Purification and properties of a novel beta-glucosidase, hydrolyzing ginsenoside Rb1 to CK, from Paecilomyces Bainier.
A novel ginsenoside-hydrolyzing beta-glucosidase was purified from Paecilomyces Bainier sp. 229 by a combination of QSepharose FF, phenyl-Sepharose CL-4B, and CHT ceramic hydroxyapatite column chromatographies. The purified enzyme was a monomeric protein with a molecular mass estimated to be 115 kDa. The optimal enzyme activity was observed at pH 3.5 and 60oC. It was highly stable within pH 3-9 and at temperatures lower than 55oC. The enzyme was specific to beta-glucoside. The order of enzyme activities against different types of beta-glucosidic linkages was beta-(1- 6)>beta-(1-2)>beta-(1-4). The enzyme converted ginsenoside Rb1 to CK specifically and efficiently. An 84.3% amount of ginsenoside Rb1, with an initial concentration of 2 mM, was converted into CK in 24 h by the enzyme at 45 degrees and pH 3.5. The hydrolysis pathway of ginsenoside Rb1 by the enzyme was Rb1-->Rd-->F2-->CK. Five tryptic peptide fragments of the enzyme were identified by a newly developed de novo sequencing method of post-source decay (PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. By comparing the five identified peptide sequences with the NCBI database, this purified beta-glucosidase proves to be a new protein that has not been reported before.
Yan
Qin
Q
School of Pharmacy and Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China.
Zhou
Xin-Wen
XW
Zhou
Wei
W
Li
Xing-Wei
XW
Feng
Mei-Qing
MQ
Zhou
Pei
P
D000595:Amino Acid Sequence; D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D036145:Ginsenosides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010143:Paecilomyces / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*
D005656:Fungal Proteins; D036145:Ginsenosides; C442759:ginsenoside Rb1; C440192:ginsenoside-beta-glucosidase; D001617:beta-Glucosidase