Identification and characterization of a ginsenoside-transforming β-glucosidase from Pseudonocardia sp. Gsoil 1536 and its application for enhanced production of minor ginsenoside Rg2(S).

Abstract

The ginsenoside Rg2(S), which is one of the pharmaceutical components of ginseng, is known to have neuroprotective, anti-inflammation, and anti-diabetic effects. However, the usage of ginsenoside Rg2(S) is restricted owing to the small amounts found in white and red ginseng. To enhance the production of ginsenoside Rg2(S) as a 100 gram unit with high specificity, yield, and purity, an enzymatic bioconversion method was developed to adopt the recombinant glycoside hydrolase (BglPC28), which is a ginsenoside-transforming recombinant β-glucosidase from Pseudonocardia sp. strain Gsoil 1536. The gene, termed bglPC28, encoding β-glucosidase (BglPC28) belonging to the glycoside hydrolase family 3 was cloned. bglPC28 consists of 2,232 bp (743 amino acid residues) with a predicted molecular mass of 78,975 Da. This enzyme was overexpressed in Escherichia coli BL21(DE3) using a GST-fused pGEX 4T-1 vector system. The optimum conditions of the recombinant BglPC28 were pH 7.0 and 37 °C. BglPC28 can effectively transform the ginsenoside Re to Rg2(S); the Km values of PNPG and Re were 6.36 ± 1.10 and 1.42 ± 0.13 mM, respectively, and the Vmax values were 40.0 ± 2.55 and 5.62 ± 0.21 µmol min-1 mg-1 of protein, respectively. A scaled-up biotransformation reaction was performed in a 10 L jar fermenter at pH 7.0 and 30°C for 12 hours with a concentration of 20 mg/ml of ginsenoside Re from American ginseng roots. Finally, 113 g of Rg2(S) was produced from 150 g of Re with 84.0 ± 1.1% chromatographic purity. These results suggest that this enzymatic method could be usefully exploited in the preparation of ginsenoside Rg2(S) in the cosmetics, functional food, and pharmaceutical industries.