Nabil Miled

Interfacial catalysis by lipases

Abstract We designed a convenient, specific, sensitive and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent. The presence of detergents above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase is linear with time and proportional to the amount of lipase added. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. We observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds.

N-terminal peptide of Rhizopus oryzae lipase is important for its catalytic properties

In a culture medium, the Rhizopus oryzae strain produces only one form of lipase, ROL32. When the concentrated culture medium was stored at 0 °C during several months or kept at 6 °C during a few days, we noticed the appearance of a second shorter form of ROL32 lacking its N‐terminal 28 amino acid (ROL29). ROL29 was purified to homogeneity and its 21 N‐terminal amino acid residues were found to be identical to the 29–49 sequence of ROL32. The cleavage of the N‐terminal peptide reduced the specific activity of ROL29 by 50% using either triolein or tributyrin as substrates. In order to explain this decrease of the specific activity of ROL29, we measured its critical surface pressure of penetration into phosphatidyl choline from egg yolk films which was found to be 10 mN/m, in contrast to a value of 23 mN/m found in ROL32. A kinetic study on the surface pressure dependency, stereoselectivity and regioselectivity of ROL29 was performed using the three dicaprin isomers spread as monomolecular films at the air–water interface. Our results showed that in contrast to ROL32, ROL29 presented a preference for the distal ester groups of one diglyceride isomer (1,3‐sn‐dicaprin). Furthermore, ROL32 was markedly more stereoselective than ROL29 for the sn‐3 position of the 2,3‐sn‐enantiomer of dicaprin. A structural explanation of the enhanced penetration capacity as well as the catalytic activity of ROL32 was proposed by molecular modeling. We concluded that the N‐terminal peptide of ROL32 can play an important role in the specific activity, the regioselectivity, the stereoselectivity and the binding of the enzyme to its substrate.

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.

Fish processing wastes for microbial enzyme production: a review

Fishery processing industries generate large amounts of by-products. The disposal of these wastes represents an increasing environmental and health problem. To avoid wasting these by-products, various disposal methods have been applied including, ensilation, fermentation, hydrolysate and fish oil production. Interestingly, fish by-products provide an excellent nutrient source for microbial growth useful in enzyme production process, which is largely governed by the cost related to the growth media. Fish wastes (heads, viscera, chitinous material, wastewater, etc.) were prepared and tested as growth substrates for microbial enzymes production such as protease, lipase, chitinolytic and ligninolytic enzymes. This new approach described in this review can reduce environmental problems associated with waste disposal and, simultaneously, lower the cost of microbial enzyme production.

A grey mullet enzyme displaying both lipase and phospholipase activities: Purification and characterization

A lipase from the golden grey mullet viscera was purified to homogeneity by ammonium sulphate precipitation, gel filtration, anionic and cation exchange chromatographies. The pure enzyme tentatively named grey mullet digestive lipase (GmDL) is a monomer having a molecular mass of about 35 kDa, as determined by SDS-PAGE analysis. No similarity was found between the NH2-terminal amino acid residues of GmDL and those of other known digestive lipases. GmDL is a serine enzyme, like all known lipases from different origins. Interestingly, GmDL has not only lipase activity but also a phospholipase activity which requires the presence of Ca(2+) and bile salts. Specific activities of 64 U/mg, 55 U/mg and 63 U/mg were measured using tributyrin, olive oil emulsion or phosphatidylcholine as substrate, respectively at pH 8 and at 50°C. GmDL is therefore a thermo-active enzyme as compared to other fish lipases studied so far. It is worth to notice that grey mullet lipase was active in the presence of salt concentrations as high as 0.8M.

Seasonal Variations in Proximate and Fatty Acid Composition of Viscera of Sardinella aurita, Sarpa salpa, and Sepia officinalis from Tunisia

Seasonal variations of the proximate composition and fatty acid profiles in viscera from Sardinella aurita, Sarpa salpa, and Sepia officinalis were studied. Significant seasonal variations were observed in the amounts of moisture, lipid, protein, and ash between species. Viscera protein content undergoes large fluctuations. This tendency is different from results observed for the edible parts or for the whole body of many marine species. Ash content also showed significant differences. Lipid contents varied with seasons, in a proportionally inverse manner to water contents. Fatty acid composition showed significant differences from October to December. Interestingly, the highest total omega-3 contents were comparable to many commercial marine fish oils.

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.

Physicochemical Characterization and Nutritional Quality of Fish By-Products:In vitro Oils Digestibility and Synthesis of Flavour Esters

Three fish species (Annular sea bream, sardine and golden grey mullet) were examined as the most Tunisian fishes consumed and could be used as a valuable bio-resource. The fillet and the pyloric caeca from these fish have been investigated for their proximate composition, minerals, nutritional quality and oil physicochemical properties. Fish fillets and viscera showed higher macro-mineral concentrations. Moreover, unsaturated fatty acids were found to be predominant over the saturated ones. The lipid health indexes and the predominance of PUFAs acids in all studied fish could meet people’s needs. Interestingly, a higher stability of polyene, peroxide values and carotenoids were observed during the storage for 30 days at -20°C, which allows higher oils stability. In vitro digestibility model showed that fish oils were efficiently hydrolyzed by pancreatic lipase, which suggests the higher assimilation of fish oils by consumers. Furthermore, fish lipases revealed an acceptable potential to produce aromatic esters.

3‐D structure modelling of the Staphylococcus simulans lipase: conformational changes, substrate specificity and novel structural features

We have modelled, using the CHARMM27 energy force field, the structures of closed and open forms of Staphylococcus simulans lipase (SSL) on the basis of the crystal structures of Bacillus stearothermophilus and Staphylococcus hyicus lipases, respectively. The models suggested the presence of a main lid and a second lid that may act with the former as a double door to control the access to the active site. Superimposition of both closed and open forms of SSL allowed us to determine the hinge regions allowing the movements of the main and the second lid upon lipase activation. The flexibility of these hinge regions was checked by molecular dynamics simulations. The SSL models also allowed us to identify key residues involved in binding substrates, calcium or zinc ions.

Biochemical and structural comparative study between bird and mammal pancreatic colipases.

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of ∼10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.