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.

Antifungal activity of lactic acid bacteria and their application in food biopreservation.

Lactic acid bacteria (LAB) are ubiquitous bacteria associated with spontaneous lactic fermentation of vegetables, dairy and meat products. They are generally recognized as safe (GRAS), and they are involved in transformation of probiotic lacto-fermented foods, highly desired for their nutraceutical properties. The antifungal activity is one of the exciting properties of LAB, because of its possible application in food bio-preservation, as alternative to chemical preservatives. Many recent research works have been developed on antifungal activity of LAB, and they demonstrate their capacity to produce various antifungal compounds, (i.e. organic acids, PLA, proteinaceous compounds, peptides, cyclic dipeptides, fatty acids, and other compounds), of different properties (hydrophilic, hydrophobic and amphiphilic). The effectiveness of LAB in controlling spoilage and pathogenic fungi, demonstrated in different agricultural and food products, can be due to the synergistic effect between their antifungal compounds of different properties; where the amphiphilic-compounds allow the contact between the target microbial cell (hydrophilic compartment) and antifungal hydrophobic-compounds. Further studies on the interaction between compounds of these three properties are to de be developed, in order to highlight more their mechanism of action, and make LAB more profitable in improving shelf life and nutraceutical properties of foods.

In vitro lipolysis and physicochemical characterization of unconventional star anise oil towards the development of new lipid-based drug delivery systems.

Lipid-based drug delivery systems are widely used for enhancing the bioavailability of poorly water-soluble drugs. However, following oral intake, lipid excipients often undergo gastrointestinal lipolysis, which drastically affects drugs solubility and bioavailability. That's why developing new lipid excipients which are resistant to digestion would be of great interest. We studied here the potential role of the unconventional Chinese star anise whole seedpod oil (CSAO) as an alternative multifunctional lipid excipient. Pancreatic lipase-mediated digestion of the extracted crude oil emulsion was assessed in vitro. Pancreatic lipase, being a strict sn-1,3-regioselective lipase, showed a high (16-fold) olive oil to CSAO activity ratio, which could be attributed to fatty acids composition and triglycerides intramolecular structure. For the sake of comparison, the non-regioselective lipase Novozyme® 435 exhibited higher activity than pancreatic lipase on CSAO emulsion, perhaps due to its ability to release fatty acids from the internal sn-2 position of TAGs. Apart counteracting lipolysis, CSAO oil also showed additional biopharmaceutical benefits including moderate antioxidant and antihypertensive activities. Altogether, these findings highlight for the first time the potential use of star anise unconventional whole seedpod oil as a multifunctional lipid excipient for the development of new lipid 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.

Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation.

Marine species have gained significant attention as potential source for a broad spectrum of bioactive proteins. Fish phospholipases A2 (PLA2) have attracted renewed interest due to their excellent properties in lipid digestion. Herein, we report for the first time the catalytic properties of two intestinal secreted PLA2 (sPLA2) identified from Diplodus sargus (IDsPLA2) and Sparus aurata (ISaPLA2). The highest sequence identity was obtained with recently isolated Sparidae digestive PLA2 (45%) and Human pancreatic PLA2 (42%). IDsPLA2 and ISaPLA2 were overexpressed in E. coli as inclusion bodies, refolded and purified. Both enzymes have improved thermostability compared to mammalian pancreatic sPLA2 since they are active and stable at 55 °C, with specific activities of 320 and 190 U mg-1 measured on phosphatidylcholine, respectively. Interestingly, IDsPLA2, but not ISaPLA2, revealed weak toxicity towards macrophages and suggests its involvement in cell membrane degradation. ISaPLA2 was found to be more active than IDsPLA2 when using the monolayer technique at 20 mN m-1. Structural models of both enzymes revealed their differences. In silico docking of phospholipids with both models allowed proposing key amino-acids in substrate binding and selectivity. Overall, these results provide insight into the enzymatic and structural properties of two novel sPLA2 with potential for future applications.

Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase.

The involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2­dimyristoyl­sn­glycero­3­phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2­dilauroyl­sn­glycero­3­phosphocholine. Significantly improvement between 1,2­dioleoyl­sn­glycero­3­phosphocholine and 1,2­distearoyl­sn­glycero­3­phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m-1) at pH 5 and pH 6 (47.5 ±â€¯1.9 mN m-1) but decreased significantly (34.2 mN m-1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme.

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.

Identification of a new natural gastric lipase inhibitor from star anise.

The identification and isolation of bioactive compounds are of great interest in the drug delivery field, despite being a difficult task. We describe here an innovative strategy for the identification of a new gastric lipase inhibitor from star anise for the treatment of obesity. After plant screening assays for gastric lipase inhibition, star anise was selected and investigated by bioactivity guided fractionation. MALDI-TOF mass spectrometry and peptide mass fingerprinting allowed the detection of an inhibitor covalently bound to the catalytic serine of gastric lipase. A mass-directed screening approach using UPLC-HRMS and accurate mass determination searching identified the flavonoid myricitrin-5-methyl ether (M5ME) as a lipase inhibitor. The inhibitory activity was rationalized based on molecular docking, showing that M5ME is susceptible to nucleophilic attack by gastric lipase. Overall, our data suggest that M5ME may be considered as a potential candidate for future application as a gastric lipase inhibitor for the treatment of obesity.

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.

Deletion the C-terminal peptides of Vibrio harveyi phospholipase D significantly improved its enzymatic properties.

A novel phospholipase D that originate from marine Vibrio harveyi (VhPLD) was recombinant expressed and biochemically characterized. Moreover, effects of C-terminal peptides on catalytic and interfacial binding properties of VhPLD were investigated by constructing two truncated mutants (VhPLD-Δ(472-483) and VhPLD-Δ(437-483)). Optimal reaction temperature and pH value for wild-type VhPLD (VhPLD-WT) was 45 °C and pH 8.0. However, optimal reaction temperature of VhPLD-Δ(437-483) increased to 50 °C. Meanwhile, catalytic efficiency (kcat/KM) of VhPLD-Δ(472-483) and VhPLD-Δ(437-483) to the 1,2-Dioctanoyl-sn-glycero-3-phosphatidyl-p-nitrophenol (PpNP) was 12.9 and 14.2 times higher than that of VhPLD-WT. However, when compare the catalytic efficiency between VhPLD-Δ(472-483) and VhPLD-Δ(437-483), no significant change can be found between the two mutants. These results strongly indicated that the C-terminal 12 amino acids (472-483) have important role on the activity of VhPLD. Effects of C-terminal peptides on the interfacial binding properties of VhPLD to different phospholipid monolayers were also investigated by using monolayer film technology. Results of the maximum insertion pressure (MIP) indicated that deletion the C-terminal segment of VhPLD improved its interfacial binding properties to different phospholipid monolayers.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus.

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

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.].