JPH0367672B2 - - Google Patents
Info
- Publication number
- JPH0367672B2 JPH0367672B2 JP59153733A JP15373384A JPH0367672B2 JP H0367672 B2 JPH0367672 B2 JP H0367672B2 JP 59153733 A JP59153733 A JP 59153733A JP 15373384 A JP15373384 A JP 15373384A JP H0367672 B2 JPH0367672 B2 JP H0367672B2
- Authority
- JP
- Japan
- Prior art keywords
- acid
- epa
- algae
- eicosapentaenoic acid
- enzymatic production
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 235000020673 eicosapentaenoic acid Nutrition 0.000 claims description 59
- 241000195493 Cryptophyta Species 0.000 claims description 15
- MBMBGCFOFBJSGT-KUBAVDMBSA-N all-cis-docosa-4,7,10,13,16,19-hexaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(O)=O MBMBGCFOFBJSGT-KUBAVDMBSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 235000020661 alpha-linolenic acid Nutrition 0.000 claims description 12
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 claims description 12
- 229960004488 linolenic acid Drugs 0.000 claims description 12
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 claims description 12
- 235000020669 docosahexaenoic acid Nutrition 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 claims description 7
- 229960005135 eicosapentaenoic acid Drugs 0.000 claims description 7
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- DPUOLQHDNGRHBS-UHFFFAOYSA-N Brassidinsaeure Natural products CCCCCCCCC=CCCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-UHFFFAOYSA-N 0.000 claims description 5
- 150000002632 lipids Chemical class 0.000 claims description 5
- ATNNLHXCRAAGJS-QZQOTICOSA-N (e)-docos-2-enoic acid Chemical compound CCCCCCCCCCCCCCCCCCC\C=C\C(O)=O ATNNLHXCRAAGJS-QZQOTICOSA-N 0.000 claims description 4
- 239000003925 fat Substances 0.000 claims description 4
- 235000019197 fats Nutrition 0.000 claims description 4
- 229940090949 docosahexaenoic acid Drugs 0.000 claims description 3
- 238000006911 enzymatic reaction Methods 0.000 claims description 3
- 239000003921 oil Substances 0.000 claims description 2
- 235000019198 oils Nutrition 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims 2
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 claims 1
- 239000003002 pH adjusting agent Substances 0.000 claims 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims 1
- 235000017557 sodium bicarbonate Nutrition 0.000 claims 1
- 150000004665 fatty acids Chemical class 0.000 description 8
- 235000014113 dietary fatty acids Nutrition 0.000 description 7
- 229930195729 fatty acid Natural products 0.000 description 7
- 239000000194 fatty acid Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 235000021323 fish oil Nutrition 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- SECPZKHBENQXJG-FPLPWBNLSA-N palmitoleic acid Chemical compound CCCCCC\C=C/CCCCCCCC(O)=O SECPZKHBENQXJG-FPLPWBNLSA-N 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 3
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 3
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 3
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 3
- 108010039731 Fatty Acid Synthases Proteins 0.000 description 3
- 239000005642 Oleic acid Substances 0.000 description 3
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 235000013402 health food Nutrition 0.000 description 3
