p-nitrophenol ⃰ᗘ (mM)

49 㓝⣲ⓗ࡞⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡢㄪ〇

3-2-7 ࡢ᪉ἲ࡟ᚑࡗ࡚ Ni-Sepharose ࡟ࡼࡿ⢭〇ࢆ⾜ࡗࡓࠊ1.35 mg/ml ࣇࢥ࢖ࢲࣥࢹ࢔ࢭ

ࢳ࣮ࣛࢮࢆ15 mlࠊ2.5%ࣇࢥ࢖ࢲࣥࢆ40 ml㸦1000 mg ࡢࣇࢥ࢖ࢲࣥ⢊ᮎࢆྵࡴ㸧ࠊ1.0 M Tris-HCl Buffer (pH 8.0)ࢆ40 mlࠊ⵨␃Ỉࢆ305 mlࢆྵࡴྜィ 400 ml ࡢ⁐ᾮࢆࠊࣅ࣮࣮࢝ෆ࡛

30 Υ࡛㍍ࡃ᧠ᢾࡋ࡞ࡀࡽ24 h཯ᛂᚋࠊྠࡌ㓝⣲ࢆ15 ml ῧຍࡋࠊࡉࡽ࡟ 24 h ཯ᛂࡉࡏࠊ 80 Υ, 10 min⇕ฎ⌮ࡋࡓࠋᚓࡽࢀࡓ཯ᛂᾮࢆ14,000 rpm, 30 min㐲ᚰࡋࠊୖΎࢆVIVASPIN 㸦VS0102ࠊศ⏬ศᏊ㔞10,000䠅䛻䜘䜚⣙ 20 ml 䛻䜎䛷⃰⦰䛧䚸⵨␃Ỉ䛻ᑐ䛧䛶୍ᬌ㏱ᯒ䛧䛯ᚋ䚸

෾⤖஝⇱䛧䚸⬺䜰䝉䝏䝹໬䝣䝁䜲䝎䞁䛸䛧䛯䚹

50

⤖ᯝ

fud ࡢ

PCR

ቑᖜ

3-2-4 ࡢ᪉ἲ࡛PCRࢆ⾜ࡗࡓ⤖ᯝࠊfudࡢሷᇶ㛗࡛࠶ࡿ1.9 kbp௜㏆࡟ࣂࣥࢻࡀ☜ㄆ࡛ࡁ

ࡓࠋᚓࡽࢀࡓfud࡜pCold IࠊpCold TFࢆNde I ࡜Sal I࡛ᾘ໬ࡋࠊpCold I࡟┦ᙜࡍࡿ4407 bp ࡢࣂࣥࢻࠊpCold TF࡟┦ᙜࡍࡿ5769 bp ࡢࣂࣥࢻ࠾ࡼࡧ fud ࡟┦ᙜࡍࡿ 1887 bp ࡢࣂ

ࣥࢻ࡟ࡘ࠸࡚ࡑࢀࡒࢀษࡾฟࡋࢆ⾜࠸ࠊGel Extraction Kitࢆ⏝࠸࡚ᢳฟࡋࠊ15 PLࡢ⵨␃Ỉ

࡛⁐ฟࡉࡏࡓࠋ

3-2-5 ࡟♧ࡋࡓ᧯స࡛pCold I࡜fud࠶ࡿ࠸ࡣࠊpCold TF࡜fudࡢࣛ࢖ࢤ࣮ࢩࣙࣥࢆ⾜࠸ࠊ

E.coli DH5Dࢥࣥࣆࢸࣥࢺࢭࣝ࡬ࡢᑟධࢆ⾜ࡗࡓࠋ37 Υ୍࡛ᬌ㟼⨨ᇵ㣴ࡋࡓ࡜ࡇࢁࠊࢥࣟࢽ

࣮ࢆ☜ㄆ࡛ࡁࡓࠋ⏕ࡌࡓࢥࣟࢽ࣮ᩘಶࡎࡘ࠿ࡽࣉࣛࢫ࣑ࢻ༢㞳ࢆ⾜࠸ࠊCᮎ➃ഃࢆSalI (pCold Iࢆ࣋ࢡࢱ࣮࡜ࡋ࡚⏝࠸ࡓࡶࡢࡣEcoRI)ࠊNᮎ➃ഃࢆNdeI࡛ᾘ໬ࡋࡓᚋ࢔࣮࢞ࣟࢫ㟁ẼὋ

ືࢆࡋࡓ⤖ᯝࢆ ᅗ3-2࡟♧ࡍࠋpCold I࡜fudࢆࣛ࢖ࢤ࣮ࢩࣙࣥࡋࡓ⤌᥮࠼ࣉࣛࢫ࣑ࢻࡣࠊ fud࡜⪃࠼ࡽࢀࡿ1887 bp࡜pCold I࡜⪃࠼ࡽࢀࡿ4407 bp௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀぢࡽࢀࡿ

ࡣࡎ࡛ࠊᅗ3-2 ࡢ࣮ࣞࣥ7 ࡟┦ᙜࡍࡿࣃࢱ࣮ࣥࡀㄆࡵࡽࢀࡓࠋ୍᪉ࠊpCold TF࡜fudࢆࣛ

࢖ࢤ࣮ࢩࣙࣥࡋࡓ⤌᥮࠼ࣉࣛࢫ࣑ࢻࡣࠊfud࡜⪃࠼ࡽࢀࡿ1887 bp࡜pCold TF࡜⪃࠼ࡽࢀࡿ

5769 bp௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀぢࡽࢀࡿࡣࡎ࡛ࠊᅗ3-2 ࡢ࣮ࣞࣥ13, 14, 16 ࡟┦ᙜࡍࡿࣃ

ࢱ࣮ࣥࡀㄆࡵࡽࢀࡓࠋࡇࢀࡽ࡟┠ⓗDNA᩿∦ࡀྵࡲࢀ࡚࠸ࡿ࡜ุ᩿ࡋࠊࡇࢀࡽࣉࣛࢫ࣑ࢻ

ࢆ⏝࠸࡚E.coli BL21(DE3) ࢆᙧ㉁㌿᥮ࡋࡓࠋ

51

ᅗ3-2. ไ㝈㓝⣲ᾘ໬࡟ࡼࡾ⤌᥮࠼ࣉࣛࢫ࣑ࢻࢆ☜ㄆࡋࡓ࢔࣮࢞ࣟࢫ㟁ẼὋື⤖ᯝ

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

E. coli BL21(DE3)/pCold I/fudࡢⓎ⌧᳨ウࢆ3-2-6࡟ᚑࡗ࡚⾜ࡗࡓࠋSDS-PAGE࡟ࡼࡿࢱࣥ

ࣃࢡ㉁ࡢ⏕⏘᳨ウ⤖ᯝࢆᅗ3-3 ࡟♧ࡍࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡇࡢⓎ⌧࣋ࢡࢱ࣮࡛ࡣࠊ┠ⓗࢱࣥࣃ

ࢡ㉁ࡢ㢧ⴭ࡞⏕⏘ࡀぢࡽࢀ࡞࠿ࡗࡓࠋ

52

ᅗ3-3. E. coli BL21(DE3)/pCold I/fud ⏤᮶C.F.EࡢSDS-PAGE⤖ᯝ

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

Ⓨ⌧࣋ࢡࢱ࣮ pCold I ࡟ࡼࡿ㑇ఏᏊⓎ⌧ࡀ࠺ࡲࡃ࠸࠿࡞࠿ࡗࡓࡢ࡛ࠊ┠ⓗࢱࣥࣃࢡ㉁࡟

ྍ⏝ᛶࢱࢢࢆ௜ຍ࡛ࡁࡿⓎ⌧࣋ࢡࢱ࣮ pCold TF ࢆ⏝࠸ࡓ᳨ウࢆ 3-2-6 ࡟ᚑࡗ࡚⾜ࡗࡓࠋ

SDS-PAGE࡟ࡼࡿࢱࣥࣃࢡ㉁ࡢ⏕⏘᳨ウ⤖ᯝࢆᅗ3-4 ࡟♧ࡍࠋ࣮ࣞࣥ2࡟࠾࠸࡚⣙122 kDa

௜㏆࡟⃰࠸ࣂࣥࢻࡀ☜ㄆ࡛ࡁࠊࡇࡢ኱ࡁࡉࡣࠊ┠ⓗ㓝⣲ࢱࣥࣃࢡ㉁ (⣙70 kDa) ࡟His-Tag

ࢆྵࡴྍ⁐໬ࢱࢢ (⣙52 kDa) ࡀ௜ຍࡋࡓศᏊ㔞࡟୍⮴ࡋ࡚࠸ࡓࠋ

53

ᅗ3-4. E. coli BL21(DE3)/pCold TF/fud ⏤᮶C.F.EࡢSDS-PAGE⤖ᯝ

E.coli BL21(DE3)/pCold TF/fud ⏤᮶

C.F.E.

୰ࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳࣛ

࣮ࢮάᛶ

E. coli BL21(DE3)/pCold TF/fudࡼࡾㄪ〇ࡋࡓC.F.E.ࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ ᐃ

ࢆ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛3-2-12 ࡟♧ࡍ᧯స࡛⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ0 h࡛ࡣ㓑㓟ࡢ᳨ฟ ࡣ࡛ࡁ࡞࠿ࡗࡓࡀࠊ1 h཯ᛂᚋ࡟ࡣ114 mg/Lࡢ㓑㓟ࡢ㐟㞳ࡀ☜ㄆ࡛ࡁࡓࠋ ᅗ3-5࡟࢖࢜ࣥ

ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡿศᯒࢳ࣮ࣕࢺࢆ♧ࡍࠋ

ࡇࡢ⤖ᯝ࠿ࡽࠊE. coli BL21(DE3)/pCold TF/fud ࡢ C. F. E. ୰࡟ࡣࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳࣛ

࣮ࢮࡀᏑᅾࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊ2-2-6 ࡟♧ࡍ᪉ἲ࡛㓝⣲άᛶ ᐃࢆ⾜ࡗࡓሙྜ࡛

ࡶἼ㛗340 nmࡢ྾ගᗘୖ᪼ࡀㄆࡵࡽࢀࠊ␗࡞ࡿ஧ࡘࡢᡭἲࢆ⏝࠸࡚ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳࣛ

࣮ࢮάᛶࢆ᭷ࡋ࡚࠸ࡿࡇ࡜ࢆ☜ㄆࡋࡓࠋࡉࡽ࡟ࠊ㓝⣲཯ᛂࢆ3᪥㛫⾜ࡗࡓᚋ࡟࢖࢜ࣥࢡ࣐ࣟ

ࢺࢢࣛࣇ࢕࣮ศᯒࢆ⾜ࡗ࡚ࡶࠊ◲㓟࢖࢜ࣥࡣ඲ࡃ᳨ฟ࡛ࡁ࡞࠿ࡗࡓࠋ

54

ᅗ3-5. E. coli BL21(DE3)/pCold TF/fud C. F. E. ࢆ⏝࠸ࡓ㓝⣲཯ᛂᚋࡢ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛࣇ

࢕࣮ศᯒࡢࢳ࣮ࣕࢺ⤖ᯝ

Ni Sepharose 6 Fast Flow

࡟ࡼࡿ㓝⣲⢭〇 (1 ᅇ┠)

E. coli BL21(DE3)/pCold TF/fudࢆ12 Lᇵ㣴ࡋ࡚ᚓࡽࢀࡓC.F.E. (3834 mg-protein) ࡢ࠺ࡕࠊ

୍㒊ࡢ C.F.E. (1046 mg-protein) ࢆ 3-2-7 ࡢ᪉ἲ࡟ᚑ࠸ࠊ┠ⓗ㓝⣲ࡢ⢭〇ࢆ⾜ࡗࡓࠋྛ

Imidazole⃰ᗘࡢ buffer ࡛⁐ฟࡋࡓ⏬ศࢆᅇ཰ࡋࠊ20 mM Tris-HCl (pH 8.0) ࡟ᑐࡋ࡚㏱ᯒᚋࠊ 㓝⣲άᛶ ᐃࢆᐇ᪋ࡋࡓ⤖ᯝࠊ50 mM Imidazoleࢆྵࡴ buffer ࡛⁐ฟࡋࡓ⏬ศ (Fraction No.

127㹼185) ࡟࠾࠸࡚ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶࢆ᭷ࡍࡿ⼥ྜࢱࣥࣃࢡ㉁ (122 kDa)ࢆ᳨

ฟࡍࡿࡇ࡜ࡀ࡛ࡁࠊࡇࢀࡽࢆᅇ཰ࡋࡓࠋ

55

HRV 3C Protease

࡛ࡢࢱࢢ㓄ิࡢษ᩿࡜

Ni Sepharose 6 Fast Flow

࡟ࡼࡿ

㓝⣲⢭〇 (2 ᅇ┠)

3-3-5 ࡛ᚓࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶࢆ᭷ࡍࡿ⼥ྜࢱࣥࣃࢡ㉁ࢆ 3-2-8 ࡢ᪉ἲ࡛

HRV 3C Protease࡟ࡼࡿ㓝⣲཯ᛂࢆ⾜࠸ࠊHis-Tagࢆྵࡴྍ⁐໬ࢱࢢࡢ㒊ศ࡜┠ⓗ㓝⣲࡜ࢆษ

᩿ࡋࡓࠋḟ࠸࡛཯ᛂᾮࢆNi Sepharose 6 FF࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࢳ࣮ࣕࢪࡋࠊHis-Tag

ࢆྵࡴྍ⁐໬ࢱࢢࡢ㒊ศࢆᶞ⬡࡟྾╔ࡉࡏࡓࠋࡑࡋ࡚ࠊ20 mM Imidazoleࢆྵࡴ buffer ࡛⁐

ฟࡋࡓ⏬ศ (Wash⏬ศࠊFraction No. 11㹼18) ࡟ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶࢆ᭷ࡍࡿ┠

ⓗ㓝⣲ࢱࣥࣃࢡ㉁ (70 kDa)ࢆᚓࡿࡇ࡜ࡀ࡛ࡁࠊࡇࢀࡽࢆᅇ཰ࡋࡓࠋSDS-PAGEࡢ⤖ᯝࢆᅗ

3-6 ࡟♧ࡍࠋ2-2-6 ࡢ᪉ἲ࡛㓝⣲άᛶࢆ ᐃࡋࡓ⤖ᯝࠊ᭱⤊⢭〇㓝⣲ࡢẚάᛶࡣ C.F.E.࡟ẚ࡭

15ಸୖ᪼ࡋࡓࡀࠊάᛶࡢᅇ཰⋡ࡣ5.7㸣࡛࠶ࡗࡓ (⾲3-12)ࠋ

ᅗ3-6. E. coli BL21(DE3)/pCold TF/fud ࡢC.F.E.࠿ࡽ⢭〇ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ

SDS-PAGE⤖ᯝ

1. C. F. E. (20 Pg), 2. 1^st Ni-Sepharose (5.2 Pg),

3. After HRC 3C protease treatment (20 Pg), 4. 2^nd Ni-Sepharose (1.1 Pg)

56

⾲3-8. E. coli BL21(DE3)/pCold TF/fud࠿ࡽ⢭〇ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇⤖ᯝ

Sample

Total protein

Specific activity

Total

activity Yield Purification [ mg ] [ mU/mg ] [ mU ] [ % ] [ fold ]

C. F. E. 1046.0 30.0 31380 100.0 1.0

1st Ni Sepharose 61.9 467.0 28907 92.1 15.5 2nd Ni Sepharose 4.07 437.0 1779 5.7 14.6

⮳㐺 ᗘࠊ⮳㐺

pH

ࡢ᳨ウ

᳨ウࡢ⤖ᯝࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⮳㐺 ᗘࡣ40 Υ㸦ᅗ3-7㸧ࠊ⮳㐺 pH ࡣ8.0㸦ᅗ

3-8㸧࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ࡞࠾ࠊάᛶࡣ2-2-6 ࡢ᪉ἲ࡟ࡼࡾホ౯ࡋࡓࠋ

ᅗ3-7. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⮳㐺 ᗘ᳨ウ⤖ᯝ

57

ᅗ3-8. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⮳㐺pH᳨ウ⤖ᯝ

⇕Ᏻᐃᛶࡢ᳨ウ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⇕Ᏻᐃᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋάᛶࡣ2-2-6 ࡢ᪉ἲ࡟ࡼࡾホ౯ ࡋࡓࠋ30 ศ⇕ฎ⌮ᚋࡢ㓝⣲⁐ᾮࢆ⏝࠸࡚཯ᛂࢆ⾜ࡗࡓ࡜ࡇࢁࠊᮍฎ⌮ࡢ㓝⣲⁐ᾮࡢάᛶࢆ

100%࡜ࡍࡿ࡜ࠊ30 Υࡲ࡛ࡣ⣙ 80㸣ࡢṧᏑάᛶࢆಖᣢࡍࡿࡶࡢࡢࠊ35 Υ௨ୖ࡛㢧ⴭ࡟άᛶ

ࡀపୗࡋࡓࡇ࡜࠿ࡽࠊ⇕࡟ᑐࡍࡿᏳᐃᛶࡣ㧗ࡃ࡞࠸ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ㸦ᅗ3-9㸧ࠋ

ᅗ3-9. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⇕Ᏻᐃᛶ᳨ウ⤖ᯝ

58

㔠ᒓ࢖࢜ࣥࠊ㓝⣲㜼ᐖ๣ࡢάᛶ࡬ࡢᙳ㡪᳨ウ

ྛ✀㔠ᒓ࢖࢜ࣥࠊ࠾ࡼࡧ㓝⣲㜼ᐖ๣ࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ࡟ཬࡰࡍᙳ㡪ࢆ᳨

ウࡋࡓࠋ࡞࠾ࠊάᛶࡣ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾホ౯ࡋࡓࠋࡑࡢ⤖ᯝࠊ㔠ᒓ࡟㛵ࡋ࡚

ࡣ㖟ࠊ㖡ࠊள㖄ࠊࢥࣂࣝࢺࠊࢽࢵࢣࣝࠊỈ㖟ࡢῧຍ࡟ࡼࡾάᛶࡢ㜼ᐖࡀぢࡽࢀࡓ㸦ᅗ 3-10㸧ࠋ

୍᪉ࠊ㜼ᐖ๣࡟㛵ࡋ࡚ࡣSH㜼ᐖ๣࡟ࡼࡿάᛶࡢ㜼ᐖࡀぢࡽࢀࡓ㸦ᅗ 3-11㸧ࠋ

ᅗ3-10. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ࡟ཬࡰࡍ㔠ᒓ࢖࢜ࣥࡢᙳ㡪

ᅗ3-11. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ࡟ཬࡰࡍ㓝⣲㜼ᐖ๣ࡢᙳ㡪

59

ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟㓄ิ࡟ࡘ࠸࡚ BLAST ᳨⣴ࢆ⾜ࡗࡓ࡜ࡇࢁࠊ Dienelactone hydrolaseࠊacetylxylan esterase࡞࡝ࡀࣄࢵࢺࡋࡓ㸦⾲2-5㸧ࠋDienelactone hydrolase

࡟㛵ࡍࡿㄽᩥ࡛ࡣࠊ᭷ᶵ㓟ࡢp-nitrophenyl࢚ࢫࢸࣝ㸦ேᕤᇶ㉁㸧ࢆ⏝࠸࡚άᛶ ᐃࡀ⾜ࢃࢀ

࡚࠸ࡓࡓࡵࠊᮏ㓝⣲ࡢேᕤᇶ㉁࡟ᑐࡍࡿάᛶࢆ᳨ウࡍࡿࡇ࡜࡟ࡋࡓ [77] ࠋࡑࡢ⤖ᯝࠊp-nitrophenyl acetateࠊp-nitrophenyl propionate ࡟ ຍ ࠼ ࠊp-nitrophenyl butyrateࠊp-nitrophenyl

hexanoate ࡟ᑐࡍࡿάᛶࡀぢࡽࢀࡓࠋࡑࢀࡒࢀࡢᇶ㉁࡟ᑐࡍࡿάᛶࢆᅗ 3-12

࡟♧ࡍࠋp-nitrophenyl acetate࡛᭱ࡶ㧗࠸άᛶࡀぢࡽࢀࠊᇶ㉁ࡢⅣ⣲㙐ࡀ㛗ࡃ࡞ࡿ࡯࡝άᛶࡀపࡃ࡞ࡿഴ

ྥࡀぢࡽࢀࡿ୍᪉ࠊp-nitrophenyl laurate࡟ᑐࡍࡿάᛶࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋ

ᅗ3-12. ேᕤᇶ㉁࡟ᑐࡍࡿࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢάᛶ᳨ウ⤖ᯝ

࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿศゎࣉࣟࢭࢫ࡬ࡢ㛵୚࡟ࡘ࠸࡚ࡢ

᳨ウ

3-2-16 ࡛㏙࡭ࡓࠊDEAE-Sepharose 0.1 M NaCl⁐ฟ⏬ศࢆ㒊ศ⢭〇ࣇࢥ࢖ࢲࣥపศᏊ໬

㓝⣲࡜ࡋ࡚⏝࠸ࠊ㡯┠3-3-6࡛ᚓࡽࢀࡓ⢭〇㓝⣲ࢆࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ࡜ࡋ࡚⏝࠸

