ᾏὒ⏤᮶ Luteolibacter algae H18 ࡢ

(1)

ᾏ

ᾏὒ⏤᮶ Luteolibacter algae H18 ࡢ

ࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲࡟㛵ࡍࡿ◊✲

2019 ᖺ 7 ᭶

㛗ᑿ㐩ᙪ

(2)

┠

┠ḟ

ᗎㄽ ... 1

1-1. ࣇࢥ࢖ࢲࣥ࡟ࡘ࠸࡚ ... 1

1-2. ࣇࢥ࢖ࢲࣥศゎᚤ⏕≀࡟ࡘ࠸࡚ ... 7

1-3. ࣇࢥ࢖ࢲࣥศゎ㓝⣲࡟ࡘ࠸࡚ ... 10

1-4. ᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ ... 15

Luteolibacter algae H18 ࠿ࡽࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇

...

17

2-1. ⥴ゝ ... 17

2-2. ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ... 18

2-2-1. ᮦᩱ࠾ࡼࡧヨ⸆ ... 18

2-2-2. Luteolibacter algae H18ࡢᇵ㣴 ... 18

2-2-3. ↓⣽⬊ᢳฟᾮ (C.F.E.) ࡢㄪ〇 ... 19

2-2-4. ࢱࣥࣃࢡ㉁⃰ᗘࡢᐃ㔞 ... 20

2-2-5. SDS࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ㟁ẼὋື (SDS-PAGE) ... 20

2-2-6. F࢟ࢵࢺࢆ⏝࠸ࡓ㓝⣲άᛶࡢ ᐃ࡜㓑㓟⃰ᗘࡢᐃ㔞 ... 20

2-2-7. DEAE-Sepharose FF ࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇 ... 21

2-2-8. Phenyl-TOYOPEARL 650M࡟ࡼࡿ㓝⣲⢭〇 ... 21

2-2-9. Q-Sepharose FF ࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇 ... 22

2-2-10. Butyl-TOYOPEARL 650M ࡟ࡼࡿ㓝⣲⢭〇... 22

2-2-11. Mono Q^®HR 10/10 ࡟ࡼࡿ㓝⣲⢭〇... 22

2-2-12. Superose 6^®HR 10/30 ࡟ࡼࡿ㓝⣲⢭〇 ... 23

2-2-13. ⢭〇㓝⣲ࢱࣥࣃࢡ㉁ࡢࣉࣟࢸ࢔࣮ࢮᾘ໬࡜ Tricine SDS ࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤ ࣝ㟁ẼὋື (Tricine SDS-PAGE) ... 23

2-2-14. Nᮎ➃࠾ࡼࡧෆ㒊࢔࣑ࣀ㓟ࢩ࣮ࢡ࢚ࣥࢫゎᯒ ... 23

2-2-15. DNAᢳฟ ... 24

2-3. ⤖ᯝ ... 26

2-3-1. ኱㔞ᇵ㣴࡜ C.F.E. ࡢㄪ〇 ... 26

2-3-3. Phenyl-TOYOPEARL 650M࡟ࡼࡿ㓝⣲⢭〇 ... 27

2-3-4. Q-Sepharose FF ࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇 ... 28

2-3-5. Butyl-TOYOPEARL 650M ࡟ࡼࡿ㓝⣲⢭〇 ... 29

2-3-6. Mono Q^®HR 10/10 ࡟ࡼࡿ㓝⣲⢭〇 ... 30

2-3-7. Superose 6^®HR 10/30 ࡟ࡼࡿ㓝⣲⢭〇 ... 32

2-3-8. H18ᰴࢤࣀ࣒DNAࡢᢳฟ࡜ࢻࣛࣇࢺࢤࣀ࣒㓄ิゎᯒ ... 34

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2-3-9. ⢭〇㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิゎᯒ ... 35

2-4. ⪃ᐹ ... 38

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢࠊ␗✀Ⓨ⌧࠾ࡼࡧ㓝⣲ ໬Ꮫⓗᛶ㉁... 40

3-1. ⥴ゝ ... 40

3-2. ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ... 41

ࢥࣥࣆࢸࣥࢺࢭࣝࡢㄪ〇 ... 41

3-2-2. ࣉࣛࢫ࣑ࢻ༢㞳࡜ࣉࣛ࢖࣐࣮ࡢタィ... 42

3-2-3. PCRቑᖜ ... 42

3-2-4. ไ㝈㓝⣲ᾘ໬ ... 43

3-2-5. ࣛ࢖ࢤ࣮ࢩࣙࣥ ... 43

3-2-6. ⤌᥮࠼኱⭠⳦࡟ࡼࡿ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘᳨ウ ... 44

3-2-7. Ni Sepharose 6 Fast Flow ࡟ࡼࡿ㓝⣲⢭〇 ... 44

3-2-8. HRV 3C Protease ࡛ࡢࢱࢢ㓄ิࡢษ᩿ ... 45

3-2-9. ⮳㐺 pH ࡢ᳨ウ ... 45

3-2-10. ⮳㐺 ᗘࡢ᳨ウ ... 46

3-2-11. ⇕Ᏻᐃᛶࡢ᳨ウ ... 46

3-2-12. ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡿ㓑㓟ࠊ◲㓟ࡢᐃ㔞 ... 46

3-2-13. 㔠ᒓ࢖࢜ࣥࡢάᛶ࡬ࡢᙳ㡪᳨ウ ... 46

3-2-14. 㓝⣲㜼ᐖ๣ࡢάᛶ࡬ࡢᙳ㡪᳨ウ ... 47

3-2-15. ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ ... 47

3-2-16. ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㛵୚ ... 48

3-2-17. 㓝⣲ⓗ࡞⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡢㄪ〇 ... 49

3-3. ⤖ᯝ ... 50

3-3-1. fud ࡢPCRቑᖜ ... 50

3-3-2. E.coli BL21(DE3)/pCold I/fud ࡟ࡼࡿ┠ⓗࢱࣥࣃࢡ㉁⏕⏘᳨ウ ... 51

3-3-3. E.coli BL21(DE3)/pCold TF/fud ࡟ࡼࡿ┠ⓗࢱࣥࣃࢡ㉁⏕⏘᳨ウ ... 52

3-3-4. E.coli BL21(DE3)/pCold TF/fud ⏤᮶ C.F.E. ୰ࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ . 53 3-3-5. Ni Sepharose 6 Fast Flow ࡟ࡼࡿ㓝⣲⢭〇 (1 ᅇ┠) ... 54

3-3-6. HRV 3C Protease ࡛ࡢࢱࢢ㓄ิࡢษ᩿࡜Ni Sepharose 6 Fast Flow ࡟ࡼࡿ㓝⣲⢭〇 (2 ᅇ┠) ... 55

3-3-7. ⮳㐺 ᗘࠊ⮳㐺 pH ࡢ᳨ウ ... 56

3-3-8. ⇕Ᏻᐃᛶࡢ᳨ウ ... 57

3-3-9. 㔠ᒓ࢖࢜ࣥࠊ㓝⣲㜼ᐖ๣ࡢάᛶ࡬ࡢᙳ㡪᳨ウ ... 58

3-3-10. ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ ... 59

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3-3-11. ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿศゎࣉࣟࢭࢫ࡬ࡢ㛵୚࡟ࡘ࠸࡚ࡢ᳨ウ ... 59

3-3-12. 㓝⣲ⓗ࡞⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡢㄪ〇⤖ᯝ ... 61

3-4. ⪃ᐹ ... 63

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢゐ፹ṧᇶࡢண ... 66

4-1. ⥴ゝ ... 66

4-2. ᐇ㦂᪉ἲ ... 67

4-2-1. DNA ᧯స ... 67

4-2-2. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண ᵓ㐀ࣔࢹࣝ ... 67

4-2-3. ࣉࣛ࢖࣐࣮ࡢタィ ... 67

4-2-4. PCRቑᖜ ... 68

4-2-5. ࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ᳨ウ ... 68

4-2-6. ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ ... 69

4-3. ⤖ᯝ ... 70

4-3-1. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண ᵓ㐀ࣔࢹࣝࡢసᡂ ... 70

4-3-2. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢCᮎ➃ഃḞᦆࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿⓎ⌧ᰴࡢᵓ⠏ ... 73

4-3-3. ⤌ࡳ᥮࠼ᰴࡢⓎ⌧᳨ウ ... 74

4-3-4. ࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿάᛶ᳨ウ ... 75

4-3-5. ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ ... 76

4-4. ⪃ᐹ ... 77

Luteolibacter algae H18 ࠿ࡽࡢࣇࢥ࢖ࢲࢼ࣮ࢮࡢ⢭〇

...

78

5-1. ⥴ゝ ... 78

5-2. ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ... 80

5-2-1. Luteolibacter algae H18ࡢᇵ㣴࡜↓⣽⬊ᢳฟᾮ (C.F.E.) ࡢㄪ〇 ... 80

5-2-2. 㓝⣲άᛶ ᐃ ... 80

5-2-4. Phenyl-TOYOPEARL 650M ࡟ࡼࡿ㓝⣲⢭〇 ... 80

5-2-5. Resource Q ࣒࢝ࣛ࡟ࡼࡿ⢭〇 ... 81

5-3. ⤖ᯝ ... 82

5-3-1. ኱㔞ᇵ㣴࡜ C.F.E. ࡢ㓝⣲ㄪ〇 ... 82

5-3-3. Phenyl-TOYOPEARL 650M ࡟ࡼࡿ㓝⣲⢭〇 ... 83

5-3-4. Resource Q ࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇 ... 84

5-3-5. ⢭〇㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิゎᯒ ... 87

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5-4. ⪃ᐹ ... 89

ࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ⌧ࠊ㓝⣲໬Ꮫⓗᛶ㉁ ... 93

6-1. ⥴ゝ ... 93

6-2. ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ... 94

6-2-1. DNAᐇ㦂ࠊ⤌᥮࠼኱⭠⳦࡟ࡼࡿ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘᳨ウ ... 94

6-2-2. ࣉࣛ࢖࣐࣮ࡢタィ ... 94

6-2-3. PCRቑᖜ ... 94

6-2-4. Ni Sepharose 6 Fast Flow ࡜ Resource Q ࡟ࡼࡿ㓝⣲⢭〇 ... 95

6-2-5. Somogyi-Nelson ἲ࡟ࡼࡿ Fct114 ࡢ㓝⣲໬Ꮫⓗㅖᛶ㉁ࡢ᳨ウ ... 95

6-2-6. ⮳㐺 ᗘࡢ᳨ウ ... 97

6-2-7. ⇕Ᏻᐃᛶࡢ᳨ウ ... 97

6-2-8. ⮳㐺 pH ࡢ᳨ウ ... 97

6-2-9. pH Ᏻᐃᛶࡢ᳨ウ ... 97

6-2-10. 㔠ᒓ࢖࢜ࣥࡢάᛶ࡬ࡢᙳ㡪 ... 98

6-2-11. 㓝⣲㜼ᐖ๣ࡀάᛶ࡟ཬࡰࡍᙳ㡪᳨ウ ... 98

6-2-12. ᇶ㉁≉␗ᛶࡢ᳨ウ ... 98

6-2-13. ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢ࢔ࢭࢳࣝᇶࡢ㛵୚ ... 99

6-3. ⤖ᯝ ... 100

6-3-1. fct114ࡢpET-21aࠊpCold IࠊpCold TF ࣋ࢡࢱ࣮࡬ࡢࢡ࣮ࣟࢽࣥࢢ ... 100

6-3-2. E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114 ࡟ࡼࡿ┠ⓗࢱࣥ ࣃࢡ㉁⏕⏘᳨ウ ... 101

6-3-3. E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114⏤᮶C.F.E.୰ࡢ ࣇࢥ࢖ࢲࢼ࣮ࢮάᛶ ... 102

6-3-4. Ni Sepharose 6 FF࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇 ... 103

6-3-5. Resource Q࡟ࡼࡿ⢭〇 ... 104

6-3-6. ⮳㐺 ᗘࠊ⮳㐺 pH ࡢ᳨ウ ... 107

6-3-7. ⇕Ᏻᐃᛶࡢ᳨ウ ... 109

6-3-8. pHᏳᐃᛶࡢ᳨ウ ... 109

6-3-9. 㔠ᒓ࢖࢜ࣥࠊ㓝⣲㜼ᐖ๣ࡢάᛶ࡬ࡢᙳ㡪᳨ウ ... 110

6-3-10. ᇶ㉁≉␗ᛶࡢ᳨ウ ... 112

6-3-11. ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢ࢔ࢭࢳࣝᇶࡢ㛵୚ ... 112

6-4. ⪃ᐹ ... 114

⥲ᣓ

...

116

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ᘬᘬ⏝ᩥ⊩

...

119

ㅰ㎡ ... 129

◊✲ᴗ⦼ ... 130

(7)

1

ᗎㄽ

ࣇࢥ࢖ࢲࣥ࡟ࡘ࠸࡚

ከ⢾㢮ࡣ㠀ᖖ࡟㔜せ࡞ࣂ࢖࣐࢜ࢫ㈨※࡛࠶ࡾࠊࡑࡢ࡯࡜ࢇ࡝ࡣ᳜≀⏤᮶࡛࠶ࡿࠋ᳜≀ࡣࠊ 㝣⏕ࡢ᳜≀࡜ᾏ⏕ࡢ᳜≀࡟኱ู࡛ࡁࠊ㝣⏕ࡢ᳜≀࠿ࡽࡢከ⢾㢮࡜ࡋ࡚ࡣࢭ࣮ࣝࣟࢫࠊ࣑࣊ࢭ

࣮ࣝࣟࢫࠊࣜࢢࢽࣥࡀ୺࡜ࡋ࡚฼⏝ࡉࢀ࡚ࡁ࡚࠸ࡿࠋࡋ࠿ࡋࠊ㝣⏕ࡢ᳜≀࡜ẚ㍑ࡋࡓሙྜࠊ ᾏ⏕ࡢ᳜≀࠿ࡽࡢከ⢾㢮ࡣ࡯࡜ࢇ࡝฼⏝ࡉࢀ࡚࠸࡞࠸ࠋᅄ㠃⎔ᾏࡢ᪥ᮏ࡛ࡣከ⢾㢮ࢆⴭ㔞

ྵ᭷ࡍࡿᾏ⸴ࡢ⏕⫱ࡀㄆࡵࡽࢀࡿࠋᾏ⸴㈨※ࡣ↓ᑾⶶ࡜⪃࠼ࡽࢀࡀࡕ࡛࠶ࡿࡀࠊࡇࢀࡣㄗࡾ

࡛࠶ࡿࠋ࡞ࡐ࡞ࡽࠊᾏ⸴ࡢ⏕⫱ࡋ࡚࠸ࡿሙᡤࡣ⸴ሙ࡜࿧ࡤࢀࡿ᭷⏝㨶௓㢮ࡢ᳇ᜥሙ࡜⪃࠼ࡽ

ࢀ࡚࠾ࡾࠊಖㆤࠊ㐀ᡂࡀ✚ᴟⓗ࡟⾜ࢃࢀ࡚࠸ࡿࠋࡇࡢࡼ࠺࡞⸴ሙࢆᵓᡂࡋ࡚࠸ࡿᾏ⸴ࢆ᥇㞟 ࡍࡿࡇ࡜ࡣỈ⏘ᴗ㛵ಀ⪅ࡢ⌮ゎࡀᚓࡽࢀ࡞࠸ࡢࡀ⌧≧࡛࠶ࡾࠊ࠶ࡿ≉ᐃࡢᾏ⸴ࢆࡑࡢࡲࡲ

