Yanai K, Nakane A, Kawate A, Hirayama M
Bio Science Laboratories, Meiji Seika Kaisha, Ltd., Sakado-shi, Saitama, Japan.
Biosci Biotechnol Biochem. 2001 Apr;65(4):766-73. doi: 10.1271/bbb.65.766.
The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.
从黑曲霉ATCC 20611中分离出编码产生低聚果糖的β-呋喃果糖苷酶的fopA基因。从核苷酸序列推导的一级结构与黑曲霉的另外两种β-呋喃果糖苷酶的结构有相当大的相似性，但fopA基因产物有几个氨基酸插入以及一个由38个氨基酸组成的额外的C末端多肽，这在另外两种酶中未发现。我们成功地在酿酒酵母中表达了fopA基因，从酿酒酵母转化体的培养上清液中获得的fopA基因产物具有与从黑曲霉ATCC 20611纯化的β-呋喃果糖苷酶相似的特性。然而，当用截短了38个氨基酸的C末端的fopA基因产物转化酿酒酵母时，我们在培养上清液或细胞裂解物中均未检测到任何β-呋喃果糖苷酶活性。在对这些样品的蛋白质免疫印迹分析中，没有与抗β-呋喃果糖苷酶抗体发生交叉反应的蛋白质产物。这些结果表明，由38个氨基酸组成的fopA基因产物的C末端区域对于该酶的产生至关重要。
