## 3 Results and Discussion
Both strains are known for the high activity of enzymes associated with cytochrome P450 [33], which explains their high tolerance in the dehalogenation reaction to various substrates. Taking into account the results obtained during the screening biotransformation, we decided to perform the preparative scale biotransformation on only two selected strains: The results of this step are presented in Table 2.  
Both strains: _C. japonica_ and _F. culmorum_, converted lactones **2-4** into the same product, which was hydroxylactone **5**. The best degree of conversion was observed for bromolactone **3** when the _F. culmorum_ stain was used as a biocatalyst (78.8% of lactone **5**). In the case of _C. japonica_, the degree of conversion was similar for all three substrates, the amount of product ranging from 55% to 60%. In the next step, an isolated yield, the enantiospecificity and optical purity of hydroxylactone **5** obtained during all preparative biotransformation were determined. The results of this step are shown in Table 3.  
As we can see in Table 3, the enantiomeric excesses of all hydroxylactones were low. Such a situation was probably caused by the structure of the substrates, in which only one steric hindrance was the methyl group at C-5 carbon. The hydroxylactone **5** with the highest enantiomeric excess (17.7%) was obtained from bromolactone **3**. In all cases, hydroxylactone was created with a slight excess of (+)-enantiomer. During the conducted biotransformation, the strong impact of the structure of lactones **2-4** on the possibility of their conversion into hydroxylactone could be observed. Because of that, the structures of these compounds were established on the basis of their spectral data (\({}^{1}\)H NMR, \({}^{13}\)C NMR, COSY, HMQC and IR) and confirmed by HRMS analysis. The absorption bands lying at 1787 (for **2**), 1799 (for **3**) and 1792 (for **4**) cm\({}^{-1}\) on the IR spectra confirmed the presence of the \(\gamma\)-lactone ring in these molecules. Analysis of the \({}^{1}\)H NMR spectra proved that the cyclohexane ring in these three lactones took a chair conformation. Wide multiplet coming from proton H-4 suggested the equatorial orientation of the CH\({}_{3}\)-9 group. The shapes of signals coming from proton H-6 (wide multiplet) and proton H-1 (small coupling constant) indicated their axial (H-6) and equatorial (H-1) orientation. It means that the C-O bond of lactone ring is located in an axial position. Signal coming from proton H-2 looked like a wide singlet suggesting its equatorial orientation and thereby indicating that the halogen atom assumed an axial position. Analysis of \({}^{1}\)H NMR spectra of halolactones **2-4** and hydroxylactone **5** proved that the cyclohexane ring in these four lactones took a chair conformation. The only difference in the structure of the substrates and product was the location of the halogen atom and hydroxy group. In the molecules of \begin{table}
\begin{tabular}{c c c c c c c} \hline
**Microorganism** & **Substrate (2)** & **Product (5)** & **Substrate (3)** & **Product (5)** & **Substrate (4)** & **Product (5)** \\ \hline _C. japonica_ AM472 & 40.0 & 60.0 & 44.8 & 55.2 & 42.5 & 57.5 \\ _F. culmorum_ AM10 & - & - & 21.1 & 78.8 & - & - \\ \hline \end{tabular}
\end{table} 
Table 2: Results of the preparative scale biotransformations of halolactones **2-4** after 7 days of incubation (in % according to GC). \begin{table}
\begin{tabular}{c c c c} \hline
**Substrate** & **Strain** & **Isolated Yield mg/\%** & _ee (\%)_ \\ \hline chlorolactone **2** & _C. japonica_ AM472 & 10/11.1 & 4.0 \\ bromolactone **3** & _F. culmorum_ AM10 & 27/37.4 & 14.9 \\ bromolactone **3** & _C. japonica_ AM472 & 16/21.9 & 17.7 \\ iodolactone **4** & _C. japonica_ AM472 & 16/25.7 & 6.4 \\ \hline \end{tabular}
\end{table} 
Table 3: Results of preparative biotransformation of lactones **2-4**. lactones **2-4**, the halogen atom assumed an axial position. In the molecule of the hydroxylactone **5**, the hydroxy group occupied an equatorial position (Figure 1).