Highly efficient biosynthesis of phosphatidylserine by the surface adsorption-catalysis in purely aqueous media and mechanism study by biomolecular simulation

Wang, Jiao; Qi, Xinyu; Yu, Wenyu; Zhang, Xiaoli; Zhang, Tiantian; Li, Binglin

doi:10.1016/j.mcat.2021.111397

Cited by 5 publications

(5 citation statements)

References 27 publications

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“…In addition, a large number of PA byproducts were generated during the transformation process . In previous studies, the hydrolysis reaction rates of PLD could be reduced by changing the reaction system , or using immobilized enzymes, − which is difficult to scale up. Herein, we employed protein engineering to enhance the transphosphatidylation efficiency by increasing the space and hydrophobicity of PLD as well as manipulating the coordination of l -Ser within the active site.…”

Section: Discussionmentioning

confidence: 99%

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Liu²,

Song

et al. 2022

J. Agric. Food Chem.

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Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A “reconstruct substrate pocket” strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (Sr MBPPLDMu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu6 produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.

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Section: Discussionmentioning

confidence: 99%

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Liu²,

Song

et al. 2022

J. Agric. Food Chem.

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“…They all had the same template, a human gastric lipase (PDB: 1HLG). The details on homology modeling have been reported previously . Generated models were further optimized in a water box of 90 × 90 × 90 Å 3 by MD simulation.…”

Section: Methodsmentioning

confidence: 99%

“…The details on homology modeling have been reported previously. 20 Generated models were further optimized in a water box of 90 × 90 × 90 Å 3 by MD simulation. The temperature was set to 30 °C.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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

Sowa

Kreuter

Sella

et al. 2022

J. Agric. Food Chem.

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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.

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“…Next, an MD simulation (NAMD v.2.14 with standard CHARMM36 force field) was used to study their kinetic behaviors [ 21 , 22 ]. The temperature and pressure were set to 37 °C and 1 atm [ 23 ]. The MD simulation time was set as 240 ns.…”

Section: Methodsmentioning

confidence: 99%

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

Broel

Sowa

Manhard³

et al. 2022

Foods

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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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Highly efficient biosynthesis of phosphatidylserine by the surface adsorption-catalysis in purely aqueous media and mechanism study by biomolecular simulation

Cited by 5 publications

References 27 publications

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

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

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

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