Altering the Chain Length Specificity of a Lipase from Pleurotus citrinopileatus for the Application in Cheese Making

Abstract

In traditional cheese making, pregastric lipolytic enzymes of animal origin are used for the acceleration of ripening and the formation of spicy flavor compounds. Especially for cheese specialities, such as Pecorino, Provolone, or Feta, pregastric esterases (PGE) play an important role. A lipase from Pleurotus citrinopileatus could serve as a substitute for these animal-derived enzymes, thus offering vegetarian, kosher, and halal alternatives. However, the hydrolytic activity of this enzyme towards long-chain fatty acids is slightly too high, which may lead to off-flavors during long-term ripening. Therefore, an optimization via protein engineering (PE) was performed by changing the specificity towards medium-chain fatty acids. With a semi-rational design, possible mutants at eight different positions were created and analyzed in silico. Heterologous expression was performed for 24 predicted mutants, of which 18 caused a change in the hydrolysis profile. Three mutants (F91L, L302G, and L305A) were used in application tests to produce Feta-type brine cheese. The sensory analyses showed promising results for cheeses prepared with the L305A mutant, and SPME-GC-MS analysis of volatile free fatty acids supported these findings. Therefore, altering the chain length specificity via PE becomes a powerful tool for the replacement of PGEs in cheese making.

Keywords: Pleurotus citrinopileatus; chain length specificity; cheese making; lipase; semi-rational design.

Figure 1

Relative specific activity [%] of PCI_Lip mutants (expression: n = 1, mutants marked with * n = 3) against selected model substrates. The relative specific activities were based on the specific activity [U mg⁻¹] of the PCI_Lip WT against pNPO (set to 100%, red line). The photometric esterase/lipase assays were performed in triplicates. The error bars represent the standard deviation of these measurements.

Figure 2

Feta-type brine cheese produced without addition of lipase, the reference PGE opti-zym z10uc, PCI_Lip WT (1 U), F91L (1 U), L302G (0.7 U), and L305A (1 U) after 30 d of ripening at 13 °C. Shown are peak areas of selected vFFA from the cheese samples after SPME-GC-MS analysis. The measurements were performed in duplicates and the error bar corresponds to the mean deviation. C2:0 = acetic acid, C4:0 = butyric acid, C6:0 = hexanoic acid, C8:0 = octanoic acid, and C10:0 = decanoic acid.