Replacement of Pregastric Lipases in Cheese Production: Identification and Heterologous Expression of a Lipase from Pleurotus citrinopileatus .
Miriam A SowaNadja KreuterNadine SellaWendell AlbuquerqueJulia ManhardAlexander SieglParviz GhezellouBinglin LiBernhard SpenglerEdgar WeichhardMartin RühlHolger ZornMartin GandPublished in: Journal of agricultural and food chemistry (2022)
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.
Keyphrases
- gas chromatography mass spectrometry
- liquid chromatography
- mass spectrometry
- ms ms
- escherichia coli
- high resolution
- gas chromatography
- solid phase extraction
- tandem mass spectrometry
- poor prognosis
- single cell
- high resolution mass spectrometry
- fatty acid
- electronic health record
- simultaneous determination
- ultra high performance liquid chromatography
- high performance liquid chromatography
- liquid chromatography tandem mass spectrometry
- binding protein
- big data
- rna seq
- cell wall
- magnetic resonance
- genome wide
- long non coding rna
- machine learning
- solid state
- staphylococcus aureus
- genetic diversity
- pseudomonas aeruginosa
- high speed
- heat shock
- contrast enhanced
- artificial intelligence
- clinical evaluation