台湾文旦柚和蜜柚之柠檬苦素葡萄糖转移酶研究

Study on Taiwan Wentan and Miyu Pummeloes Limonoid Glucosyltransferase

柑橘类果汁在加工过程,在风味上受柠檬苦素影响甚巨,本研究针对台湾柚类柠檬苦素葡萄糖转移酶基因(limonin glucosyltransferase gene, lgt),选殖出一1600 bp片段,并对其作用活性位置预测出一段转录的44胺基酸序列,得到一个完整的开放框架(open reading frame),此段功能性基因有助于未来进行构筑与选殖,应用于柑橘类果汁在风味改进的研究。本研究亦探讨台湾的文旦柚和蜜柚lgt在开花至成熟时期的表现情况。结果得知,蜜柚中仅有第二型存在,且果实中含有大量lgt表现,而文旦同时具有两型lgt存在,但表现量相较于蜜柚低,此结果可应证前人之结论,即文旦柚于果实成长后期采收,因 lgt表现量不足,而导致其果实中仍含有大量苦味化合物仍有存在,导至于加工过程中苦味化合物的生成。本研究利用內果皮以快速蛋白纯化系统(FPLC) HiTrap Q管柱进行纯化,得分子量大约40 kDa之LGTase,并以2,2'-二辛可宁酸(bicinchoninic acid)定量蛋白质为0.6,1 mg/ml,此方法有助提高柚类加工过程中果皮废弃物的利用性。

Influence of limonin glusocyltransferease displays much influence on citrus juice bitterness, espe-cially in pumeloes. In this study, we have cloned a 1600 bp lgt from pumeloes limonin glucosyl-transferase’s active site, a 44 amino acids open reading frame was determined. This opening reading frame would be useful for genetic construction and cloning in upcoming study for citrus juice enhancement in flavor. This study also investigated Wentan and Miyu lgt during flowering and maturing. The results indicated that type II lgt was found only in Miyu and the gene expression was actively found in fruit. Wentan have both type I and type II lgt but the expression level is much lower than Miyu. These findings were similar to past researches which indicated that matured Wentan had less lgt expression in their fruit thus much bitter compounds appearance during its processing. We also used FPLC to purify LGTase, an approximately 40 kDa protein was then obtained and quantitated at 0.6 and 1 mg/ml. The enzyme can be used to reduce bitterness in pummeloes and further increase utility of peel wastes in processing.