CONICET | Buscador de Institutos y Recursos Humanos

Fruits of Bromelia hieronymi Mez (Bromeliaceae) contain high amounts of cysteine peptidases (CPs), which have been characterized from the biochemical and kinetic point of view. These peptidases are capable to hydrolyze food proteins, releasing bioactive peptides with both antioxidant and antihypertensive activities. The aim of the present work included cloning and sequencing of several CPs from RNA obtained from unripe fruits. The total RNA was extracted from 0.2 g of fruit crushed with liquid nitrogen. Retrotranscription reaction was carried out to synthesize the cDNA. Then, a specific primer designed from conserved N-terminal sequences of CPs was used in a PCR in order to amplify and select peptidase cDNAs. The amplified products about of 990 bp were cloned into pGEM-T Easy vector and transformed into competent Top 10 Escherichia coli cells. Ten clones were selected, and sequences corresponding to three new peptidases were obtained and named Bh-CP 3, 4, and 5. Their deduced amino-acid sequences encoded three proteins of 230 residues each one, with molecular masses of 24781.82, 24787.92, and 24793.88 kDa, respectively. The predicted pI was a unique value (8.41), and the aliphatic index presented values of 71.65, 74.22, and 73.35, respectively. All sequences displayed the characteristic primary structure of plant CPs (including four residues of the catalytic site, and six Cys residues involved in disulfide bonds), and did not present possible N-glycosylation sites. Bh-cp 3, 4, and 5 showed 97, 99, and 97% of identity with Bh-CP1, other peptidase sequenced from B. hieronymi, and all of them showed 87 % identity with fruit bromelain from Ananas comosus. This work is the first step to express these enzymes, which might be promising biocatalysts for food and pharmaceutical industries.

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