JPH02261389A - Production of 5-aminolevulinic acid - Google Patents
Production of 5-aminolevulinic acidInfo
- Publication number
- JPH02261389A JPH02261389A JP8221989A JP8221989A JPH02261389A JP H02261389 A JPH02261389 A JP H02261389A JP 8221989 A JP8221989 A JP 8221989A JP 8221989 A JP8221989 A JP 8221989A JP H02261389 A JPH02261389 A JP H02261389A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- ala
- aminolevulinic acid
- bacterial cells
- changes
- 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.)
- Granted
Links
- ZGXJTSGNIOSYLO-UHFFFAOYSA-N 88755TAZ87 Chemical compound NCC(=O)CCC(O)=O ZGXJTSGNIOSYLO-UHFFFAOYSA-N 0.000 title claims description 54
- 229960002749 aminolevulinic acid Drugs 0.000 title claims description 54
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 241000193459 Moorella thermoacetica Species 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 239000007787 solid Substances 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 abstract description 7
- 150000001875 compounds Chemical class 0.000 abstract description 3
- 230000002363 herbicidal effect Effects 0.000 abstract description 2
- 239000004009 herbicide Substances 0.000 abstract description 2
- JOOXCMJARBKPKM-UHFFFAOYSA-N 4-oxopentanoic acid Chemical compound CC(=O)CCC(O)=O JOOXCMJARBKPKM-UHFFFAOYSA-N 0.000 description 46
- 229940040102 levulinic acid Drugs 0.000 description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 230000001580 bacterial effect Effects 0.000 description 17
- 239000002609 medium Substances 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 7
- QSHWIQZFGQKFMA-UHFFFAOYSA-N Porphobilinogen Natural products NCC=1NC=C(CCC(O)=O)C=1CC(O)=O QSHWIQZFGQKFMA-UHFFFAOYSA-N 0.000 description 4
- YPHQRHBJEUDWJW-UHFFFAOYSA-N porphobilinogen Chemical compound NCC1=NC=C(CCC(O)=O)[C]1CC(O)=O YPHQRHBJEUDWJW-UHFFFAOYSA-N 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910017052 cobalt Inorganic materials 0.000 description 3
- 239000010941 cobalt Substances 0.000 description 3
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 3
- 229910052742 iron Inorganic materials 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 2
- 239000004158 L-cystine Substances 0.000 description 2
- 235000019393 L-cystine Nutrition 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960003067 cystine Drugs 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 239000011777 magnesium Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000696 methanogenic effect Effects 0.000 description 2
- 230000000243 photosynthetic effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 229910021654 trace metal Inorganic materials 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 102000004867 Hydro-Lyases Human genes 0.000 description 1
- 108090001042 Hydro-Lyases Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 229930195722 L-methionine Natural products 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 1
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 1
- 239000011768 flavin mononucleotide Substances 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
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- 239000011664 nicotinic acid Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
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- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 229960001153 serine Drugs 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、除草剤として効果のある５−アミノレブリン
酸（ＡＬＡ）の製法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing 5-aminolevulinic acid (ALA), which is effective as a herbicide.
従来、５−アミノレブリン酸（ＡＬＡ）は、光合成細菌
や、メタン生成細菌を培養する際に、培地中にレブリン
酸（ＬＡ）を添加することによって、生成させることが
提案されていた。Conventionally, it has been proposed that 5-aminolevulinic acid (ALA) be produced by adding levulinic acid (LA) to a culture medium when culturing photosynthetic bacteria or methanogenic bacteria.