࡚ࠊE.coli BL21(DE3)/pCold TF/fud࡛⏕⏘ࡉࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢜࢟ࢼ࣡ࣔࢬ ࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢ㛵୚ࢆ᳨ウࡋࡓࠋ㡯┠3-2-16ࡢ᪉ἲ࡛㓝⣲཯ᛂࢆ⾜ࡗࡓ཯

60

ᛂᾮࢆ⏝࠸࡚ࠊHPLCศᯒࢆ⾜ࡗࡓ⤖ᯝࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡔࡅࢆῧຍࡋࡓ཯ᛂ ᾮ࡛ࡣࠊ཯ᛂࡢ๓ᚋ࡛ࣆ࣮ࢡࡢࣜࢸࣥࢩࣙࣥࢱ࢖࣒࡟㢧ⴭ࡞㐪࠸ࡣࡳࡽࢀ࡞࠿ࡗࡓ㸦ᅗ

3-13 (C)㸧ࠋ୍᪉ࠊపศᏊ໬㓝⣲ࡔࡅࢆῧຍࡋࡓ཯ᛂᾮ࡛ࡣⱝᖸࡢపศᏊ໬ࡀㄆࡵࡽࢀ㸦ᅗ

3-13 (B)㸧ࠊపศᏊ໬㓝⣲࡜ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ୧᪉ࢆῧຍࡋࡓ཯ᛂᾮ࡛ࡣࠊ㢧ⴭ࡟

పศᏊ໬ࡉࢀࡓࣇࢥ࢖ࢲࣥࡢࣆ࣮ࢡࢆ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡓ㸦⤖ᯝ♧ࡋ࡚࠸࡞࠸㸧ࠋࡇࡢ ࡇ࡜࠿ࡽࠊᮏ◊✲࡛ᚓࡽࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡀࣇࢥ࢖ࢲࣥపศᏊ໬࡟኱ࡁࡃ㛵

୚ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࡢ࡛ࠊḟ࡟2ࡘࡢ㓝⣲཯ᛂ㡰ᗎ࡟ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆඛ࡟ῧຍࡋࠊ⇕ฎ⌮ᚋࠊపศᏊ໬㓝⣲ࢆῧຍࡋࡓሙྜࠊపศ Ꮚ໬ࣇࢥ࢖ࢲࣥࡢࣆ࣮ࢡࢆ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡓ㸦ᅗ 3-13 (E)㸧ࠋ୍᪉࡛ࠊపศᏊ໬㓝⣲ࢆ

ඛ࡟ῧຍࡋࠊ⇕ฎ⌮ᚋࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆῧຍࡋࡓሙྜ࡛ࡣࠊᚋẁࡢ཯ᛂ๓ᚋ࡛

ࣆ࣮ࢡࡢࣜࢸࣥࢩࣙࣥࢱ࢖࣒࡟㢧ⴭ࡞㐪࠸ࡣぢࡽࢀ࡞࠿ࡗࡓ㸦ᅗ 3-13 (B, D)㸧ࠋ௨ୖࡢ⤖ᯝ

࠿ࡽࠊᮏ⳦ᰴ࡟ࡼࡗ࡚ࣇࢥ࢖ࢲࣥࡀศゎࡉࢀࡿ㝿ࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡀࠊ᭱ึ࡟ࣇ

ࢥ࢖ࢲࣥ࡟స⏝ࡋࡓᚋࠊࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲࡟ࡼࡗ࡚ࣇࢥ࢖ࢲࣥࡀపศᏊ໬ࡉࢀࡿ࡜

࠸࠺ࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ

61

ᅗ3-13. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ࡜పศᏊ໬㓝⣲ࢆ⏝࠸ࡓ㓝⣲཯ᛂ⁐ᾮࡢHPLCศᯒ

㓝⣲ⓗ࡞⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡢㄪ〇⤖ᯝ

3-2-17 ࡢ᪉ἲ࡟ࡼࡾࠊ1 g ࡢ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࢆཎᩱ࡟ࡋ࡚⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖

ࢲࣥࢆ1 gᚓࡿࡇ࡜ࡀ࡛ࡁࡓࠋ㓝⣲཯ᛂ๓ᚋࡢࢧࣥࣉࣝࡢศᏊ㔞࡟ኚ໬ࡣ࡞ࡃࠊಖᣢࡋ࡚࠸

ࡿ◲㓟ᇶࡶ཯ᛂ๓ᚋ࡛ 15㸣๓ᚋ࡜ኚ໬ࡀ࡞࠿ࡗࡓࠋ୍᪉ࠊ3-2-17 ࡢ㓝⣲཯ᛂᾮࢆ࢖࢜ࣥࢡ

62

࣐ࣟࢺࢢࣛࣇ࢕࣮ࠊF ࢟ࢵࢺ࡛ศᯒࡋࡓ࡜ࡇࢁࠊ㓑㓟࢖࢜ࣥࡢᏑᅾࡀ᫂ࡽ࠿࡟ㄆࡵࡽࢀࡓ (ᅗ3-14, 3-15) ࠋ

ᅗ3-14. 㓝⣲཯ᛂ๓ᚋ࡟࠾ࡅࡿ㓑㓟࢖࢜ࣥ⃰ᗘ⤖ᯝ

ᅗ3-15. 㓝⣲཯ᛂ๓ᚋ࡟࠾ࡅࡿ◲㓟ᇶྵ᭷⋡

63

⪃ᐹ

ᮏ❶࡛ࡣࠊ➨ 2 ❶࡛ᚓࡽࢀࡓ㑇ఏᏊ࠾ࡼࡧ࢔࣑ࣀ㓟㓄ิࢆᇶ࡟࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲ

ࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮ㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ 㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆ࠾ࡇ࡞ࡗࡓࠋ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲ

ࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㑇ఏᏊ࠾ࡼࡧ㓝⣲ࢱࣥࣃࢡ㉁࡟ࡘ࠸࡚

ࡣࠊㄪ࡭ࡓ㝈ࡾ࡟࠾࠸࡚ࠊࡑࡢሗ࿌౛ࡣ࡞࠸ࠋ

㑇ఏᏊⓎ⌧࡟࠾࠸࡚ࠊࢹ࣮ࢱࢆ♧ࡋ࡚ࡣ࠸࡞࠸ࡀࠊpET࣋ࢡࢱ࣮⣔࡛ࡣⓎ⌧ࡉࡏࡿࡇ࡜ࡀ

࡛ࡁ࡞࠿ࡗࡓࠋࡉࡽ࡟ࠊప ࡛ຠ⋡ⓗ࡟Ⓨ⌧ྍ⬟࡛࠶ࡿ pCold ࣋ࢡࢱ࣮ࢆ⏝࠸࡚ࡶ㑇ఏᏊ ࡢⓎ⌧ࡣぢࡽࢀ࡞࠿ࡗࡓ㸦ᅗ 3-5㸧ࠋࡑࡇ࡛ࢺ࣮ࣜ࢞ࣇ࢓ࢡࢱ࣮࡜ࡢ⼥ྜࢱࣥࣃࢡ㉁࡜ࡋ࡚

㑇ఏᏊⓎ⌧ࢆ⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࠊpCold TFⓎ⌧࣋ࢡࢱ࣮ࢆ౑⏝ࡋࡓࠋࡑࡢ⤖ᯝࠊᅗ3-4࡟♧

ࡍࡼ࠺࡟ࠊ⼥ྜࢱࣥࣃࢡ㉁ࡀ↓⣽⬊ᢳฟᾮ୰࡟㐣๫࡟⏕⏘ࡉࢀࠊࡉࡽ࡟ࡑࡢ㓝⣲ࢆ⏝࠸࡚ࣇ

ࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࡓ㓝⣲཯ᛂࢆ⾜࠺ࡇ࡜࡟ࡼࡾࠊ㓑㓟ࡢ㐟㞳ࢆ☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋ⤌

᥮࠼኱⭠⳦ࡼࡾ⏕⏘ࡋࡓ㓝⣲ࡣࠊ㔠ᒓ࣮࢟ࣞࢺ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊ

HRV 3C Protease࡟ࡼࡾࢺ࣮ࣜ࢞ࣇ࢓ࢡࢱ࣮ࡢษ᩿ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝᚓࡽࢀࡓ⢭〇㓝⣲ࡢ

ࢧࣈࣘࢽࢵࢺࡢศᏊ㔞ࡣࠊ㔝⏕ᰴ⏤᮶ࡢ㓝⣲ࡢࡶࡢ࡜୍⮴ࡋ࡚࠸ࡓࠋ

௒ᅇ⢭〇ᶆရࡀᚓࡽࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ࡜㒊ศ⢭〇ࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲

ࢆ⏝࠸ࡓ㓝⣲཯ᛂࡢ⤖ᯝࡼࡾࠊH18ᰴ࡟ࡼࡿࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫࢆᅗ3-16࡟ᥦၐࡍࡿࠋ ࡍ࡞ࢃࡕࠊࡲࡎࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࡀ㉳ࡇࡾࠊࡑࡢᚋࠊపศᏊ໬཯ᛂࡀ㐍⾜ࡍࡿ

ࡶࡢ࡜⪃࠼ࡽࢀࡓࠋࡓࡔࠊH18 ᰴ࡟ࡼࡿࣇࢥ࢖ࢲࣥศゎࡢ㐣⛬࡛◲㓟ࡢ㐟㞳ࡶㄆࡵࡽࢀ࡚

࠾ࡾࠊᮏ⳦ᰴࡢࣇࢥ࢖ࢲࣥศゎ࡛ࡣ⬺◲㓟໬㓝⣲ࡶస⏝ࡍࡿ࡜⪃࠼ࡽࢀࡿࡀࠊࡇࡢ㓝⣲࡟㛵 ࡍࡿ᳨ウࡣ௒ᚋࡢㄢ㢟࡛࠶ࡿࠋ

64

ᅗ3-16. ᝿ᐃࡉࢀࡿH18ᰴ࡟ࡼࡿ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ

㒊ศ⢭〇ࡋࡓࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࢆ⏝࠸ࡓ㝿ࠊࣇࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂࡀࢃࡎ࠿࡟

㐍⾜ࡍࡿ࡜࠸࠺⌧㇟ࡀぢࡽࢀࡓ (ᅗ3-13 (B)) ࠋࡇࡢཎᅉ࡜ࡋ࡚ࠊᑡ㔞ࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭ

65

ࢳ࣮ࣛࢮࡀ㒊ศ⢭〇㓝⣲ᶆရ࡟ΰධࡋ࡚࠾ࡾࠊࡑࡢ⤖ᯝࠊ⏕ᡂࡋࡓ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ

࡟పศᏊ໬㓝⣲ࡀస⏝ࡋࡓྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊFormosa algae⏤᮶ࡢࣇࢥ࢖ࢲࣥప ศᏊ໬㓝⣲ࡣࠊFucus evanescens⏤᮶ࡢኳ↛ࣇࢥ࢖ࢲࣥࡼࡾࡶࡑࡢ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ

ࢆຠ⋡ⓗ࡟ศゎࡋࡓ࡜࠸࠺ሗ࿌౛ࡶ࠶ࡿ [47] ࠋࡇࡢࡇ࡜࠿ࡽࠊࣇࢥ࢖ࢲࣥࡢపศᏊ໬࡟㛵

୚ࡍࡿ㓝⣲ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࢡ࣮ࣟࢽࣥࢢࡀᚲ㡲࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ

ᮏ◊✲࡟ࡼࡾᚓࡽࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡣࠊࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚⬺࢔ࢭࢳࣝ໬ά ᛶࢆ᭷ࡋ࡚࠸ࡿࡀࠊ⬺◲㓟໬άᛶࢆ♧ࡉ࡞࠸ࡓࡵࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆ⏝࠸ࡿࡇ

࡜࡛ࠊ◲㓟ᇶࡢྵ᭷㔞࠾ࡼࡧศᏊ㔞ࡢῶᑡࢆࡉࡏࡿࡇ࡜࡞ࡃ⬺࢔ࢭࢳࣝ໬࢜࢟ࢼ࣡ࣔࢬࢡ

ࣇࢥ࢖ࢲࣥࢆㄪ〇ࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ࢔ࢭࢳࣝᇶ࡜⏕⌮άᛶ

࡜ࡢ┦㛵ᛶࡣ᫂ࡽ࠿࡟ࡉࢀ࡚࠸࡞࠸ࡢࡀ⌧≧࡛࠶ࡾࠊᮏ◊✲࡛ᚓࡽࢀࡓ⬺࢔ࢭࢳࣝ໬ࣇࢥ

࢖ࢲࣥ࡜ኳ↛ࣇࢥ࢖ࢲࣥ࡜ࢆẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾ✀ࠎࡢ⏕⌮άᛶࡢ㐪࠸ࢆ᳨ウࡍࡿࡇ࡜ࡀ

ྍ⬟࡜࡞ࡗࡓ࡜⪃࠼ࡽࢀࡿࠋ

66

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢゐ፹ṧᇶࡢ ண 

⥴ゝ

➨ 3 ❶࡛ࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮ㑇 ఏᏊࡢࢡ࣮ࣟࢽࣥࢢཬࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆ

⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ኱⭠⳦࡟࠾࠸࡚ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⏕⏘ࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧

၀ࡉࢀࠊࡉࡽ࡟⏕⏘ࡉࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆ⏝࠸ࡿࡇ࡜࡛࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ

࢖ࢲࣥࡢศᏊ㔞ࢆῶᑡࡉࡏࡿࡇ࡜࡞ࡃ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࢆㄪ〇ࡍࡿࡇ࡜ࡀྍ⬟࡟࡞

ࡗࡓࠋࡲࡓࠊࡇࡢࢹ࢔ࢭࢳ࣮ࣛࢮࡣ㧗ศᏊ㔞ࡢࣇࢥ࢖ࢲࣥ࠿ࡽ㓑㓟ࢆ㐟㞳ࡉࡏࡿࡔࡅ࡛ࡣ࡞

ࡃࠊపศᏊ㔞ࡢ p-ࢽࢺࣟࣇ࢙ࢽࣝ࢔ࢭࢸ࣮ࢺ➼ࡢேᕤᇶ㉁ࡢຍỈศゎࡶゐ፹࡛ࡁࡓࠋ୍᪉

➨ 2 ❶࡛ࡣࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟㓄ิ୰࡟ G-X1-S-X2-G ࣔࢳ࣮ࣇࡸ

L(Q)T(F)F(Y)DDPG ࡜ HTW(Y)S(AD)H ࢆྵࡴ 2 ࡘࡢࣔࢳ࣮ࣇࢆ☜ㄆࡍࡿࡇ࡜ࡀฟ᮶࡞࠿

ࡗࡓࡇ࡜࠿ࡽࠊᮏ㓝⣲ࡀ࢔ࢭࢳࣝࢢࣝࢡࣟ࢟ࢩࣛࣥࡢ⬺࢔ࢭࢳࣝ໬ࢆゐ፹ࡍࡿ㓝⣲ [75] ࡸ

࢟ࢳࣥࢹ࢔ࢭࢳ࣮ࣛࢮ [76]࡜ࡣ␗࡞ࡿゐ፹ࣔࢳ࣮ࣇࢆ᭷ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

ࡑࡇ࡛ࠊᮏ❶࡛ࡣ➨ 2 ❶࡛ᚓࡽࢀࡓ࢔࣑ࣀ㓟㓄ิࢆᇶ࡟ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண  ᵓ㐀ࡢసᡂࠊゐ፹ṧᇶࡢண ࠾ࡼࡧࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ࡟ᚲせ࡞㡿ᇦࡢ᥈⣴ࢆ࠾ࡇ࡞ࡗ ࡓࠋ

67

ᐇ㦂᪉ἲ

DNA

᧯స

DNA ࢆᑐ㇟࡜ࡍࡿᐇ㦂᪉ἲࡣ2❶ࠊ3❶࡟グ㍕ࡋࡓ᪉ἲ࡜ྠࡌ࡛࠶ࡿࠋ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண ᵓ㐀ࣔࢹࣝ

SWISS-MODEL (http://swissmodel.expasy.org/) ࡟ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟㓄

ิࢆධຊࡋࠊࣄࢵࢺࡋࡓࢸࣥࣉ࣮ࣞࢺࢆ⏝࠸࡚ண ᵓ㐀ࣔࢹࣝࡢసᡂࢆ⾜ࡗࡓࠋ

ࣉࣛ࢖࣐࣮ࡢタィ

pCold TF࣋ࢡࢱ࣮࡟Cᮎ➃㓄ิࢆ๐㝖ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࢆࢥ࣮ࢻࡍࡿ㑇ఏ

Ꮚࢆࢡ࣮ࣟࢽࣥࢢࡍࡿ㝿ࠊ┠ⓗ DNA ᩿∦ࢆ PCR ቑᖜࡍࡿࡓࡵ࡟ࠊForward ࣉࣛ࢖࣐࣮࠾

ࡼࡧReverseࣉࣛ࢖࣐࣮ࡢタィࢆ⾜ࡗࡓࠋࡑࢀࡒࢀࡢࣉࣛ࢖࣐࣮ࡢሷᇶ㓄ิࢆ⾲4-1࡟ࢆ♧

ࡍࠋForwardࣉࣛ࢖࣐࣮ fudF1 ࡟ࡣNde I ไ㝈㓝⣲ㄆ㆑㓄ิࢆࠊReverseࣉࣛ࢖࣐࣮ fudR1

㹼fudR6࡟ࡣSal Iไ㝈㓝⣲ㄆ㆑㓄ิࢆᣢࡓࡏࠊࡑࢀࡒࢀࢆኴᏐ࡛♧ࡋࡓࠋእὀࡋࠊ㏦ࡽࢀ

࡚ࡁࡓࣉࣛ࢖࣐࣮࡟⁛⳦Ỉࢆῧຍࡋ࡚100 PMࡢ⁐ᾮࢆㄪ〇ࡋࠊࡑࢀࢆ⁛⳦Ỉ࡛ᕼ㔘ࡋࡓ1 PMࡢࡶࡢࢆPCR ᐇ㦂࡟⏝࠸ࡓࠋ

68

⾲4-1. ྛࣉࣛ࢖࣐࣮㓄ิ

ࣉࣛ࢖࣐࣮ 㓄ิ

fudF1 (Forwardࣉࣛ࢖࣐࣮) CCACGGCATATGAAAATCCTCAAAATC

fudR1 CAACACGTCGACTCAGATCTCGAAGCTACC

fudR2 AATTAAGTCGACTCACTCCCTCGAGGCATC

fudR3 AATTACGTCGACTCAATGCCAGAAACGCG

fudR4 ACAGTCGACTCAATTGAAGCGATCCGTG

fudR5 AATGTCGACTCAGACCTTGTGGGTCGC

fudR6 ACAGTCGACTCAGCTTTCGAAGTCTGC

ࣉࣛ࢖࣐࣮ࡢ⤌ࡳྜࢃࡏࡣ࠙Fud664-1ࠚfudF1 ̽ fudR1ࠊ࠙Fud664-2ࠚfudF1 ̽ fudR2ࠊ

࠙Fud664-3ࠚfudF1 ̽ fudR3ࠊ࠙Fud664-4ࠚfudF1 ̽ fudR4ࠊ࠙Fud664-5ࠚfudF1 ̽ fudR5ࠊ

࠙Fud664-6ࠚfudF1 ̽ fudR6

PCR

ቑᖜ

⾲3-4࡟♧ࡍ⤌ᡂ࡛PrimeSTAR Max DNA polymerase (TaKaRa Bio.)ࢆྵࡴ཯ᛂᾮࢆㄪ〇ᚋࠊ TaKaRa Thermal Cycler Dice^® Touchࢆ⏝࠸࡚PCRࢆ⾜ࡗࡓࠋPCRቑᖜ࡛ࡣࡲࡎࠊ94 ℃࡛2 ศ㛫ຍ⇕ࡋࠊḟ࡟30ࢧ࢖ࢡࣝ (98 Υ࡛10 ⛊㛫ࠊ63 Υ (ࡲࡓࡣ65 Υ) ࡛5 ⛊㛫ࠊ࠾ࡼࡧ 72 Υ࡛ 1.5 ศ㛫) ࡢఙ㛗཯ᛂࢆ⾜ࡗࡓࠋ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື࡜ษࡾฟࡋࢆ⾜࠸ࠊ

NucleoSpin^® Gel and PCR Clean-upࢆ⏝࠸࡚௜ᒓࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚⢭〇ࡋࡓࠋ

ࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ᳨ウ

2 mg/ml ↓⣽⬊ᢳฟᾮࠊ0.25% ࣇࢥ࢖ࢲࣥࠊ100 mM Tris-HCl buffer (pH 8.0) ࢆྵࡴ཯ᛂᾮ

400 Plࢆ30Υ, 24 h࢖࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ80 Υ, 5 min⇕ฎ⌮ࡋࠊ࢖࢜ࣥࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮

࡟ࡼࡗ࡚㓑㓟⃰ᗘࢆ ᐃࡋࡓࠋ

69

ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ

0.1 mg/ml ↓⣽⬊ᢳฟᾮࠊ1 mM ேᕤᇶ㉁ࠊ100 mM Tris-HCl buffer (pH 7.0) ࢆྵࡴ཯ᛂᾮ 1 ml ࢆ 30 Υ࡛࢖࣮ࣥ࢟ࣗ࣋ࢺ (p-nitrophenyl acetate ࡢሙྜࡣ 20 minࠊࡑࡢ௚ࡢᇶ㉁ࠊp-nitrophenyl propionate, p-minࠊࡑࡢ௚ࡢᇶ㉁ࠊp-nitrophenyl butyrate, p-minࠊࡑࡢ௚ࡢᇶ㉁ࠊp-nitrophenyl hexanoateࡢሙྜࡣ60 min) ᚋࠊ

