title: Bioconversion of ginsenoside Rc into Rd by a novel α-L-arabinofuranosidase, Abf22-3 from Leuconostoc sp. 22-3: cloning, expression, and enzyme characterization.
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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea.
authors.forename: Qing-Mei
authors.initials: QM
authors.lastname: Liu
authors.forename: Hae-Min
authors.initials: HM
authors.lastname: Jung
authors.forename: Chang-Hao
authors.initials: CH
authors.lastname: Cui
authors.forename: Bong-Hyun
authors.initials: BH
authors.lastname: Sung
authors.forename: Jin-Kwang
authors.initials: JK
authors.lastname: Kim
authors.forename: Song-Gun
authors.initials: SG
authors.lastname: Kim
authors.forename: Sung-Taik
authors.initials: ST
authors.lastname: Lee
authors.forename: Sun-Chang
authors.initials: SC
authors.lastname: Kim
authors.forename: Wan-Taek
authors.initials: WT
authors.lastname: Im
mesh_terms: D001711:Biotransformation; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005516:Food Microbiology; D036145:Ginsenosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007723:Korea; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D036145:Ginsenosides; D011994:Recombinant Proteins; C044462:ginsenoside Rc; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; C049863:ginsenoside Rd