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 102100022089 Acyl-[acyl-carrier-protein] hydrolase Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 235000021314 Palmitic acid Nutrition 0.000 description 2
- 235000021319 Palmitoleic acid Nutrition 0.000 description 2
- 235000021355 Stearic acid Nutrition 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- SECPZKHBENQXJG-UHFFFAOYSA-N cis-palmitoleic acid Natural products CCCCCCC=CCCCCCCCC(O)=O SECPZKHBENQXJG-UHFFFAOYSA-N 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 2
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 2
- 235000021313 oleic acid Nutrition 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 239000008117 stearic acid Substances 0.000 description 2
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000000452 Acetyl-CoA carboxylase Human genes 0.000 description 1
- 108010016219 Acetyl-CoA carboxylase Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000199897 Alaria esculenta Species 0.000 description 1
- 241001310494 Ammodytes marinus Species 0.000 description 1
- 241000512259 Ascophyllum nodosum Species 0.000 description 1
- 108010018763 Biotin carboxylase Proteins 0.000 description 1
- 241001474374 Blennius Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- 240000009108 Chlorella vulgaris Species 0.000 description 1
- 241000555825 Clupeidae Species 0.000 description 1
- 241001149724 Cololabis adocetus Species 0.000 description 1
- 241000571268 Cryptomonas erosa Species 0.000 description 1
- URXZXNYJPAJJOQ-UHFFFAOYSA-N Erucic acid Natural products CCCCCCC=CCCCCCCCCCCCC(O)=O URXZXNYJPAJJOQ-UHFFFAOYSA-N 0.000 description 1
- 241000195619 Euglena gracilis Species 0.000 description 1
- 102000015303 Fatty Acid Synthases Human genes 0.000 description 1
- 241000004455 Fucus spiralis Species 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000920475 Marvania coccoides Species 0.000 description 1
- 241000195659 Neodesmus pupukensis Species 0.000 description 1
- 241000212297 Pelvetia Species 0.000 description 1
- 241000206608 Pyropia tenera Species 0.000 description 1
- 241000206613 Pyropia yezoensis Species 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 241000206572 Rhodophyta Species 0.000 description 1
- 241000983746 Saccharina latissima Species 0.000 description 1
- 241000269821 Scombridae Species 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 241000196251 Ulva arasakii Species 0.000 description 1
- 241000196245 Ulva intestinalis Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- IQLUYYHUNSSHIY-HZUMYPAESA-N eicosatetraenoic acid Chemical compound CCCCCCCCCCC\C=C\C=C\C=C\C=C\C(O)=O IQLUYYHUNSSHIY-HZUMYPAESA-N 0.000 description 1
- DPUOLQHDNGRHBS-KTKRTIGZSA-N erucic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCCCC(O)=O DPUOLQHDNGRHBS-KTKRTIGZSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 230000004136 fatty acid synthesis Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 229940013317 fish oils Drugs 0.000 description 1
- 238000001640 fractional crystallisation Methods 0.000 description 1
- 235000021588 free fatty acids Nutrition 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 235000020640 mackerel Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Description
