Studies On Nitrite Reductase From Boletus Edulis

Nitrite is widely distributed in the environment. Food is the main source of nitrite intake for human beings. As a strong oxidizing agent, extensive nitrite intake could cause intoxication. In the acidic environment produced by gastric acid, nitrite is converted into carcinogenic nitrosamine. The safety of nitrite present in food has attracted the attention of researchers. However, there are yet no reports on nitrite-reducing activity of macrofungi. Boletus edulis, a wild mushroom known worldwide, is rich in nutrition and has high value for food and medical purposes. Based on the fact that its fruiting body was capable of lowering the nitrite content in pickles fermentation, a series of experiment was conducted to clarify the mechanism of its nitrite-reducing effect. The results showed that this activity was attributed to the NiR in the fruiting body. This NiR was purified, and the enzymology property was studied in vivo and in vitro. Furthermore, the NiR was cloned in both a prokaryotic expression vector and an eukaryotic expression vector. This research not only enriched the resource pool of nitrite reductase, but also provide a new opportunities and theoretical basis for dealing with the therats to public health brought by nitrite.A 15.5-fold purification of the B. edulis NiR was achieved after the fruiting body extract had been subjected to (NH4)2SO4 precipitation, anion exchange chromatography and molecular sieve chromatography. This NiR was a homodimer and possesses a molecular of 90 kDa. The purified NiR exhibited a specific activity of 11.8 U/mg and the yield was 1.2%. Its optimum temperature was 45-50 ℃ while the optimum pH was 6.8. Fe3+ and Cu2+ ions could strongly affect the NiR activity. Fe3+ ions promoted the activity and the higher concentration of Fe3+, the higher activity of NiR. The lower concertration of Cu2+ inhibited whereas high concentration promoted NiR activity. The kinetic parameters of B. edulis NiR were determined using different concentrations of nitrite. The Km value for nitrite was 19.73 mM while the Vmax value was 2.95 U/mg of protein.To test if the NiR had nitrite-reducing activity in vivo, different treatments of NaNO2 to mice were conducted. Mice intoxicated with the low dose of nitrite (0.015 g/100 g) were all alive while those treated with the intermediate (0.025 g/100 g) or high (0.036 g/100 g) dose of nitrite died in 18±9.5 min and 11 ± 3.7 min, respectively. However, when treated concurrently with nitrite and the NiR extract, the symptoms of intoxication could be relieved and the mice could live significantly longer than control mice and some of them could survive the poisoning. Increasing the dosage of NiR extract brought about better detoxication. These results manifested that B. edulis NiR was capable of prolonging the lifespan of intoxicated mice indicating that it had the action of an antidote. B. edulis NiR could also reduce the nitrite content in the blood of rats which received NaNO2 by intragastric administration. In the experimental group that received B. edulis NiR, there was a shift in the peak. This shift and the smaller peak were most likely due to the elimination of nitrite by B. edulis NiR. As the dosage of NiR administered was elevated, the peak of blood nitrite level disappeared. For a longer duration of action of nitrite reductase in vivo, this NiR was packaged into capsules to protect the enzyme from gastric degradation. When introducing NiR in the form of capsules to rats by intragastric administration, we found the nitrite reduction time was postponed to 2 h later. Furthermore, the nitrite reduction period continued from 120 min to 360 min, which was substantially longer than 100 min in the control group.Through BLAST search of the 15 amino acids at the N-terminal, the DNA sequence and protein sequence were obtained from the database of B. edulis genome which is accessible at Joint Genome Institute (JGI). Using PCR amplification method with DNA from the material as template, the cDNA sequence was obtained by sequencing, which revealed a similarity of about 90% with the hit protein. We cloned the synthetic DNA sequence into a prokaryotic expression vector and an eukaryotic expression vector. The recombinant plasmid pET-28a-nir and pPIC9K-nir were successfully transformed into Escherichia coli BL21 (DE3) and Pichia pastoris GS115, respectively. For determination of expression product of pET-28a-nir/BL21(DE3), by inducing with IPTG, the target band appeared as expectlly on the SDS-PAGE profile and the target protein appeared as an inclusion body. The Fermented supernatant of pPIC9K-nir/GS115 induced by methanol did not show the target band.