결합 친화력과 셀룰로스 분해 활성 향상을 위한 셀룰라아제에 : 탄수화물 결합모듈 융합효소의 구축

다국어 초록 (Multilingual Abstract)

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 has both endo- and exoglucanase activities were fused with two types of carbohydrate binding modules (CBM). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima xyn 10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50℃, while it increased to 60℃ for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (KM),for which KMdecreased from 104 mg/ml to 35.1–33.9 mg/ml for CMC, and from 115 mg/ml to 80.3–55.5 mg/ml for Avicel, respectively. The catalytic efficiency (kcat/KM) also increased from 4.80(mg/ml)-1sec-1 to 5.36–6.46 (mg/ml)-1sec-1 for CMC, and from 1.77 (mg/ml)-1sec-1 to 2.40–4.45 (mg/ml)-1sec-1 for Avicel, respectively, by addition of CBM3 and CBM9.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1 Cellulose 1
• 1.2 Chemical Composition, Structure and Morphology of Cellulose Fibers 1
• 1.3 Sources of Cellulase Enzymes 7
• 1.3.1 Fungal Enzyme Systems 7
• 1.3.2 Bacterial Enzyme Systems 7
• 1.4 General Classification of Cellulase Enzymes 9
• 1.4.1 Endocellulases 9
• 1.4.2 Exocellulases or cellobiohydrolases 9
• 1.4.3 Cellobiases or beta-glucosidases 10
• 2. Materials and Method 14
• 2.1 Bacterial strains, growth media and Plasmid 14
• 2.2 Construction of Fusion Enzyme Expression Vector 14
• 2.3 Expression and Purification 17
• 2.4 Cellulase Activity Assay 18
• 2.5 Biochemical Properties and Enzyme Kinetics 18
• 2.6 Analysis Method 19
• 2.7 Test of Cellulase Activity 20
• 2.8 Application of Fusion of GH5-CBM3-CBM9 in Cellulose Hydrolysis 20
• 3. Results 21
• 3.1 Production of Fusion Enzymes 21
• 3.2 Biochemical Properties of the Enzymes 27
• 3.3 Kinetics and Binding Abilities of the Enzymes 33
• 3.4 Visualization of Cellulase activity 35
• 3.5 Application of Fusion of GH5-CBM3-CBM9 in Cellulose Hydrolysis 37
• 4.Discussion 39
• References 43