【発明の詳細な説明】
（発明の利用分野）
本発明はエイコサペンタエン酸（以下「EPA」
と略称する）の酵素的生産方法、さらに詳述すれ
ば、藻類中の多不飽和脂肪酸合成酵素による
EPA前駆体からのEPAの特異的合成活性を利用
して、ドコセン酸及びドコサヘキサエン酸夾雑量
の少ない高純度のEPA含有油脂を酵素的に生産
する方法に関する。
（従来技術）
EPAに代表される多不飽和脂肪酸は、生体膜
の構成成分として重要な役割を担つている。ま
た、胆汁酸の分泌を促進し、中性脂肪の合成を抑
制し、血漿コレステロールの低下作用を有する。
さらに、プロスタグランジン一族の生成に際し基
質となり、ヒトを含む高等哺乳動物の体内で必須
的な機能を発揮する。特にEPAはタイプ３のプ
ロスタグランジンの生成の際の基質として重要で
あつて、ドコサヘキサエン酸（以下「DHA」と
略称する）と共に血小板の凝集抑制作用があり、
血栓症の治療及び予防剤としての応用が検討され
ている。さらにEPAは、血漿コレステロールレ
ベルの低下に寄与する多不飽和脂肪酸の中でも特
にその活性が高く、通常の植物油中に含まれるリ
ノール酸などよりも遥に有効である。
このように、EPAがその血栓防止作用に基づ
く健康食品あるいは医薬品としての可能性がデン
マークのダイヤーベルグ（Am.J.Clin.Nutr，28，
958頁、1975年）の疫学的調査により明らかにさ
れて以来、わが国においてもEPAを多く含有す
るイワシ、サバ、サンマ及びイカナゴ等の魚の摂
食が推奨されるようになつてきた。
今日、健康食品として市販されているEPAは、
煮取法によつて得られた魚油の分別物であつて、
そのEPA含量は10〜30％であり、同時に含まれ
るDHAのそれと合計しても40％を越えるものは
少ない〔注〕。煮取法によつて抽出される魚油は
構成脂肪酸として多種類の脂肪酸を含む混合グリ
セリドであつて、抽出時の熱及び水との接触等に
よりEPA、DHA等の多不飽和脂肪酸の変質の恐
れや、抽出中に若干量残存する水分による酸化
（過酸化）等の不安がある。さらに、これら魚油
EPAの分別に使用されたアセトン、メチルエチ
ルケトンなど各種の有機溶剤は通常減圧下に除去
されるが、その完全除去は技術的及びコスト的に
問題点が多い。
（注） 中性脂肪（主としてトリグリセリド）の
まま分別、濃縮してもEPAの含量を30〜40％
以上に高めるのは理論的に困難であろうと推定
される。
医薬品としてのEPAは、様々な方法によつて
抽出された魚油を酵素的に若しくはアルカリ条件
下で処理して遊離脂肪酸まで加水分解するか又は
該遊離酸をメチル若しくはエチルエステルに変じ
た後、これらを低温分別結晶法、尿素付加法、減
圧蒸留法又は逆相クロマト法等によりさらに精製
してEPA濃度を90％以上としたものが多い。し
かし、これらの方法を用いて得られたEPA濃縮
物は、工程中に（アセトンなどの）各種の有機溶
媒が使用されたり又は200℃近い高熱を加えられ
たりするため、有機溶媒の残留やEPAの重合、
異性化及び／又は酸化等による変質の懸念をはら
んでいる。さらに、魚油等をEPAの原料として
用いた場合、心臓疾患の原因の一つとして疑われ
ているドコセン酸（慣用名、エルシン酸）の除去
が困難であるため、健康食品、医薬品等に利用す
る上で問題点を含む。加えて、魚油中に多く含ま
れているDHAとEPAの相互分離も困難であつ
て、最終商品中にすらDHAが相当量混在してい
るのが実情である。因にDHAの生理学上の意義
は、EPAに類似していると考えられてはいるも
のの、EPAそのものを目的とした商品中に他種
類の脂肪酸が多量に混在していることは必ずしも
望ましいことではない。
（本発明の解決せんとする問題点）
以上述べてきたように、健康食品又は医薬品と
して考えられているEPAには幾つかの問題点が
あることから、本発明者らは残留溶媒や変質の心
配のない安全なEPAを高濃度に得る方法を鋭意
研究した結果、藻類中に多く含まれているEPA
合成酵素を利用して、ドコセン酸、DHA等を殆
ど含まない高純度EPAを酵素的に生産するのに
成功した。
（問題点を解決するための手段）
本発明は、以上述べた通り、藻類中に多く含ま
れているEPA合成酵素を利用して酵素反応的に
高純度のEPAを生産することを骨子とするもの
である。ここに「EPA合成酵素」とは、後に説
明するように、各種のEPA前駆体を相互交換し
て最終的にEPAの合成に至るまでに関与する一
切の酵素の集合体を意味する。一般に、植物によ
る脂肪酸合成酵素活性は動物の1/100以下と言わ
れているが、本発明者の知見によれば、意外なこ
とに、藻類におけるEPA合成酵素活性は著しく
高く、充分実用化能であることが判明した。
ところで、藻類中に含まれる脂質含量は、通常
乾物中約0.2〜3.0％と少ないが、スサビノリ
（Porphyra yezoensis）、ナラワスサビノリ（P.
y.narawaensis、アサクサノリ（Ｐ．tenera）等
の紅藻類中には、その全脂肪酸の実に40〜60％も
のEPAが含まれている。緑藻類のアオノリ
（Enteromorpha intestinalis）やアオサ（Ulva
Lactuca）、褐藻類のコンブ（Laminaria
Saccharina）、アイヌワカメ（Alaria
esculenta）、ヒバマタ（Fucus spiralis）及びエ
ゾイシゲ（Pelvetia canaliculate）等にも、全
脂肪酸中、季節的及び地域的変化を考慮に入れて
も、５〜30％ものEPAが含まれており、いずれ
もドコセン酸、DHA等を殆ど含んでいない。ま
た、微小藻類、例えばクロレラ（Chlorella
minutissima，C.vulgaris，Nannochloris
coccoides Nauman）、カゲヒゲムシ
（Cryptomonas erosa EHRENBERG）及び
ユーグレナ（Euglena gracilis）などでは、
EPA含量は全脂肪酸中約５〜40％に達する。
従つて、EPAを高純度に含有する藻類から脂
質を直接抽出しても、魚油由来のEPA製品と同
等かそれ以上の商品価値を有するものが得られる
筈であるが、元来天然の藻類中に含まれる脂質含
量そのものが先述の通り少量であるから、抽出設
備、ランニングコスト及び藻類自体の価格等の要
因を考慮すれば、EPAの生産を目的として藻類
中の脂質を抽出することはあまり得策とは言えな
い。
とまれ、前述にように、通常の高等植物及び微
生物等にはEPAは全く含まれていないが、単細
胞及び多細胞藻類中には、特異的に多量のEPA
を含むものがある。上述の藻類はいずれも強力な
多不飽和脂肪酸合成酵素、特にEPA合成酵素系
を有するので、本発明に用いる酵素源として使用
できるが、EPA合成酵素活性はEPA含量の多い
藻類程強くなる傾向が認められた。
最近になつて脂肪酸の初生合成の機構がよく研
究され、脂肪酸の鎖長延長、不飽和化等の機作に
ついてもかなり明らかにされてきた。高等植物に
おける脂肪酸合成は一般にアセチルCoAカルボ
キシラーゼ、アシル運搬タンパク質（ACP）の
関与する飽和脂肪酸の合成酵素及びメチル基側へ
の不飽和化酵素（植物型）によつて行なわれ、主
としてオレイン酸からリノール酸及びリノレン酸
へと変換する。しかし、藻類においては、さらに
リノレン酸からEPAへの変換経路（動物型）が
存在すると考えられる。
本発明者らはスサビノリを用いて試験管内及び
