Cloning and molecular characterisation of cdna encoding plant aspartic proteases with milk clotting activity from Cynara scolymus and Arctium minus

COLOMBO, MA.L.; CIMINO, C.; LIGGIERI, C.; BRUNO, M.; VAIRO CAVALLI, S.

Córdoba

Congreso; V Congreso Internacional de Ciencia y Tecnología de los Alimentos 2014; 2014

Subsecretaría de Innovación y Vinculación Tecnológica - Gobierno de Córdoba

Successful rennet of plant origin largely employed in the Mediterranean region for the manufacture of traditional high quality cheeses at farm level is obtained from Cynara cardunculus L. (Asteraceae) flowers. These flowers are a rich source of aspartylendopeptidases (APs) with milk clotting-activity. All species included within the tribe: Cardueae Cass., Asteraceae family contain APs in their flowers with milk-clotting activity but without excessive proteolytic action. From aqueous extracts of Cynara scolymus L. and Arctium minus (Hill) Bernh. flowers we have isolated and characterised these type of enzymes. The development of synthetic rennet based on recombinant enzymes would emerge as a solution for large-scale production. To study the possible suitability of C. scolymus and A. minus APs as rennet substitute we have cloned its cDNA using total RNA isolated from young flower buds. RNA was used as PCR template with primers designed from conserved regions of plant APs. The amplified products of 1515 bp (C. scolymus) and 1530 bp (A. minus) were cloned and sequenced. Analyzes of the resulting translated sequences reveal a high level of sequence identity among each other and other typical plant APs available in databases. The protein products encoded preproenzymes with a hydrophobic signal peptide, a pro-segment, and an amino acid-long polypeptide interrupted by a plant-specific insert (PSI) domain. According to conventional nomenclature for APs we have named the cloned enzymes from A. minus and C. scolymus arctiumsin and cardosin s, respectively. The amino acid sequence of cardosin s showed high degree of similarity (identities: 97%, positives: 98%) with cardosin a fromC. cardunculus. While arctiumsin had 73% similarity with cirsin from Cirsium vulgare (identities: 96%, positives: 98%). Both proteins have a vacuolar sorting domain, the C-terminal peptide VGFAEAA, whereas cardosin s also presents an RGD motif, which has been proposed to be related to the adhesion-mediated proteolytic mechanisms associated with pollen-tube extension. Structural models were constructed by homology modeling using the "swiss-model" program and prophytepsin (Hordeum vulgare) structure was taken as reference. Procardosin s and proarctiumsin have an identity percentage of 61.6% and 71% with prophytepsin, respectively. Phylogenetic relationship analysis among several thistle-flower APs was carried out by the neighbour-joining method, phytepsin precursor and pepsinogen (Homo sapiens sapiens) were also included. On the basis of thistle-AP family tree, the APs can be divided into two groups. Proarctiumsin and procardosin s belong to separate groups. It has been proposed that an AP ancestral gene duplicated and gave rise to cyprosins and cardosins during evolution of C. cardunculus and that thereafter duplications occurred within both groups.