Abstract

Rhamnogalacturonan endolyase (RG-lyase; PL4 family; EC number 4.2.2.23) is an enzyme with a catalytic beta-elimination mechanism acting on the alfa-1,4-glycosidic bond between rhamnose (Rha) and galacturonic acid (GalA) that exists in the main backbone of rhamnogalacturonan-I (RG-I). RG-I is a type of pectin present in the cell wall of fruit tissues, which is subjected to disassembling during softening of Fragaria chiloensis fruit. FcRGL4 was isolated from F. chiloensis with a length of 2028 basepairs; the protein sequence has 676 aminoacids and a molecular weight of ~75,5 kDa without posttranslational modifications. Relative expression analyses indicate the increase in the level of transcripts of FcRGL4 throughout the ripening of F. chiloensis fruit, with maximum values at the turning and ripe fruit stages. The coding sequence was heterologous expressed in the methylotrophic yeast Pichia pastoris. The recombinant protein was purified through cation exchange (CMC) and affinity (His-tag) consecutive chromatographies. Enzymatic activity assays were performed with RG-I from potato as substrate, detecting the formation of a double bond in GalA at 235 nm. Furthermore, the recombinant RG-lyase enzyme is able to breakdown the mucilage of A. thaliana germinating seeds, which is rich in slightly branched RG-I. The role of FcRGL4 is discussed in terms of its participation during softening of F. chiloensis fruit.