JPS6317439B2 - - Google Patents
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- Publication number
- JPS6317439B2 JPS6317439B2 JP14526878A JP14526878A JPS6317439B2 JP S6317439 B2 JPS6317439 B2 JP S6317439B2 JP 14526878 A JP14526878 A JP 14526878A JP 14526878 A JP14526878 A JP 14526878A JP S6317439 B2 JPS6317439 B2 JP S6317439B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction solution
- reaction
- atp
- ifo
- μmoles
- 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
- 238000006243 chemical reaction Methods 0.000 claims description 55
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 30
- 230000001580 bacterial effect Effects 0.000 claims description 22
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 229930024421 Adenine Natural products 0.000 claims description 12
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 12
- 229960000643 adenine Drugs 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 11
- 241000588698 Erwinia Species 0.000 claims description 10
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims description 9
- 230000000694 effects Effects 0.000 claims description 9
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 9
- 229940045145 uridine Drugs 0.000 claims description 9
- 108091000080 Phosphotransferase Proteins 0.000 claims description 8
- 102000020233 phosphotransferase Human genes 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 7
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims description 6
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims description 6
- 241000235070 Saccharomyces Species 0.000 claims description 6
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims description 6
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 claims description 5
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 claims description 5
- 229930010555 Inosine Natural products 0.000 claims description 5
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 229940029575 guanosine Drugs 0.000 claims description 5
- 229960003786 inosine Drugs 0.000 claims description 5
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 4
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 108030006122 Nucleoside ribosyltransferases Proteins 0.000 claims description 4
- 241000186216 Corynebacterium Species 0.000 claims description 3
- 150000001720 carbohydrates Chemical class 0.000 claims description 2
- 241000588722 Escherichia Species 0.000 claims 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 9
- 229910000160 potassium phosphate Inorganic materials 0.000 description 9
- 235000011009 potassium phosphates Nutrition 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 229930091371 Fructose Natural products 0.000 description 6
- 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 6
- 239000005715 Fructose Substances 0.000 description 6
- 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 6
- 239000008103 glucose Substances 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- 239000001177 diphosphate Substances 0.000 description 5
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 5
- 235000011180 diphosphates Nutrition 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 description 4
- 241000588701 Pectobacterium carotovorum Species 0.000 description 4
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 description 4
- 108010009099 nucleoside phosphorylase Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 241000798438 Aroideae Species 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 2
- 241001123227 Saccharomyces pastorianus Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 159000000009 barium salts Chemical class 0.000 description 2
- TZCXTZWJZNENPQ-UHFFFAOYSA-L barium sulfate Chemical compound [Ba+2].[O-]S([O-])(=O)=O TZCXTZWJZNENPQ-UHFFFAOYSA-L 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 229940023064 escherichia coli Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- -1 for example Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000004254 Ammonium phosphate Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001430228 Clavibacter sepedonicus Species 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000588702 Pectobacterium carotovorum subsp. carotovorum Species 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- 229910000148 ammonium phosphate Inorganic materials 0.000 description 1
- 235000019289 ammonium phosphates Nutrition 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 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
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019264 food flavour enhancer Nutrition 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000137 polyphosphoric acid Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明は、酵素反応により、アデノシン三リン
酸を製造する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing adenosine triphosphate by an enzymatic reaction.
生理活性を有するアデノシン三リン酸（以下
ATPとする）は、生化学、臨床試験における反
応基質として、また食品の栄養、風味の強化剤と
して、さらには医薬品として極めて重要な物質で
ある。従来、このATPは発酵法によつて一部生
産されているが、従来の製造方法では、原料面ま
たは発酵工程面で生産コストが高くつくために、
新しい安価な工業的製造法の開発が強く期待され
ている。 Physiologically active adenosine triphosphate (hereinafter referred to as
ATP) is an extremely important substance as a reaction substrate in biochemistry and clinical trials, as a nutritional and flavor enhancer for foods, and as a pharmaceutical agent. Conventionally, this ATP has been partially produced by fermentation, but the conventional production method requires high production costs in terms of raw materials and fermentation process.
There are strong expectations for the development of new inexpensive industrial manufacturing methods.
本発明者らは、種々の発酵生産方式について研
究した結果、ATPの安価な工業的製造法を見出
し、本発明を完成するに至つた。 As a result of research on various fermentation production methods, the present inventors discovered an inexpensive industrial production method for ATP and completed the present invention.
