Three new cysteine peptidases cloned and sequenced from fruits of Bromelia hieronymi Mez (Bromeliaceae).

MARIA LAURA COLOMBO; AGUSTINA FERNANDEZ; CONSTANZA LIGGIERI; SANDRA ELIZABETH VAIRO CAVALLI; MARIELA BRUNO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias LIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2017

Sociedades de BioCiencias

Fruits of Bromelia hieronymi Mez (Bromeliaceae) contain high amounts ofcysteine peptidases (CPs), which have been characterized from thebiochemical and kinetic point of view. These peptidases are capable tohydrolyze food proteins, releasing bioactive peptides with both antioxidant andantihypertensive activities. The aim of the present work included cloning andsequencing of several CPs from RNA obtained from unripe fruits. The total RNAwas extracted from 0.2 g of fruit crushed with liquid nitrogen. Retrotranscriptionreaction was carried out to synthesize the cDNA. Then, a specific primerdesigned from conserved N-terminal sequences of CPs was used in a PCR inorder to amplify and select peptidase cDNAs. The amplified products about of990 bp were cloned into pGEM-T Easy vector and transformed into competentTop 10 Escherichia coli cells. Ten clones were selected, and sequencescorresponding to three new peptidases were obtained and named Bh-CP 3, 4,and 5. Their deduced amino-acid sequences encoded three proteins of 230residues each one, with molecular masses of 24781.82, 24787.92, and24793.88 kDa, respectively. The predicted pI was a unique value (8.41), andthe aliphatic index presented values of 71.65, 74.22, and 73.35, respectively. Allsequences displayed the characteristic primary structure of plant CPs (includingfour residues of the catalytic site, and six Cys residues involved in disulfidebonds), and did not present possible N-glycosylation sites. Bh-cp 3, 4, and 5showed 97, 99, and 97% of identity with Bh-CP1, other peptidase sequencedfrom B. hieronymi, and all of them showed 87 % identity with fruit bromelainfrom Ananas comosus. This work is the first step to express these enzymes,which might be promising biocatalysts for food and pharmaceutical industries.