Expression and characterization of an organic solvent tolerant recombinant lipase from Staphylococcus capitis SH6 for food wastewater treatment.

The study illustrated here aims on an organic solvent tolerant lipase from Staphylococcus capitis (SCL). The gene part, encoding the mature lipase, was cloned and sequenced. The concluded polypeptide sequence, equivalent to the protein, consist of 388 amino acid residues with a molecular mass of about 45 kDa. A structure-based alignment of the SCL amino acid sequence shows high identities with those many staphylococcal lipases. From this alignment of sequences, the catalytic triad (Ser 117, Asp 308 and His 347) of SCL could be identified. The mature part of the SCL was expressed in Escherichia coli and the recombinant lipase (r-SCL) was purified to homogeneity. The purified r-SCL presented a quite interesting stability at low temperatures (< 30 °C) and the enzyme was found to be highly stable in polar organic solvent and at a pH ranging from 3 to 12. After that, we have demonstrated that the recombinant enzyme may be implicated in the biodegradability of oily wastewater from effluents of fast-food restaurants; the maximum conversion yield into fatty acids obtained at 30 °C, was 65%.

Functional Characterization and Anti-Tumor Effect of a Novel Group II Secreted Phospholipase A2 from Snake Venom of Saudi Cerastes cerates gasperetti.

Secreted phospholipases A2 are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A2 with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A2 (Cc-PLA2-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA2 ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA2-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.

Integrin-mediated human glioblastoma cells adhesion, migration and invasion by native and recombinant phospholipases of Scorpio maurus venom glands.

Integrins are a large family of cell surface receptors mediating the interaction of cells with their microenvironment and they play an important role in glioma biology. In the present work, we reported the anti-tumor effect of Sm-PLGV a phospholipase A2 from Tunisian scorpion venom glands-as well as its recombinant forms expressed in Escherichia coli-through interference with integrin receptor function in malignant glioma cells U87. These phospholipases inhibited in a dose dependent manner the adhesion, migration and invasion onto fibrinogen and fibronectin without any cytotoxicity. We showed that Sm-PLGV and its recombinant constructs blocked U87 migration by reducing their velocity and directional persistence. The inhibitory effect was related to a blockage of the integrins αvß3 and α5ß1 function. Inactivation of the enzymatic activity of Sm-PLGV by chemical modification with p-bromophenacyl bromide did not affect its anti-tumor properties, suggesting the presence of 'pharmacological sites' distinct from the catalytic site in scorpion venom phospholipases A2.

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.

Novel Stable Protease Inhibitor from Phoenix dactylifera(L.) Flowers with Antimicrobial and Antitumoral Activities.

A novel protease inhibitor isolated from date palm Phoenix dactylifera(L.) flowers (PIDF) was purified and characterized. A heat and acidic treatment step followed by ethanol precipitation and reverse-phase high-performance chromatography was applied to purify this natural protease inhibitor to homogeneity with a single band of about 19 kDa. The stability study depicted that PIDF was fully stable at 40 °C and retained 65% of its initial activity after heating at 50 °C for 24 h. Its thermal stability at 70 °C was markedly enhanced by adding calcium, bovine serum albumin, and sorbitol as well as by metal divalent cations, especially Mg2+ and Hg2+. This protease inhibitor showed high inhibitory activity against therapeutic proteases, including pepsin, trypsin, chymotrypsin, and collagenase, and acted as a potent inhibitor of some commercial microbial proteases from Aspergillus oryzae, Bacillus. sp, and Bacillus licheniformis. Moreover, a potent antibacterial spectrum against Gram (+) and Gram (-) bacterial strains and an efficient antifungal effect were observed. Its cytotoxicity toward human colorectal cancer cell LoVo and HCT-116 lines suggested that PIDF could serve as a new therapeutic target inhibiting human colorectal cancer.

Synthesis and Biological Evaluation of Some New 3-Aryl-2-thioxo-2,3-dihydroquinazolin-4(1H)-ones and 3-Aryl-2-(benzylthio)quinazolin-4(3H)-ones as Antioxidants; COX-2, LDHA, α-Glucosidase and α-Amylase Inhibitors; and Anti-Colon Carcinoma and Apoptosis-Inducing Agents.

