Polysaccharide from Thymelaea hirsuta L. leaves: Structural characterization, functional properties and antioxidant evaluation.

In this study, we successfully extracted a new polysaccharide from Thymelaea hirsuta L., which we named THP, with a yield of 13.94 % through aqueous extraction. The polysaccharide comprises 46.83 % neutral sugars and 7.08 % uronic acids. This heteropolysaccharide contains glucose, glucuronic acid, rhamnose, arabinose, and galactose with relative molar ratios of 29.68: 25.73: 21.31: 13.47 and 9.8, respectively. Analysis of glycosylation positions via methylation and structural features using Fourier transform infrared (FT-IR) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy revealed that THP has a repeating unit (1 → 3 and 1 → 4)-linked ß-d-glucopyranosyl backbone, with the d-glucopyranosyl residue as the branch point at O-6. The scanning electron microscopy (SEM) examination showed ellipsoidal granules with smooth surface. This polysaccharide also has good foaming capacity and emulsion stability, and a water holding capacity of 1.81 g/g. The THP dispersion at 1 % showed shear thinning behavior, demonstrating that it is a promising natural additive in various food formulations. The polysaccharide also demonstrated significant antioxidant properties, with a total antioxidant capacity of 315 mg α-tocopherol equivalents/g and an IC50 value of 8.01 mg/mL using the ß-carotene bleaching method, surpassing those of synthetic antioxidants. Additionally, when added at a concentration of 0.13 % to an oil/water emulsion system, THP effectively delayed lipid oxidation during storage at 37 °C. The kinetic study of THP-stabilized oil/water emulsion is anticipated to provide valuable insights for its future applications in food and pharmaceutical emulsions.

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.

Pistacia atlantica Desf. roots extract: LC-ESI-MS Analysis, antioxidant activity and gastroprotective effect on experimentally-induced ultrastructural gastric ulcers in mice.

Pistacia atlantica Desf. (Atlas Pistachio) is an Anacardiaceae tree traditionally used in Tunisia for the treatment of ophthalmic, stomatitis, and digestive tract diseases. In the present study, the Pistacia atlantica Desf. roots extract (PR) was phytochemically analyzed, for the first time, by LC-ESI-MS for phenolic and flavonoid contents, in vitro tested for its potential antioxidant activity based on the 2.2-diphenyl-1-picrylhydrazyl (DPPH) and the reduced power essays (FRAP), and in vivo tested for its ability to shield against ethanol-induced gastric ulcer in mice. The LC-ESI-MS analysis proved the identification of 12 compounds, including Quinic, Gallic, and Protocatechuic, as major phenolic acids and high levels of flavonoids, such as Catechin, Epicatechin, and Cirsiliol. PR also exhibited a mild in vitro antioxidant activity when compared with ascorbic acid. In vivo pretreatment of ethanol-ulcerated mice with PR doses 50 mg/kg and 100 mg/kg body weight (b.w) significantly reduced (P< .05) gastric lesions at a rate of 20.10% and a rate of 40.90%, respectively, when compared with 60.70% rate of sucralfate (50 mg/kg b.w) evidenced by a dose-dependent manner increase in the gastric mucosa enzymatic (SOD, CAT, GPx) antioxidant levels, the decline of the lipid peroxidation, and the preservation of normal gastric superficial epithelium. The underlying mechanism of PR antiulcerogenic activity could be due to a synergistic effect of phenolic acids and flavonoid contents which enhances the gastric antioxidant defense system.Abbreviations: BHT: butylated hydroxytoluene, b.w: body weight, CAT: catalase, DPPH:1-Diphenyl-2-picrylhydrazyl, DW: dry weight, EtOH: ethanol, FRAP: Ferric reducing antioxidant power, GAE: gallic acid equivalents, GPx: Glutathione peroxidase, QE: quercetin equivalents, LC-ESI-MS: Liquid chromatography-Electrospray Ionization-Tandem Mass Spectrometry, MDA: malondialdehyde, PR: Pistacia root, TBA: thiobarbituric acid reagent, TBARS: thiobarbituric acid reactive substances, TCA: trichloroacetic acid, SOD: Superoxide dismutase.

Studies of crab digestive phospholipase acting on phospholipid monolayers: Activation by temperature.

