Dehidroepiandrosteron bileşiğinin aspergıllus glaucus küfü ile biyotransformasyonu = Bıiotransformation of dehydroepiandrosterone by aspergıllus glaucus

Abstract

Doğal ürünler bulundukları canlılara daha iyi hayat şartları sağlayan ve buna ilaveten günümüzde özellikle diğer canlılar üzerindeki etkileriyle ilgi odağına yerleşen kimyasal maddelerdir. Genelde doğal ürünleri terpenoidler, alkaloidler, steroidler, poliketidler, fenilpropanoidler, yağ asitleri ile yağ asitlerinin türevleri, özelleşmiş peptidler, özelleşmiş karbonhidratlar olarak sınıflandırma eğilimi söz konusudur. Enzimlerin substratları olmayan kimyasal maddelerde katalizledikleri kimyasal reaksiyonlar biyotransformasyonlar olarak adlandırılır. Biyotransformasyonlarda rol oynayan enzimler sabitlenmiş olarak, serbest olarak ya da çeşitli biyolojik sistemlerin yapısında fonksiyonlarını gerçekleştirebilirler. Biyotransformasyonların uygulanmasında genelde küfler, mayalar ve bakteriler gibi mikroorganizmalar biyolojik sistemler olarak kullanılır. Mikroorganizmalarla gerçekleştirilen biyotransformasyonlar bilinen kimyasal sentez yöntemlerine kıyasla çok sayıda avantajlar sağlamaktadır. Mikrobiyal biyotransformasyonlar mikroorganizmaların serbest olarak veya belirli yüzeylere sabitlenmesiyle uygulanabilir. . Özelikle son yıllarda küf enzimlerinin yüksek regio ve stereoseçicilikleri nedeniyle küflerle gerçekleştirilen mikrobiyal steroid biyotransformasyonları çok sayıda önemli ilaç ve benzeri maddelerin sentezi için yaygın olarak uygulanmaktadır. Bu çalışmada DHEA olarak da bilinen dehidroepiandrosteron (9) bileşiğinin Aspergillus glaucus MRC 200914 izolatında nasıl metabolize edildiğinin incelenmesi hedeflenmiştir. Bahsedilen hedef doğrultusunda biyotransformasyon deneyi öncesinde Aspergillus glaucus MRC 200914 izolatı için belli aralıklarla taze alt kültürler hazırlandı. İzolat için besiyeri hazırlandıktan sonra erlenlere dağıtılıp otoklavda sterilize edildi. Erlenlere en taze alt kültürdeki izolat steril şartlarda inoküle edildikten sonra erlenler 3 gün inkübe edildi. İzolatın geliştiği bu erlenlere DHEA (9) steril şartlarda eklenerek 5 gün inkübe edildi. İnkübasyon sonrasında besiyeri filtre edildi ve içerisindeki steroidler etil asetat ile ekstrake edildi. Ekstraktlarının evaporatörde uçurulrulmasıyla oluşan kalıntıdaki steroidler ise bir kolon kromatografisi çalışması ile ayrıldı. Elde edilen steroidlerin yapı tayinleri ise erime noktaları, NMR ve IR spektumları alındıktan sonra substratınkiler ile karşılaştırılarak belirlendi ve Aspergillus glaucus MRC 200914 ile DHEA (9) biyotransformasyonunun 3β,11α-dihidroksiandrost-5-en-17-on (16) ve 3β,7α-dihidroksiandrost-5-en-17-on (18) metabolitlerini verdiği anlaşıldı.