本発明は、アデニンにウリジン、シチジン、イ
ノシンまたはグアノシンと無機リン酸とを反応さ
せてATPを製造するに際し、ヌクレオシドリボ
シル基転移酵素活性を有するエルウイニア
（Erwinia）属、エシエリチア（Escherichia）
属、バチルス（Bacillus）属あるいはコリネバク
テリウム（Corynebacterium）属に属する微生物
菌体処理物と、リン酸転移酵素活性を有するサツ
カロミセス（Saccharomyces）属に属する微生
物またはその菌体処理物とを、前者の１量（蛋白
量として）に対して後者を１〜４倍量（乾燥菌体
重量として）の割合で存在させ、さらに発酵性糖
類を存在させて、反応液を微アルカリ性に保つこ
とを特徴とするATPの製造法である。 The present invention uses ATP produced by reacting adenine with uridine, cytidine, inosine, or guanosine and inorganic phosphoric acid.
A microorganism treated with microorganisms belonging to the genus Bacillus or Corynebacterium, and a microorganism belonging to the genus Saccharomyces having phosphotransferase activity or a treated product thereof, The latter is present at a ratio of 1 to 4 times the amount (as dry bacterial weight) per 1 amount (as protein amount), and fermentable sugars are also present to keep the reaction solution slightly alkaline. This is a method for producing ATP.
ウリジン、シチジン、イノシンまたはグアノシ
ンは調味料工業で大量に副生される。 Uridine, cytidine, inosine or guanosine are by-produced in large quantities in the seasoning industry.
無機リン酸としては、たとえばリン酸カリウ
ム、リン酸ナトリウム、リン酸アンモニウムなど
のリン酸塩またはリン酸やポリリン酸が使用され
る。 As the inorganic phosphoric acid, for example, phosphates such as potassium phosphate, sodium phosphate, and ammonium phosphate, phosphoric acid, and polyphosphoric acid are used.
本発明においては、ヌクレオシドリボシル基転
移酵素活性を有する微生物菌体処理物とリン酸転
移酵素活性を有する微生物またはその菌体処理物
を使用するのであるが、ヌクレオシドリボシル基
転移酵素活性を有する微生物菌体処理物として
は、Escherichiacoli IFO 3806、Erwinia
carotovora IFO 3057、Erwinia aroideae IFO
3830、Bacillus sphericus IFO 3525、
Corynebacterium spedonicum IFO 12188など
の菌体磨砕物、乾燥菌体、超音波処理菌体、溶剤
処理菌体、無細胞抽出液、部分精製酵素標品、固
定化酵素標品が用いられ、菌株は本酵素活性を有
する菌であればいずれの菌でもよい。 In the present invention, a microbial cell treatment product having nucleoside ribosyltransferase activity and a microorganism having phosphotransferase activity or a bacterial cell treatment product thereof are used. Escherichiacoli IFO 3806, Erwinia
carotovora IFO 3057, Erwinia aroideae IFO
3830, Bacillus sphericus IFO 3525,
Corynebacterium spedonicum IFO 12188 and other bacterial cell crush, dried bacterial cells, sonicated bacterial cells, solvent-treated bacterial cells, cell-free extracts, partially purified enzyme preparations, and immobilized enzyme preparations are used. Any active bacteria may be used.
リン酸転移酵素活性を有する微生物またはその
菌体処理物としては、Saccharomyces
cerevisiae IFO 635、Saccharomyces
carlsbergensis IFO 641、Saccharomyces sake
kyokaiNo.７、Saccharomyces sake kyokaiNo.８
などの乾燥菌体、菌体磨砕物、超音波処理菌体、
溶剤処理菌体が用いられ、本酵素活性を有する菌
株であればいずれの菌でも使用できる。 As microorganisms having phosphotransferase activity or their bacterial cell products, Saccharomyces
cerevisiae IFO 635, Saccharomyces
carlsbergensis IFO 641, Saccharomyces sake
kyokaiNo.7, Saccharomyces sake kyokaiNo.8
dried bacterial cells, ground bacterial cells, sonicated bacterial cells, etc.
Solvent-treated bacterial cells are used, and any bacterial strain having this enzyme activity can be used.
