformations often involve entire microorganisms since the majority of the needed enzyme systems are bound to the membrane and hard to remove. Bacteria, yeasts, molds, and microalgae are the four most common microorganisms employed in biotransformations. Microorganisms use non-specific enzyme systems to perform a variety of reactions on both natural and synthetic substrates. Among these reactions, microbial hydroxylations are the most common and preferred. The importance of this process was first recognized in 1952 when microbial hydroxylation helped solve an important problem related to the synthesis of cortical steroids. Due to the isolation of the position from other functional groups, the addition of an oxygen function to C-11 was an extremely time-consuming, expensive and challenging procedure. Rhizopus arrhizus effectively solved this problem by microbial hydroxylation. Following microbial hydroxylation, microbial biotransformations became prominent. Since then, various substrate groups, including steroids, have been widely used for microbial biotransformations. Because of their high regioselectivity and stereoselectivity, microbial steroid biotransformations are widely used to synthesize several key steroid hormones and medicines. Many microbial steroid biotransformations have been described in recent years. Tons of outstanding efforts are still being made to increase the efficiency of microbial biotransformations and discover new, valuable microbes and reactions. Since the initial description of microbial hydroxylation in 1952, various fungi have consistently been one of the most studied whole-cell systems for biotransformation processes. Various fungi have biotransformed numerous steroid species. Several intriguing results have been obtained from these biotransformations, including microbial hydroxylations, Baeyer-Villiger oxidations and 5α-reduction. In this study, DHEA (9) and pregnenolone (3) compounds were biotransformed separately by Penicillium hirsitum MRC 500372 mold. After preparing the medium for the growth of the mold, it was dispensed into flasks. An autoclave was used for sterilization. P. hirsitum MRC 500372 mold was inoculated in sterile flasks under appropriate conditions and incubated for 3 days. DHEA (9) and pregnenolone (3) were added separately to the molds in the sterile flasks and incubated for another 5 days. After incubation, steroids were filtered from the medium and extracted with ethyl acetate. The residue obtained by evaporation of the ethyl acetate in an evaporator was subjected to column chromatography to purify steroids. Using structural methods such as NMR, IR spectroscopy and melting point determination, incubation of DHEA (9) and pregnenolone (3) substrates with P. hirsitum MRC 500372 resulted in metabolites of androst-4-en-3,17-dione (5) from DHEA (9) and progesterone (2) from pregnenolone (3).