生体内条件下におけるEPA合成を研究し、いず
れも市販品として入手できる 14Cラベルの酢酸、
酪酸、ミリスチン酸、パルミチン酸、パルミトオ
レイン酸、ステアリン酸、オレイン酸、リノール
酸及びリノレン酸中の放射能がEPA中に有意的
に取り込まれることを確認したが、最も効率良く
EPA中に取り込まれる基質はリノレン酸であつ
た。即ち下図に示されるように、EPAの直接の
前駆体はリノレン酸由来のエイコサテトラエン酸
であるので、リノレン酸はEPA合成のため試験
に供した基質中では最も効率の良い基質といえる
が、リノレン酸の前駆体でもある酢酸、酪酸、ミ
リスチン酸、パルミチン酸、パルミトオレイン
酸、ステアリン酸、オレイン酸及びリノール酸な
ども最終的に種々の生化学的変換過程を経てリノ
レン酸を介してEPA中に取り込まれる。
【表】
↓ ↓
ω−3,6,9,12−エイコサテトラエン酸
(C 20：4ω3)
Detailed Description of the Invention (Field of Application of the Invention) The present invention relates to eicosapentaenoic acid (hereinafter referred to as "EPA")
(abbreviated as ), and more specifically, by using polyunsaturated fatty acid synthase in algae.
The present invention relates to a method for enzymatically producing highly pure EPA-containing fats and oils with a small amount of docosenoic acid and docosahexaenoic acid contamination by utilizing the specific synthesis activity of EPA from EPA precursors. (Prior Art) Polyunsaturated fatty acids represented by EPA play an important role as a constituent of biological membranes. It also promotes the secretion of bile acids, suppresses the synthesis of neutral fats, and has the effect of lowering plasma cholesterol.
Furthermore, it serves as a substrate for the production of the prostaglandin family, and exerts essential functions in the bodies of higher mammals, including humans. In particular, EPA is important as a substrate for the production of type 3 prostaglandin, and together with docosahexaenoic acid (hereinafter abbreviated as "DHA"), it has an inhibitory effect on platelet aggregation.
Application as a therapeutic and preventive agent for thrombosis is being considered. Furthermore, EPA is particularly active among polyunsaturated fatty acids that contribute to lowering plasma cholesterol levels, and is far more effective than linoleic acid, which is found in ordinary vegetable oils. In this way, the possibility of EPA as a health food or medicine based on its antithrombotic effect was demonstrated by the Danish company Am.J.Clin.Nutr, 28 .
Since the discovery of EPA in an epidemiological study (p. 958, 1975), it has become recommended in Japan to consume fish such as sardines, mackerel, saury, and sand eel, which contain large amounts of EPA. EPA, which is commercially available as a health food today, is
A fractionated product of fish oil obtained by the boiling method,
Its EPA content is 10 to 30%, and even when combined with the DHA contained at the same time, it rarely exceeds 40% [Note]. Fish oil extracted by the boiling method is a mixed glyceride containing many types of fatty acids as constituent fatty acids, and there is a risk that polyunsaturated fatty acids such as EPA and DHA may deteriorate due to heat during extraction and contact with water. There are concerns about oxidation (peroxidation) due to a small amount of water remaining during extraction. Additionally, these fish oils
The various organic solvents used in EPA separation, such as acetone and methyl ethyl ketone, are usually removed under reduced pressure, but their complete removal poses many technical and cost problems. (Note) Even if the neutral fats (mainly triglycerides) are separated and concentrated, the EPA content will be reduced by 30-40%.
