Purification and Characterization of Glutamine Synthetase from Sugar Beet

摘要

More than one-third of the sugar (sucrose) consumed by human is obtained from sugar beet (Beta vulgaris L.), and beet root is highly regarded as a potential biomass feedstock for fuel-ethanol production. Applying nitrogen (N) fertiliser is one of important measures to improve the yields of sugar beet, however, unreasonable application of N fertilizer not only lead to beet yield and quality decreasing, but also bring about low renewable energy output/fossil fuel input ratio and a series of current environmental issues. So, continued research and improved understanding of the chemistry of N in soils and the biochemistry of N uptake and assimilation, may assist in improving management practices for better yields and high quality in sugar beet planting, accomplishing high energy output/input ratio and low environmental effects. Glutamine synthetase (GS;EC: 6.3.1.2) plays a key role in the assimilation of nitrogen in sugar beet. GS from leaves (GS2) and roots (GS1) of sugar beet have been purified to homogeneity by ion exchange and gel giltration on Sephadex G-200 HR. The isoforms of the enzyme show a differential distribution in leaf and root tissues. Denaturing SDS-PAGE experiments revealed GS is composed of eight subunits with a molecular weight of SO kD each. Moreover, determined results of GS characterization showed When the seedlings reached 31-d-old, only GS2 was present in the leaf of sugar beet, but both GS1 and GS2 were observed in the root. The activity of GS was highest at Mg2+concentration of 0.2 M at pH 7.2, and at temperatures of 35°C in the leaf and 40°C in the root. A Lineweaver-Burk plot showed that the Km for Glu-Na was 7.3 mM and for NH2OH was S.6 mM for sugar beet leaf GS. For root GS, the Km for Glu-Na was 5.8 mM and for NH2OH the Km was 7.1 mM. We identified not only the characteristics of GS in sugar beet, but also optimized the enzyme assay for GS. The study indicated GS was regulated by nitrogen form. These results will be used for theoretical basis of N fertilization practices efficiency in sugar beet cultivation, aid in evaluation of sugar beet germplasm and selecting high ammonia-assimilating lines, and promote the sustainable development of beet sugar manufacturing, energy and ecological environment