Title Page
ABSTRACT
Contents
I. INTRODUCTION 13
II. MATERIALS AND METHODS 19
2.1. Strains and Media 19
2.2. Construction of recombinant hmCA and Cyn plasmids 22
2.3. Expression and purification of enzymes 27
2.4. SDS-PAGE and western blot analyses 28
2.5. Thermostability Test 29
2.6. Enzymatic Conversion of Carbon Dioxide 31
2.7. Enzyme assays 32
III. RESULTS AND DISCUSSION 36
3.1. Expression of subunits and the assembly of hmCA complex 36
3.2. Thermostability of cyclic and polymeric hmCA 39
3.3. Enzymatic Conversion of Carbon Dioxide 43
3.4. Design of recombinant cyanase and hmCA 45
3.5. Expression of subunits and the assembly of complexes 50
3.6. Binding effect of cyanase and hmCA on cyanate degradation 53
IV. CONCLUSION 56
REFERENCES 58
ABSTRACT IN KOREAN (국문 요약) 66
Table 1. List of microbial strains and plasmids used in this study 20
Table 2. List of primers used in this study 23
Table 3. Proportions of reaction samples tested in this study. 33
Table 4. Proportions of reaction samples tested in this study. 34
Fig. 1. Schematic diagram of cyanate detoxification of cyanase and carbonic anhydrase. Cyanase relies on HCOLSUB3LSUP- to convert the... 17
Fig. 2. Comparison of SDS-PAGE profiles and activities of recombinant enzymes. (A) Results of western blot after protein SDS-PAGE treatment.... 37
Fig. 3. Comparison of thermal stability of three types of hmCA. (A) In short-term reaction, wild hmCA shows a sharp decrease in recovery from... 40
Fig. 4. Comparison of the efficacy of converting CO₂ into CaCO₃ of three hmCAs. When the amount of synthesized CaCO₃ is calculated, polymeric... 44
Fig. 5. Analysis of primary PCR results of enzyme genes by agarose gel electrophoresis. 5 μL of PCR reaction is blended with 1 μL of 6X Loading... 46
Fig. 6. Analysis of final PCR results of recombinant genes by agarose gel electrophoresis. 5 μL of PCR reaction is blended with 1 μL of 6X Loading... 47
Fig. 7. Analysis of PCR results of recombinant genes by agarose gel electrophoresis. 5 μL of PCR reaction is blended with 1 μL of 6X Loading... 48
Fig. 8. SDS-PAGE profile of recombinant enzymes. It can be confirmed that all of the purified proteins form a band at the expected position, and... 51
Fig. 9. (A) Spy + Clop (B) Snoop + Clop (Cyanase-Tag) (C) Snoop + Clop (Tag-Cyanase) (D) Spy + Clop (Catcher-hm CA, Tag-Cyanase)... 54