葡糖澱粉酵素 (glucoamylase, 1,4-α-D-glucan glucohydrolase, EC 3.2.1.3)，為水解酶的一種，可以催化多醣類的裂解。其作用由 -1,4和 -1,6鍵結的非還原端開始，進而水解出一個個的 -葡萄糖。此酵素來源相當廣泛，從動物、植物和微生物中皆可分離而取得，其中又以製備最為容易的微生物為主要的市場來源。葡糖澱粉酵素在工業上之應用已有多年歷史，例如澱粉的加工、糖漿的製作及酒精飲料的製成等等，而其他新的用途與應用也被不斷地開發與研究中。在本實驗中我們使用麵包酵母 (Saccharomyces cerevisiae)來表現根黴菌 (Rhizopus oryzae)之葡糖糖澱粉酵素。根黴菌的葡糖糖澱粉酵基因包含604個胺基酸，其構造大致可分為三個部分：一是具有吸附澱粉能力的區域，一是具有催化能力的區域，另一個則是負責連接這兩個功能性區域的連接片段。首先，我們將重組之葡糖澱粉酵素置於不同宿主中進行表現，篩選最佳的表現宿主、培養條件及純化方法，結果發現在適當的培養及純化下可得到大量的蛋白質產物。其次，在蛋白質功能性分析的實驗中，我們構築了一系列於澱粉結合區及連接片段縮短的葡糖澱粉酵素之突變株，分別名為wt-GA (WT)、GAΔ26-131 (L)、GAΔ26-145 (d1)、GAΔ26-160 (d2)和 GAΔ26-167 (cd)。藉由觀察各個突變株在功能及穩定上性質的改變以釐清該酵素不同區域的真實功能與關聯性。實驗結果顯示連接片段的長度的確會影響該酵素的表現及功能，此外其對蛋白質的結構也有很大的影響，完整的連接片段具有穩定酵素催化區二級結構的功能，至於更詳細的機制與功能分析我們將進一步繼續探討與研究。

Fungal glucoamylases (1,4-a-D-glucan glucohydrolase, EC 3.2.1.3, GA) are exo-acting starch hydrolase releasing -D-glucose from the nonreducing ends of starch and related substrates. Glucoamylases are glycoproteins that are secreted, and widely used in the production of sweeteners and ethanol. The Rhizopus GA gene encodes 604 amino acids and the mature GA consists of 579 amino acids with a starch binding domain and a catalytic domain connected by a putative highly glycosylated linker region. The function and the structure of the catalytic and starch binding domains have been thoroughly investigated, so in our study we focus on the roles that the linker length and the degree of glycosylation played on the enzymatic function. We have constructed a series of truncated mutants in the linker region and expressed the recombinant enzymes in Saccharomyces strain. In addition, we further investigated the positions of the glycans in GA employing N-terminal peptide sequencing and mass spectrometry.

Table of Contents
Abstract (Chinese)……………i
Abstract (English) ……………ii
Acknowledgement ……………iii
Table of Contents ……………iv
List of Tables ……………vi
List of Figures ……………vii
Abbreviations ……………ix
1. Introduction...1
1.1. Glucoamylase
2. Materials and Methods...3
2.1. Microbial strains and plasmid
2.2. Culture media
2.3. Construction of GA mutants with a series of internal deletions in the linker region
2.4. Competent cell preparation and transformation
2.5. Yeast transformation
2.6. Culture conditions for glucoamylase production
2.7. Glucoamylase activity detection and assay
2.7.1 Plate assay for GA activity
2.7.2 Quantitatively assay for GA activity
2.8. Polyacrylamide gel electrophoresis
2.9. Western blotting analysis
2.10 Purification of recombinant GAs
2.11 Protein concentration
2.12 N-terminal peptide sequence analysis
2.13 pI determination
2.14 Posttranslational modification
2.14.1 Determination of N-linked glycosylation
2.14.2 Determination of O-linked glycosylation
2.15 Circular Dichroism spectrometry
2.16 Effect of temperature and pH on enzyme stability and activity
2.17 Mass determination
2.18 Inhibition of GA by acarbose
2.19 Crystallization and X-ray measurements
3. Results and Discussion...18
3.1. Construction of GA mutants
3.2. Expression and secretion of glucoamylas
3.3. Production and purification of GA
3.4. Protein identification by SDS-PAGE and Western blot
3.5. Glucoamylase activity test
3.5.1 Plate assay for GA activity
3.5.2 Quantitative assay for GA activity
3.6. Circular Dichroism Spectroscopy and Thermal Stability
3.7. Glycoprotein analysis
3.7.1 Determining the molecular weight of GA
3.7.2 Glycosylation site mapping of GA
3.7.3 Determining N-linked glycosylation
3.7.4 Determining O-linked glycosylation
3.7.5 Identification of glycosylation sites in GA
3.8 Inhibition of GA by acarbose
3.9 Structure of glucoamylase
4. References...32
5. Tables...35
6. Figures...40

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