Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

Evolution and properties of alanine racemase from Synechocystis sp. PCC6803.

Alanine racemase (EC 5.1.1.1) depends on pyridoxal 5'-phosphate and catalyses the interconversion between L- and D-Ala. The enzyme is responsible for the biosynthesis of D-Ala, which is an essential component of the peptidoglycan layer of bacterial cell walls. Phylogenetic analysis of alanine racemases demonstrated that the cyanobacterial enzyme diverged before the separation of gram-positive and gram-negative enzymes. This result is interesting considering that the peptidoglycans observed in cyanobacteria seem to combine the properties of those in both gram-negative and gram-positive bacteria. We cloned the putative alanine racemase gene (slr0823) of Synechocystis sp. PCC6803 in Escherichia coli cells, expressed and purified the enzyme protein and studied its enzymological properties. The enzymatic properties of the Synechocystis enzyme were similar to those of other gram-positive and gram-negative bacterial enzymes. Alignment of the amino acid sequences of alanine racemase enzymes revealed that the conserved tyrosine residue in the active centre of most of the gram-positive and gram-negative bacterial enzymes has been replaced with tryptophan in most of the cyanobacterial enzymes. We carried out the site-directed mutagenesis involving the corresponding residue of Synechocystis enzyme (W385) and revealed that the residue is involved in the substrate recognition by the enzyme.

Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering.

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.

Production of active manganese peroxidase in Escherichia coli by co-expression of chaperones and in vitro maturation by ATP-dependent chaperone release.

Manganese peroxidase (MnP) is a fungal heme-containing enzyme which oxidizes Mn2+ to Mn3+, a diffusible and strong non-specific oxidant capable of attacking bulky phenolic substrates. Therefore, MnP is indispensable in the polymer and paper industries. Previous attempts of MnP expression in Escherichia coli resulted in the formation of inclusion bodies which required in vitro refolding. Aiming to investigate the bacterial production of MnP, we have revealed an interesting mechanism underlying chaperone-assisted maturation of this enzyme to its active form. Since we previously found that in vitro expression of MnP in E. coli system depends on disulfide bond isomerase DsbC, we chose SHuffle T7 Express, an E. coli constitutively expressing DsbC, as the host for in vivo expression of MnP. Initially, only a low amount of the enzyme was present in the soluble fraction, with no detectable peroxidase activity. Co-expression of MnP with different chaperone revealed that DnaK, DnaJ, and GrpE contributed the most to the solubility improvement, however, remained in a complex with the MnP, preventing the enzyme to assume its active conformation. We resolved this by in vitro maturation, involving incubation of the MnP-chaperone complex with hemin, ATP, and ATP regeneration system. While ATP enables the chaperones to finish the refolding cycle and release the MnP in its correctly folded form, hemin supports the formation of the holo-enzyme with fully recovered peroxidase activity. We believe that the findings of this paper will serve as an important clue for establishing the bacterial production of fungal peroxidases in the future.

Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein-coupled receptor and TMEM16 scramblases.

Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([3H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [3H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [3H]PI hydrolysis increased, indicating that [3H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.

Salt-induced increase in the yield of enzymatically synthesized phosphatidylinositol and the underlying mechanism.

The purpose of this study was to improve the efficiency of enzymatic synthesis of phosphatidylinositol (PI) from phosphatidylcholine (PC) and myo-inositol in a phospholipase D (PLD)-mediated transphosphatidylation. A conventional biphasic reaction system consisting of ethyl acetate and an aqueous buffer afforded PI with a yield of 14 mol%. In contrast, the reaction performed in the presence of high concentration (0.8-4.3 M) of NaCl in the aqueous phase showed improved PI yield in a NaCl concentration-dependent manner. At 4.3 M NaCl, PI yield of as much as 35 mol% was achieved. The increase in the PI yield offered by other tested salts varied; however, we observed that some salts caused inactivation of the enzyme when used at high concentrations. Although NaCl at high concentration increased the apparent hydrolytic activity on aggregated PC, it decreased the activity towards monomeric PC, indicating that high concentration of salt intrinsically inhibits the enzyme. Binding assays revealed that PLD re-localized from the aqueous phase to the solvent-buffer interface, where the enzymatic reaction takes place, in the presence of both, the salt and PC. Hence, we concluded that improvement of the PI synthesis in the presence of salt occurs mainly due to the accumulation of the enzyme at the interface by strengthening the hydrophobic interactions, by which the apparent activation outweighs the salt-induced inhibitory effect. Using this improved system, several PI with defined structures, namely sn-1, 2-dioleoyl-PI, sn-1-palmitoyl-2-oleoyl-PI, and sn-1-stearoyl-2-arachidonoyl-PI, were successfully synthesized with overall yields of 25-37%, and PI isomeric purities of 91-96%.

Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D.

Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo-inositol as an alcohol acceptor, our previous study started with the wild-type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI-synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1-PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1-PI and 3-PI in the ratio of 76/24, by subjecting four residues of its acceptor-binding site to saturation mutagenesis. Subsequent screening pointed at NYR-186T and NYR-186L as the most improved variants producing PI with a ratio of 1-/3-PI = 93/7 and 87/13, respectively, at 37°C. Lowering the reaction temperature further improved the specificity of both variants to 1-/3-PI > 97/3 at 20°C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor-binding site, thus limiting the possible orientations of myo-inositol. The two newly isolated PLDs are promising for future application in large-scale 1-PI production.

Extracellular production of Pseudozyma (Candida) antarctica lipase B with genuine primary sequence in recombinant Escherichia coli.

An Escherichia coli expression system was established to produce recombinant extracellular Pseudozyma (Candida) antarctica lipase B (CALB). With the aim of producing the genuine CALB without additional amino acid residues, the mature portion of the CALB gene was fused seamlessly to a pelB signal sequence and expressed in E. coli BL21(DE3) using the pET system. Inducing gene expression at low temperature (20°C) was crucial for the production of active CALB; higher temperatures caused inclusion body formation. Prolonged induction for 48 h at 20°C allowed for the enzyme to be released into the culture medium, with more than half of the activity detected in the culture supernatant. A catalytically inactive CALB mutant (S105A) protein was similarly released, suggesting that the lipid-hydrolyzing activity of the enzyme was not the reason for the release. The CALB production level was further improved by optimizing the culture medium. Under the optimized conditions, the CALB in the culture supernatant amounted to 550 mg/L. The recombinant CALB was purified from the culture supernatant, yielding 5.67 mg of purified CALB from 50 mL of culture. N-terminal sequencing and ESI-MS analyses showed proper removal of the pelB signal sequence and the correct molecular weight of the protein, respectively, confirming the structural integrity of the recombinant CALB. The kinetic parameters towards p-nitrophenylbutyrate and the enantiomeric selectivity on rac-1-phenylethylacetate of the recombinant CALB were consistent with those of the authentic CALB. This is the first example of E. coli-based extracellular production of a CALB enzyme without extra amino acid residues.

Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae.

The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites. Therefore, no extra amino acids are inserted at the N-terminus of the target protein that might affect its function or conformational stability. As a model system, ß-galactosidase (LacA) from Aspergillus oryzae was used as a target protein for secretion from Saccharomyces cerevisiae. In total, 60 SPs were selected from S. cerevisiae secretory proteins and utilized to generate the SP library. While many of the SP-LacA fusions were not secreted, several of the SPs, AGA2, CRH1, PLB1, and MF(alpha)1, were found to enhance LacA secretion compared to the WT sequence. Our results indicate that SPOT is a valuable method for optimizing the bioproduction of any target protein, and could be adapted to many host strains.

Deletion of a dynamic surface loop improves stability and changes kinetic behavior of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

Supplementary phosphatidylinositol (PI) was shown to improve lipid metabolism in animals, thus it is interesting for pharmaceutical and nutritional applications. Homogenous PI can be produced in transphosphatidylation of phosphatidylcholine (PC) with myo-inositol catalyzed by phospholipase D (PLD). Only bacterial enzymes able to catalyze PI synthesis are Streptomyces antibioticus PLD (SaPLD) variants, among which DYR (W187D/Y191Y/Y385R) has the best kinetic profile. Increase in PI yield is possible by providing excess of solvated myo-inositol, which is achievable at high temperatures due to its highly temperature-dependent solubility. However, high-temperature PI synthesis requires the thermostable PLD. Previous site-directed combinatorial mutagenesis at the residues of DYR having high B-factor yielded the most improved variant, D40H/T291Y DYR, obtained by the combination of two selected mutations. D40 and T291 are located within dynamic surface loops, D37-G45 (termed D40 loop) and G273-T313. Thus, in this work, thermostabilization of DYR SaPLD was attempted by rational design based on deletion of the D40 loop, generating two variants, Δ37-45 DYR and Δ38-46 DYR PLD. Δ38-46 DYR showed highest thermostability as its activity half-life at 70°C proved 11.7 and 8.0 times longer than that of the DYR and Δ37-45 DYR, respectively. Studies on molecular dynamics predicted Δ38-46 DYR to have the least average RMSD change as temperature dramatically increases. At 60 and 70°C, both mutants synthesized PI in a twofold higher yield compared to the DYR, while at the same time produced less of the hydrolytic side-product, phosphatidic acid.

A chromogenic substrate for solid-phase detection of phospholipase A2.

A method for solid-phase detection of phospholipase A2 (PLA2) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA2. The substrate is hydrolyzed by PLA2 into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA2 spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA2 were distinguishable from those producing an inactive mutant PLA2, facilitating high-throughput screening in directed evolution of the enzyme.

