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。
【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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2011年第6期1077-1084,共8页
Scientia Agricultura Sinica
基金
山东省自然科学基金(ZR2010CM034)
山东省高等学校科技计划(J10LC63)
泰山学院引进人才项目(Y2008-2-6)
国家"十一五"科技支撑计划项目(2006BAD04B01)
关键词
小麦
淀粉去分支酶
异淀粉酶聚合体
热稳定性
wheat
starch debranching-enzyme
isoamylase-oligomer
thermal stability
