Title Page
ABSTRACT
Contents
I. INTRODUCTION 11
II. MATERIALS AND METHODS 17
2.1. Strains, plasmids and media 17
2.2. DNA manipulation and plasmid construction 20
2.3. Expression and purification of the recombinant proteins 24
2.4. Ulvan lyase and UGL complex assembly and CBM-based enzyme recycling 25
2.5. Ulvan extraction from green algae 26
2.6. Measurement of enzymatic activities 27
2.7. Effects of FaUL and UGL on the growth of L.casei in MRS medium 28
III. RESULTS AND DISCUSSION 29
3.1. Chimeric ulvan degrading enzyme design for enzyme complex assembly 29
3.2. Protein subunits for the construction ulvan-degrading complexes 32
3.3. Confirmation of assembly of ulvan enzyme complex using CBM purification 35
3.4. Effect of ulvan degrading enzyme complex 37
3.5. Effect of ulvan degradation products on the growth of L. casei 42
IV. CONCLUSION 45
REFERENCES 46
ABSTRACT IN KOREAN (국문 요약) 50
Table 1. Microbial strains and plasmids used in this study 18
Table 2. List of oligonucleotide primers used in this study. 21
Fig. 1. Chemical structure of the ulvan repeat disaccharide 12
Fig. 2. Schematic illustrations of ulvan depolymerization 15
Fig. 3. Strategies for generating cFaUL, cUGL, and mCbpA expression vectors. Images show the cFaUL (A), cUGL (B), and... 31
Fig. 4. Individual protein expression was confirmed using SDS-PAGE and Western blot of cFaUL, cUGL, and mCbpA. lanes: 1,... 33
Fig. 5. Ulvan depolymerase complex formed by purification using CBM. The assembly of the ulvan depolymerase complex was... 36
Fig. 6. Decomposition of ulvan extracted from Ulva powder using the constructed Ulvan lyase enzyme; (A) control (B) cFaUL Data show... 38
Fig. 7. Decomposition of ulvan extracted from Ulva biomass using the constructed Ulvan degrading enzyme; (A) cFaUL, (B)... 40
Fig. 8. L. casei growth using ulvan degradation products of the constructed enzyme complex; (A) control, (B) ulvan, (C)... 44