410 nmࡢ྾ගᗘࢆ ᐃࡋࠊάᛶࢆồࡵࡓࠋ᳨㔞⥺ࡣ100 mM Tris-HCl buffer (pH 7.0) ࢆྵࡴ

0.02 mM㹼0.2 mMࡢp-nitrophenolࢆ⏝࠸࡚సᡂࡋࡓࠋ

70

⤖ᯝ

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண ᵓ㐀ࣔࢹࣝࡢసᡂ

SWISS-MODEL (http://swissmodel.expasy.org/) ࡛ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟㓄ิ

ࢆ᳨⣴ࡋࡓ࡜ࡇࢁࠊࢸࣥࣉ࣮ࣞࢺ࡜ࡋ࡚acetyl xylan esteraseࠊcephalosporin C deacetylase࡞

࡝ࡀࣄࢵࢺࡋࡓࠋࡑࡢ୰ࡢacetyl xylan esterase ࡜࢔ࣀࢸ࣮ࢩࣙࣥࡉࢀ࡚࠸ࡿTM0077ࢆࢸ

ࣥࣉ࣮ࣞࢺ࡜ࡋࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢᵓ㐀ࣔࢹࣝࢆసᡂࡋࡓ (ᅗ4-1) ࠋ

ᅗ4-1. SWISS-MODEL࡟ࡼࡿࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢண ᵓ㐀ࣔࢹࣝ

TM0077ࡣࠊacetyl xylan esterase࡜࢔ࣀࢸ࣮ࢩࣙࣥࡉࢀ࡚࠸ࡿࡀࠊ࢔ࢭࢳࣝ࢟ࢩࣛࣥ࡟ᑐ

ࡍࡿάᛶࡣᣢࡓ࡞࠸࡜ሗ࿌ࡉࢀ࡚࠸ࡿ [78]ࠋࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢNᮎ➃㡿ᇦࡀࠊ

TM0077ࡢ඲㛗࡜❧యᵓ㐀ࡀ㢮ఝࡋ࡚࠾ࡾࠊࡇࡢ㒊ศࡢᵓ㐀ࣔࢹࣝࢆసᡂࡋࡓࠋCᮎ➃ഃࡣ

ࢸࣥࣉ࣮ࣞࢺࡀぢࡘ࠿ࡽ࡞࠿ࡗࡓࡓࡵࠊᵓ㐀ࢆண ࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠿ࡗࡓࠋTM0077࡜ࣇ

71

ࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢᵓ㐀ࢆ㔜ࡡྜࢃࡏࡓ࡜ࡇࢁࠊTM0077ࡢゐ፹ṧᇶ࡜᝿ᐃࡉࢀ࡚࠸

ࡿSerࠊAspࠊHisࡢ఩⨨ࡀࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ࡜୍⮴ࡋ࡚࠾ࡾࠊࡇࢀࡽࡢṧᇶࡀࣇࢥ

࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢゐ፹ṧᇶ࡛࠶ࡿ࡜ண᝿ࡉࢀࡓ (ᅗ4-2)ࠋ

༙㏱᫂ࡢ㒊ศ㸸TM0077 (ࢸࣥࣉ࣮ࣞࢺ)

ᅗ4-2. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ᝿ᐃࡉࢀࡿゐ፹ṧᇶ

Fud ࡜⛠ࡋࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ࡜ TM0077 ࡢ࢔࣑ࣀ㓟㓄ิࡢẚ㍑ࢆᅗ 4-3 ࡟♧

ࡍࡀࠊࡑࡢ┦ྠᛶࡣ 16% ࡛࠶ࡿࠋFud ࡢNᮎ➃ഃ࡜TM0077 ࡟Ꮡᅾࡍࡿ3ࡘࡢ᥎ᐃゐ፹ ṧᇶࡢ఩⨨ࢆ㉥Ꮠ࡛♧ࡋ࡚࠸ࡿࠋ

72

ᅗ4-3. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮ (Fud) ࡜TM0077ࡢ࢔࣑ࣀ㓟㓄ิẚ㍑

73

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ

C

ᮎ➃ഃḞᦆࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿⓎ

⌧ᰴࡢᵓ⠏

ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢCᮎ➃ഃ࡟ࡣࠊTM0077࡟ࡣぢࡽࢀ࡞࠸㡿ᇦ (ᅗ4-3ࡢ㟷Ⰽ ࡢⅬ⥺࡛ᅖࡲࢀࡓ㒊ศ) ࡀᏑᅾࡍࡿࠋࡇࡢ㡿ᇦࢆḞᦆࡉࡏࡓࢱࣥࣃࢡ㉁ࢆ⏕⏘ࡋࠊCᮎ➃㡿 ᇦࡀάᛶ࡟ཬࡰࡍᙳ㡪ࢆ᳨ウࡋࡓࠋ௒ᅇࠊᅗ4-4࡟♧ࡍࡼ࠺࡟ࠊCᮎ➃ഃࡢ㛗ࡉࢆኚ࠼ࡓ6

✀㢮ࡢࢱࣥࣃࢡ㉁ࢆFud-1 ~ Fud-6࡟ࡘ࠸᳨࡚ウࡋࡓࠋ

ᅗ4-4. Cᮎ➃ഃࢆḞᦆࡉࡏࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟ṧᇶᩘ

⾲4-1ࡢࣉࣛ࢖࣐࣮ࢆ⏝࠸࡚ࠊ㡯┠4-2-4ࡢ㏻ࡾ࡟PCRࢆ⾜ࡗࡓ⤖ᯝࠊfud-1 ~ fud-6ࡢሷ ᇶ㛗࡛࠶ࡿ1059 bpࠊ1143 bpࠊ1233 bpࠊ1395 bpࠊ1557 bpࠊ1725 bp௜㏆࡟ࣂࣥࢻࡀ☜ㄆࡉࢀ

ࡓࠋᚓࡽࢀࡓ DNA ᩿∦࡜ pCold TF ࣋ࢡࢱ࣮ࢆNdeI࡜SalI࡛ไ㝈㓝⣲ᾘ໬ࡋࠊࣛ࢖ࢤ࣮

ࢩࣙࣥࢆ⾜࠸ࠊE. coli DH5Dࢆᙧ㉁㌿᥮ࡋࡓࠋ37Υ୍࡛ᬌ㟼⨨ᇵ㣴ࡋࠊ⏕ࡌࡓࢥࣟࢽ࣮࠿ࡽ

ࣉࣛࢫ࣑ࢻ༢㞳ࢆ⾜࠸ࠊไ㝈㓝⣲࡛ᾘ໬ࡋࡓᚋ࢔࣮࢞ࣟࢫ㟁ẼὋືࢆࡋࡓࠋࡑࡢ࠺ࡕࠊ fud-1㹼fud3 ࡢ⤖ᯝࢆ ᅗ4-5࡟♧ࡍࠋfud-1 ~ fud-6ࠊpCold TFࡢሷᇶ㛗௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀ ぢࡽࢀࡓࡢ࡛ࠊᚓࡽࢀࡓࣉࣛࢫ࣑ࢻ୰࡟┠ⓗDNA᩿∦ࡀྵࡲࢀ࡚࠸ࡿ࡜ุ᩿ࡋࠊࡇࡢࣉࣛ

ࢫ࣑ࢻࢆ⏝࠸࡚E.coli BL21(DE3) 䜢ᙧ㉁㌿᥮䛧䛯䚹

74

ᅗ4-5. fud-1㹼fud-3 ࢆࢡ࣮ࣟࢽࣥࢢࡋࡓࣉࣛࢫ࣑ࢻࢆไ㝈㓝⣲ᾘ໬ࡋࡓᚋࡢ

࢔࣮࢞ࣟࢫ㟁ẼὋື⤖ᯝ

⤌ࡳ᥮࠼ᰴࡢⓎ⌧᳨ウ

㻌 ⤌䜏᥮䛘ᰴ࡟ࡼࡿ┠ⓗ㑇ఏᏊࡢⓎ⌧࡟ࡘ࠸࡚ࠊSDS-PAGE࡟ࡼࡾ᳨ウࡋࡓ㸦ᅗ4-6㸧ࠋྍ⁐

ᛶࠊ୙⁐ᛶ࡝ࡕࡽࡶ┠ⓗࢱࣥࣃࢡ㉁ࡢศᏊ㔞࡛࠶ࡿ91 kDaࠊ94 kDaࠊ98 kDaࠊ104 kDaࠊ110

kDaࠊ116 kDa௜㏆࡟ࡍ࡭࡚ࡢ㑇ఏᏊࡢ㧗Ⓨ⌧ࡀ☜ㄆ࡛ࡁࡓࠋ

75

ᅗ4-6. fud-1㹼fud-6ࢆ᭷ࡍࡿ⤌ࡳ᥮࠼ᰴࡢ↓⣽⬊ᢳฟᾮࡢSDS-PAGE⤖ᯝ

ࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿάᛶ᳨ウ

㻌 㡯┠4-3-3䛷ᚓ䜙䜜䛯↓⣽⬊ᢳฟᾮ䜢⏝䛔䛶䚸ࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࡓ㓝⣲཯ᛂࢆ⾜࠸ࠊ㓑

㓟⃰ᗘࢆ ᐃࡋࡓ࡜ࡇࢁࠊFud-1~4࡛ࡣ㓑㓟ࡀ᳨ฟࡉࢀࡎࠊFud-5, 6࡛ࡣࢃࡎ࠿࡟㓑㓟ࡀ᳨

ฟࡉࢀࡓࡀࠊ඲㛗ࡢFud࡜ẚ㍑ࡍࡿ࡜ࠊάᛶࡣ࡯࡜ࢇ࡝↓࠸࡜⪃࠼ࡽࢀࡓ㸦ࢹ࣮ࢱࡣ♧ࡉ࡞

࠸㸧ࠋ

76

ேᕤᇶ㉁࡟ᑐࡍࡿάᛶ᳨ウ

㡯┠4-3-3࡛ᚓࡽࢀࡓ↓⣽⬊ᢳฟᾮࢆ⏝࠸࡚ࠊேᕤᇶ㉁࡟ᑐࡍࡿάᛶࢆ ᐃࡋࡓ⤖ᯝࢆᅗ

4-7 ࡟♧ࡍࠋCᮎ➃ࢆ๐ࡗࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ୰࡛᭱ࡶ㛗࠸ Fud-6 ࡛ࡶ Fud ࡟ ẚ࡭࡚ࢃࡎ࠿࡞άᛶࡋ࠿ぢࡽࢀࡎࠊCᮎ➃㡿ᇦࡣάᛶ࡟ᚲせ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ

ᅗ4-7. Fud1㹼6ࡢேᕤᇶ㉁࡟ᑐࡍࡿẚάᛶ

77

⪃ᐹ

ᮏ❶࡛ࡣ➨2❶࡛ᚓࡽࢀࡓࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ࢔࣑ࣀ㓟㓄ิࢆ SWISS-MODEL

࡟ࡼࡾゎᯒࡋࠊࣄࢵࢺࡋࡓTM0077ࡢ❧యᵓ㐀ࢆࢸࣥࣉ࣮ࣞࢺ࡜ࡋ࡚ࠊ3ḟᵓ㐀ࡢண ࢆ࠾

ࡇ࡞ࡗࡓࠋTM0077 ࡣࠊCE7 ࣇ࢓࣑࣮ࣜ࡟ᒓࡋ࡚࠸ࡿ࢔ࢭࢳ࢚ࣝࢫࢸ࣮ࣛࢮ࡛࠶ࡾࠊacetyl

xylan esterase࡜࢔ࣀࢸ࣮ࢩࣙࣥࡉࢀ࡚ࡣ࠸ࡿࡶࡢࡢࠊ࢔ࢭࢳࣝ࢟ࢩࣛࣥ࡟ᑐࡍࡿάᛶࡣぢࡽ

ࢀࡎࠊࢭࣇ࢓ࣟࢫ࣏ࣜࣥࡢ⬺࢔ࢭࢳࣝ໬άᛶࢆ᭷ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ [78]ࠋ୧⪅ࡢ

࢔ࣛ࢖࣓ࣥࢺࢆྲྀࡿ࡜ࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ C ᮎ➃㡿ᇦ⣙ 300 ࢔࣑ࣀ㓟ṧᇶࡣ

TM0077 ࡟ࡣᏑᅾࡏࡎࠊNᮎ➃㒊ศ࡜ TM0077ࡢ୍ḟ㓄ิࡢ┦ྠᛶࡣ16㸣࡛࠶ࡗࡓࡀࠊຍ

Ỉศゎ㓝⣲ࡢ඾ᆺⓗ࡞ゐ፹㒊఩࡛࠶ࡿࢭࣜࣥࠊ࢔ࢫࣃࣛࢠࣥ㓟ࠊࣄࢫࢳࢪࣥࡣಖᏑࡉࢀ࡚࠸

ࡓࠋࡉࡽ࡟ࠊᵓ㐀ࣔࢹࣝ࡟࠾࠸࡚ࡇࢀࡽ3ࡘࡢṧᇶࡢ✵㛫ⓗ࡞఩⨨ࡣࠊࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ

࣮ࣛࢮ࡜ TM0077 ࡢ㛫୍࡛⮴ࡋ࡚࠸ࡓࠋᚑࡗ࡚ࡇࢀࡽࡢṧᇶࡀゐ፹ṧᇶ࡛࠶ࡿྍ⬟ᛶࡀ㧗

࠸࡜᝿ᐃࡉࢀࡓࡓࡵࠊTM0077 ࡟ࡣᏑᅾࡋ࡞࠸ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ C ᮎ➃㡿ᇦࡢ ๐㝖ࢆ⾜ࡗ࡚ࡳࡓࠋࡋ࠿ࡋࠊCᮎ➃㡿ᇦࢆ๐㝖ࡋࡓࢱࣥࣃࢡ㉁࡟ࡣάᛶࡀ࡯࡜ࢇ࡝ぢࡽࢀ࡞

࠿ࡗࡓࠋࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ C ᮎ➃㡿ᇦࡣࠊゐ፹ṧᇶࢆྵࡲ࡞࠸࡜ண᝿ࡉࢀࡓ࡟

ࡶ㛵ࢃࡽࡎࠊ㓝⣲άᛶ࡟ᙳ㡪ࢆཬࡰࡋࡓࡇ࡜࠿ࡽࠊᇶ㉁ࡢ࢔ࢭࢳࣝᇶࢆྲྀࡾ㎸ࡴ㝿࡟ C ᮎ

➃㡿ᇦࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ

78

Luteolibacter algae H18 ࠿ࡽࡢࣇࢥ࢖ࢲࢼ

࣮ࢮࡢ⢭〇

⥴ゝ

ࣇࢥ࢖ࢲࣥࡢ◲㓟໬ࣇࢥ࣮ࢫṧᇶ㛫ࡢࢢࣜࢥࢩࢻ⤖ྜࡢ㛤⿣ࢆゐ፹ࡍࡿ㓝⣲ࡣࣇࢥ࢖ࢲ ࢼ࣮ࢮ࡜࿧ࡤࢀࢢࣜࢥࢩࢲ࣮ࢮ࡟ᒓࡋ࡚࠸ࡿ (EC␒ྕ 3.2.1.) ࠋኳ↛ࡢࣇࢥ࢖ࢲࣥࡣࠊ࣊ࢸ

ࣟ◲㓟໬ከ⢾㢮࡛࠶ࡾࠊ」㞧࡞ᵓ㐀ࢆ᭷ࡋ࡚࠸ࡿࡓࡵࠊࣇࢥ࢖ࢲࣥ࡟ᑐࡋゐ፹άᛶࢆ᭷ࡍࡿ

㓝⣲ࢆศ㢮ࡍࡿࡇ࡜ࡣ㠀ᖖ࡟㞴ࡋ࠸࡜ࡉࢀ࡚࠸ࡿࠋࣇࢥ࢖ࢲࣥࡢᵓ㐀࡟㛵ࡍࡿ᝟ሗࡀஈࡋ

࠸࠺࠼࡟ࠊ㓝⣲άᛶ࡟㛵ࡍࡿࢹ࣮ࢱࡀ୙ṇ☜࡞ࡓࡵࠊࡇࢀࡽࡢ㓝⣲ࢆศ㢮ࡍࡿୖ࡛ΰ஘ࢆᣍ ࡃሙྜࡀ࠶ࡿࠋ౛࠼ࡤࠊFucus vesiculosus ࠿ࡽ༢㞳ࡉࢀࡓࣇࢥ࢖ࢲࣥࡢᵓ㐀ࡣࠊ1Ѝ2-D-L-ࣇ

࢝ࣥ࡜ㄗࡗ࡚Ỵᐃࡉࢀࠊࡑࢀࡀࣇࢥ࢖ࢲࢼ࣮ࢮࡢㄗศ㢮 EC␒ྕ 3.3.1.44.ࢆࡶࡓࡽࡋࡓࠋᚋ

࡟ࡇࡢᵓ㐀ࡣࠊD-1Ѝ3- ࠾ࡼࡧ D-1Ѝ4-⤖ྜࡢࣇࢥ࣮ࢫṧᇶ࡜ࡋ࡚ಟṇࡉࢀࡓ [35] ࠋࡋ࠿

ࡋ࡞ࡀࡽࠊ㓝⣲ࡢศ㢮ࡣࡲࡔኚ᭦ࡉࢀ࡚ࡣ࠸࡞࠸ࠋࣇࢥ࣮ࢫṧᇶࡢࡳ࠿ࡽᵓ⠏ࡉࢀࡓከ⢾㢮

࡛࠶ࡿࣇ࢝ࣥ୰ࡢࢢࣜࢥࢩࢻ⤖ྜࡢຍỈศゎࢆゐ፹ࡍࡿ㓝⣲ࡣࠊࣇ࢝ࢼ࣮ࢮ࡜ࡋ࡚࿨ྡࡉ

ࢀࡿ࡭ࡁ࡛࠶ࡾࠊࣇࢥ࢖ࢲࢼ࣮ࢮ࡜ࡋ࡚࿨ྡࡉࢀࡿ࡭ࡁ࡛ࡣ࡞࠸࡜࠸࠺ࡇ࡜࡟␃ពࡋࡓ࠸ࠋ

Henrissatࡽ࡟ࡼࡗ࡚ᵓ⠏ࡉࢀࡓ⢾㉁㛵㐃㓝⣲ࡢࢹ࣮ࢱ࣮࣋ࢫ࡛࠶ࡿCAZy (ww.cazy.org)ࡣ

[65, 79, 80]ࠊᮍ▱ࡢ≉ᛶࢆᣢࡘ࣌ࣉࢳࢻࢆศ㢮ࡍࡿࡇ࡜ࡶ࡛ࡁࠊ᫬࡟ࡣCAZy࡟ࡼࡗ࡚㓝⣲

ࡢゐ፹ⓗ࠾ࡼࡧศᏊⓗᛶ㉁ࡢỴᐃ [81]ࠊ㛤⿣ࡉࢀࡿ࡭ࡁࢢࣜࢥࢩࢻ⤖ྜࡢᙧ≧ࢆண ࡍࡿ

ࡇ࡜࡟฼⏝ࡉࢀ࡚࠸ࡿ [82] ࠋCAZy࡛ࡣ⢾㉁ຍỈศゎ㓝⣲ࢆ➹㢌࡟ࠊ⢾㌿⛣㓝⣲ࠊከ⢾㢮

ࣜ࢔࣮ࢮࠊ⢾㉁࢚ࢫࢸ࣮ࣛࢮࡀࡑࢀࡒࢀศ㢮ࡉࢀ࡚࠾ࡾࠊ࡞࠿࡛ࡶ⢾㉁ຍỈศゎ㓝⣲ࡣ2019 ᖺ1᭶⌧ᅾࠊ156 ࡢ⢾㉁ຍỈศゎ㓝⣲ࣇ࢓࣑࣮ࣜ (glycoside hydrolase family; GHࣇ࢓࣑ࣜ

79

࣮) ࡟ศ㢮ࡉࢀ࡚࠸ࡿࠋ࠸ࡃࡘ࠿ࡢ⢾㉁ຍỈศゎ㓝⣲ࡣ」ᩘࡢࣇ࢓࣑࣮ࣜ࡟ᒓࡋࠊࡑࢀࡒࢀ

ᑡ࡞ࡃ࡜ࡶ1ࡘ௨ୖࡢ⢾㉁⤖ྜࣔࢪ࣮ࣗࣝࢆ᭷ࡋ࡚࠸ࡿࠋ

ࡇࡢศ㢮࡟ࡼࢀࡤࠊᗎㄽ࡛ࡶ㏙࡭ࡓࡼ࠺࡟ࠊࣇࢥ࢖ࢲࢼ࣮ࢮࡣGH 107ࣇ࢓࣑࣮ࣜ࡟ᒓࡋ

࡚࠸ࡿࠋࡇࡢࣇ࢓࣑࣮ࣜ࡟᭱ึ࡟Ⓩ㘓ࡉࢀࡓࡢࡣࠊᾏὒᛶ⣽⳦ Mariniflexile fucanivorans

SW5^T⏤᮶ࡢFcnA (CAI47003.1)࡛࠶ࡿࠋࣇࢥ࢖ࢲࢼ࣮ࢮFcnAࡢ㓝⣲཯ᛂ࡟ࡘ࠸࡚ࠊPelvetia

canaliculata⏤᮶ࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋ࡚ヲ⣽࡞᳨ウࡀ⾜ࢃࢀࡓ⤖ᯝࠊFcnAࡣከ⢾ࣜ࢔࣮ࢮ

