Moriyama Satoshi, Tanaka Hidenori, Uwataki Masato, Muguruma Michio, Ohta Kazuyoshi
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan.
J Biosci Bioeng. 2003;96(4):324-31. doi: 10.1016/S1389-1723(03)90131-9.
A genomic DNA segment and cDNAs encoding an extracellular exoinulinase from Aspergillus niger strain 12 were cloned and sequenced. Southern blot analysis indicated that the exoinulinase gene (inuE) was present as a single copy in the genome. An open reading frame of 1611 by was interrupted by a single intron of 60 bp, and encoded a 19-amino acid signal peptide and a 518-amino acid mature protein. The mature protein contained a single Cys residue and nine potential N-linked glycosylation sites. Three distinct transcription start points were observed at positions -41 (A), -35 (A), and -31 (A) from the start codon. The 5'-noncoding region had a putative TATA at position -75 (TATAAA). Transcription of the inuE gene was induced by inulin or sucrose and repressed by fructose or glucose. The inuE cDNA was functionally expressed under the control of the alcohol oxidase gene promoter in the methylotrophic yeast Pichia pastoris. The deduced amino acid sequence of the inuE gene product was 91% identical to that of an exoinulinase from Aspergillus awamori. A neighbor-joining tree showed that exo- and endoinulinases found in Aspergillus and Penicillium spp. have independently evolved the respective hydrolytic activities toward terminal and internal beta-2,1-fructofuranosidic linkages in inulin.
克隆并测序了来自黑曲霉12菌株的细胞外菊粉酶的基因组DNA片段和cDNA。Southern印迹分析表明，菊粉酶基因（inuE）在基因组中以单拷贝形式存在。一个1611bp的开放阅读框被一个60bp的单一内含子打断，编码一个19个氨基酸的信号肽和一个518个氨基酸的成熟蛋白。成熟蛋白含有一个半胱氨酸残基和九个潜在的N-糖基化位点。在起始密码子上游-41（A）、-35（A）和-31（A）位置观察到三个不同的转录起始点。5'-非编码区在-75（TATAAA）位置有一个推定的TATA框。inuE基因的转录受菊粉或蔗糖诱导，受果糖或葡萄糖抑制。inuE cDNA在甲基营养酵母毕赤酵母中受醇氧化酶基因启动子控制下进行功能表达。inuE基因产物的推导氨基酸序列与泡盛曲霉的一种菊粉酶的氨基酸序列有91%的同一性。一个邻接法树状图表明，在曲霉属和青霉属中发现的外切和内切菊粉酶已分别独立进化出对菊粉中末端和内部β-2,1-呋喃果糖苷键的水解活性。
