Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Synergistic effect of polysaccharides, betalain pigment and phenolic compounds of red prickly pear (Opuntia stricta) in the stabilization of salami.

The aim of this work is to try to substitute some synthetic additives by a natural extract from red prickly pear (Opuntia stricta) which known by its richness on bioactive polysaccharides mainly consisting of galactose, rhamnose and galacturonic acid. This natural fruit has a high content of carbohydrates above 18.81% FM. It contains also a high level of polyphenols 152.25 ±â€¯0.26 µg QE/mg PPE and flavonoids about 370.60 ±â€¯0.12 µg GAE/mg of PPE. In addition, prickly pear extract (PPE) displayed a strong antioxidant and antimicrobial activities. These activities are likely due to its phenolic, flavonoid and carbohydrate contents. Moreover, the addition of 2.5% of PPE, as a natural colorant and antimicrobial agent in salami formulation, causes a decrease in hardness and chewiness of the formulated salami. Interestingly, PPE inhibited bacterial growth in salami stored at 4 °C over 30 days. Sensorial analysis shows that the color, taste and texture of salami prepared with 2.5% of PPE are markedly more appreciated by panelists. Our results suggest that the betalain pigment, carbohydrate and phenolic compounds present in PPE could be used as a natural colorant, antioxidant and antimicrobial agent without change of the sensory characteristics.

Efficient heterologous expression, functional characterization and molecular modeling of annular seabream digestive phospholipase A2.

Here we report the cDNA cloning of a phospholipase A2 (PLA2) from five Sparidae species. The deduced amino acid sequences show high similarity with pancreatic PLA2. In addition, a phylogenetic tree derived from alignment of various available sequences revealed that Sparidae PLA2 are closer to avian PLA2 group IB than to mammals' ones. In order to understand the structure-function relationships of these enzymes, we report here the recombinant expression in E.coli, the refolding and characterization of His-tagged annular seabream PLA2 (AsPLA2). A single Ni-affinity chromatography step was used to obtain a highly purified recombinant AsPLA2 with a molecular mass of 15kDa as attested by gel electrophoresis and MALDI-TOF mass spectrometry data. The enzyme has a specific activity of 400U.mg-1 measured on phosphatidylcholine at pH 8.5 and 50°C. The enzyme high thermo-activity and thermo-stability make it a potential candidate in various biological applications. The 3D structure models of these enzymes were compared with structures of phylogenetically related pancreatic PLA2. By following these models and utilizing molecular dynamics simulations, the resistance of the AsPLA2 at high temperatures was explained. Using the monomolecular film technique, AsPLA2 was found to be active on various phospholipids spread at the air/water interface at a surface pressure between 12 and 25dyncm-1. Interestingly, this enzyme was shown to be mostly active on dilauroyl-phosphatidylglycerol monolayers and this behavior was confirmed by molecular docking and dynamics simulations analysis. The discovery of a thermo-active new member of Sparidae PLA2, provides new insights on structure-activity relationships of fish PLA2.

Biochemical characterization, cloning and molecular modeling of a digestive lipase from red seabream (Pagrus major): Structural explanation of the interaction deficiency with colipase and lipidic interface.

Red seabream digestive lipase (RsDL) was purified from fresh pyloric caeca. Pure RsDL has an apparent molecular mass of 50 kDa. The RsDL is more active on short-chain triacylglycerols (TC4), and enzymatic activity decreases when medium (TC8) or long-chain (olive oil) triacylglycerols were used as substrates. The specific activities of RsDL are very weak as compared to those obtained with classical pancreatic lipases. No colipase was detected in the red seabream pyloric caeca. Furthermore, the RsDL was not activated by a mammal colipase. Similar results were reported for annular seabream lipase. In order to explain structurally the discrepancies between sparidae and mammal lipases, genes encoding mature RsDL and five other lipases from sparidae fish species were cloned and sequenced. Phylogenetic studies indicated the closest homology of sparidae lipases to bird pancreatic ones. Structural models were built for annular seabream and RsDL under their closed and open forms using mammal pancreatic lipases as templates. Several differences were noticed when analyzing the amino acids corresponding to those involved in HPL binding to colipase. This is likely to prevent interaction between the fish lipase and the mammalian colipase and may explain the fact that mammalian colipase is not effective in activating sparidae lipases. In addition, several hydrophobic residues, playing a key role in anchoring pancreatic lipase onto the lipid interface, are replaced by polar residues in fish lipases. This might explain the reason why the latter enzymes display weak activity levels when compared to mammalian pancreatic lipases.