࡛ࡣ࡞ࡃࠊ࢚ࣥࢻᆺຍỈศゎ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓ [66, 83]ࠋࡉࡽ࡟㏆ᖺࠊᮏ㓝⣲࡟

ࡘ࠸࡚ࠊX⥺⤖ᬗᵓ㐀ゎᯒ࡜NMR࡟ࡼࡗ࡚ᵓ㐀Ỵᐃࡀ࡞ࡉࢀࡓ [84]ࠋGH 107ࣇ࢓࣑࣮ࣜ

࡟ࡣFcnA௨እ࡟ࠊAlteromonas sp. SN-1009⏤᮶ࡢFda1 (AAO00508.1)࡜Fda2 (AAO00509.1) [85]ࠊShewanella violacea DSS12^T⏤᮶㑇ఏᏊSVI_0379 (BAJ00350.1) [40] ࡞࡝ࠊ2019ᖺ1᭶

⌧ᅾࠊྜィ16ಶࡢࢹ࣮ࢱࡀⓏ㘓ࡉࢀ࡚࠸ࡿࠋ

ⴭ⪅ࡽࡢࢢ࣮ࣝࣉ࡛ࡣ࢜࢟ࢼ࣡ࣔࢬࢡCladosiphon okamuranus⏤᮶ࣇࢥ࢖ࢲࣥࢆศゎࡋࠊ

Ⅳ⣲※࡜ࡋ࡚฼⏝࡛ࡁࡿLuteolibacter algae H18ࢆ༢㞳ࡋࠊศゎ࡟㛵୚ࡍࡿ㓝⣲࡟ࡘ࠸࡚ࡢ

◊✲ࢆ㐍ࡵ࡚࠸ࡿࠋ➨4❶ࡲ࡛࡟ࠊH18ᰴࡀ᭷ࡍࡿࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬཯ᛂࢆゐ፹ࡍ

ࡿࢹ࢔ࢭࢳ࣮ࣛࢮ࡟ࡘ࠸࡚㏙࡭࡚ࡁࡓࡀࠊᮏ❶࡛ࡣࣇࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂࢆゐ፹ࡍࡿ

ࣇࢥ࢖ࢲࢼ࣮ࢮ࡟ࡘ࠸࡚ゝཬࡍࡿࠋࡲࡎࠊH18ᰴ࠿ࡽࣇࢥ࢖ࢲࢼ࣮ࢮࢆ✀ࠎࡢࢡ࣐ࣟࢺࢢࣛ

ࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊ㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิࢆỴᐃࡋࡓࠋࡑࡋ࡚ࠊ᫂ࡽ࠿࡟ࡋࡓH18 ᰴࡢࢻ

ࣛࣇࢺࢤࣀ࣒᝟ሗࢆᇶ࡟ࠊࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊࢆ≉ᐃࡋࠊ᫂ࡽ࠿࡟࡞ࡗࡓᮏ㓝⣲ࡢ࢔࣑ࣀ

㓟㓄ิ࡟ࡘ࠸࡚⪃ᐹࢆ⾜ࡗࡓࠋ

80

ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ

Luteolibacter algae H18ࡢᇵ㣴࡜↓⣽⬊ᢳฟᾮ (C.F.E.) ࡢㄪ〇

H18ᰴࡢᇵ㣴࠾ࡼࡧ C.F.E.ࡢㄪ〇࡟㛵ࡍࡿᐇ㦂᪉ἲࡣ2❶࡟グ㍕ࡋࡓ᪉ἲ࡜ྠࡌ࡛࠶ࡿࠋ

㓝⣲άᛶ ᐃ

⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡣ 3-2-19 ࡢ᪉ἲ࡟ᚑࡗ࡚ࠊ㓝⣲ⓗ࡟ㄪ〇ࡋࡓࠋ0.25% ⬺࢔ࢭࢳ

ࣝ໬ࣇࢥ࢖ࢲࣥࠊ100 mM Tris-HCl buffer (pH 8.0)ࠊࡑࡋ࡚㒊ศ⢭〇㓝⣲ࢆྵࡴ㓝⣲཯ᛂᾮࢆ

඲㔞200 Pl ㄪ〇ࡋ30 Υࠊ24 h࡛࢖࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ85 Υࠊ5 min⇕ฎ⌮࡟ࡼࡗ࡚཯ᛂ೵

Ṇࡉࡏࡓࠋࡑࡢ཯ᛂᾮࢆ⏝࠸࡚ࠊ3-2-18࡟♧ࡍ࣏ࣥࣉ࡜᳨ฟჾࢆ⏝࠸࡚HPLCࢆ⾜࠸ࠊᇶ㉁

ࡢపศᏊ໬ࢆホ౯ࡋࡓࠋ

DEAE-Sepharose FF

࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇

42 Lࡢᇵ㣴ᾮ࠿ࡽㄪ〇ࡋࡓH18ᰴC.F.E. (ࢱࣥࣃࢡ㉁㔞 6250 mg) ࢆ20 mM Tris-HCl buffer (pH 8.0) ࡛ᖹ⾮໬ࡋࡓDEAE-Sepharose FF࣒࢝ࣛ㸦ᶞ⬡㔞 800 mL, 40™6.0 cm㸧࡟࢔ࣉࣛ࢖

ࡋࡓࠋὶ㏿ࡣ3.0 mL/minࠊศ⏬ࢧ࢖ࢬࢆ18.5 mL࡟タᐃࡋࡓࠋ20 mM Tris-HCl buffer (pH 8.0)

࡛Ὑίᚋࠊ0.1, 0.2 M NaClࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡛⁐ฟࢆ⾜࠸ࠊ0.1 M NaCl

ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡛⁐ฟࡉࢀࡓ⏬ศࡢ࠺ࡕࠊ280 nm ࡢ྾཰ࡀ0.5௨ୖ

ࡢ⏬ศ࡟ࡘ࠸࡚ࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࡢάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

Phenyl-TOYOPEARL 650M

࡟ࡼࡿ㓝⣲⢭〇

2.0 M (NH4)2SO4 ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡛DEAE-Sepharose 0.1 M⁐ฟ⏬ศ 㸦ࢱࣥࣃࢡ㉁㔞3460 mg㸧ࢆ୍ᬌ㏱ᯒࡋࡓ࡜ࡇࢁࠊỿẊࡀ⏕ࡌࡓࠋỿẊࢆྲྀࡾ㝖࠸ࡓୖ⃈ࡳ

ᾮ ࢆ 2.0 M (NH4)2SO4 ࢆ ྵ ࡴ 20 mM Tris-HCl buffer (pH 8.0) ࡛ ᖹ ⾮ ໬ ࡋ ࡓ

Phenyl-81

TOYOPEARL 650M㸦ᶞ⬡㔞350 mL, 12.5×3.0 cm㸧࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ1.5 mL/minࠊศ⏬

ࢧ࢖ࢬࢆ3 mL࡟タᐃࡋࡓࠋ2.0 M (NH4)2SO4 ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0) ࡛Ὑί ᚋࠊ2.0, 1.0, 0.5 M (NH4)2SO4ࢆྵࡴ20 mM Tris-HCl buffer (pH 8.0)࡛⁐ฟࢆ⾜ࡗࡓࠋ1.0 M (NH4)2SO4 ⁐ฟ⏬ศ࡛ࡣ280 nmࡢ྾཰ࡀ0.2௨ୖࡢ⏬ศ࡟ࡘ࠸࡚ࠊ0.5 M (NH4)2SO4 ⁐ฟ⏬

ศ࡛ࡣ 280 nm ࡢ྾཰ࡀ 0.05 ௨ୖࡢ⏬ศ࡟ࡘ࠸࡚ࣇࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࡢάᛶ ᐃࢆ⾜

࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋ

Resource Q

࣒࢝ࣛ࡟ࡼࡿ⢭〇

5-2-4࡛ᚓࡽࢀࡓ0.5, 1.0 M (NH4)2SO4 ⁐ฟ⏬ศࢆVivaspin Turbo15 (Sartorius) ࢆ⏝࠸࡚20

mM Tris-HCl buffer (pH 8.0) ࡟ࡼࡾࠊูࠎ࡟⬺ሷฎ⌮࡜⃰⦰ฎ⌮ࢆྠ᫬࡟⾜ࡗࡓࠋࡑࢀࡒࢀ

ࡢ⁐ฟ⏬ศ࠿ࡽㄪ〇ࡋࡓ㒊ศ⢭〇㓝⣲㸦0.5 M (NH4)2SO4 ⁐ฟ⏬ศࡣࢱࣥࣃࢡ㉁㔞4.22 mgࠊ 1.0 M (NH4)2SO4 ⁐ฟ⏬ศࡣࢱࣥࣃࢡ㉁㔞4.21 mg㸧ࢆࠊ20 mM Tris-HCl buffer (pH 8.0) ࡛ᖹ

⾮໬ࡋࡓResource Q㸦ᶞ⬡㔞6 mL, 3.9×2.2 cm㸧࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ0.2 mL/minࠊศ⏬ࢧ

࢖ࢬࢆ2 mL࡟タᐃࡋࡓࠋ20 mM Tris-HCl buffer (pH 8.0) ࡛Ὑίᚋࠊࢢࣛࢪ࢚ࣥࢺ⃰ᗘࡣ0

M࠿ࡽ0.15 M NaCl࡟タᐃࡋࠊÄKTAࢩࢫࢸ࣒ࢆ⏝࠸࡚⁐ฟࢆ⾜ࡗࡓࠋྛࣇࣛࢡࢩࣙࣥࡢࣇ

ࢥ࢖ࢲࣥపศᏊ໬㓝⣲ࡢάᛶ ᐃࢆ⾜࠸ࠊάᛶ⏬ศࢆ㞟ࡵࡓࠋᚓࡽࢀࡓ⢭〇㓝⣲ࡢ Nᮎ➃

࢔࣑ࣀ㓟㓄ิࡣ2-2-15 ࡢ᪉ἲ࡛Ỵᐃࡋࡓࠋ

82

⤖ᯝ

኱㔞ᇵ㣴࡜ C.F.E. ࡢ㓝⣲ㄪ〇

H18 ᰴࡢᇵ㣴ᾮ42 L࠿ࡽ120 gࡢ‵⳦యࡀᚓࡽࢀࠊ6250 mgࡢࢱࣥࣃࢡ㉁ࢆྵࡴ C. F.

E. ࢆㄪ〇ࡋࡓࠋ

DEAE-Sepharose FF

࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇

C.F.E. (3460 mg-protein) ࢆ20 mM Tris-HCl buffer (pH 8.0) ࡟ᑐࡋ࡚㏱ᯒᚋࠊDEAE-Sepharose FF࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛNaCl⃰ᗘ࡛⁐ฟࡋࡓ⏬ศࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆᐇ᪋ࡋࡓ

⤖ᯝࠊ0.1 M NaCl⁐ฟ⏬ศ࡟࠾࠸࡚άᛶࡀ᳨ฟࡉࢀࠊFraction No. 420㹼437 ࢆᅇ཰ࡋࡓ (ᅗ 5-1) ࠋ

ᅗ 5-1. DEAE-Sepharose FF ࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ⏬ࡋࡓࢱࣥࣃࢡ㉁ࡢ⁐ฟࣃࢱ࣮

ࣥ

83

࡞࠾ࠊ㓝⣲άᛶ࡟ࡘ࠸࡚ࡣࠊᇶ㉁࡛࠶ࡿ㧗ศᏊ㔞ࣇࢥ࢖ࢲࣥࡢపศᏊ໬ࢆ HPLC ࡢࣜࢸ

ࣥࢩࣙࣥࢱ࢖࣒ኚ໬࡛ホ౯ࡍࡿࡓࡵ࡟㓝⣲άᛶࢆᩘ್໬࡛ࡁࡎࠊᮏ❶ࡢࢡ࣐ࣟࢺࢢࣛࣇ࢕

࣮ࡢ⁐ฟࣃࢱ࣮ࣥ୰࡟ࡣ♧ࡋ࡚࠸࡞࠸ࠋ

Phenyl-TOYOPEARL 650M

࡟ࡼࡿ㓝⣲⢭〇

DEAE-Sepharose 0.1 M NaCl⁐ฟ⏬ศࢆ2.0 M (NH4)2SO4 ࢆྵࡴ20 mM Tris-HCl buffer (pH

8.0) ࡟ᑐࡋ୍࡚ᬌ㏱ᯒᚋࠊPhenyl-TOYOPEARL ࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛ(NH4)2SO4 ⃰ᗘ

࡛⁐ฟࡋࡓ⏬ศࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗࡓ⤖ᯝࠊ1.0 M (NH4)2SO4ࠊ0.5 M (NH4)2SO4⁐ ฟ⏬ศࡢ୧᪉࡟άᛶࡀ᳨ฟࡉࢀࡓࡢ࡛ࠊFraction No. 157㹼191ࠊFraction No. 218㹼233ࢆࡑࢀ

ࡒࢀᅇ཰ࡋࡓ (ᅗ5-2) ࠋ

ᅗ5-2. Phenyl-TOYOPEARL࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡛ศ⏬ࡋࡓࢱࣥࣃࢡ㉁ࡢ⁐ฟࣃࢱ࣮

ࣥ

84

Resource Q

࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇

Phenyl-TOYOPEARL 0.5, 1.0 M (NH4)2SO4 ⁐ฟ⏬ศࢆࠊࡑࢀࡒࢀ Vivaspin spin Turbo15 (Sartorius)ࢆ⏝࠸࡚20 mM Tris-HCl buffer (pH 8.0)࡟ࡼࡾࠊ⬺ሷฎ⌮࡜⃰⦰ฎ⌮ᚋࠊResource Q࣒࢝ࣛ࡟࢔ࣉࣛ࢖ࡋࡓࠋྛNaCl⃰ᗘ࡛⁐ฟࡋࡓྛศࢆᅇ཰ᚋࠊ㓝⣲άᛶ ᐃࢆ⾜ࡗࡓ⤖

ᯝࠊ࡝ࡕࡽࡶFraction No. 28㹼35 ࢆᅇ཰ࡋࡓ (ᅗ5-3, 5-4) ࠋ

ᅗ5-3. Phenyl-TOYOPEARL 0.5 M (NH4)2SO4⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࡓResource Q࣒࢝ࣛࢡࣟ

࣐ࢺࢢࣛࣇ࢕࣮ࡢࢱࣥࣃࢡ㉁ࡢ⁐ฟࣃࢱ࣮ࣥ

85

ᅗ5-4. Phenyl-TOYOPEARL 1.0 M (NH4)2SO4⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࡓResource Q࣒࢝ࣛࢡࣟ

࣐ࢺࢢࣛࣇ࢕࣮ࡢࢱࣥࣃࢡ㉁ࡢ⁐ฟࣃࢱ࣮ࣥ

Resource Q࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ᚋࡢࣇࣛࢡࢩࣙࣥࢆ⏝࠸࡚SDS-PAGE ศᯒࢆ⾜ࡗ

ࡓ(ᅗ 5-5, 5-6) ࠋ ࣇ ࢥ࢖ ࢲࣥప ศᏊ໬ άᛶ ࡜ SDS-PAGE ࡢ⤖ᯝ ࢆẚ࡭ ࡿ࡜ࠊ

Phenyl-TOYOPEARL 1 M (NH4)2SO4 ⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࡓࠊResource Qᚋࡢ⁐ฟ⏬ศ࡛ࡣࠊ㓝⣲

άᛶࡢᙉࡉ࡜SDS-PAGE ࡟࠾ࡅࡿࠊ≉ᐃࡢࣂࣥࢻࡢ⃰ࡉࡀ୍⮴ࡋ࡞࠿ࡗࡓࡓࡵ㸦ᅗ4, 5-6㸧ࠊ࡝ࡢࣂࣥࢻࡀ┠ⓗࢱࣥࣃࢡ㉁࡛࠶ࡿ࠿≉ᐃ࡛ࡁ࡞࠿ࡗࡓࠋ୍᪉ࠊPhenyl-TOYOPEARL

0.5 M (NH4)2SO4 ⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࡓࠊ Resource Q ᚋࡢ⁐ฟ⏬ศ࡛ࡣࠊάᛶࡢᙉ࠸

86

Fraction No. 30㹼32࡟ࡣࠊ100 kDa௜㏆࡟㉥୸࡛♧ࡋࡓࠊ≉᭷ࡢࣂࣥࢻࡀぢࡽࢀࡿࡇ࡜࠿ࡽ

㸦ᅗ5-3, 5-5㸧 ࠊࡇࡢࣂࣥࢻࢆษࡾฟࡋࠊNᮎ➃࢔࣑ࣀ㓟㓄ิࢆỴᐃࡋࡓࠋ

ᅗ5-5. Phenyl-TOYOPEARL 0.5 M (NH4)2SO4 ⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࠊResource Q࣒࢝ࣛࢡࣟ

࣐ࢺࢢࣛࣇ࢕࣮࡛ศ⏬ࡋࡓࣇࣛࢡࢩࣙࣥࡢSDS-PAGE⤖ᯝ

ᅗ5-6. Phenyl-TOYOPEARL 1.0 M (NH4)2SO4 ⁐ฟ⏬ศࢆࢳ࣮ࣕࢪࡋࠊResource Q࣒࢝ࣛࢡࣟ

࣐ࢺࢢࣛࣇ࢕࣮࡛ศ⏬ࡋࡓࣇࣛࢡࢩࣙࣥࡢSDS-PAGE⤖ᯝ

87

⢭〇㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิゎᯒ

⢭〇㓝⣲ࢆSDS-PAGEࡢࢤࣝ࠿ࡽPVDF⭷࡟㌿෗ࡋࠊࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢫゎᯒ⿦⨨

࡛࢚ࢻ࣐ࣥศゎἲ࡟ࡼࡿ N ᮎ➃࢔࣑ࣀ㓟㓄ิࡢゎᯒࢆ⾜ࡗࡓ⤖ᯝࠊAEVSELMPLP ࡜࠸࠺

㓄ิࡀᚓࡽࢀࡓࠋ┠ⓗ㑇ఏᏊࡢྠᐃ࡟ࡣゎᯒࢯࣇࢺin silico Molecular Cloningࢆ⏝࠸ࡓࠋࡑ ࡢ⤖ᯝࠊࡍ࡛࡟᫂ࡽ࠿࡟ࡋ࡚࠸ࡿH18ᰴࡢࢻࣛࣇࢺࢤࣀ࣒ୖ࡟ࡇࡢNᮎ➃࢔࣑ࣀ㓟㓄ิࢆ

ࢥ࣮ࢻࡍࡿORFࢆぢฟࡋࠊࡑࡢ඲㛗ࡣ3078 bpࠊࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ࡢศᏊ㔞ࡣ112 kDa

࡛࠶ࡗࡓ (ᅗ5-7)ࠋࡇࡇ࡛ࠊぢฟࡉࢀࡓORF඲㛗ࢆfct114 full ࡜⛠ࡍࡿࡇ࡜࡜ࡋ, ࢚ࢻ࣐ࣥ

ศゎἲ࡟ࡼࡾ᳨ฟࡉࢀࡓ࢔࣑ࣀ㓟㓄ิࡼࡾୗὶࢆࢥ࣮ࢻࡍࡿDNA᩿∦ࢆfct114࡜ࡋࡓࠋࡑ

ࡋ࡚ࠊfct114 fullࡀࢥ࣮ࢻࡍࡿ࢔࣑ࣀ㓟㓄ิ࡟ᑐࡋ࡚BLAST᳨⣴ࢆ⾜ࡗࡓ࡜ࡇࢁࠊ┦ྠᛶࡀ

㧗࠸ࢱࣥࣃࢡ㉁ࡢከࡃࡣᶵ⬟ᮍ▱࡛࠶ࡾࠊ࢔ࣀࢸ࣮ࢩࣙࣥࡉࢀ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡢ୰࡛᭱

ࡶ┦ྠᛶࡀ㧗࠸ࡢࡣglycosyl hydrolase [Sphingomonas sanguinis] (┦ྠᛶ26%)࡛࠶ࡗࡓ㸦⾲ 5-1㸧ࠋ

ᅗ5-7. H18ᰴ⏤᮶ࣇࢥ࢖ࢲࢼ࣮ࢮࡢ࢔࣑ࣀ㓟㓄ิ

Nᮎ➃࢔࣑ࣀ㓟㓄ิࡢゎᯒ࡛ᚓࡽࢀࡓ㓄ิࢆኴኴᏐ࡛♧ࡍࠋ

88

⾲5-1. Fct114 fullࡢBLAST᳨⣴⤖ᯝ

ࢱࣥࣃࢡ㉁ ⳦ᰴ ┦ྠᛶ (㸣) Accession No

hypothetical protein Rubritalea marina 50 WP_018970822.1

hypothetical protein Formosa agariphila 40 WP_038529980.1

hypothetical protein Lentisphaera araneosa 39 WP_007280829.1 hypothetical protein Parabacteroides timonensis 38 WP_075559901.1 hypothetical protein Sunxiuqinia elliptica 34 WP_093919541.1 hypothetical protein Rhodopirellula sallentina 32 WP_008689565.1 glycosyl hydrolase

