The enzyme nitrate reductase (NR) catalyzes the reduction of nitrate to nitrite and controls the rate of nitrate assimilation. The in vitro assay of NR was optimized for the wild strain (brown, MA), and the phycoerythrin-deficient strain (light-green, VC) of Hypnea musciformis. Both strains were cultured at temperature of 23 ± 2°C, photoperiod of 14h, irradiance of 60-90 µmol photons m-2s-1, with medium composed by sterilized seawater (salinity 30 psu) with 50% von Stosch's enrichment solution (VSES/2). The optimal conditions for in vitro assay of NR were: 40µM of NADH; 10min of incubation of crude extracts (EB), and 100µL of EB to both strains. Optimal activity of NR occurred at 4 and 2mM of nitrate to the VC and MA strains, respectively. The VC and MA strains showed, respectively, Michaelis-Menten constants (K M) for NADH of 0.2068 and 0.0837µM, and K M for nitrate of 0.0492 and 0.0294mM. The results indicate that the NR of MA strain has higher affinity by the substrate than the NR of VC strain of H. musciformis. Experiments on the effects of availabilities of nitrate (5 to 105µM) and nitrate and phosphate (0.5 to 25.5µM, with a N:P relation of 4:1) showed that NR activity of VC and MA strain did not increase with the addition of nitrate to the medium, what can be related with their nutritional state. The NR activity was higher in treatments with phosphate addition than those with only nitrate addition, indicating that this nutrient is important to metabolic processes related to the NR activity.
colour strain; Hypnea; nitrate; nitrate reductase; phosphate
