Cleaner degreasing of sheepskins by the Yarrowia lipolytica LIP2 lipase as a chemical-free alternative in the leather industry.

Conventional degreasing of skins and hides in the leather industry requires high amounts of organic solvents and detergents that cause environmental issues. In this study, the LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was shown to be effective in degreasing sheepskins, thus reducing the amount of harmful chemicals. Using 6 mg of lipase/kg of raw skin, successful degreasing was achieved in only 15 min at pH 8 and 30°C. ToF-SIMS mass spectra of chemically and enzymatically treated sheepskins are consistent with a selective elimination process for the enzymatic treatment. Comparative SEM microscopy, ATR-FTIR spectroscopy and physicochemical analyses showed better properties of the enzymatically treated leather than those of the chemical treatment. Effluent physicochemical parameters showed that the enzymatic treatment is a cleaner degreasing operation. Altogether, this work opens new horizons to use the YLLIP2 lipase as a more efficient alternative in the leather industry.

Oil from hake (Merluccius merluccius): Characterization, antioxidant activity, wound healing and anti-inflammatory effects.

The aim of this work was to evaluate some biological properties of hake head oil (HHO) as well its lipid composition. The fatty acid profiles showed a dominance of unsaturated fatty acids overtaking 55% of the total fatty acids. Omega-3 polyunsaturated fatty acid profiles exhibited a dominance of EPA (eicosapentaenoic acid) (3.96%) and DHA (docosahexaenoic acid) (25.39%). The antioxidant activity was determined through two different assays: DPPH scavenging activity and ß-carotene bleaching by linoleic acid assay. Eighteen mice were excised on their back and divided into 3 groups, treated with sterile saline, commercial healing cream and HHO, respectively. The wound closure rate, the hydroxyproline contents and the histopathology evolution in skin tissue were elaborated. Also, the anti-inflammatory activity was studied by carrageenan-induced mouse paw edema. Mice were divided into 3 groups treated respectively with sterile saline, anti inflammatory drug reference and HHO. The anti-inflammatory evaluation of HHO in mice exhibited an important inhibition of carrageenan-induced hind paws edema, as confirmed by the histological analysis, the superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) level. HHO displayed a significant wound healing effect probably due to the anti-inflammatory and antioxidant activities of its EPA and DHA contents. The overall results proved that HHO might be favorable drugs who exert a great therapeutic potential wound healing and anti-inflammatory effects in animal model.

The extremely halophilic archaeon Halobacterium salinarum ETD5 from the solar saltern of Sfax (Tunisia) produces multiple halocins.

The extremely halophilic archaeon Halobacterium salinarum strain ETD5 was previously isolated from the solar saltern of Sfax (Tunisia) and shown to encode and express halocin S8. The Hbt. salinarum ETD5 culture supernatant was shown here to exhibit high antimicrobial activity against several halophilic archaea and bacteria of different genera, showing a cross-domain inhibition. The antimicrobial activity was destroyed by proteases, thus pointing to halocins. A bioguided purification procedure was applied using two chromatography steps and antimicrobial assays directed against Halorubrum chaoviator ETR14. In-gel screening assay showed the presence of two antimicrobial bands of approximately 8 and 14 kDa, for which characterization was investigated by N-terminal sequencing and mass spectrometry. The full-length form of halocin S8 that contains 81 amino acids and differs from the 36 amino acid short-length halocin S8 previously described from an uncharacterized haloarchaeon S8a, was identified in the 8 kDa halocin band. A novel halocin that we termed halocin S14 was found in the 14 kDa band. It exhibits amino acid sequence identities with the N-terminally truncated region of the archaeal Mn-superoxide dismutase. These results show that Hbt. salinarum ETD5 produces multiple halocins, a feature that had not been described until now in the domain Archaea.

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.

Characterization of monoacylglycerols and diacylglycerols rich in polyunsaturated fatty acids produced by hydrolysis of Musteleus mustelus liver oil catalyzed by an immobilized bacterial lipase.

The use of an immobilized Serratia. sp W3 lipase as a replacement for the standard pancreatic lipases in the hydrolysis of liver oil from the Musteleus mustelus was studied. Monoacylglycerols (MAGs) and diacylglycerols (DAGs) containing ω-3 polyunsaturated fatty acids, namely eicosapentaenoic and docosahexaenoic acids were produced in hexane solvent at reaction temperatures reaching 55 °C with a molar triacylglycerol conversion over than 75 ± 5% in 24 h showing excellent hydrolysis characteristics. The favorable conditions for the hydrolysis reaction allowed fats with higher melting points to be analyzed facilitating the coupling of the hydrolysis reaction to the later steps in the analytical protocol. The lipid composition was elucidated for the first time by employing a highly efficient UHPLC-MS method with a novel embedded linear retention index approach. MAGs and DAGs obtained during the enzymatic hydrolysis could be used for the production of glycerol based emulsifiers of nutritional interest.