Oxidative stress, COX-2, LDHA and hyperglycemia are interlinked contributing pathways in the etiology, progression and metastasis of colon cancer. Additionally, dysregulated apoptosis in cells with genetic alternations leads to their progression in malignant transformation. Therefore, quinazolinones 3a-3h and 5a-5h were synthesized and evaluated as antioxidants, enzymes inhibitors and cytotoxic agents against LoVo and HCT-116 cells. Moreover, the most active cytotoxic derivatives were evaluated as apoptosis inducers. The results indicated that 3a, 3g and 5a were efficiently scavenged DPPH radicals with lowered IC50 values (mM) ranging from 0.165 ± 0.0057 to 0.191 ± 0.0099, as compared to 0.245 ± 0.0257 by BHT. Derivatives 3h, 5a and 5h were recognized as more potent dual inhibitors than quercetin against α-amylase and α-glucosidase, in addition to 3a, 3c, 3f and 5b-5f against α-amylase. Although none of the compounds demonstrated a higher efficiency than the reference inhibitors against COX-2 and LDHA, 3a and 3g were identified as the most active derivatives. Molecular docking studies were used to elucidate the binding affinities and binding interactions between the inhibitors and their target proteins. Compounds 3a and 3f showed cytotoxic activities, with IC50 values (µM) of 294.32 ± 8.41 and 383.5 ± 8.99 (LoVo), as well as 298.05 ± 13.26 and 323.59 ± 3.00 (HCT-116). The cytotoxicity mechanism of 3a and 3f could be attributed to the modulation of apoptosis regulators (Bax and Bcl-2), the activation of intrinsic and extrinsic apoptosis pathways via the upregulation of initiator caspases-8 and -9 as well as executioner caspase-3, and the arrest of LoVo and HCT-116 cell cycles in the G2/M and G1 phases, respectively. Lastly, the physicochemical, medicinal chemistry and ADMET properties of all compounds were predicted.

Efficient heterologous expression in Pichia pastoris, immobilization and functional characterization of a scorpion venom secreted phospholipase A2.

Industrial processes have expanded with the ability to clone and express recombinant immobilized enzymes in microorganisms such as Pichia pastoris that have commercially attractive amounts of the appropriate genes. This report describes the overexpression in Pichia pastoris, immobilization, and functional characterization of a secreted phospholipase A2 from scorpion venom Scorpio maurus: rPLA2(-5). After 48 h of culture, the recombinant rPLA2(-5) was secreted into the culture medium and expressed at about 9 mg/L. Comparative analyses of the kinetics and hydrolysis of rPLA2(-5) monolayers at various surface pressures were conducted with the same form produced in Escherichia coli. As a second part of the study, rPLA2(-5) overexpressed in Pichia pastoris was immobilized by adsorption on CaCO3, with about 78 percent of the activity. In comparison to the free enzyme, rPLA2(-5) was studied for stability. Immobilization improved the thermal stability of rPLA2(-5) and even the stability at acidic pH. Moreover, we found that the immobilization improved the stability of rPLA2(-5) towards bile salts, Tween 80, Triton X-100, and SDS, as well as its stability towards many organic solvents. Until now, this is the first study to describe the overexpression and immobilization of a scorpion venom phospholipase A2 that possesses an interesting stability characteristic that makes it useful for a wide range of biotechnological applications.

Spirulina platensis, Punica granatum peel, and moringa leaves extracts in cosmetic formulations: an integrated approach of in vitro biological activities and acceptability studies.

The selection of suitable natural raw materials in the cosmetic research and development is a key point, in order not only to obtain the expected results but also to avoid undesirable side effects. In this study, spirulina platensis, pomegranate (Punica granatum) peel, and moringa leaves alone were evaluated for anti-oxidant and antimicrobial properties. The chemical composition (moisture, dry matter, protein, lipid, and ash) and total polyphenols, flavonoids, and carotenoids content were evaluated in the three extracts. Total antioxidant capacity and ferric reducing activity power of extracts were also studied. Using agar diffusion method, the anti-Micrococcus luteus, Staphylococcus aureus, E. coli, Listeria monocytogenes, Salmonella typhimurium, and Enterococus faecalis activities were measured. Interestingly, after combinations, pomegranate peel/spirulina (A), and moringa/spirulina (B): 25%/75% and 50%/50%, we have found that pomegranate peel can be incorporated into cosmetic formulations as an excellent preservative due to its exceptionally amount of phenolic compounds, powerful antioxidant activity, and its antibacterial activity against pathogenic strains.

Scorpion venom phospholipases A2: A minireview.

