J.C. Gripon

Le système protéolytique de Penicillium roqueforti: III. — Purification, propriétés et spécificité d'une protéase inhibée par l'E.D.T.A.

Summary An extracellular protease of Penicillium roqueforti , differing from the acid protease, has been isolated from culture media by ammonium precipitation, gel filtration and CM-cellulose chromatography. The purified preparation was homogenous on polyacrylamide gel electrophoresis at pH 9.0, 9.2 and 4.3. The molecular weight was estimated to be 20 000 daltons by gel filtration. Optimum pH for casein and hemoglobin digestion was respectively 5.5 and 4.2. The enzyme was thermostable (residual activity: 25 p. cent by the treatment at 100°C for 20 min pH 6.0) but it was very unstable at 65°C. Cobalt ions promoted an activation of 100 p. cent on casein hydrolysis. Chelating agents inhibited the enzyme activity completely. D.F.P., sulfhydryl reagents, D.A.N. had no effect on the activity. The protease II displays a specificity which is different from that found for neutral proteases sensitive to metal chelators, Disubstituted peptides which are classical substrates for these enzymes, (Z-X-Y-NH 2 , Y = Leu or Phe), are not split by the protease II. Two peptide bonds (Ala 14 -Leu 15 , Tyr 16 -Leu 17 ) are hydrolysed at a good rate on the oxydized insulin B-chain. The others peptide bonds usually hydrolysed by metalloproteases (His 5 -Leu 6 , His 10 -Leu 11 , Gly 23 -Phe 24 , Phe 24 -Phe 25 ) are not attacked by protease II.

Le système protéolytique de Penicillium roqueforti: II - Purification et propriétés de la protéase acide

Summary An extracellular protease has been isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by precipitation with ammonium sulfate, filtration on Bio-gel P 100 columns and chromatography on D.E.A.E.-cellulose columns The purified preparation was homogenous by gel filtration on Bio-gel P 60 and electrophoretical analysis at pH 9.0 and 5.0. The protease exhibited the properties of an acid protease: the optimum pH was 3.5 for casein or hemoglobin hydrolysis and for bovine trypsinogen activation; at 40°C, the enzyme is most stable in the range of pH 3.5 to 5.5; the optimum temperatures was 50°C. E.D.T.A., D.F.P. and sulfhydryl reagents induced no inhibition. The enzyme exhibited a milk clotting activity that was fifty times weaker that the activity of rennin. Its molecular weight, estimated by gel filtration was 33 400. Amino acid composition is: Lys15, His2, Arg1, Trp5, Asp33, Thr27, Glu16, Pro10, Gly40, Ala25, Cys2, Val21, Ile20, Leu21, Tyr14, Phe19. The properties of this protease were closely similar to that of P. janthinellum and Aspergillus oryzae.

Separation and identification of hydrophilic peptides in dairy products using FMOC derivatization

Small hydrophilic di- and tripeptides from food products are not separated by reversed-phase high-performance liquid chromatography (RP-HPLC). A simple method using precolumn derivatization with 9-fluorenylmethoxycarbonyl chloride (FMOC) of hydrophilic peptides followed by RP-HPLC separation is presented. Peptides can subsequently be identified by Edman degradation after deprotection of the peptide derivatives with piperidine. Fifteen peptides (ten dipeptides, four tripeptides and one tetrapeptide) were sequenced from the water-soluble fraction of an enzyme-modified cheese model. Some synthetic peptides (Ile-Asn, Val-Thr, Ala-Pro, Val-Gln, Thr-Gln and Gly-Gly) corresponding to purified peptides were sensory tested.

Purification and Caracterization of a Cell-Wall Protease from Streptococcus Lactis

Mesophilic streptococci have complex nitrogen requirements and the concentration of free amino-acids and peptides in milk is too low to support an optimal growth. Cell-wall proteinases are essential for casein hydrolysis and peptides production. The present communication describes the purification and characterization of a cell-wall proteinase from Streptococcus lactis.