Substrate specificity, purification and identification of a novel pectinase with the specificity of hydrolyzing the α-1,4-glycosyl residue in steroidal saponin

It is known that the sugar chain linked to steroidal frame plays an important role in physiological and pharmacological activities. In the previous research, we found and confirmed that the terminal C3-O-α-1,2-rhamnosyl moiety linked to the C-3 of steroidal saponin is the key group of platelet aggregation and cytotoxicity. In order to make a complete approach for the structure–activity relationship, we have tried to find the specific enzymes modifying the structure of C3-sugar chain. In the present paper, we describe a novel enzyme from, Klerzyme-150 (K-150), which is specifically capable of hydrolyzing the α-1,4-glycosyl residues at C-3 postion of steroidal saponins. 15 steroidal saponins with different monosaccharides at C3-sugar chains were chosen as substrates to investigate the substrate specificity of K-150. The results showed, based on TLC, HPLC and spectra data analyses, that all products were determined as secondary saponins with the α-1, 4-glycosyl residues removed, which indicated that the enzyme exhibited strict regioselectivity and stereoselectivity. The novel enzyme was purified from K-150 to apparent homogeneity and its structure was identified as rhamnogalacturonan lyase A (Rgl A). The molecular mass of the purified enzyme was 52.08 kDa.