臺灣博碩士論文加值系統

從Neocallimastix frontalis（NfrBGS）篩選出來的纖維雙糖酶，屬於糖苷水解酶第三家族(GH 3)，其胺基酸有769個且該酵素之分子量為84.246 kDa，能夠將纖維雙糖水解成葡萄糖之功能。以纖維雙糖做為受質，在0.1M磷酸緩衝溶液（pH 6）及溫度40°C下，反應10分鐘至48小時後，利用LC-RI進行產物分析。發現在反應時間16小時之後，纖維雙糖已全部被水解成葡萄糖。由LC-RI分析葡萄糖濃度，並換算得到kcat為1275.87±33.97s-1、Km為4.62±0.23 mg·ml-1。本實驗也利用BLAST (Basic Local Alignment Search Tool)進行序列分析，將NfrBGS的胺基酸序列和GH3家族已知結構且屬於β-glucosidase (EC 3.2.1.21)的蛋白質做序列比對，得知β-glucosidase of Trichoderma reesei QM9414 (PDB:3ZYZ)的序列與NfrBGS最相似。再以其做結構模擬，並分析活性區域；結果顯示NfrBGS是由兩個受質催化結構域(Glyco_Hydro_3（殘基：68-342）、Glyco_Hydro_3C（殘基：343-570)以及一個受質吸附模塊sigma70（殘基：704-765)組成。經由結構模擬，推測NfrBGS的主要功能性胺基酸是以Glu446作為質子受體，Asp244作為親核性胺基酸，在活性催化區域進行水解反應。此論文將討論NfrBGS催化纖維雙糖的實驗與結構模擬分析。

The β-glucosidase identify from Neocallimastix frontalis (NfrBGS), belongs to the glycoside hydrolase family three (GH3), and has 769 amino acids with the molecular weight of 84.246 kDa, which hydrolyzes the cellobiose into glucose. The enzymatic products were analyzed by using liquid chromatography with refractive index detector (LC-RI). The reaction time was 10 minutes to 48 hours in pH 6.0, 0.1M sodium phosphate buffer solution at 40°C. It was found that cellobiose had been hydrolyzed to glucose after 16 hours of reaction time. Based on the amount of glucose from the LC-RI product, we analyzed the Km was 4.62±0.23 mg·ml-1 and the kcat was 1275.87±33.97 s-1.
In addition, we built a structural model of NfrBGS based on the results from sequence alignment analyses analyzed by BLAST (Basic Local Alignment Search Tool), using β-glucosidase of Trichoderma reesei QM9414 (pdb: 3ZYZ) as a model. The modeled NfrBGS structure is composed mainly of three domains, two derived catalytic modules (Glyco_Hydro_3: 68-342 , Glyco_Hydro_3C : 343-570 ) and an acceptor module (sigma70 : 704-765 ). Homologous structural modeling of NfrBGS suggested that Glu446 as proton acceptor and Asp244 as nucleophilic amino acid are at the cleft site located between the two derived catalytic modules, which are the putative substrate-binding site and catalytic center. We also discussed the hydrolysis of lactose by NfrBGS .

目錄

摘 要 i
Abstract ii
致謝 iii
目錄 iv
表目錄 v
圖目錄 vi
第一章 緒論 1
1.1 前言 1
1.2 纖維素 3
1.3 纖維素酶 4
1.4 雙糖水解酶 9
1.5 文獻回顧 12
1.6 纖維素酶與雙糖酶的應用 12
1.7 研究動機與目的 13
第二章 材料與儀器 15
2.1 材料 15
2.2 儀器 18
第三章 實驗方法 19
3.1 NfrBGS的菌種保存 19
3.2 NfrBGS的培養 19
3.3 水解產物分析方法 21
3.4 酵素動力學 22
3.5 軟體結構模擬分析 23
第四章 實驗結果 25
4.1 NfrBGS的表現及純化 25
4.2 NfrBGS的水解產物分析 26
4.3 NfrBGS的酵素動力學分析 29
4.4 NfrBGS的序列與模擬結構分析 32
第五章 討論與結論 41
參考文獻 43
附錄 47
附錄一 GH3與NfrBGS蛋白質活性區域胺基酸分析整理 47
附錄二 DNS染色法酵素動力學分析資料 48
附錄三 LC-RI分析系統酵素動力學分析資料 50

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