Secreted phospholipases A2 (sPLA2) are water-soluble lipolytic enzymes that act at the interface of organized lipid substrates, where the catalytic step is coupled to various interfacial phenomena as enzyme penetration, solubilisation of reaction products, lateral packing and loss of mechanical stability of organized assemblies of phospholipid molecule, among others. Using the monomolecular film technique, we compared the interfacial properties of crab digestive sPLA2 (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two sPLA2 was performed using monomolecular films of three different substrates: di C12-PC (1.2-dilauroyl-sn-glycerol-3-phosphocholine); di C12-PG (1.2-dilauroyl-sn-glycerol-3-phosphoglycerol) and di C12-PE (1.2-dilauroyl-sn-glycerol-3-phosphoethanolamine). The use of a substrate in monolayer state, during the catalytic reactions, allows us to monitor the effect of several physicochemical parameters by altering the "quality of interface". The effect of temperature on the hydrolysis rate of these substrates was also checked. Our results show that activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of sPLA2. The hyperbolic catalytic behaviour observed was coherent with hopping mode of action, one of the two characteristic mechanisms of interfacial catalysis of sPLA2.

Antibacterial, anti-adherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4.

The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL-1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL-1 and 1.17 mg mL-1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.

Valorization of the peel of pea: Pisum sativum by evaluation of its antioxidant and antimicrobial activities.

This study deals with evaluating antioxidant and antimicrobial activities of the peel of pea (Pisum sativum), with particular attention to the content of some bioactive compounds. Total content of polyphenols and flavonoids of Pisum sativum peel extracts, including a crude aqueous extract, a methanolic extract and an ethyl acetate extract was carried out according to the standard methods to assess their corresponding antioxidant activities. The organic solvents extracts antioxidant activities, determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, ferric reducing (FRAP) assay and 2,2 azinobis 3-ethylbenzo-thiozoline-6-sulfonic acid (ABTS) assay, were relatively high. The highest activity was found in ethyl acetate extract. The antimicrobial activities of extracts were also assessed. The highest MIC value was occurred with E.Coli (850 µg/ml) when using ethyl acetate extract. From the results obtained, Pisum sativum peel can be considered as a very good source of health promoting compounds.

Lipase pre-hydrolysis enhance anaerobic biodigestion of soap stock from an oil refining industry.

A novel alcalophilic Staphylococcus haemolyticus strain with the lipolytic activity was used to perform enzymatic hydrolysis pretreatment of soap stock: a lipid rich solid waste from an oil refining industry. The culture liquid of the selected bacteria and an enzymatic preparation obtained by precipitation with ammonium sulphate from a filtrate of the same culture liquid were used for enzymatic pretreatment. The hydrolysis was carried with different incubation concentrations (10, 20 and 30%) of soap stock and the pretreatment efficiency was verified by running comparative biodegradability tests (crude and treated lipid waste). All pretreated assays showed higher reaction rate compared to crude lipid waste, which was confirmed by the increased levels of biogas production. The pretreatment of solutions containing 10% emulsified soap stock was optimized for 24 h hydrolysis time, enabling high-biogaz formation (800 ml). The use of enzymatic pre-treatment seemed to be a very promising alternative for treating soap stock having high fat contents.

Lipolytic activity levels and colipase presence in digestive glands of some marine animals.

Studies on the digestive secretions in aquatic animals can elucidate certain aspects of their nutritive physiology. The aim of the present study was to compare the digestive lipase and phospholipase activities in ten marine species belonging to four classes following the taxonomic classification of marine organisms. All aquatic digestive tissues tested are equipped with lipase and phospholipase activities, assuming the hydrolysis of fat-rich food. The lipolytic activities determined in the pancreases of cartilaginous fishes were greater than those in bony fishes, molluscs and crustaceans. This finding might be explained by the strong digestive utilization of fat-rich macronutrients by these carnivorous fishes. A trend of activities and stabilities at different pH and temperatures for crude lipases and phospholipases from these aquatic animals suggests that the optimum pH and temperature for marine lipases are species dependent. Interestingly, the sardine caecal lipase and phospholipase were found to be mostly stable in a broad range of acidic pH values. The maximum activities of lipolytic enzymes from the hepatopancreases of Hexaplex trunculus (molluscs) and Carcinus mediterranus (crustaceans) were found to be 50 and 60 °C, respectively, whereas the optimal temperature of lipolytic enzymes for the other species was classically around 40 °C. Thermoactivity of molluscs' lipolytic preparations makes them potential candidates in industrial applications. Among digestive glands studied, only pancreas (cartilaginous fish) contained the classically known colipase. Regarded as the most primitive living jawed vertebrates, cartilaginous fishes represented by sharks and rays could be considered as the oldest vertebrates possessing a complex digestive system like that of mammals.

Characterization of a novel biosurfactant produced by Staphylococcus sp. strain 1E with potential application on hydrocarbon bioremediation.

