From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.
In work reported here, a proteolytic extract prepared from Maclura pomifera latex was employed to hydrolyze bovine caseins. Densitograms of Tricine-sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) indicated that the caseins were considerably degraded after a 10-min reaction. The degree of hydrolysis determined by the 2,4,6-trinitrobenzenesulfonic-acid method was 17.1±0.7% after 180min of digestion. The concentration of small peptides increased with hydrolysis time, and analysis by reverse-phase high-performance liquid chromatography (RP HPLC) and mass spectrometry, revealed a virtually unchanged peptide profile. These results suggested that those proteases were highly specific, as only certain peptide bonds were cleaved. The hydrolysate of 180min displayed the highest inhibition of angiotensin-converting enzyme (ACE) showing an IC of 1.72±0.25mg/mL, and the analysis of the peptide fractionation in this hydrolysate by RP HPLC exhibited two peaks responsible for that activity. Fragmentation analysis through the use of iterated matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) with the aid of bioinformatics tools enabled us to deduce two peptide sequences-one, YQEPVLGPVRGPFPIIV, having been previously reported as an ACE-inhibitor; the other, RFFVAPFPE, as yet undescribed. The presence of bioactive peptides in these casein hydrolysates argues for their potential use in the development of functional foods.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.