Cloning, sequencing and expression of an α-L-arabinofuranosidase from Aspergillus sojae
Cloning, sequencing and expression of an α-L-arabinofuranosidase from Aspergillus sojae
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DOI：
10.1016/s1389-1723(00)88830-1
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发表时间：
2000-03-01
影响因子：
2.8
通讯作者：
Tajima, S
中科院分区：
文献类型：
--
作者：
Kimura, I;Yoshioka, N;Tajima, S
从大豆曲霉的cDNA中克隆了阿拉伯呋喃糖苷酶基因，该基因含有一个由984个碱基对组成的开放阅读框，编码328个氨基酸残基。测序结果表明，成熟酶的前面有一个26-AA信号序列，预测其相对分子质量为32,749 Da。大豆芽孢杆菌阿拉伯呋喃糖苷酶由单一的催化域组成；它不具有特定的底物结合域，如报道的变铅青链霉菌阿拉伯呋喃糖苷酶中的木聚糖结合域(文森特，P.等人：生物化学。J.，322,845-852,1997)。该成熟酶催化结构域的氨基酸序列与天蓝色链霉菌(74%)、黑曲霉(75%)、变铅青链霉菌(74%)和图宾曲霉(75%)的催化结构域具有广泛的同源性，属于糖基水解酶62家族。克隆的AFdase基因在大肠杆菌BL21(DE3)pLysS中以纤维素结合域标签融合蛋白的形式表达。纯化的重组酶的比活力为18.6单位/mg蛋白，是大豆曲霉固体培养纯化酶的1/4。
The arabinofuranosidase gene was cloned from the cDNA of Aspergillus sojae, It was found to contain an open reading frame composed of 984 base pairs (bp) and to encode 328 amino acid residues (aa). The cDNA sequence suggested that the mature enzyme is preceded by a 26-aa signal sequence and the molecular mass was predicted to be 32,749 Da. The A. sojae arabinofuranosidase consists of a single catalytic domain; it does not have a specific substrate-binding domain such as the xylan-binding domain reported in an arabinofuranosidase from Streptomyces lividans (Vincent, P. et al.: Biochem. J., 322, 845-852, 1997). The deduced amino acid sequence of the catalytic domain of the mature enzyme exhibits extensive identity with the catalytic domains of Streptomyces coelicolor (74%), Aspergillus niger (75%), S. lividans (74%), and Aspergillus tubingensis (75 %), which are enzymes that belong to family 62 of the glycosyl hydrolases. The cloned AFdase gene was expressed in Escherichia coli BL21 (DE3) pLysS as a cellulose-binding domain tag fusion protein. The specific activity of the purified recombinant enzyme was 18.6 units/mg protein, which is one-fourth that of the enzyme purified from a solid-state culture of A. sojae.