つまり、一般に光合成細菌やメタン生成細菌は、単に培
養するだけでは、菌体内での中間生成物としての５−ア
ミノレブリン酸（ＡＬＡ）が蓄積されず、そのために、
ＡＬＡ脱水酵素の阻害剤として知られているレブリン酸
（■、Ａ）によって、５−アミノレブリン酸（ＡＬＡ）
からポルホビリノーゲン（ＰＢＧ）に至る生合成を阻害
して、５−アミノレブリン酸（ＡＬＡ）を採取すること
が考えられていた。In other words, in general, photosynthetic bacteria and methanogenic bacteria do not accumulate 5-aminolevulinic acid (ALA) as an intermediate product within the bacterial cells simply by culturing them, and therefore,
5-Aminolevulinic acid (ALA) is produced by levulinic acid (■, A), which is known as an inhibitor of ALA dehydratase.
It has been considered to inhibit biosynthesis from PBG to porphobilinogen (PBG) to collect 5-aminolevulinic acid (ALA).
しかし、それらの菌の培養に際し、培地にレブリン酸（
ＬＡ）を添加しなければならないために、コスト高にな
るという欠点があるだけでなく、菌体が活性を失なって
増殖率が低下するという欠点があっな。However, when culturing these bacteria, levulinic acid (
Since it is necessary to add LA), there is not only the disadvantage that the cost is high, but also the disadvantage that the bacterial cells lose their activity and the growth rate decreases.
本発明の目的は、菌体を増殖させながらレブリン酸を添
加せずとも５−アミノレブリン酸を生産できるようにす
る点にある。An object of the present invention is to make it possible to produce 5-aminolevulinic acid without adding levulinic acid while growing bacterial cells.
本発明は、ホモ酢酸菌であるところのクロストリデイウ
ム・サーモアセチカム（Ｃ１ｏｓｔｒｉｄｉｕｍｔｈｅ
ｒｎ＋ｏａｃｅｔｉｃｕｓ） ＡＴＣＣ３１４９０が、
単に培養するだけで５−アミノレブリン酸（ＡＬＡ）を
生成して蓄積することを見出すに至り、その本発明にお
ける５−アミノレブリン酸（ＡＬＡ）の製法の特徴手段
は、クロストリデイウム・サーモアセチカム（Ｃｌｏｓ
ｔｒｉｄｉｕｍ ｔｈｅｒｍｏａｃｅｔｉｃｕｍ）ＡＴ
ＣＣ３１４９０を、絶対嫌気状態で培養し、培養後の処
理液を固液分離して分離液を取出し、前記分離液から５
−アミノレブリン酸（ＡＬＡ）を採取することにあり、
その作用効果は、次の通りである。The present invention relates to Clostridium thermoaceticum, which is a homoacetic acid bacterium.
rn+oaceticus) ATCC31490 is
It has been discovered that 5-aminolevulinic acid (ALA) can be produced and accumulated simply by culturing, and the characteristic means of the method for producing 5-aminolevulinic acid (ALA) in the present invention is based on Clostridium thermoaceticum. (Clos
tridium thermoaceticum)AT
CC31490 was cultured in an absolute anaerobic state, and the treated solution after the culture was separated into solid and liquid to take out the separated solution.
- To collect aminolevulinic acid (ALA),
Its effects are as follows.
つまり、クロストリデイウム・サーモアセチカム（ＣＩ
ｏｓｔｒｊｄｉｕａ＋ ｔｈｅｒｍｏａｃｅｔｉｃｕｓ
＋）ＡＴＣＣ３１４９０を、絶対嫌気状態で培養するこ
とによって、菌体の増殖に連動して、レブリン酸（ＬＡ
）を添加しなくとも酢酸以外に多量の５−アミノレブリ
ン酸（ＡＬＡ）を、菌体外に多く蓄積する。In other words, Clostridium thermoaceticum (CI)
ostrjdiua+ thermoaceticus
+) By culturing ATCC31490 under absolute anaerobic conditions, levulinic acid (LA
) in addition to acetic acid, a large amount of 5-aminolevulinic acid (ALA) is accumulated outside the bacterial cells.
