Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Sci Rep. 2018 May 29;8(1):8340. doi: 10.1038/s41598-018-26721-w.
Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as "alternan". The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40 °C and 5.0, respectively, and its' activity was stimulated up to 2.4-fold by the presence of Mn. Kinetic studies of LcALT showed a high transglycosylation activity, with K 32.2 ± 3.2 mM and kcat 290 ± 12 s. Alternan generated by LcALT (Lc-alternan) harbours partially alternating α-1,6 and α- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90 nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.
交替蔗糖酶催化蔗糖分子上的葡萄糖残基顺序转移到另一个蔗糖分子上，形成长链聚合物，称为“交替聚糖”。从柠檬明串珠菌 ABK-1（Leuconostoc citreum ABK-1）中成功克隆并在大肠杆菌中表达了交替蔗糖酶编码基因（Lcalt）。Lcalt 编码 2057 个氨基酸残基的 LcALT；该酶的最适温度和 pH 值分别为 40°C 和 5.0，Mn 的存在可将其活性提高 2.4 倍。LcALT 的动力学研究表明，其具有较高的转糖基活性，K m 值为 32.2±3.2mM，k cat 值为 290±12s。LcALT 生成的交替聚糖（Lc-alternan）具有部分交替的α-1,6 和α-1,3 糖苷键，通过 NMR 光谱、甲基化分析和 Lc-alternan 产物的部分水解得到证实。与之前报道的交替聚糖不同，Lc-alternan 可以自组装，在溶液中形成平均粒径为 90nm 的纳米颗粒。在浓度高于 15%（w/v）时，Lc-alternan 纳米颗粒会解体并形成高粘度溶液，而该聚合物一旦干燥就会形成透明薄膜。
