Background: Substrate specificity determinants of mammalian haloacid dehalogenase (HAD) phosphatases are poorly understood. Results: AUM (aspartate-based, ubiquitous, Mg 2ϩ -dependent phosphatase) is a novel tyrosine phosphatase and paralog of the serine/threonine-and pyridoxal 5Ј-phosphate phosphatase chronophin. Conclusion: Conserved cap residues in AUM or chronophin determine phosphatase substrate specificity. Significance: These findings provide a starting point for structure-based development of HAD phosphatase inhibitors.
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.
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.
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