㣗⏝࡜ࡋ࡚⏝࠸࡚࠸ࡿ௨እࠊࡑࡢ࡯࡜ࢇ࡝ࡣ฼⏝ࡉࢀ࡚࠸࡞࠸ࡢ࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ㏆

ᖺࠊỈ⏘ᴗ⏺࡛ࡣࠊ࠿ࡘ࡚⋓ࢀ࡚࠸ࡓࡶࡢࡀ⋓ࢀ࡞࠸ࠊ࠿ࡘ࡚⫱ࡗࡓࡶࡢࡀ⫱ࡓ࡞࠸ࠊ࡜࠸

࠺ࡼ࠺࡞⎔ቃࡢኚ໬࡟ࡼࡾỈ⏘㈨※ࡢ㣗ရ௨እ࡛ࡢ⏝㏵ࡢ㛤Ⓨࡀὀ┠ࡉࢀ࡚࠸ࡿࠋୡ⏺ⓗ

࡞ほⅬ࠿ࡽࡍࡿ࡜ࠊ୍㒊ࡢᾏ⸴ࡀᕤᴗཎᩱ࡜ࡋ࡚⏝࠸ࡽࢀ࡚࠸ࡿࠋ౛࠼ࡤࠊLessoniaᒓࡢ〓

⸴࠿ࡽ࢔ࣝࢠࣥ㓟ࡀ〇㐀ࡉࢀ࡚࠸ࡿࠋ࢔ࣝࢠࣥ㓟ࡣ㧗࠸⢓ᛶ࡜✀ࠎࡢ㔠ᒓሷ࡜≉ᚩࡢ࠶ࡿ

ࢤࣝࢆసࡿࡓࡵ་⒪ࡸ㣗ရࡢศ㔝࡛ࠊᗈࡃ⏝࠸ࡽࢀ࡚࠾ࡾࠊୡ⏺࡛ࡢ⏕⏘㔞ࡣᖺ㛫⣙20000 ࢺࣥ௨ୖ࡜᥎ᐃࡉࢀࡿࠋ᪥ᮏ࡛ࡣࠊ1 ♫ࡀ⣙ 1500 ࢺࣥ⛬〇㐀ࡋ࡚࠸ࡿ [1]ࠋ〓⸴࡟ྵࡲࢀ

ࡿከ⢾㢮࡜ࡋ࡚ࡣࠊ࢔ࣝࢠࣥ㓟௨እ࡟ࠊ㈓ⶶከ⢾㢮࡛࠶ࡿ࣑ࣛࢼࣛࣥࠊ⣽⬊ቨከ⢾㢮࡛࠶ࡿ

ࢭ࣮ࣝࣟࢫࠊࣇࢥ࢖ࢲࣥ࡞࡝ࡀ▱ࡽࢀ࡚࠸ࡿࠋ୰࡛ࡶࣇࢥ࢖ࢲࣥࡣࠊ〓⸴㢮 (࢜࢟ࢼ࣡ࣔࢬ ࢡࠊ࢞ࢦ࣓ࢥࣥࣈࠊ࣓࢝ࣈ) ࡢᡂ⇍ࡋࡓᡂయ࡟ྵࡲࢀࡿ◲㓟໬ከ⢾࡛࠶ࡿ [2]ࠋࣇࢥ࢖ࢲࣥ

ࡣࠊࣇࢥ࣮ࢫࡀD-1, 3⤖ྜ࡛㐃⤖ࡋࡓIᆺ࡜ࠊD-1, 3࠾ࡼࡧD-1, 4⤖ྜࢆ஺஫࡟⧞ࡾ㏉ࡍII

(8)

2

ᆺࡢ 2 ✀㢮ࡀ࠶ࡿ࡜ࡉࢀࠊࡇࢀࡽࡢᇶᮏᵓ㐀௨እ࡟ࡶഃ㙐ࡸᯞศ࠿ࢀᵓ㐀ࢆ᭷ࡍࡿ」㞧࡞

࣊ࢸࣟ◲㓟໬ከ⢾࡛࠶ࡿ [3]ࠋࡉࡽ࡟ࠊ〓⸴ࡢ✀㢮࡟ࡼࡗ࡚ྵ᭷ࡉࢀࡿࣇࢥ࢖ࢲࣥࡣࠊᵓᡂ

⢾ࠊ◲㓟໬ᗘࢆࡣࡌࡵ࡜ࡍࡿ໬Ꮫᵓ㐀࠾ࡼࡧࠊᶵ⬟ᛶࡀ኱ࡁࡃ␗࡞ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ

[4]ࠋ

ࣇࢥ࢖ࢲࣥࡣࠊ1913 ᖺ࡟ࢫ࢙࣮࢘ࢹࣥࡢ࢘ࣉࢧࣛ኱Ꮫࡢ Kylin ࡽ࡟ࡼࡗ࡚ึࡵ࡚ሗ࿌ࡉ

ࢀ [5]ࠊࣇࢥ࣮ࢫࢆ୺ᵓᡂ⢾࡜ࡋ࡚◲㓟ᇶࡸ࢘ࣟࣥ㓟ṧᇶࡀ⤖ྜࡋࡓከ⢾㢮࡜ᐃ⩏ࡉࢀࡿࠋ

ࡲࡓࠊ୍⯡ⓗ࡟౑⏝ࡉࢀࡿࣇࢥ࢖ࢲࣥ࡜࠸࠺ྡ⛠ࡣࠊྠ୍ࡢᵓ㐀ࡢ≀㉁࡟௜ࡅࡽࢀࡓࡶࡢ࡛

ࡣ࡞ࡃࠊ୺ᡂศࡀࣇࢥ࣮ࢫ࡛࠶ࡿከ⢾㢮ࡢ⥲⛠࡛࠶ࡿࠋᚑࡗ࡚ࠊࣇࢥ࣮ࢫ௨እ࡟ᵓᡂ⢾࡜ࡋ

࡚ࠊ࢞ࣛࢡࢺ࣮ࢫࠊ࣐ࣥࣀ࣮ࢫࠊ࢟ࢩ࣮ࣟࢫ࡞࡝ࡀᏑᅾࡍࡿሙྜࡀ࠶ࡿࠋ୰࡛ࡶࠊᚋ㏙ࡍࡿ

ࡼ࠺࡟ࠊ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢᵓ㐀ࡣヲ⣽࡟◊✲ࡉࢀ࡚࠾ࡾࠊࢢࣝࢡࣟࣥ㓟ࢆྵࡴ

U-ࣇࢥ࢖ࢲࣥࠊ◲㓟໬ࣇࢥ࣮ࢫࡢࡳ࠿ࡽ࡞ࡿF-ࣇࢥ࢖ࢲࣥࠊ࢞ࣛࢡࢺ࣮ࢫࢆྵࡴG-ࣇࢥ࢖

ࢲࣥࢆྵ᭷ࡋ࡚࠸ࡿ࡜ࡉࢀ࡚࠸ࡿ [6, 7]ࠋࡑࡋ࡚ࠊ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࡢU-ࣇࢥ࢖ࢲࣥࡀࡀࢇ

⣽⬊ࢆ࢔࣏ࢺ࣮ࢩࢫࡉࡏࡿస⏝ࡀぢฟࡉࢀ[8]ࠊࡇࡢሗ࿌௨㝆ࠊࣇࢥ࢖ࢲࣥࡢ⏕⌮άᛶࡀ୍

㌍ὀ┠ࢆᾎࡧࡿࡼ࠺࡟࡞ࡗࡓࠋ

ᾏ⸴⏤᮶ࡢࣇࢥ࢖ࢲࣥࡢ≉ᛶࡣᗈ⠊࡟◊✲ࡉࢀ࡚࠾ࡾࠊ㏆ᖺࠊ࢖ࣥࣇ࢚ࣝࣥࢨ࢘࢕ࣝࢫ➼

࡟ᑐࡍࡿᢠ࢘࢖ࣝࢫస⏝ [9]ࠊᢠ࢞ࣥస⏝ [10, 11]ࠊᢠจ⾑స⏝ [12, 13, 14]࡜࠸ࡗࡓ⏕⌮ά ᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊ࣓ࢱ࣎ࣜࢵࢡࢩࣥࢻ࣮࣒ࣟ࡟ᑐࡍࡿࣇࢥ࢖ࢲࣥࡢ⏕⌮άᛶࡶ㠀 ᖖ࡟ከࡃሗ࿌ࡉࢀ࡚࠾ࡾࠊ㧗⬡⫫㣗ㄏⓎ⫧‶࣐࢘ࢫ࡟࠾ࡅࡿᢠ⫧‶ࠊయ㔜ቑຍᢚไ࠾ࡼࡧ⬡

⫫㔞ࡢῶᑡ [15]ࠊᢠ㧗⬡⾑⑕ຠᯝ [16]ࠊ㧗⬡⫫㣗ㄏⓎᛶࡢࢢࣝࢥ࣮ࢫ୙⪏ᛶ࠾ࡼࡧ࢖ࣥࢫ

ࣜࣥ᢬ᢠᛶࢆᨵၿࡍࡿᢠ⢾ᒀ⑓స⏝ [17] ࡉࡽ࡟ࡣࠊᢠ㧗⾑ᅽຠᯝ [18, 19] ࡞࡝ࡀሗ࿌ࡉࢀ

࡚࠸ࡿࠋࡇࡢᢠ㧗⾑ᅽຠᯝࡢせᅉ࡜࡞ࡿ࣓࢝ࢽࢬ࣒ࡢ୍ࡘ࡜ࡋ࡚ࠊ⾑₢୰ࡢ࢔ࣥࢪ࢜ࢸࣥࢩ

ࣥIIࡢ⏘⏕ࢆ㜼ᐖࡍࡿࡇ࡜ࡀᥦၐࡉࢀ࡚࠸ࡿࠋ࢔ࣥࢪ࢜ࢸࣥࢩࣥIIࡣࡑࡢཷᐜయ࡟⤖ྜࡋ

࡚⾑⟶ࢆ཰⦰ࡉࡏࠊ⾑ᅽࢆୖࡆࡿస⏝ࢆ♧ࡍࠋࣇࢥ࢖ࢲࣥࡣࠊ࢔ࣥࢪ࢜ࢸࣥࢩࣥ II ࡢ⏘⏕

ࢆ㜼ᐖࡋࠊࡑࡢ⤖ᯝࠊཷᐜయ࡬ࡢ⤖ྜࡍࡿ㔞ࢆῶᑡࡉࡏࡿࡇ࡜࡛ࠊ⾑ᅽࡢୖ᪼ࢆᢚไࡋ࡚࠸

(9)

3

ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ [20]ࠋ୍᪉ࠊ࣐࢘ࢫ࡟⤒ཱྀᢞ୚ࡋࡓࣇࢥ࢖ࢲࣥࡀࠊච␿㞀ᐖ࡟ࡼࡗ࡚ᘬ ࡁ㉳ࡇࡉࢀࡿ࢔ࣞࣝࢠ࣮⑕≧ࢆᢚไࡍࡿ࡜࠸࠺ᢠ࢔ࣞࣝࢠ࣮స⏝ࡶሗ࿌ࡉࢀ࡚࠸ࡿ [21- 23]ࠋ௒ࡲ࡛࡟ᵝࠎ࡞ᢠ࢔ࣞࣝࢠ࣮స⏝࡟ࡘ࠸࡚ࡢ▱ぢࡀ࠶ࡿࡀࠊ⤒ཱྀᢞ୚ࡋࡓሙྜࡢᢠ࢔

ࣞࣝࢠ࣮࣓࢝ࢽࢬ࣒࡟㛵ࡍࡿሗ࿌౛ࡣᑡ࡞࠸ࠋࡇࡢሙྜࠊࣇࢥ࢖ࢲࣥࡣࠊ⭠࠿ࡽࡢ྾཰ࢆ࡯

࡜ࢇ࡝࠶ࡿ࠸ࡣ඲ࡃ♧ࡉ࡞࠸ࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡢ࡛ [14, 24, 25]ࠊ⭠⟶ච␿ࢆ㈿ά໬ࡍ

ࡿࡇ࡜࡟ࡼࡾࠊ࢔ࣞࣝࢠ࣮࡟ᑐࡋ࡚ከᵝ࡞άᛶࢆ♧ࡍ࡜᥎ ࡉࢀࡿࠋࡉࡽ࡟ࠊࣇࢥ࣮ࢫ࡜◲

㓟໬ࣇࢥ࣮ࢫ࠿ࡽ࡞ࡿ࣐ࢥࣥࣈ (Saccharina japonica) ⏤᮶ࡢ F-ࣇࢥ࢖ࢲࣥࡀࠊᢠ⾑ᰦస⏝

ࢆ♧ࡍࡶࡢࡢࠊ᏶඲࡟྾཰ࡉࢀࡿࡇ࡜࡞ࡃ௦ㅰࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [14]ࠋ ࣇࢥ࢖ࢲࣥࢆྵ᭷ࡍࡿ〓⸴ࡢ୍✀࡛࠶ࡿࣔࢬࢡ㢮ࡣ〓⸴⥙ࢼ࣐࢞ࢶࣔ┠ࡢᾏ⸴࡛ࠊᯞศ

࠿ࢀࡋࡓ⣽࠸⣒≧࡛⢓ᾮ࡟ᐩࢇࡔయࢆᣢࡕࠊྂࡃ࠿ࡽ㣗⏝࡜ࡉࢀ࡚ࡁࡓࠋ୺࡞ࣔࢬࢡ㢮ࡣࠊ

࢜࢟ࢼ࣡ࣔࢬࢡ (Cladosiphon okamuranus)ࠊࣔࢬࢡ (Nemacystus decipines)ࠊࣇࢺࣔࢬࢡ (Tinocladia crassa)ࠊ࢟ࢩࣗ࢘ࣔࢬࢡ (Cladosiphon umezakii) ࡛࠶ࡿࠋࡑࡢ୰࡛ࠊ࢜࢟ࢼ࣡ࣔ

ࢬࢡࡣࠊ୺࡟᪥ᮏ࡛㣗ࡉࢀࠊࡶࡎࡃ㓑࡜ࡋ࡚ᩱ⌮ࡉࢀࡿሙྜࡀከࡃࠊࣔࢬࢡ㢮ࡢ୰࡛᭱ࡶ⏕

⏘㔞ࡀከ࠸࡜ྠ᫬࡟ࠊࣇࢥ࢖ࢲࣥࡢཎᩱ࡜ࡋ࡚ࡶ᭱ࡶከࡃ฼⏝ࡉࢀ࡚࠸ࡿࠋ࢜࢟ࢼ࣡ࣔࢬࢡ ࡣࠊ዆⨾኱ᓥࢆ໭㝈ࠊඵ㔜ᓥㅖᓥࢆ༡㝈࡜ࡍࡿ༡すㅖᓥᅛ᭷ࡢ㣗⏝ᾏ⸴࡛࠶ࡾࠊ1970 ᖺ௦