(GH family 2) Sphingomonas sanguinis 26 WP_058717281.1

89

⪃ᐹ

ⴭ⪅ࡽࡢࢢ࣮ࣝࣉ࡛ࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ㈨໬ᛶ⣽⳦ L. algae H18 ᰴࡣࠊࣇࢥ

࢖ࢲࣥศゎ㓝⣲ࢆ⣽⬊ෆ࡟⏕⏘ࡋࠊ」ᩘࡢ㓝⣲཯ᛂ࡟ࡼࡾࣇࢥ࢖ࢲࣥࡢศゎࡀ㐍⾜ࡍࡿࡇ

࡜ࢆ᫂ࡽ࠿࡟ࡋ࡚࠸ࡿ [46]ࠋᮏ❶࡛ࡣࠊࡑࡢ୰࡛ࠊࣇࢥ࢖ࢲࣥࡢపศᏊ໬ࢆゐ፹ࡍࡿࣇࢥ࢖

ࢲࢼ࣮ࢮࢆH18ᰴ࠿ࡽ⢭〇ࡋࠊNᮎ➃࢔࣑ࣀ㓟㓄ิࢆỴᐃࡍࡿࡇ࡜ࡀ࡛ࡁࡓࡇ࡜ࢆ㏙࡭ࡓࠋ ࡇࢀࡲ࡛ࠊ4✀㢮ࡢࣇࢥ࢖ࢲࣥศゎ㓝⣲ࡀࠊFucobacter marina SA-0082 [64]ࠊM. fucanivorans SW5 [66]ࠊFormosa algae KM3553 [47]ࠊ࠾ࡼࡧSphingomonas paucimobilis PF-1 [86]࠿ࡽ࡯ࡰ

ᆒ୍࡟࡞ࡿࡲ࡛⢭〇ࡉࢀ࡚࠸ࡿࠋࡉࡽ࡟ຍ࠼࡚ࠊKM3553ᰴ⏤᮶ࡢFFA2ࡀࠊࢤࣀ࣒᝟ሗ࡟

ᇶ࡙࠸࡚⤌᥮࠼኱⭠⳦ࢆ⏝࠸࡚ㄪ〇ࡉࢀ࡚࠸ࡿ [67]ࠋࡓࡔࠊFFA2 ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ

(WP_057784219.1) ࡣࠊࡑࢀࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡓࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊ࡜ẚ㍑ࡍࡿࡇ࡜࡟

ࡼࡗ࡚Ỵᐃࡉࢀ࡚࠾ࡾࠊ㔝⏕ᰴ KM3553 ࠿ࡽㄪ〇ࡉࢀࡓ㓝⣲࡟ࡘ࠸࡚ࡣ࢔࣑ࣀ㓟㓄ิࡀỴ ᐃࡉࢀ࡚࠸࡞࠸ࡓࡵࠊKM3553ᰴࡢ୧㓝⣲ࡀྠ୍࡛࠶ࡿ࠿࡝࠺࠿ࡣ᫂ࡽ࠿࡛ࡣ࡞࠸ [47, 67]ࠋ

ࡲࡓࡇࢀ௨እ࡟ࡶࠊࣇࢥ࢖ࢲࣥศゎ㓝⣲ࡢ㒊ศ⢭〇㓝⣲ࡀࠊAlteromonas sp. SN-1009࠿ࡽᚓ

ࡽࢀ࡚࠸ࡿ [32]ࠋᮏㄽᩥ࡛ᢅࡗ࡚࠸ࡿH18ᰴࢆྵࡴࡇࢀࡽ⣽⳦ࡣ࠸ࡎࢀࡶᾏὒ⎔ቃ࠿ࡽศ 㞳ࡉࢀࡓࡶࡢ࡛࠶ࡿࠋࣇࢥ࢖ࢲࣥࡣ〓⸴㢮࡟≉ᚩⓗ࡞ከ⢾㢮࡛࠶ࡿࡓࡵࠊࣇࢥ࢖ࢲࣥࡢపศ Ꮚ໬㓝⣲ࢆᾏὒ⎔ቃ⏤᮶ࡢᚤ⏕≀ࡀ᭷ࡍࡿࡇ࡜ࡣጇᙜ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ ࡇࢀࡽࡢ୰࡛ࠊ H18 ᰴ࠾ࡼࡧ KMM3553 ᰴࡢ㓝⣲ࡣ⣽⬊ෆ㓝⣲࡜ࡋ࡚ᚤ⏕≀ᰴࡢ↓⣽⬊ᢳฟ≀࠿ࡽ⢭〇ࡉ

ࢀࡓࡢ࡟ᑐࡋ࡚ࠊ௚ࡢ㓝⣲ࡣᇵ㣴ୖΎ࠿ࡽㄪ〇ࡉࢀࡓࠋ࠾ࡑࡽࡃ๓⪅ࡢ2ᰴ࡟ࡣࠊ㧗ศᏊ໬

ྜ≀࡛࠶ࡿࣇࢥ࢖ࢲࣥ⣽⬊ෆ࡟ྲྀࡾ㎸ࡴࡓࡵࡢ≉ู࡞ࢩࢫࢸ࣒ࡀ࠶ࡿࡶࡢ࡜᥎ᐹࡉࢀࡿࠋ ࡇࢀࡽࡢ㓝⣲࡟ࡘ࠸࡚ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡣ◊✲ࡉࢀ࡚࠾ࡾࠊH18ᰴ⏤᮶ࡢ㓝⣲࡜௚ࡢ㓝⣲࡜

ࡢ㛫࡟ࡣࠊヲ⣽ࡣࡇࡇ࡛ࡣ㏙࡭࡞࠸ࡀࠊ࠸ࡃࡘ࠿ࡢ㢮ఝⅬࠊ┦㐪Ⅼࡀ࠶ࡿࠋ౛࠼ࡤࠊH18ᰴ

⏤᮶ࡢࣇࢥ࢖ࢲࢼ࣮ࢮ㸦Fct114㸧ࡢศᏊ㔞ࡣࠊ௒ࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿࣇࢥ࢖ࢲࣥศゎ㓝⣲

ࡢศᏊ㔞࡜࡯ࡰྠࡌ 100 kDa࡛࠶ࡾࠊSA-0082⏤᮶ࡢ㓝⣲ (67 kDa)ࡼࡾࡶ኱ࡁ࠸ࠋ

90

୍᪉ࠊM. fucanivorans SW5 [66] ࡜ F. algae KMM3553 [67] ⏤᮶ࡢࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊ

fcnA (AJ877239) ࠾ࡼࡧffa2ࡣࢡ࣮ࣟࢽࣥࢢࡉࢀࠊ኱⭠⳦ෆ࡛Ⓨ⌧ࡉࢀ࡚࠸ࡿࠋࡉࡽ࡟ࠊ3ࡘ

ࡢࣇࢥ࢖ࢲࢼ࣮ࢮࠊFda1 (AAO00508.1)ࠊFda2 (AAO00509.1)ࠊ࠾ࡼࡧSVI_0379 (BAJ00350.1) ࡢ࢔࣑ࣀ㓟㓄ิࡀDDBJ ࢹ࣮ࢱ࣮࣋ࢫ࡟බ㛤ࡉࢀ࡚࠸ࡿࠋFct114 ࡶྵࡵࠊࡇࢀࡽ 6ࡘࡢ࢔

࣑ࣀ㓟㓄ิࡢNᮎ➃㡿ᇦࡢ࢔ࣛ࢖࣓ࣥࢺࢆྲྀࡗࡓࡢࡀᅗ 5-8 ࡛࠶ࡿࠋFct114 ௨እࠊࡍ࡭࡚

ᗎㄽ࡛ࡶゐࢀࡓGH107 ࣇ࢓࣑࣮ࣜ࡟ᒓࡋ࡚࠾ࡾࠊࡇࢀࡽ5ࡘࡢ㓄ิ࡟ࡣᅗ5-8࡟♧ࡍࡼ࠺

࡞࠸ࡃࡘ࠿ࡢಖᏑࡉࢀࡓࣔࢳ࣮ࣇࡀN ᮎ➃㡿ᇦ࡟ほᐹࡉࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊFct114 ࡣࡑ ࡢࡼ࠺࡞ࣔࢳ࣮ࣇࢆḞ࠸࡚࠸ࡿࠋࡲࡓࠊFct114ࡢ࢔࣑ࣀ㓟㓄ิࡣࠊ௚ࡢ5ࡘࡢࣇࢥ࢖ࢲࣥศ ゎ㓝⣲ࡢ㓄ิ࡜ 25%ᮍ‶ࡢ┦ྠᛶࡋ࠿♧ࡉ࡞࠿ࡗࡓࠋBLAST ᳨⣴ࡢ⤖ᯝࠊ⾲ 5㸫1 ࡟♧ࡍ

ࡼ࠺࡟ࠊ᭱ࡶ┦ྠᛶࡢ㧗࠸ࢱࣥࣃࢡ㉁ࡣhypothetical protein࡛࠶ࡾࠊ࢔ࣀࢸ࣮ࢩࣙࣥࡉࢀ࡚

࠸ࡿࢱࣥࣃࢡ㉁ࡢ୰࡛᭱ࡶ┦ྠᛶࡀ㧗࠸ࢱࣥࣃࢡ㉁ࡣࠊS. sanguinis⏤᮶࡛ GH ࣇ࢓࣑࣮ࣜ

2 ࡟ᒓࡍࡿglycoside hydrolase࡛࠶ࡗࡓࠋ௨ୖࡢࡇ࡜ࡼࡾࠊᮏ㓝⣲ࡣ᪂ࡋ࠸ࢱ࢖ࣉࡢࢢࣜࢥ

ࢩࢻࣄࢻ࣮ࣟࣛࢮ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ

91

ᅗ5-8. L. algae H18 (Fct114)ࠊM. fucanivorans SW5 (FcnA)ࠊF. algae KMM3553 (FFA2)ࠊ Schewanella violacea DSS12 (SVI_0379)ࠊ࠾ࡼࡧAlteromonas sp. SN-1009 (Fda1,

Fda2)⏤᮶ࣇࢥ࢖ࢲࢼ࣮ࢮࡢNᮎ➃㡿ᇦࡢ࢔࣑ࣀ㓟㓄ิࡢ࢔ࣛ࢖࣓ࣥࢺ

▮༳ࡣࠊFct114ࢆ㝖࠸࡚ࣇࢥ࢖ࢲࢼ࣮ࢮࡢ㛫࡛ಖᏑࡉࢀ࡚࠸ࡿࣔࢳ࣮ࣇࢆ♧ࡍࠋ

࡞࠾ࠊ௒ᅇぢฟࡉࢀࡓ ORF ࡟ࡣࠊN ᮎ➃࢔࣑ࣀ㓟㓄ิࡢゎᯒ⤖ᯝ࡟ࡼࡗ࡚ᚓࡽࢀࡓ

AEVSELMPLP࡜࠸࠺㓄ิࡢୖὶ࡟ࠊMHPLRFPITFPKLPALGAALVLSLLAATAPLKA࡜࠸࠺

㓄ิࡀ♧ࡉࢀࡓࠋࡇࡢ㓄ิࡀSignalP 4.1 Server࡟ࡼࡾࢩࢢࢼࣝ㓄ิ࡛࠶ࡿ࠿ㄪ࡭ࡓ࡜ࡇࢁࠊ ᅗ5-9࡟♧ࡍࡼ࠺࡟ࢩࢢࢼࣝ㓄ิ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀ࡚࠸ࡿࠋ

92

ᅗ5-9. Fct114fullࡢࢩࢢࢼࣝ㓄ิࡢ᳨ウ⤖ᯝ

Cleavage site between pos. 33 and 34: LKA-AE D=0.831 D-cutoff=0.570 Networks=SignalP-noTM

93

ࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ

⌧ࠊ㓝⣲໬Ꮫⓗᛶ㉁

⥴ゝ

➨5 ❶࡛ࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮࢆ L.

algae H18 ࡢ↓⣽⬊ᢳฟᾮ࠿ࡽ✀ࠎࡢࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟ࡼࡾ⢭〇ࡋࠊ㓝⣲ࡢNᮎ➃࢔࣑

ࣀ㓟㓄ิࢆỴᐃࡋࡓࠋࡑࡋ࡚ࡍ࡛࡟᫂ࡽ࠿࡟ࡋ࡚࠸ࡓH18ᰴࡢࢻࣛࣇࢺࢤࣀ࣒᝟ሗࢆᇶ࡟ࠊ

ࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊ㸦fct114㸧ࢆ≉ᐃࡋࠊᮏ㓝⣲ࡢ࢔࣑ࣀ㓟㓄ิࢆỴᐃࡋࡓࠋࡉࡽ࡟ᮏ㓝

⣲࡜ࡍ࡛࡟࢔࣑ࣀ㓟㓄ิࡀሗ࿌ࡉࢀ࡚࠸ࡿ M. fucanivorans SW5 ⏤᮶ࡢ FcnAࠊF. algae KMM3553⏤᮶ࡢ FFA2ࠊSchewanella violacea DSS12 ⏤᮶ࡢSVI_0379ࠊ࠾ࡼࡧAlteromonas

sp. SN-1009 ⏤᮶ࡢFda1 ࡜Fda2ࡢ6ࡘࡢ Nᮎ➃㡿ᇦࡢ࢔࣑ࣀ㓟㓄ิࢆẚ㍑ࡋࡓ࡜ࡇࢁࠊ

Fct114 ௨እࡢ5✀ࡢ㛫࡟㓄ิࡢ┦ྠᛶࡣぢࡽࢀࡓࡶࡢࡢࠊFct114 ࡜ࡢ┦ྠᛶࡣ࠸ࡎࢀࡶㄆ

ࡵࡽࢀ࡞࠿ࡗࡓࠋ

ࡇࢀࡲ࡛⾲ 1-4 ࡟ࡇࢀࡲ࡛࡟ሗ࿌ࡉࢀ࡚࠸ࡿࣇࢥ࢖ࢲࣥศゎ㓝⣲ࡢㅖᛶ㉁ࢆࡲ࡜ࡵࡓࠋ

⣽⳦⏤᮶ࡢ㓝⣲ࡢከࡃࡀ Zn²⁺ࡸ Cu²⁺࡟ࡼࡗ࡚㓝⣲άᛶࡀ㜼ᐖࡉࢀ࡚࠸ࡿ࡯࠿ࠊ⮳㐺 ᗘࡸ

⮳㐺pH࡞࡝ࡀ␗࡞ࡗ࡚࠸ࡿࠋࡑࡇ࡛ࠊᮏ❶࡛ࡣࠊ➨5❶࡛᫂ࡽ࠿࡟࡞ࡗࡓࠊ࢜࢟ࢼ࣡ࣔࢬ ࢡࣇࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊ fct114 ࡢࢡ࣮ࣟࢽࣥࢢࠊ࠾

ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆᐇ᪋ࡋࠊ᪤▱ࡢࣇࢥ࢖

ࢲࢼ࣮ࢮ࡜ࡢẚ㍑᳨ウࢆ⾜ࡗࡓࠋ

94

ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ

DNA

ᐇ㦂ࠊ⤌᥮࠼኱⭠⳦࡟ࡼࡿ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘᳨ウ

DNAࢆᢅ࠺ᐇ㦂᪉ἲࠊࡍ࡞ࢃࡕࠊࢥࣥࣆࢸࣥࢺࢭࣝࡢㄪ〇ࠊᙧ㉁㌿᥮ࠊࣉࣛࢫ࣑ࢻ༢㞳ࠊ ไ㝈㓝⣲ᾘ໬ࠊࣛ࢖ࢤ࣮ࢩࣙࣥ࡟㛵ࡍࡿᐇ㦂᪉ἲ࡞ࡽࡧ࡟ࠊ኱⭠⳦࡟ࡼࡿ㓝⣲ࢱࣥࣃࢡ㉁ࡢ

⏕⏘᳨ウ࡟㛵ࡍࡿᐇ㦂᪉ἲࡣࠊ3❶࡟グ㍕ࡋࡓ᪉ἲ࡜ྠࡌ࡛࠶ࡿࠋ

ࣉࣛ࢖࣐࣮ࡢタィ

ࣇ ࢥ ࢖ ࢲ ࢼ ࣮ ࢮ 㑇 ఏ Ꮚ ࠊ fct114 ࢆ PCR ቑ ᖜ ࡍ ࡿ ࡓ ࡵ ࡟ ࠊForward ࣉ ࣛ ࢖ ࣐ ࣮ ࢆ

ATTATGCTCATATGGCGGAGGTTTCCGAGCTG (Nde I ไ㝈㓝⣲ㄆ㆑㓄ิࡣኴኴᏐ࡛♧ࡉࢀࠊ

ୗ ⥺ 㒊 ࡀ ATG 㛤 ጞ ࢥ ࢻ ࣥ ࢆ ♧ ࡍ ) ࠊ Reverse ࣉ ࣛ ࢖ ࣐ ࣮ ࢆ

GTGAGGTAGAATTCGGGTTTTGGCTGAAGGTTG (Eco RIไ㝈㓝⣲ㄆ㆑㓄ิࢆኴኴᏐ࡛♧ࡋ

࡚࠸ࡿ) ࡜タィࡋࡓࠋእὀࡋࠊ㏦ࡽࢀ࡚ࡁࡓࣉࣛ࢖࣐࣮࡟⁛⳦Ỉࢆῧຍࡋ࡚100 PMࡢ⁐ᾮ

ࢆㄪ〇ࡋࠊࡑࢀࢆ⁛⳦Ỉ࡛ᕼ㔘ࡋࡓ1 PMࡢࡶࡢࢆPCR ᐇ㦂࡟⏝࠸ࡓࠋ

PCR

ቑᖜ

PCR ቑᖜࡢ㝿ࡢኚᛶ཯ᛂࠊఙ㛗཯ᛂࡢ ᗘ࠾ࡼࡧ཯ᛂ᫬㛫࡟ࡘ࠸࡚ࡣࠊ94℃࡛ 2ศ㛫ຍ

⇕ࡋࠊḟ࡟30ࢧ࢖ࢡࣝ (98 Υ࡛10⛊㛫ࠊ68 Υ࡛3ศ㛫) ࡛⾜ࡗࡓࠋ࢔࣮࢞ࣟࢫࢤࣝ㟁Ẽ Ὃື࡜ษࡾฟࡋࢆ⾜࠸ࠊQIAGEN Gel Extraction Kitࢆ⏝࠸࡚௜ᒓࡢࣉࣟࢺࢥ࣮ࣝ࡟ᚑࡗ࡚⢭

〇ࡋࡓࠋⓎ⌧࣋ࢡࢱ࣮ࡣ pET-21aࠊpCold IࠊpCold TF ࢆ⏝࠸ࠊEco RIࠊNde I ࡛ᾘ໬ࡋࠊPCR

⏘≀࡜ࣛ࢖ࢤ࣮ࢩࣙࣥࡋࡓࠋ

95

Ni Sepharose 6 Fast Flow

࡜

Resource Q

࡟ࡼࡿ㓝⣲⢭〇

⤖ྜBuffer࡜ࡋ࡚ࠊ50 mM Tris-HCl buffer (pH 7.4), 500 mM NaCl, 20 mM Imidazoleࢆ⏝࠸

ࡓࠋࡇࡢ⤖ྜ Buffer ୍࡛ᬌ㏱ᯒࡋࡓࠊ9 L ࡢᇵ㣴ᾮ࠿ࡽᚓࡽࢀࡓ E.coli BL21(DE3)/pCold I/fct114 ⏤᮶ࡢC.F.E. (ࢱࣥࣃࢡ㉁㔞 901 mg) ࢆࠊ⤖ྜBuffer࡛ᖹ⾮໬ࡋࡓNi Sepharose 6 FF

