- 學位論文
2-acetyl-1-pyrroline體外合成模式之建立
Establishment of in vitro model of 2-acetyl-1-pyrroline synthesis and expression of its biosynthesis-related enzymes
摘要
2-acetyl-1-pyrroline (2-AP) 是麵包和香米中主要的香氣來源。食品中2-AP之生合成主要是藉由梅納反應。當食品含有鳥胺酸或脯胺酸時，能與糖類代謝物在高熱的條件下產生2-AP。Δ1-pyrroline-5-carboxylate (P5C)為鳥胺酸的代謝產物， methylglyoxal (MG) 則是葡萄糖的代謝產物。因此本實驗室推測P5C可與MG反應形成2-AP。我們利用鳥胺酸當做反應的基質，再加入MG並於25℃下反應形成2-AP已成功建立體外合成2-AP之模式，並利用GC/MS進行定性、定量分析。體外合成2-AP其濃度最高可達到0.4 μg/mL。在時間點方面，2-AP之濃度在第三天達到最高 (0.43 μg/mL)。在濃度點方面，當鳥胺酸或MG的濃度越高時，2-AP的生成量也隨之下降。然而生物體中2-AP之生成不是依靠梅納反應。有文獻推論B. cereus可藉由體內酵素之作用將1-pyrroline乙醯化生成2-AP。為了探討2-AP在生物體的生合成路徑，故分別自納豆菌與酵母菌選殖出甘油磷酸去氫酶 glycerol-3-phosphate dehydrogenase (G3PDH)、MG合成酶methylglyoxal synthase (MGS) 及乙醯基轉移酶acetyltransferase (MPR1) 等2-AP生合成相關基因並大量表現於Escherichia coli BL21且利用Ni2+ 親和管柱分離純化蛋白並分析其酵素活性。MPR1、G3PDH、MGS的分子量分別為26、66、15 kDa。MPR1及G3PDH的比活性分別為0.035、 0.0089 U/mg。
並列摘要
2-acetyl-1-pyrroline (2-AP) had been proposed as a character impact odorant causing the roasty note of bread crust and aromatic rice. The synthesis of 2-AP associated with amount of ornithine or proline in food and ornithine could reacted with carbohydrate degradation products during heating. The product of ornithine metabolism was Δ1-pyrroline-5-carboxylate (P5C) and methylglyoxal (MG) was end product of glucose degradation. We hypothesized that P5C reacted with MG result in synthesis of 2-AP. We also established the in vitro model of 2-AP synthesis successfully using ornithine and MG at 25℃, identification and quantification of 2-AP by GC/MS. The concentration of 2-AP synthesis about 0.4 μg/mL. In the time-dependent, values of 2-AP was the highest (0.43 μg/mL) in third day. In dose-dependent, the values of 2-AP synthesis decreased with increasing ornithine or MG concentrations. However, there were no relationship between maillard reaction and 2-AP biosynthesis in vivo. The reports suggested that the produ ction of 2-AP occurs via acetylation of 1-pyrroline by B. cereus. In order to study the pathway of 2-AP biosynthesis in vitro. In this study, the genes hypothesized to be involved in 2-AP biosynthesis, including glycerol-3-phosphate dehydrogenase (G3PDH), methylglypxal synthase (MGS) and acetyltransferase (MPR1), were cloned from Bacillus subtilis ssp. natto and Saccharomyces cerevisiae. These genes were overexpressed in Escherichia coli BL21 (DE3) and purified by Ni2+-affinity column. The molecular mass for MPR1, G3PDH and MGS of the expressed recombinant proteins were 26 kDa, 66 kDa and 15 kDa, respectively. The specific activity of MPR1 and G3PDH were 0.035, 0.0086 U/mg respectively.