Akimoto H, Kiyota N, Kushima T, Nakamura T, Ohta K
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, Japan.
Biosci Biotechnol Biochem. 2000 Nov;64(11):2328-35. doi: 10.1271/bbb.64.2328.
A genomic DNA segment and cDNAs encoding an extracellular endoinulinase of Penicillium sp. strain TN-88 were cloned and sequenced. Southern blot analysis indicated that the endoinulinase gene (inuC) was present as a single copy in the genome. An open reading frame, consisting of 1,545 bp, was not interrupted by introns, and it encoded a 25 amino acid signal peptide and a 490 amino acid mature protein. The mature protein contained three Cys residues and ten potential N-linked glycosylation sites. Three distinct transcriptional start points were observed at positions -242 (A), -215 (A), and -75 (C) from the start codon. The 5'-noncoding region had a putative TATA box at position -120 (TATATATA) and two contiguous CAAT sequences at -159 to -151. The deduced amino acid sequence showed 72 and 85% identities with those of Aspergillus niger and Penicillium purpurogenum endoinulinase genes, respectively. A neighbor-joining tree showed that fungal endoinulinases form a distinct cluster from other members of the beta-fructofuranosidase superfamily and that they are more closely related to bacterial levanases than to a fungal fructosyltransferase, yeast invertases, or a yeast exoinulinase.
克隆并测序了编码青霉属菌株TN - 88胞外菊粉内切酶的基因组DNA片段和cDNA。Southern杂交分析表明，菊粉内切酶基因（inuC）在基因组中以单拷贝形式存在。一个由1545 bp组成的开放阅读框未被内含子打断，它编码一个25个氨基酸的信号肽和一个490个氨基酸的成熟蛋白。成熟蛋白含有三个半胱氨酸残基和十个潜在的N - 糖基化位点。在距起始密码子-242（A）、-215（A）和-75（C）位置观察到三个不同的转录起始点。5' - 非编码区在-120位置（TATATATA）有一个假定的TATA框，在-159至-151有两个相邻的CAAT序列。推导的氨基酸序列与黑曲霉和产紫青霉菊粉内切酶基因的氨基酸序列分别具有72%和85%的同一性。邻接法树显示，真菌菊粉内切酶与β - 呋喃果糖苷酶超家族的其他成员形成一个独特的聚类，并且它们与细菌果聚糖酶的关系比与真菌果糖基转移酶、酵母转化酶或酵母外切菊粉酶的关系更密切。
