A new chymotrypsin-like serine protease involved in dietary protein digestion in a primitive animal, Scorpio maurus: purification and biochemical characterization

<p>Abstract</p> <p>Background</p> <p>Most recent works on chymotrypsins have been focused on marine animals and insects. However, no study was reported in chelicerate.</p> <p>Results</p> <p>Scorpion chymotrypsin-like protease (SCP) was purified to homogeneity from delipidated hepatopancreases. The protease NH₂-terminal sequence exhibited more than 60% monoacids identity with those of insect putative peptidases. The protease displayed no sequence homology with classical proteases. From this point of view, the protease recalls the case of the scorpion lipase which displayed no sequence homology with known lipases. The scorpion amylase purified and characterized by our time, has an amino-acids sequence similar to those of mammalian amylases. The enzyme was characterized with respect its biochemical properties: it was active on a chymotrypsin substrate and had an apparent molecular mass of 25 kDa, like the classically known chymotrypsins. The dependence of the SCP activity and stability on pH and temperature was similar to that of mammalian chymotrypsin proteases. However, the SCP displayed a lower specific activity and a boarder pH activity range (from 6 to 9).</p> <p>Conclusion</p> <p>lower animal have a less evaluated digestive organ: a hepatopancreas, whereas, higher ones possess individualized pancreas and liver. A new chymotrypsin-like protease was purified for the first time from the scorpion hepatopancreas. Its biochemical characterization showed new features as compared to classical chymotrypsin-higher-animals proteases.</p

Immobilized Rhizopus oryzae lipase catalyzed synthesis of palm stearin and cetyl alcohol wax esters: Optimization by Response Surface Methodology

<p>Abstract</p> <p>Background</p> <p>Waxes are esters of long-chain fatty acids and long-chain alcohols. Their principal natural sources are animals (sperm whale oil) and vegetables (jojoba) which are expensive and not easily available. Wax esters synthesized by enzymatic transesterification, using palm stearin as raw material, can be considered as an alternative to natural ones.</p> <p>Results</p> <p>Palm stearin is a solid fraction obtained by fractionation of palm oil. Palm stearin was esterified with cetyl alcohol to produce a mixture of wax esters. A non-commercial immobilized lipase from <it>Rhizopus oryzae </it>was used as biocatalyst. Response surface methodology was employed to determine the effects of the temperature (30-50°C), the enzyme concentration (33.34-300 IU/mL), the alcohol/palm stearin molar ratio (3-7 mol/mol) and the substrate concentration (0.06-0.34 g/mL) on the conversion yield of palm stearin. Under optimal conditions (temperature, 30°C; enzyme concentration, 300 IU/mL; molar ratio 3 and substrate concentration 0.21 g/mL) a high conversion yield of 98.52% was reached within a reaction time of 2 h.</p> <p>Conclusions</p> <p>Response surface methodology was successfully applied to determine the optimum operational conditions for synthesis of palm stearin based wax esters. This study may provide useful tools to develop economical and efficient processes for the synthesis of wax esters.</p

Purification and biochemical characterization of a secreted group IIA chicken intestinal phospholipase A2

<p>Abstract</p> <p>Background</p> <p>Secretory phospholipase A2 group IIA (IIA PLA2) is a protein shown to be highly expressed in the intestine of mammals. However, no study was reported in birds.</p> <p>Results</p> <p>Chicken intestinal group IIA phospholipase A₂(ChPLA₂-IIA) was obtained after an acidic treatment (pH.3.0), precipitation by ammonium sulphate, followed by sequential column chromatographies on Sephadex G-50 and mono-S ion exchanger. The enzyme was found to be a monomeric protein with a molecular mass of around 14 kDa. The purified enzyme showed a substrate preference for phosphatidylethanolamine and phosphatidylglycerol, and didn't hydrolyse phosphatidylcholine. Under optimal assay conditions, in the presence of 10 mM NaTDC and 10 mM CaCl_2,a specific activity of 160 U.mg^-1for purified ChPLA₂-IIA was measured using egg yolk as substrate. The fifteen NH2-terminal amino acid residues of ChPLA₂-IIA were sequenced and showed a close homology with known intestinal secreted phospholipases A₂. The gene encoding the mature ChPLA₂-IIA was cloned and sequenced. To further investigate structure-activity relationship, a 3D model of ChPLA₂-IIA was built using the human intestinal phospholipase A₂structure as template.</p> <p>Conclusion</p> <p>ChPLA2-IIA was purified to homogeneity using only two chromatographic colomns. Sequence analysis of the cloned cDNA indicates that the enzyme is highly basic with a pI of 9.0 and has a high degree of homology with mammalian intestinal PLA₂-IIA.</p

Purification and characterization of the first recombinant bird pancreatic lipase expressed in Pichia pastoris: The turkey

