CLONING AND SEQUENCING OF A NEW CYSTEINEPEPTIDASE FROM FRUITS OF Bromelia hieronymi MEZ

COLOMBO, M. L.; LIGGIERI, C.; FERNÁNDEZ, A.; VAIRO CAVALLI, S.; BRUNO, M.

Córdoba

Congreso; LII Reunión Annual de la SAIBBM; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Fruits of Bromelia hieronymi Mez (Bromeliaceae) possess highcontent of cysteine peptidases with potential uses in both foodand pharmaceutical industries. The aim of this work included cloning and sequencing of a new cysteine peptidase (CP) from this species. Unripe fruits were crushed in a mortar and pestleunder liquid nitrogen and 0.2 g were employed to extract totalRNA. cDNA was synthesized by retrotranscription and used asPCR template with specific primers designed from conserved N-terminalsequences of CPs. The amplified product of 989 bpwas cloned into pGEM-TEasy vector and transformed intochemically competent E. coli (Top10) cells. Several clones were sequenced. The deduced amino acid sequence (named BhCP2) encoded an enzyme of 229 residues with predictedmolecular mass of 24.6816 kDa, pI 5.71, and extinction molarcoefficient 58,705 M−1 cm−1. BhCP2sequence displays thecharacteristic primary structure of plant CPs in addition toconserved catalytic residues as well as six Cys residues, involved in the formation of disulfide bonds. The peptidase shows 93% of identity with BhCP1, other peptidase from B. hieronymi, and ca. 80 % with Ananas comosus peptidases. This work is the first step to express this enzyme, which might be a promising biocatalyst for industrial processes.