Testosteron bileşiğinin aspergıllus glaucus küfü ile blyotramsformasyonu
Biotransformation of testosterone by aspergillus glaucus
Yazar: ALI ABDULHUSSEIN ALI ALI
Danışman: PROF. DR. KUDRET YILDIRIM
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Biyokimya Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Biyokimya = Biochemistry

Özet:
Doğal ürünler canlıların çoğalma ve gelişmesi için elzem olmayan bileşiklerdir. Bu
bileşikler bulundukları canlılara bazı avantajlar sağladıkları ve daha çok diğer canlılara
olan etkileri ile dikkat çeken kimyasal maddelerdir. Doğal ürünler genelde
terpenoidler, alkaloidler, steroidler, poliketidler, fenilpropanoidler, özelleşmiş
peptidtler, özelleşmiş karbonhidratlar ve yağ asitleri ile yağ asitlerinin türevleri olarak
gruplandırılırlar.
Enzimlerin subtratları olmayan yabancı olan maddelerde gerçekleştirdikleri kimyasal
değişimler biyotransformasyonlar olarak adlandırılır. Biyotransformasyonlarda rol
oynayan enzimler serbest, sabitlenmiş veya çeşitli biyolojik sistemlerin bünyesinde
mevcut olarak etki gösterebilirler. Biyotransformasyonların gerçekleştirildiği
biyolojik sistemlerin en çok kullanılanları küfler, mayalar ve bakteriler gibi mikrobiyal
hücrelerdir. Küfler, mayalar ve bakteriler gibi mikroorganizmalar ile gerçekleştirilen
mikrobiyal biyotransformasyonlar klasik kimyasal sentez yöntemlerine göre daha
fazla avantajlar sağlamaktadır. Mikrobiyal biyotransformasyonların uygulanması için
genelde çeşitli mikroorganizmalar serbest veya belirli bir yüzeye sabitlenmiş olarak
kullanılabilir.
Günümüzde küfler ile gerçekleştirilen steroid biyotransformasyonları küf enzimlerinin
yüksek regio ve stereoseçicilikleri olmaları nedeni ile birçok önemli bileşiğin elde
edilmesinde yaygın olarak uygulanmaktadır.
Bu çalışmada testosteron (6) steroidinin Aspergillus glaucus MRC 200914 küflünde
nasıl metabolize edildiğinin incelenmesi amaçlanmıştır. Bahsedilen bu amaç
doğrultusunda biyotransformasyon çalışmaları öncesinde Aspergillus glaucus küfüne
ait stok kültürlerinden periyodik olarak taze alt kültürler hazırlandı. Daha sonra
biyotransformasyon deneyi için besiyeri hazırlanıp erlenlere dağıtılarak otoklavda
sterilizasyonu gerçekleştirildi. Erlenlere en taze alt kültürden steril şartlarda küfün
nakli gerçekleştirildikten sonra bu erlenler 3 gün inkübasyona bırakıldı. İçerisinde
yeterince küf gelişen bu erlenlere ise testosteron (6) steril şartlarda ilave edilip 5 gün
daha inkübasyona devam edildi. İnkübasyon sonrasında besiyeri filtrasyona maruz
bırakıldıktan sonra steroidler etil asetat ekstraksiyonu ile organik faza geçirildi. Etil
asetat ekstraktlarının susuz Na2SO4 ile kurutulup evaporatörde uzaklaştırıldı. Elde
edilen kalıntı içerisindeki steroidler ise kolon kromatografisi yöntemi ile ayrıldı.
Biyotransformasyondan kaynaklanan steroidtlerin yapı tayinleri, erime noktaları tayini
ve bazı spektroskopik yöntemler kullanılarak gerçekleştirildi. Yapı tayinleri
neticesinde Aspergillus glaucus ile testosteron (6) biyotransformasyonunun 11α,17βdihidroksiandrost-4-en-3-on (13) ve 6β,17β-dihidroksiandrost-4-en-3-on (14)
metabolitleri ile neticelendiği anlaşıldı.

Abstract:
Compounds that are not essential for the reproduction and development of living things are called natural products. Natural products are chemical substances that provide benefits to the living things and attract more attention due to their effects on other living things. Natural products are generally clasisified under groups such as terpenoids, alkaloids, steroids, polyketides, peptides, phenylpropanoids, specialized amino acids, specialized carbohydrates and fatty acids and their derivatives.
Biological systems have the ability to perform chemical changes on ksenobiotics. The uses of biological systems for this purpose are called biotransformations. Biotransformations are carried out by free or immobilised enzymes and biological systems with enzymes. Some common biological systems widely used for biotransformations are cell cultures, tissue cultures, organ cultures, microsomes, microorganisms and spores of microorganisms.
Enzymes catalyze almost all reactions in living organisms. Enzymes lower an energy of activation (EA). Enzymes reduce the time to reach the reaction equilibrium. Enzymes are not consumed or changed by the reaction. Enzymes do not change the G and the equilibrium position of the reaction. More than 3200 enzymes have been identified by the International Union of Biochemistry. It is believed that nature may offer 25000 enzymes. 