࡟㣴Ṫᢏ⾡ࡀ☜❧ࡉࢀࡓἈ⦖┴ࡢ୺せỈ⏘≀࡛ࠊᅜෆ⏕⏘ࡢ 90%௨ୖࢆ༨ࡵࡿ [26]ࠋ࢜࢟

ࢼ࣡ࣔࢬ⏤᮶ࣇࢥ࢖ࢲࣥࡢᵓ㐀ࡣ 1999ᖺ࡟㛗ᒸࡽ࡟ࡼࡗ࡚᥎ᐃ໬Ꮫᵓ㐀ࡀⓎ⾲ࡉࢀ [27]ࠊ 2003 ᖺ࡟㓇஭ࠊຍ⸨ࡽࡣ࢜࢟ࢼ࣡ࣔࢬ⏤᮶ࣇࢥ࢖ࢲࣥࢆ≉␗ⓗ࡟ศゎࡍࡿ㓝⣲ࢆᾏὒ⣽⳦

࠿ࡽ᪂ࡋࡃⓎぢࡋࠊࡇࢀࢆ฼⏝ࡋ࡚࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥࡢ୺࡞໬Ꮫᵓ㐀ࢆỴᐃ

ࡋࡓ [28]ࠋࡑࡢᵓ㐀ࡣࠊࣇࢥ࣮ࢫࡢD-1, 3⤖ྜࡢ⧞ࡾ㏉ࡋᵓ㐀ࢆᇶᮏ࡟ࠊࣇࢥ࣮ࢫࡢ4఩Ⅳ

⣲ࡢ⣙60%࡟◲㓟ᇶࡀ⤖ྜࡋࠊࣇࢥ࣮ࢫ6ศᏊ࡟1ಶࡢ๭ྜ࡛ࢢࣝࢡࣟࣥ㓟ࡀD-1, 2⤖ྜࡋ ࡓᵓ㐀࡛࠶ࡿ (ᅗ 1-1) [27, 28]ࠋࡉࡽ࡟2009ᖺ࡟⏣ᖾࡽ࡟ࡼࡗ࡚ࠊ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ

࢖ࢲࣥ࡟ࡣࠊ࢔ࢭࢳࣝᇶࡀྵࡲࢀ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡓ (ᅗ 1-2) [29]ࠋ

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4

ᅗ1-1. ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥࡢ᥎ᐃᵓ㐀ᘧ-1 [27, 28]

ᅗ 1-2. ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥࡢ᥎ᐃᵓ㐀ᘧ-2 [29]

࢜࢟ࢼ࣡ࣔࢬࢡ௨እ࡟ࣇࢥ࢖ࢲࣥࢆྵ᭷ࡍࡿ〓⸴࡜ࡋ࡚◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࡢࡀࠊ࢞

ࢦ࣓ࢥࣥࣈ(Kjellmaniella crassifolia)࡛࠶ࡿࠋ࢞ࢦ࣓ࢥࣥࣈࡣࠊ〓⸴㢮ࢺࣟࣟࢥࣥࣈᒓࡢ㣗

⏝ࡢᾏ⸴࡛࠶ࡾࠊࡑࡢ⏕⫱ࡣ໭ᾏ㐨ภ㤋࿘㎶ࡢᆅᇦ࡟㝈ࡽࢀ࡚࠸ࡿࠋ໭࠿ࡽࡢᐮὶࡀ༡࠿

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5

ࡽࡢᬮὶࡢᨭὶ࡜஺ᕪࡍࡿ㝈ࡽࢀࡓᾏᇦࡢỈ῝10࣓࣮ࢺࣝ௨ୖࡢ࡜ࡇࢁ࡟⏕⫱ࡍࡿᕼᑡ

࡞ࢥࣥࣈ࡛࠶ࡾࠊ⢓ࡾࡀᙉ࠸ࡇ࡜ࡀ≉ᚩ࡛࠶ࡿࠋ࢞ࢦ࣓ࡢྡ๓ࡣࠊࢥࣥࣈ⾲㠃ࡢฝพᶍᵝ ࡢ⡲┠ (࠿ࡈࡵ) ࡟⏤᮶ࡋ࡚࠾ࡾࠊ࡜ࢁࢁ᪻ᕸࡸᯇ๓ₕࡅࡢᮦᩱ࡜ࡋ࡚㛗ࡃ㣗ࡉࢀ࡚ࡁࡓ [26]ࠋ࢞ࢦ࣓ࢥࣥࣈࡣࠊࢥࣥࣈࡢ୰࡛ࡶࣇࢥ࢖ࢲࣥࢆ㇏ᐩ࡟ྵࡳࠊඛ࡟ࡶ㏙࡭ࡓࡼ࠺࡟ࠊ

◲㓟໬ࣇࢥ࣮ࢫࢆ୺ᡂศ࡜ࡍࡿF-ࣇࢥ࢖ࢲࣥࠊࢢࣝࢡࣟࣥ㓟ࢆྵࡴU-ࣇࢥ࢖ࢲࣥࠊ࢞ࣛࢡ ࢺ࣮ࢫࢆྵࡴG-ࣇࢥ࢖ࢲࣥࡢ3✀㢮ࡢ໬Ꮫᵓ㐀ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ (ᅗ1-3) [28, 30- 32]ࠋ

ᅗ1-3. ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢᵓ㐀ᘧ [31, 32]

௨ୗࡢ⾲࡟ࡣࠊྛ✀ᾏ⸴ࡢࣇࢥ࢖ࢲࣥྵ᭷㔞 (⾲ 1-1) ࠾ࡼࡧ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ

࢖ࢲࣥ࡜࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢᵓᡂ⢾ࠊ◲㓟᰿ࠊ࢝ࢳ࢜ࣥࡢศᯒ౛ (⾲ 1-2) ࡟ 㛵ࡍࡿ᝟ሗࢆグ㍕ࡋࡓࠋ

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6

⾲ 1-1. ྛ✀ᾏ⸴ࡢࣇࢥ࢖ࢲࣥྵ᭷㔞 [33]

〓⸴㢮 ࣇࢥ࢖ࢲࣥྵ᭷㔞 (g / kg ஝⇱㔜㔞)

࢜࢟ࢼ࣡ࣔࢬࢡ 250

࢞ࢦ࣓ࢥࣥࣈ 40

࣓࣡࢝ (⬊Ꮚⴥ㒊 (࣓

࢝ࣈ) )

80

ࣔࢬࢡ 250

࣐ࢥࣥࣈ 15

࢔࣓ࣛ 70

Ecklonia maxima 40

Leossonia nigrescens 46

Fucus vesiculosus 70

Ascophyllum nodosum 110

⾲ 1-2. ࢜࢟ࢼ࣡ࣔࢬࢡ࠾ࡼࡧ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢᵓᡂ⢾ࠊ◲㓟᰿ࠊ࢝ࢳ࢜ࣥ

ࡢศᯒ್ [26]

ᵓᡂᡂศ ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥ

ศᯒ್ (%)

࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥ

ศᯒ್ (%)

ࣇࢥ࣮ࢫ 46.8 37.7

࢞ࣛࢡࢺ࣮ࢫ - 1.3

ࢢࣝࢡࣟࣥ㓟 8.7 2.5

࣐ࣥࣀ࣮ࢫ - 1.8

࣒ࣛࣀ࣮ࢫ - < 0.5

࢟ࢩ࣮ࣟࢫ - 0.5

◲㓟᰿ 16.0 33.2

࢝ࢳ࢜ࣥ 5.7 7.2

ྜ ィ 77.2 84.2

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7

ࣇࢥ࢖ࢲࣥศゎᚤ⏕≀࡟ࡘ࠸࡚

ࣇࢥ࢖ࢲࣥࡣࠊ㣗≀⧄⥔ࡢ୍✀࡛࠶ࡾࠊ࢔ࣝࢠࣥ㓟ࠊᐮኳࠊ࢝ࣛࢠ࣮ࢼࣥ࡜ྠࡌࡼ࠺࡟ࣄ

ࢺࡢయෆ࡛ࡣᾘ໬࣭྾཰ࡉࢀࡎࠊࡑࡢࡲࡲࢫࢺ࣮ࣞࢺ࡟యእ࡟᤼ฟࡉࢀࡿࠋࡑࡢ୍᪉࡛ࠊ࠶

ࡿ✀ࡢỈ⏘⏕≀ࡸࠊᾏὒ⎔ቃ୰࡟Ꮡᅾࡍࡿᚤ⏕≀ࡣࣇࢥ࢖ࢲࣥࢆศゎࡍࡿ㓝⣲ࢆ᭷ࡋ࡚࠸

ࡿࠋ1996 ᖺ࡟ึࡵ࡚ࣇࢥ࢖ࢲࣥࡢ໬Ꮫᵓ㐀ࡀ᫂ࡽ࠿࡟ࡉࢀࡓ㝿ࡶࠊࡇࢀࡽ㓝⣲ࡀ⏝࠸ࡽࢀ

࡚࠸ࡿ [30]ࠋࡇࡢࡼ࠺࡞㓝⣲ࢆ⏕⏘ࡍࡿࣇࢥ࢖ࢲࣥ㈨໬ᛶᚤ⏕≀࡟㛵ࡍࡿ◊✲࡟ࡘ࠸࡚ࡣࠊ 1959ᖺ࡟ึࡵ࡚Yapheࡽ࡟ࡼࡗ࡚ሗ࿌ࡉࢀࡓ [34]ࠋࡇࡢሗ࿌ࡣࠊ࢝ࣛࢠ࣮ࢼࣥศゎ㓝⣲⏕

⏘⳦࡜ࡋ࡚༢㞳ࡉࢀࡓ Pseudomonasᒓ2ᰴࡀࠊ⳦యእ࡟ࣇࢥ࢖ࢲࣥศゎ㓝⣲ࢆ⏕⏘ࡍࡿ࡜

࠸࠺ࡶࡢ࡛࠶ࡗࡓ [34]ࠋࡑࡢᚋࠊ1967ᖺ࡟Thanassi ࡽ࡟ࡼࡗ࡚ࠊ↓⬨᳝ື≀࡛࠶ࡿ Haliotus ᒓ (ࢼ࣐ࢥ) ࡢ⫢⮅୰࠿ࡽࡢ㓝⣲⢭〇࡟㛵ࡍࡿ◊✲ࡢ୰࡛ࠊࣇࢥ࢖ࢲࣥ࠿ࡽ◲㓟ሷࢆ㐟㞳ࡉ ࡏࡎ࡟D-1, 2-L-ࣇࢥࢩࢻ⤖ྜࢆ࢚ࣥࢻᆺ࡛ຍỈศゎࡍࡿ㓝⣲ࠊࣇࢥ࢖ࢲࢼ࣮ࢮ (Fucoidanase, EC 3. 2. 1. 44. ) ࡢᏑᅾࡀሗ࿌ࡉࢀ࡚࠸ࡿ [35]ࠋࡉࡽ࡟ࠊ1992ᖺ࡟ྂᕝࡽ࡟ࡼࡗ࡚ࠊVibrio ᒓ 5ᰴ࠿ࡽࠊ3✀ࡢࣇࢥ࢖ࢲࢼ࣮ࢮࡀ༢㞳ࡉࢀࡓ [36]ࠋ

ࣇࢥ࢖ࢲࣥศゎ㓝⣲࡟ࡘ࠸࡚ࠊ௒ࡲ࡛࡟᭱ࡶ⢭ຊⓗ࡟◊✲ࢆ㐍ࡵࡓࡢࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡ

࠾ࡼࡧ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢ໬Ꮫᵓ㐀ࢆ᫂ࡽ࠿࡟ࡋࡓ㓇஭ࡽࡢࢢ࣮ࣝࣉ࡛࠶ࡿࠋ ᙼࡽࡣ࢞ࢦ࣓ࢥࣥࣈ࠿ࡽࣇࢥ࢖ࢲࣥࢆ஝⇱㔜㔞࡛3.8 %ࡢ཰⋡࡛ㄪ〇ࡋࠊࡇࢀࢆ⏝࠸࡚ࠊ࢞

ࢦ࣓ࣇࢥ࢖ࢲࣥࢆศゎࡍࡿᾏὒᚤ⏕≀ࡢࢫࢡ࣮ࣜࢽࣥࢢࢆ⾜ࡗࡓ⤖ᯝࠊFlavobacterium࡟ᒓ

ࡋࠊ16SrDNA㓄ิ࠿ࡽᬻᐃⓗ࡟࿨ྡࡋࡓFucobacter marina SA-0082ࢆ༢㞳ࡋࡓ [37]ࠋࡇࡢ

ᰴࡣࠊ࣓࣡࢝ࡸLessonia nigrescens⏤᮶ࡢࣇࢥ࢖ࢲࣥࡶ㒊ศⓗ࡟฼⏝ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚

࠸ࡿ [37]ࠋࡲࡓ㓇஭ࡽࡣࠊSA-0082 ᰴ௨እ࡟ Fucophilus fucoidanolyticus SI-1234 [38]ࠊ

Alteromonas SN-1009 [32] ࡜࿨ྡࡋࡓࣇࢥ࢖ࢲࣥศゎ⳦ᰴࡶ༢㞳ࡋ࡚࠾ࡾࠊ≉࡟๓⪅ࡣ⣽⳦

ศ㢮ୖࠊVerrucomicrobia 㛛࡜࠸࠺௒ࡲ࡛࡯࡜ࢇ࡝ᇵ㣴ᐇ⦼ࡢ࡞࠸㛛࡟ᒓࡍࡿᚤ⏕≀࡛࠶ࡿ

ࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋࡑࡢᚋࠊ 2000ᖺ࡟ Bakuninaࡽ࡟ࡼࡗ࡚ Cytophaga ᒓࠊAlteromonas

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8

ᒓࠊPseudoalteromonas ᒓ⣽⳦ࡀࣇࢥ࢖ࢲࢼ࣮ࢮࢆ᭷ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿ࡉࢀࡓ [39]ࠋ௨㝆ࠊ ከࡃࡢ◊✲⪅ࡽ࡟ࡼࡗ࡚ࣇࢥ࢖ࢲࣥ㈨໬ᛶ⣽⳦ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [40]ࠋࣇࢥ࢖ࢲࣥ㈨໬⬟

ࡢ࠶ࡿ⳦㢮ࠊ↓⬨᳝ື≀࡜ྜࢃࡏ᪤࡟ሗ࿌ࡉࢀ࡚࠸ࡿࣇࢥ࢖ࢲࣥ㈨໬ᛶ⣽⳦࡜ࡑࡢᇶ㉁࡜

࡞ࡿࣇࢥ࢖ࢲࣥࢆ᭷ࡍࡿᾏ⸴ྡࢆ⾲ 1-3 ࡟ࡲ࡜ࡵࡓࠋ

⾲ 1-3. ࣇࢥ࢖ࢲࣥ㈨໬ᛶᚤ⏕≀࡜ᇶ㉁࡜࡞ࡿࣇࢥ࢖ࢲࣥࡢศ㞳※

Organism Origin of fucoidan References

Bacteria

Vibrio sp. No. 5 Kjellmaniella crassifolia Furukawa et al. (1992) [36]

Pseudoalteromonas citrea KMM3296, KMM3298

Fucus evanescens

Chorda filum Bakunina et al. (2002) [41]

Fucobacter marina SA-0082 K. crassifolia Sakai et al. (2002) [37]

Fucophilus fucoidanolyticus SI-1234

C. okamuranus Sakai et al. (2003) [38]

Alteromonas sp. SN-1009 K. crassifolia Sakai et al. (2004) [32]

Mariniflexile fucanivorans SW5^T

Pelvetia canaliculata Descamps et al. (2006) [42]

Sphingomonas paucimobilis PF-1

Undaria pinnatifida

Kim et al. (2008) [43]