発酵性糖類としては、たとえばグルコース、ガ
ラクトース、フルクトース、乳糖、マンノース、
リボースおよびこれらのリン酸エステル化合物
（たとえば果糖二リン酸）が使用される。 Examples of fermentable sugars include glucose, galactose, fructose, lactose, mannose,
Ribose and their phosphate ester compounds (eg fructose diphosphate) are used.
前記ヌクレオシドリボシル基転移酵素活性を有
する微生物菌体処理物と、リン酸転移酵素活性を
有する微生物またはその菌体処理物との割合は、
前者の１量（酵素蛋白量として）に対して後者を
１〜４倍量（乾燥菌体重量として）にすることが
必要である。これ以外の割合では反応が進まない
か、あるいは反応が進行しても収率が非常に悪
い。 The ratio of the microorganism-treated product having nucleoside ribosyltransferase activity to the microorganism having phosphotransferase activity or its microbial-processed product is:
It is necessary to use 1 to 4 times the amount of the latter (as dry bacterial weight) per 1 amount of the former (as the amount of enzyme protein). If the ratio is other than this, the reaction will not proceed, or even if the reaction does proceed, the yield will be very poor.
反応液は微アルカリ性に保つことが必要であ
り、PH7.2〜8.5が好ましい。ヌクレオシドリボシ
ル基転移酵素（ヌクレオシドホスホリラーゼ）
は、中性から微アルカリ性で強く作用するが、酸
性側では作用せず、一方、リン酸転移酵素は酸性
側で強く作用するが、中性から微アルカリ性では
その作用はゆるやかである。したがつて、反応液
を微アルカリ性にすることにより、ヌクレオシド
ホスホリラーゼを強く作用させつつ、リン酸転移
酵素をゆるやかに作用させることができ、これに
よつてATPの収率を高めることができる。 It is necessary to keep the reaction solution slightly alkaline, preferably PH7.2 to 8.5. Nucleoside ribosyltransferase (nucleoside phosphorylase)
acts strongly in neutral to slightly alkaline conditions, but does not act in acidic conditions, while phosphotransferase acts strongly in acidic conditions, but its action is gradual in neutral to slightly alkaline conditions. Therefore, by making the reaction solution slightly alkaline, it is possible to cause the nucleoside phosphorylase to act strongly and the phosphotransferase to act slowly, thereby increasing the yield of ATP.
反応温度は10〜45℃、反応時間は５〜40時間が
好ましく、反応は振盪もしくは静置条件下で行な
う。 The reaction temperature is preferably 10 to 45°C, the reaction time is preferably 5 to 40 hours, and the reaction is carried out under shaking or standing conditions.
反応に必要な各成分は、すべて最初から添加混
合して反応させることができるが、好ましい実施
条件としては、ヌクレオシドホスホリラーゼ反応
（リボシル基転移反応）の進行途中において、発
酵性糖類とリン酸転移酵素活性を有する微生物あ
るいはその菌体処理物を添加するのがよく、この
ようにすることにより、ATPの収率を高めるこ
とができる。 All the components necessary for the reaction can be added and mixed from the beginning, but as a preferred implementation condition, during the progress of the nucleoside phosphorylase reaction (ribosyl group transfer reaction), the fermentable saccharide and the phosphotransferase are mixed together. It is preferable to add active microorganisms or their bacterial cell products, and by doing so, the yield of ATP can be increased.
生産されたATPの分離、精製は、一般に知ら
れている分離方法、たとえばイオン交換樹脂法、
活性炭吸着法、溶剤抽出沈澱法などの諸方法を併
用して分離、精製することができる。 The produced ATP can be separated and purified using generally known separation methods, such as the ion exchange resin method,
Separation and purification can be performed using various methods such as activated carbon adsorption method and solvent extraction precipitation method.
本発明によれば、調味料工業で大量に副生する
ウリジン、シチジン、イノシンまたはグアノシン
をリボース源とし、他方、工業上安価に供給され
る無機リン酸および発酵性糖類をそれぞれリン酸
源およびエネルギー源として、アデニンから
ATPを効率よく製造することができる。 According to the present invention, uridine, cytidine, inosine, or guanosine, which are by-produced in large quantities in the seasoning industry, are used as the ribose source, and on the other hand, inorganic phosphoric acid and fermentable sugars, which are supplied at low cost in the industry, are used as the phosphate source and energy source, respectively. From adenine as a source
ATP can be produced efficiently.