It is presumed that it would be theoretically difficult to increase it above this level. EPA as a pharmaceutical product is produced by treating fish oil extracted by various methods enzymatically or under alkaline conditions to hydrolyze it to free fatty acids, or converting the free acids into methyl or ethyl esters. In many cases, the EPA concentration is further purified by low-temperature fractional crystallization, urea addition, vacuum distillation, reversed phase chromatography, etc. to reach an EPA concentration of 90% or higher. However, the EPA concentrate obtained using these methods uses various organic solvents (such as acetone) or is heated to nearly 200°C during the process, resulting in residual organic solvents and EPA. polymerization of
There are concerns about deterioration due to isomerization and/or oxidation. Furthermore, when fish oil is used as a raw material for EPA, it is difficult to remove docosenoic acid (commonly known as erucic acid), which is suspected to be one of the causes of heart disease. Including the problems above. In addition, it is difficult to separate DHA and EPA, which are abundantly contained in fish oil, and the reality is that a considerable amount of DHA is mixed even in the final product. Incidentally, although the physiological significance of DHA is thought to be similar to that of EPA, it is not necessarily desirable for products intended for EPA to contain large amounts of other types of fatty acids. do not have. (Problems to be solved by the present invention) As stated above, there are several problems with EPA, which is considered as a health food or medicine. As a result of intensive research into a method to obtain a high concentration of safe and worry-free EPA, we found that EPA, which is abundant in algae,
Using a synthetic enzyme, we succeeded in enzymatically producing high-purity EPA containing almost no docosenoic acid, DHA, etc. (Means for Solving the Problems) As described above, the main point of the present invention is to produce highly pure EPA through an enzymatic reaction using EPA synthase, which is abundantly contained in algae. It is something. Here, the term "EPA synthase" refers to a collection of all enzymes involved in the mutual exchange of various EPA precursors and the final synthesis of EPA, as will be explained later. It is generally said that the fatty acid synthase activity in plants is less than 1/100 of that in animals, but according to the findings of the present inventors, surprisingly, the EPA synthase activity in algae is extremely high and is sufficient for practical use. It turned out to be. By the way, the lipid content contained in algae is usually low at about 0.2 to 3.0% based on dry matter, but it is found in algae such as Porphyra yezoensis and P.
Red algae such as Y. narawaensis and P. tenera contain 40 to 60% EPA of their total fatty acids. The green algae Enteromorpha intestinalis and Ulva
Lactuca ), brown algae kelp ( Laminaria
Saccharina), Ainu seaweed ( Alaria
esculenta), Fucus spiralis , and Pelvetia canaliculate , etc., also contain 5 to 30% EPA in total fatty acids, even taking into account seasonal and regional variations, and all of them contain docosen. Contains almost no acids, DHA, etc. It also contains microalgae, such as Chlorella ( Chlorella
minutissima, C.vulgaris , Nannochloris
coccoides Nauman), Cryptomonas erosa EHRENBERG , and Euglena gracilis .
The EPA content amounts to about 5-40% of the total fatty acids. Therefore, even if lipids are directly extracted from algae that contain high-purity EPA, it should be possible to obtain products with commercial value equal to or greater than EPA products derived from fish oil. Since the lipid content itself contained in algae is small as mentioned above, it is not a good idea to extract lipids from algae for the purpose of producing EPA, considering factors such as extraction equipment, running costs, and the price of the algae itself. It can not be said. As mentioned above, normal higher plants and microorganisms do not contain any EPA, but unicellular and multicellular algae specifically contain large amounts of EPA.
