| Summary: | 从南极深海底泥中筛选得到一株中度嗜盐菌Halomonas sp.Nj223,利用PCR技术,以该菌株基因组为模板,扩增出ectC基因。将目的基因的PCR扩增产物克隆至表达载体pET-his。经酶切、PCR鉴定、测序验证结果表明,目的基因插入的位置、大小和读码框均正确,表达载体构建成功。经SDS-PAGE分析,出现预期大小的目的蛋白条带。分离纯化复性的ectoine合成酶后测定其酶活力,在体外验证了ectoine的部分生物合成途径。进一步分析了pH和温度对酶活的影响发现,该酶最适pH为8.0,最适温度为25℃。 A moderately halophilic bacterium Halomonas sp. NJ223 was isolated from Antarctica deep-sea sediment. This bacterium accumulates ectoine as the main compatible solute in response to severe osmotic stress. Ecoine synthase catalyzes circulation of γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) to ectoine in the last step of the three enzymatic steps. The gene of ectoine synthase from this strain was amplified by PCR and the DNA sequence of a 393-bp segment was sequenced. The amino acid sequences of this enzyme present high homology to the known sequence. The significance of this gene was proved by the expression in Escherichia coli. Thus, the amplified fragment was cloned into the expression vector pET-his. The insert position, the size and the reading frame were identified by PCR, restriction digestion and the sequence analysis of the recombinant plasmids. SDS-PAGE shows that the relative molecular mass of the expression product was 15kDa as predicted, which indicated that the recombinant plasmids could express the gene of ectoine synthase. The biosynthetic pathway of ectoine was partially elucidated by renaturation and enzyme activity detection of purified ectoine synthase in vitro. Determination of effect of pH and temperature on enzyme activity shows that the optimal reaction condition of pH was 8.0 and temperature was 25℃. 大洋专项(DY105-04-02-06)~
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