Flavobacterium sp. F31 C. okamuranus Ohshiro et al. (2010) [44]

Flavobacteriaceae CZ1127 Acaudina molpadioides Chang et al. (2010) [45]

Luteolibacter algae H18 C. okamuranus Ohshiro et al. (2012) [46]

Formosa algae KM3553^T F. evanescens Silchenko et al. (2013) [47]

Invertebrates

Haliotis sp. F. gardneri Thanassi and Nakada

(1967) [35]

Patinopecten yessoensis Nemacytus decipieus Kitamura et al. (1992) [48]

Pecten maximus Ascophyllum nodosum Berteau et al. (2002) [49]

Littorina kurila F. distichus Bilan et al. (2005) [50]

Lambis sp. F. evanescens Silchenko et al. (2014) [51]

Fungi

Dendryphiella arenaria TM94 F. vesiculosus Wu et al. (2011) [52]

Fusarium sp. LD8 F. vesiculosus Wu et al. (2011) [53]

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9

ࡇ ࢀ ࡲ ࡛ ࡟ ⴭ ⪅ ࡽ ࡢ ࢢ ࣝ ࣮ ࣉ ࡛ ࡣ ࢜ ࢟ ࢼ ࣡ ࣔ ࢬ ࢡ ࣇ ࢥ ࢖ ࢲ ࣥ ࢆ ㈨ ໬ ࡍ ࡿ ᚤ ⏕ ≀ Flavobacterium sp. F31ࠊLuteolibacter algae H18ࡀ༢㞳ࡉࢀ࡚࠸ࡿ [44, 46]ࠋ ≉࡟H18ᰴࡣࠊ ࢢ࣒ࣛ㝜ᛶࡢ㠀㐠ືᛶᚤ⏕≀࡛࠶ࡾࠊࡑࡢࢥࣟࢽ࣮ࡣ㯤Ⰽࡃᖹ⁥࡛࠶ࡗࡓࠋ16S rRNA㓄ิ

ศᯒࡢ⤖ᯝࠊᅗ1-4࡟♧ࡍࡼ࠺࡟Luteolibacter algae A5J-40㸦AB331894-1㸧ࡢ㓄ิ࡜᭱ࡶ㧗

࠸㢮ఝᛶ㸦97㸣㸧ࢆ♧ࡋࠊLuteolibacter algae H18࡜࿨ྡࡉࢀࡓࠋ᭦࡟ࡑࡢ16S rRNA㓄ิࡣࠊ

᪤▱ࡢࣇࢥ࢖ࢲࣥศゎᚤ⏕≀࡛࠶ࡿ Mariniflexile fucanivorans SW5^T (AJ628046)࡜ 70%ࠊ Fucobacter marina SA-0082 (AB057592)࡜71%ࠊFucophilus fucoidanolyticus SI-1234 (AB073978)

࡜ 79%ࠊSphingomonas paucimobilis PF-1 (AM237364)࡜ 72%ࠊAlteomonos sp. SN-1009 (AB544005)࡜ 73%ࠊࡑࡋ࡚ Flavobacterium sp. F31 (AB544005)࡜ 71%ࡢ㢮ఝᛶࡀ♧ࡉࢀࡓ [46]ࠋ

ᅗ1-4. Luteolibacter algae H18 ࡢ16S rRNA 㓄ิ࡟ᇶ࡙ࡃ⣔⤫ᶞ [46]

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10

ࣇࢥ࢖ࢲࣥศゎ㓝⣲࡟ࡘ࠸࡚

ᾏ⸴㢮࡟ྵࡲࢀࡿከ⢾㢮ࡢ௦⾲ⓗ࡞ࡶࡢ࡜ࡋ࡚ࠊᐮኳࠊ࢝ࣛࢠ࣮ࢼࣥࠊࣇࢥ࢖ࢲࣥࠊ࢔

ࣝࢠࣥ㓟ࡀ࠶ࡿࠋࡇࢀࡽከ⢾㢮ࢆศゎࡍࡿ㓝⣲ࡣࡑࢀࡒࢀ࠸ࡃࡘ࠿ሗ࿌ࡉࢀ࡚࠸ࡿࡀࠊࡑ ࡢ୰࡛ࡶ࢔ࣝࢠࣥ㓟ศゎ㓝⣲࡟㛵ࡍࡿ◊✲ࡀヲ⣽࡟⾜ࢃࢀ࡚࠸ࡿࠋ࢔ࣝࢠࣥ㓟ࡣ〓⸴㢮ࡢ

⣽⬊ቨࢆᵓᡂࡍࡿከ⢾࡛ࠊᵓᡂ⢾࡜ࡋ࡚ E-D-࣐ࣥࢾࣟࣥ㓟࡜ࡑࡢC5-epimer࡛࠶ࡿ D-L- ࢢࣝࣟࣥ㓟࠿ࡽ࡞ࡿࣈࣟࢵࢡ࣏࣐࣮࡛ࣜ࠶ࡿࠋᅗ1-5 ࡟♧ࡍࡼ࠺࡟ E-D-࣐ࣥࢾࣟࣥ㓟ࡀE-

1,4-⤖ྜࡋࡓ┤㙐ࡢ࣐ࣥࢾࣟࣥ㓟ࣈࣟࢵࢡ (Mࣈࣟࢵࢡ) ࡜ D-L-ࢢࣝࣟࣥ㓟ࡀD-1,4-⤖ྜࡋ

ࡓ᤬ࡌࢀࡓ࣏ࢣࢵࢺᵓ㐀ࢆ᭷ࡍࡿ┤㙐ࡢࢢࣝࣟࣥ㓟ࣈࣟࢵࢡ (Gࣈࣟࢵࢡ) ࠾ࡼࡧࠊE-D-

࣐ࣥࢾࣟࣥ㓟࡜D-L-ࢢࣝࣟࣥ㓟ࡀ୙つ๎࡟1,4-⤖ྜࡋࡓMG ࣛࣥࢲ࣒ࣈࣟࢵࢡࡢᏑᅾࡀ᫂

ࡽ࠿࡟࡞ࡗ࡚࠸ࡿ [54, 55] ࠋMࣈࣟࢵࢡ࡜Gࣈࣟࢵࢡࡢẚ⋡࡟ࡼࡾ≉ᛶࡀᕥྑࡉࢀࠊG

ࣈࣟࢵࢡࡢ࣏ࢣࢵࢺᵓ㐀࡟ከ౯࢖࢜ࣥࡀࢺࣛࢵࣉࡉࢀࡿ࡜ࠊࡑࡇࢆᯫᶫⅬ࡜ࡋ࡚ࢤࣝࡀᙧ ᡂࡉࢀࡿࠋࡍ࡞ࢃࡕࠊGࣈࣟࢵࢡࡢẚ⋡ࡀ㧗࠸⛬ࠊᯫᶫⅬࡀከࡃ࡞ࡿࡓࡵࠊࡼࡾ◳ᗘࡢ㧗

࠸ࢤࣝ࡟࡞ࡿࡇ࡜ࡀࢃ࠿ࡗ࡚࠸ࡿ [56, 57] ࠋ

ᅗ1-5. ࢔ࣝࢠࣥ㓟ࡢGࣈࣟࢵࢡ, Mࣈࣟࢵࢡࡢᵓ㐀 [57]

(a) Gࣈࣟࢵࢡ (b) Mࣈࣟࢵࢡ

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11

࢔ࣝࢠࣥ㓟ࢆศゎࡍࡿ㓝⣲ࠊ࢔ࣝࢠࣥ㓟ࣜ࢔࣮ࢮࡣࠊྂࡃࡣ࢔࣡ࣅ࡞࡝ࡢ᳜㣗ື≀ࡢෆ

⮚࡟ከࡃྵࡲࢀ࡚࠸ࡿࡇ࡜ࡀ኱ᓥ࡟ࡼࡗ࡚ぢฟࡉࢀ [58]ࠊ࢔࣡ࣅ⛶㈅ࡢෆ⮚୰ࡢศゎ㓝⣲

࡟ࡘ࠸࡚ࡣᒣ⏣ࡽࡢሗ࿌ࡀ࠶ࡿ [59]ࠋ21ୡ⣖࡟࡞ࡾࠊࢱࣥࣃࢡ㉁ࡢ࢔࣑ࣀ㓟㓄ิ᝟ሗ㔞ࡀ 㣕㌍ⓗ࡟ቑຍࡋࡓ⤖ᯝࠊ┦஫ࡢ㓄ิ㢮ఝᛶࡀ㧗࠸ࢱࣥࣃࢡ㉁ࡢ୍⩌ࡣࣇ࢓࣑࣮ࣜ࡜ศ㢮ࡉ

ࢀࡿࡼ࠺࡟࡞ࡗࡓࠋ࢔ࣝࢠࣥ㓟ࣜ࢔࣮ࢮࡣࠊከ⢾ࣜ࢔࣮ࢮFamily 5, 6, 7, 14, 15, 17, 18࡟ᒓ ࡍࡿࡇ࡜ࡀᥦၐࡉࢀ࡚࠾ࡾ[60]ࠊ୺࡟࣏ࣜGࣈࣟࢵࢡ࠾ࡼࡧMGࣛࣥࢲ࣒ࣈࣟࢵࢡ࡟ᑐࡋ

࡚≉␗ⓗ࡞࣏ࣜGࣜ࢔࣮ࢮ㸦EC 4.2.2.11㸧ࡲࡓࡣࠊMࣈࣟࢵࢡ࡟ᑐࡋ࡚≉␗ⓗ࡞࣏ࣜMࣜ

࢔࣮ࢮ㸦EC 4.2.2.3㸧ࡢ࠸ࡎࢀ࠿࡟ศ㢮ࡉࢀࡿࠋࡉࡽ࡟ࠊࡇࢀࡽࡢ㓝⣲ࡣࠊ࢚࢟ࢯᆺ (Family 6, 15, ࡑࡋ࡚17)ࠊ࢚ࣥࢻᆺ (Family 5࡜7) ࡟ศࡅࡽࢀࡿࡀ [61, 62]ࠊ࠸ࡎࢀࡶ4-

O-ࢢࣜࢥࢩࢻ⤖ྜࢆษ᩿ࡋ࡚C-4࡜C-5ࡢ㛫࡟஧㔜⤖ྜࢆᙧᡂࡍࡿࠋ࢔ࣝࢠࣥ㓟ࡢศゎᵝ

ᘧࡣࠊࢹࣥࣉࣥࠊࢭ࣮ࣝࣟࢫ➼ࡢ௚ࡢከ⢾࡜ྠࡌࡃࠊࡲࡎ࢚ࣥࢻᆺ࢔ࣝࢠࣥ㓟ࣜ࢔࣮ࢮ࡟

ࡼࡗ࡚࢔ࣝࢠࣥ㓟ෆ㒊ࢆศゎࡋ࡚࢜ࣜࢦ⢾࡜ࡋࠊḟ࡟࢚࢟ࢯᆺ࢔ࣝࢠࣥ㓟ࣜ࢔࣮ࢮ࡟ࡼࡗ

࡚࢜ࣜࢦ⢾ࢆศゎࡋࠊ༢⢾ࢆᙧᡂࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ [62] ࠋ

୍᪉ࠊࣇࢥ࢖ࢲࣥࡣࠊ࢔ࣝࢠࣥ㓟࡜ẚ㍑ࡋ࡚ᵓ㐀ࡀ」㞧࡛࠶ࡾࠊࣇࢥ࢖ࢲࣥࡢศゎ㓝⣲

࡜ࡋ࡚ࡣࠊࣇࢥ࢖ࢲࣥࡢ୺㙐ࢆศゎࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮࠊഃ㙐ࡢ◲㓟ᇶࢆ⬺㞳ࡍࡿࢫࣝࣇ

࢓ࢱ࣮ࢮࠊ࢔ࢭࢳࣝᇶࢆ⬺㞳ࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮ࡜࠸ࡗࡓ㓝⣲ࡀᏑᅾࡍࡿࡇ࡜ࡀ௒ࡲ࡛ࡢ

◊✲࡛᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ[46, 63] ࡀࠊᮍゎ᫂࡞㒊ศࡀከ࠸ࡢࡀ⌧≧࡛࠶ࡿࠋFurukawaࡽ ࡣࠊࣇࢥ࢖ࢲࣥࡢᏑᅾࡍࡿᇵᆅ࡛ࠊVibrioᒓ⣽⳦࡜ྠᐃࡉࢀࡓN-5ᰴࡢ⳦యෆ࡟ࣇࢥ࢖ࢲ ࢼ࣮ࢮ࡜ࣇࢥ࢖ࢲࣥࢫࣝࣇ࢓ࢱ࣮ࢮࢆㄏᑟⓗ࡟⏕⏘ࡉࡏࡓ [63]ࠋࣇࢥ࢖ࢲࣥࢫࣝࣇ࢓ࢱ࣮

ࢮࡣࠊࣇࢥ࢖ࢲࢼ࣮ࢮ࡟ᑡࡋ㐜ࢀ࡚⏕⏘ࡉࢀࡓࠋ୧㓝⣲ࡢᇶ㉁≉␗ᛶ࡟㛵ࡋ࡚ࠊࣇࢥ࢖ࢲ ࢼ࣮ࢮࡣ࢞ࢦ࣓ࢥࣥࣈ࠾ࡼࡧࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ࠊࣇࢥ࢖ࢲࣥࢫࣝࣇ࢓ࢱ࣮ࢮࡣࠊ࢞ࢦ࣓

ࢥࣥࣈࠊࣔࢬࢡ࠾ࡼࡧࣇࢺࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟స⏝ࡋ࡚ࠊࡑࢀࡒࢀ㑏ඖ⢾࡜◲㓟࢖࢜ࣥࢆ

⏕ᡂࡍࡿࡇ࡜ࢆ☜ㄆࡋࡓ [63] ࠋࡉࡽ࡟ࠊFurukawaࡽࡣࠊVibrio ᒓࡢ⳦ᰴ࠿ࡽࢹ࢕ࢫࢡ- PAGE࡟࠾࠸࡚༢୍࡟ࡲ࡛⢭〇ࡋࡓ3✀ࡢࣇࢥ࢖ࢲࢼ࣮ࢮࡀࠊ࢞ࢦ࣓ࢥࣥࣈࣇࢥ࢖ࢲࣥࢆ

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12

ຍỈศゎࡋࠊ୺࡞⏕ᡂ≀࡜ࡋ࡚◲㓟ᇶࢆ᭷ࡍࡿపศᏊ㔞ࡢ࢜ࣜࢦ⢾ࢆ⏕ᡂࡍࡿࡇ࡜ࢆ☜ㄆ ࡋ࡚࠸ࡿ [36] ࠋ

୍᪉๓㏙ࡢࡼ࠺࡟ࠊ㓇஭ࡽࡣ࢞ࢦ࣓ࢥࣥࣈ⏤᮶ࣇࢥ࢖ࢲࣥࡢ໬Ꮫᵓ㐀ࢆ᫂ࡽ࠿࡟ࡋࡓ ࡀࠊࡑࡢ㝿ࠊᵓ㐀Ỵᐃ࡟㈉⊩ࡋࡓࡢࡀࠊࣇࢥ࢖ࢲࣥࢆ࢜ࣜࢦ⢾༢఩࡟ࡲ࡛≉␗ⓗ࡟ษ᩿࡛

ࡁࡿࣇࢥ࢖ࢲࣥศゎ㓝⣲࡛࠶ࡿࠋࡇࢀࡽ㓝⣲ࡢ⏕⏘⳦ᰴࡣࠊᾏὒᚤ⏕≀ࢆࢫࢡ࣮ࣜࢽࣥࢢ ࡋ࡚ᚓࡽࢀࡓࡶࡢ࡛࠶ࡿ [32, 37, 38] ࠋሗ࿌ࡢ୰࡛Alteromonas sp. SN-1009 ࡜ Fucobacter marina SA-0082ࠊࡀ᭷ࡍࡿ㓝⣲ࡣ㝈ࡽࢀࡓࣇࢥ࢖ࢲࣥ࡟ࡋ࠿స⏝ࡋ࡞࠸ࡢ࡟ᑐࡋ [32, 64]ࠊ

