Enzyme extraction and purification were conducted at 4 °C. A ratio of 1:6 (w/v) cold extraction buffer (50 mM NaOH-Bicine pH 8.2, 20 mM MgCl2, 1 mM EDTA, 2 mM Benzamidine, 5 mM aminocaproic acid, 50 mM 2-mercaptoethanol, 10 mM DL-dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)) was added to samples of 40 mg (1:12.5 w/v) of GMF- or NNMF-exposed Lima beans that were previously grinded in liquid nitrogen. The homogenate was then centrifuged at 14,000× g for 5 min at 4 °C. The supernatant was transferred to a new ice-cold tube and immediately used for the RuBisCO activity assay.
The assay was carried out in a Microplate reader (NB-12-0035 Neo Biotech, Nanterre, FR). In flat bottom 96-well polystyrene microplates, 159.9 µL of mQ water for the blank and 153.9 µL for the samples were pipetted into each well, followed by a 35.1 µL assay mix (100 mM NaOH-Bicine pH 8.2, 20 mM MgCl2, 10 mM NaHCO3, 20 mM KCl, 5 mM DTT, 2 UI 3-Phosphoglyceric phosphokinase, 0.4 UI α-Glycerophosphate Dehydrogenase, 24 UI Triosephosphate isomerase, 2.8 UI Glyceraldehyde 3-Phosphate Dehydrogenase, 3 mM ATP, 1 mM NADH) avoiding exposure to light. Next, 5 µL of a sample supernatant were added to the wells. Then, 6 µL 20 mM RuBP (ribulose-1,5-bisphosphate) were added to start RubisCO activity. Then, 5 µL of the sample supernatant were added to the wells. The Microplate reader was set at 30 °C and absorbance was read at 340 nm. The calculation of RubisCO activity was carried out according to Sales and co-workers [80 ].
The assay was carried out in a Microplate reader (NB-12-0035 Neo Biotech, Nanterre, FR). In flat bottom 96-well polystyrene microplates, 159.9 µL of mQ water for the blank and 153.9 µL for the samples were pipetted into each well, followed by a 35.1 µL assay mix (100 mM NaOH-Bicine pH 8.2, 20 mM MgCl2, 10 mM NaHCO3, 20 mM KCl, 5 mM DTT, 2 UI 3-Phosphoglyceric phosphokinase, 0.4 UI α-Glycerophosphate Dehydrogenase, 24 UI Triosephosphate isomerase, 2.8 UI Glyceraldehyde 3-Phosphate Dehydrogenase, 3 mM ATP, 1 mM NADH) avoiding exposure to light. Next, 5 µL of a sample supernatant were added to the wells. Then, 6 µL 20 mM RuBP (ribulose-1,5-bisphosphate) were added to start RubisCO activity. Then, 5 µL of the sample supernatant were added to the wells. The Microplate reader was set at 30 °C and absorbance was read at 340 nm. The calculation of RubisCO activity was carried out according to Sales and co-workers [80 ].
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