Purification of Balansain I, an Endopeptidase from Unripe Fruits of Bromelia balansae Mez (Bromeliaceae)
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- 作者：Marcelo F. Pardo ; Laura M. I. Ló ; pez ; Francesc Canals ; Francesc X. Avilé ; s ; Claudia L. Natalucci ; and Né ; stor O. Caffini
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2000
- 出版时间：September 2000
- 年：2000
- 卷：48
- 期：9
- 页码：3795 - 3800
- 全文大小：138K
- 年卷期：v.48,no.9(September 2000)
- ISSN：1520-5118
文摘
A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae).Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanolprecipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionicstrength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 Msodium chloride solution at 37 
C), and exhibited high thermal stability (inactivation required heatingfor 60 min at 75 C). Anion exchange chromatography allowed the isolation of a fraction purified tomass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 andmolecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yieldis high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteasesrepresent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrowerpH profile than that obtained for REP, with a maximum pH value ~9.0 and was inhibited by E-64and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic typesof peptidases. The alanine and glutamine derivatives of N-
-carbobenzoxy-L-amino acid p-nitrophenylesters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a veryhigh homology (85-90%) with other known Bromeliaceae endopeptidases.Keywords: Plant endopeptidase; Bromelia balansae; Bromeliaceae; purification
C), and exhibited high thermal stability (inactivation required heatingfor 60 min at 75 C). Anion exchange chromatography allowed the isolation of a fraction purified tomass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 andmolecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yieldis high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteasesrepresent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrowerpH profile than that obtained for REP, with a maximum pH value ~9.0 and was inhibited by E-64and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic typesof peptidases. The alanine and glutamine derivatives of N--carbobenzoxy-L-amino acid p-nitrophenylesters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a veryhigh homology (85-90%) with other known Bromeliaceae endopeptidases.Keywords: Plant endopeptidase; Bromelia balansae; Bromeliaceae; purification