Protodioscin-Glycosidase-1 Hydrolyzing 26-O-Beta-D-Glucoside And 3-O-(1 -> Aeuro Parts Per Thousand 4)-Alpha-L-Rhamnoside Of Steroidal Saponins From Aspergillus Oryzae

A novel protodioscin-(steroidal saponin)-glycoside hydrolase, named protodioscin-glycosidase-1 (PGase-1), was purified and characterized from the Aspergillus oryzae strain. The molecular mass of this enzyme was determined to be about 55 kDa based on SDS-polyacrylamide gel electrophoresis. PGase-1 was able to hydrolyze the terminal 26-O-beta-d-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1 -> aEuro parts per thousand 4)-alpha-l-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1 -> aEuro parts per thousand 2)-alpha-l-rhamnopyranoside of progenin III, 3-O-beta-d-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the alpha-d-galactopyranoside, beta-d-glucopyranoside, and beta-d-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the alpha-d-mannopyranoside, alpha-l-arabinopyranoside, alpha-d-glucopyranoside, beta-d-xylopyranoside, and alpha-l-rhamnopyranoside of p-nitrophenyl-glycosides. These new properties of PGase-1 were significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB. The gene (termed as pgase-1) encoding PGase-1 was cloned, sequenced, and expressed in Pichia pastoris GS115. The complete nucleotide sequence of pgase-1 consists of 1,725 bp. The recombinant PGase-1 from recombinant P. pastoris GS115 strain also showed the activity hydrolyzing glycosides of steroidal saponins which was similar to that of the wild-type PGase-1 from A. oryzae. The PGase-1 gene is highly similar to Aspergilli alpha-amylase (EC 3.2.1.1), and PGase-1 should be classified as glycoside hydrolase family 13 by the method of gene sequence-based classification. But the enzyme properties of PGase-1 are different from those of alpha-amylase in this family.