Biochemical characterization of an alkaline and detergent-stable Lipase from Fusarium annulatum Bugnicourt strain CBS associated with olive tree dieback.

This work describes a novel extracellular lipolytic carboxylester hydrolase named FAL, with lipase and phospholipase A1 (PLA1) activity, from a newly isolated filamentous fungus Ascomycota CBS strain, identified as Fusarium annulatum Bunigcourt. FAL was purified to about 62-fold using ammonium sulphate precipitation, Superdex® 200 Increase gel filtration and Q-Sepharose Fast Flow columns, with a total yield of 21%. The specific activity of FAL was found to be 3500 U/mg at pH 9 and 40°C and 5000 U/mg at pH 11 and 45°C, on emulsions of triocanoin and egg yolk phosphatidylcholine, respectively. SDS-PAGE and zymography analysis estimated the molecular weight of FAL to be 33 kDa. FAL was shown to be a PLA1 with a regioselectivity to the sn-1 position of surface-coated phospholipids esterified with α-eleostearic acid. FAL is a serine enzyme since its activity on triglycerides and phospholipids was completely inhibited by the lipase inhibitor Orlistat (40 µM). Interestingly, compared to Fusarium graminearum lipase (GZEL) and the Thermomyces lanuginosus lipase (Lipolase®), this novel fungal (phospho)lipase showed extreme tolerance to the presence of non-polar organic solvents, non-ionic and anionic surfactants, and oxidants, in addition to significant compatibility and stability with some available laundry detergents. The analysis of washing performance showed that it has the capability to efficiently eliminate oil-stains. Overall, FAL could be an ideal choice for application in detergents.

Preparation, characterization, immobilization, and molecular docking analysis of a novel detergent-stable subtilisin-like serine protease from Streptomyces mutabilis strain TN-X30.

We recently described the production of a detergent-biocompatible crude protease from Streptomyces mutabilis strain TN-X30. Here, we describe the purification, characterization, and immobilization of the serine alkaline protease (named SPSM), as well as the cloning, sequencing, and over-expression of its corresponding gene (spSM). Pure enzyme was obtained after ammonium sulphate precipitation followed by heat-treatment and Sephacryl® S-200 column purification. The sequence of the first 26 NH2-terminal residues of SPSM showed a high sequence identity to subtilisin-like serine proteases produced by actinobacteria. The spSM gene was heterologously expressed in Escherichia coli BL21(DE3)pLysS and E. coli BL21-AI™ strains using pTrc99A (rSPSM) and Gateway™ pDEST™ 17 [(His)6-tagged SPSM] vectors, respectively. Results obtained indicated that the (His)6-tagged SPSM showed the highest stability. The SPSM was immobilized using encapsulation and adsorption-encapsulation approaches and three different carriers. Features of SPSM in soluble and immobilized forms were analyzed by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode, X-ray diffraction (XRD), zeta potential measurements, and field emission scanning electron microscopy (FE-SEM). The white clay and kaolin used in this study are eco-friendly binders to alginate-SPSM and show great potential for application of the immobilized SPSM in various industries. Molecular modeling and docking of N-succinyl-l-Phe-l-Ala-l-Ala-l-Phe-p-nitroanilide in the active site of SPSM revealed the involvement of 21 amino acids in substrate binding.

Purification, Biochemical and Kinetic Characterization of a Novel Alkaline sn-1,3-Regioselective Triacylglycerol Lipase from Penicilliumcrustosum Thom Strain P22 Isolated from Moroccan Olive Mill Wastewater.

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicilliumcrustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH2-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations.

Phospholipase D inhibitors screening: Probing and evaluation of ancient and novel molecules.

