Proterties Of Protodioscin-glycosidase-1and Molecular Cloning,Expression Of Protodioscin-glycosidase-1Gene

The steroidal saponins with long sugar chains are physiologically active ingredients in medicinal plant of Dioscorea L., but these natural steroidal saponins generally containing long sugar chains have low activity and low absorption in human bodies. In this paper, the enzyme from microorganism was used for converting the steroidal saponins in Dioscorea nipponica Makino to more active and easy-absorbing steroidal saponins with low sugar chains. In addition, a novel steroidal saponin-glycosidase was purified, characterized, cloned and expressed in Pichia pastoris GS115.Firstly, the total steroidal saponins of250g were obtained from4kg dried roots of D. nipponica Makino after by ethanol extraction, desugaration and decoloration. The extraction yield was about6.3%. Tow main steroidal saponins were separated by silica gel chromatography, and the structure of these tow steroidal saponins was identified as protodioscin, i. e.,26-O-β-D-glucopyranosyl-25(R)-22-hydroxyl-5-ene-furostane-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-glucopyrano side, and dioscin dioscin, i.e., diosgenin-3-O-α-L-rhamnopyranosyl-(1â†'2)-[α-L-rhamnopyranosyl-(1â†'4)]-β-D-glucopyranoside, respectively.Aspergillus oryzae DLFCC-38was screened as the enzyme producing microorganism, the enzyme from this microorganism could almost convert the total steroidal saponins of Dioscorea nipponica Makino to major progenin â…¢ and gracillin, and small amount of diosgenin. The optimal culture conditions for enzyme production by A. oryzae DLFCC-38were investigated. For enzyme production, the strain was cultured for72h at30℃, in the medium containing2%(v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for9h, at50℃and pH5.0, with the20mg/ml of substrate. In the scale-up preparation of progenin â…¢,117g of crude product was obtained from160g of substrate.117g of the crude product was purified with silica gel column to obtain60.3g progenin with the yield of51.5%,11.54g gracillin with the yield of9.86%, and3.01g diosgenin with the yield of2.57%.After3-steps, a protodioscin-glycosidase was purified from the A. oryzae DLFCC-38culture by the methods of molecular sieve chromatography and ion exchange chromatography. The molecular mass of this enzyme was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH of the enzyme was5.0and50℃, respectively, and the enzyme was stable at pH3.0-8.0, below60 ℃. The activity of this enzyme was not obviously affected by K+, Na+, Mg2+and Ca2+ions, but slightly inhibited by Zn2+and strictly inhibited Cu2+and Fe3+ions.The purified protodioscin-glycosidase was able to hydrolyze the terminal26-O-β-D-glucopyranoside of protodioscin to produce dioscin, and then further hydrolyze the terminal3-O-(1â†'4)-α-L-rhamnopyranoside of dioscin to form progenin â…¢. The Km and Vmax for protodioscin were4.59mM and3.86mM/h, respectively. The Km and Vmax for dioscin were42.6mM and0.15mM/h, respectively. In addition, this enzyme also could hydrolyze the a-D-galactopyranoside, β-D-glucopyranoside and β-D-galactopyranoside of p-nitrophenyl-glycosides. These new properties of the protodioscin-glycosidase are significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB where typically one enzyme hydrolyzes one type of glycoside. It therefore represents the advent of a novel enzyme, and the new enzyme was named protodioscin-glycosidase-1(PGase-1).The complete gene sequence of protodioscin-glycosidase-1was amplified by the methods of RT-PCR and RACE, and the sequence length is1725bp. The full-length cDNA contains a1497-bp open reading frame (ORF) which encoding498amino acid residues, and20amino acids were cleaved from the N terminus of the mature form and seemed to be a signal sequence. The protodioscin-glycosidase-1gene cloned from A. oryzae was subcloned into the vector pPIC9K and then transformed into Pichia pastoris GS115. The optimal culture condition of recombinant P. pastoris GS115was identified as:the culture time was144h and the final methnol content was0.5%. The recombinant protodioscin-glycosidase-1from recombinant P. pastoris GS115also showed the activity hydrolyzing glycosides of steroidal saponins and the molecular mass of recombinant protodioscin-glycosidase-1was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis, which were both similar to that of the wild-type protodioscin-glycosidase-1from A. oryzae. The results show that gene has been successfully expressed in P. pastoris GS115. The protodioscin-glycosidase-1gene is highly similar to a-amylase (EC3.2.1.1), and the protodioscin-glycosidase-1should be classified as glycoside hydrolase family13by the method of gene sequence-based classification. But the enzyme properties of this enzyme are different from those of a-amylase in this family. Therefore, gene sequence of the enzyme can not completely represent enzyme properties.