Enzymes provide some advantages for their users. Enzymes are very effitive  catalysts, For example, the reaction rates of enzymatic reactions are accelerated by a factor of 108-1010. This can even exceed a value of 1012, that is impossible with other catalysts. Enzymes are environmentally acceptable since they are totally degradable. Most other chemical reagents cause environmental problems. Enzymes usually act under mild conditions (around pH 7, 30 °C and 1 atm). This minimises some problems (isomerisation, racemisation, rearrangements, decomposition). Since enzymes are compatible with each other, enzymes usually function under the same or similar conditions. Therefore, in one flask several reactions can be performed. This is achieved by using multienyme systems. Some enzymes are bound to their natural role. However some enzymes exhibit a high substrate telorance. They can accept a large variety of natural or unnatural compounds. Enzymes can catalyse a broad spectrum of reactions. There is an enzyme-catalysed reaction equivalent to almost every organic reaction. 
Enzymes are chemoselective, regioselective and enantioselective. Enzymes are chemoselective and generally act on just one single type of functional group and other functionalities remain unchanged. Therefore, reactions generally tend to be cleaner. Enzymes are regioselective and can distinguish between functional groups that are chemically located in different regions of the same substrate molecule. Enzymes can perform this due to their complex three-dimensional structures. Enzymes are enantioselective and are chiral catalysts as they are made from L-amino acids. As a consequense,  any type of chrality on substrate molecule is recognised by enzymes. A prochiral substrate can be converted into an optically active product and both enantiomers in a racemic substrate generally react at different rates, yielding a kinetic resulotion.  
However, there are some disadvantages for using enzymes. Enzymes are provided by nature as a one type of enantiomer. When the other type of enantiomeric product is required, an enzyme with exactly the opposite stereochemical selectivity is needed. Unfortunately, this is often impossible. Enzymes require narrow operation parameters. Working under mild conditions sometimes causes trouble since elevated temperatures and extreme pH lead to inhibition of enzymes. Enzymes display 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 are only poorly soluble in aqueous media. Therefore, shifting enzymatic reaction from an aqueous medium to an organic medium would be highly desired. Unfortunately, this may cause some loss of catalytic activity. Enzymes are bound to their natural cofactors. Although enzymes are extremely flexible for acepting unnatural substrates they are almost exclusively bound to their natural cofactors such as NADH or NADPH. However, tthese molecules are relatively unstable and too expensive to use in stoichiometric amounts. Unfortunately, they can not be replaced by their more economical man-made substitutes. Enzymes are prone to inhibition phenomena. Many enzymatic reactions are prone to substrate or product inhibition which forces enzymes to stop working at higher substrate and/or product concentrations. Some enzymes may cause allergies. Although enzymes can cause allergic reactions this can be minimised by regarding enzymes as chemicals and using them with care.
Biotransformations are generally performed either by isolated enzyme systems or by intact whole microorganisms. There are more than 300 commercially available isolated enzyme systems. As many enzyme systems that are involved are membrane bound and difficult to isolate, intact whole microorganisms are often used for biotransformations. Main groups of microorganisms widely used for biotransformations are molds, yeasts, bacteria and microalgae. 
Microorganisms perform a number of reactions with both natural and synthetic substrates by utilizing their non-specific enzyme systems.  Microbial hydroxylations are very common and are favourite among these reactions. The value of microbial hydroxylation was first noticed in 1952 when it helped to overcome a major problem in the cortical steroid synthesis. The insertion of an oxygen function at C-11 was a very difficult and expensive process since that position was remote from other functional groups. This insertion was efficiently performed by Rhizopus arrhizus via microbial hydroxylation. Microbial biotransformations became popular with this microbial hydroxylation. Since then, steroids and a number of other different substrate groups have been widely used for microbial biotransformations. Microbial steroid biotransformations have found world wide application for the preparation 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 tremendous attempts to increase the efficiency of microbial biotransformations and to find new useful microorganisms and reactions. Since first microbial hydroxylation was reported in 1952, fungi have routinely been one of the most studied whole cell systems for biotransformation reactions. Different fungi have been used for the biotransformations of many steroids. These biotransformations afforded some interesting results, such as microbial hydroxylations, Baeyer-Villiger oxidations and 5α-reduction.
In this work, testosterone (6) has been incubated with Aspergillus glaucus MRC 200914 in order to see how this fungus metabolise the substrate. The liquid medium was prepared for the fungus in 1 L of distilled water. The medium was evenly disrubuted into 10 erlenmeyer flasks of 250 mL were 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. Ethyl acetate extracts were dried over sodium sulfate anhydrous. The solvent evaporated in vacuo to give a brown gum that was then chromatographed on silica gel 60. 11α-hydroxyandrost-4-ene-3,17-dione (13) and 6β-hydroxyandrost-4-ene-3,17-dione (14) were obtained from the chromatography work. The structure determination of the steroids was carried out by comparing melting points, NMR and IR spectra of the starting material with those of metabolites.