## 3 Results
### Substrate Specificity of Y. lipolytica Extracellular Lipases in Crude and Purified Lipase Solutions
It was assessed whether there may be a correlation between the production of extracellular lipases by the lipolytic yeast species and the level of free fatty acids present in the plant oil used as the sole carbon source in the medium. The acid values were then determined, and the results of these measurements are presented in Table 3. The acid values of the oils (free fatty acid content) were higher for olive oil (0.66) and peanut oil (0.55) than for rice oil (0.39) and corn oil (0.25 mg KOH/g). The effect of the yeast strain used, the type of plant oil in the culture medium, and the type of the medium were investigated in relation to the extracellular activity of lipases synthesized by _Y. lipolytica_ yeast cells using a Latin square design. The results of the experiment are presented in the form of main effects plots in Figure 4. The analysis of variance of the experimental data showed no significant effect of the two factors, i.e., plant oil and yeast strain, on the extracellular hydrolytic activity and specific activity. Only the type of medium used was statistically significant. The next stage of the current work was the determination of the substrate specificity of the enzymes in the supernatant (cell-free extract) from the cultures of three _Y. lipolytica_ strains. It is worth mentioning that the ATCC 18942 strain, due to its very low production of lipolytic enzymes (Figure 3), was excluded from further research. After 48 h of yeast cultivation in the YPO medium, the activities of supernatants towards different _p_-nitrophenyl esters were measured. At this stage, proteins with hydrolytic activity were not purified and thus their activity was determined in a mixture of various enzymes, including hydrolytic ones, secreted by yeast cells into the medium. The esters used for this part of the research were derivatives of fatty acids: butyric acid (C4:0), lauric acid (C12:0), and oleic acid (C18:1). Derivatives of these three fatty acids were selected because they enabled the determination  
Figure 4: Main effects plot for _Y. lipolytica_ extracellular lipase activity after 65 h shaken cultures. The first third of the figure shows lipase activity obtained when the medium was supplemented with plant oils; the second third of the figure shows lipase activity in two media—YP rich medium or YNB minimal medium; and the third part of the figure shows lipase activity according to four different yeast strains. of the affinity of enzyme proteins for short-, medium-, and long-chain carbon sources. Analyzing the data presented in Figure 5, it can be observed that the highest hydrolytic activities were obtained for the two yeast strains of _Y. lipolytica_, i.e., KKP 379 and W29. Both of these strains showed a high affinity for the substrates used, but the highest was noted for esters of medium-chain fatty acids. The hydrolytic activity for the C12 ester was 0.199 U/mL and 0.143 U/mL for the KKP 379 and W29 strains, respectively. _Y. lipolytica_ KKP 379 also showed an activity of 0.107 and 0.100 U/mL for the butyric and oleic acid esters. In the case of the W29 strains, these activities were slightly lower, at 0.071 and 0.088 U/mL, respectively. In the case of the ATCC 90812 strain, the hydrolytic enzymes present in this supernatant had the highest activity towards _p_-nitrophenyl butyrate (0.139 U/mL), but in the other two cases (for laurate and oleate esters), the activity was significantly lower compared to other strains. It can therefore be concluded that the cell-free extract of this strain was dominated by esterases (EC 3.1.1.1) and/or the amount of lipolytic proteins was small, while for the strains KKP 379 and W29, lipases (EC 3.1.1.3) were in the majority and/or the activity of these enzymes was relatively high. Due to the observed high activity about the oleic acid ester, comparable to the activity of the W29 strain, and the higher activity about the lauric acid derivative, the KKP 379 strain was selected for the next step, the purpose of which was to purify lipolytic enzymes.