従って、従来のように、培地にレブリン酸（ＬＡ）を添
加して、５−アミノレブリン酸（ＡＬＡ）からボルホビ
リノーゲン（ＰＢＧ）に至る生合成を阻害せずとも、単
に通常の培地で培養するだけで５−アミノレブリン酸が
蓄積するために、培養のための材料費を安くでき、しか
も、菌体外に蓄積するために、採取のための作業手間を
少なくでき、生産コストを低く経済性を高めることがで
きるようになった。Therefore, instead of adding levulinic acid (LA) to the medium and inhibiting the biosynthesis from 5-aminolevulinic acid (ALA) to borphobilinogen (PBG) as in the past, it is possible to simply culture in a normal medium. Because 5-aminolevulinic acid accumulates just by doing this, the cost of materials for culturing can be reduced.Furthermore, since it accumulates outside the bacterial cells, the labor required for collection can be reduced, reducing production costs and making it more economical. It is now possible to increase the
その上、レブリン酸等によって菌体の活性が抑えられる
、ということがないために、増殖率を高く維持しながら
連続的に５−アミノレブリン酸を生産することができや
すくなった。Furthermore, since the activity of the bacterial cells is not suppressed by levulinic acid or the like, it has become easier to continuously produce 5-aminolevulinic acid while maintaining a high growth rate.
次に、本発明の詳細な説明する。 Next, the present invention will be explained in detail.
クロストリデイウム・サーモアセチカム（リユ５ｔｒｉ
ｄｉｕ＋ｓ ｔｈｅｒｍｏａｃｅｔｉｃｕｎ＋）は、好
熱性絶対嫌気性菌で、一般に、グルコース、フラクトー
ス、キシロース等の糖質１モルから３モルの酢酸を生成
することが知られている。Clostridium thermoaceticum (Ryu 5tri)
diu+s thermoaceticun+) is a thermophilic obligate anaerobic bacterium, and is generally known to produce 3 moles of acetic acid from 1 mole of carbohydrates such as glucose, fructose, and xylose.
そして、本発明者は、特にクロストリデイウム・サーモ
アセチカム（Ｃ１ｏｓｔｒｉｄｉｕｓ＋Ｌｈｅｒｍｏａ
ｃｅ ｔ ｉｃｕｍ）ＡＴＣＣ３１４９０が、酢酸以外
にビタミンＢ＋ｚ及びその前駆体としての５−アミノレ
ブリン酸 （ＡＬＡ）を、菌体内だけでなく、菌体外に
も多く生成して蓄積することを見出すに至った。In particular, the present inventor has discovered that Clostridium thermoaceticum (C1ostridius+Lhermoa
It was discovered that ATCC 31490 produces and accumulates vitamin B+z and its precursor, 5-aminolevulinic acid (ALA), in addition to acetic acid, not only inside the bacteria but also outside the bacteria. .
そこで、前述の５−アミノレブリン酸
（ＡＬＡ）の製法は、クロストリデイウム・サーモアセ
チカム（Ｃｌｏｓｔｒｉｄｉｕｍ ｔｈｅｒｍｏａｃｅ
ｔｉｃｕｍ）ＡＴＣＣ３１４９０を、少なくとも炭素（
Ｃ）、カリウム（Ｘ）、リン（Ｐ）、イオウ（Ｓ）、マ
グネシウム（Ｍｇ）、及び、チッ素（Ｎ）を含有元素と
する培地中において、培養温度４７°Ｃ〜６５℃（望ま
しくは６０°Ｃ）、常圧ｐ Ｈ４，５〜８、絶対嫌気状
態という培養条件で培養し、培養後の処理液を遠心分離
器によって固液分離して、分離液として上清液を取出し
、その上清液を、陽イオン交換樹脂（商品名Ｄｏｗｅｘ
）に対する吸着及び脱着操作によって、５−アミノレブ
リン酸（ＡＬＡ）を採取する。Therefore, the method for producing 5-aminolevulinic acid (ALA) described above is based on Clostridium thermoaceticum (Clostridium thermoaceticum).
ticum) ATCC 31490 with at least carbon (
C), potassium (X), phosphorus (P), sulfur (S), magnesium (Mg), and nitrogen (N) at a culture temperature of 47°C to 65°C (preferably 60°C), normal pressure pH 4.5 to 8, and absolute anaerobic conditions. The treated solution after culture is separated into solid and liquid using a centrifuge, and the supernatant is taken out as a separated solution. The supernatant liquid was treated with a cation exchange resin (trade name: Dowex).
