Cloning and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Lactobacillus brevis That Transforms Ginsenosides Rb1 and F2 into Ginsenoside Rd and Compound K원문보기
Zhong, Fei-Liang
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department of Chemistry, College of Science, Yanbian University)
,
Ma, Rui
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department of Chemistry, College of Science, Yanbian University)
,
Jiang, Mingliang
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department of Chemistry, College of Science, Yanbian University)
,
Dong, Wei-Wei
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department of Chemistry, College of Science, Yanbian University)
,
Jiang, Jun
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department of Chemistry, College of Science, Yanbian University)
,
Wu, Songquan
(Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, and Department)
,
Li, Donghao
,
Quan, Lin-Hu
The ginsenoside-hydrolyzing β-glucosidase gene (bgy2) was cloned from Lactobacillus brevis. We expressed this gene in Escherichia coli BL21(DE3), isolated the resulting protein, and then utilized the enzyme for the biotransformation of ginsenosides. The bgy2 gene contains 2,223 bp, and encodes a pro...
The ginsenoside-hydrolyzing β-glucosidase gene (bgy2) was cloned from Lactobacillus brevis. We expressed this gene in Escherichia coli BL21(DE3), isolated the resulting protein, and then utilized the enzyme for the biotransformation of ginsenosides. The bgy2 gene contains 2,223 bp, and encodes a protein of 741 amino acids that is a member of glycosyl hydrolase family 3. β-Glucosidase (Bgy2) cleaved the outer glucose moieties of ginsenosides at the C-20 position, and the inner glucose at the C-3 position. Under optimal conditions (pH 7.0, 30℃), we used 0.1 mg/ml Bgy2 in 20 mM sodium phosphate buffer (PBS) for enzymatic studies. In these conditions, 1.0 mg/ml ginsenoside Rb1 and ginsenoside F2 were converted into 0.59 mg/ml ginsenoside Rd and 0.72mg/ml compound K, with molar conversion productivities of 69% and 91%, respectively. In pharmaceutical and commercial industries, this recombinant Bgy2 would be suitable for producting ginsenoside Rd and compound K.
The ginsenoside-hydrolyzing β-glucosidase gene (bgy2) was cloned from Lactobacillus brevis. We expressed this gene in Escherichia coli BL21(DE3), isolated the resulting protein, and then utilized the enzyme for the biotransformation of ginsenosides. The bgy2 gene contains 2,223 bp, and encodes a protein of 741 amino acids that is a member of glycosyl hydrolase family 3. β-Glucosidase (Bgy2) cleaved the outer glucose moieties of ginsenosides at the C-20 position, and the inner glucose at the C-3 position. Under optimal conditions (pH 7.0, 30℃), we used 0.1 mg/ml Bgy2 in 20 mM sodium phosphate buffer (PBS) for enzymatic studies. In these conditions, 1.0 mg/ml ginsenoside Rb1 and ginsenoside F2 were converted into 0.59 mg/ml ginsenoside Rd and 0.72mg/ml compound K, with molar conversion productivities of 69% and 91%, respectively. In pharmaceutical and commercial industries, this recombinant Bgy2 would be suitable for producting ginsenoside Rd and compound K.
In conclusion, Bgy2 is a member of the GH 3 family from L. brevis.
MBP-Bgy2 was purified by MBP-bound amylose resin, and then the supernatant from the cell lysates and the purified enzyme were analyzed by SDS-PAGE. The molecular mass of the MBP-Bgy2 predicted from the amino acid sequence was 123 kDa, which was the mass observed by SDS-PAGE(Fig. 1).
Therefore, Bgy2 exhibits substrate specificity for ginsenosides Rb1 and F2 with glucose moieties at the C-20 and C-3 positions, and illustrates specific affinity to the outer C-20 glucose and C-3 glucose. The transformation pathway illustrated in Fig.
3D). When compared with the reference standard ginsenoside Rd, metabolite 1 had an equal retention time and ESI-MS/MS fragmentation patterns (Figs. 3A and 3C).
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