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

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A novel alpha-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 A degrees 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 Rb-1 and Rb-2. Over-expressed recombinant enzymes were purified by two steps with amylose-affinity and DEAE-cellulose chromatography and then characterized. The kinetic parameters for alpha-l-arabinofuranosidase showed apparent K-m and V-max values of 0.95 +/- A 0.02 mu M and 1.2 +/- A 0.1 mu mol min(-1) mg of protein(-1) against p-nitrophenyl-alpha-l-arabinofuranoside, respectively. Using a purified MBP-Abf22-3 (10 mu g/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.
Publisher
SPRINGER
Issue Date
2013-04
Language
English
Article Type
Article
Keywords

THERMOSTABLE BETA-GLYCOSIDASE; COMPOUND K; DIFFERENT PARTS; PANAX-GINSENG; GLUCOSIDASE; RB-1; BIOTRANSFORMATION; TRANSFORMATION; PURIFICATION; RG(1)

Citation

ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY, v.103, no.4, pp.747 - 754

ISSN
0003-6072
DOI
10.1007/s10482-012-9856-2
URI
http://hdl.handle.net/10203/173783
Appears in Collection
BS-Journal Papers(저널논문)
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