5-aminolevulinic acid (ALA) is collected by adsorption and desorption operations on ).
尚、採取された５−アミノレブリン酸
（ＡＬＡ）は、第１図に示すように、薄層クロマトグラ
フィー（ＴＬＣ）による同定によって確認した。The collected 5-aminolevulinic acid (ALA) was identified by thin layer chromatography (TLC) as shown in FIG.
〔実験例１〕 次に各種培地による培養結果を示す。[Experiment example 1] Next, the culture results using various media are shown.
次の表１に示すように、７種類の組成の培地で夫々培養
した。As shown in Table 1 below, the cells were cultured in media with seven different compositions.
Ｒ１
用いた培地の組成億イオン水１１に対して）■〜■まで
の複合培地は、既知の組成で、特に酵母エキスやトリプ
トンが含まれ、高価で成分の確定困難なところがあり、
これに対し、■〜■の合成培地と称するものは、今回新
規に酵母エキスやトリプトンの特に存在しない安価な培
地で、それらの培地による培養結果は、表２に示す。R1 Composition of the medium used The complex media from ■ to ■ (relative to ionized water 11) have known compositions, especially yeast extract and tryptone, and are expensive and difficult to determine the ingredients.
On the other hand, the synthetic media referred to as ① to ② are inexpensive media that do not particularly contain yeast extract or tryptone, and the culture results using these media are shown in Table 2.
尚、表１中の前記微量金属溶液は、ｌｌ中に次の成分が
含まれる。In addition, the trace metal solution in Table 1 contains the following components in 11.
ＭｎＣ１ｇ ・４ＨｚＯＯ，５ｇ
ＮａｔＳｅ０３ １７．２１ｍｇＨ，ＢＯｌ
１０ｍｇＺｎＣＩｇ
５ｍｇ
ＡＩＫ（５０４） ｚ・１２１ｈ０ １０＋ｗｇＮｉ
Ｃ１ｚ・６８ｚＯ２１１１ｇ
ＣＬＩＣＩ！ ・２８！ＯＬＩｌｇ
ＥＤＴＡ ０．５ｍｇ前記ビタミン溶
液としては、ビオチン、ＦＭＮ、葉酸、ニコチン酸、パ
ントテン酸、Ｐ−アミノ安息香酸、チアミンピロリン酸
が、各々２ｍｇ／ｌ含まれる。MnC1g ・4HzOO, 5g NatSe03 17.21mgH, BOl
10mg ZnCIg
5mg AIK (504) z・121h0 10+wgNi
C1z・68zO2111g CLICI!・28! OLIlg EDTA 0.5 mg The vitamin solution contains 2 mg/l each of biotin, FMN, folic acid, nicotinic acid, pantothenic acid, P-aminobenzoic acid, and thiamine pyrophosphate.
前記アミノ酸溶液としては、Ｌ−アラニン、Ｌ−アルギ
ニン、Ｌ−アスパラギン、Ｌ−システィン、Ｌ−シスチ
ン、Ｌ−グルタミン酸、Ｌヒスチジン、Ｌ−ロイシン、
Ｌ−リジン、Ｌ−メチオニン、Ｌ−フェニルアラニン、
Ｌ−セリン、Ｌ−スレオニン、Ｌ−トリプトファン、Ｌ
−バリン、が、各々２ｍｇ／ｌ含まれる。The amino acid solution includes L-alanine, L-arginine, L-asparagine, L-cystine, L-cystine, L-glutamic acid, L-histidine, L-leucine,
L-lysine, L-methionine, L-phenylalanine,
L-serine, L-threonine, L-tryptophan, L
-valine, each containing 2 mg/l.