Fucophilus fucoidanolyticus SI-1234⏤᮶ࡢ㓝⣲ࡣࠊㄪ࡭ࡓ㝈ࡾࡍ࡭࡚ࡢᾏ⸴⏤᮶ࣇࢥ࢖ࢲࣥ

ࢆ࠶ࡿ⛬ᗘ㈨໬ࡋࡓࡇ࡜࠿ࡽ [38]ࠊከ⢾㢮ࡢࣇࢥ࢖ࢲࣥศゎ㓝⣲⏕⏘⳦࡜ࡋ࡚฼⏝࡛ࡁࡿ

ྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡇࢀࡽ㓝⣲࡟ࡘ࠸࡚ࡣࡍ࡛࡟኱⭠⳦ࢆ⏝࠸ࡓ⤌᥮࠼㓝⣲࡜ࡋ࡚ࡢ⏕

⏘⣔ࢆ☜❧ࡋࠊ≉チࢆᐆ࣊ࣝࢫࢣ࢔ᰴᘧ఍♫ࡀྲྀᚓࠊಖ᭷ࡋ࡚࠸ࡿࠋ

Henrissatࡽࡣ170,000ࢆ㉸࠼ࡿ⭾኱࡞ᩘࡢ⢾㉁ຍỈศゎ㓝⣲ࡢ㓄ิࢆࡑࢀࡽࡢ㓄ิ㢮ఝ

ᛶࢆࡶ࡜࡟ࠊ130⛬ᗘࡢ⢾㉁ຍỈศゎ㓝⣲ࣇ࢓࣑࣮ࣜ (glycoside hydrolase family; GHࣇ࢓

࣑࣮ࣜ) ࡜ࡋ࡚ศ㢮ࡋ࡚࠸ࡿ [65]ࠋࡇࡢ㓝⣲ศ㢮࡟ࡼࡿ࡜ࠊ௒ࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿࣇࢥ

࢖ࢲࢼ࣮ࢮࡣࡍ࡭࡚GHࣇ࢓࣑࣮ࣜ107 ࡟ศ㢮ࡉࢀ࡚࠾ࡾࠊ᭱ึ࡟GHࣇ࢓࣑࣮ࣜ107࡟ ศ㢮ࡉࢀࡓࡢࡣࠊᾏὒᛶ⣽⳦ Mariniflexile fucanivorans SW5^T ࡼࡾศ㞳⢭〇ࡉࢀࡓࣇࢥ࢖ࢲ

ࢼ࣮ࢮFcnA࡛࠶ࡿ [66]ࠋࡇࡢ㓝⣲㑇ఏᏊfcnAࡣࢡ࣮ࣟࢽࣥࢢࡉࢀࠊ኱⭠⳦࡛␗✀Ⓨ⌧ࡉ

ࢀ࡚࠾ࡾࠊfcnAࡢሷᇶ㛗ࡣ3021 bpࠊFcnAࡢศᏊ㔞ࡣࠊ1007 amino acids ࡛࠶ࡗࡓ [66] ࠋ

ࡉࡽ࡟ࠊSilchenko ࡽ࡟ࡼࡗ࡚ࡶ Formosa algae KMM3553 ᰴ࠿ࡽࣇࢥ࢖ࢲࢼ࣮ࢮ FFA2

ࡀࡢ㑇ఏᏊࡀࢡ࣮ࣟࢽࣥࢢࠊ␗✀Ⓨ⌧ࡉࢀ࡚࠾ࡾࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿ

[67]ࠋࡇࢀࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࡢㅖᛶ㉁ࢆ⾲1-4 ࡟♧ࡍࠋ

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13

SourceMode of actionT-optimum, ΥpH- optimumpH- stabilitypIInhibitorsActivatorsMr, kDaReferences Bacteria Vibrio sp. No. 5exo38-456.04.0-9.05.80Hg2+ , Fe3+ , Ag+ Co2+ (2 mM)39.5Furukawa et al. (1992) [36] endon.d.6.5-7.0n.d.n.d.n.d.n.d.n.d. Fucobacter marina SN-0082endo407.5n.d.n.d.Ag+ , Zn2+ , Cu2+0.4 M NaCl 67Sakai et al. (2003) [64] Alteromonas sp. SN-1009endo30-356.5-8.0n.d.n.d.Zn2+ , Cu2+ 0.4 M NaCl, Ca2+100Sakai et al. (2004) [32] Mariniflexile fucanivorans SW5Tendo20-257.5n.d.n.d.n.d.Ca2+105Colinet al . (2006) [66] Sphingomonas paucimobilis PF-1endon.d.n.d.n.d.n.d.n.d.n.d.n.d.Kimet al. (2008) [43] Formosa algae KM3553Tendon.d.6.5-9.0n.d.n.d.Zn2+ , Cu2+ Mg2+ , Ca2+ , Ba2+96Silchenkoet al. (2013) [47] Invertebrates Haliotus sp.endo385.42.0-10.0n.d.Hg2+ , Mn2+ , Ag+ (0.001 M)Mg2+ (0.01 M)100-200Thanassi and Nakada (1967) [35] Pecten maximusendon.d.5.5n.d.7.4n.d.n.d.85Berteauet al. (2002) [49] Littorina sitkanaendon.d.5.4n.d.n.d.n.d.0.2 M NaCln.d.Bilan et al. (2005) [50] Lambis sp.endo455.0n.d.n.d.Hg2+ , Zn2+ , Cu2+n.d.50Silchenkoet al. (2014) [51] Fungi Dendryphiella arenaria TM94endo506.05.0-7.04.4n.d.n.d.180Wu et al. (2011) [52] Fusarium sp. LD8endo606.06.04.5n.d.n.d.64Wu et al. (2011) [53]

⾲ 1-4.ࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࡢㅖᛶ㉁ Pseudoalteromonas citrea KMM3296, KMM3297, KMM3298Bakuninaet al. (2002) [41]

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14

ⴭ⪅ࡽࡢࢢ࣮ࣝࣉ࡛ࡣ࢜࢟ࢼ࣡ࣔࢬࢡCladosiphon okamuranus⏤᮶ࣇࢥ࢖ࢲࣥࡢ⏕⌮ά ᛶᶵᵓࡢゎ᫂ࢆ┠ᣦࡋ࡚ࠊ㓝⣲ⓗ࡞పศᏊ໬࡟ྲྀࡾ⤌ࢇ࡛࠸ࡿࠋࡍ࡛࡟༢㞳ࡋ࡚࠸ࡿࣇࢥ

࢖ࢲࣥศゎ⳦Luteolibacter algae H18ࡣࠊࣇࢥ࢖ࢲࣥศゎ㓝⣲ࢆ⣽⬊ෆ࡟᭷ࡋ࡚࠾ࡾࠊࣇࢥ

࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࡢᚋ࡟పศᏊ໬཯ᛂࡀ㉳ࡇࡿࡇ࡜ࢆᥦၐࡋ࡚࠸ࡿ[46]ࠋࣇࢥ࢖

ࢲࣥࡢᵓ㐀ࡢከᵝᛶࡣࠊ◲㓟ᇶࡸ࢔ࢭࢳࣝᇶࡢ㔞ⓗࠊ఩⨨ⓗ࡞ᕪ࡟ຍ࠼࡚ࠊ㦵᱁ᵓ㐀ࠊഃ

㙐ᵓ㐀ࠊᵓᡂ⢾ࡢ✀㢮ࡲ࡛࡟ཬࡪࡓࡵࠊ඲ࡃู✀ࡢศᏊࡀఱ✀㢮ࡶᏑᅾࡍࡿࠋࡑࡋ࡚ࠊࡑ

ࢀࡽከᵝ࡞ࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚స⏝ࡍࡿ㓝⣲࡟ࡘ࠸࡚ࡣࠊ๓㏙ࡢ⾲1-4 ࡟♧ࡍࡶࡢሗ࿌ࡀ

࡞ࡉࢀ࡚࠸ࡿࡀࠊࡇࢀࡣࠊ୍㒊࡟ࡋ࠿㐣ࡂࡎࠊᮍࡔ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸㓝⣲ࡶᏑᅾࡋ࡚

࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋࡍ࡞ࢃࡕࠊࣇࢥ࢖ࢲࣥࢆపศᏊ໬ࡍࡿ㓝⣲࡟㛵ࡍࡿሗ࿌౛ࡣ࠶ࡿࡶࡢ ࡢࠊ⬺࢔ࢭࢳࣝ໬ࠊ⬺◲㓟໬࡟㛵୚ࡍࡿ㓝⣲࡟ࡘ࠸࡚ࡢ◊✲ሗ࿌ࡣ࡯࡜ࢇ࡝࡞࠸ࡢࡀ⌧≧

࡛࠶ࡿࠋᚑࡗ࡚ࠊࣇࢥ࢖ࢲࣥศゎ࡟㛵୚ࡍࡿ㓝⣲ࢆⴭ㔞⏕⏘ࡍࡿࡇ࡜ࡀ࡛ࡁࢀࡤࠊࡑࢀࢆ

ࣇࢥ࢖ࢲࣥ࡟స⏝ࡉࡏࡿࡇ࡜࡟ࡼࡾࠊ◲㓟ᇶࢆᣢࡘ✀ࠎࡢ࢜ࣜࢦ⢾ࠊ◲㓟ᇶࢆᣢࡓ࡞࠸

✀ࠎࡢ࢜ࣜࢦ⢾࡞࡝ࢆㄪ〇ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࡾࠊࡑࡢᵓ㐀ࢆỴᐃࡍࡿࡇ࡜࡟ࡼࡾࠊࣇࢥ

࢖ࢲࣥࡢᵓ㐀ཬࡧ㓝⣲ࡢᇶ㉁≉␗ᛶࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜࡛ࡁࠊࡉࡽ࡟ࡣࣇࢥ࢖ࢲࣥࢆ㓝⣲

ⓗ࡟ศゎࡋ࡚⏕ᡂࡋࡓᵝࠎ࡞ᵓ㐀ࡢࣇࢥ࢖ࢲࣥศゎ≀ࡢ⏕⌮άᛶࢆホ౯ࡍࡿࡇ࡜࡟ࡼࡾࠊ

ࣇࢥ࢖ࢲࣥᵓ㐀࡜⏕⌮άᛶ࡜ࡢ┦㛵ᛶࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

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15

ᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ

ࡇࢀࡲ࡛ࡢ◊✲࡛ࠊLuteolibacter algae H18 ࡢ⣽⬊ෆ㓝⣲࡟ࡼࡗ࡚ࣇࢥ࢖ࢲࣥࡀపศᏊ໬

ࡉࢀࡿ㝿ࠊ⬺࢔ࢭࢳࣝ໬཯ᛂࡀ㉳ࡇࡗࡓᚋ࡟పศᏊ໬཯ᛂࡀ㐍ࡴࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ [46]ࠋ ࡑࡢ୍᪉࡛ࠊH18 ᰴࡢࢤࣀ࣒DNA᝟ሗࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋࡑࡇ࡛ᮏ◊✲࡛ࡣࠊ

⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࠊపศᏊ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖

ࢲࢼ࣮ࢮࢆH18 ᰴ࠿ࡽ㧗ᗘ࡟⢭〇ࡋࠊࡑࢀࡽࡢ࢔࣑ࣀ㓟㓄ิࢆỴᐃᚋࠊH18ᰴࡢࢤࣀ࣒᝟

ሗࢆ฼⏝ࡋ࡚ࠊ㓝⣲㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊࡑࡋ࡚

ࡑࢀࡽ㓝⣲ࡢㅖᛶ㉁ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜࡟ࡋࡓࠋ ᮏᏛ఩ㄽᩥࡣ8❶࡟ࡼࡾᵓᡂࡉࢀࡿࠋ

➨ 1 ❶ࠕᗎㄽ࡛ࠖࡣࠊᮏ◊✲ࡢ⫼ᬒ࡜ࡋ࡚ࣇࢥ࢖ࢲࣥࠊࣇࢥ࢖ࢲࣥ㈨໬ᛶᚤ⏕≀ࠊࡑࡋ

࡚ࣇࢥ࢖ࢲࣥศゎ࡟㛵୚ࡍࡿ㓝⣲࡟ࡘ࠸࡚㏙࡭ࠊࡲࡓᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ࡟ࡘ࠸࡚㏙

࡭ࡓࠋ

➨ 2 ❶ ࠕLuteolibacter algae H18࠿ࡽࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇࡛ࠖࡣࠊ࢜࢟ࢼ

࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆ✀ࠎࡢࢡ

࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊᮏ㓝⣲ࡢ㒊ศ࢔࣑ࣀ㓟㓄ิࢆỴᐃࡋࡓ࠺࠼࡛ࠊᮏ㓝⣲㑇ఏ ᏊࢆH18ᰴࡢࢤࣀ࣒᝟ሗ࠿ࡽ᥈⣴ࡋࠊྠᐃࡋࡓࠋ

➨ 3 ❶ ࠕࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ⌧ࠊ㓝⣲໬Ꮫⓗᛶ㉁࡛ࠖ

ࡣࠊ᫂ࡽ࠿࡟ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㑇ఏᏊࢆࢡ࣮ࣟࢽࣥࢢࡋࠊ኱⭠⳦࡛Ⓨ⌧ࡉࡏࠊ 㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲ࡢㅖᛶ㉁᳨ウࢆ⾜ࡗࡓࠋ

➨ 4 ❶ࠕࢹ࢔ࢭࢳ࣮ࣛࢮࡢゐ፹άᛶṧᇶࡢண ࡛ࠖࡣࠊSWISS-MODELࢆ฼⏝ࡋࠊࣇࢥ

࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡀ࡝ࡢࡼ࠺࡞ᵓ㐀ࠊάᛶ㒊఩ࢆᣢࡘࡇ࡜ࡀண᝿ࡉࢀࡿ࠿ࢆㄪ࡭ࡓࠋ

➨ 5 ❶ࠕLuteolibacter algae H18࠿ࡽࡢࣇࢥ࢖ࢲࢼ࣮ࢮࡢ⢭〇࡛ࠖࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇ

ࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮࢆ✀ࠎࡢࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭

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〇ࡋࠊᮏ㓝⣲ࡢ㒊ศ࢔࣑ࣀ㓟㓄ิࢆỴᐃࡋࡓࠋ

➨ 6 ❶ࠕࣇࢥ࢖ࢲࢼ࣮ࢮࡢ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ⌧࡛ࠖࡣࠊ᫂ࡽ࠿࡟ࡋࡓࣇࢥ࢖ࢲࢼ