࣒࢝ࣛ (ᶞ⬡㔞 25 mL, 16.0™1.8 cm) ࡟࢔ࣉࣛ࢖ࡋࡓࠋὶ㏿ࡣ0.5 mL/minࠊศ⏬ࢧ࢖ࢬࢆ5 mL࡟タᐃࡋࡓࠋ⤖ྜBuffer࡛Ὑίᚋࠊ⁐ฟBuffer࡜ࡋ࡚50 mM Tris-HCl buffer (pH 7.0), 500

mM NaCl, 50 mM Imidazole࡛⁐ฟࢆ⾜ࡗࡓࠋ280 nmࡢ྾཰ࢆ᭷ࡍࡿ⏬ศ࡟ࡘ࠸࡚SDS-PAGE

ࢆ⾜࠸ࠊ┠ⓗ㓝⣲ࢱࣥࣃࢡ㉁ࢆྵࡴ⏬ศࢆ㞟ࡵࠊVivaspin Turbo 15ࢆ⏝࠸࡚⤖ྜBuffer࡟ࡼ

ࡾࠊ⬺ሷฎ⌮࡜⃰⦰ฎ⌮ࢆྠ᫬࡟⾜ࡗࡓࠋࡑࡢᚋࠊ20 mM Tris-HCl buffer (pH 8.0) ࡛ᖹ⾮໬

ࡋࡓResource Q㸦ᶞ⬡㔞6 mL, 3.9×2.2 cm㸧࡟࢔ࣉࣛ࢖ࡋࠊὶ㏿ࡣ0.2 mL/minࠊศ⏬ࢧ࢖ࢬ

ࢆ2 mL࡟タᐃࡋࡓࠋ20 mM Tris-HCl buffer (pH 8.0) ࡛Ὑίᚋࠊࢢࣛࢪ࢚ࣥࢺ⃰ᗘࡣ0 M࠿

ࡽ0.175 M NaCl࡟タᐃࡋࠊÄKTAࢩࢫࢸ࣒ࢆ⏝࠸࡚⁐ฟࢆ⾜ࡗࡓࠋ

Somogyi-Nelson

ἲ࡟ࡼࡿ Fct114 ࡢ㓝⣲໬Ꮫⓗㅖᛶ㉁ࡢ᳨ウ

3-2-19ࡢ᪉ἲ࡟ᚑࡗ࡚ࠊ㓝⣲ⓗ࡟ㄪ〇ࡋࡓ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࠊ⢾ࡢ㑏ඖ

ᮎ➃⃰ᗘࢆẚⰍ ᐃࡍࡿࡇ࡜ࡀ࡛ࡁࡿ Somogyi-Nelson ἲ [87] ࢆ⏝࠸࡚ࠊFct114 ࡛㓝⣲཯

ᛂࡉࡏࡓᚋࡢ㑏ඖᮎ➃⃰ᗘࢆ⟬ฟࡋࠊ㓝⣲άᛶࢆᐃ㔞ⓗ࡟ ᐃࡋࡓࠋSomogyi-Nelson ἲ࡛

ࡣࠊ㖡ヨ⸆ (⾲6-2, ⾲6-3) ࡜Nelsonヨ⸆ (⾲6-4) ࢆ⏝࠸ࡓࠋ㖡ヨ⸆ࡢ (a) ヨ⸆100 PL࡜

(b) ヨ⸆2.5 mLࡢΰྜᾮࢆㄪ〇ࡋࡓᚋࠊࡑࡇ࠿ࡽ100 PL᥇ྲྀࡋࠊ㓝⣲཯ᛂᾮ100 ȣLࢆΰ

ࡐࠊἛ㦐‮ᾎ୰ (100Υ)࡛ 10 min ࡢ⇕ฎ⌮ࢆ⾜ࡗࡓࠋࡑࡢᚋᛴ෭ࡋࠊNelsonヨ⸆ (⾲ 6-4) 100 PLࢆΰࡐຍ࠼ࠊ30 minᐊ ࡛ᨺ⨨ࡋࡓࠋࡑࡋ࡚ࠊࡑࡢ⁐ᾮࢆ2.5 mLࡢMilliQ࡛ᕼ㔘ࡋ ࡓᚋࠊA660࡛྾ගᗘࢆ ᐃࡋࡓࠋ᳨㔞⥺ࡣL-ࣇࢥ࣮ࢫࢆ⏝࠸࡚సᡂࡋࡓ㸦ᅗ 6-1㸧ࠋ

96

⾲6-2. 㖡ヨ⸆ (a) ヨ⸆⤌ᡂ

15% ◲㓟㖡(II)஬Ỉ࿴≀ 15 g

Total 100 mL

⾲6-3. 㖡ヨ⸆ (b) ヨ⸆⤌ᡂ

↓ỈⅣ㓟ࢯ࣮ࢲ 12.5 g 㓇▼㓟ࢼࢺ࣒࣒ࣜ࢘࢝ࣜ࢘ᅄỈ࿴≀ 12.5 g

Ⅳ㓟Ỉ⣲ࢼࢺ࣒ࣜ࢘ 10 g

↓Ỉ◲㓟ࢯ࣮ࢲ 100 g

Total 500 mL

⾲6-4. Nelsonヨ⸆⤌ᡂ

୐ࣔࣜࣈࢸࣥ㓟භ࢔ࣥࣔࢽ࣒࢘ᅄỈ࿴≀ 12.5 g ࡦ⣲Ỉ⣲஧ࢼࢺ࣒ࣜ࢘୐Ỉ࿴≀ 12.5 g

⃰◲㓟 10 g

Total 500 mL

ᅗ6-1. Somogyi-Nelsonἲ࡟࠾ࡅࡿL-ࣇࢥ࣮ࢫࡢ᳨㔞⥺

0 0.5 1 1.5 2 2.5

0 1 2 3 4 5 6 7

A

660

䝣䝁䞊䝇⃰ᗘ (mM)

97

⮳㐺 ᗘࡢ᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ 11.7 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ 2%ࠊ100

mM ࣜࣥ㓟buffer (pH 7.4) ࢆྵࡴ཯ᛂᾮ 200 Plࡢ᮲௳࡛10㹼70 Υࠊ10 min཯ᛂᚋࠊ85Υࠊ

5 minฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋFct114ࡢ⮳㐺 ᗘࢆ᳨ウࡋࡓࠋ

⇕Ᏻᐃᛶࡢ᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ (11.7 Pg/ml) ࢆ25㹼60 Υ࡛30 minࣉࣞ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓᚋࠊ⬺

࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ 2%ࠊ100 mM ࣜࣥ㓟buffer (pH 7.4) ࢆྵࡴ཯ᛂᾮ 200 Plࡢ᮲௳࡛

30 Υࠊ10 min཯ᛂᚋࠊ85 Υࠊ5 minฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋFct114 ࡢ⇕Ᏻᐃᛶࢆ᳨ウࡋࡓࠋ

⮳㐺 pH ࡢ᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ11.7 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ2%ࠊ100

mM buffer ࢆྵࡴ཯ᛂᾮ200 Plࡢ᮲௳࡛30 Υࠊ10 min཯ᛂᚋࠊ85Υࠊ5 minฎ⌮ࡋࠊ6-2-5

ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋFct114ࡢ⮳㐺pHࢆ᳨ウࡋࡓࠋ࡞࠾ࠊpH 5.0 ~ 7.3࡛ࡣ

ࣜࣥ㓟࣒࢝ࣜ࢘bufferࠊpH 7.7, pH 8.2࡛ࡣ࣍࢘㓟bufferࠊpH 9.9 ࡛ࡣNaHCO3/NaOH bufferࠊ ࡑࡋ࡚ࠊpH 10.5࡛ࡣNa2HPO4/NaOH bufferࢆ⏝࠸࡚཯ᛂࢆ⾜ࡗࡓࠋ

pH

Ᏻᐃᛶࡢ᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ (11.7 Pg/ml) ࢆpH 4.5 㹼 pH 10.0 ࡢ⠊ᅖ࡛30 minࣉࣞ࢖ࣥ࢟ࣗ࣋

࣮ࢺࡋࡓᚋࠊ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ 2%ࠊ100 mM ࣜࣥ㓟buffer (pH 7.4) ࢆྵࡴ཯ᛂᾮ 200 Plࡢ᮲௳࡛30 Υࠊ10 min཯ᛂᚋࠊ85 Υࠊ5 minฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃

ࢆᐃ㔞ࡋFct114ࡢpHᏳᐃᛶࢆ᳨ウࡋࡓࠋ

98 㔠ᒓ࢖࢜ࣥࡢάᛶ࡬ࡢᙳ㡪

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ11.7 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ2%ࠊ100

mM ࣜࣥ㓟 buffer (pH 7.4)ࠊྛ㔠ᒓ࢖࢜ࣥࢆ1 mMࢆྵࡴ཯ᛂᾮ200 Plࢆ30 Υ࡛10 min࢖

࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ85 Υ, 5 min⇕ฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋࡓࠋ⏝

࠸ࡓ໬ྜ≀ࡣࠊNaClࠊKClࠊAgNO3ࠊMgCl2ࠊBaCl2ࠊCaCl2ࠊAlCl3ࠊFeCl2ࠊCuCl2ࠊMnCl2ࠊ ZnCl2ࠊCoCl2ࠊNiCl2࡛࠶ࡿࠋ

㓝⣲㜼ᐖ๣ࡀάᛶ࡟ཬࡰࡍᙳ㡪᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ23.4 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ2%ࠊ100

mM ࣜࣥ㓟 buffer (pH 7.4)ࠊྛ㓝⣲㜼ᐖ๣ࢆ1 mMࢆྵࡴ཯ᛂᾮ200 Plࢆ30 Υ࡛10 min࢖

࣮ࣥ࢟ࣗ࣋ࢺᚋࠊ85 Υ, 5 min⇕ฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋࡓࠋ⏝

࠸ࡓ໬ྜ≀ࡣࠊEDTAࠊp-Chloromercuribenzoate (PCMB)ࠊ8-QuinolinolࠊN-Ethylmaleimideࠊ N-Bromosuccinimideࠊ2,2’-Bipyridineࠊ1,10-Phenanthroline monohydrateࠊDiethyldithiocarbamic acid sodium salt trihydrateࠊ5,5’-Dithiobis (2-nitrobenzoic acid) (DTNB)࡛࠶ࡿࠋ

ᇶ㉁≉␗ᛶࡢ᳨ウ

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ177 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ100 mM ࣜࣥ㓟 buffer (pH 7.4)ࠊྛ

ࣇࢥ࢖ࢲࣥ࠶ࡿ࠸ࡣᾏὒ⏤᮶ከ⢾ 2% ࢆྵࡴ཯ᛂᾮ200 Plࢆ30 Υ࡛10 min࢖࣮ࣥ࢟ࣗ࣋

ࢺᚋࠊ85 Υ, 5 min⇕ฎ⌮ࡋࠊ6-2-5 ࡢ᪉ἲ࡛⏕ᡂࡋࡓ㑏ඖᮎ➃ࢆᐃ㔞ࡋࡓࠋ⏝࠸ࡓ໬ྜ≀

ࡣࠊࣇࢥ࢖ࢲࣥ࡟㛵ࡋ࡚ࡣࠊ⬺࢔ࢭࢳࣝ໬࢜࢟ࢼ࣡ࣔࢬࢡ (C. okamuranus) ࣇࢥ࢖ࢲࣥࠊ࢜

࢟ࢼ࣡ࣔࢬࢡ (C. okamuranus) ࣇࢥ࢖ࢲࣥࠊ࢔࢝ࣔࢡ (Sargassum horneri) ࣇࢥ࢖ࢲࣥࠊࣄࣂ

࣐ࢱ (Fucus vesiculosus) ࣇࢥ࢖ࢲࣥࠊ࣓࣡࢝ (Undaria pinnatifida) ࣇࢥ࢖ࢲࣥࠊ࢜࢜࢘࢟ࣔ

(Macrocystis pyrifera) ࣇࢥ࢖ࢲࣥࠊࢥࣥࣈ (Kjellmaniella crassifolia) ࣇࢥ࢖ࢲࣥࠊࢼ࣐ࢥ

99

(Holothuroidea sp.) ࣇࢥ࢖ࢲ࡛ࣥ࠶ࡿࠋᾏὒ⏤᮶ከ⢾࡟㛵ࡋ࡚ࡣࠊ࢔ࣝࢠࣥ㓟ࠊ࣑ࣛࢼࣜࣥࠊ

࢝ࣛࢠ࣮ࢼࣥࠊࢥࣥࢻࣟ࢖ࢳࣥ◲㓟ࠊࣄ࢔ࣝࣟࣥ㓟࡛࠶ࡿࠋ

࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢ࢔ࢭࢳࣝᇶࡢ㛵୚

6-2-4࡛ᚓࡓ⢭〇㓝⣲ࢆ117 Pg/ml࡟࡞ࡿࡼ࠺࡟ຍ࠼ࠊ100 mM ࣜࣥ㓟 buffer (pH 7.4)ࠊ⬺

࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ࠶ࡿ࠸ࡣࣇࢥ࢖ࢲࣥ 2% ࢆྵࡴ཯ᛂᾮ200 Plࢆ30 Υ࡛10 min࢖ࣥ

࣮࢟ࣗ࣋ࢺᚋࠊ85 Υ, 5 min⇕ฎ⌮ࡋࠊHPLCศᯒࢆ⾜ࡗࡓࠋ

100

⤖ᯝ

fct114ࡢ

pET-21a

ࠊ

pCold I

ࠊ

pCold TF

࣋ࢡࢱ࣮࡬ࡢࢡ࣮ࣟࢽࣥࢢ

6-2-2࠾ࡼࡧ6-2-3 ࡢ᪉ἲ࡛PCRࢆ⾜ࡗࡓ⤖ᯝࠊfct114ࡢሷᇶ㛗࡛࠶ࡿ3.0 kbp௜㏆࡟ࣂ

ࣥࢻࡀ☜ㄆ࡛ࡁࡓࠋᚓࡽࢀࡓ fct114 ࡜pET-21aࠊpCold IࠊpCold TFࢆNde I ࡜EcoRI࡛ ᾘ໬ࡋࠊpET-21a࡟┦ᙜࡍࡿ5443 bpࡢࣂࣥࢻࠊpCold I ࡟┦ᙜࡍࡿ 4407 bp ࡢࣂࣥ

ࢻࠊpCold TF࡟┦ᙜࡍࡿ579 bpࡢࣂࣥࢻ࠾ࡼࡧ fct114 ࡟┦ᙜࡍࡿ 3078 bp ࡢࣂࣥࢻ࡟

ࡘ࠸࡚ࡑࢀࡒࢀษࡾฟࡋࢆ⾜࠸ࠊGel Extraction Kitࢆ⏝࠸࡚ᢳฟࡋࠊ25PLࡢ௜ᒓࡢbuffer

࡛⁐ฟࡉࡏࡓࠋ

fct114࡜pET-21aࠊpCold I࠶ࡿ࠸ࡣ pCold TF࡜ࡢࣛ࢖ࢤ࣮ࢩࣙࣥࢆ⾜࠸ࠊE.coliDH5Dࢥ

ࣥࣆࢸࣥࢺࢭࣝ࡬ࡢᑟධࢆ⾜ࡗࡓࠋ37Υ୍࡛ᬌ㟼⨨ᇵ㣴ࡋࡓ࡜ࡇࢁࠊࢥࣟࢽ࣮ࢆ☜ㄆ࡛ࡁ ࡓࠋ⏕ࡌࡓࢥࣟࢽ࣮ᩘಶࡎࡘ࠿ࡽࣉࣛࢫ࣑ࢻ༢㞳ࢆ⾜࠸ࠊCᮎ➃ഃࢆEcoRIࠊNᮎ➃ഃࢆ

NdeI࡛ᾘ໬ࡋࡓᚋ࢔࣮࢞ࣟࢫ㟁ẼὋືࢆࡋࡓ⤖ᯝࢆ ᅗ6-2 ࡟♧ࡍࠋpET-21a࡜fct114ࢆࣛ

࢖ࢤ࣮ࢩࣙࣥࡋࡓ⤌᥮࠼ࣉࣛࢫ࣑ࢻࡣࠊfct114࡜⪃࠼ࡽࢀࡿ3078 bp࡜pET-21a࡜⪃࠼ࡽࢀ

ࡿ5443 bp௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀぢࡽࢀࡿࡣࡎ࡛ࠊᅗ6-2 ࡢ࣮ࣞࣥ1 ࡟┦ᙜࡍࡿࣃࢱ࣮

ࣥࡀㄆࡵࡽࢀࡓࠋࡑࡋ࡚ࠊpCold I࡜fct114ࢆࣛ࢖ࢤ࣮ࢩࣙࣥࡋࡓ⤌᥮࠼ࣉࣛࢫ࣑ࢻࡣࠊ

fct114࡜⪃࠼ࡽࢀࡿ3078 bp࡜pCold I࡜⪃࠼ࡽࢀࡿ4407 bp௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀぢࡽ

ࢀࡿࡣࡎ࡛ࠊᅗ6-2 ࡢ࣮ࣞࣥ2 ࡟┦ᙜࡍࡿࣃࢱ࣮ࣥࡀㄆࡵࡽࢀࡓࠋ୍᪉ࠊpCold TF࡜

fct114ࢆࣛ࢖ࢤ࣮ࢩࣙࣥࡋࡓ⤌᥮࠼ࣉࣛࢫ࣑ࢻࡣࠊfct114࡜⪃࠼ࡽࢀࡿ3078 bp࡜pCold TF

࡜⪃࠼ࡽࢀࡿ5769 bp௜㏆࡟ࢩࣥࢢࣝࣂࣥࢻࡀぢࡽࢀࡿࡣࡎࡔࡗࡓࡀࠊᅗ6-2 ࡢ࣮ࣞࣥ3

࡟♧ࡍ㏻ࡾࠊ┦ᙜࡍࡿࣃࢱ࣮ࣥࡀㄆࡵࡽ࡞࠿ࡗࡓࠋpCold TF௨እࡢpET-21a࠾ࡼࡧpCold I

࡟┠ⓗDNA᩿∦ࡀྵࡲࢀ࡚࠸ࡿ࡜ุ᩿ࡋࠊࡇࢀࡽࣉࣛࢫ࣑ࢻࢆ⏝࠸࡚E.coliBL21(DE3)

ࢆᙧ㉁㌿᥮ࡋࡓࠋ

101

ᅗ6-2. ไ㝈㓝⣲ᾘ໬࡟ࡼࡾ⤌᥮࠼ࣉࣛࢫ࣑ࢻࢆ☜ㄆࡋࡓ࢔࣮࢞ࣟࢫ㟁ẼὋື⤖ᯝ

M. Markers, 1. pET-21a/fct114, 2. pCold I/fct114, 3. pCold TF/fct114, 4. pET-21a.

E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114 ࡟ࡼ

ࡿ┠ⓗࢱࣥࣃࢡ㉁⏕⏘᳨ウ

E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114࡟ࡼࡿ┠ⓗ㑇ఏᏊࡢⓎ

⌧᳨ウࢆ⾜ࡗࡓࠋSDS-PAGE࡟ࡼࡿࢱࣥࣃࢡ㉁ࡢ⏕⏘᳨ウ⤖ᯝࢆᅗ6-3 ࡟♧ࡍࠋ┠ⓗࢱࣥࣃ

ࢡ㉁࡛࠶ࡿFct114ࡢศᏊ㔞ࡣࠊ108 kDa࡛࠶ࡾࠊࡑࡢ௜㏆ࡢศᏊ㔞ࡢࣂࣥࢻࢆぢࡿ࡜࣋ࢡࢱ

࣮ࡢࡳࢆᙧ㉁㌿᥮ࡋࡓ኱⭠⳦ (࣮ࣞࣥ5, 6) ࡟ࡣ↓࠸ࣂࣥࢻࡀᏑᅾࡋࡓࡓࡵ㸦࣮ࣞࣥ1ࠊ3㸧ࠊ

Fct114 ࡀ⏕⏘ࡉࢀ࡚࠸ࡿࡇ࡜ࡀ SDS-PAGE ࡢ⤖ᯝࡼࡾ♧၀ࡉࢀࡓࠋࡲࡓࠊE.coli BL21

(DE3)/pET-21a/fct114ࡢC.F.E.ࡼࡾE.coli BL21 (DE3)/pCold I/fct114ࡢC.F.E.ࡢ࡯࠺ࡀ┠ⓗࢱࣥ

ࣃࢡ㉁㑇ఏᏊࢆከࡃⓎ⌧ࡋ࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡛࠶ࡗࡓࡓࡵࠊFct114 ࢆ኱㔞࡟ᚓࡿࡓࡵ

E.coli BL21 (DE3)/pCold I/fct114ࢆ኱㔞ᇵ㣴ࡍࡿࡇ࡜࡜ࡋࡓࠋ

102

ᅗ6-3. E.coli BL21(DE3)/pET-21a/fct114࠾ࡼࡧE. coli BL21(DE3)/pCold I/fct114 ⏤᮶C.F.Eࡢ

SDS-PAGE⤖ᯝ

M. Markers,

1. E. coli BL21(DE3)/pET-21a/fct114 cell-free extracts, 2. E. coli BL21(DE3)/pET-21a/fct114 insoluble fraction, 3. E. coli BL21(DE3)/pCold I/fct114 cell-free extracts, 4. E. coli BL21(DE3)/pCold I/fct114 insoluble fraction, 5. E. coli BL21(DE3)/pCold I/fct114 cell-free extracts, 6. E. coli BL21(DE3)/pCold I/fct114 insoluble fraction, 7. E. coli BL21(DE3)/pET-21a cell-free extracts, 8. E. coli BL21(DE3)/pCold I cell-free extracts

E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114⏤᮶

C.F.E.