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.

Physicochemical properties, antioxidant activity and in vitro gastrointestinal digestion of purified proteins from black cumin seeds.

The main purpose of this work was to investigate some physiochemical and antioxidant properties of purified proteins of 18 kDa from black cumin seeds. The structural properties of purified proteins were characterized using Fourier Transform-Infrared spectroscopy (FT-IR) and Circular dichroism (CD) spectroscopy analysis. The Tunisian purified protein exhibited a α-helix structure and the Turkish purified protein adopted a ß-sheet conformation. The thermal properties were also evaluated by differential scanning calorimetry (DSC). The Tunisian purified protein presented two endothermic peaks, the first one was at 76.71 °C and the second one was at 131.32 °C. However, only one endothermic peak was found for the Turkish purified protein at 157.63 °C. Both Tunisian and Turkish purified proteins were very stable towards heat-induced denaturation. In addition, the effect of pH, salt (NaCl and Na2SO4) and temperature on functional properties was investigated. The Tunisian black cumin seeds pure protein exhibited better antioxidant activity than that of the Turkish one at all tested concentrations (0.2 to 1 mg/mL) and temperatures (50 °C, 75 °C and 100 °C), as well as after gastrointestinal digestion simulation.

Helix aspersa gelatin as an emulsifier and emulsion stabilizer: functional properties and effects on pancreatic lipolysis.

Emulsions are widely used in food and pharmaceutical applications for the encapsulation, solubilization, entrapment, and controlled delivery of active ingredients. In order to fulfill the increasing demand for clean label excipients, natural polymers could be used to replace the potentially irritative synthetic surfactants used in emulsion formulation. In the present study, we have studied the properties of oil-in-water emulsions prepared with land snail gelatin (LSG) as the sole emulsifying agent, extracted and described for the first time. LSG was evaluated in terms of proximate composition, oil and water holding capacity, emulsifying and foaming properties, color and amino acid composition. Emulsions of trioctanoylglycerol (TC8) and olive oil were made at different gelatin/oil ratios and changes in droplet-size distribution were determined. The superior emulsifying properties of LSG, the susceptibility of gelatin protein emulsions increasing flocculation on storage, and the coalescence of gelatin emulsions following centrifugation were demonstrated. Furthermore, the effect of LSG on the activity of turkey pancreatic lipase (TPL) was evaluated through the pH-stat methodology with TC8 and olive oil emulsions. The LSG affected the TPL activity in a concentration-dependent way. Our results showed that LSG, comparably to gum arabic, increases the pancreatic lipase activity and improves its stability at the oil-water interface.

Cactus juice as bioflocculant in the coagulation-flocculation process for industrial wastewater treatment: a comparative study with polyacrylamide.

Most industries in the world treat their wastewaters with a conventional coagulation-flocculation process using alum as coagulant, polyacrylamide (PAM) as flocculant and lime as coagulant aid. To reduce the use of chemical products in the process, experiments were conducted to substitute the PAM with cactus juice (CJ) as flocculant. From the obtained data, it was concluded that the substitution of PAM with CJ in the coagulation-flocculation process was very effective, compared with PAM. Depending on the wastewater's origin, the bioflocculant showed removal efficiencies of 83.3-88.7% for suspended solids (SS) and 59.1-69.1% for chemical oxygen demand (COD). Lime addition enhanced the coagulation-flocculation process in the presence of CJ similarly to the PAM with efficiencies greater than 90% for both SS and COD. The CJ powder's infrared (IR) spectrum showed the main functional groups present in PAM. It was concluded that CJ as a flocculant fits well with the definition of sustainability and it is appropriate for countries that have regions where cactuses grow naturally.

Essential oil of the leaves of Ricinus communis L.: in vitro cytotoxicity and antimicrobial properties.

BACKGROUND: The aim of the present study was to appraise the antimicrobial activity of Ricinus communis L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines. METHODS: The agar disk diffusion method was used to study the antibacterial activity of Ricinus communis L. essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of Ricinus communis L. essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test. RESULTS: The essential oil from the leaves of Ricinus communis L. was analyzed by GC-MS and bioassays were carried out. Five constituents of the oil were identified by GC-MS. The antimicrobial activity of the oil was investigated in order to evaluate its efficacy against twelve bacteria and four fungi species, using disc diffusion and minimum inhibitory concentration methods. The essential oil showed strong antimicrobial activity against all microorganisms tested with higher sensitivity for Bacillus subtilis, Staphylococcus aureus and Enterobacter cloacae. The cytotoxic and apoptotic effects of the essential oil on HeLa cell lines were examined by MTT assay. The cytotoxicity of the oil was quite strong with IC50 values less than 2.63 mg/ml for both cell lines. CONCLUSION: The present study showed the potential antimicrobial and anticarcinogenic properties of the essential oil of Ricinus communis L., indicating the possibilities of its potential use in the formula of natural remedies for the topical treatment of infections.