There are some that include. All of the above-mentioned algae have strong polyunsaturated fatty acid synthases, especially the EPA synthase system, so they can be used as enzyme sources for the present invention, but the EPA synthase activity tends to be stronger in algae with higher EPA content. Admitted. Recently, the mechanism of initial biosynthesis of fatty acids has been extensively studied, and the mechanisms of fatty acid chain elongation, desaturation, etc. have also been largely clarified. Fatty acid synthesis in higher plants is generally carried out by acetyl CoA carboxylase, a saturated fatty acid synthesizing enzyme involving acyl transport protein (ACP), and a methyl group desaturase (plant type), mainly converting oleic acid to linoleic acid. Converts to acid and linolenic acid. However, in algae, it is thought that there is an additional conversion pathway (animal type) from linolenic acid to EPA. The present inventors studied the synthesis of EPA under in vitro and in vivo conditions using C. japonica, and found that 14 C-labeled acetic acid, which is commercially available,
It was confirmed that radioactivity in butyric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid was significantly incorporated into EPA, but most efficiently.
The substrate incorporated into EPA was linolenic acid. In other words, as shown in the figure below, the direct precursor of EPA is eicosatetraenoic acid derived from linolenic acid, so linolenic acid can be said to be the most efficient substrate among the substrates tested for EPA synthesis. The precursors of linolenic acid, such as acetic acid, butyric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid, are finally converted to linolenic acid through various biochemical conversion processes. Incorporated into EPA. [Table] ↓ ↓
ω-3,6,9,12-eicosatetraenoic acid
(C 20：4ω3)
Claims (1)
としてエイコサペンタエン酸を含有する藻類中の
エイコサペンタエン酸合成酵素を作用させること
を特徴とするエイコサペンタエン酸の酵素的生産
方法。 ２ 酵素反応液中に該リノレン酸の４〜80重量％
量の酢酸を加える特許請求の範囲第１項記載のエ
イコサペンタエン酸の酵素的生産方法。 ３ 酵素反応液の最終的な水素イオン濃度をPH３
〜10に調整するに当り、PH調整剤として炭酸水素
ナトリウムを使用する特許請求の範囲第１項又は
第２項記載のエイコサペンタエン酸の酵素的生産
方法。 ４ 特許請求の範囲第１項記載のリノレン酸を基
質とする方法において、生産物として得られる総
脂質中に含まれるドコセン酸及びドコサヘキサエ
ン酸の含有量が共に3.0重量％以下であることを
特徴とするエイコサペンタエン酸の酵素的生産方
法。[Scope of Claims] 1. A method for the enzymatic production of eicosapentaenoic acid, which comprises causing eicosapentaenoic acid synthase in algae containing eicosapentaenoic acid to act on linolenic acid or fats and oils containing linolenic acid as a substrate. 2 4 to 80% by weight of the linolenic acid in the enzyme reaction solution
A method for the enzymatic production of eicosapentaenoic acid as claimed in claim 1, wherein an amount of acetic acid is added. 3 Set the final hydrogen ion concentration of the enzyme reaction solution to PH3.
3. The method for enzymatic production of eicosapentaenoic acid according to claim 1 or 2, wherein sodium hydrogen carbonate is used as a pH adjuster in adjusting the pH to 10 to 10. 4. The method using linolenic acid as a substrate according to claim 1, characterized in that the content of docosenoic acid and docosahexaenoic acid contained in the total lipid obtained as a product is 3.0% by weight or less. A method for enzymatic production of eicosapentaenoic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59153733A JPS6131092A (en) | 1984-07-23 | 1984-07-23 | Method for enzymic production of eicosapentaenoic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59153733A JPS6131092A (en) | 1984-07-23 | 1984-07-23 | Method for enzymic production of eicosapentaenoic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6131092A JPS6131092A (en) | 1986-02-13 |
| JPH0367672B2 true JPH0367672B2 (en) | 1991-10-23 |
Family
ID=15568905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59153733A Granted JPS6131092A (en) | 1984-07-23 | 1984-07-23 | Method for enzymic production of eicosapentaenoic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6131092A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU602376B2 (en) * | 1986-12-26 | 1990-10-11 | Sagami Chemical Research Center | Process for production of eicosapentaenoic acid |
| JPS63185390A (en) * | 1987-01-27 | 1988-07-30 | Suntory Ltd | Production of eicosapentaenoic acid by algae |
| JPS63185389A (en) * | 1987-01-27 | 1988-07-30 | Suntory Ltd | Production of highly unsaturated fatty acid by microbial conversion |
| WO2015011418A1 (en) * | 2013-07-25 | 2015-01-29 | Roquette Freres | Method for optimising the production efficiency, organoleptic quality and stability over time of a protein-rich microalgae biomass |
-
1984
- 1984-07-23 JP JP59153733A patent/JPS6131092A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6131092A (en) | 1986-02-13 |
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