Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH, UK.
Plant Mol Biol. 2018 Mar;96(4-5):417-427. doi: 10.1007/s11103-018-0707-z. Epub 2018 Jan 29.
Cloning of two isoamylase genes, MeISA1 and MeISA2, from cassava (Manihot esculenta Crantz) tubers, accompanied by their co-expression in E. coli demonstrates a requirement for heteromeric complex formation to achieve debranching activity. Starch debranching enzyme (DBE) or isoamylase (ISA) (EC.3.2.1.68), an important enzyme in starch metabolism, catalyses the hydrolysis of α-1,6 glycosidic linkages of amylopectin. Isoforms of ISAs have been reported in higher plants and algae (Fujita et al. in Planta 208:283-293, 1999; Hussain et al. in Plant Cell 15:133-149, 2003; Ishizaki et al. in Agric Biol Chem 47:771-779, 1983; Mouille et al. in Plant Cell 8:1353-1366, 1996). In the current work, cassava ISA genes were isolated from cDNA generated from total RNA from tubers of Manihot esculanta Crantz cultivar KU50. MeISA1 and MeISA2 were successfully amplified and cloned into a pETDuet1 vector. The putative MeISA1 and MeISA2 proteins comprised 763 and 882 amino acids, with substantial similarity to StISA1 and StISA2 from potato (84.4% and 68.9%, respectively). Recombinant MeISA1 and MeISA2 were co-expressed in Escherichia coli SoluBL21 (DE3). Histrap-Purified rMeISA1 and rMeISA2 showed approximate molecular weights of 87 and 99 kDa, respectively, by SDS-PAGE. Debranching activity was only detectable in the column fractions where both recombinant ISA isoforms were present. The heteromeric DBE from crude extracts of 4-5 h induced cultures analysed by gel filtration chromatography and western blot showed combinations of rMeISA1 and rMeISA2 at ratios of 1:1 to 4:1. Pooled fractions with DBE activity were used for enzyme characterisation, which showed that the enzyme was specific for amylopectin, with optimum activity at 37 °C and pH 7.0. Enzyme activity was enhanced by Co, Mg and Ca, but was strongly inhibited by Cu. Debranched amylopectin products showed chain length distributions typical of plant DBE.
从木薯（Manihot esculenta Crantz）块茎中克隆出两个同工淀粉酶基因 MeISA1 和 MeISA2，并在大肠杆菌中共同表达，证明了异源寡聚体形成是实现脱支酶活性所必需的。淀粉分支酶（DBE）或同工淀粉酶（ISA）（EC.3.2.1.68）是淀粉代谢中的一种重要酶，可催化支链淀粉中α-1,6 糖苷键的水解。已在高等植物和藻类中报道了 ISA 的同工酶（Fujita 等人，Planta 208：283-293，1999；Hussain 等人，Plant Cell 15：133-149，2003；Ishizaki 等人，Agric Biol Chem 47：771-779，1983；Mouille 等人，Plant Cell 8：1353-1366，1996）。在本研究中，从木薯品种 KU50 的块茎总 RNA 生成的 cDNA 中分离出木薯 ISA 基因。成功扩增并克隆 MeISA1 和 MeISA2 到 pETDuet1 载体中。推定的 MeISA1 和 MeISA2 蛋白分别由 763 和 882 个氨基酸组成，与马铃薯中的 StISA1 和 StISA2 具有高度相似性（分别为 84.4%和 68.9%）。重组 MeISA1 和 MeISA2 在大肠杆菌 SoluBL21（DE3）中共同表达。Histrap-Purified rMeISA1 和 rMeISA2 通过 SDS-PAGE 显示出约 87 和 99 kDa 的分子量。仅在柱级分中可检测到同时存在两种重组 ISA 同工酶时才具有脱支酶活性。通过凝胶过滤色谱和 western blot 分析，从 4-5 h 诱导培养物的粗提取物中检测到的异源 DBE 显示 rMeISA1 和 rMeISA2 的组合比为 1:1 至 4:1。具有 DBE 活性的合并级分用于酶特性分析，表明该酶特异性作用于支链淀粉，最适活性为 37°C 和 pH 7.0。酶活性被 Co、Mg 和 Ca 增强，但被 Cu 强烈抑制。脱支支链淀粉产物的链长分布具有植物 DBE 的典型特征。
