利用液相層析串聯質譜儀法結合酵素水解分析糯性玉米澱粉之分支點區塊結構特徵

Abstract

支鏈澱粉由糯玉米澱粉的主要組成，其物理性質除了鏈長以外，也受其分支點區塊結構的影響，但此區塊的分析目前尚無有效的分析工具可以檢視其結構，本研究嘗試使用兩種澱粉酵素結合液相層析串聯質譜法，來建構一個有效的分析平台。利用二甲亞碸(DMSO)完全分散糯玉米澱粉分子後，進行β-amylase水解完全去除澱粉分子的外鏈，再以α-amylase進行水解以得到β,α-limit dextrin (β,α-LD)，此β,α-LD即是糯玉米澱粉分支點區塊，為具有α- (1→6) 分支點的短鏈麥芽糊精(dextrin)。為分析此一群複雜β,α-LD的聚合度與分支點區塊結構，使用多孔石墨碳液相層析管柱 (Hypercarb™ Porous Graphitic Carbon) 分離個別的β,α-LD分子，再利用串聯質譜偵測器，分析其聚合度與推斷其分子特徵。結果顯示以0.1%氨水為修飾劑，不會影響石墨碳管柱分離β,α-LD分子的效能，但可有效地提高麥芽糊精的游離感度，其聚合度主要分布在3~9範圍，並含有更大的聚合分佈。經由質譜撞碎機制，可得到多種α- (1→4)與α-(1→6) 的特徵斷片異構物，藉由多次質譜的高比例醣苷鍵B型和跨醣基環0,4A，以及低比例的醣苷鍵C型的特徵斷片，來作為判斷分支的位置，並以異潘諾醣 (6-o-a-D-Glucosyl-maltose) 為分析模式標準品，可以推測23種以上分支點區塊結構，做為觀察糯玉米澱粉分子特徵的工具。

Waxy corn starch is mainly composed of amylopectin. The physical property is not only affected by chain length distribution but also its branching point blocks. However, there are barely effective analysis methods of the research in branching point blocks. To build an effective analysis method, our study try to use two enzymes to hydrolyze starch and analyze through HPLC-MS method. We use dimethyl sulfoxide to dissolve waxy corn starch, and then cut its external chain through β-amylase, which is called β-limit dextrin. Then, we use α-amylse to hydrolyze β-limit dextrin and finally get β,α-limit dextrin. These β,α-limit dextrin which have α-(1→6) branching point can represent branching point blocks. To analyze these complicated β,α-limit dextrin, first, we choose graphitic carbon chromatography column (Hypercarb™ Porous Graphitic Carbon) to separate each isomer in β,α-limit dextrin. Second, we connect mass spectrometry to detect the degree of polymerization (DP), linkage position and structure. The result shows that using 0.1% ammonium hydroxide as modifier dose not affect the effiency of seperation but can enhance the ionization sensitivity of β,α-limit dextrin. The DP range is mainly around 3-9 and has few higher DP distribution. Through the mechanism of mass collision, we can get various characteristic fragments of α-(1→4) and α-(1→6) linkage. With high ratio fragment B, 0,4A and low ratio fragment C, we may know the position of branching point. Using standard like maltotriose, isomaltotriose, panose, isopanose, maltodextrin to analyze fragment pattern, we can successfully elucidate 23 structures of branching blocks.