Antibacterial, anti-chlamydial, and cytotoxic activities of a marine snail (Hexaplex trunculus) phospholipase A2: an in vitro study.

In order to report pharmacological characterization of marine snail (Hexaplex trunculus) hepatopancreatic phospholipase A(2) (mSDPLA(2)), we have talked for the first time the antimicrobial activity against different pathogenic bacterial strains, anti-chlamydial activity as well as its cytotoxic activity against McCoy cell lines. mSDPLA(2), showed a high level of activity towards Gram-positive bacteria as Staphylococcus aureus and Staphylococcus epidermidis. Whereas Gram-negative bacteria, unfortunately, exhibited a higher resistance, mSDPLA(2) was also found to have a strong cytotoxic activity, causing significant morphological alterations of the McCoy cell lines surfaces and to be a hinder to the proliferation. Moreover, mSDPLA(2) proved to have a very potent anti-chlamydial activity. Over 95 % inhibition of chlamydial inclusions were obtained at a concentration of 10 µg/ml of mSDPLA(2) after 24 h postinfection. Interestingly, at a concentration of 10 µg/ml of mSDPLA(2), the proliferation of McCoy cells was not affected. Approximately 50 % inhibition of cell growth was obtained with a concentration of 37 µg/mL of mSDPLA(2). mSDPLA(2) could be considered as an excellent candidate for the development of a new anti-infective agent. This enzyme showed significant antimicrobial activities.

A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters.

A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively.

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.

Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells.

BACKGROUND: Among the digestive enzymes, phospholipase A2 (PLA2) hydrolyzes the essential dietary phospholipids in marine fish and shellfish. However, we know little about the organs that produce PLA2, and the ontogeny of the PLA2-cells. Accordingly, accurate localization of PLA2 in marine snails might afford a better understanding permitting the control of the quality and composition of diets and the mode of digestion of lipid food. RESULTS: We have previously producted an antiserum reacting specifically with mSDPLA2. It labeled zymogen granules of the hepatopancreatic acinar cells and the secretory materials of certain epithelial cells in the depths of epithelial crypts in the hepatopancreas of snail. To confirm this localization a laser capture microdissection was performed targeting stained cells of hepatopancreas tissue sections. A Western blot analysis revealed a strong signal at the expected size (30 kDa), probably corresponding to the PLA2. CONCLUSIONS: The present results support the presence of two hepatopancreatic intracellular and extracellular PLA2 in the prosobranchs gastropods molluscs, Littorina littorea and Buccinum undatum and bring insights on their localizations.

The in-vitro evaluation of antibacterial, antifungal and cytotoxic properties of Marrubium vulgare L. essential oil grown in Tunisia.

BACKGROUND: In order to validate its antiseptic and anticancer properties with respect to traditional uses, we have screened for the first time the antimicrobial activity of aerial parts of M. vulgare L. essential oil against different pathogenic microorganisms and the cytotoxic activity against HeLa cell lines. METHODS: The agar disk diffusion method was used to study the antibacterial activity of M. vulgare essential oil against 12 bacterial and 4 fungi strains. The disc diameters of zone of inhibition (DD), the minimum inhibitory concentrations (MIC) and the concentration inhibiting 50% (IC50) were investigated to characterize the antimicrobial activities of this essential oil. The in vitro cytotoxicity of M. vulgare essential oil was examined using a modified MTT assay; the viability and the IC50 were used to evaluate this test. RESULTS: The antimicrobial activity of the essential oil was investigated in order to evaluate its efficacy against the different tested microorganisms. The present results results showed a significant activity against microorganisms especially Gram (+) bacteria with inhibition zones and minimal inhibitory concentration values in the range of 6.6-25.2 mm and 1120-2600 µg/ml, respectively, whereas Gram (-) bacteria exhibited a higher resistance. As far as the antifungal activity, among four strains tested, Botrytis cinerea exhibited the strongest activity with inhibition zones of 12.6 mm. However, Fusarium solani, Penicillium digitatum and Aspergillus niger were less sensitive to M. vulgare essential oil. About the citotoxicity assay, this finding indicate the capability of this essential oil to inhibited the proliferation of HeLa cell lines under some conditions with IC50 value of 0.258 µg/ml. CONCLUSION: This investigation showed that the M. vulgare essential oil has a potent antimicrobial activity against some Gram (+) pathogenic bacteria and Botrytis cinerea fungi. The present studies confirm the use of this essential oil as anticancer agent. Further research is required to evaluate the practical values of therapeutic applications.

Kinetic properties of pancreatic and intestinal sPLA2 from chicken and mammals using the monomolecular film technique.

The interfacial kinetic and binding data for the pancreatic and intestinal sPLA2 from bird and mammals show that these enzymes have dramatically different ability to bind and hydrolyse phospholipids. The main conclusions from our experimental data indicate that phosphatidylcholine monolayers (PC), in contrast to phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), were resistant to the hydrolysis by human intestinal sPLA2. Conversely, chicken intestinal sPLA2 was found to be able to hydrolyse all the phospholipids tested, including PC. The experiments show also that the interfacial penetrating ability of chicken sPLA2 (from intestine and pancreas) was higher than their mammalian's orthologs. This observation is confirmed by the activity of pancreatic chicken PLA2 measured on PC film showing that the interfacial pressure window that permits sPLA2 activity was very large, between 5 and 20 dynes cm(-1), compared with the porcine pancreatic sPLA2-IB which was inactive at pressure above 15 dynes cm(-1). In trying to establish a structure-function relationship, we examined the surface electrostatic potentials of the various sPLA2 from chicken and mammals. We reported in this study that the binding, orientation and persistence of sPLA2 at the lipid-water interface is probably governed by the electrostatic and hydrophobic forces operative at this surface. These variations argue strongly that these enzymes are not isoforms and that they are expected to have functions other than the release of lipid mediators for the biosynthesis of the eicosanoids.

Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells.

BACKGROUND: Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. RESULTS: The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. CONCLUSION: The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids.

Nutrient composition of the marine snail (Hexaplex trunculus) from the Tunisian Mediterranean coasts.

BACKGROUND: Marine snail (Hexaplex trunculus) presents increasing nutritional, commercial and economical importance, being widely consumed in northern Africa, particularly in Mediterranean countries. From a nutritional point of view there is still limited information on the chemical composition of edible tissues (meat and hepatopancreas) of this species. Therefore, the aims of the present work were to study the proximate chemical composition, fatty acid and amino acid profiles of H. trunculus from the Tunisian Mediterranean coasts. RESULTS: Fatty acid profiles showed that the polyunsaturated fatty acids (PUFA) content is higher than the saturated fatty acids (SFA). The yields of PUFA and SFA present in the meat fat were 68.2% and 33.4% of the total fatty acids, respectively. Similar values were obtained in the hepatopancreatic lipidic fraction. Snail tissues contain valuable concentrations of PUFA, especially n-6 and n-3 with chain lengths of 20 and 22 carbons. All edible tissues were valuable sources of essential amino acids. Aspartic acid is the major amino acids present in the meat and hepatopancreas. The concentrations of nutrients were also determined in the hepatopancreas and meat of H. trunculus. Significantly high concentrations of minerals and trace elements were found in these tissues. CONCLUSION: This study suggests that H. trunculus is an important source of protein and essential amino acids. Furthermore, the snail lipidic fraction contains high proportions of polyunsaturated fatty acids benefical for human health.

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities.

A marine snail digestive phospholipase A2 (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A2, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 degrees C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6mM CaCl2. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases. Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA2. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A2 from venoms and another class of non toxic pancreatic phospholipase A2 from mammals.

Enzymatic synthesis of eugenol benzoate by immobilized Staphylococcus aureus lipase: optimization using response surface methodology and determination of antioxidant activity.

The ability of a non-commercial immobilized Staphylococcus aureus lipase to catalyze the esterification of eugenol with benzoic acid was checked and the antioxidant power of the ester formed was evaluated. Response surface methodology based on four variables (the reaction temperature, the amount of lipase, the benzoic acid/eugenol molar ratio and the volume of solvent) was used to optimize the experimental conditions of eugenol benzoate synthesis. The maximum conversion yield (75%) was obtained using 240 IU of immobilized lipase, a benzoic acid/eugenol molar ratio of 1.22 dissolved in 4.6 ml chloroform at 41 degrees Celsius. The antioxidant activities of eugenol and its ester were evaluated. Compared to BHT, used as a model synthetic antioxidant, the eugenol benzoate showed a higher antioxidative activity. The IC(50) value for 1,1-diphenyl-2-picrylhydrazyl was found to be 18.2 microg/ml versus 20.2 microg/ml for eugenol and eugenol benzoate.