Many venomous species, including snakes, bees and scorpions, contain a variety of secreted phospholipases A2 (sPLA2) that contribute to prey digestion and venom toxicity. Based on their primary structures, the different venom sPLA2 have been classified into four groups I, II, III and IX. While the structure of sPLA2 groups II and I has been well characterized, only one crystal structure of group III sPLA2 from bee venom was described. Scorpion venom sPLA2 belong to group III with a particular heterodimeric structure composed of a long enzymatic chain linked by a disulfide bridge to a Short one after the release of five residues (penta-peptide) during the maturation processes. The function of the Short chain is still not clear. Its sequence varies in composition and in length between different scorpion species. Available studies of the structure-function relationships of scorpion venom sPLA2 are limited. Some biological activities of these enzymes such as neurotoxicity, myotoxicity, along with the hemolytic, anticoagulant, anti-tumoral and anti-angiogenic activities have been investigated. In this review, we tentatively summarize the last findings about the biochemical properties, the structure-function relation and the biological activities of scorpion venom phospholipases A2. In addition to expanding the database of structures for these sPLA2 and understanding their properties and functions, this survey is intended to explore the molecular mechanisms and signaling pathways of the described biological activities.

Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment.

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

Purification and biochemical characterization of an organic solvent-tolerant and detergent-stable lipase from Staphylococcus capitis.

A mesophilic bacterial culture, producing an extracellular alkaline lipase, was isolated from the gas-washing wastewaters generated from the Sfax phosphate plant of the Tunisian Chemical Group and identified as Staphylococcus capitis strain. The lipase, named S. capitis lipase (SCL), has been purified to homogeneity from the culture medium. The purified enzyme molecular weight was around 45 kDa. Specific activities about 3,900 and 500 U/mg were measured using tributyrin and olive oil emulsion as substrates, respectively at 37°C and pH 8.5. Interestingly, the SCL maintained more than 60% of its initial activity over a wide pH values ranging from 5 to 11 with a high stability between pH 9 and 11 after 1 hr of incubation at room temperature. The lipase activity was enhanced in the presence of 2 mM of Mg2+ , Ca2+ , and K+ . SCL showed significant stability in the presence of detergents and organic solvents. Altogether, these features make the SCL useful for industrial applications. Besides, SCL was compatible with commercially available detergents, and its incorporation increases lipid degradation performances making it a potential candidate in detergent formulation.

Anti-angiogenic effect of phospholipases A2 from Scorpio maurus venom glands on Human Umbilical Vein Endothelial Cells.

In a previous study, we purified Sm-PLGV an heterodimeric phospholipase A2, from the venom glands of the Tunisian scorpion Scorpio maurus. This enzyme contains a Long chain, a penta-peptide insertion, which is cut out during the maturation process, followed by a short chain. A disulfide bridge links the two chains. Three recombinant forms of this enzyme were produced in Escherichia coli: rPLA2(+5) with a penta-peptide insert, rPLA2(-5) without the penta-peptide, and the Long chain alone without the short one. In the present study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(-5) displayed more potent anti-angiogenic activity in vitro than the recombinant Long chain and the short one obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of Human Umbilical Vein Endothelial Cells. Using Matrigel™, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(-5) significantly inhibited tubulogensesis. We also showed a clear dissociation between the anti-angiogenic effect of Sm-PLGV and its catalytic activity.

Native and recombinant phospholipases A2 of Scorpio maurus venom glands impair angiogenesis by targeting integrins α5ß1 and αvß3.

We recently purified an heterodimeric phospholipase A2 named Sm-PLGV from the venom glands of scorpion Scorpio maurus containing a Long chain, a penta-peptide insertion, which is cut out during the maturation, followed by a short chain. Three recombinant forms of Sm-PLGV were produced in Escherichia coli: rPLA2(+5) containing the full-length sequence including the penta-peptide insert, rPLA2(-5) a fused continuous chain of the Long and the short chains without the penta-peptide and the Long chain alone without the short one. In this study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(-5) displayed more potent anti-angiogenic properties than the recombinant Long chain and the short chain obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of human microvascular endothelial cells through the alteration of α5ß1 and αvß3 integrins function. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(-5) significantly inhibited both in vitro and in vivo angiogenesis. We also showed a clear dissociation of the anti-angiogenic effect of Sm-PLGV and its catalytic activity. This is the first study describing an anti-angiogenic effect for recombinant scorpion venom enzymes.

Biological properties of Alsidium corallinum and its potential protective effects against damage caused by potassium bromate in the mouse liver.