୰ࡢࣇࢥ࢖ࢲࢼ࣮ࢮάᛶ

E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114ࡼࡾㄪ〇ࡋࡓC.F.E.ࡢ

ࣇࢥ࢖ࢲࢼ࣮ࢮάᛶࡢ᭷↓ࢆ᳨ウࡋࡓࠋ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࠊHPLCࢆ⏝࠸

࡚ 5-2-2 ࡟ ♧ ࡍ ᧯ స ࡜ ྠ ᵝ ࡢ ᪉ ἲ ࡛ ⾜ ࡗ ࡓ ࠋ ࡑ ࡢ ⤖ ᯝ ࢆ ᅗ 6-4 ࡟ ♧ ࡍ ࠋE. coli

BL21(DE3)/pCold I/fct114 ࡼࡾㄪ〇ࡋࡓ C.F.E.࡟࠾࠸࡚ࣇࢥ࢖ࢲࢼ࣮ࢮάᛶࢆ☜ㄆࡍࡿࡇ࡜

103

ࡀฟ᮶ࡓࠋ୍᪉࡛ࠊ E. coli BL21(DE3)/pET-21a/fct114ࡼࡾㄪ〇ࡋࡓC.F.E.࡛ࡣࣇࢥ࢖ࢲࢼ࣮

ࢮάᛶࡀぢࡽࢀ࡞࠿ࡗࡓࠋ

Black㸸Negative control

Red㸸E.coli BL21(DE3)/pET-21a/fct114 cell-free extracts (Including substrate)

Blue㸸E.coli BL21(DE3)/pCold I/fct114 cell-free extracts (Including substrate)

ᅗ6-4. E. coli BL21(DE3)/pET-21a/fct114࡜E. coli BL21(DE3)/pCold I/fct114 ⏤᮶C.F.Eࢆ

⏝࠸ࡓ㓝⣲཯ᛂᚋࡢHPLCࡢࢳ࣮ࣕࢺ⤖ᯝ

Ni Sepharose 6 FF

࣒࢝ࣛ࡟ࡼࡿ㓝⣲⢭〇

E. coli BL21(DE3)/pCold I/fct114ࢆ9 Lᇵ㣴ࡋ࡚ᚓࡽࢀࡓC.F.E. (900.9 mg-protein) ࢆ 6-2-4 ࡢ᪉ἲ࡟ᚑ࠸ࠊ┠ⓗ㓝⣲ࡢ⢭〇ࢆ⾜ࡗࡓࠋྛImidazole⃰ᗘࡢ buffer ࡛⁐ฟࡋࡓ⏬ศࢆᅇ཰

ࡋࠊ20 mM Tris-HCl (pH 8.0) ࡟ᑐࡋ࡚㏱ᯒᚋࠊ㓝⣲άᛶ ᐃࢆᐇ᪋ࡋࡓ⤖ᯝࠊ100 mM Imidazoleࢆྵࡴ buffer ࡛⁐ฟࡋࡓ⏬ศ㸦Fraction No. 1㹼40㸧࡟࠾࠸࡚ࣇࢥ࢖ࢲࢼ࣮ࢮάᛶ

ࢆ᭷ࡍࡿࢱࣥࣃࢡ㉁ (100 kDa௜㏆) ࢆ᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࠊࡇࢀࡽࢆᅇ཰ࡋࡓࠋ

104

Resource Q

࡟ࡼࡿ⢭〇

6-3-4 ࡛ᚓࡓࣇࢥ࢖ࢲࢼ࣮ࢮάᛶࢆ᭷ࡍࡿ⁐ฟ⏬ศࢆࠊࡑࢀࡒࢀ Vivaspin Turbo 15

(Sartrius) ࢆ⏝࠸࡚20 mM Tris-HCl buffer (pH 8.0) ࡟ࡼࡾࠊ⬺ሷฎ⌮࡜⃰⦰ᚋࠊࡑࡢ㒊ศ⢭

〇㓝⣲㸦ࢱࣥࣃࢡ㉁㔞94.8 mg㸧ࢆࠊ6-2-4 ࡢ᪉ἲ࡟ᚑ࠸ࠊResource Q ࡟ࡼࡾ⢭〇ࡋࡓࠋࡑ ࡢ㝿ࡢ⁐ฟࣃࢱ࣮ࣥࢆᅗ 6-5 ࡟♧ࡍࠋࢱࣥࣃࢡ㉁ࡢ⁐ฟࡀㄆࡵࡽࢀࡓࣇࣛࢡࢩࣙࣥ࡟ࡘ࠸

࡚ SDS-PAGE ゎᯒࢆࡋࡓ⤖ᯝࢆᅗ 6-6 ࡟♧ࡍࠋ

ᅗ6-5. Resource Q࣒࢝ࣛࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮ࡢ⁐ฟࣃࢱ࣮ࣥ

105

ᅗ6-6. Resource Q࡛⁐ฟࡉࢀࡓྛࣇࣛࢡࢩࣙࣥࡢSDS-PAGE⤖ᯝ

ᅗ6-6࡛ぢࡽࢀࡿࡼ࠺࡟Resource Q࣒࢝ࣛࢆ㏻ࡍࡇ࡜࡟ࡼࡾࠊNi-Sepharose࡛⢭〇ࡋࡓ࡜

ࡁࡼࡾࡶࣂࣥࢻࡀởࡃ࡞ࡿ࡜࠸࠺⌧㇟ࡀぢࡽࢀࡓࠋࡇࡢ㝿ࠊ࡝ࡢࣇࣛࢡࢩࣙࣥࡶྠࡌࡼ࠺࡞

ࣂࣥࢻࣃࢱ࣮ࣥࢆ♧ࡋ 100 kDa ௜㏆ࡢࣂࣥࢻࡢ⃰ᗘ࡟౫Ꮡࡋ࡚࠸ࡿ࠿ࡢࡼ࠺࡟௚ࡢࣂࣥࢻ ࡢ⃰ᗘࡶⷧࡃ࡞ࡗ࡚࠸ࡿࡇ࡜࠿ࡽࠊࡇࢀࡣࠊFct114ࡀ SDS-PAGE๓ࡢ⇕ฎ⌮࡟ࡼࡗ࡚⇕ศ ゎࡉࢀ࡚࠸ࡿࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼ࠊ⇕ฎ⌮᫬㛫ࢆኚ᭦ࡋ࡚෌ᗘ SDS-PAGEࢆ⾜ࡗࡓ (ᅗ 6-7) ࠋࡇࡢ⤖ᯝࠊFct114 ࡣ⇕ฎ⌮࡟ࡼࡗ࡚ศゎࡉࢀ࡚࠸ࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋࡑࡇ࡛ࠊ⇕ฎ⌮

᫬㛫ࢆ15 sec࡟ࡋࠊResource QᚋࡢFraction No. 10~22ࢆᅇ཰ࡋ࡚ࠊSDS-PAGEࢆ⾜ࡗࡓ࡜

ࡇࢁࠊ࡯ࡰ༢୍࡟⢭〇ࡉࢀ࡚࠸ࡿ (ᅗ 6-8) ࡇ࡜ࡀࢃ࠿ࡗࡓࡓࡵࠊࡇࡢ㓝⣲⁐ᾮࢆ⏝࠸࡚௒

ᚋࡢᐇ㦂ࢆ⾜࠺ࡇ࡜࡟ࡋࡓࠋࡇࡢࢧࣥࣉࣝࡢࢱࣥࣃࢡ㉁⃰ᗘࡣ1.77 mg/mL࡛࠶ࡗࡓࠋ

106

ᅗ6-7. ⇕ฎ⌮᫬㛫ࢆኚ᭦ࡋࡓFraction No. 15ࡢ⁐ฟ⏬ศࡢSDS-PAGE⤖ᯝ

107

ᅗ6-8. E. coli BL21(DE3)/pCold I/fct114 ࡢC.F.E.࠿ࡽ⢭〇ࡋࡓࣇࢥ࢖ࢲࢼ࣮ࢮࡢ

SDS-PAGE⤖ᯝ

1. C. F. E. of E.coli BL21(DE3)/pCold I (23.5 Pg), 2. C. F. E. of E.coli BL21(DE3)/pCold I/fct114 (5.0 Pg), 3. Pooled fractions after Resource Q chromatography (1.8 Pg).

⮳㐺 ᗘࠊ⮳㐺 pH ࡢ᳨ウ

᳨ウࡢ⤖ᯝࠊࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢ⮳㐺 ᗘࡣ40Υ (ᅗ6-9)ࠊ⮳㐺 pH 7.3 (ᅗ6-10) ࡛

࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ࡞࠾ࠊάᛶࡣ 6-2-5 ࡢ᪉ἲ࡟ࡼࡾホ౯ࡋࡓࠋ

108

ᅗ6-9. ࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢ⮳㐺 ᗘ᳨ウ⤖ᯝ

ᅗ6-10. ࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢ⮳㐺pH᳨ウ⤖ᯝ

109

⇕Ᏻᐃᛶࡢ᳨ウ

ࣇࢥ࢖ࢲࢼ࣮ࢮ Fct114 ࡢ⇕Ᏻᐃᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋάᛶࡣ 6-2-5 ࡢ᪉ἲ࡟ࡼࡾホ౯ࡋ ࡓࠋᮍฎ⌮ࡢ㓝⣲⁐ᾮࡢάᛶࢆ100%࡜ࡍࡿ࡜ࠊ40 Υࡲ࡛ࡣ⣙ 80㸣ࡢṧᏑάᛶࢆಖᣢࡍࡿ

ࡶࡢࡢࠊ40 Υ௨ୖ࡛㢧ⴭ࡟άᛶࡀపୗࡋࡓ (ᅗ6-11) ࠋ

ᅗ6-11. ࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢ⇕Ᏻᐃᛶ᳨ウ⤖ᯝ

pH

Ᏻᐃᛶࡢ᳨ウ

ࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢpHᏳᐃᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋάᛶࡣ6-2-5ࡢ᪉ἲ࡟ࡼࡾホ౯ࡋ

ࡓࠋpH 7.0 ࡢ㓝⣲⁐ᾮࡢάᛶࢆ100%࡜ࡍࡿ࡜ࠊFct114ࡣpH 6.0~8.5ࡢ㛫࡛Ᏻᐃ࡛࠶ࡿࡇ࡜

ࡀ♧၀ࡉࢀࡓ (ᅗ6-12) ࠋ

110

ᅗ6-12. ࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢpHᏳᐃᛶ᳨ウ⤖ᯝ

㔠ᒓ࢖࢜ࣥࠊ㓝⣲㜼ᐖ๣ࡢάᛶ࡬ࡢᙳ㡪᳨ウ

ྛ✀㔠ᒓ࢖࢜ࣥࠊ࠾ࡼࡧ㓝⣲㜼ᐖ๣ࡢࣇࢥ࢖ࢲࢼ࣮ࢮάᛶ࡟ཬࡰࡍᙳ㡪ࢆ᳨ウࡋࡓࠋ࡞࠾ࠊ άᛶࡣ 6-2-5 ࡢ᪉ἲ࡟ࡼࡾホ౯ࡋࡓࠋࡑࡢ⤖ᯝࠊ Ba²⁺, Ca²⁺, Fe²⁺, Al³⁺ ࡛㓝⣲άᛶࡀୖ᪼ࡋ ࡓࠋࡇࡢࡇ࡜࠿ࡽࠊᮏ㓝⣲ࡣ㔠ᒓ㓝⣲࡛࠶ࡿࡇ࡜ࡀࢃ࠿ࡗࡓ (ᅗ 6-13) ࠋ୍᪉ࠊ㜼ᐖ๣࡟㛵 ࡋ࡚ࡣSH㜼ᐖ๣࡟ࡼࡿάᛶࡢ㜼ᐖࡀぢࡽࢀࡓ (ᅗ 6-14) ࠋ

111

ᅗ6-13. ࣇࢥ࢖ࢲࢼ࣮ࢮάᛶ࡟ཬࡰࡍ㔠ᒓ࢖࢜ࣥࡢᙳ㡪

ᅗ6-14. ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮάᛶ࡟ཬࡰࡍ㓝⣲㜼ᐖ๣ࡢᙳ㡪

112 ᇶ㉁≉␗ᛶࡢ᳨ウ

᳨ウࡢ⤖ᯝࠊࣇࢥ࢖ࢲࢼ࣮ࢮ Fct114 ࡣࠊ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࡢ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ

࡟ᑐࡋ࡚᭱ࡶ㧗࠸άᛶࢆ♧ࡋࡓࡀࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚ࡶ 50㸣ࡢάᛶࢆ♧

ࡋࡓࠋᑐ↷ⓗ࡟ᮏ㓝⣲ࡣ௚ࡢᾏ⸴✀⏤᮶ࡢࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚ࡣάᛶࢆ♧ࡉࡎࠊ௒ᅇヨࡋࡓ ᾏὒᛶከ⢾࡛࠶ࡿ࢔ࣝࢠࣥ㓟ࠊ࣑ࣛࢼࣛࣥࠊ࢝ࣛࢠ࣮ࢼࣥࠊࢥࣥࢻࣟ࢖ࢳࣥ◲㓟ࠊࣄ࢔ࣝࣟ

ࣥ㓟࡟ᑐࡋ࡚ࡶάᛶࢆ♧ࡉ࡞࠿ࡗࡓ (ᅗ6-15) ࠋ

ᅗ6-15. ௚ࡢᇶ㉁࡟ᑐࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮFct114ࡢάᛶ᳨ウ⤖ᯝ

࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥศゎࣉࣟࢭࢫ࡬ࡢ࢔ࢭࢳࣝᇶࡢ㛵୚

6-2-13ࡢ᪉ἲ࡛ 10ศ㛫㓝⣲཯ᛂࢆ⾜ࡗࡓ཯ᛂᾮࢆ⏝࠸࡚ࠊHPLCศᯒࢆ⾜ࡗࡓ⤖ᯝࠊ⬺

࢔ࢭࢳࣝ໬ࡋ࡚࠸ࡿ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ᪉ࡀࠊ⬺࢔ࢭࢳࣝ໬ࡋ࡚࠸࡞࠸ࡶࡢ࡟ẚ

࡭ࠊపศᏊ໬ࢆಁ㐍ࡋ࡚࠸ࡿࡇ࡜ࡀࢃ࠿ࡗࡓ (ᅗ6-16, 6-17) ࠋࡑࡋ࡚ࠊ⬺࢔ࢭࢳࣝ໬ࡋ࡚࠸

࡞࠸ࣇࢥ࢖ࢲࣥࢆ⏝࠸ࡓ㓝⣲཯ᛂࢆ3᪥㛫⾜ࡗ࡚ࡶࠊᅗ6-16 ࡢࣃࢱ࣮ࣥࡣᅗ6-17 ࡢࣃࢱ

࣮ࣥ࡟ኚࢃࡿࡇ࡜ࡣ࡞࠿ࡗࡓࠋ

113

ᅗ6-16. ⬺࢔ࢭࢳࣝ໬ࡋ࡚࠸࡞࠸࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࡓ㝿ࡢHPLCࡢ

ࣃࢱ࣮ࣥ (㯮⥺㸸㓝⣲↓ῧຍࠊ㉥⥺㸸㓝⣲ῧຍ)

ᅗ6-17. ⬺࢔ࢭࢳࣝ໬ࡋࡓ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࢆᇶ㉁࡜ࡋࡓ㝿ࡢ HPLC ࡢࣃࢱ࣮ࣥ

(㯮⥺㸸㓝⣲↓ῧຍࠊ㉥⥺㸸㓝⣲ῧຍ)

114

⪃ᐹ

ᮏ❶࡛ࡣࠊ➨ 5 ❶࡛ᚓࡽࢀࡓሷᇶ㓄ิࢆᇶ࡟࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢపศᏊ໬཯ᛂ

ࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ 㓝⣲໬Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆ࠾ࡇ࡞ࡗࡓࠋ௒ࡲ࡛࡟ࣇࢥ࢖ࢲࣥࡢᚤ⏕≀㓝⣲࡟ࡼࡿศゎ࡟

ࡘ࠸࡚ࡣ࠸ࡃࡘ࠿ࡢ◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࡀࠊࢹ࢔ࢭࢳ࣮ࣛࢮ࡟ࡼࡗ࡚⬺࢔ࢭࢳࣝ໬ࡉࢀࡓ ᚋࠊపศᏊ໬㓝⣲࡟ࡼࡗ࡚పศᏊ໬ࡀಁ㐍ࡉࢀࡿ࡜࠸ࡗࡓศゎࣉࣟࢭࢫ࡟ࡘ࠸࡚ࡣࠊㄪ࡭ࡓ 㝈ࡾ࡟࠾࠸࡚ࠊሗ࿌౛ࡣ࡞࠸ࠋ

⢾ຍỈศゎ㓝⣲ࡢάᛶࡢከࡃࡣࠊᇶ㉁ࡢຍỈศゎ࡟ࡼࡗ࡚⏕⏘ࡉࢀࡿ㑏ඖᮎ➃⃰ᗘࢆẚ Ⰽ ᐃࡍࡿࡇ࡜࡟ࡼࡗ࡚᥎ᐃࡉࢀࡿࠋࡋࡓࡀࡗ࡚ᮏ◊✲࡟࠾࠸࡚ࡶྠᵝ࡟ Somogyi-Nelson

ἲ [87] ࢆ⏝࠸࡚Fct114ࡢ㓝⣲άᛶࢆỴᐃࡋࡓࠋ ࡋ࠿ࡋ࡞ࡀࡽࠊࡇࡢࡼ࠺࡞㑏ඖᮎ➃⃰ᗘ

ࢆ฼⏝ࡋࡓάᛶ ᐃ᪉ἲࡣࠊFucobacter marina SA-0082 ⏤᮶ࣇࢥ࢖ࢲࣥศゎ㓝⣲ [64]ࠊM.

fucanivorans SW5⏤᮶ Fcn A [66]ࠊFormosa algae KM3553⏤᮶ FFA2 [47, 67]ࠊ࡜࠸ࡗࡓࣇࢥ

࢖ࢲࣥศゎ㓝⣲࡟ࡣ㐺⏝࡛ࡁ࡞࠿ࡗࡓࠋ ᚑࡗ࡚ࠊࡇࢀࡽࡢ㓝⣲άᛶࡣࠊศගගᗘィ࡟ࡼࡿ

᪉ἲ [64] ࡸ㟁ẼὋືἲ [47, 66, 67] ࡟ࡼࡾホ౯ࡉࢀ࡚࠸ࡿࠋࡇࡢࡼ࠺࡟㓝⣲άᛶࢆᐃ㔞ࡍ

ࡿࡓࡵ࡟␗࡞ࡿᡭ㡰ࡸᡭἲࢆ⏝࠸ࡿࡢࡣࠊᇶ㉁࡜ࡋ࡚౑⏝ࡉࢀࡿࣇࢥ࢖ࢲࣥࡢ໬Ꮫᵓ㐀ࡀ ศ㞳ඖ࡜࡞ࡿᾏ⸴࡟ࡼࡗ࡚␗࡞ࡿࡇ࡜࡟㉳ᅉࡋ࡚࠸ࡿ࡜᥎ᐹࡉࢀࡿࠋ

Fct114ࡣࠊBa²⁺ࠊFe²⁺ࠊ࠾ࡼࡧCa²⁺࡞࡝ࡢ஧౯࢝ࢳ࢜ࣥ࡟ࡼࡗ࡚㓝⣲άᛶࡀቑᙉࡉࢀࡓࡀࠊ

Co²⁺ࠊCu²⁺ࠊMn²⁺ࠊ࠾ࡼࡧZn²⁺ࡣᢚไຠᯝࢆ♧ࡋࡓࠋF. algae KMM3553࡜Alteromonas sp.⏤ ᮶ࣇࢥ࢖ࢲࢼ࣮ࢮࡣࠊάᛶࢆ᭷ࡍࡿࡓࡵ࡟ࡣ㔠ᒓ࢖࢜ࣥࢆᚲせ࡜ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗ

࡚࠸ࡿ [32, 47, 67] ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊ㔠ᒓ࢖࢜ࣥࡀᇶ㉁⤖ྜࡲࡓࡣゐ፹άᛶ࡟ᙳ㡪ࢆ୚࠼

ࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ

Fct114ࡣࠊ⬺࢔ࢭࢳࣝ໬ࡉࢀࡓ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢࡳ࡞ࡽࡎࠊኳ↛ࡢ࢜࢟ࢼ࣡ࣔ

ࢬࢡࣇࢥ࢖ࢲࣥࡶຍỈศゎ࡛ࡁࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋᗎㄽ࡛ࡶ㏙࡭ࡓࡼ࠺࡟ⴭ⪅ࡽࡢ ࢢ࣮ࣝࣉ࡛ࡣࠊH18ᰴ࡟ࡼࡾ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࣇࢥ࢖ࢲࣥࡀศゎࡉࢀࡿ㝿ࠊ⬺࢔ࢭࢳࣝ໬

115

཯ᛂࡢᚋ࡟పศᏊ໬཯ᛂࡀ㉳ࡇࡿࡇ࡜ࢆᥦၐࡋ࡚࠸ࡓࡀࠊ௒ᅇ⢭〇㓝⣲ࢆ⏝࠸ࡓ᳨ウࡢ⤖

ᯝࠊF. algae KMM3553 [47] ⏤᮶ࡢ㓝⣲࡜ྠᵝ࡟ኳ↛ࡢࣇࢥ࢖ࢲࣥࡶࡺࡗࡃࡾ࡜ศゎࡍࡿࡇ

࡜ࡀ࡛ࡁࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓࠋࡋ࠿ࡋࠊᅗ6-16, 6-17 ࡀ♧ࡍࡼ࠺࡟ࠊᜍࡽࡃࡣ࢔ࢭࢳࣝᇶ࡟

