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

• Published in: Antonie van Leeuwenhoek 2013-04, Vol.103 (4), p.747
• Main Authors: Liu, Qing-mei, Jung, Hae-min, Cui, Chang-hao, Sung, Bong-hyun, Kim, Jin-kwang, Kim, Song-gun, Lee, Sung-taik, Kim, Sun-chang, Im, Wan-taek
• Format: Article
• Language: English
• Subjects: Amino acids; Cellulose; Cloning; E coli; Hydrolysis
• ISSN: 0003-6072; 1572-9699
• DOI: 10.1007/s10482-012-9856-2

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 Abf22-3â[euro](TM)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 Rb^sub 1^ and Rb^sub 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^sub m^ and V^sub max^ values of 0.95 ±Â 0.02 [mu]M and 1.2 ±Â 0.1 [mu]mol min^sup â '1^ mg of protein^sup â '1^ against p-nitrophenyl-[alpha]-l-arabinofuranoside, respectively. Using a purified MBPâ[euro]"Abf22-3 (10 [mu]g/ml), 0.1 % of ginsenoside Rc was completely converted to ginsenoside Rd within 20 min.[PUBLICATION ABSTRACT]