## 3 Results
### The Analysis of the Selected Conditions for Hexanal Biosynthesis
#### 3.2.1 Lipooxygenase Activity and Hexanal Production in the Cellular Extract and Supernatant  
One of the key parameters in the synthesis of hexanal by microorganisms is the activity of lipooxygenase, an enzyme that catalyzes the conversion of linoleic acid into 13-hydroperoxy-9,11-octadecadienoic acid. Figure 4 shows the data on the growth of yeast cells expressed in dry cell weight and the data on lipoxygenase activity in the supernatant and cellular extracts. LOX activity was measured at 12 h intervals for a period of 96 h during the growth of _Yarrovia lipolytica_ yeast on media supplemented with linoleic acid or olive oil (5 g/100 mL). Olive oil was used for a comparative purpose to test how active LOX was in the presence of a mixture of fatty acids in the medium. According to [PERSON] and [PERSON] [36], olive oil contains approximately 6.2% linoleic acid, the precursor of hexanal. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Inoculum Volume (mL/100 mL of Medium)**} & \multicolumn{5}{c}{**Concentration of Gamma-Decalactone (g/L)**} \\ \cline{2-6}  & \multicolumn{5}{c}{**Culture Time (days)**} \\ \cline{2-6}  & **1** & **2** & **3** & **5** & **7** \\ \hline
0.25 mL & 0.15 \(\pm\) 0.02 & 0.48 \(\pm\) 0.02 & 0.67 \(\pm\) 0.01 & 1.00 \(\pm\) 0.11 & 1.92 \(\pm\) 0.14 \\
0.5 mL & 0.14 \(\pm\) 0.01 & 0.31 \(\pm\) 0.03 & 0.45 \(\pm\) 0.04 & 1.32 \(\pm\) 0.14 & 2.10 \(\pm\) 0.04 \\
1 mL & 0.13 \(\pm\) 0.04 & 0.45 \(\pm\) 0.03 & 0.57 \(\pm\) 0.02 & 1.71 \(\pm\) 0.07 & 3.01 \(\pm\) 0.15 \\
2 mL & 0.11 \(\pm\) 0.03 & 0.25 \(\pm\) 0.09 & 0.64 \(\pm\) 0.07 & 0.97 \(\pm\) 0.01 & 1.29 \(\pm\) 0.06 \\
5 mL & 0.08 \(\pm\) 0.01 & 0.21 \(\pm\) 0.07 & 0.58 \(\pm\) 0.10 & 0.95 \(\pm\) 0.06 & 1.37 \(\pm\) 0.08 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Effect of inoculum size on the concentration of gamma-decalactone in the medium during the 7-day culture. The data shown in Figure 4 indicate that the yeast showed lipooxygenase activity both on a medium enriched with pure linoleic acid and on a medium with added olive oil. On the first day of the reaction, the LOX activity in the cell extract or the supernatant was comparable in both media. After 24 h, this activity varied significantly. Much higher LOX activity (2.5-5 fold) was observed in the linoleic acid medium. This is probably due to the fact that in the medium with olive oil, linoleic acid as a LOX activator was not directly available, but had to be released from triacylglycerlycerols through lipase-mediated hydrolysis. In addition, the efficiency of the olive oil hydrolysis process should also be considered in order to optimize its concentration in the culture medium. In case of the medium with the addition of linoleic acid, the activity of lipoxygenase in the cell extract after 2 days of reaction was about 2.2-2.5 times higher than the activity in the supernatant. In the analyzed period, up to 96 h of culture, the cells were in the logarithmic growth phase--the dry cell mass yield increased during this period from 2.67 \(\pm\) 0.24 g/L to 10.33 \(\pm\) 0.30 g/L. The highest LOX activity (2228.37 \(\pm\) 222.03 U/mL in the cell extract and 1002.26 \(\pm\) 153.71 U/mL in the supernatant) was obtained for this medium after 96 h of culture. The mutant yeast strain was active in converting linoleic acid to green aldehyde (Table 3). The higher activities of lipoxygenase in the cell extracts corresponded to the higher contents of hexanal in this fraction (the maximum concentration of hexanal was approximately 16 times higher relative to the supernatant fraction: 352.09 \(\pm\) 22.69 mg/L and 21.71 \(\pm\) 2.33 mg/L, respectively). In the initial period of cultivation, the concentration of hexanal in the cell extract increased, reaching its maximum concentration after about 48 h. In the supernatant, the highest concentration of hexanal was obtained in the first day of the reaction. After this period, its content decreased.