Construction And Application Of Screening Model Of Asparagine Synthetase B In Vitro

Asparagine synthetase B (AS-B, EC 6.3.5.4) widely exists in prokaryotes and eukaryotes, and it is the primary enzyme involved in the production of asparagine. The important physiological roles of AS-B make this enzyme a putative site for potential inhibitor, and now it has been found to be the target of cinmethylin.The innovations of this program were the expression of plant AS target protein which exists in soybean and construction of screening model in vitro, which constructed prokaryotic expression system. The screening model was identified with the herbicide cinmethylin and 1, 4-cineole, and the metabolites of four actinomycetes screened by our lab were screened by AS-B. And the results were as follows:(1). AS-B gene which was obtained by PCR amplification was cloned into prokaryotic expression vector pET30a (+) in correct ORF and then was transformed into Rosetta (DE3) pLysS. The best fusion expression conditions were obtained by optimizing inducing conditions such as the concentration of IPTG and inducing time. The results indicated that the yield of fusion protein reached tiptop when induced with 0.5 mM IPTG at 16℃for 14 h, and about 60% of the total fusion protein was soluble.(2). The expressed fusion protein AS-B in E.coli was purified in two chromatographic steps on a Ni-Sepharose affinity column followed by a Sephadex LH-20 gel filtration column, and the soluble fraction was obtained at more than 90% purity. The specific activity of the purified AS-B was determined by HPLC as 2.32μmol/min·mg and 2.6μmol/min·mg when glutamine and NH4Cl were nitrogen sources, respectively.(3). The screening model of AS-B was identified by the herbicide cinmethylin and 1, 4-cineole. The results showed that 1, 4-cineole inhibited AS-B activity with an I50 of 0.03μg/mL, whereas cinmethylin did not inhibit AS-B significantly. The results are consistent with previous reports, and this proved that the screening model of AS-B has been construced successfully.(4). The metabolites of four actinomycetes were screened by the screening model of AS-B. The results showed that the metabolism of No.1 actinomycete can inhibit the activity of AS-B, meanwhile the supernatant showed higher inhibition than the mycelium extract to AS-B. The identification of 16S rDNA shows that No.1 actinomycete belongs to streptomyces.