尚、前記菌体量は、乾燥重量法で調べ、前記酢酸量は、
ガスクロマトグラフィー（Ｆ Ｉ Ｄ）で測定した。The amount of bacterial cells was determined by the dry weight method, and the amount of acetic acid was
Measured by gas chromatography (FID).
表−２より酵母エキスやトリプトンの含まれない培地■
〜■でも、使用グルコースに対する酢酸の生成率が良い
ことを示す。From Table 2, medium that does not contain yeast extract or tryptone■
~■ also indicates that the production rate of acetic acid relative to the glucose used is good.
〔実験例２〕
次に、第２図に、培地■での培養時間による各種測定値
の変化を示す。[Experimental Example 2] Next, FIG. 2 shows changes in various measured values depending on the culture time in medium ①.
これによると、１モルのグルコースより約２．６モルの
酢酸が生成され、７２時間後が最大値を示し、また、生
成酢酸の増加に伴なってｐ Ｈ値が低下するために、菌
体量及び消費グルコースが、約８４時間後に最大値に達
し、それ以後は菌体の活性が低下することが判る。According to this, approximately 2.6 moles of acetic acid are produced from 1 mole of glucose, reaching a maximum value after 72 hours, and as the pH value decreases as the amount of acetic acid produced increases, the bacterial cell It can be seen that the amount and consumed glucose reached the maximum value after about 84 hours, and the activity of the bacterial cells decreased thereafter.
〔実験例３〕
次に、第３図に示すように、培養時間の変化に伴う菌体
及び５−アミノレブリン酸（ＡＬＡ）の量の変化を、レ
ブリン酸（ＬＡ）無添加状態で調べた。[Experimental Example 3] Next, as shown in FIG. 3, changes in the amounts of bacterial cells and 5-aminolevulinic acid (ALA) with changes in culture time were investigated without the addition of levulinic acid (LA).
尚、培地は、グルコースを２０ｇ／ ｌ含有させた前記
培地■を使用してスタートし、７０時間後に更にＬｏｇ
／ １のグルコースを追加して培養した。In addition, the medium was started using the above-mentioned medium (■) containing 20 g/l of glucose, and after 70 hours, the medium was further
/1 glucose was added and cultured.
つまり、第３図より時間の経過に伴って菌体及び５−ア
ミノレブリン酸（ＡＬＡ）が増加し、１２０時間後には
ＡＬＡが約１６Ｂ／βの蓄積層に達した。That is, as shown in FIG. 3, bacterial cells and 5-aminolevulinic acid (ALA) increased with the passage of time, and ALA reached an accumulation layer of about 16 B/β after 120 hours.
〔実験例４〕
次に、実験例３にひき続き、レブリン酸（ＬＡ）を追加
して１２０時間培養し、そのレフリン酸（ＬＡ）の添加
量の変化に伴う菌体１と５−アミノレブリン酸（ＡＬＡ
）の量の変化を調べ、第４図に示した。[Experimental Example 4] Next, following Experimental Example 3, levulinic acid (LA) was added and cultured for 120 hours, and bacterial cells 1 and 5-aminolevulinic acid were cultured as the amount of levulinic acid (LA) added changed. (ALA
) was investigated and shown in Figure 4.
つまり、第４図より、レブリン酸（Ｌ Ａ ）の添加に
よって５−アミノレブリン酸（ＡＬＡ）は増加して約２
１ｍｇ／ｆｆにもなったが、菌体量が減少することが判
った。In other words, from Figure 4, 5-aminolevulinic acid (ALA) increases by about 2 by adding levulinic acid (LA).
Although the concentration was as high as 1 mg/ff, it was found that the amount of bacterial cells decreased.