࣮ࢮࡢ࢔࣑ࣀ㓟㓄ิࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࢆࠊH18ᰴࡢࢤࣀ࣒᝟ሗ࠿ࡽ᥈⣴ࡋࠊࡑࡢ㓝⣲㑇ఏ Ꮚࡢࢡ࣮ࣟࢽࣥࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲ࡢㅖᛶ㉁᳨ウࢆ⾜ࡗࡓࠋ ➨ 7 ❶ ࠕ⥲ᣓ࡛ࠖࡣࠊᚤ⏕≀࡟ࡼࡿࣇࢥ࢖ࢲࣥศゎ࡟㛵ࢃࡿᮏ◊✲ࡢࡲ࡜ࡵࢆㄽࡌࡓࠋ

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Luteolibacter algae H18 ࠿ࡽࡢࣇࢥ࢖ࢲࣥ

ࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇

⥴ゝ

〓⸴㢮࡟࠾ࡅࡿࣇࢥ࢖ࢲࣥࡢᵓ㐀ࡣࠊศ㞳※࡜࡞ࡿ✀㢮࡟౫Ꮡࡍࡿ [2] ࠋ୰࡛ࡶ࢜࢟ࢼ

࣡ࣔࢬࢡ (Cladosiphon okamuranus TOKIDA) ⏤᮶ࣇࢥ࢖ࢲࣥ࡟ࡘ࠸࡚ࡣࠊ⏣ᖾࡽ࡟ࡼࡗ࡚

ヲ⣽࡟◊✲ࡀ⾜ࢃࢀ࡚࠾ࡾ [29, 68, 69] ࠊ௚ࡢࣇࢥ࢖ࢲࣥ࡟ࡣぢࡽࢀ࡞࠸࢔ࢭࢳࣝᇶࡀྵࡲ

ࢀ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀ࡚࠸ࡿࠋࡉࡽ࡟ࠊ㓇஭ࡽࡢ◊✲࡟ࡼࡾࠊ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇ

ࢥ࢖ࢲࣥࡣࠊD-1, 3 ⤖ྜࡢL-ࣇࢥ࣮ࢫṧᇶࢆ୺㙐࡜ࡋࠊഃ㙐࡜ࡋ࡚୺㙐ࡢL-ࣇࢥ࣮ࢫṧᇶ

ࡢ C-2 ࡟ D-ࢢࣝࢡࣟࣥ㓟ṧᇶࡀ⨨᥮ࡋ࡚࠾ࡾࠊࡲࡓࠊ◲㓟ᇶࡀ D-ࢢࣝࢡࣟࣥ㓟ṧᇶࡀ⨨

᥮ࡋ࡚࠸࡞࠸୺㙐ࡢ L-ࣇࢥ࣮ࢫṧᇶࡢ C-4 ࡟⨨᥮ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗ࡚࠸ࡿ

[28] ࠋࡑࡋ࡚ࠊ⏣ᖾࡽࡢ◊✲࡟ࡼࡾ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥ࡟ࡣࠊ࢔ࢭࢳࣝᇶࡀྵࡲ

ࢀ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡓ (ᅗ 1-2) [29] ࠋ

୍᪉ࠊⴭ⪅ࡽࡢࢢ࣮ࣝࣉ࡛ࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥศゎ⳦ Luteolibacter algae H18ࢆ༢㞳ࡋ࡚࠾ࡾࠊᮏ⳦ᰴ࡛ࡣࣇࢥ࢖ࢲࣥࡀపศᏊ໬ࡉࢀࡿ㝿ࠊ㓝⣲ⓗ࡟ࣇࢥ࢖ࢲࣥࡢ⬺

࢔ࢭࢳࣝ໬཯ᛂࡀ㉳ࡇࡿࡇ࡜ࢆᥦၐࡋ࡚࠸ࡿ [46] ࠋ

ࡑࡇ࡛ࠊᮏ❶࡛ࡣ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࣥ

ࢹ࢔ࢭࢳ࣮ࣛࢮࢆࠊH18ᰴ࠿ࡽ✀ࠎࡢࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊ㓝⣲ࡢ࢔࣑ࣀ㓟㓄

ิࢆỴᐃࡋࡓࠋࡑࡋ࡚ࠊ᫂ࡽ࠿࡟ࡋࡓ H18 ᰴࡢࢻࣛࣇࢺࢤࣀ࣒᝟ሗࢆᇶ࡟ࠊࣇࢥ࢖ࢲࣥࢹ

࢔ࢳ࣮ࣛࢮ㑇ఏᏊࢆ≉ᐃࡋࠊ᫂ࡽ࠿࡟࡞ࡗࡓᮏ㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิ࡟ࡘ࠸࡚⪃ᐹࢆ⾜ࡗࡓࠋ

(24)

18

ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ

ᮦᩱ࠾ࡼࡧヨ⸆

㧗ศᏊࣇࢥ࢖ࢲࣥ (SEA ALGA-F, ᰴᘧ఍♫ᾏ⏘≀ࡢࡁࡴࡽࡸ, ቃ ᕷ, ᪥ᮏ) ࡣࠊᕝᮏࡽ

ࡢ◊✲࡟ࡼࡾ☜❧ࡉࢀࡓ᪉ἲ [70] ࡟ࡼࡾ࢜࢟ࢼ࣡ࣔࢬࢡ࠿ࡽᢳฟ࠾ࡼࡧ⢭〇ࡉࢀࡓࡶࡢ

ࢆ౑⏝ࡋࡓࠋࡑࡢ௚ࠊᮏ◊✲࡛⏝࠸ࡓヨ⸆ࡣ࠸ࡎࢀࡶᕷ㈍ရ࡛࠶ࡾࠊ௨㝆ࡢᮏㄽᩥ࡛㏙࡭ࡿ

඲࡚ࡢᮦᩱࡶྠᵝ࡛࠶ࡿࠋ

Luteolibacter algae H18ࡢᇵ㣴

H18 ᰴࡢᇵ㣴࡟ࡣ༢୍Ⅳ⣲※࡜ࡋ࡚ࣇࢥ࢖ࢲࣥࢆ0.5%ྵࡴ᏶඲ྜᡂᇵᆅ (⾲ 2-1, 2-2, 2- 3) ࢆ౑⏝ࡋࡓࠋ⾲2-1࡟♧ࡍ᏶඲ྜᡂᇵᆅ5 mL ࡟⥅௦ᇵ㣴ࡋ࡚࠸ࡿH18ᰴࢆ1%᳜⳦ࡋࠊ 30 Υࠊ100 min^-1࡛72 h᣺࡜࠺ࡋࠊ๓ᇵ㣴ࢆ⾜ࡗࡓࠋࡑࡢᚋࠊ500 mLࡢྠ⤌ᡂᇵᆅࢆྵࡴ2 Lᐜ㔞ࡢᆏཱྀࣇࣛࢫࢥ࡟๓ᇵ㣴ᾮࢆ2%᳜⳦ࡋࠊ30 Υࠊ100 min^-1࡛72 h᣺࡜࠺ᇵ㣴ࢆ⾜ࡗ ࡓࠋ

⾲2-1. 0.5% ࣇࢥ࢖ࢲࣥᇵᆅ⤌ᡂ

0.5% Fucoidan Medium

Fucoidan 5.00 g

K2HPO4 4.00 g

KH2PO4 0.50 g

(NH4)2SO4 2.00 g

MgSO4࣭7H2O 0.41 g

Metal sol. 10.00 mL

Vitamin mix. 1.00 mL

Total 1.00 L

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19

⾲2-2. Metal solution⤌ᡂ Metal sol.

NaCl 1.00 g

CaCl2 2.00 g

FeSO4 0.50 g

ZnSO4 0.50 g

MnCl2䞉4H2O 0.50 g

CuSO4 0.05 g

NaMoO4䞉2H2O 0.10 g

Na2WO4䞉2H2O 0.05 g

Total

1.00 L

⾲2-3. Vitamin mixture⤌ᡂ Vitamin mix.

Biotin 100 mg

Thiamin-HCl (pH 8.0) 100 mg

Riboflavin 100 mg

Ca-Pantothenate 100 mg

Pyridoxal phosphate 100 mg

Nicotinamide 100 mg

Folic acid 100 mg

p-Aminobenzoic acid 20 mg

Vitamin B12 10 mg

Lipoic acid 10 mg

Total

1 L

↓⣽⬊ᢳฟᾮ (C.F.E.) ࡢㄪ〇

2-2-2 ࡢ᧯స࡛ᚓࡽࢀࡓᇵ㣴ᾮࢆ㐲ᚰศ㞳 (HITACHI CR21/CR20ࠊR10A rotorࠊ8,000 rpmࠊ

60 minࠊ4 Υ) ࡋࠊ‵⳦యࢆᚓࡓࠋᚓࡽࢀࡓ⳦యࢆ‵⳦య㔜㔞ࡢ 4 ಸ㔞ࡢ 20 mM Tris-HCl

buffer (pH 8.0) ࡟ᠱ⃮ࡋࠊOut put 3ࠊDuty cycle 30 ࠊ30 min ࡟࡚㉸㡢Ἴ◚○ (BRANSON SONIFIER 450) ࠊ㐲ᚰศ㞳 (KUBOTA 3780ࠊAF-5004Ch rotorࠊ8,000 rpmࠊ60 minࠊ4Υ) ࢆ

(26)

20

⾜࠸ࠊ㐲ᚰୖΎࢆ୍ᬌ20 mM Tris-HCl buffer (pH 8.0)࡟ᑐࡋ࡚㏱ᯒࡋࡓࡶࡢࢆ↓⣽⬊ᢳฟᾮ

(C.F.E.) ࡜ࡋ࡚ᅇ཰ࡋࡓࠋ

ࢱࣥࣃࢡ㉁⃰ᗘࡢᐃ㔞

ࢱࣥࣃࢡ㉁⃰ᗘࡣBio-Radࡢࣉࣟࢸ࢖ࣥ࢔ࢵࢭ࢖ἲ࡛ᐃ㔞ࡋࡓࠋ⵨␃Ỉ࡛5ಸ࡟ᕼ㔘ࡋࡓ

ࣉࣟࢸ࢖ࣥ࢔ࢵࢭ࢖ᾮࢆ3 mLࢆヨ㦂⟶࡟ศὀࡋ࡚ࠊヨᩱ60 PLࢆῧຍࡋࠊ15 minᐊ ࡛཯

ᛂࡉࡏࡓࠋ⣸እྍどศගගᗘィ (SHIMADZUࠊUV-1700) ࢆ⏝࠸࡚ 595 nmࡢἼ㛗ࡢ྾཰ࢆ

ᐃࡋࡓࠋࡲࡓࠊ0.1㹼0.8 mg/mLࡢᶆ‽࢔ࣝࣈ࣑ࣥ⁐ᾮࢆ⏝࠸࡚సᡂࡋࡓ᳨㔞⥺࡟ࡼࡾࢱࣥ

ࣃࢡ㉁⃰ᗘࢆ⟬ฟࡋࡓ [71] ࠋ

SDS

࣏ࣜ࢔ࢡࣜࣝ࢔࣑ࢻࢤࣝ㟁ẼὋື

(SDS-PAGE)

SDS-PAGE 㟁ẼὋືࡣࠊ࢔࢘ࣝࣞࢰ࣐ࣝࢵࢡࢫ஧㐃࣑ࢽࢫࣛࣈ(ATTOࠊAE-6410)ࢆ⏝࠸

12.5%ࡢࢤࣝࢆస〇ࡋࠊTris/glycine/SDS⦆⾪ᾮ⣔࡛ࠊ࣑ࢽࢫࣛࣈ1ᯛ࡟ࡘࡁ150 V࡛ࠊ⣙2

h㟁ẼὋືࢆ⾜ࡗࡓࠋࢧࣥࣉࣝ࡜SDSฎ⌮⦆⾪ᾮࢆ1:1ࡢ๭ྜ࡛ຍ࠼ࠊ100 Υ࡛3 min⇕ฎ

⌮ࢆ⾜ࡗࡓࡶࡢࢆSDS⏝ヨᩱ⁐ᾮ࡜ࡋࡓࠋὋືᚋࡢࢤࣝࡣࠊ50%࣓ࢱࣀ࣮ࣝ-10%㓑㓟࡟⁐

ゎࡋࡓࢡ࣮࣐ࢩ࣮ࣈࣜࣜ࢔ࣥࢺࣈ࣮ࣝG-250 ࡛ᰁⰍࡍࡿࡇ࡜࡟ࡼࡾࢱࣥࣃࢡ㉁ࣂࣥࢻࢆྍ

ど໬ࡋࠊ30㸣࣓ࢱࣀ࣮ࣝ-10㸣㓑㓟࡛⬺Ⰽࡋࠊ஝⇱ࡉࡏࡓ [72] ࠋศᏊ㔞࣐࣮࣮࢝ࡣࠊGE Healthcareࡼࡾ㉎ධࡋࡓࠋ

F

࢟ࢵࢺࢆ⏝࠸ࡓ㓝⣲άᛶࡢ ᐃ࡜㓑㓟⃰ᗘࡢᐃ㔞

ᕷ㈍ࡉࢀ࡚࠸ࡿ㣗ရ୰ࡢ㓑㓟⃰ᗘࢆᐃ㔞ࡍࡿ F ࢟ࢵࢺ(Roche Diagnostics, Mannheim,

Germany) ࢆ⏝࠸࡚ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶࡢ ᐃ࠾ࡼࡧࢧࣥࣉࣝ୰࡟ྵࡲࢀࡿ㓑㓟⃰ᗘࡢᐃ

㔞ࢆ⾜ࡗࡓࠋࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㓝⣲άᛶࢆ ᐃࡍࡿ㝿ࡣࠊ2.5 %ࣇࢥ࢖ࢲࣥ⁐ᾮࢆ35 PLࠊ1 M Tris-HCl buffer ( pH 8.0 )ࢆ60 PLࠊࣜࣥࢦ㓟ࢆྵࡴ F ࢟ࢵࢺࡢ⁐ᾮ Iࢆ180 PLࠊATP, CoA,

(27)

21

NADࢆྵࡴF ࢟ࢵࢺࡢ⁐ᾮ Ⅱࢆ36 PLࠊCitrate synthase, Malate dehydrogenaseࢆྵࡴF ࢟

ࢵࢺࡢ⁐ᾮ Ⅲࢆ1.5 PLࠊAcetyl-CoA-synthetase ࢆྵࡴF ࢟ࢵࢺࡢ⁐ᾮ Ⅳࢆ2.5 PLࠊ⵨␃

Ỉࢆ280 PLࢆΰྜࡋࠊ᭱ᚋ࡟άᛶࢆ ᐃࡍࡿ㓝⣲⁐ᾮ5 PLῧຍࡋࠊ30 Υ࡛5 minࠊA340ࡢ

྾ගᗘࡢቑຍࢆ ᐃࡋ㓝⣲άᛶ್ࢆ⟬ฟࡋࡓࠋNADH ࡢศᏊ྾ගಀᩘࢆ 6220 ࡜ࡋࠊ⬺࢔

ࢭࢳࣝ໬άᛶ 1 unitࢆࠊ1 min࡟1 Pmolࡢ㓑㓟ࢆ㐟㞳ࡍࡿࡓࡵ࡟ᚲせ࡞㓝⣲㔞࡜ᐃ⩏ࡋࡓࠋ

୍᪉ࠊࢧࣥࣉࣝ୰࡟ྵࡲࢀࡿ㓑㓟⃰ᗘࢆᐃ㔞ࡍࡿሙྜࡣࠊୖグ࡜ྠࡌ཯ᛂᾮ⤌ᡂ࡛340 nm ࡢ྾ගᗘୖ᪼ࡀぢࡽࢀ࡞ࡃ࡞ࡿࡲ࡛཯ᛂࢆ⾜࠸ࠊศᯒ㛤ጞ๓࡜ࡢ྾ගᗘࡢᕪ࠿ࡽ㓑㓟ࡢᐃ 㔞ࢆ⾜ࡗࡓࠋ