Phospholipase D (PLD) is a ubiquitous enzyme that cleaves the distal phosphoester bond of phospholipids generating phosphatidic acid (PA). In plants, PA is involved in numerous cell responses triggered by stress. Similarly, in mammals, PA is also a second messenger involved in tumorigenesis. PLD is nowadays considered as a therapeutic target and blocking its activity with specific inhibitors constitutes a promising strategy to treat cancers. Starting from already described PLD inhibitors, this study aims to investigate the effect of their structural modifications on the enzyme's activity, as well as identifying new potent inhibitors of eukaryotic PLDs. Being able to purify the plant PLD from Vigna unguiculata (VuPLD), we obtained a SAXS model of its structure. We then used a fluorescence-based test suitable for high-throughput screening to review the effect of eukaryotic PLD inhibitors described in the literature. In this regard, we found that only few molecules were in fact able to inhibit VuPLD and we confirmed that vanadate is the most potent of all with an IC50 around 58 µM. Moreover, the small-scale screening of a chemical library of 3120 compounds allowed us to optimize the different screening's steps and paved the way towards the discovery of new potent inhibitors.

Plant phospholipase D mining unravels new conserved residues important for catalytic activity.

Phospholipase D (PLD) is a key enzyme involved in numerous processes in all living organisms. Hydrolysis of phospholipids by PLD allows the release of phosphatidic acid which is a crucial intermediate of multiple pathways and signaling reactions, including tumorigenesis in mammals and defense responses in plants. One common feature found in the plant alpha isoform (PLDα), in some PLD from microbes and in all PLD from eukaryotes, is a duplicated motif named HKD involved in the catalysis. However, other residues are strictly conserved among these organisms and their role remains obscure. To gain further insights into PLD structure and the role of these conserved residues, we first looked for all the plant PLDα sequences available in public databases. With >200 sequences retrieved, a generic sequence was constructed showing that 138 residues are strictly conserved among plant PLDα, with some of them identical to residues found in mammalian PLDs. Using site-directed mutagenesis of the PLDα from Arabidopsis thaliana, we demonstrated that mutation of some of these residues abolished the PLD activity. Moreover, mutation of the residues around both HKD motifs enabled us to re-define the consensus sequence of these motifs. By sequential deletions of the N-terminal extremity, the minimum length of the domain required for catalytic activity was determined. Overall, this work furthers our understanding of the structure of eukaryotic PLDs and it may lead to the discovery of new regions involved in the catalytic reaction that could be targeted by small molecule modulators of PLDs.

Production, purification and biochemical characterization of a thermoactive, alkaline lipase from a newly isolated Serratia sp. W3 Tunisian strain.

A newly isolated Serratia sp. W3 strain was shown to secrete a non-induced lipase in the culture medium. Lipolytic activity was optimized using the response surface methodology (RSM) and the extracellular lipase from Serratia sp. W3 (SmL) was purified to homogeneity with a total yield of 10% and its molecular mass was estimated of about 67 kDa by SDS-PAGE. The amino acid sequence of the first 7 N-terminal residues of SmL revealed a high degree of homology with other Serratia lipase sequences. The purified SmL can be considered as thermoactive lipase, its maximal specific activity measured at pH 9 and 55 °C was shown to be 625 U/mg and 300 U/mg using tributyrin and olive oil emulsion as substrate, respectively. In contrast to other described Serratia lipases, SmL was found to be stable at a large scale of pH between pH 5 and pH 12. SmL was also able to hydrolyze its substrate in presence of various oxidizing agents as well as in presence of surfactants and some commercial detergents. Then, considering the overall biochemical properties of SmL, it can be considered as a potential candidate for industrial and biotechnological applications, such as synthesis of biodiesel and in the detergent industry.

Phospholipases: An Overview.

Phospholipases are lipolytic enzymes that hydrolyze phospholipid substrates at specific ester bonds. Phospholipases are widespread in nature and play very diverse roles from aggression in snake venom to signal transduction, lipid mediator production, and metabolite digestion in humans. Phospholipases vary considerably in structure, function, regulation, and mode of action. Tremendous advances in understanding the structure and function of phospholipases have occurred in the last decades. This introductory chapter is aimed at providing a general framework of the current understanding of phospholipases and a discussion of their mechanisms of action and emerging biological functions.