Role of disulfide bond isomerase DsbC, calcium ions, and hemin in cell-free protein synthesis of active manganese peroxidase isolated from Phanerochaete chrysosporium.

A cell-free protein synthesis system can produce various types of proteins directly from DNA templates such as PCR products, and therefore attracts great attention as an alternative protein synthesis system especially for high-throughput functional screening of proteins. Here, we report successful expression of active Phanerochaete chrysosporium manganese peroxidase (MnP) in an Escherichia coli cell-free protein synthesis system, wherein reaction conditions such as the concentrations of hemin, calcium ions, and disulfide bond isomerase were optimized to increase the solubility and activity of the synthesized enzyme. Moreover, cell-free synthesized MnP purified using the hemagglutinin tag showed higher specific activity than the commercial wild-type enzyme, suggesting that the cell-free system can be used as a preparative method for efficient synthesis of disulfide bond-containing metalloenzymes such as MnP. We believe that our system is a solid foundation for the development of a high-throughput screening method for the directed evolution of these enzymes.

Phospholipase D as a catalyst: application in phospholipid synthesis, molecular structure and protein engineering.

Phospholipase D (PLD) is a useful enzyme for its transphosphatidylation activity, which enables the enzymatic synthesis of various phospholipids (PLs). Many reports exist on PLD-mediated synthesis of natural and tailor-made PLs with functional head groups, from easily available lecithin or phosphatidylcholine. Early studies on PLD-mediated synthesis mainly employed enzymes of plant origin, which were later supplanted by ones from microorganisms, especially actinomycetes. Many PLDs are members of the PLD superfamily, having one or two copies of a signature sequence, HxKxxxxD or HKD motif, in the primary structures. PLD superfamily members share a common core structure, and thereby, a common catalytic mechanism. The catalysis proceeds via two-step reaction with the formation of phosphatidyl-enzyme intermediate. Both of the two catalytic His residues are critical in the reaction course, where one acts as a nucleophile, while the other functions as a general acid/base. PLD is being engineered to improve its activity and stability, alter head group specificity and further identify catalytically important residues. Since the knowledge on PLD enzymology is constantly expanding, this review focuses on recent advances in the field, regarding PLD-catalyzed synthesis of bioactive PLs, deeper understanding of substrate recognition and binding mechanism, altering substrate specificity, and improving thermostability. We introduced some of our recent results in combination with existing facts to further deepen the story on the nature of this useful enzyme.

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli.

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.

Metabolic profiling of ß-cryptoxanthin and its fatty acid esters by supercritical fluid chromatography coupled with triple quadrupole mass spectrometry.

In this study, a high-throughput and high-sensitivity profiling system for ß-cryptoxanthin (ßCX) and ß-cryptoxanthin fatty acid ester (ßCXFA) was constructed by supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry (QqQMS). ßCX and nine ßCXFAs were successfully separated within 20 min using a column packed with octadecylsilyl-bonded silica particles. The limit of detection was 540 fmol for the free form and 32-130 fmol for the esterified forms. These results demonstrate that both the throughput and the sensitivity of this SFC-QqQMS system are considerably higher than those of conventional methods. When this system was applied for the analysis of Citrus unshiu, ßCX and five ßCXFAs were directly detected with much simpler sample pre-preparation. The analysis of other citrus fruits indicated that the ßCXFA profiles varied with their breed variety. Furthermore, gas chromatography-mass spectrometry was used to analyze total fatty acid profiles in C. unshiu, and the results revealed that the profiles of fatty acids located in ßCXFA were distinct. This is the first report on the analysis of ßCX and its fatty acid derivatives by SFC-QqQMS. The profiling system developed in this study will be a powerful tool for investigating xanthophyll fatty acid esters.

Synthesis of phospholipids containing polyunsaturated fatty acids by phospholipase A(2)-mediated esterification with food-compatible reagents.

Enzymatic synthesis of phospholipids (PLs) containing polyunsaturated fatty acids (PUFAs) was studied. The main purpose was to establish an efficient production method for PLs containing docosahexaenoic acid or eicosapentaenoic acid using only food-compatible reagents. Phospholipase A(2) (PLA(2))-mediated ester synthesis was employed to introduce the PUFAs into the sn-2 position of lysophospholipid (LPL) to yield PUFA-containing PLs. When LPL and the fatty acids were reacted in glycerol in the presence of porcine pancreas PLA(2), the reaction was not very effective. However, it was found that addition of certain kinds of amino acids such as glycine or L-alanine in the reaction mixture improved the reaction. After the reaction, the synthesized PLs were extracted selectively with ethanol and n-hexane, leaving the unreacted LPL, amino acids and the enzyme remained in the glycerol layer. It was confirmed that the enzyme remained in the glycerol layer could be reused by adding fresh substrates for the subsequent reactions.