〔実験例５〕
次に、微量金属元素（Ｃｏ、 Ｆｅ、 Ｍｏ、 Ｓｅ、
Ｗ、Ｎｉ）化合物のうちの特にコバル） （Ｃｏ）と
、鉄（Ｆｅ）を培地に添加することによる影響を調べて
、菌体量と５−アミノレブリン酸（ＡＬＡ）の量の変化
として第５図及び第６図に示した。[Experimental Example 5] Next, trace metal elements (Co, Fe, Mo, Se,
We investigated the effects of adding W, Ni) compounds, especially cobal (Co), and iron (Fe) to the medium, and found that changes in the amount of bacterial cells and the amount of 5-aminolevulinic acid (ALA) It is shown in FIG.
つまり、第５図では、前記培地■中のコバルト（Ｃｏ）
１度を変化させた時の菌体■及び５−アミノレブリン酸
（ＡＬＡ）の生成量の変化を示し、培養時間は、全部で
１２０時間で、スタートから７０時間後に、ＩＯｇ／
１のグルコースを培地に追加しである。That is, in FIG. 5, cobalt (Co) in the medium
It shows the changes in the production amount of bacterial cells and 5-aminolevulinic acid (ALA) when the temperature is changed by 1 degree.The culture time was 120 hours in total, and 70 hours after the start, IOg/
1 of glucose was added to the medium.
尚、レブリン酸（ＬＡ）は無添加である。Note that levulinic acid (LA) was not added.
前記第５図から、コバルト濃度が、１００〜２００μ−
の範囲がＡＬＡ生成量、の最大値を示すものである。From FIG. 5, the cobalt concentration is 100 to 200 μ-
The range indicates the maximum amount of ALA produced.
また、第６図では、前記培地、■中の鉄（Ｆｅ”）濃度
を変化させた時の菌体量及び５−アミノレブリン酸（Ａ
ＬＡ）の生成量の変化を示し、培養条件は、前記第５図
の場合と同様である。Figure 6 also shows the amount of bacterial cells and 5-aminolevulinic acid (A
The culture conditions are the same as those in FIG. 5 above.
つまり、第６図より、鉄（Ｆｅ”）は菌体の増殖、及び
、５−アミノレブリン酸（ＡＬＡ）の生産にわずかしか
効果がないことが判った。In other words, from FIG. 6, it was found that iron (Fe'') had only a slight effect on the growth of bacterial cells and the production of 5-aminolevulinic acid (ALA).
［実験例６］
次に、培地■に添加する還元剤の影響を、調べて表３に
示した。尚、培養時間は９０時間である。[Experimental Example 6] Next, the influence of the reducing agent added to the medium (1) was investigated and shown in Table 3. Note that the culture time was 90 hours.
なくとも、通常は、他の培地材料から不純物として少し
でも混入してくるために、ＡＬＡの生成は成されるもの
である。At the very least, ALA is usually produced due to even a small amount of impurities being mixed in from other medium materials.
本発明に係る５−アミノレブリン酸において第１図はＡ
ＬＡを同定する薄層クロマトグラフィー、第２図は時間
変化に基づく各種測定値変化グラフ、第３図は時間変化
に基づく各種測定値変化グラフ、第４図はＬＡ濃度変化
に基づく各種測定値変化グラフ、第５図はコバルト濃度
変化に基づく各種測定値変化グラフ、第６図は鉄濃度変
化に基づく各種測定値変化グラフである。In 5-aminolevulinic acid according to the present invention, FIG.
Thin layer chromatography to identify LA, Figure 2 is a graph of changes in various measured values based on time changes, Figure 3 is a graph of changes in various measured values based on changes in time, Figure 4 is a graph of changes in various measured values based on changes in LA concentration. FIG. 5 is a graph of changes in various measured values based on changes in cobalt concentration, and FIG. 6 is a graph of changes in various measured values based on changes in iron concentration.