In the course of searching for hepatoprotective agents from natural sources, the protective effect of chemical constituents of the marine red alga Alsidium corallinum (A. corallinum) against potassium bromate (KBrO3)-induced liver damage in adult mice was investigated. The in vitro antioxidant and antibacterial properties of A. corallinum were firstly investigated. Then, A. corallinum was tested in vivo for its potential protective effects against damage caused by KBrO3 in mice models divided into four groups: controls, KBrO3, KBrO3 + A. corallinum, and A. corallinum. Our results demonstrated the rich composition of A. corallinum in antioxidant compounds like phenolics, flavonoids, anthocyanins, polysaccharides, chlorophyll and carotenoids. Its antioxidant activity was also confirmed using ß-carotene bleaching by linoleic acid assay, reducing sugar test and trolox equivalent antioxidant capacity. The ethanolic extract of A. corallinum also showed good inhibition of the tested bacteria. The coadministration of the red alga associated to the KBrO3 alleviated hepatotoxicity as monitored by the improvement of hepatic oxidative stress biomarkers and plasma biochemical parameters, when compared to the KBrO3-treated mice. These results were confirmed by the improvement of histological and molecular changes. Treatment with A. corallinum prevented liver damage induced by KBrO3, thus protecting the body against free radicals and reducing inflammation and hypercholesterolemia risks.

Biochemical and monolayer characterization of Tunisian snake venom phospholipases.

The present study investigated the kinetic and interfacial properties of two secreted phospholipases isolated from Tunisian vipers'venoms: Cerastes cerastes (CC-PLA2) and Macrovipera lebetina transmediterranea (MVL-PLA2). Results show that these enzymes have great different abilities to bind and hydrolyse phospholipids. Using egg-yolk emulsions as substrate at pH 8, we found that MVL-PLA2 has a specific activity of 1473U/mg at 37°C in presence of 1mM CaCl2. Furthermore the interfacial kinetic and binding data indicate that MVL-PLA2 has a preference to the zwitterionic phosphatidylcholine monolayers (PC). Conversely, CC-PLA2 was found to be able to hydrolyse preferentially negatively charged head group phospholipids (PG and PS) and exhibits a specific activity 9 times more important (13333U/mg at 60°C in presence of 3mM CaCl2). Molecular models of both CC-PLA2 and MVL-PLA2 3D structures have been built and their electrostatic potentials surfaces have been calculated. A marked anisotropy of the overall electrostatic charge distribution leads to a significantly difference in the dipole moment intensity between the two enzymes explaining the great differences in catalytic and binding properties, which seems to be governed by the electrostatic and hydrophobic forces operative at the surface of the two phospholipases.

A mineral and antioxidant-rich extract from the red marine Algae Alsidium corallinum exhibits cytoprotective effects against potassium bromate-induced erythrocyte oxidative damages in mice.

The present study was carried out to investigate potassium bromate toxicity in mice and the corrective effects of marine algae Alsidium corallinum. The red algae demonstrated its rich composition in phenols, triterpenes, flavonoids, alkaloids, tropolones, sodium, potassium, calcium, magnesium, iron, copper, and zinc. To confirm its antioxidant potential, an in vivo study was performed on adult mice. The animals were divided into four groups: group I were used as controls, group II received potassium bromate (0.5 g/L) via drinking water, group III received potassium bromate (0.5 g/L) by the same route as group II and 7% of A. corallinum ethanolic extract via their diet, and group IV received only 7% of algae. The potassium bromate-treated group showed a significant decrease in erythrocyte, platelet, hemoglobin, and hematocrit values and a significant increase in total white blood cells, compared to those of controls. While, superoxide dismutase, catalase, glutathione, and vitamin C values were decreased by potassium bromate treatment, lipid peroxidation (as malondialdehyde) and erythrocyte osmotic fragility values were increased. Interestingly, potassium bromate treatment showed significant genotoxic effects, as demonstrated by DNA degradation. These changes were confirmed by blood smears histopathological observations which were marked by a necrosis and a decrease of erythrocytes number. A. corallinum extract appeared to be effective against hematotoxic and genotoxic changes induced by potassium bromate, as evidenced by the improvement of the parameters cited above.

A thermoactive secreted phospholipase A2 purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity.

A lipolytic activity was located in the scorpion venom glands (telsons), from which a phospholipase A2 (Sm-PLVG) was purified. Like known phospholipases A2 from scorpion venom, which are 14-18 kDa proteins, the purified Scorpio maurus-Phospholipase from Venom Glands (Sm-PLVG) has a molecular mass of 17 kDa containing long and short chains linked by disulfide bridge. It has a specific activity of 5500 U/mg measured at 47 °C and pH 8.5 using phosphatidylcholine as a substrate in presence of 8 mM NaTDC and 12 mM CaCl2. The NH2-terminal amino acid sequences of the purified Sm-PLVG showed similarities with those of long and short chains of some previously purified phospholipases from venom scorpions. Moreover, the Sm-PLVG exhibits hemolytic activity toward human, rabbit or rat erythrocytes. This hemolytic activity was related to its ability to interact with phospholipids' monolayer at high surface pressure. These properties are similar to those of phospholipases isolated from snake venoms.