Comparative analysis of the expression level of recombinant ginsenoside-transforming β-glucosidase in GRAS hosts and mass production of theginsenoside Rh2-Mix

Abstract

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A β-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium gluta-micum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E colipGEX 4T-1 expression system. The highly expressed BglPm-C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm-C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E coli expression system.