| 摘要: | 茶胺酸為茶葉特有的胺基酸，使茶葉帶有特殊的甘味。根據研究茶胺酸在臨床上也可能有一些特殊的用途，如降低先天性高血壓老鼠的血壓、影響腦中神經傳遞物的代謝、及增加抗癌藥物的抗癌活性或降低毒性等。
本論文的研究目標，主要先探討麩醯胺酶的活性測定方法及發展快速的茶胺酸分析方法的可行性，進而嘗試分離具有轉移γ-glutamyl基性質的麩醯胺酶，並藉以進行合成茶胺酸試驗。
首先，利用逆向分析管柱 (C18) 開發完成「離子對高效能液相層析法」(ion-pair HPLC)，配合OPA (ο-Phthalaldehyde) 與glutamate反應所產生之具螢光衍生物，進行麩醯胺酶活性測定；此法之靈敏度相當高，只需1μM的麩胺酸存在即可被檢出。本研究也發展出一套利用HPLC快速檢出茶胺酸的程序，藉由偵測195nm的吸光值進行定量，並利用雙極真空管檢測器紀錄各成分的吸收光譜，每次分析只要五分鐘即可，且沒有明顯的干擾現象；不過此法之靈敏度較低，茶胺酸被檢出之最低濃度約為100μM。
麩醯胺酶的來源是一種寄生於茶葉中經初步鑑定為Xanthomonas屬菌所分泌。菌體先經純化，再殖入於150 mL含 NB的培養液中，利用震盪培養箱以30℃及100 rpm進行震盪培養。
取菌體外的培養基部分，進行麩醯胺酶的純化，在經由硫銨沉澱、膠體層析管柱等純化步驟，得到較純的酵素。經過純化的酵素其水解能力的最適pH為8.0，分子量約為68,000。在茶胺酸合成的試驗方面，較適當的反應條件為使用66 mM 麩醯胺及0.1 M 乙基胺在pH 11的硼酸緩衝液下，於35℃進行反應。由結果顯示，在反應1小時後即有24.2 mM/U的茶胺酸生成，至7小時候則有79.4 mM/U的茶胺酸生成。此項催化能力與現有文獻所報導的最大值相仿。
我們在本論文中尚未詳細的對胞內和胞外麩醯胺酶的特性進行比較，但我們能確定的是，此菌會產生麩醯胺酶，此麩醯胺酶同時具有水解麩醯胺和轉醯胺基的能力。由於膠質物的干擾，此酶目前尚無法大量製備以應用於茶胺酸生產，不過我們確認本酵素可由培養液中萃取而得，卻是過去文獻所未報導者，很值得進一步加以探討。
Theanine is a unique amino acid found almost solely in tea plants and the main component responsible for the exotic taste of tea. Researches on the pharmacological effects indicated that theanine could reduce the blood pressure in spontaneous hypertensive rats, influence the level of neurotransmitters in the brain, increase the drug-induced antitumor activity, and ameliorate the toxicities of drugs.
In this study, systems used for the assays of the glutaminase activity and the theanine formation were developed first. The glutaminase activity was measured by an ion-pair liquid chromatography equipped with a fluorescence detector after the formation of ο-phthalaldehyde, derivative of L-glutamate. Theanine production in the reaction mixture was estimated from the absorbance at 195 nm after fractionation by high performance liquid chromatography. The results indicated that the sensitivity of assay method for glutaminase was so high that only 1μΜ of glutamate was needed for the evaluation. On the other hand, the detection time of theanine was less then 5 minutes, although 100μΜ of theanine was required for quantification by the procedure described above.
A glutaminase-producing gram-negative bacterium, which might belong to Xanthomonas species from a preliminary characterization, was isolated and mass cultured. The growth medium contained 8 g of NB and 10 g of glutamate per liter of culture. Bacteria were grown in Erlenmeyer flasks at 30℃ under aerobic conditions with shaking at a speed of 100 rpm.
Purification procedures of the extracellular glutaminase from culture medium included concentration, ammonium sulfate precipitation and gel filtrations. The molecular weight of this enzyme was estimated to be about 68,000 by gel filtration on a Sephacryl S-200 HR column. The optimal pHs for glutamine hydrolysis and theanine formation were determined to be 8.0 and 11, respectively. The results indicated that when a mixture containing 66 mM glutamine and 0.1 M ethylamine was incubated with glutaminase (1.67 mU/mL) at 35℃ in borate buffer (50 mM, pH 11), 24.2 mM/U and 79.4 mM/U of theanine were formed within 1 and 7 hours respectively.
Although the results are still not good enough for industrial uses at present time, however, it was the first time that an extracellular glutaminase from bacterium was demonstrated. It seems worthwhile to do further studies about this glutaminase. The species identification is undergoing by the FIRDI.
Keywords: bacterium, extracellular glutaminase, theanine formation, ion-pair HPLC, ο-phthalaldehyde |
