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

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.

Replacement of Pregastric Lipases in Cheese Production: Identification and Heterologous Expression of a Lipase from Pleurotus citrinopileatus.

Traditionally produced piquant cheeses such as Feta or Provolone rely on pregastric lipolytic enzymes of animal origin to intensify flavor formation during ripening. Herein, we report a novel fungal lipase, derived from the phylum Basidiomycota to replace animal-derived products. A screening of 31 strains for the desired hydrolytic activities was performed, which revealed a promising fungal species. The secretome of an edible golden oyster mushroom, Pleurotus citrinopileatus, provided suitable enzymatic activity, and the coding sequence of the corresponding enzyme was identified by combining transcriptome and liquid chromatography high-resolution electrospray tandem mass spectroscopy (LC-HR-ESI-MS/MS) data. Recombinant expression in Escherichia coli BL21 (DE3) using chaperones GroES-GroEL and DnaK-DnaJ-GrpE was established. The recombinant lipolytic enzyme was purified and biochemically characterized in terms of thermal and pH stability, optimal reaction conditions, and kinetic data toward p-nitrophenyl esters. An application in the microscale production of Feta-type brine cheese revealed promising sensory properties, which were confirmed by headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses in comparison with the reference enzyme opti-zym z10uc from goat origin. Supplementation with 2.3 U of the heterologously expressed fungal lipase produced the most comparable free fatty acid profile after 30 days of ripening. The flavor and texture formed during the application of the new lipase from P. citrinopileatus proved to be competitive to the use of pregastric lipases and could therefore replace the products of animal origin.

Evolutionary and structural analyses of mammalian haloacid dehalogenase-type phosphatases AUM and chronophin provide insight into the basis of their different substrate specificities.

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg(2+)-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5'-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.