A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria : Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl-protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and PP1 positions. Its activity is optimal at 95³C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.z 1998 Federation of European Biochemical Societies.

Since P-lactoglobulin is resistant to peptic hydrolysis in physiological conditions, the increase of its digestibility by this enzyme was sought by the destabilization of its folding using m e t W that do not influence the biological value of protein, such as high pressure, medium polarity changes (alcohol addition), and esteri3cation (ethylation). For example, the rate of hydrolysis of 8-lactoglobulin by pepsin (negligible at 0.1 MPa) increased considerably with pressure up to 300 MPa. l h susceptibility of all potential @-lactoglobulin proteolytic sites to peptic cleavage remained constant over the pressure range that was studied. The aa'dition of alcohols decreases the bulk diekctric constant of the medium and, according to CD measuremnts, increases significantly the proportion of helical structure in /3-lactoglobulin while increasing susceptibility to peptic hydrolysis. In the presence of alcohols (ethanol, ethylene glycol), &lactoglobulin hydrolysis by pepsin was initiated when its secondary structure began to change and diversijied peptic peptide populations were obtained. llre chemical modijkation of /3luctoglobulin by mild estenjcation yieldr a 40%-ethyiuted 8-lactoglobulin derivative that is rapidly hydrolyzed by pepsin. As compared with peptic hydrolysis of P-lactoglobulin in aqueous ethanol, 22 new sites of pepsin cleavage were induced by esterijkation of the protein. 439 440 CHOBERT, BRIAND, DUFOUR, DIB, DALGALARRONDO and HAERTLE PEPTIC HYDROLYSIS OF B-LACTOGLOBULIN 441Various proteases are used as tools in primary structure determination or to probe protein conformation changes. It is well known that in most cases, the cleavage of all the peptide bonds does not occur,at the same rate and/or at the same time. It is influenced by the specificity and the accessibility of the enzyme molecule to the cleavable peptide bond. The huge amounts of P-lactoglobulin that are generated every year stimulate questions about its alternative uses. The established uses of this protein are limited, mostly because of its partial or total resistance to hydrolysis by proteases. In the present review, different ways affecting the folding of this protein (high pressure, media containing alcohols, or esterification), and leading it to become susceptible to peptic attack are described.

No abstract

The addition of ethylene glycol, and 1,2‐ and 1,3‐propanediol, decreases the bulk dielectric constant of the medium, and according to CD measurements, increases significantly the proportion of helical structure in β‐lactoglobulin. The medium‐induced folding changes followed by limited peptic hydrolysis show that the cleavage of β‐lactoglobulin by pepsin is triggered by structural transformations induced by ethylene glycol only and not by 1,2‐ and 1,3‐propanediol. Density measurements, at constant chemical potential and constant molality, demonstrate that all diols are present in the immediate domain of the protein. They are engaged in hydrophobic interactions with the amino acids of β‐lactoglobulin core inducing the formation of additional α‐helices. © 1996 John Wiley & Sons, Inc.

Styrax officinalis is a deciduous large shrub that grows in many Mediterranean countries. Active ingredients are localized in the fruit pericarp. Primary phytochemical analysis showed the presence of saponins, tannins and triterpenes and absence of alkaloids and flavonoids. No antimicrobial, but strong ichthyotoxic and molluscicidal effects were observed in the saponin-rich extract .Toxicity tests with the snail Cornu aspersum proved to be a potent contact molluscicide. Allowing the snail to creep on leaves and surfaces sprayed with a 1% (w/v) of pericarp extract resulted in severe dehydration and foaming through their membranes, which resulted in their death after 30 min. When the snails were fed lettuce leaves treated with the same extract solution, it did not cause any observable effect. These findings show that S. officinalis is a promising natural source of a potent contact molluscicide with no visible effect on the snail upon ingestion.