次に実施例を示す。 Next, examples will be shown.
実施例 １
反応液１ml当り、グルコース600μmoles、リン
酸カリウム（PH7.4）600μmoles、ウリジン
100μmoles、アデニン50μmoles、
MgSO410μmoles、Erwinia cartovora IFO
3057の無細胞標品７mg（蛋白質量として）および
Saccharomyces cerevisiae IFO 635の乾燥菌体
25mgを含むように調製した。この反応液２mlを28
℃、24時間振盪させ反応を行なつたところ、反応
液１ml当りATP28μmolesを得た。Example 1 Per 1 ml of reaction solution, 600 μmoles of glucose, 600 μmoles of potassium phosphate (PH7.4), uridine
100μmoles, adenine 50μmoles,
MgSO4 10μmoles, Erwinia cartovora IFO
7 mg of cell-free preparation of 3057 (as protein amount) and
Dried bacterial cells of Saccharomyces cerevisiae IFO 635
It was prepared to contain 25 mg. 2 ml of this reaction solution
When the reaction was carried out by shaking at ℃ for 24 hours, 28 μmoles of ATP was obtained per ml of the reaction solution.
以上の組成で反応液1000ml中にグルコース108
ｇ、リン酸カリウム95ｇ、ウリジン24.4ｇ、アデ
ニン6.8ｇ、MgSO41.2ｇをそれぞれ溶かし、
Erwinia cartovora IFO 3057微工研菌寄第9525
号の無細胞標品７ｇ（蛋白質量として）および
Saccharomyces cerevisiae IFO 635微工研菌寄
第9522号の乾燥菌体25ｇを懸濁させ、28℃、24時
間反応させたところ、ATPが14ｇ生成された。 With the above composition, 108 glucose in 1000ml of reaction solution
Dissolve g, 95 g of potassium phosphate, 24.4 g of uridine, 6.8 g of adenine, and 1.2 g of MgSO 4 , respectively.
Erwinia cartovora IFO 3057 Microtechnical Research Institute No. 9525
7g of cell-free preparation (as protein amount) and
When 25 g of dried microbial cells of Saccharomyces cerevisiae IFO 635 Microtechnical Laboratory No. 9522 were suspended and reacted at 28°C for 24 hours, 14 g of ATP was produced.
反応終了後、反応液を短時間加熱処理して固形
分を除去する。上澄液をPH3.5とし、活性炭カラ
ムを通過させ生成ATPを吸着させる。水洗後、
アンモニヤ性50％エタノール溶液で溶出し、
ATP含有区分を分取し、濃縮してPH８に調整し
た。 After the reaction is completed, the reaction solution is heated for a short time to remove solids. The supernatant liquid is adjusted to pH 3.5 and passed through an activated carbon column to adsorb the produced ATP. After washing with water,
Elute with ammoniacal 50% ethanol solution,
The ATP-containing fraction was separated, concentrated, and adjusted to pH 8.
次に、あらかじめCl型に調整したダウエツクス
１×２樹脂に濃縮液を吸着せしめ、水洗したあと
HCl−NaClの溶媒系で溶出を行ない、ATPの区
分を採取し、活性炭処理で脱塩し、溶液に塩化バ
リウムを加えてバリウム塩として沈澱させ分離し
た。バリウム塩は硫酸ソーダ溶液に溶かしこみ、
バリウムを硫酸バリウムとして除去し、ATPを
ソーダ塩として単離した。収量は、ATP12.6ｇ
（遊離として）であつた。 Next, the concentrated solution was adsorbed onto Dowex 1×2 resin, which had been adjusted to Cl type in advance, and after washing with water,
Elution was performed with a solvent system of HCl-NaCl, a fraction of ATP was collected, desalted by activated carbon treatment, and barium chloride was added to the solution to precipitate and separate the barium salt. The barium salt is dissolved in a sodium sulfate solution.
Barium was removed as barium sulfate and ATP was isolated as soda salt. Yield: ATP12.6g
(as free).
