Bioconversion of ginsenoside Rc into Rd by a novel α-l-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization

• DOI: 10.1007/s10482-012-9856-2
• Publication Date: 2012-12-06T05:17:15Z
• Authors: Qing-Mei Liu; Hae-Min Jung; Chang-Hao Cui; Bong-Hyun Sung; Jin-Kwang Kim; Song-Gun Kim; Sung-Taik Lee; Sun-Chang Kim; Wan-Taek Im
• Keywords: DNA, Bacterial; 0303 health sciences; Korea; Ginsenosides; Glycoside Hydrolases; Sequence Homology, Amino Acid; Molecular Sequence Data; Temperature; Sequence Analysis, DNA; Hydrogen-Ion Concentration; Recombinant Proteins; Molecular Weight; Kinetics; 03 medical and health sciences; Enzyme Stability; Escherichia coli; Food Microbiology; Cloning, Molecular; Biotransformation; Leuconostoc; Chromatography, Liquid

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.