Abstract
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-β-d-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1 → 4)-α-l-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1 → 2)-α-l-rhamnopyranoside of progenin III, 3-O-β-d-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the α-d-galactopyranoside, β-d-glucopyranoside, and β-d-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the α-d-mannopyranoside, α-l-arabinopyranoside, α-d-glucopyranoside, β-d-xylopyranoside, and α-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 α-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 α-amylase in this family.
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Acknowledgments
M.Q. Tang and X.D. Yuan (TaKaRa Biotechnology (Dalian) Co., Ltd. China) are thanked for sequencing determination. This work was supported by Program for Liaoning Innovative Research Team in University (LNIRT: 2009 T007) and National Science of Foundation of P.R. China (NSFC).
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Liu, T., Yu, H., Liu, C. et al. Protodioscin-glycosidase-1 hydrolyzing 26-O-β-d-glucoside and 3-O-(1 → 4)-α-l-rhamnoside of steroidal saponins from Aspergillus oryzae . Appl Microbiol Biotechnol 97, 10035–10043 (2013). https://doi.org/10.1007/s00253-013-4791-3
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DOI: https://doi.org/10.1007/s00253-013-4791-3