Kang Hee Kyoung, Seo Mi Young, Seo Eun Seong, Kim Doman, Chung Seon Yong, Kimura Atsuo, Day Donal F, Robyt John F
Engineering Research Institute, Chonnam National University, Gwang-Ju, 500-757, South Korea.
Biochim Biophys Acta. 2005 Jan 21;1727(1):5-15. doi: 10.1016/j.bbaexp.2004.10.012. Epub 2005 Jan 4.
Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1.
肠系膜明串珠菌B - 512 FMC利用蔗糖产生右旋糖酐和果聚糖。由于右旋糖酐和由右旋糖酐蔗糖酶（肠系膜明串珠菌的一种聚糖蔗糖酶）合成的低聚糖在工业上具有重要意义，人们对右旋糖酐蔗糖酶了解很多，包括其基因的表达和调控。然而，关于另一种在工业上具有重要意义的肠系膜明串珠菌聚糖蔗糖酶——果聚糖蔗糖酶及其基因，尚无详细报道。在本文中，我们首次报道了肠系膜明串珠菌果聚糖蔗糖酶基因（m1ft）的分离及其分子特征。基因m1ft由1272个核苷酸组成，编码一个含有424个氨基酸残基的蛋白质，计算分子量为47.1 kDa。基于SDS - PAGE，纯化后的蛋白质估计约为51.7 kDa，包括一个His标签。在非变性SDS - PAGE上，它在103 kDa处显示出一条活性带，表明活性M1FT为二聚体形式。通过NMR和用抗果聚糖抗体进行的斑点印迹分析证实了M1FT果聚糖的结构。M1FT将150 mM蔗糖转化为果聚糖（18%）、蔗果三糖（17%）、蔗果四糖（11%）和1,1,1 - 蔗果五糖（7%），同时释放出葡萄糖。M1FT酶以麦芽糖为受体产物产生了松二糖[O - α - D - 吡喃葡萄糖基 - (1→4) - O - α - D - 吡喃葡萄糖基 - (1→2) - β - D - 呋喃果糖苷]。该酶形成果聚糖的最佳温度和pH分别为30℃和pH 6.2。1 mM Hg2 +或Ag2 +可完全消除M1FT果聚糖蔗糖酶的活性。果聚糖蔗糖酶的Km和Vmax值经计算分别为26.6 mM和126.6 μmol min - 1 mg - 1。