実施例 ２
反応液１ml当り、Erwinia cartovoraの代りに
Escherichia coli IFO 3806の無細胞標品８mg
（蛋白質量として）を添加し、反応時間を22時間
とした以外は実施例１に準じた条件下で反応を行
なつた。その結果、反応液１ml当り
ATP10μmolesを得た。Example 2 Per 1 ml of reaction solution, instead of Erwinia cartovora
Escherichia coli IFO 3806 cell-free preparation 8mg
The reaction was carried out under the same conditions as in Example 1, except that (in terms of protein amount) was added and the reaction time was 22 hours. As a result, per ml of reaction solution
Obtained 10 μmoles of ATP.
実施例 ３
反応液１ml当り、Erwinia cartovoraの代りに
Bacillus sphericus IFO 3525の無細胞標品を８
mg（蛋白質量として）添加した以外は実施例１に
準じた条件下で反応を行なつた。その結果、反応
液１ml当りATP20μmolesを得た。Example 3 Per ml of reaction solution, instead of Erwinia cartovora
8 cell-free preparations of Bacillus sphericus IFO 3525
The reaction was carried out under the same conditions as in Example 1, except that mg (in terms of protein amount) was added. As a result, 20 μmoles of ATP was obtained per ml of reaction solution.
実施例 ４
反応液１ml当り、Erwinia cartovoraの代りに
Corynebacterium sepedonicum IFO 12188の微
工研菌寄第9524号の無細胞標品７mg（蛋白質量と
して）添加した以外は実施例１に準じた条件で反
応を行なつた。その結果、反応液１ml当り
ATP23μmolesを得た。Example 4 Per ml of reaction solution, instead of Erwinia cartovora
The reaction was carried out under the same conditions as in Example 1, except that 7 mg (in terms of protein amount) of a cell-free standard of Corynebacterium sepedonicum IFO 12188, Fiber Science and Technology Research Institute No. 9524, was added. As a result, per ml of reaction solution
Obtained 23 μmoles of ATP.
実施例 ５
反応液１ml当り、Saccharomyces cerevisiae
の代りにSaccharomyces sake KyokaiNo.７の乾
燥菌体25mgを添加した以外は実施例１に準じた条
件下で反応を行なつた。その結果、反応液１ml当
りATP25μmolesを得た。Example 5 Saccharomyces cerevisiae per 1 ml of reaction solution
The reaction was carried out under the same conditions as in Example 1, except that 25 mg of dried Saccharomyces sake Kyokai No. 7 cells was added instead. As a result, 25 μmoles of ATP was obtained per 1 ml of the reaction solution.
実施例 ６
反応液１ml当り、グルコース600μmoles、リン
酸カリウム（PH7.8）400μmoles、シチジン
100μmoles、アデニン50μmoles、
MgSO410μmoles、Erwinia、carotovora IFO
3057の無細胞標品10mg（蛋白質量として）および
Saccharomyces cerevisiae IFO 635の乾燥菌体
25mgを含む反応液３mlを28℃、12時間振盪反応を
行なつた。その結果、反応液１ml当り
ATP25μmolesを得た。Example 6 Per 1 ml of reaction solution, 600 μmoles of glucose, 400 μmoles of potassium phosphate (PH7.8), cytidine
100μmoles, adenine 50μmoles,
MgSO4 10μmoles, Erwinia, carotovora IFO
10 mg of cell-free preparation of 3057 (as protein amount) and
Dried bacterial cells of Saccharomyces cerevisiae IFO 635
3 ml of the reaction solution containing 25 mg was subjected to a shaking reaction at 28°C for 12 hours. As a result, per ml of reaction solution
Obtained 25μmoles of ATP.
実施例 ７
リン酸カリウム（PH7.2）600μmoles、アデニ
ン75μmoles、ウリジン100μmoles、
MgSO410μmoles、Erwinia carotovora IFO
3057の無細胞標品10mg（蛋白質量として）を含む
反応液0.9mlを28℃、４時間反応させた後、果糖
二リン酸（FDP）100μmolesを添加して１mlとな
し、さらにSaccharomyces carlsbergensis IFO
641微工研菌寄第9523号の乾燥菌体25mgを加えて、
16時間（総反応時間20時間）反応を行なつた。そ
の結果、反応液１ml当りATP32μmolesを得た。Example 7 Potassium phosphate (PH7.2) 600μmoles, adenine 75μmoles, uridine 100μmoles,
MgSO4 10μmoles, Erwinia carotovora IFO
After reacting 0.9 ml of the reaction solution containing 10 mg (as protein amount) of the cell-free preparation of 3057 at 28°C for 4 hours, 100 μmoles of fructose diphosphate (FDP) was added to make 1 ml, and then Saccharomyces carlsbergensis IFO
Add 25 mg of dried bacterial cells of 641 Microtechnical Laboratory No. 9523,
The reaction was carried out for 16 hours (total reaction time 20 hours). As a result, 32 μmoles of ATP was obtained per ml of reaction solution.
