Activity detection kit

Nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthetase (GOGAT), and glutamate dehydrogenase (GDH) were determined using activity detection kits (SolarBio, Beijing, China). NR activity was determined by soaking in inducer for 2 h, drying by filter paper, and freezing at − 20 °C for 30 min. After the filter paper was aspirated to dryness, 0.1 g sample was weighed, and 1 mL extract was added. The sample was ground in an ice bath, centrifuged at 4000 rpm and 4 °C for 10 min, and the supernatant was placed on ice for testing. NiR and GOGAT activity was determined by weighing 0.1 g sample, adding 1 mL extract, grinding in an ice bath, centrifuging at 10,000 rpm and 4 °C for 10 min, and then, the supernatant was removed and placed on ice for testing.
For determination of the GS and GDH activity, 0.1 g sample was weighed, and 1 mL extract was added. The sample was ground in ice bath, centrifuged at 8000 rpm and 4 °C for 10 min, and the supernatant was then removed and placed on ice for testing. Deionized water was used as the reference solution. The colorimetric wavelengths of NR, GDH, and GOGAT are 340 nm, while those of NiR and GS are 540 nm.

Ammonia concentration was determined in the supernatant using an automated phenol-hypochlorite method (Broderick and Kang, 1980 (link)). The concentration of urea was determined using a urea assay kit (Sigma-Aldrich) based on the manufacturer’s protocol. The concentration of total free α-amino acids in the supernatant of the mixed amino acid-grown cultures was analyzed using ninhydrin as previously described (Jones et al., 2002 (link)). Microbial crude protein (MCP) was determined by the Folin phenol method (Makkar et al., 1982 (link)). Urease and glutamine synthase (GS) activity were determined using urease and glutamine synthase assay kits (Sigma-Aldrich), respectively, and glutamate dehydrogenase (GDH) and glutamate synthetase (GOGAT) activity were measured using activity detection kits (Solarbio, Beijing, China) according to the manufacturer’s instructions.

POD, SOD, and CAT activity levels of the prepared supernatant were determined using the respective activity detection kits (Solarbio, Beijing, China) in accordance with the manufacturer’s instructions.