de Pascale D, Di Lernia I, Sasso M P, Furia A, De Rosa M, Rossi M
Department of Biological Chemistry, University of Naples Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Extremophiles. 2002 Dec;6(6):463-8. doi: 10.1007/s00792-002-0283-6. Epub 2002 Aug 10.
In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins. In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S. solfataricus MT4 have been cloned and expressed in E. coli (Rb791). Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps. We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C. Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.
近年来，已发现许多硫化叶菌属的嗜热微生物能从淀粉和糊精中产生海藻糖。在我们实验室中，编码嗜热栖热菌MT4的海藻糖基糊精形成酶（TDFE）和海藻糖形成酶（TFE）的基因已被克隆并在大肠杆菌（Rb791）中表达。在此我们报告通过融合TDFE和TFE编码序列获得的一种新蛋白质的构建，该蛋白质能够通过连续的酶促步骤在高温下从糊精中产生海藻糖。我们证明这种双功能融合酶能够在75摄氏度下从麦芽寡糖开始产生海藻糖。此外，我们从无细胞细菌提取物和不溶性部分中部分纯化了重组融合蛋白，在这些不溶性部分中，融合蛋白也以聚集体形式存在于包涵体中。
