Abstract
Ginsenoside compound K has been used as a key nutritional and cosmetic component because of its anti-fatigue and skin anti-aging effects. β-Glycosidase from Sulfolobus solfataricus (SS-BGL) is known as the most efficient enzyme for compound K production. The hydrolytic pathway from ginsenoside Rb1 to compound K via Rd and F2 is the most important because Rb1 is the most abundant component in ginseng extract. However, the enzymatic conversion of ginsenoside Rd to F2 is a limiting step in the hydrolytic pathway because of the relatively low activity for Rd. A V209 residue obtained from error-prone PCR was related to Rd-hydrolyzing activity, and a docking pose showing an interaction with Val209 was selected from numerous docking poses. W361F was obtained by rational design using the docking pose that exhibited 4.2-fold higher activity, 3.7-fold higher catalytic efficiency, and 3.1-fold lower binding energy for Rd than the wild-type enzyme, indicating that W361F compensated for the limiting step. W361F completely converted Rb1 to compound K with a productivity of 843 mg l−1 h−1 in 80 min, and showed also 7.4-fold higher activity for the flavanone, hesperidin, than the wild-type enzyme. Therefore, the W361F variant SS-BGL can be useful for hydrolysis of other glycosides as well as compound K production from Rb1, and semi-rational design is a useful tool for enhancing hydrolytic activity of β-glycosidase.
Highlights
Ginsenosides, which are the active components of the valuable herb ginseng
Determination of a residue related to ginsenoside Rd‐hydrolyzing activity by screening of mutant library produced from error‐prone The hydrolytic activity of pNP-β-d-glucopyranoside is determined using a microplate reader, while ginsenoside Rd is determined using an HPLC system, indicating that the assay of pNP-β-d-glucopyranoside is more convenient and rapid
V209A variant enzyme was selected with the highest activity by screening of mutant library produced from error-prone PCR
Summary
Ginsenosides, which are the active components of the valuable herb ginseng A. Meyer), have been used as traditional herbal medicines in Asian countries (Lu et al 2009). Meyer), have been used as traditional herbal medicines in Asian countries (Lu et al 2009) They have diverse beneficial biological properties such as anti-cancer (Lee et al 2009), anti-oxidant (Cho et al 2006), anti-inflammatory (Wang et al 2012), anti-allergic (Bae et al 2002), anti-fatigue (Yoshikawa et al 2003), and anti-skin aging (Kang et al 2009) activities. Because compound K is absent in wild ginseng, it has been produced from ginseng extract by enzymatic reactions using recombinant
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