1. Bioconversion of ginsenoside Rc into Rd by a novel α-l-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization
- Author
-
Wan-Taek Im, Song-Gun Kim, Qing-Mei Liu, Jin-Kwang Kim, Bong-Hyun Sung, Hae-Min Jung, Sun Chang Kim, Sung-Taik Lee, and Chang-Hao Cui
- Subjects
DNA, Bacterial ,Ginsenosides ,Glycoside Hydrolases ,Stereochemistry ,Molecular Sequence Data ,medicine.disease_cause ,Microbiology ,Hydrolysis ,chemistry.chemical_compound ,Enzyme Stability ,Escherichia coli ,medicine ,Leuconostoc ,Glycoside hydrolase ,Cloning, Molecular ,Molecular Biology ,Biotransformation ,chemistry.chemical_classification ,Korea ,Chromatography ,Sequence Homology, Amino Acid ,biology ,Molecular mass ,Temperature ,Sequence Analysis, DNA ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant Proteins ,Amino acid ,Molecular Weight ,Kinetics ,Enzyme ,chemistry ,Ginsenoside ,Food Microbiology ,Chromatography, Liquid - Abstract
A novel α-L-arabinofuranosidase (Abf22-3) that could biotransform ginsenoside Rc into Rd was obtained from the ginsenoside converting Leuconostoc sp. strain 22-3, isolated from the Korean fermented food kimchi. The gene, termed abf22-3, consisting of 1,527 bp and encoding a protein with a predicted molecular mass of 58,486 Da was cloned into the pMAL-c2x (TEV) vector. A BLAST search using the Abf22-3's amino acid sequence revealed significant homology to that of family 51 glycoside hydrolases. The over-expressed recombinant Abf22-3 in Escherichia coli BL21 (DE3) catalyzed the hydrolysis of the arabinofuranoside moiety attached to the C-20 position of ginsenoside Rc under optimal conditions of pH 6.0 and 30 °C. This result indicated that Abf22-3 selectively converts ginsenoside Rc into Rd, but did not catalyze the hydrolysis of glucopyranosyl groups from Rc or other ginsenosides such as Rb1 and Rb2. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for α-L-arabinofuranosidase showed apparent Km and Vmax values of 0.95 ± 0.02 μM and 1.2 ± 0.1 μmol min(-1) mg of protein(-1) against p-nitrophenyl-α-L-arabinofuranoside, respectively. Using a purified MBP-Abf22-3 (10 μg/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.
- Published
- 2012