DEAE-Sepharose FF

࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇

117.5 L ࡢᇵ㣴ᾮ࠿ࡽㄪ〇ࡋࡓ H18 ᰴ C.F.E. (ࢱࣥࣃࢡ㉁㔞 8622 mg)ࢆ 20 mM Tris-HCl buffer (pH 8.0)࡛ᖹ⾮໬ࡋࡓDEAE-Sepharose FF࣒࢝ࣛ (ᶞ⬡㔞 900 mL, 40×6.0 cm) ࡟࢔ࣉ

ࣛ࢖ࡋࡓࠋὶ㏿ࡣ3.0 mL/minࠊศ⏬ࢧ࢖ࢬࢆ20 mL࡟タᐃࡋࡓࠋ20 mM Tris-HCl buffer (pH 8.0) ࡛Ὑίᚋࠊ0.1, 0.2, 0.3 M NaClࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0)࡛⁐ฟࢆ⾜ࡗࡓࠋ

280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ࠸࡚ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

Phenyl-TOYOPEARL 650M

࡟ࡼࡿ㓝⣲⢭〇

2.0 M (NH4)2SO4 ࢆྵࡴ 20 mM Tris-HCl buffer (pH 8.0)࡟ᑐࡋ୍࡚ᬌ㏱ᯒࡋࡓ DEAE-

Sepharose 0.2 M ⁐ฟ⏬ศ (ࢱࣥࣃࢡ㉁㔞 1866 mg) ࢆࠊྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛ᖹ⾮໬ࡋࡓ

Phenyl-TOYOPEARL 650M (ᶞ⬡㔞200 mL, 50×3.0 cm)࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ1.5 mL/minࠊศ

⏬ࢧ࢖ࢬࢆ7 mL࡟タᐃࡋࡓࠋྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛Ὑίᚋࠊ2.0, 1.5, 1.0, 0.5 M (NH4)2SO4ࢆ

ྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡛⁐ฟࢆ⾜ࡗࡓࠋ280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ࠸࡚

ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

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22

Q-Sepharose FF

20 mM Tris-HCl buffer (pH 8.0) ࡟ᑐࡋ୍࡚ᬌ㏱ᯒࡋࡓPhenyl-TOYOPEARL 1.0 M⁐ฟ⏬ศ

(ࢱࣥࣃࢡ㉁㔞427 mg) ࢆࠊྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛ᖹ⾮໬ࡋࡓQ-Sepharose FF࣒࢝ࣛ (ᶞ⬡

㔞50 mL, 12×2.7 cm) ࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ2.0 mL/minࠊศ⏬ࢧ࢖ࢬࢆ3 mL࡟タᐃࡋࡓࠋ

ྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛Ὑίᚋࠊ0.1, 0.2, 0.3, 0.4, 0.5 M NaClࢆྵࡴ20 mM Tris-HCl buffer (pH

8.0) ࡛⁐ฟࢆ⾜ࡗࡓࠋ280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ࠸࡚ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊ

άᛶ⏬ศࢆ㞟ࡵࡓࠋ

Butyl-TOYOPEARL 650M ࡟ࡼࡿ㓝⣲⢭〇

2.0 M (NH4)2SO4ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡟ᑐࡋ୍࡚ᬌ㏱ᯒࡋࡓQ-Sepharose

0.2 M ⁐ฟ⏬ ศ(ࢱࣥࣃࢡ ㉁㔞 54 mg) ࢆࠊ ྠ⤌ ᡂࡢࣂࢵ ࣇ࢓࣮ ࡛ ᖹ⾮ ໬ࡋࡓ Butyl-

TOYOPEARL 650M (ᶞ⬡㔞6.0 mL, 30×1.0 cm) ࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ0.3 mL/minࠊศ⏬ࢧ࢖

ࢬࢆ0.8 mL࡟タᐃࡋࡓࠋྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛Ὑίᚋࠊ2.0, 1.5, 1.0, 0.5 M (NH4)2SO4ࢆྵࡴ

20 mM Tris-HCl buffer (pH 8.0)࡛⁐ฟࢆ⾜ࡗࡓࠋ280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ࠸࡚ࢹ࢔ࢭ

ࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

Mono Q^®HR

10/10

㝈እࢁ㐣⭷ࢆ⏝࠸࡚⬺ሷࡋࠊ0.76 mg/mLࡲ࡛⃰⦰ࡋࡓButyl-TOYOPEARL 1.0 M⁐ฟ⏬ศ (ࢱࣥࣃࢡ㉁㔞1.52 mg) ࢆࠊ20 mM Tris-HCl buffer (pH 8.0) ࡛ᖹ⾮໬ࡋࡓMono Q^®HR 10/10 (GE Healthcare) (ᶞ⬡㔞8.0 mL, 10×1.0 cm) ࡟2 mLࡎࡘ2ᅇ࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ0.3 mL/minࠊ ศ⏬ࢧ࢖ࢬࢆ1.15 mL࡟タᐃࡋࡓࠋྠ⤌ᡂࡢࣂࢵࣇ࢓࣮࡛Ὑίᚋࠊ1ᅇ┠ࡣ0~0.5 M NaClࢆ

ྵࡴ20 mM Tris-HCl buffer (pH 8.0)࡛ࠊ2ᅇ┠ࡣ0.2~0.5 M NaClࢆྵࡴ20 mM Tris-HCl buffer

(pH 8.0) ࡛ࢢࣛࢪ࢚ࣥࢺ⁐ฟࢆÄKTAࢩࢫࢸ࣒ࢆ⏝࠸࡚⾜ࡗࡓࠋ280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬

ศ࡟ࡘ࠸࡚ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

(29)

23

Superose 6^®HR

10/30

Mono-Q࡟ࡼࡿ2ᅇ┠ࡢ⁐ฟࣇࣛࢡࢩࣙࣥNo. 37 (ࢱࣥࣃࢡ㉁㔞 0.1 mg) ࢆࠊ20 mM Tris- HCl buffer (pH 8.0) ࡛ᖹ⾮໬ࡋࡓSuperose 6 ^®HR 10/30 (GE Healthcare) (ᶞ⬡㔞24 mL, 30×1.0 cm) ࡟⃰⦰ࡏࡎ࡟0.5 mL ࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ0.2 mL/minࠊศ⏬ࢧ࢖ࢬࢆ1.0 mL࡟タᐃࡋ

ࡓࠋୖグbuffer࡛ÄKTAࢩࢫࢸ࣒ࢆ⏝࠸࡚⁐ฟࢆ⾜ࡗࡓࠋ280 nm ࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ

࠸࡚ࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

⢭〇㓝⣲ࢱࣥࣃࢡ㉁ࡢࣉࣟࢸ࢔࣮ࢮᾘ໬࡜

Tricine SDS

࣏ࣜ࢔ࢡࣜࣝ࢔

࣑ࢻࢤࣝ㟁ẼὋື (Tricine SDS-PAGE)

Tricine SDS-PAGE㟁ẼὋືࡣࠊࣞࢰ࣐ࣝࢵࢡࢫ஧㐃࣑ࢽࢫࣛࣈ (ATTO, AE-6410) ࢆ⏝࠸ࠊ

⃰⦰ࢤࣝ㸦16.2%T, 6%C㸧ࠊࢫ࣮࣌ࢧ࣮ࢤࣝ㸦10%T, 3%C㸧ࠊศ㞳ࢤࣝ㸦4%T, 3%C㸧ࡢ୕ᒙ࠿

ࡽ࡞ࡿࢤࣝࢆస〇ࡋࡓࠋTris/Tricine/SDS⦆⾪ᾮ⣔࡛ࠊ150 V࡛2 hὋືࢆ⾜ࡗࡓࠋ

⢭〇㓝⣲ࡢࣉࣟࢸ࢔࣮ࢮᾘ໬ࡣ௨ୗࡢᡭ㡰࡛⾜ࡗࡓࠋ᭱⤊⢭〇㓝⣲60 PL (70 Pg) ࡟10%

SDS 1.22 PL ࢆຍ࠼ࠊ100 Υ, 5 min ⇕ฎ⌮ࡋ࡚㧗ḟᵓ㐀ࢆቯࡋࡓᚋࠊV8 ࣉࣟࢸ࢔࣮ࢮ㸦1

mg/mL㸧ࢆ1 PLῧຍࡋ37 Υ, 24 h཯ᛂࡉࡏࡓࠋࡇࡢࢧࣥࣉࣝ10 PL࡟SDSฎ⌮⦆⾪ᾮࢆ

10 PLຍ࠼ࠊ100 Υ࡛3 min⇕ฎ⌮ࢆ⾜ࡗࡓࡶࡢࢆTricine SDS⏝ヨᩱ⁐ᾮ࡜ࡋࡓࠋ

Nᮎ➃࠾ࡼࡧෆ㒊࢔࣑ࣀ㓟ࢩ࣮ࢡ࢚ࣥࢫゎᯒ

SDS-PAGE ࡢࢤࣝ࠿ࡽ PVDF ⭷࡬ࢱࣥࣃࢡ㉁ࢆ㌿෗ࡍࡿࡓࡵ࡟ࠊࣈࣟࢵࢸ࢕ࣥࢢ⁐ᾮA

⁐ᾮ (300 mM Tris-HCl buffer (pH 7.0)ࠊ5% ࣓ࢱࣀ࣮ࣝ)ࠊB⁐ᾮ (25 mM Tris-HCl buffer (pH 7.0)ࠊ5% ࣓ࢱࣀ࣮ࣝ)ࠊC⁐ᾮ (25 mM Tris-HCl buffer (pH 7.0)ࠊ40 mM 6-࢔࣑ࣀ࣊࢟ࢧࣥ㓟ࠊ 5% ࣓ ࢱ ࣀ ࣮ ࣝ) ࢆ ㄪ〇 ࡋ ࡓ ࠋSDS-PAGE ࡢ ࢤ ࣝ ࡜ ྠ ࡌ ࢧ ࢖ ࢬ ࡢ PVDF ࣓ ࣥ ࣈ ࣞ ࣥ

(MilliporeࠊImmobilon) ࢆ 100%࣓ࢱࣀ࣮ࣝ࡟20~30 ⛊㛫ᾐࡋࡓᚋࠊࣈࣟࢵࢸ࢕ࣥࢢ⁐ᾮ B

࡟ỿࡵࠊ30 min௨ୖ᣺࡜࠺ࡋࡓࠋࢤࣝ࡜ྠࡌࢧ࢖ࢬࡢࢁ⣬ࢆ6ᯛ⏝ពࡋࠊࣈࣟࢵࢸ࢕ࣥࢢ⁐

(30)

24

ᾮA࡟2ᯛࠊB࡟1ᯛࠊC࡟3ᯛࠊ⣙10 minᾐࡋࡓࠋࣈࣟࢵࢸ࢕ࣥࢢ⁐ᾮA࡟ᾐࡋࡓ2ᯛ

ࡢࢁ⣬ࢆࢺࣛࣥࢫࣇ࢓࣮⿦⨨ (ATTOࠊAE-6675) ࡢୗ㒊㟁ᴟᯈࡢୖ࡟✚ᒙࡋࡓࠋࣈࣟࢵࢸ࢕

ࣥࢢ⁐ᾮB࡟ᾐࡋࡓࢁ⣬ࢆ1ᯛࡑࡢୖ࡟✚ᒙࡋࠊࡑࡢୖ࡟࣓ࣥࣈࣞࣥࢆ㍕ࡏࠊࣈࣟࢵࢸ࢕

ࣥࢢ⁐ᾮBࢆᑡ㔞ࡓࡽࡋࡓࠋ᭱⤊⢭〇ࢧࣥࣉࣝࢆὋືࡋࡓSDS-PAGEࡢࢤࣝࢆ୍▐ࣈࣟࢵ

ࢹ࢕ࣥࢢ⁐ᾮB࡟ᾐࡋࡓᚋࠊ࣓ࣥࣈࣞࣥ࡟㍕ࡏࠊࣈࣟࢵࢸ࢕ࣥࢢ⁐ᾮC࡟ᾐࡋࡓ3ᯛࡢࢁ

⣬ࢆୖ࡟✚ᒙࡋࡓࠋ඲యࢆᢲࡋࡘࡪࡍࡼ࠺࡟ᅽ╔ࡋࠊୖ࠿ࡽࣈࣟࢵࢸ࢕ࣥࢢ⁐ᾮ C ࢆᑡ㔞 ࡓࡽࡋࡓࠋࢺࣛࣥࢫࣇ࢓࣮⿦⨨ࡢ⵹ࢆ㛢ࡵࠊୖ㒊㟁ᴟᯈࢆ㟼࠿࡟ࢁ⣬࡟ᐦ╔ࡉࡏࠊ100 mA ᐃ㟁ὶࡢ㏻㟁᮲௳࡛4 Υࠊ2 hࠊ㌿෗ࡋࡓࠋࡑࡢᚋ࣓ࣥࣈࣞࣥࢆྲྀࡾฟࡋࠊࢡ࣮࣐ࢩ࣮ࣈࣜ

ࣜ࢔ࣥࢺࣈ࣮ࣝᰁⰍ㸦ᰁⰍᾮࡣ㏻ᖖࡢ SDS-PAGE ࡜ྠࡌ㸧ࢆ10 min⾜ࡗࡓࠋḟ࡟ࠊ㏻ᖖࡢ

SDS-PAGE࡜ࡣ⤌ᡂࡢ␗࡞ࡿ⬺Ⰽᾮ (࣓ࢱࣀ࣮ࣝ㸸㓑㓟㸻 9㸸1) ࡛⬺Ⰽࡋࠊ࣓ࣥࣈࣞࣥࢆ

࢔࣑ࣝ࣍࢖ࣝࡢୖ࡛஝࠿ࡋࡓࠋ┠ⓗࡢࣂࣥࢻࢆ࢝ࢵࢱ࣮࡛ษࡾฟࡋࠊ⭷∦ࢆ㫽ྲྀ኱Ꮫ࣋ࣥࢳ

ࣕ ࣮ ࣅ ࢪ ࢿ ࢫ ࣛ ࣎ ࣛ ࢺ ࣜ ࣮ ࡢ ࣉ ࣟ ࢸ ࢖ ࣥ ࢩ ࣮ ࢡ ࢚ ࣥ ࢫ ゎ ᯒ ⿦ ⨨ (PPSQ-31A Protein Sequencer, Shimadzu) ࡛ゎᯒࢆ⾜ࡗࡓࠋ

