Cloning and sequencing of a new cysteine peptidase from fruits of Bromelia hieronymi Mez

MARIA LAURA COLOMBO; CONSTANZA LIGGIERI; AGUSTINA FERNÁNDEZ; MARIELA BRUNO

Córdoba

Congreso; LII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Fruitsof Bromelia hieronymi Mez(Bromeliaceae) possess a high content of cysteine peptidases with potentialuses in both food and pharmaceutical industries. The aim of this work wascloning and sequencing a peptidase from this species. Unripe fruits werecrushed in a mortar with liquid nitrogen and 0.2 g were employed to extract totalRNA and then. Retrotranscription and cDNA amplification was achieved usingspecifics primers. Amplification products were analyzed on 1% agarose gelcontaining gel red to visualize DNA bands on UV trans-illuminator. Selectedbands in agarose gel were cut out and the cDNA was gel-purified, ligated intothe pGEM-T Easy vector, and transformed into competent E. coli (XL1-Blue)cells. A consensus sequence was obtained, containing 989 bp, where 687 firstones codifying for a hypothetical polypeptide chain of the mature peptidase(229 amino acids), named Bh-CP2 (molecular mass 24.6816 kDa, pI 5.71,extinction molar coefficient 58,705 M−1 cm−1). Bh-CP2 sequence shows 93% ofidentity with Bh-CP1, other peptidase from B. hieronymi, and values from around80 % for Ananas comosus peptidases. Presence of highly preserved residues isobserved, like those forming the catalytic site, as well as six Cys residues, involvedin the formation of disulfide bounds. This work is the first step to expressthis enzyme, which is a promising biocatalyst for industrial processes.