Claims (1)
ｉｃｕｍ￥）ＡＴＣＣ３１４９０を、絶対嫌気状態で培
養し、培養後の処理液を固液分離して分離液を取出し、
前記分離液から５−アミノレブリン酸（ＡＬＡ）を採取
する５−アミノレブリン酸の製法。[Claims] Clostridium thermoaceticum
icum￥) ATCC31490 is cultured in an absolute anaerobic state, the treated solution after culture is separated into solid and liquid, and the separated solution is taken out.
A method for producing 5-aminolevulinic acid, in which 5-aminolevulinic acid (ALA) is collected from the separated liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8221989A JPH0655147B2 (en) | 1989-03-31 | 1989-03-31 | Process for producing 5-aminolevulinic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8221989A JPH0655147B2 (en) | 1989-03-31 | 1989-03-31 | Process for producing 5-aminolevulinic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02261389A true JPH02261389A (en) | 1990-10-24 |
| JPH0655147B2 JPH0655147B2 (en) | 1994-07-27 |
Family
ID=13768303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8221989A Expired - Lifetime JPH0655147B2 (en) | 1989-03-31 | 1989-03-31 | Process for producing 5-aminolevulinic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0655147B2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06141875A (en) * | 1992-11-08 | 1994-05-24 | Cosmo Sogo Kenkyusho:Kk | Production of 5-aminolevulinic acid by microorganism |
| US5763235A (en) * | 1994-12-20 | 1998-06-09 | Cosmo Research Institute | 5-aminolevulinic acid producing microorganism and process for producing 5-aminolevulinic acid |
| WO2007034673A1 (en) * | 2005-09-21 | 2007-03-29 | Cosmo Oil Co., Ltd. | Process for producing 5-aminolevulinic acid hydrochloride |
| JP2012121918A (en) * | 2012-03-08 | 2012-06-28 | Cosmo Oil Co Ltd | Method for producing 5-aminolevulinic acid hydrochloride |
| WO2014148539A1 (en) * | 2013-03-22 | 2014-09-25 | コスモ石油株式会社 | Method for producing 5-aminolevulinic acid or salt thereof |
| JP2014207882A (en) * | 2013-03-22 | 2014-11-06 | コスモ石油株式会社 | Method for producing 5-aminolevulinic acid or salt thereof |
-
1989
- 1989-03-31 JP JP8221989A patent/JPH0655147B2/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06141875A (en) * | 1992-11-08 | 1994-05-24 | Cosmo Sogo Kenkyusho:Kk | Production of 5-aminolevulinic acid by microorganism |
| US5763235A (en) * | 1994-12-20 | 1998-06-09 | Cosmo Research Institute | 5-aminolevulinic acid producing microorganism and process for producing 5-aminolevulinic acid |
| WO2007034673A1 (en) * | 2005-09-21 | 2007-03-29 | Cosmo Oil Co., Ltd. | Process for producing 5-aminolevulinic acid hydrochloride |
| JP2007084466A (en) * | 2005-09-21 | 2007-04-05 | Cosmo Oil Co Ltd | Method for producing 5-aminolevulinic acid hydrochloride |
| US8148574B2 (en) | 2005-09-21 | 2012-04-03 | Cosmo Oil Co., Ltd. | Method for producing 5-aminolevulinic acid hydrochloride |
| JP2012121918A (en) * | 2012-03-08 | 2012-06-28 | Cosmo Oil Co Ltd | Method for producing 5-aminolevulinic acid hydrochloride |
| WO2014148539A1 (en) * | 2013-03-22 | 2014-09-25 | コスモ石油株式会社 | Method for producing 5-aminolevulinic acid or salt thereof |
| JP2014207882A (en) * | 2013-03-22 | 2014-11-06 | コスモ石油株式会社 | Method for producing 5-aminolevulinic acid or salt thereof |
| US9963724B2 (en) | 2013-03-22 | 2018-05-08 | Neo Ala Co., Ltd. | Method for producing 5-aminolevulinic acid or salt thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0655147B2 (en) | 1994-07-27 |
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