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Title Page
Contents
LIST OF ABBREVIATIONS 7
Abstract 8
Introduction 10
MATERIALS AND METHODS 13
Materials 13
Isolation of P450 BM3 variants 13
Method 14
1. Production of catalase 15
2. Voges-Proskauer reaction 15
3. Growth temperature 15
4. Hydrolysis of starch 15
5. Acid and gas from carbohydrates 16
6. Width of rod 1.0 ㎛ or greater 16
Polymerase chain reaction and cloning of P450 BM3 variants 17
Expression and purification of P450 BM3 variants 17
Distribution of hydroxylated products of fatty acids for P450 BM3 variants 18
Kinetic parameters of activities for fatty acids supported by BM3 variants 19
RESULTS 20
Isolation of BM3 variants 20
1. Production of catalase 20
2. Voges-Proskauer reaction 20
3. Growth temperature 24
4. Hydrolysis of starch 24
5. Acid and gas from carbohydrates 27
Relevant properties of the amino acids sequences of P450 BM3 variants. 27
Sequence homology and variation of P450 BM3 variants 27
Turnover numbers of fatty acids by P450 BM3 variants 38
Kinetic parameters of fatty acids by P450 BM3 variants 38
DISCUSSION 45
REFERENCES 48
초록 55
Table 1. Simplified key for the tentative identification of Bacillus megaterium. 14
Table 2. Relevant properties of Bacillus megaterium. 30
Table 3. Nucleotide sequence homology among P450 BM3 variants. 32
Table 4. Amino acid sequence homology among P450 BM3 variants. 33
Table 5. Sequence variations of P450 BM3 enzymes. 34
Table 6. Variation frequency of amino acid sequences of P450 BM3 enzymes. 37
Table 7. Distribution of hydroxylated products of fatty acids for P450 BM3 variants. 39
Table 8. Kinetic parameters of fatty acids by P450 BM3 variants. 43
Figure 1. Bubbles formation test by catalase using hydrogen peroxide. 21
Figure 2. Acetylmethylcarbinol reaction with Barritt’s reagent. 22
Figure 3. Voges-Proskauer reaction. 23
Figure 4. Condition for growable temperature. 25
Figure 5. Hydrolysis of starch using Gram’s iodine. 26
Figure 6. Carbohydrate fermentation test. 28
Figure 7. Comparison of distinct regions of 16S rRNA gene sequence (A) and ITS (B) from B. megaterium . 29
Figure 8. Fe2+·CO-difference spectra of P450 BM3 variants.(이미지참조) 31
Figure 9. Turnover numbers of fatty acids by P450 BM3 variants. 40
Figure 10. Kinetic parameters of lauric acid by P450 BM3 variants. 41
Figure 11. Kinetic parameters of myristic acid by P450 BM3 variants. 42
Figure 12. Turnover numbers of palmitic acid by P450 BM3 variants. 44
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