実施例 ８
反応液１ml当り、リン酸カリウム（PH7.4）
600μmoles、アデニン60μmoles、シチジン
100μmoles、MgSO410μmoles、Erwinia
aroideae IFO 3830の無細胞標品10mg（蛋白質量
として）を含む反応液0.9mlを28℃、８時間反応
させた後、果糖二リン酸100μmolesを添加して１
mlとし、さらにSaccharomyces cerevisiae
IFO635の乾燥菌体25mgを加えて16時間反応を行
なつた。その結果、反応液１ml当り
ATP20μmolesを得た。Example 8 Potassium phosphate (PH7.4) per 1 ml of reaction solution
600μmoles, adenine 60μmoles, cytidine
100μmoles, MgSO 4 10μmoles, Erwinia
After reacting 0.9 ml of a reaction solution containing 10 mg (as protein amount) of a cell-free preparation of aroideae IFO 3830 at 28°C for 8 hours, 100 μmoles of fructose diphosphate was added.
ml and further Saccharomyces cerevisiae
25 mg of dried IFO635 cells were added and the reaction was carried out for 16 hours. As a result, per ml of reaction solution
Obtained 20 μmoles of ATP.
実施例 ９
リン酸二カリウム（PH7.6）400μmoles、アデ
ニン75μmoles、ウリジン100μmoles、
MgSO410μmoles、Erwinia carotovora IFO
3057の無細胞抽出物のポリアクリルアミドゲル固
定化標品10mg（蛋白質量として）を含む反応液
0.9mlを28℃、４時間反応を行させた後、果糖二
リン酸100μmoles添加して１mlとなし、さらに
Saccharomyces cerevisiae IFO 635の乾燥菌体
25mgを加えて16時間反応を行なつた。その結果、
反応液１ml当りATP15μmolesを得た。Example 9 Dipotassium phosphate (PH7.6) 400 μmoles, adenine 75 μmoles, uridine 100 μmoles,
MgSO4 10μmoles, Erwinia carotovora IFO
Reaction solution containing 10 mg (as protein amount) of polyacrylamide gel-immobilized sample of cell-free extract of 3057
After reacting 0.9ml at 28℃ for 4 hours, add 100μmoles of fructose diphosphate to make 1ml, and then
Dried bacterial cells of Saccharomyces cerevisiae IFO 635
25 mg was added and the reaction was carried out for 16 hours. the result,
15 μmoles of ATP was obtained per 1 ml of reaction solution.
実施例 10
リン酸カリウム（PH7.8）400μmoles、アデニ
ン30μmoles、ウリジン50μmoles、
MgSO410μmoles、Erwinia carotovora IFO
3057から部分精製したヌクレオシドホスホリラー
ゼのポリアクリルアマイドゲル固定化標品８mg
（蛋白質量として）を含む反応液0.9mlを28℃、４
時間反応させた後、果糖二リン酸100μmoles添加
して１mlとなし、さらにSaccharomyces
carlsbergensis IFO 641の乾燥菌体25mgを加えて
16時間反応を行なつた。その結果、反応液１ml当
りATP8μmolesを得た。Example 10 Potassium phosphate (PH7.8) 400μmoles, adenine 30μmoles, uridine 50μmoles,
MgSO4 10μmoles, Erwinia carotovora IFO
8 mg of polyacrylamide gel-immobilized specimen of nucleoside phosphorylase partially purified from 3057
0.9ml of the reaction solution containing (as protein amount) was heated at 28℃ for
After reacting for an hour, 100 μmoles of fructose diphosphate was added to make 1 ml, and then Saccharomyces
Add 25mg of dried bacterial cells of carlsbergensis IFO 641
The reaction was carried out for 16 hours. As a result, 8 μmoles of ATP was obtained per ml of reaction solution.
