Sakurai H, Yokota A, Tomita F
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Biosci Biotechnol Biochem. 1997 Jan;61(1):87-92. doi: 10.1271/bbb.61.87.
The gene encoding an extracellular inulin fructotransferase (depolymerizing) (inulase II) (EC 2.4.1.93), designated ift gene, was cloned from the genomic DNA of Arthrobacter sp. H65-7, and expressed in Escherichia coli for the first time. Sequence analysis showed a single open reading frame consisting of 1314 base pairs that encoded a signal peptide of 32 amino acids and a mature protein of 405 amino acids. The primary structure showed a homology of 49.8% with that of the inulin fructotransferase (DFA I-producing) (EC 2.4.1.200) from Arthrobacter globiformis S14-3. E. coli cells carrying the ift gene produced the active enzyme under control of the lac promoter. The expression of the ift gene was improved by a plasmid, pIFT-B, in which the ift gene was immediately downstream from the lac promoter. An E. coli transformant carrying pIFT-B expressed twice as much activity of inulase II as that of the original strain, Arthrobacter sp. H65-7. Most of the enzyme activity existed within the cells.
编码一种胞外菊粉果糖转移酶（解聚）（菊粉酶II）（EC 2.4.1.93）的基因，命名为ift基因，从节杆菌属菌株H65 - 7的基因组DNA中克隆得到，并首次在大肠杆菌中表达。序列分析显示有一个由1314个碱基对组成的单一开放阅读框，其编码一个32个氨基酸的信号肽和一个405个氨基酸的成熟蛋白。一级结构与球形节杆菌S14 - 3的菊粉果糖转移酶（产生DFA I）（EC 2.4.1.200）的一级结构具有49.8%的同源性。携带ift基因的大肠杆菌细胞在lac启动子的控制下产生活性酶。ift基因的表达通过质粒pIFT - B得到改善，在该质粒中ift基因紧接在lac启动子下游。携带pIFT - B的大肠杆菌转化体所表达的菊粉酶II活性是原始菌株节杆菌属菌株H65 - 7的两倍。大部分酶活性存在于细胞内。