DNAᢳฟ

Luteolibacter algae H18 ࡢࢻࣛࣇࢺࢤࣀ࣒㓄ิỴᐃࡣࠊᮾி㎰ᴗ኱Ꮫࡢ୰ᕝᩍᤵ࡟౫㢗ࡋ

ࡓࠋࡑࡢࡓࡵࡢ DNA ᢳฟࠊ⢭〇ࡣ௨ୗࡢ㏻ࡾ⾜ࡗࡓࠋࣇࢥ࢖ࢲࣥࢆ0.5%ྵࡴ᏶඲ྜᡂᇵ ᆅ (⾲2-1) 5 mL࡛30 Υࠊ72 h᣺࡜࠺ᇵ㣴ࡋࡓᇵ㣴ᾮࢆ࣐࢖ࢡࣟࢳ࣮ࣗࣈ࡟⛣ࡋࠊ4 Υ࡛

12000 rpm, 5 min㐲ᚰࡋ࡚ỿẊࢆᅇ཰ࡋࡓࠋDNAᢳฟ࡟ࡣPromega DNA Purification Kitࢆ౑

⏝ࡋࡓࠋỿẊࢆ50 mM EDTA (pH 7.5) 480 PL࡟ᠱ⃮ࡋࠊlytic enzyme 120 PLࢆຍ࠼37 Υ࡛

30~60 min཯ᛂࡋࡓࠋ15,000 rpm࡛2 min㐲ᚰࡋࠊୖΎࢆᤞ࡚ࡓỿẊ࡟Nuclei lysis solution 600 PLࢆຍ࠼ࠊᠱ⃮ࡋࠊ80 Υ࡛5 minಖ ࡋࡓࠋRNase solution (QIAGEN) 3 PLࢆຍ࠼࡚ΰ

ྜࡋࠊ37 Υ࡛15~60 min཯ᛂࡋࠊᐊ ࡲ࡛෭ࡸࡋࡓࠋḟ࡟ࠊProtein precipitation solution 200 PLࢆຍ࠼࡚ΰྜࡋࠊịୖ࡛5 minᨺ⨨ࡋࡓࠋ15,000 rpm࡛3 min㐲ᚰࡋࠊୖΎࢆ⁛⳦࣐࢖ࢡ

(31)

25

ࣟࢳ࣮ࣗࣈ࡟⛣ࡋࠊ࢖ࢯࣉࣟࣃࣀ࣮ࣝ600 PLຍ࠼࡚ΰྜࡋࡓࠋ15,000 rpm࡛5 min㐲ᚰࡋ

࡚ୖΎࢆᤞ࡚ࠊ70%࢚ࢱࣀ࣮ࣝ600 PLࢆຍ࠼ΰྜࡋࠊ15,000 rpm࡛2 min㐲ᚰࡋ࡚ୖΎࢆᤞ

࡚ࡓࠋ᭱ᚋ࡟ࠊࢹࢩࢣ࣮ࢱ࣮࡛15 min஝⇱ࡋࠊ Rehydration solution 100 PLຍ࠼ࠊ65 Υ࡛1 hಖ ࡋࡓᚋࡢ⁐ᾮࢆ DNA ࢧࣥࣉࣝ࡜ࡋࡓࠋ

(32)

26

⤖ᯝ

኱㔞ᇵ㣴࡜ C.F.E. ࡢㄪ〇

H18 ᰴࡢᇵ㣴ᾮ117.5 L࠿ࡽ473 gࡢ‵⳦యࡀᚓࡽࢀࠊ8622 mgࡢࢱࣥࣃࢡ㉁ࢆྵࡴ C. F.

E. ࢆㄪ〇ࡋࡓࠋ

DEAE-Sepharose FF

C.F.E. ࢆ20 mM Tris-HCl buffer (pH 8.0) ࡟ᑐࡋ࡚㏱ᯒᚋࠊDEAE-Sepharose FF࣒࢝ࣛ࡟࢔

ࣉࣛ࢖ࡋࡓࠋྛ NaCl ⃰ᗘ࡛⁐ฟࡋࡓ⏬ศࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆᐇ᪋ࡋࡓ⤖ᯝࠊ0.2 M NaCl⁐ฟ⏬ศ࡟࠾࠸࡚άᛶࡀ᳨ฟࡉࢀࠊFraction No. 669㹼689 ࢆᅇ཰ࡋࡓ (ᅗ2-1) ࠋ

ᅗ2-1. DEAE-Sepharose FF࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⁐ฟࣃࢱ࣮ࣥ

(33)

27

Phenyl-TOYOPEARL 650M

DEAE-Sepharose 0.2 M⁐ฟ⏬ศࢆ2.0 M (NH4)2SO4ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0)

࡟ᑐࡋ୍࡚ᬌ㏱ᯒᚋࠊPhenyl-TOYOPEARL࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛ(NH4)2SO4⃰ᗘ࡛⁐

ฟࡋࡓ⏬ศࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ1.0 M (NH4)2SO4⁐ฟ⏬ศ࡟࠾࠸࡚άᛶ ࡀ᳨ฟࡉࢀࠊFraction No. 412㹼465 ࢆᅇ཰ࡋࡓ (ᅗ2-2) ࠋ

ᅗ2-2. Phenyl-TOYOPEARL࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

(34)

28

Q-Sepharose FF

Phenyl-TOYOPEARL 1.0 M⁐ฟ⏬ศࢆ20 mM Tris-HCl buffer (pH 8.0) ࡟ᑐࡋ୍࡚ᬌ㏱ᯒ ᚋࠊQ-Sepharose FF࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛNaCl⃰ᗘ࡛⁐ฟࡋࡓྛศࢆᅇ཰ᚋࠊ㓝⣲ά ᛶ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ0.2 M NaCl⁐ฟ⏬ศ࡟࠾࠸࡚άᛶࡀ᳨ฟࡉࢀࠊFraction No. 602㹼666

ࢆᅇ཰ࡋࡓ (ᅗ2-3) ࠋ

ᅗ2-3. Q-Sepharose FF࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

(35)

29

Butyl-TOYOPEARL 650M

Q-Sepharose 0.2 M⁐ฟ⏬ศࢆ2.0 M (NH4)2SO4ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ୍ᬌ

㏱ᯒᚋࠊButyl -TOYOPEARL࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛ(NH4)2SO4⃰ᗘ࡛⁐ฟࡋࡓྛศࢆ

ᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ1.0 M (NH4)2SO4⁐ฟ⏬ศ࡟࠾࠸࡚άᛶࡀ᳨ฟࡉࢀࠊ Fraction No. 386㹼420 ࢆᅇ཰ࡋࡓ (ᅗ2-4) ࠋ

ᅗ2-4. Butyl-TOYOPEARL࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

(36)

30

Mono Q

^®HR

10/10

Butyl-TOYOPEARL 1.0 M⁐ฟ⏬ศࢆ㝈እࢁ㐣⭷ࢆ⏝࠸࡚⬺ሷ⃰⦰ࡋࠊ20 mM Tris-HCl

buffer (pH 8.0) ࡟bufferࢆኚ࠼ࡓࢧࣥࣉࣝ࡟ࡘ࠸࡚ࠊMono Q^® HR 10/10࣒࢝ࣛ࡟࢔ࣉࣛ࢖

ࡋࡓࠋ⁐ฟࡋࡓࣇࣛࢡࢩࣙࣥࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ1ᅇ┠ࡣFraction No.

44, 45ࠊ2ᅇ┠ࡣFraction No. 37࡟࠾࠸࡚άᛶࡀ᳨ฟࡉࢀྛࣇࣛࢡࢩࣙࣥࢆᅇ཰ࡋࡓ (ᅗ2-

5, 2-6) ࠋ

ᅗ2-6. Mono Q ^®HR 10/10 ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ1ᅇ┠ࡢ⁐ฟࣃࢱ࣮ࣥ

(37)

31

ᅗ2-6. Mono Q ^®HR 10/10 ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ2ᅇ┠ࡢ⁐ฟࣃࢱ࣮ࣥ

(38)

32

Superose 6

^®HR

10/30

Mono-Q⁐ฟࣇࣛࢡࢩࣙࣥࡢ࠺ࡕ2ᅇ┠ࡢFraction No. 37࡟ࡘ࠸࡚Superose 6 ^®HR 10/30

࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࢆ⾜࠸ࠊ⁐ฟࡋࡓࣇࣛࢡࢩࣙࣥࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗ

ࡓ⤖ᯝࠊFraction No. 12࡟άᛶࡀ᳨ฟࡉࢀࡓࡢ࡛ᅇ཰ࡋࡓ (ᅗ2-7) ࠋ᭱⤊⢭〇㓝⣲ࡢẚά

ᛶࡣC.F.E.࡟ẚ࡭81ಸୖ᪼ࡋࡓࡀࠊάᛶࡢᅇ཰⋡ࡣ0.07㸣࡛࠶ࡗࡓ (⾲2-4) ࠋ㓝⣲⢭〇

ྛẁ㝵࡟࠾ࡅࡿSDS-PAGEศᯒࡢ⤖ᯝࢆᅗ2-8࡟♧ࡍࠋ

ᅗ2-7. Superose 6 ^®HR 10/30 ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ㞳ࡋࡓ

(39)

33

⾲2-4. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇⤖ᯝ

Sample Total protein [mg]

Specific activity [mU/mg]

Total activity [mU]

Purification [fold]

Yield [%]

C.F.E. 8622.00 55.2 476000 1.00 100.00

DEAE-Sepharose 2791.00 58.9 164395 1.07 34.5000

Phenyl-

TOYOPEARL 411.00 144.0 59184 2.61 12.4000

Q-Sepharose 54.20 442.0 23956 8.01 5.0300

Butyl-

TOYOPEARL 3.80 1076.0 4090 19.50 0.8590

Mono-Q 0.35 4666.0 1633 84.50 0.3430

Superose 0.07 4686.0 328 84.90 0.0689

ᅗ2-8. H18ᰴ࠿ࡽ⢭〇ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢSDS-PAGEศᯒ

(40)

34

H18

ᰴࢤࣀ࣒

DNA

ࡢᢳฟ࡜ࢻࣛࣇࢺࢤࣀ࣒㓄ิゎᯒ

ᢳฟࡋࡓ H18 ᰴ DNA ࡢ⃰ᗘ࡜A260/A280್ࡣࠊ450 Pg/mLࠊ1.8 ࡛࠶ࡾࠊࢻࣛࣇࢺࢤࣀ࣒

ゎᯒ࡟㐺ࡋࡓࢧࣥࣉ࡛ࣝ࠶ࡗࡓࠋᅗ2-9࡟DNA ࢧࣥࣉࣝࡢ࢔࣮࢞ࣟࢫ㟁ẼὋືࡢࣃࢱ࣮ࣥ

ࢆ♧ࡍ㸦࡞࠾ࠊࡇࡇ࡛ࡣྠ᫬࡟ DNA ᢳฟࡋࡓ௚ࡢ⳦ᰴࡢ DNA ࢧࣥࣉࣝࡶὋືࡋ࡚࠸ࡿࠋ㸧 ḟୡ௦ࢩ࣮ࢡ࢚ࣥࢧ࣮࡟ࡼࡿゎᯒࡢ⤖ᯝࠊH18 ᰴࡢ඲ࢤࣀ࣒ DNA ࡢ඲㛗ࡣ 4155 kb ࡛࠶

ࡾࠊࢤࣀ࣒ゎᯒ࡟ࡣゎᯒࢯࣇࢺin silico Molecular Cloningࢆ౑⏝ࡋࡓࠋ

ᅗ2-9. H18ᰴ඲DNAࡢ࢔࣮࢞ࣟࢫ㟁ẼὋື⤖ᯝ

(41)

35

⢭〇㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิゎᯒ

⢭〇㓝⣲ࢆ SDS-PAGEࡢࢤࣝ࠿ࡽ PVDF ⭷࡟㌿෗ࡋࠊࡑࡢ࢔࣑ࣀ㓟㓄ิࡢỴᐃࢆヨࡳ

ࡓࡀࠊ᭷ព࡞ࢩࢢࢼࣝࢆᚓࡿࡇ࡜ࡀ࡛ࡁ࡞࠿ࡗࡓࠋࡑࡇ࡛ࠊV8ࣉࣟࢸ࢔࣮ࢮ࡟ࡼࡿࢱࣥࣃ

ࢡ㉁ࢆ⾜ࡗࡓᚋ࡟ SDS-PAGE ࡛ศ㞳ࡋࠊ෌ᗘࠊPVDF ⭷࡬㌿෗ࡋࠊ࢔࣑ࣀ㓟㓄ิࡢỴᐃ

ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊV(LS)SAEPN?MAIGL࠾ࡼࡧLQLED(GE)?Tࡢ㓄ิ᝟ሗࡀᚓࡽࢀ

ࡓࠋࡇࢀࡽࡢ㓄ิࢆH18ᰴࡢࢤࣀ࣒᝟ሗ࡟ᑐࡋ᳨࡚⣴ࡋࡓ⤖ᯝࠊ1ࡘࡢ ORFࡀぢࡘ࠿

ࡾࠊࡇࡢ ORFࠊࡍ࡞ࢃࡕࠊ┠ⓗࡢ࢔࣑ࣀ㓟㓄ิࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࢆ fud ࡜⛠ࡍࡿࡇ࡜

࡟ࡋࡓࠋᅗ 2-10 ࡟ࡣ fud ࠿ࡽ⏕ᡂࡍࡿࢱࣥࣃࢡ㉁ࡢ࢔࣑ࣀ㓟㓄ิࢆࠊᅗ2-11 ࡟ࡣࠊFud ࡢ࢔࣑ࣀ㓟㓄ิ࠾ࡼࡧࠊBLAST᳨⣴ࡢ⤖ᯝࠊ┦ྠᛶࡢ㧗࠸㓝⣲ࢱࣥࣃࢡ㉁࡜ࡋ࡚ࣄࢵࢺࡋ ࡓRhodopirellula sallentina⏤᮶ Dienelactone hydrolase ࡜Singulisphaera acidiphila⏤᮶ acetyl xylan deacetylase ࡜ࡢ࢔ࣛ࢖࣓ࣥࢺࢆ♧ࡋࡓࠋ

ᅗ2-10. Fud࠿ࡽ⏕ᡂࡍࡿࢱࣥࣃࢡ㉁ࡢ࢔࣑ࣀ㓟㓄ิ

ኴ

ኴᏐࡣࠊෆ㒊࢔࣑ࣀ㓟㓄ิゎᯒ࡛ᚓࡽࢀࡓ㓄ิ࡜୍⮴ࡋࡓ㓄ิࢆ♧ࡍࠋ

(42)

36

ᅗ2-12. H18ᰴ⏤᮶ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ (LC209227, Fud)ࠊRhodopirellula sallentina

⏤᮶ dienelactone hydrolase (WP_044303235.1, DH)ࠊSingulisphaera acidiphila⏤᮶ acetyl xylan deacetylase (WP 015248495.1, AE) ࡢ࢔࣑ࣀ㓟㓄ิ࢔ࣛ࢖࣓ࣥࢺ

⣽࠸ᐇ⥺ࡣ⢭〇ࢱࣥࣃࢡ㉁࠿ࡽᚓࡽࢀࡓෆ㒊࢔࣑ࣀ㓟㓄ิࢆࠊኴ࠸ᐇ⥺ࡣ඾ᆺⓗ࡞࢚ࢫࢸ࣮ࣛ

ࢮࣔࢳ࣮ࣇ࡛࠶ࡿG-X1-S-X2-Gࢆࠊᫍ༳ࡣẚ㍑ࡋࡓ3ࡘࡢ࢔࣑ࣀ㓟㓄ิ඲࡚ࡢ㛫࡛ྠ୍࡛࠶ࡿ

࢔࣑ࣀ㓟ṧᇶࢆ♧ࡍࠋ