中国农业科学 ›› 2011, Vol. 44 ›› Issue (6): 1077-1084 .

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

小麦籽粒异淀粉酶聚合体的结构及酶学性质

  

  1. (泰山学院生物系)
  • 收稿日期:2010-07-20 修回日期:2010-08-24 出版日期:2011-03-15 发布日期:2011-03-15
  • 通讯作者: 王宪泽

The Constructure and Enzymatic Characterization of Isoamylase-Oligomer from Wheat Endosperm


  

  1. (泰山学院生物系)
  • Received:2010-07-20 Revised:2010-08-24 Online:2011-03-15 Published:2011-03-15
  • Contact: WANG Xian-ze

摘要:

【目的】阐明小麦淀粉去分支酶(Debranching enzyme, DBE)的结构和酶学特性,为品质改良提供理论依据。【方法】以小麦品种糯麦2号为材料,利用硫酸铵分级沉淀、超滤、阴离子交换柱层析和葡聚糖凝胶层析法对异淀粉酶(Isoamylase, ISA)同工酶聚合体进行分离纯化,采用非变性聚丙烯酰胺凝胶电泳(Native-PAGE)和SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)对ISA聚合体的结构和组成成分进行分析。【结果】小麦籽粒胚乳中异淀粉酶(ISA)形成2种不同聚合体,即ISA1同源四聚体和由4个ISA1与1个ISA2组成的异源五聚体,分子量分别约为320 kD和380 kD。ISA1同源四聚体的最适催化温度为30℃,40℃处理10 min后完全失去活性;ISA1/ISA2异源五聚体的最适温度为35℃,50℃处理10 min后仍有50%的活性。纯化后的ISA1和ISA2进行Native-PAGE检测证明,ISA2单独存在没有催化活性,将ISA2加入到ISA1中可检测到与ISA1/ISA2异源聚合体相同的ISA活性。【结论】小麦胚乳中存在2种形式的ISA多亚基聚合体;在40℃和50℃高温条件下,ISA1/ISA2异源聚合体的热稳定性高于ISA1同源聚合体;ISA2可显著增强ISA1聚合体的酶活性,可能在支链淀粉合成中起关键作用。

关键词: 小麦, 淀粉去分支酶, 异淀粉酶聚合体, 热稳定性

Abstract:

【Objective】Isoamylases are debranching enzymes that hydrolyze α-1, 6 linkages in α-1, 4/α-1, 6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. 【Method】The isoamylase-oligomer were fractionated by salting out with ammonium sulphate, DEAE-cellulose column chromatograthy, and gel filtration using sephadex G-200. The constructure and component of ISA complexes were studied by using native polyacrylamide gel electrophoresis (Native-PAGE) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 Results of the study showed that there are two distinct polymeric forms of isoamylase (ISA) in wheat endosperm: Presumably a homo-tetramer of ISA1 and a hetero-oligomer composed of four ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers were approximately 320 and 380 kD, respectively. The ISA1 homo-oligomer optimum temperature was 30℃, after 10 min 40℃ treatment, the activity was completely lost, while the optimum temperature of ISA1/ISA2 heter-oligomer was 35℃, which was active even when incubated at 50℃ for 10 min. Native-PAGE showed that ISA2 not existed separate activity,but when the ISA1 homo-oligomer was incubated with the ISA2, it had the same bands as ISA1/ISA2 hetro-oligomer, indicating that ISA1 and ISA2 combined to form a hetero-oligomer. 【Conclusion】Two multimeric forms of ISA are present in wheat endosperm. The hetero-oligomer was found to be more stable at high temperatures of 40℃ and 50℃. These results indicate that ISA2 plays an important role in amylopectin biosynthesis by enhancing the activity of the ISA1 homo-oligomer and facilitating the binding of the hetero-oligomer to polyglucan substrates.

Key words: wheat, starch debranching-enzyme, isoamylase-oligomer, thermal stability