고세균 유래 호열성 α-glucosidase와 glycosynthase를 이용한 배당체의 합성 연구 = Enzymatic Synthesis of Glycosides Using α-Glucosidase and Glycosynthase from Thermophilic Archaeon

다국어 초록 (Multilingual Abstract)

Oligosaccharides in the form of glyco-conjugates and glycosides have a range of important functions in biological systems and therefore have great potential as therapeutics. The chemical synthesis of oligosaccharides is limited because of the complex structure including the stereo-specificity and the regio-specificity of the products, whereas the enzymatic synthesis can control the stereo- and regio-specificity of the products. Glycoside hydrolases (glycosidase) are used to form glycosidic bonds through reverse hydrolysis or transglycosylation (especially, retaining glycosidase can catalyze the transfer of a glycosyl moiety from donor to acceptor). In this way they can be used to synthesize glycosides, but the yields are typically low because the product itself is a substrate for the enzyme and undergoes hydrolysis. Glycosynthase obtained from glycosidase by modifying catalytic nucleophile is capable of synthesizing oligosaccharide derivatives without hydrolysis of the product. Most of reported glycosynthases are β-glycosynthases derived from retaining type β-glycosidase, on the other hand, α-glycosynthase is just one derived from Schizosaccharomyces pombe retaining type α-glucosidase.
We tried to construct the α-type glycosynthase using thermostable α-glucosidase for efficient synthesis of α-type glycosides. At first, an α-glucosidase from thermophilic archaeon T. acidophilum DSM1728 was cloned and expressed in Escherichia coli, and efficiently purified using heat treatment and Ni-NTA affinity chromatography. The purified enzyme which has a 82.5 kDa protein showed strong maltose-hydrolyzing activity. Optimal activity was measured at 80℃ within a broad pH range from 5.0 to 6.0. The hydrolytic patterns of various α-type substrates revealed that the enzyme is typical of α-glucosidase (EC 3.2.1.20). Interestingly, aesculin (Ki = 4.3 μM) inhibited T. acidophilum α-glucosidase by mixed type fashion while acarbose (Ki = 3.0 μM) competitively inhibited the enzyme. The ability of synthesis α-type glycosides was examined using arbutin and maltose as an acceptor and a donor, respectively. Three transfer products were observed by thin-layer chromatography and recycling preparative high performance liquid chromatography. The yields of three transfer products were not high enough because they were re-hydrolyzed by α-glucosidase. To increase the yields of transfer products a glycosynthase was constructed by site-directed mutagenesis. Two approachs were accomplished to increase the yield of products with p-nitrophenyl-α-glucoside as an acceptor. First method was that using β-glucosyl fluoride as a substrate and second method was using sodium formate as an external nucleophiles with maltose as a normal substrate. The yield of transfer product was 18.5% when using first method, on the other hand, the yield was increased to 42.5% when using second method. As summarized our result, the method using sodium formate was efficient to increasing yield of transfer product.

목차 (Table of Contents)

• Ⅰ. 서 론 1
• Ⅱ. 실험 재료 및 방법 5
• 1. 실험 재료 5
• 1-1. 사용 균주 및 플라스미드 5
• 1-2. 효소와 시약 5
• 2. 실험 방법 7
• 2-1. α-glucosidase (aglA;Ta0298) 유전자의 클로닝과 발현 7
• 2-2. 재조합 효소의 정제 8
• 2-3. α-glucosidase 역가 측정 9
• 2-4. Kinetic parameters의 결정 10
• 2-5. 당전이 반응을 통한 배당체 합성 및 반응산물 분석 11
• 2-6. 돌연변이체의 구축 및 glycosynthase 변이체의 발현 12
• Ⅲ. 실험 결과 15
• 1. 재조합 α-glucosidase (AglA)의 발현과 정제 15
• 2. AglA의 활성에 미치는 pH와 온도의 영향 19
• 3. 기질특이성과 AglA의 가수분해 작용 특성 분석 22
• 4. AglA의 기질에 대한 kinetic parameters 22
• 5. 저해제에 대한 저해상수 결정 26
• 6. 당전이 반응을 통한 arbutin 유도체 생산 29
• 7. Glycosynthase 변이체의 발현 및 정제 32
• 8. Glycosynthase 변이체를 이용한 당전이 반응 32
• Ⅳ. 고 찰 37
• Ⅴ. 참고문헌 42
• Abstract 49