Haraguchi K, Mori S, Hayashi K
National Institute of Agro-Environmental Sciences, 3-1-1 Kannondai, Tsukuba-shi, Ibaraki 305-8604, Japan.
J Biosci Bioeng. 2000;89(6):590-5. doi: 10.1016/s1389-1723(00)80062-6.
A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.
克隆了球形节杆菌C11 - 1中编码菊粉果糖转移酶（产生DFA III）[EC 2.4.1.93]的基因，并测定了其核苷酸序列。克隆片段包含一个1353 bp的开放阅读框。起始密码子估计是一个不寻常的密码子GTG。该基因编码一个用于分泌的信号肽（40个氨基酸残基）。根据测序数据计算，天然酶的分子量为43,400 Da。该酶推导的氨基酸序列与球形节杆菌H65 - 7的菊粉果糖转移酶（产生DFA III）的氨基酸序列具有74.0%的同源性。它与球形节杆菌S14 - 3的菊粉果糖转移酶（产生DFA I）[EC 2.4.1.200]也具有45.1%的同源性。在大肠杆菌克隆的培养上清液中产生的酶被纯化至电泳纯阶段。肉汤中分泌的克隆酶的N端氨基酸序列与球形节杆菌C11 - 1的天然酶相同。因此，对于这种酶，估计球形节杆菌C11 - 1和大肠杆菌JM109用于分泌的信号肽酶的切割位点是相同的。
