Biochemical, Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary.

Secretory group V phospholipase A2 (PLA2-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA2, the production of PLA2-V from dromedary. The obtained DrPLA2-V has an absolute requirement for Ca2+ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that Kcat/Kmapp is 2.6 ± 0.02 mM-1 s-1. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.

A Proteinaceous Alpha-Amylase Inhibitor from Moringa Oleifera Leaf Extract: Purification, Characterization, and Insecticide Effects against C. maculates Insect Larvae.

The main objective of the current study was the extraction, purification, and enzymatic characterization of a potent proteinaceous amylase inhibitor from Moringa oleifera. The antimicrobial potential and insecticide effects against C. maculates insect larvae were also studied. The α-amylase inhibitor was extracted in methanol (with an inhibitory activity of 65.6% ± 4.93). Afterwards, the inhibitor αAI.Mol was purified after a heat treatment at 70 °C for 15 min followed by one chromatographic step of Sephadex G-50. An apparent molecular weight of 25 kDa was analyzed, and the N-terminal sequence showed the highest identity level (89%) with the monomeric α-amylase inhibitor from Triticum dicoccoides. αAI.Mol was found to tolerate pH values ranging from 5.0 to 11.0 and showed maximal activity at pH 9.0. Thermal stability was remarkably important, since the inhibitory activity was maintained at 55% after 1 h of incubation at 70 °C and at 53% after an incubation of 45 min at 80 °C. The potency of the current purified inhibitor against amylases from different origins indicates that αAI.Mol seems to possess the highest affinity toward human salivary α-amylase (90% inhibitory activity), followed by the α-amylase of insects Callosobruchus maculatus and Tribolium confusum (71% and 61%, respectively). The kinetic parameters were also calculated, and the Kmax and Vmax of the digestive amylase were estimated at 185 (mmol/min/mg) and 0.13 mM, respectively. The inhibitor possesses a strong bactericidal effect against Gram+ and Gram- strains, and the MIC values were >1 against B. cereus but >6 against E. coli. Interestingly, the rates of survival and pupation of C. maculates insect larvae were remarkably affected by the purified αAI.Mol from Moringa oleifera.

Functional characterization and FTIR-based 3D modeling of full length and truncated forms of Scorpio maurus venom phospholipase A2.

BACKGROUND: Heterodimeric phospholipase A2 from venom glands of Tunisian scorpion Scorpio maurus (Sm-PLGV) had been purified. It contains long and short chains linked by a disulfide bridge. Sm-PLGV exhibits hemolytic activity towards human erythrocytes and interacts with phospholipid monolayers at high surface pressure. The investigation of structure-function relationships should provide new clues to understand its activity. METHODS: Molecular cloning of Sm-PLGV and heterologous expression in Escherichia coli of three recombinant forms was used to determine the role of the short chain on enzymatic activity. Infrared spectroscopy assisted 3D model building of the three recombinant constructs (phospholipases with and without the penta-peptide and Long chain only) allowed us to propose an explanation of the differences in specific activities and their interaction with various phospholipids. RESULTS: Nucleotide sequence of Sm-PLGV encodes 129 residues corresponding to the Long chain, the penta-peptide and the short chain. Although recombinant phospholipases without and with the penta-peptide have different specific activities, they display a similar substrate specificity on various phospholipid monolayers and similar bell-shaped activity profiles with maxima at high surface pressure. The absence of the short chain reduces significantly enzymatic and hemolytic activities. The 3D models pointed to an interaction of the short chain with the catalytic residues, what might explain the difference in activities of our constructs. CONCLUSION: Infrared spectroscopy data and 3D modeling confirm the experimental findings that highlight the importance of the short chain for the Sm-PLGV activity. GENERAL SIGNIFICANCE: New informations are given to further establish the structure-function relationships of the Sm-PLGV.

Biochemical and molecular characterization of a lipase from an Algerian isolated Staphylococcus aureus strain.

A Staphylococcus aureus strain, isolated from an Algerian biotope, secretes a non-induced lipase in the culture medium. The S. aureus lipase (SAL) was purified to homogeneity. Pure SAL is a monomeric protein (43 kDa). The 20 N-terminal amino acid residues showed a high degree of homology with other staphylococcal lipase sequences. SAL presents specific activities of about 1600 and 555 U mg-1 using tributyrin and olive oil emulsion as substrates, respectively. In contrast to other staphylococcal lipases previously characterized, SAL was stable at a pH range from 6 to 9 after 1 h incubation, and retained 50% of its activity after 10 min incubation at 50 °C. The purified enzyme was also characterized using monolayer technique. Lipase activity can be measured only when the surface pressure exceeds 15 mN m-1 . The critical surface pressure (πc ) measured with egg-PC films was estimated at 33 mN m-1 . SAL showed a preference for the distal ester groups of the diglyceride isomers at low surface pressure, for the adjacent ester groups at high surface pressure and a preference for the sn-3 position of the 2,3-sn-enantiomer of dicaprin. Cloned and sequenced gene part, encoding the mature lipase shows, in comparison with S. aureus lipase 3 (SAL3), a deletion of three residues (LKA) at the N-terminal extremity and a substitution of glycine 208 and isoleucine 226 with an arginine and leucine, respectively.

The galactolipase activity of Fusarium solani (phospho)lipase.

The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.

Kinetic properties of a novel Fusarium solani (phospho)lipase: a monolayer study.