<p>Abstract</p> <p>Background</p> <p>The turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Some biochemical properties and kinetic studies were determined using emulsified system and monomolecular film techniques. Those studies have shown that despite the accumulation of free fatty acids at the olive oil/water interface, TPL continues to hydrolyse efficiently the olive oil and the TC₄in the absence of colipase and bile salts, contrary to most classical digestive lipases which denaturate rapidly under the same conditions. The aim of the present study was to express TPL in the methylotrophic yeast <it>Pichia pastoris </it>in order to get a large amount of this enzyme exhibiting interesting biochemical properties, to purify and characterize the recombinant enzyme.</p> <p>Results</p> <p>The recombinant TPL was secreted into the culture medium and the expression level reached about 15 mg/l after 4 days of culture. Using Q-PCR, the number of expression cassette integrated on <it>Pichia </it>genomic DNA was estimated to 5. The purified rTPL, with molecular mass of 50 kDa, has a specific activity of 5300 U/mg on emulsified olive oil and 9500 U/mg on tributyrin. The optimal temperature and pH of rTPL were 37°C and pH 8.5. The stability, reaction kinetics and effects of calcium ions and bile salts were also determined.</p> <p>Conclusions</p> <p>Our results show that the expressed TPL have the same properties as the native TPL previously purified. This result allows us the use of the recombinant enzyme to investigate the TPL structure-function relationships.</p

Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca

<p>Abstract</p> <p>Background</p> <p>Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes.</p> <p>Results</p> <p>A marine stingray phospholipase A₂(SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl₂using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry.</p> <p>Conclusions</p> <p>SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.</p

In vitro study of the PLA2 inhibition and antioxidant activities of Aloe vera leaf skin extracts

<p>Abstract</p> <p>Background</p> <p>In the present work we determined the total phenolic content of <it>Aloe vera </it>leaf skin (AVLS) extracts by using various solvents (hexane, chloroform-ethanol (1/1), ethyl acetate, butanol and water). We have also evaluated the antioxidant and the anti-PLA2 properties of these extracts by measuring their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA).</p> <p>Results</p> <p>The water extract exhibits the highest inhibitory effect with an IC₅₀= 0.22 mg/ml and interestingly no effect was observed on the digestive phospholipase A2 (group IB) even at a concentration of 5 mg/ml. Antioxidant activities were also analyzed and the most active extracts were observed when using chloroform ethanol (1/1) and ethyl acetate (IC₅₀= 0.274 and 0.326 mg/ml, respectively). Analysis of the total phenolic content reveals that the water extract, with the best anti-PLA2 effect, was poor in phenolic molecules (2 mg GAE/g). This latter value has to be compared with the chloroform-ethanol and the ethyl acetate extracts (40 and 23.8 mg GAE/g, respectively), mostly responsible for the antioxidant activity.</p> <p>Conclusion</p> <p>A significant correlation was established between the total phenolic content and the antioxidant capacity but not with the anti PLA2 activity. Results from phytochemical screening suggest that the anti PLA2 molecules were probably catechin tannins compounds.</p

Synthesis of lipophilic tyrosyl esters derivatives and assessment of their antimicrobial and antileishmania activities

<p>Abstract</p> <p>Background</p> <p>Preparation of tyrosyl lipophilic derivatives was carried out as a response to the food, cosmetic and pharmaceutical industries' increasing demand for new lipophilic antioxidants.</p> <p>Results</p> <p>A large series of tyrosyl esters (<b>TyC₂</b>to <b>TyC_18:1</b>) with increasing lipophilicity was synthesized in a good yield using lipase from <it>Candida antarctica </it>(Novozyme 435). Spectroscopic analyses of purified esters showed that the tyrosol was esterified on the primary hydroxyl group. Synthetized compounds were evaluated for either their antimicrobial activity, by both diffusion well and minimal inhibition concentration (MIC) methods, or their antileishmanial activity against <it>Leishmania major </it>and <it>Leishmania infantum </it>parasite species.</p> <p>Among all the tested compounds, our results showed that only <b>TyC₈</b>, <b>TyC₁₀</b>and <b>TyC₁₂</b>exhibited antibacterial and antileishmanial activities. When MIC and IC₅₀values were plotted against the acyl chain length of each tyrosyl derivative, <b>TyC₁₀</b>showed a parabolic shape with a minimum value. This nonlinear dependency with the increase of the chain length indicates that biological activities are probably associated to the surfactant effectiveness of lipophilic derivatives.</p> <p>Conclusion</p> <p>These results open up potential applications to use medium tyrosyl derivatives surfactants, antioxidants, antimicrobial and antileishmanial compounds in cosmetic, food and pharmaceutical industries.</p

Bipolar Disorder and Multiple Sclerosis: A Case Series

Background. The prevalence of psychiatric disturbance for patients with multiple sclerosis (MS) is higher than that observed in other chronic health conditions. We report three cases of MS and bipolar disorder and we discuss the possible etiological hypothesis and treatment options. Observations. All patients fulfilled the McDonald criteria for MS. Two patients were followed up in psychiatry for manic or depressive symptoms before developing MS. A third patient was diagnosed with MS and developed deferred psychotic symptoms. Some clinical and radiological features are highlighted in our patients: one manic episode induced by high dose corticosteroids and one case of a new orbitofrontal MRI lesion concomitant with the emergence of psychiatric symptoms. All patients needed antipsychotic treatment with almost good tolerance for high dose corticosteroids and interferon beta treatment. Conclusions. MRI lesions suggest the possible implication of local MS-related brain damage in development of pure “psychiatric fits” in MS. Genetic susceptibility is another hypothesis for this association. We have noticed that interferon beta treatments were well tolerated while high dose corticosteroids may induce manic fits