Enzyme Activity Assays for Ginsenoside Biosynthesis

Amylase (AMY, EC 3.1.1.2) activity was detected by the 3,5-dinitrosalicylic acid colorimetric method (Hao et al., 2007 ). Malate dehydrogenase (MDH, EC 1.1.1.37) activity was examined as described by Husted and Schjoerring (1995 (link)), with some modifications. Ten microliter samples were added to a 3 ml reaction mixture containing 0.17 mM oxalacetic acid and 0.094 mM β-NADH disodium salt in 0.1 M Tris buffer, pH 7.5. The reaction was measured by the decrease in absorbance at 340 nm for 180s in a spectrophotometer (Hitachi U-2001 Japan), the same reaction system only with sample buffer added in was used as a blank. Superoxide dismutase (SOD, EC 1.14.1.1) activity was measured according to the method of Zhang and Kirkham (1996 (link)), and Xu and Huang (2004 (link)). One unit of SOD activity is defined as the amount of SOD required to cause 50% inhibition of nitroblue tetrazolium (NBT) reduction at 560 nm min-1. Catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC.1.11.1.7) activity were determined based on the method of Chance and Maehly (1955 (link)) as described in detail for creeping bentgrass in Xu and Huang (2004 (link)). Enzyme activities were based on the absorbance change of the reaction solution per minute at a given wavelength for each enzyme: CAT at 240 nm and POD at 470 nm.
The activities of farnesyl diphosphate synthase (FDPS, EC. 2.5.1.10), cycloartenol synthase (CAS, EC. 5.4.99.8), squalene epoxidases (SE, EC:1.14.13.132), and squalene synthase (SS, EC. 2.5.1.21) involved in ginsenosides biosynthesis, were quantified by an indirect competitive enzyme-linked immunosorbent assay (ELISA). The optical density (OD) values of each sample were read by a BioTek ELx800 microplate reader at 450 nm. The primary concentration of each test sample was calculated from the linear regression equation based on the OD values of the standards.