Abstract：
Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) is a highly conserved enzyme that is involved in glycolysis and gluconeogenesis. In this study, we cloned the fructose-1,6-bisphosphate aldolase gene from Euphausia superba (EsFBA). The full-length cDNA sequence of EsFBA is 1098 bp long and encodes a 365-amino-acid protein. The fructose-1,6-bisphosphate aldolase gene was expressed in Escherichia coli (E. coli). A highly purified protein was obtained using HisTrap HP affinity chromatography and size-exclusion chromatography. The predicted three-dimensional structure of EsFBA showed a 65.66% homology with human aldolase, whereas it had the highest homology (84.38%) with the FBA of Penaeus vannamei. Recombinant EsFBA had the highest activity at 45 °C and pH 7.0 in phosphate buffer. By examining the activity of metal ions and EDTA, we found that the effect of metal ions and EDTA on EsFBA\'s enzyme activity was not significant, while the presence of borohydride severely reduced the enzymatic activity; thus, EsFBA was confirmed to be a class I aldolase. Furthermore, targeted mutations at positions 34, 147, 188, and 230 confirmed that they are key amino acid residues for EsFBA.
摘要：
果糖-1,6-二磷酸醛缩酶（EC4.1.2.13）是一种高度保守的酶，参与糖酵解和糖异生。在这项研究中,我们从Euphausiasuperba（EsFBA）中克隆了果糖-1,6-二磷酸醛缩酶基因。EsFBA的全长cDNA序列长1098bp，编码365个氨基酸的蛋白质。果糖-1,6-二磷酸醛缩酶基因在大肠杆菌中表达（E。大肠杆菌）。使用HisTrapHP亲和色谱和尺寸排阻色谱获得高度纯化的蛋白质。预测的EsFBA三维结构与人醛缩酶有65.66%的同源性,与南美白对虾FBA的同源性最高(84.38%)。重组EsFBA在45°C和pH7.0的磷酸盐缓冲液中具有最高的活性。通过检测金属离子和EDTA的活性，发现金属离子和EDTA对EsFBA酶活性的影响不显著,而硼氢化物的存在严重降低了酶活性；因此，EsFBA被确认为I类醛缩酶。此外，在位置34、147、188和230的靶向突变证实它们是EsFBA的关键氨基酸残基。
