小麦籽粒灌浆过程中淀粉去分支酶的类型、活性及其纯化

Type,Activity,and Purification of Starch-Debranching Enzymes during Grain Filling Period in Wheat

以小麦品种鲁麦21和烟优361(低支链淀粉含量)、鲁麦1号和济宁13(中支链淀粉含量)、济南16和糯麦2号(高支链淀粉含量)为材料,采用分光光度、聚丙烯酰胺凝胶电泳和高效液相色谱法,对小麦籽粒灌浆成熟过程中异淀粉酶和极限糊精酶的活性变化、类型特征及酶解产物进行了分析,并利用硫酸铵沉淀、葡聚糖凝胶层析(G-100)和阴离子交换柱层析(DEAE)分别对两种淀粉去分支酶进行了分离纯化。结果表明,两种淀粉去分支酶均存在于淀粉合成过程中;在灌浆成熟过程中,6个品种的两种淀粉去分支酶活性都在15 d左右达最高值,且异淀粉酶活性呈单峰曲线,而极限糊精酶活性呈波浪形曲线,在25 d左右又出现另一峰值;纯化后的异淀粉酶和极限糊精酶分子量分别为83和100 kD。高效液相色谱图谱表明,以支链淀粉为底物时生成葡萄糖和糊精,以普鲁蓝为底物时只生成麦芽三糖。

Amylopectin is a major constituent of starch in plant tissues, physical and chemical analysis of granular starch have led to a widely accepted model for amylopeetin structure called ＂cluster model＂, in which amorphous and crystalline regions alternate with a defined periodicity. Within amylopectin the crystalline component is composed of parallel arrays of linear chains packed tightly in double helices. Branch linkages, which account for approximate 5 % of the glucosyl linkages in amylopectin, are located at the root of each cluster in the amorphous region. This periodic clustering of branches allows for the alignment of the intervening linear chains and their dense packing into crystalline regions, thus providing an efficient mechanism for nutrient storage. Biochemical analysis of su-1 mutants of maize （ Zea mays L. ） and rice （ Oryza sativa L. ） endosperm indicate that debranching enzymes （DBE） are proposed to play an essential role in the final determination of amylopectin structure. The precise way in which debranching enzymes involved in amylopectin synthesis is difficult to assess because of the lack of information about the occurrence and nature of these enzymes in starch-synthesizing organs and relatively little is known about these enzymes in higher plants. To provide more information about the occurrence and possible role of debranching enzymes in starch synthesis, six wheat （ Tritieum aestivum L. ） cultivars corresponding to Lumai 21 and Yanyou 361 （low amylopectin）, Lumai 1 and Jining 13 （medium amylopectin）, and Jinan 16 and Nuomai 2 （high amylopectin） were used in the present study. Activity changes, activity difference and types of DBE were studied during grain filling period by using spectrophotometer, native-PAGE and HPLC. The DBEs were also purified by salting out with ammonium sulphate, DEAE-cellulose column chromatograthy, and gel filtration using sephadex G-100. The results showed that two types of DBE were identified during starch synthesis and they all reached the highest activity at the 15th day after anthesis, the activity changes of isoamylase appeared as a single-peak curve, but those of pullulanase appeared as an undulatory curve, with another high activity around the 25th day after flowering. The molecular weights of isoamylase and pullulanase were determined to be 83 and 100 kD by SDS-PAGE respectively. HPLC analysis showed that activities of starch debranching enzymes to amylopectin and pullulan were similar among the six wheat cultivars. Glucose and dextrin were identified as the major hydrolysis products of amylopectin by isoamylase and mahotriose was identified as a major hydrolysis product of pullulan by pullulanase.