Using the monomolecular film technique, we studied interfacial properties of Fusarium solani lipase (FSL). This lipolytic enzyme was found to be unique among the fungal lipases possessing not only a lipase activity but also a high phospholipase one.The FSL was able to hydrolyze dicaprin films at various surface pressures. The surface pressure dependency, the stereospecificity, and the regioselectivity of FSL were performed using optically pure stereoisomers of diglyceride (1,2-sn- dicaprin and 2,3-sn-dicaprin) and a prochiral isomer (1,3-sn-dicaprin) spread as monomolecular films at the air-water interface. The FSL prefers adjacent ester groups of the diglyceride isomers (1,2-sn-dicaprin and 2,3-sn-dicaprin) at low and high surface pressures. Furthermore, FSL was found to be markedly stereospecific for the sn-1 position of the 1,2-sn-enantiomer of dicaprin at both low and high surface pressures.Moreover, FSL shows high activities on phospholipids monolayers. However, this enzyme displays high preference to zwitterionic phospholipids compared to the negatively charged ones.

Kinetic behaviour of recombinant Fusarium solani lipases using monomolecular films: Effect of the heterologous expression.

Two lipases from Fusarium solani, FSL and FSL2, were efficiently expressed in Pichia pastoris. To check the influence of the expression on interfacial properties of FSL and to study kinetic properties of FSL2, interfacial parameters of FSL2, native FSL, untagged recombinant and tagged recombinant forms of FSL were compared using the monomolecular film technique. Kinetic study on the dependence of the stereoselectivity of these lipases on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The FSL2 seems to have an important penetration power with a preference for adjacent ester groups and the heterologous expression accompanied or not with the N-His-tag extension on the FSL were found to modify the pressure preference and increase the catalytic hydrolysis rate of three dicaprin isomers. The heterologous expression was found to preserve the FSL regioselectivity without affecting its stereospecificity at high and low surface pressure. The evaluation of the recombinant expression Effects on Catalysis (REC), the N-Tag Effects on Catalysis (TEC), and the N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression was more efficient than the presence of the N-terminal tag extension on the FSL.

Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling.

The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZαA vector containing the Saccharomyces cerevisiae α-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37°C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40°C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca2+ and inhibited by EDTA or Cu2+ and partially by Mg2+ or K+. In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions.

Heterologous overexpression and biochemical characterization of the (galactophospho)lipase from Fusarium solani in Pichia pastoris that is expressed in planta.

High-level extracellular production of Fusarium solani (galactophospho)lipase, named FSL, was achieved using a Pichia pastoris X33 expression system. The (galactophospho) lipase encoding gene was cloned into pGAPZαA with the Saccharomyces cerevisiae α-factor signal sequence by two different ways. The two constructs consist of an additional sequence of a (His)6-tag of the vector fused to the N-terminus of this enzyme (tFSL) while the other expression vector was constructed without any additional sequence (rFSL). Compared to the native enzyme (nFSL) (18.75 mg/L), a high level secretion of rFSL (310 mg/L) and tFSL (240 mg/L) was achieved providing an important improvement in enzyme production. Biochemical characterization showed that pure recombinant proteins (rFSL and tFSL) presented similar behaviour towards triglycerides, phospholipid and galactolipid. Like the nFSL, rFSL and tFSL are active at high concentration of bile salts (4mM) and calcium ions enhanced lipase activity. During plant infection, transcripts of this fungal lipase gene were detected 3, 7 and 10 days post infection.

Evaluation of the recombinant turkey pancreatic lipase phospholipase activity: A monolayer study.

Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols.

Purification and biochemical characterization of a halotolerant Staphylococcus sp. extracellular lipase.

We have isolated a lipolytic halotolerant bacterium, designated as CJ3, that was identified as a Staphylococcus sp. Culture conditions were optimized and the highest extracellular lipase production amounting to 5 U/ml was achieved after 24 h of cultivation. The extracellular lipase was purified 24-fold by ammonium sulfate precipitation and a Sephacryl S-200 chromatography, and its molecular mass was found to be around 38 kDa, as revealed by SDS-PAGE and gel filtration. The lipase substrate specificity was investigated using short (tributyrin) and long (olive oil) chain triglyceride substrates. The lipase was inhibited by submicellar concentrations of Triton X-100, and maximum specific activities were found to be 802 U/mg on tributyrin and 260 U/mg on olive oil at pH 8.0 and 45 °C. The lipase was fairly stable in the pH range from 6.0 to 9.0, and about 69% of its activity was retained after incubation at 45 °C for 60 min. The enzyme showed a high tolerance to a wide range of salt concentration and a good stability in organic solvents, especially in long-chain alcohols.

Purification and biochemical characterization of a novel alkaline (phospho)lipase from a newly isolated Fusarium solani strain.

An extracellular lipase from Fusarium solani strain (F. solani lipase (FSL)) was purified to homogeneity by ammonium sulphate precipitation, gel filtration and anion exchange chromatography. The purified enzyme has a molecular mass of 30 kDa as estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 12 NH(2)-terminal amino acid residues showed a high degree of homology with a putative lipase from the fungus Necteria heamatoccocae. It is a serine enzyme, like all known lipases from different origins. Interestingly, FSL has not only lipase activity but also a high phospholipase activity which requires the presence of Ca(2+) and bile salts. The specific activities of FSL were about 1,610 and 2,414 U/mg on olive oil emulsion and egg-yolk phosphatidylcholine as substrates, respectively, at pH 8.0 and 37 °C. The (phospho)lipase enzyme was stable in the pH range of 5-10 and at temperatures below 45 °C.