Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)(8)-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.

将编码淀粉蔗糖酶（amylosucrase）的多糖奈瑟菌（Neisseria polysaccharea）基因进行亚克隆，并在大肠杆菌（Escherichia coli）中表达。测序结果显示，推导得到的氨基酸序列与此前已发表的序列存在显著差异。将该序列与α-淀粉酶家族（α-amylase family）的酶序列进行比对后，可预测其包含一个(β/α)₈-桶状结构域。淀粉水解酶的8个高度保守区域中，有6个存在于该淀粉蔗糖酶中，其中4个区域构成了α-淀粉酶的活性位点。这些位点在葡萄糖基转移酶（glucosyltransferases）和葡聚糖蔗糖酶（dextransucrases）的序列中也同样保守。然而，进化树分析并未显示二者之间存在较强的同源性。研究人员采用亲和层析法，利用谷胱甘肽S-转移酶-淀粉蔗糖酶融合蛋白（glutathione S-transferase–amylosucrase）与谷胱甘肽琼脂糖凝胶4B（glutathione-Sepharose 4B）之间的特异性相互作用对淀粉蔗糖酶进行纯化。纯化后的酶可使糖原的部分支链发生线性延伸，平均聚合度达到75。