Research Centre for Chemistry, Indonesian Institute of Sciences, Jl. Sangkuriang, Bandung, Indonesia.
Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
J Antibiot (Tokyo). 2014 Feb;67(2):137-41. doi: 10.1038/ja.2013.95. Epub 2013 Oct 16.
The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 °C, optimum pH between 5.5 and 6.0 and remained stable up to 70 °C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.
从 Nonomuraea sp. ID06-A0189 的基因组 DNA 中扩增、测序并在大肠杆菌中表达了菊粉果糖基转移酶（DFA III 形成）（EC 4.2.2.18）基因。该基因被命名为 Nsp-ift，全长 1326bp，编码一个由推测的 37 个氨基酸信号肽和 404 个氨基酸成熟蛋白组成的蛋白。在起始密码子上游 12 个碱基处鉴定到一个推定的核糖体结合序列。然而，通过计算机分析未能找到典型的细菌启动子。该酶的推导氨基酸序列与 Frankia sp. EAN1pec 的菊粉果糖基转移酶（DFA I 形成）最为相似。推导氨基酸序列的系统发育分析表明，Nonomuraea sp. ID06-A0189 和 Frankia sp. EAN1pec 的菊粉果糖基转移酶与 Arthrobacter sp. H65-7、A. globiformis 和 Bacillus sp. snu-7 的菊粉果糖基转移酶形成了一个独特的分支，分别与 Nsp-ift 的相似度为 57%、56%和 56%。不含推定信号肽的 Nsp-ift 成功在大肠杆菌中表达，并通过 His 标签亲和层析进行了部分纯化。重组酶的最适温度在 65-70°C 之间，最适 pH 在 5.5-6.0 之间，在 70°C 下仍保持稳定。这些性质与原始酶相同。在通过 Southern 杂交、酶活性测定和 PCR 对 10 种 Nonomuraea 物种进行测试中，只有 Nonomuraea sp. ID06-A0189 具有 Nsp-ift 基因，表明 Nsp-ift 在该属中不是高度保守的。