A biosurfactant-producing bacterium (Staphylococcus sp. strain 1E) was isolated from an Algerian crude oil contaminated soil. Biosurfactant production was tested with different carbon sources using the surface tension measurement and the oil displacement test. Olive oil produced the highest reduction in surface tension (25.9 dynes cm(-1)). Crude oil presented the best substrate for 1E biosurfactant emulsification activity. The biosurfactant produced by strain 1E reduced the growth medium surface tension below 30 dynes cm(-1). This reduction was also obtained in cell-free filtrates. Biosurfactant produced by strain 1E showed stability in a wide range of pH (from 2 to 12), temperature (from 4 to 55 °C) and salinity (from 0 to 300 g l(-1)) variations. The biosurfactant produced by strain 1E belonged to lipopeptide group and also constituted an antibacterial activity againt the pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis. Phenanthrene solubility in water was enhanced by biosurfactant addition. Our results suggest that the 1E biosurfactant has interesting properties for its application in bioremediation of hydrocarbons contaminated sites.

Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain.

BACKGROUND: Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. RESULTS: This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C). The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry), was used as the sole carbon source. CONCLUSION: A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment.

A newly high alkaline lipase: an ideal choice for application in detergent formulations.

BACKGROUND: Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. RESULTS: A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1) were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. CONCLUSIONS: These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.

An organic-solvent-tolerant esterase from turkey pharyngeal tissue.

Stability is a crucial factor for the application of enzymes in biotechnology. Investigation of esterase activity in the pharyngeal tissue of turkey (Meleagris gallopavo), showed that optimum catalytic conditions of pure enzyme were 50 degrees C and pH 8.5. Turkey pharyngeal esterase (TPE) retained 75% of its maximum activity after incubation for 1h at 50 degrees C. Thermostability of the esterase was enhanced in the presence of an analogous substrate: phosphatidylcholine. TPE had a wide pH range of stability (pH 4.0-10.0). Esterase activity was compatible with the presence of organic solvents. Furthermore, the hydrolysis was found to be slightly activated by Ca(2+), but drastically reduced by Zn(2+) and Cu(2+). Phenylmethanesulphonyl fluoride (PMSF) a serine-specific inhibitor, strongly inhibited the esterase activity, whereas beta-mercaptoethanol, a thiol group inhibitor, did not show any effect on the activity. Esterase activity in the presence of organic solvents, as well as in acidic and alkaline pHs and at high temperatures makes it a good candidate for its application in non-aqueous biocatalysis.

Crab digestive phospholipase: a new invertebrate member.

Crab digestive phospholipase (CDPL) was purified from the hepatopancreas of Carcinus mediterraneus crabs. Homogeneous enzyme was obtained after two chromatography steps: anion exchange and size exclusion HPLC column. Homogeneous CDPL has a molecular mass of 14 kDa as determined by SDS/PAGE analysis. Unlike known digestive phospholipases like porcine PLA(2) (PPPL), CDPL displayed its maximal activity at 50 degrees C and not at 37 degrees C. A specific activity of 40 U/mg for the purified CDPL was measured using PC as substrate under optimal conditions (pH 8 and 50 degrees C) in the presence of 8 mM sodium deoxycholate (NaDC) and 10 mM CaCl(2). In contrast to PPPL, purified CDPL was completely inactivated at 60 degrees C. The N-terminal sequence was determined by automatic Edman degradation. No similarity between 12 N-terminal amino acid residues of CDPL was found with those of known digestive phospholipases. CDPL appears to be a new member of invertebrate phospholipases, and it is potentially useful for treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds which can be used in the food industry.

Occurrence of pancreatic lipase-related protein-2 in various species and its relationship with herbivore diet.

The occurrence of classical pancreatic lipase (PL) and pancreatic lipase-related proteins 1 and 2 (PLRP1s and 2) in the pancreas of ten mammalian species (humans, pig, rat, guinea pig, coypu, rabbit, horse, ox, goat and sheep) and two bird species (ostrich and turkey) was investigated. The lipases were purified from delipidated pancreas and identified based on the results of Western blotting analysis with anti-human PLRP2 serum, the catalytic properties and N-terminal microsequencing data. PLRP2s were detected in the pancreas of monogastric herbivorous animals (guinea pig, coypu, rabbit and horse) but not in that of ruminant herbivorous animals (ox, goat and sheep). The pancreas of carnivorous animals (dogs and cats) does not have any detectable PLRP2, but contains high levels of PL and PLRP1. By contrast, the pancreas of omnivorous animals (humans and rats) contains PL, PLRP1 and PLRP2, with the exception of porcine pancreas, where no PLRP2 was detected. In the case of bird (ostrich and turkey) pancreases, only classical PL was detected. The substrate specificity of PLRP2s was investigated using phospholipid micelles and synthetic monomolecular galactolipid films. Like human PLRP2, rabbit and horse PLRP2s are galactolipases. In polygastric herbivorous animals (ruminants), however, galactolipids are hydrolyzed via microbial enzymatic processes (involving galactolipases). The absence of galactolipids in carnivorous animals' diet may explain why no PLRP2s were detected here in the pancreas of these species.

