黑曲霉耐酸性α-淀粉酶基因真核表达载体的构建及酶学性质研究

Characterization and expression vector construction of an acid-stable α-amylase from Aspergillus niger

以报道的黑曲霉耐酸性α-淀粉酶基因(ANasamy)为基础设计两条引物,通过PCR等分子生物学手段克隆了一段全长为1975 bp的ANasamy基因,构建了关于ANasamy的表达质粒pPTH-ANasamy,并在黑曲霉菌株中获得表达。分离、纯化重组耐酸性α-淀粉酶并对该酶的基本酶学性质进行研究,该酶蛋白质分子量约为70 kDa,在65℃及pH 4.5条件下催化活力最高,具有较高的热稳定性及低pH耐受性。Cu^(2+)和Fe^(3+)对该淀粉酶活力有较强的抑制作用。

An acid-stable a-amylase gene （ANasamy） of Aspergillus niger was cloned, sequenced and expressed by a series of molecular method. An expressing plasmid pPTH-ANasamy carrying the ANasamy was constructed and expressed in A. niger. The molecular weight of the purified α-amylase was about 70 kDa. Enzyme characterization revealed that the a-amylase had an optimal temperature of 65℃ and an optimal pH of 4.5, and had prominent temperature stability and low pH stability. The purified ANasamy was strongly inhibited by Cu^2＋ and Fe^3＋ .