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.

Substrate specificities of baking lipases for use in fine bakery goods

“…The FA substrate specificities of lipases can be modified by altering the amino acid sequence of the protein causing, e.g., a steric hindrance for long-chain FA. This approach is commonly applied and has been used, e.g., for the design of a lipase for cheese making [30]. However, the design of tailored lipases is expensive and time-consuming.…”

“…For the analysis of lipase substrate specificity, only methods for the determination of lipase activity depending on FA chain length (FA substrate specificity) have been established so far. For this means, the use of the aforementioned p-nitrophenyl assay is common and has been applied by a range of groups [30,31,105] . The p-nitrophenyl esters are available with FA residues of varying chain lengths.…”

“…Therefore, their FA substrate specificity is important: while short-chain FA like butyric acid cause off-flavours at high concentrations, unsaturated long-chain FA are prone to oxidation and can also lead to undesired volatile compounds. Depending on the desired flavour, lipases with matching FA substrate specificities are therefore applied or newly designed, as done for the manufacturing of cheese [30]. For baking purposes, different effects on the baking quality of breads depending on the alcohol moiety substrate specificity of lipases were shown by Melis et al [32].…”

Rhodamine B‐based lipase activity assay with natural substrates—development and application

“…While the preference for certain fatty acids (fatty acid substrate specificity, FASS) is used to modify sensory properties, the preference for the specific alcohol moieties (AMSS) is decisive for the technological properties of the resulting product. As an example, FASS can be used to modify aromas and flavours of cheeses (Broel et al ., 2022). For baking applications, the AMSS of lipids is decisive (Melis et al ., 2017; Stemler, 2023): while lipases with suitable reaction patterns can improve the baking quality of bread and cakes, the release of different alcohol moieties does not affect the properties of baked goods.…”