Crab digestive lipase acting at high temperature: purification and biochemical characterization.

In recent years, recovery and characterization of enzymes from fish and aquatic invertebrates have taken place and this had led to the emergence of some interesting new applications of these enzymes. However, much less is known about lipases from crustaceans. A lipolytic activity was located in the crab digestive glands (hepatopancreas), from which a crab digestive lipase (CDL) was purified. Pure CDL has a molecular mass of 65kDa as determined by SDS/PAGE analysis. Unlike known digestive lipases, CDL displayed its maximal activity on long and short-chain triacylglycerols at a temperature of 60 degrees C. A specific activity of 500U/mg or 130U/mg was obtained with TC(4) or olive oil as substrate, respectively. Only 10% of the maximal activity was detected at 37 degrees C. The enzyme retained 80% of its maximal activity when incubated during 10 min at 60 degrees C, and was completely inactivated at a temperature higher than 65 degrees C. Interestingly, neither colipase, nor bile salts were detected in the crab hepatopancreas. Which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produce bile salts. No similarity between the 13 N-terminal amino acid residues of CDL was found with those of known other digestive lipases.

Purification and biochemical characterization of a turkey preduodenal esterase.

A preduodenal esterase was purified to homogeneity from turkey (Meleagris gallopavo) pharyngial tissue. Pure turkey pregastric esterase (TPrE) was obtained after anion exchange chromatography (DEAE-cellulose), Sephacryl S-200 gel filtration, anion exchange chromatography (Mono-Q sepharose) and affinity chromatography (Blue-Gel Affi Gel). The pure enzyme has an apparent molecular mass of 50 kDa, as determined by SDS/PAGE analysis. The optimum pH and temperature for enzyme activity using tributyrin as substrate were 8.5 and 48 degrees C, respectively. Under these conditions, the specific activity measured was 650 U/mg. No significant lipolytic activity was found when was tested on triolein or liprocil as substrates or with monolayer dicaprin with TPrE. In contrast, TPrE displayed a maximal activities of 800, 680 and 520 U/mg with vinyl acetate, vinyl propionate and vinyl butyrate, respectively. This enzyme retained 75% of its maximal activity when incubated for 30 min at pH 4 and 50 degrees C, but it was completely inactivated after an incubation for 10 min at 60 degrees C. The TPrE N-terminal amino acid sequence showed similarities to the N-terminal sequence of a thioesterase from mallard duck, but no similarity with known preduodenal lipases was found.

In vitro sulfation of N-acetyllactosaminide by soluble recombinant human beta-Gal-3'-sulfotransferase.

Membrane-bound beta-Gal-3'-sulfotransferase (GP3ST) was expressed and used for in vitro sulfation of Tamm-Horsfall glycoprotein. Further, the regioselective transfer of sulfate to an N-acetyllactosamine derivative could be realised with soluble chimeric GP3ST, also in combination with Lac transglycosylation by means of beta-galactosidase. Two alternative straightforward chemical syntheses for the target compound could be elaborated.

Purification and characterization of a novel lipase from the digestive glands of a primitive animal: the scorpion.

Higher animal's lipases are well characterized, however, much less is known about lipases from primitive ones. We choose the scorpion, one of the most ancient invertebrates, as a model of a primitive animal. A lipolytic activity was located in the scorpion digestive glands, from which a scorpion digestive lipase (SDL) was purified. Pure SDL, a glycosylated protein, has a molecular mass of 50 kDa, it presents the interfacial activation phenomenon. It was found to be more active on short-chain triacylglycerols than on long-chain triacylglycerols. SDL is a serine enzyme and possesses one accessible sulfhydryl group which is not essential for the catalysis. Among the NH2-terminal 33 residues, a 17 amino acids sequence shows similarities with sequence of Drosophila melanogaster putative lipase. Interestingly, neither colipase, nor bile salts were detected in the scorpion hepatopancreas. This indicates that colipase evolved in vertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. Furthermore, polyclonal antibodies directed against SDL failed to recognise the classical digestive lipases. Altogether, these results suggest that SDL is a member of a new group of digestive lipases belonging to invertebrates.

Synthesis of a molecular mimic of the Glc1Man9 oligoside as potential inhibitor of calnexin binding to DeltaF508 CFTR protein.

Deletion of phenylalanine at position 508 of the CFTR protein is associated with a severe form of cystic fibrosis. Biosynthetic arrest of the misfolded DeltaF508 CFTR protein in the endoplasmic reticulum is due to prolonged interaction with protein chaperones. In order to overcome this retention and thereby restore the delivery of the protein to the plasma membrane, a molecular mimic of the glycoprotein oligoside moiety has been designed and synthesized. Ability of this mimic to inhibit the binding of the natural Glc1Man9GlcNAc oligoside to calnexin has been measured.