実施例 11
反応液１ml当り、リン酸カリウム（PH7.2）
600μmoles、アデニン60μmoles、イノシン
100μmoles、MgSO410μmoles、Erwinia
aroideae IFO 3830の無細胞標品10mg（蛋白質量
として）を含む反応液を28℃、８時間反応させた
後、グルコース100μmolesを添加して、さらに
Saccharomyces cerevisiae IFO 635の乾燥菌体
25mgを加えて15時間反応を行なつた。その結果、
反応液１ml当りATP18μmolesを得た。Example 11 Potassium phosphate (PH7.2) per 1 ml of reaction solution
600μmoles, adenine 60μmoles, inosine
100μmoles, MgSO 4 10μmoles, Erwinia
After reacting a reaction solution containing 10 mg (as protein amount) of aroideae IFO 3830 cell-free preparation at 28°C for 8 hours, 100 μmoles of glucose was added and further
Dried bacterial cells of Saccharomyces cerevisiae IFO 635
25 mg was added and the reaction was carried out for 15 hours. the result,
18 μmoles of ATP was obtained per 1 ml of reaction solution.
実施例 12
反応液１ml当り、リン酸カリウム（PH7.5）
600μmoles、アデニン50μmoles、グアノシン
70μmoles、MgSO410μmoles、Erwinia
carotovora IFO 3057の無細胞標品７mg（蛋白質
量として）を含む反応液を28℃、８時間反応させ
た後、グルコース100μmolesを添加して、さらに
Saccharomyces cerevisiae IFO 635の乾燥菌体
30mgを加えて16時間反応を行なつた。その結果、
反応液11ml当りATP27μmolesを得た。Example 12 Potassium phosphate (PH7.5) per 1ml of reaction solution
600μmoles, adenine 50μmoles, guanosine
70μmoles, MgSO4 10μmoles, Erwinia
After reacting a reaction solution containing 7 mg (as protein amount) of a cell-free preparation of carotovora IFO 3057 at 28°C for 8 hours, 100 μmoles of glucose was added and further
Dried bacterial cells of Saccharomyces cerevisiae IFO 635
30 mg was added and the reaction was carried out for 16 hours. the result,
27 μmoles of ATP was obtained per 11 ml of reaction solution.
Claims (1)
たはグアノシンと無機リン酸とを反応させアデノ
シン三リン酸を製造するに際し、ヌクレオシドリ
ボシル基転移酵素活性を有するエルウイニア
（Erwinia）属、エシエリチア（Escherichia）
属、バチルス（Bacillus）属あるいはコリネバク
テリウム（Corynebacterium）属に属する微生物
菌体処理物と、リン酸転移酵素活性を有するサツ
カロミセス（Saccharomyces）属に属する微生
物またはその菌体処理物とを、前者の１量（蛋白
量として）に対して後者を１〜４倍量（乾燥菌体
重量として）の割合で存在させ、さらに発酵性糖
類を存在させ、反応液を微アルカリ性に保つこと
を特徴とするアデノシン三リン酸の製造法。1. When producing adenosine triphosphate by reacting adenine with uridine, cytidine, inosine or guanosine and inorganic phosphoric acid, Erwinia genus and Escherichia which have nucleoside ribosyl transferase activity are used.
A microorganism treated with microorganisms belonging to the genus Bacillus or Corynebacterium, and a microorganism belonging to the genus Saccharomyces having phosphotransferase activity or a treated product thereof with the former The latter is present at a ratio of 1 to 4 times the amount (as dry bacterial weight) per 1 amount (as protein amount), and fermentable saccharides are further present to maintain the reaction solution slightly alkaline. Method for producing adenosine triphosphate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14526878A JPS5571495A (en) | 1978-11-27 | 1978-11-27 | Preparation of adenine nucleotide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14526878A JPS5571495A (en) | 1978-11-27 | 1978-11-27 | Preparation of adenine nucleotide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5571495A JPS5571495A (en) | 1980-05-29 |
| JPS6317439B2 true JPS6317439B2 (en) | 1988-04-13 |
Family
ID=15381194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14526878A Granted JPS5571495A (en) | 1978-11-27 | 1978-11-27 | Preparation of adenine nucleotide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5571495A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60210995A (en) * | 1984-04-04 | 1985-10-23 | Kyowa Hakko Kogyo Co Ltd | Preparation of adenosine-5'-triphosphate |
-
1978
- 1978-11-27 JP JP14526878A patent/JPS5571495A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5571495A (en) | 1980-05-29 |
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