ࡼࡿ❧య㞀ᐖࡢࡓࡵࠊFct114 ࡣ࢔ࢭࢳࣝᇶࡀ௜ຍࡋࡓᵓ㐀࡟ᑐࡋ࡚స⏝࡛ࡁ࡞࠸ࡇ࡜ࡀ᥎ ᐹࡉࢀࡓࠋ

࢜࢟ࢼ࣡ࣔࢬࢡ (C. okamuranus) ࠊ࢔࢝ࣔࢡ (Sargassum horneri)ࠊࣄࣂ࣐ࢱ (Fucus vesiculosus)ࠊ ࣡ ࢝ ࣓ (Undaria pinnatifida)ࠊ ࢜ ࢜ ࢘ ࢟ ࣔ (Macrocystis pyrifera)ࠊ ࢥ ࣥ ࣈ (Kjellmaniella crassifolia)ࠊࢼ࣐ࢥ (Holothuroidea sp.) ⏤᮶ࡢ7✀ࡢࣇࢥ࢖ࢲࣥ࡟ᑐࡍࡿFct114 ࡢάᛶࢆㄪ࡭ࡓ࡜ࡇࢁࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ࡢࡳ≉␗ⓗ࡟άᛶࢆ♧ࡋࡓࠋᑡ࡞ࡃ࡜

ࡶ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢᵓ㐀ࡣࠊ௚ࡢᾏ⸴⏤᮶ࣇࢥ࢖ࢲࣥ࡜ࡣ␗࡞ࡗ࡚࠸ࡿ࡜⪃࠼

ࡽࢀ࡚࠸ࡿࠋྠᵝ࡟F. algae KMM3553࡜Alteromonas sp. SN-1009࠿ࡽ⢭〇ࡉࢀࡓ㓝⣲ࡣࠊ ࡑࢀࡒࢀࡢ⳦ᰴࡀ༢㞳ࡉࢀࡓᾏ⸴ࡢࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚ࡢࡳάᛶࢆ♧ࡋࡓࠋᚑࡗ࡚ࠊࡇࢀࡽ

ࡢᇶ㉁≉␗ᛶࡣࠊྛ㓝⣲ࡀࠊࣇࢥ࢖ࢲࣥ㈨໬ᛶᚤ⏕≀ࡀ௜╔ࡋ࡚࠸ࡓ⎔ቃ࡟ᛂࡌ࡚ࠊ≉ᐃࡢ

ࣇࢥ࢖ࢲࣥᵓ㐀ࢆ≉␗ⓗ࡟ศゎࡍࡿࡼ࠺࡟㐍໬ࡋࡓࡢ࡛ࡣ࡞࠸࠿࡜⪃࠼ࡽࢀࡿࠋ

116

⥲ᣓ

ᮏ◊✲࡛ࡣ➨2❶࡛Luteolibacter algae H18࠿ࡽࡢࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ⢭〇ࠊ➨

3 ❶࡛ࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࡢ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ⌧ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡢホ౯ࡋࠊ

➨4❶࡛ࢹ࢔ࢭࢳ࣮ࣛࢮࡢゐ፹ṧᇶࡢண ࠊࡉࡽ࡟➨5❶࡛Luteolibacter algae H18࠿ࡽࡢ

ࣇࢥ࢖ࢲࢼ࣮ࢮࡢ⢭〇ࠊ➨6❶࡛ࣇࢥ࢖ࢲࢼ࣮ࢮࡢ㑇ఏᏊྠᐃ࠾ࡼࡧ␗✀Ⓨ⌧ࠊ㓝⣲໬Ꮫⓗ

ᛶ㉁ࡢホ౯ࢆ⾜ࡗࡓࠋᮏ❶࡛ࡣࠊ௨ୖࢆ㋃ࡲ࠼࡚ࠊLuteolibacter algae H18⏤᮶ࡢࢹ࢔ࢭࢳࣛ

࣮ࢮFud࠾ࡼࡧࣇࢥ࢖ࢲࢼ࣮ࢮ Fct114࡟ࡘ࠸࡚㏙࡭ࠊࡉࡽ࡟Luteolibacter algae H18ࡢ࢜࢟

ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ࡘ࠸࡚ࡢศゎࣉࣟࢭࢫ࡟ࡘ࠸࡚㏙࡭ࡿࠋ

➨2❶࠿ࡽ➨4❶ࡢ⤖ᯝࡼࡾࠊLuteolibacter algae H18⏤᮶ࡢࢹ࢔ࢭࢳ࣮ࣛࢮFudࡣ௨ୗ

࡟♧ࡍᛶ㉁ࢆ᭷ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ࢔࣑ࣀ㓟㓄ิ࠿ࡽィ⟬ࡉࢀࡿࢱࣥࣃࢡ㉁ࡢศ

Ꮚ㔞ࡣ69,789 Da࡛࠶ࡾࠊࡇࢀࡣSDS-PAGE࠿ࡽࡢศᏊ㔞࡜࡯ࡰ୍⮴ࡋ࡚࠸ࡓࠋࡑࡋ࡚ࡑࡢ

࢔࣑ࣀ㓟㓄ิࢆࢥ࣮ࢻࡍࡿORFࢆᇶ࡟࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ⬺࢔ࢭࢳࣝ໬ࢆゐ፹ࡍ

ࡿࢹ࢔ࢭࢳ࣮ࣛࢮ㑇ఏᏊࡢࢡ࣮ࣟࢽࣥࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲໬

Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊFudࡣࠊ⮳㐺 ᗘࡣ40 Υࠊ⮳㐺pH 8.0࡛࠶ࡾࠊ

⇕Ᏻᐃᛶ࡟ࡘ࠸࡚ࡣࠊ35 Υ௨ୖ࡛㢧ⴭ࡟άᛶࡀపୗࡋࡓࡓࡵࠊ⇕࡟ᑐࡍࡿᏳᐃᛶࡣ㧗ࡃ࡞

࠸ࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ㔠ᒓ࢖࢜ࣥࡢάᛶ࡟ᑐࡍࡿᙳ㡪ࡣࠊAg⁺, Cu²⁺, Zn²⁺, Ni²⁺, Hg²⁺࡟ᑐࡋ࡚

άᛶ㜼ᐖࡀぢࡽࢀࠊ㜼ᐖ๣࡟ࡘ࠸࡚ࡣࠊSH㜼ᐖ๣࡟ࡼࡿάᛶࡀぢࡽࢀࡓࠋ୍᪉࡛ࠊ

SWISS-MODEL࡟ࡼࡿண ᵓ㐀ࣔࢹࣝ࡟࠾࠸࡚ゐ፹ṧᇶ࡜ࡋ࡚ࡣࠊAsp299, His329, Ser238ࡀ᝿ᐃࡉ

ࢀࡓࠋFudࡢCᮎ➃㡿ᇦ⣙ 300 ࢔࣑ࣀ㓟ṧᇶࡣࠊ❧యᵓ㐀ࡢࢸࣥࣉ࣮ࣞࢺ࡜ࡋࡓTM0077

࡟ࡣᏑᅾࡋ࡞࠸ࡓࡵࠊࡇࡢ C ᮎ➃㡿ᇦࢆ๐㝖ࡋάᛶ᳨ウࢆ⾜ࡗࡓ࡜ࡇࢁࠊゐ፹ṧᇶࢆྵࡲ

࡞࠸࡟ࡶ㛵ࢃࡽࡎࠊ㓝⣲άᛶ࡟ᙳ㡪ࢆཬࡰࡋࡓࠋᇶ㉁ࡢ࢔ࢭࢳࣝᇶࢆྲྀࡾ㎸ࡴ㝿࡟ Cᮎ➃ 㡿ᇦࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ

➨5❶࡜➨6❶ࡢ⤖ᯝࡼࡾࠊLuteolibacter algae H18⏤᮶ࡢࣇࢥ࢖ࢲࢼ࣮ࢮ Fct114ࡣ௨ୗ

117

࡟♧ࡍᛶ㉁ࢆ᭷ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ࢔࣑ࣀ㓟㓄ิ࠿ࡽィ⟬ࡉࢀࡿࢱࣥࣃࢡ㉁ࡢศ

Ꮚ㔞ࡣ112 kDa࡛࠶ࡾࠊࡇࢀࡣ SDS-PAGE࠿ࡽࡢศᏊ㔞࡜࡯ࡰ୍⮴ࡋ࡚࠸ࡓࠋ࡞࠾ࠊ௒ᅇ

ぢฟࡉࢀࡓORF࡟ࡣࠊNᮎ➃࢔࣑ࣀ㓟㓄ิࡢゎᯒ⤖ᯝ࡟ࡼࡗ࡚ᚓࡽࢀࡓAEVSELMPLP࡜

࠸࠺㓄ิࡢୖὶ࡟ࠊMHPLRFPITFPKLPALGAALVLSLLAATAPLKA࡜࠸࠺㓄ิࡀ♧ࡉࢀࡓࠋ ࡇࡢ㓄ิࡀ SignalP 4.1 Server ࡟ࡼࡾࢩࢢࢼࣝ㓄ิ࡛࠶ࡿ࠿ㄪ࡭ࡓ࡜ࡇࢁࠊࢩࢢࢼࣝ㓄ิ࡛

࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡑࡋ࡚ࡑࡢ࢔࣑ࣀ㓟㓄ิࢆࢥ࣮ࢻࡍࡿORFࢆᇶ࡟࢜࢟ࢼ࣡ࣔࢬࢡ

⏤᮶ࡢ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࡢపศᏊ໬ࢆゐ፹ࡍࡿࣇࢥ࢖ࢲࢼ࣮ࢮ㑇ఏᏊࡢࢡ࣮ࣟࢽࣥ

ࢢ࠾ࡼࡧ኱⭠⳦࡛ࡢ㓝⣲ࢱࣥࣃࢡ㉁ࡢ⏕⏘ࠊ㓝⣲໬Ꮫⓗᛶ㉁ࡢ≉ᚩỴᐃࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ

Fct114ࡣࠊ⮳㐺 ᗘࡣ40 Υࠊ⮳㐺pH 7.3 ࡛࠶ࡾࠊ⇕Ᏻᐃᛶ࡟ࡘ࠸࡚ࡣࠊ40 Υ௨ୖ࡛㢧ⴭ

࡟άᛶࡀపୗࡋࡓࠋpHᏳᐃᛶࡣࠊpH 7.0 ࡢάᛶࢆ100%࡜ࡍࡿ࡜ࠊpH 6.0㹼8.5ࡢ㛫࡛Ᏻᐃ

࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ㔠ᒓ࢖࢜ࣥࡢάᛶ࡟ᑐࡍࡿᙳ㡪ࡣࠊBa²⁺, Ca²⁺, Fe²⁺, Al³⁺ ࡛㓝⣲ά ᛶࡀୖ᪼ࡋࠊ≉࡟஧౯ࡢ࢝ࢳ࢜ࣥ࡟ࡼࡾάᛶࡀቑᙉࡉࢀࡓࡀࠊCo²⁺ࠊCu²⁺ࠊMn²⁺ ࠾ࡼࡧ Zn²⁺

ࢆῧຍࡋࡓሙྜࡣάᛶࡀᢚไࡉࢀࡓࠋࡇࡢࡇ࡜࠿ࡽࠊᮏ㓝⣲ࡣ㔠ᒓ㓝⣲࡛࠶ࡿࡇ࡜ࡀࢃ࠿ࡗ ࡓࠋ୍᪉ࠊ㜼ᐖ๣࡟㛵ࡋ࡚ࡣࠊSH㜼ᐖ๣࡟ࡼࡿάᛶࡀぢࡽࢀࡓࠋࡲࡓࠊᇶ㉁≉␗ᛶࡢ᳨ウ

ࢆ⾜ࡗࡓ⤖ᯝࠊFct114ࡣ௚ࡢᾏ⸴⏤᮶ࡢࣇࢥ࢖ࢲࣥࡸᾏὒᛶከ⢾㢮࡛࠶ࡿ࢔ࣝࢠࣥ㓟ࠊ࣑ࣛ

ࢼࣛࣥࠊ࢝ࣛࢠ࣮ࢼࣥࠊࢥࣥࢻࣟ࢖ࢳࣥ◲㓟ࠊࣄ࢔ࣝࣟࣥ㓟࡟ᑐࡋ࡚άᛶࢆ♧ࡉࡎࠊ࢜࢟ࢼ

࣡ࣔࢬࢡࣔࢬࢡࣇࢥ࢖ࢲࣥ࡟ᑐࡋ࡚≉␗ⓗ࡟పศᏊ໬ࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡉࡽ࡟࢜࢟

ࢼ࣡ࣔࢬࢡ⏤᮶ࡢ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥ࡜࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ୧᪉࡟άᛶࢆ♧

ࡋࡓࡀࠊ⬺࢔ࢭࢳࣝ໬ࡋࡓࣇࢥ࢖ࢲࣥࡢ᪉ࡀࠊࡼࡾపศᏊ໬ࢆಁ㐍ࡍࡿࡇ࡜ࡀࢃ࠿ࡗࡓࠋ ௨ୖࡢࡼ࠺࡟ࠊᮏ◊✲࡛ࡣࣇࢥ࢖ࢲࣥࢹ࢔ࢭࢳ࣮ࣛࢮࠊࣇࢥ࢖ࢲࢼ࣮ࢮࡢ㓝⣲≉ᛶࢆ᫂ࡽ

࠿࡟ࡋࡓࠋࡇࢀࡽ2ࡘࡢ㓝⣲ࢆ⏝࠸ࡿࡇ࡜࡛ࠊ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࠊ࢜࢟ࢼ࣡ࣔࢬࢡ

⏤᮶ࡢ⬺࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࠊ࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶ࡢ⬺࢔ࢭࢳ࣭ࣝపศᏊ໬ࣇࢥ࢖ࢲࣥࠊ

࢜࢟ࢼ࣡ࣔࢬࢡ⏤᮶పศᏊ໬ࣇࢥ࢖ࢲࣥࢆㄪ〇ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࡗࡓࠋ≉࡟㓝⣲ⓗ࡟⬺

࢔ࢭࢳࣝ໬ࣇࢥ࢖ࢲࣥࢆㄪ〇ࡍࡿ᪉ἲࡣࠊ⌧ᅾࡲ࡛࡟ሗ࿌౛ࡀ࡞࠸ࠋࡇࢀࡽ4✀㢮ࡢᵓ㐀ࡢ

118

ࣇࢥ࢖ࢲࣥࢆㄪ〇࡛ࡁࡿࡼ࠺࡟࡞ࡗࡓࡇ࡜࡛ࠊ௒ࡲ࡛࡟ሗ࿌౛ࡢ࠶ࡿ࢖ࣥࣇ࢚ࣝࣥࢨ࢘࢕

ࣝࢫ➼࡟ᑐࡍࡿᢠ࢘࢖ࣝࢫస⏝ [9]ࠊᢠ࢞ࣥస⏝ [10, 11]ࠊᢠจ⾑స⏝ [12, 13, 14]࡜࠸ࡗࡓ

ࣇࢥ࢖ࢲࣥࡢ⏕⌮άᛶ࡜ᵓ㐀࡜ࡢ┦㛵ᛶࡢゎ᫂࡬࡜⧅ࡀࡿ࡜⪃࠼ࡽࢀࡿࠋከ⢾ࡢ㓝⣲ⓗศ ゎࡣࠊከ⢾ࡢᕤᴗⓗ฼⏝ࡸ་⸆ရࡢ㛤Ⓨ࡞࡝࡟࠾࠸࡚᭷⏝࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࡀࠊ௒ᚋࠊከ

⢾ࡢᵓ㐀࡜⏕⌮άᛶ࡜ࡢ┦㛵ᛶ࡟ࡘ࠸࡚᭦࡞ࡿ◊✲ࡀᚲせ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ᭱ᚋ࡟ࠊᮏ

◊✲ࡀ࢜࢟ࢼ࣡ࣔࢬࢡࣇࢥ࢖ࢲࣥࡢ⏝㏵㛤Ⓨࡸ㟂せᣑ኱࡬ࡢ୍ຓ࡜࡞ࡿࡇ࡜ࢆᮇᚅࡍࡿࠋ

119

ᘬ

ᘬ⏝ᩥ⊩

すฟⱥ୍、ᾏ⸴ᕤᴗ_, ᇼ㍤୕࣭኱㔝ṇኵ࣭ᇼཱྀ೺㞝⦅ࠕ21ୡ⣖ึ㢌ࡢ⸴Ꮫࡢ⌧ἣࠖ, ᪥ ᮏ⸴㢮Ꮫ఍、ᒣᙧ、2002, 123-125.

Li, B., Lu, F., Wei, X., & Zhao, R., Fucoidan: Structure and bioactivity. Molecules, 2008,13:

1671–1695.

Cumashi, A., Ushakova, N. A., Preobrazhenskaya, M. E., D’incecco, A., Piccoli, A., Totani, L., Nicola, N., Morozevich, G. E., Berman, A. E., Bilan, M. I., Usov, A. I., Ustyuzhanina, N. E., Grachev, A. A., Sanderson, C. J., Kelly M., Rabinovich, G. A., Iacobelli, S., & Nifantiev, N. E.,A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds.Glycobiology, 2007, 17: 541–552.

Shan, X., Liu, X., Hao, J., Cai, C., Fan, F., Dun, Y., Zhao, X., Liu, X., Li, C., & Yu, G.,In vitro andin vivohypoglycemic effects of brown algal fucoidans.International Journal of Biological Macromolecules, 2016, 82: 249-255.

Kylin, H., Analysis of the biochemistry of the seaweed.H. Z. Physiol. Chem., 1913, 83: 171-197.

6. ຍ⸨㑳அ㐍ࠊ㓇஭Ṋࠊబᕝ⿱❶ࠊᾏ⸴ࡢᢠࡀࢇస⏝. ᭷⏝ᾏ⸴ㄅࠊ኱㔝ṇኵ⦅ⴭࠊෆ⏣

⪁㭯ᅡࠊ2004ࠊ477-490.

7. ຍ⸨㑳அ㐍ࠊబᕝ⿱❶ࠊᾏⲡ㣗≀⧄⥔࡟ࡼࡿ⒴ࣜࢫࢡ㍍ῶ. ⸴㢮ࠊ2000ࠊ48: 13-19.

8. Yu, F., Kitano, H., Sakai, T., Katayama, K., Nakanishi, Y., Ikai, K., & Kato, I., Apotosis of human carcinoma cell lines induced by fucoidan (sulfated fucose-containing polysaccharide) and its degraded fragments by fucoidanase and endo-fucoidan-lyase. Abstracts of XVIIIth Jap.

Carbohydr. Sympo., 1996, 93-94.

9. Jung-Bum, L., Hayashi, K., Hashimoto, K., Nakano, M., & Hayashi, T., Novel antiviral fucoidan from Sporophyll of Undaria pinnatifida(Mekabu). Chem. Pharm. Bull., 2004, 52: 1091-1094.

120

10. Ermakova, S., Sokolova, R., Kim, S-M., Um, B-H., Isakov, V., & Zvyagintseva, T., Fucoidans from brown seaweeds Sargassum hornery, Eclonia cava, Costaria costata: Structural characteristics and anticancer activity.Applied Biochemistry and Biotechnology, 2011, 164: 841-850.

11. Vishchuk, O. S., Ermakova, S. P., & Zvyagintseva, T. N., Sulfated polysaccharides from brown seaweeds Saccharina japonicaandUndaria pinnatifida: Isolation, structural characteristics, and antitumor activity.Carbohydrate Research, 2011, 346: 2769-2776.

12. Jin, W., Zhang, Q., Wang, J., & Zhang, W., A comparative study of the anticoagulant activities of eleven fucoidans.Carbohydrate Polymers, 2013, 91: 1-6.

13. Athukorala, Y., Jung, W-K., Vasanthan, T., & Jeon, Y-J., An anticoagulative polysaccharide from an enzymatic hydrolysate ofEcklonia cava.Carbohydrate Polymers, 2006,66: 184–191.

14. Ren, R., Azuma, Y., Ojima, T., Hashimoto, T., Mizuno, M., Nishitani, Y., Yoshida, M., Azuma, T.,

& Kanazawa, K., Modulation of platelet aggregation-related eicosanoid production by dietary F-fucoidan from brown alga Laminaria japonicain human subjects.Br. J. Nutr., 2013, 110: 880–890.

15. Kim, M-J., Jeon, J., & Lee, J-S.,Fucoidan prevents high-fat diet-induced obesity in animals by suppression of fat accumulation.Phytotherapy Research, 2014,28: 137-143.

16. Lee, S-J., Kim, C-W., Jang, H-J., Cho, S-Y., & Choi, J-W., Anti-hyperlipidemia and anti-arteriosclerosis effects of Laminaria japonica in Sprague-Dawley rats. Fisheries and Aquatic Sciences, 2011, 14: 235-241.

17. Shan, X., Liu, X., Hao, J., Cai, C., Fan, F., Dun, Y., Zhao, X., Liu, X., Li, C., & Yu, G., In vitro andin vivo hypoglycemic effects of brown algal fucoidans.International Journal of Biological Macromolecules, 2016, 82: 249-255.

18. Li, X., Li, J., Li, Z., Sang, Y., Niu, Y., Zhang, Q., Ding, H., & Yin, S., Fucoidan from Undaria pinnatifida prevents vascular dysfunction through PI3K/Akt/eNOS-dependent mechanisms in the

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