Although natutral products are compounds that are not essential for the reproduction and development of living things, they provide benefits to the living things and attract more attention due to their effects on other living things. These compounds are usually divided into some groups such as terpenoids, alkaloids, steroids, polyketides, peptides, phenylpropanoids, specialized peptides, specialized carbohydrates, fatty acids and their derivatives. Enzymes or enzymes within biological systems can fulfil chemical changes on ksenobiotics and these changes are called biotransformations. Enzymes or biological systems are used as free or immobilised apparatus to fulfill biotransformations. Cell cultures, tissue cultures, organ cultures, microsomes, microorganisms and spores of microorganisms are usually used as common biological systems for biotransformations. By decreasing the energy of activation (EA), enzymes can fulfill almost every reaction in living things. Although enzymes decrease the time to obtain the reaction equilibrium, enzymes are not vanished or deforrmed by the reaction and they differentiete the DG and the equilibrium position of the reaction. The International Union of Biochemistry has been announced the presence of more then 3200 enzymes and it is estimated that nature can provide 25000 enzymes. Enzymes give some advantages for their users since they are very potent catalysts, For example, enzymatic reaction rates are expedited by a factor of 108-1010 and this can even reach a value of 1012. Enzymes are environmentally friendly since they are made of amino acids and are completely degradable. Although most other chemical reagents cause environmental problems, enzymes usually work under mild conditions (around pH 7, 30 °C and 1 atm) and this reduces some problems such as, isomerisation, racemisation, rearrangements, decomposition. In a multienzyme system enzymes are compatible with each other and usually function under the same or similar conditions. Therefore, by using multienyme systems in a one flask several reactions can be fulfilled. Some enzymes are obligated to their natural role although some enzymes have a high substrate telorance. These enzymes can react with a large variety of natural or unnatural compounds. Enzymes can fulfill a broad spectrum of reactions and there is an enzyme-catalysed reaction alternative to almost every known reaction. Enzymes are chemoselective, regioselective and enantioselective molecules due to their complex three-dimensional structures. As enzymes are chemoselective, they usally reaact with just one single type of functional group and other functionalities stay unchanged. That is why enzymatic reactions generally give no by products. Since enzymes are regioselective, they can differeniate between functional groups which are chemically located in different regions of the same substrate. Enzymes are enantioselective and are chiral catalysts since they are only made from L-amino acids. Therefore, any type of chrality on substrate molecule is sensed by enzymes. A prochiral substrate can be turned into a chiral product and both enantiomers in a racemic substrate generally can react at different rates, resulting in a kinetic resulotion. Unfortunately, there are also some restrictions for using enzymes. Nature limits enzymes to one of enantiomers. When the other type of enantiomeric product is needed, an enzyme with exactly the opposite stereochemical selectivity is required. However, this is usually impossible. Enzymes usually have narrow operation parameters. Fulfilling under mild conditions often result in obstacles as raised up temperatures and extreme pH lead to enzyme inhibition. Although enzymes have their highest catalytic activity in water, water is usually the least suitable solvent for most organic reactions due to its high boiling point and high heat of vaporisation. Moreover, most organic compounds show very low solubilities in aqueous media. That is why shifting enzymatic reaction from an aqueous medium to an organic medium might be higly desired. However, this can result in loss of catalytic activity due to enzyme denaturation. Enzymes are very boun to their natural cofactors. Although enzymes are exceedingly malleable for reacting unnatural substrates they are almost completely bound to their natural cofactors such as NADH and NADPH. However, these molecules are relatively unstable and too expensive to use in stoichiometric amounts and are not changeable by their more economical man-made substitues. Enzymes are liable to inhibition phenomena. Many enzymatic reactions are responsive to substrate or product inhibition which makes enzymes stop working at higher substrate and / or product concentrations. Enzymes can also disturb their users due to allergies. Although enzymes can cause allergies this can be diminished by considering enzymes as chemicals and using with the same care. Biotransformations are usally fulfilled either by isolated enzyme systems or by intact whole microorganisms. It is thought that there are more than 300 commercially available isolated enzyme systems. As many enzyme systems which are involved are membrane bound and difficult to obtain, intact whole microorganisms are generally used for biotransformations. Molds, yeasts, bacteria and microalgae are usually used for microbial biotransformations. Microorganisms have some non-specific enzyme systems. That is why microornanisms fulfill a number of reactions on both natural and synthetic substrates. Microbial hydroxylations are the most common and are favourite among these reactions. The importance of microbial hydroxylation was first observed in 1952 when it solved a major problem in the cortical steroid synthesis. The addition of an oxygen function at C-11 was a very long difficult and expensive work as this position was remote from any other functional groups. This obstacle was effectively terminated by Rhizopus arrhizus by microbial hydroxylation. This microbial hydroxylation made microbial biotransformations popular. Since then, different types of steroids have been genereally used for microbial biotransformations. Microbial steroid biotransformations have widely applied for the production of some important steroid hormones and drugs due to their high regio- and stereoselectivity. A number of microbial steroid biotransformations have been reported in recent years. There are still a lot of efforts to increase the effectiveness of known microbial biotransformations and to obtain new handy microorganisms and reactions. Since first microbial hydroxylation was reported in 1952, a lot of different fungi have consistently been one of the most investigated whole-cell systems for biotransformation reactions. Different fungi have been used for the biotransformations of many types of steroids. These biotransformations gave some interesting results, such as microbial hydroxylations, Baeyer-Villiger oxidations and 5α-reduction. In this work, dehydroepiandrosterone (9), which is also known as DHEA, was incubated with Aspergillus glaucus MRC 200914 in order to see how this fungus metabolises the substrate. The medium was prepared for the fungus in 1 L of distilled water. The medium was evenly disrubuted into 10 erlenmeyer flasks of 250 mL and sterilized by an autoclave. These flasks were inoculated by the fungus. The flasks were incubated for 3 days at 25 ºC on a shaker and the substrate in DMF was added aseptically into these flasks. All flasks were further incubated for 5 days under the same conditions. After incubation, the fungal mycellium was separated from the broth by filtration under the vacuum. The mycellium was rinsed with ethyl acetate and the broth was then extracted with ethyl acetate. The extracts were dried over sodium sulfate anhydrous and evaporated in vacuo to give a brown gum that was then chromatographed on silica gel 60. 3β,11α-dihydroxyandrost-5-ene-17-one (16) and 3β,7α-dihydroxyandrost-5-ene-17-one (18) were obtained from the chromatography work. The structures of these compounds were determined by comparing melting points, NMR and IR spectra of the starting material with those of metabolites.