An L213A variant of β-glycosidase from Sulfolobus solfataricus with increased α-L-arabinofuranosidase activity converts ginsenoside Rc to compound K.

Ji-Hyeon Choi,Kyung-Chul Shin,Deok-Kun Oh,Kyung-Jin Min

doi:10.1371/journal.pone.0191018

Abstract

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β-d-glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α-l-arabinofuranoside in these ginsenosides. To determine candidate residues involved in α-l-arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α-l-arabinofuranosidase activity. A L213A variant β-glycosidase with increased α-l-arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α-l-arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α-l-arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides.

Highlights

PPD-type ginsenosides contain no sugar, one, or two β-D-glucopyranoses linked to C-3; and no sugar, inner β-D-glucopyranose, or outer α-Larabinopyranose, α-L-arabinofuranose, or β-D-xylopyranose linked to C-20 [8]
Ginsenoside standards purchased from Ambo Institute (Seoul, Korea) and BTGin (Daejeon, Korea) were Rb1, Rc, F2, compound O (C-O), compound Y (C-Y), compound Mc (C-Mc), compound Mc1 (C-Mc1), Compound K (C-K), Rb2, and Rd
Only two enzymes could convert ginsenoside Rc to C-K. β-Glycosidase from P. furiosus produced C-K as an intermediate, which was completely hydrolyzed to aglycone protopanaxadiol (APPD) [24], and β-glycosidase from S. solfataricus produced C-K as an end product, which hydrolyzes two glucose in C3, but does not hydrolyze arabinose in C-20 at Rc

Summary

Introduction

Compound K (C-K, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol) is one molecule of β-D-glucopyranose linked to C-20 in the dammarane skeleton and can be produced from the hydrolysis of glycosylated PPD-type ginsenosides such as Rb1, Rb2, and Rc. C-K has demonstrated beneficial pharmaceutical properties such as anti-allergic, anti-arthritic, anti-carcinogenic, anti-diabetic and anti-inflammatory activities [6, 10,11,12,13,14,15,16]. C-K has been used as an essential ingredient in cosmetics and traditional medicine Diverse methods, such as heating, acid hydrolysis, alkali treatment, and microbial and enzymatic transformation, for obtaining C-K have been carried out due to the absence of C-K in ginseng [21]. Β-Glycosidase from Sulfolobus solfataricus has been an efficient C-K-producing enzyme using glycosylated PPD-type ginsenosides as substrates because of its broad hydrolysis activity, including β-D-glucopyranosidase, β-D-galactopyranosidase, β-D-xylopyranosidase, and α-Larabinopyranosidase activity [22]. A β-glycosidase variant with increased α-L-arabinofuranosidase activity significantly improved by using the L213A variant enzyme with increased α-L-arabinofuranosidase activity

Materials and methods

Analytical methods

Results and discussion