Enzyme Activity Assays in Microplates

Enzyme activities were determined in a 96-well microplate reader (BioTek Instruments) from protein extracts. GS activity was measured monitoring the formation of γ-glutamilhydroxamate (γ-GHM) in a semibiosynthetic assay (Setién et al., 2013 (link)). GDH, PEPC, ICDH, and NAD-ME activities were measured monitoring the evolution of NAD(P)H monitored at 340 nm. GDH activity was determined in aminating sense (Setién et al., 2013 (link)). For ryegrass, wheat, and clover, novel data of carbon enzymes activities (PEPC, ICDH, and NAD-ME) were determined as previously described by Vega-Mas et al. (2015) (link) or Sarasketa et al. (2016) (link).