A Continuous and Sensitive Spectrophotometric Assay for Lipase and Phospholipase A Activities Using α-Eleostearic Acid-Containing Substrates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen lipase and phospholipase A (PLA) activities. Here, a new ultraviolet spectrophotometric assay for lipase or PLA was developed using natural triglycerides or synthetic glycerophosphatidylcholines containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and consequently confers strong UV absorption properties of this free fatty acid as well as of lipid substrates harboring it. The substrate was coated into the wells of a microplate, and the lipolytic activities were measured by the absorbance increase at 272 nm due to the transition of α-eleostearic acid moiety from the adsorbed to the soluble state. This continuous assay is compatible with a high-throughput screening method and can be applied specifically to the screening of new potential lipase, PLA1 and PLA2 inhibitors.

Direct and Continuous Measurement of Phospholipase D Activities Using the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

Phospholipase D (PLD) hydrolyzes phospholipids to form phosphatidic acid (PA) and the corresponding headgroup. To date, PLD has been linked to several pathologies, such as cancer, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released upon phosphatidylcholine (PC) hydrolysis. Therefore, we designed a PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline. This assay exhibits a strong fluorescence signal upon Ca2+ complexation with the PLD-generated PA and is not limited to PC as the substrate but allows the use of natural phospholipids with various headgroups. Besides, this easy-to-handle assay allows to monitor prokaryotic and eukaryotic PLD activities in a continuous way and on a microplate scale.

Heterologous Expression and Functional Characterization of Sparidae Fish Digestive Phospholipase A2.

In this study, we have produced for the first time a fish phospholipase (PLA2) in heterologous system (E. coli). The Diplodus annularis PLA2 (DaPLA2) was then refolded from inclusion bodies and purified by Ni-affinity chromatography. We used the pH-stat method (with emulsified phosphatidylcholine as substrate) and the monomolecular film technique (using various glycerophospholipids substrates spread in the form of monomolecular films at the air-water interface) to access the biochemical and kinetic properties of the recombinant DaPLA2. The DaPLA2 was found to be active and stable at higher temperatures (37-50 °C) than expected. Interestingly, DaPLA2 hydrolyzes efficiently both purified phosphatidylglycerol and phosphatidylethanolamine at 20 mN/m. These analytical results corroborate with the fact that the catalytic activity of DaPLA2, measured with the pH-stat using egg yolk as substrate, is mainly due to the hydrolysis of the PE fraction present in egg yolk, whereas the phosphatidylglycerol is a hallmark substrate for the most secreted PLA2-IB.

Expression and Purification of Recombinant Vigna unguiculata Phospholipase D in Pichia pastoris for Structural Studies.

The production of pure enzymes in high quantities is a proven strategy to study the catalytic mechanism as well as the solving of structure at the atomic scale for therapeutic or industrial purposes. Phospholipase D (PLD, EC 3.1.4.4) is found in a wide majority of living organisms and has been shown to be involved in signal transduction, vesicle trafficking, and membrane metabolism processes. Located at the membrane-cytoplasm interface, plant PLDs are soluble but also bear an evident hydrophobic aspect making challenging its expression and its purification in large quantity. So far there is no high-resolution three-dimensional structure for a eukaryotic PLD. The protocols herein describe the cloning of the eukaryotic recombinant PLDα of Vigna unguiculata (cowpea) into the yeast expression system Pichia pastoris and its two-step purification process. This allowed us to purify to homogeneity hundreds of micrograms of highly pure protein to conduct in fine structural studies.

Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

Here we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg-1 measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA2. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA2 at high temperatures was explained. Using the monomolecular film technique, AsPLA2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm-1. Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA2, provides new insights on structure-activity relationships of fish PLA2.

Correction: Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

[This corrects the article DOI: 10.7717/peerj.3524.].

Screening of phospholipase A activity and its production by new actinomycete strains cultivated by solid-state fermentation.

Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to Streptomyces (73%) and Micromonospora (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity. Then, a colorimetric HTS assay for general PLA activity detection (cHTS-PLA) using enriched PC (≈60%) as substrate and cresol red as indicator was developed and applied; this cHTS-PLA assay was validated with known PLAs. For the first time, actinomycete strains were cultivated by solid-state fermentation (SSF) using PC as inductor and sugar-cane bagasse as support to produce high PLA activity (from 207 to 2,591 mU/g of support). Phospholipase activity of the enzymatic extracts from SSF was determined using the implemented cHTS-PLA assay and the PC hydrolysis products obtained, were analyzed by TLC showing the presence of lyso-PC. Three actinomycete strains of the Streptomyces genus that stood out for high accumulation of lyso-PC, were selected and analyzed with the specific substrate 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC) in order to confirm the presence of PLA activity in their enzymatic extracts. Overall, the results obtained pave the way toward the HTS of PLA activity in crude microbial enzymatic extracts at a larger scale. The cHTS-PLA assay developed here can be also proposed as a routine assay for PLA activity determination during enzyme purification,directed evolution or mutagenesis approaches. In addition, the production of PLA activity by actinomycetes using SSF allow find and produce novel PLAs with potential applications in biotechnology.

α-Eleostearic acid-containing triglycerides for a continuous assay to determine lipase sn-1 and sn-3 regio-preference.

Lipases are essentially described as sn-1 and sn-3 regio-selective. Actually few methods are available to measure this lipase regio-selectivity, moreover they require chiral chromatography analysis or specific derivations which are discontinuous and time consuming. In this study we describe a new, convenient, sensitive and continuous spectrophotometric method to screen lipases regio-selectivity using synthetic triglycerides (TG) containing α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) either at the sn-1 position [1-α-eleostearoyl-2,3-octadecyl-sn-glycerol (sn-EOO)] or at the sn-3 position [1,2-octadecyl-3-α-eleostearoyl-sn-glycerol (sn-OOE)] and coated onto the wells of microtiter plates. A non-hydrolysable ether bond, with a non UV-absorbing alkyl chain, was introduced at the other sn positions to prevent acyl chain migration during TG synthesis or lipolysis. The synthesis of TG containing α-eleostearic acid was performed from S-glycidol in six steps to obtain sn-EOO and in five steps to sn-OOE. The α-eleostearic acid conjugated triene constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the TG harboring it. The lipase activity on coated sn-EOO or sn-OOE was measured by the increase in the absorbance at 272nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. Human and porcine pancreatic lipases, guinea pig pancreatic lipase related protein 2, Thermomyces lanuginosus lipase, Candida antarctica lipase A and Candida antarctica lipase B were all used to validate the assay. This continuous high-throughput screening method could determine directly without any processes after lipolysis the regio-selectivity of various lipases.

Functional and Structural Characterization of a Thermostable Phospholipase A2 from a Sparidae Fish (Diplodus annularis).

Novel phospholipase (PLA2) genes from the Sparidae family were cloned. The sequenced PLA2 revealed an identity with pancreatic PLA2 group IB. To better understand the structure/function relationships of these enzymes and their evolution, the Diplodus annularis PLA2 (DaPLA2) was overexpressed in E. coli. The refolded enzyme was purified by Ni-affinity chromatography and has a molecular mass of 15 kDa as determined by MALDI-TOF spectrometry. Interestingly, unlike the pancreatic type, the DaPLA2 was active and stable at higher temperatures, which suggests its great potential in biotechnological applications. The 3D structure of DaPLA2 was constructed to gain insights into the functional properties of sparidae PLA2. Molecular docking and dynamic simulations were performed to explain the higher thermal stability and the substrate specificity of DaPLA2. Using the monolayer technique, the purified DaPLA2 was found to be active on various phospholipids ranging from 10 to 20 mN·m-1, which explained the absence of the hemolytic activity for DaPLA2.

A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Reassessing the Potential Activities of Plant CGI-58 Protein.

Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

Functional Characterization of the N-Terminal C2 Domain from Arabidopsis thaliana Phospholipase Dα and Dß.

Most of plant phospholipases D (PLD) exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca2+-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDα from Arabidopsis thaliana (AtPLDα) in the yeast Pichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDα was found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, in E. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDα along with its equivalent C2 domain of the AtPLDß, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca2+-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca2+. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca2+-binding site within the C2 domain of AtPLDα.

Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 µM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale.