## 3 Results
### Crude Extracellular Lipase Preparations as Potential Biocatalysts  
Various studies have confirmed that the enzymes produced by _Y. lipolytica_ yeast can be used as biocatalysts in enzymatic reactions. Both purified and crude extracellular lipases, as well as whole yeast cells, have been applied in, e.g., biodiesel synthesis, ring-opening polymerization, kinetic resolution, and flavor ester synthesis [6,10]. To compare the activity of enzymes obtained from shaken cultures of _Y. lipolytica_ KKP 379, the hydrolytic activity of proteins in the model reaction of _p_-nitrophenyl laurate hydrolysis was evaluated. The following fractions of lipases were applied: extracellular lipases present in the crude solution (unpurified supernatant), and intracellular lipases from the yeast biomass both before and after cell disintegration, as well as the extract of intracellular enzymes obtained after cell disintegration. The results of the hydrolytic activity measurements are presented in Table 4.  
Intracellular enzymes, namely cell-bound ones and those from the cytosol, showed a very high enzymatic activity of 146.70 U/g (Table 4). As a result of cell disintegration using the glass beads, the cell structure was damaged, and the enzymes were released into the solution, which is reflected in the reduced activity of the yeast biomass after disintegration (54.02 U/g) compared to the initial state. This is also confirmed by the activity of the supernatant obtained after the disintegration process, which also showed a hydrolytic activity of 0.37 U/mL. The preparation obtained by physical adsorption of the supernatant after the disintegration of cells on Lewatit VP OC 1065 resin also exhibited satisfactory  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{1}{c}{**Source of Lipases**} & \multicolumn{1}{c}{**Hydrolytic Activity**} & \multicolumn{1}{c}{**Hydrolytic Activity**} \\  & **[U/mL]** & **[U/g DM]** \\ \hline Crude enzyme solution (supernatant) & 0.07 \(\pm\) 0.09 \({}^{\text{B}}\) & - \\ \hline Yeast biomass before disintegration & - & 146.70 \({}^{\text{b}}\)\(\pm\) 1.50 \\ \hline Yeast biomass after disintegration & - & 54.02 \({}^{\text{a}}\)\(\pm\) 6.32 \\ \hline Enzyme solution after yeast cell & 0.37 \(\pm\) 0.08 \({}^{\text{A}}\) & - \\ disintegration & & 232.00 \({}^{\text{c}}\)\(\pm\) 5.20 \\ \hline \hline \end{tabular}
\end{table}
Table 4: The hydrolytic activity of crude enzyme solution and yeast biomass of _Y. lipolytica_. Means with the same capital letter (A,B) or lowercase letter (a–c) did not differ significantly (\(\alpha\) = 0.05).  
Figure 7: The specific activity of extracellular lipase from _Y. lipolytica_ KKP 379 (fraction 60) towards different fatty acids esters of _p_-nitrophenol. Means with the same letter (a–d) did not differ significantly (\(\alpha\) = 0.05).  
hydrolytic activity, and 232.00 U/g was achieved. Enzymatic kinetics was also evaluated for crude extracellular lipase immobilized on the support (Figure 8). It was revealed that the enzymatic preparation retained 50% of its activity after 60 min of the hydrolysis reaction, in comparison to the activity measured after 15 min (80% after 30 min and 63% after 40 min).