JPH02200192A - Production of aldehydes - Google Patents
Production of aldehydesInfo
- Publication number
- JPH02200192A JPH02200192A JP5511289A JP5511289A JPH02200192A JP H02200192 A JPH02200192 A JP H02200192A JP 5511289 A JP5511289 A JP 5511289A JP 5511289 A JP5511289 A JP 5511289A JP H02200192 A JPH02200192 A JP H02200192A
- Authority
- JP
- Japan
- Prior art keywords
- group
- formula
- enzyme
- carbon atoms
- tables
- 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
- 238000004519 manufacturing process Methods 0.000 title claims description 16
- 150000001299 aldehydes Chemical class 0.000 title abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 claims abstract description 40
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 16
- 241000589516 Pseudomonas Species 0.000 claims abstract description 15
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 125000003118 aryl group Chemical group 0.000 claims abstract description 8
- 125000000753 cycloalkyl group Chemical group 0.000 claims abstract description 7
- 125000003545 alkoxy group Chemical group 0.000 claims abstract description 6
- 229930182470 glycoside Natural products 0.000 claims abstract description 6
- 150000002338 glycosides Chemical class 0.000 claims abstract description 6
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims abstract description 5
- 125000003011 styrenyl group Chemical class [H]\C(*)=C(/[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims abstract description 5
- 125000001424 substituent group Chemical group 0.000 claims abstract description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 19
- 230000000694 effects Effects 0.000 claims description 14
- 102000016680 Dioxygenases Human genes 0.000 claims description 13
- 108010028143 Dioxygenases Proteins 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 8
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 6
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 235000012141 vanillin Nutrition 0.000 abstract description 9
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 abstract description 9
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 abstract description 9
- 241000894006 Bacteria Species 0.000 abstract description 5
- 238000012258 culturing Methods 0.000 abstract description 4
- 238000006555 catalytic reaction Methods 0.000 abstract 1
- 150000003440 styrenes Chemical class 0.000 description 16
- 239000005977 Ethylene Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000012064 sodium phosphate buffer Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- IXWOUPGDGMCKGT-UHFFFAOYSA-N 2,3-dihydroxybenzaldehyde Chemical compound OC1=CC=CC(C=O)=C1O IXWOUPGDGMCKGT-UHFFFAOYSA-N 0.000 description 2
- YQUVCSBJEUQKSH-UHFFFAOYSA-N 3,4-dihydroxybenzoic acid Chemical group OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 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 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 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 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- BJIOGJUNALELMI-ARJAWSKDSA-N cis-isoeugenol Chemical compound COC1=CC(\C=C/C)=CC=C1O BJIOGJUNALELMI-ARJAWSKDSA-N 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- 238000006114 decarboxylation reaction Methods 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 210000003495 flagella Anatomy 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000007524 organic acids Chemical class 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- RUVINXPYWBROJD-ONEGZZNKSA-N trans-anethole Chemical compound COC1=CC=C(\C=C\C)C=C1 RUVINXPYWBROJD-ONEGZZNKSA-N 0.000 description 2
- 238000002525 ultrasonication Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- CDBKCIOGROFMEO-NSCUHMNNSA-N (E)-3-(3-formyl-4-hydroxy-5-methoxyphenyl)prop-2-enoic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(C=O)=C1O CDBKCIOGROFMEO-NSCUHMNNSA-N 0.000 description 1
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- QWBAFPFNGRFSFB-UHFFFAOYSA-N 2-hydroxy-3-oxopropanoic acid Chemical compound O=CC(O)C(O)=O QWBAFPFNGRFSFB-UHFFFAOYSA-N 0.000 description 1
- KQPXJFAYGYIGRU-ONEGZZNKSA-N 3,3'-dimethoxy-trans-stilbene-4,4'-diol Chemical group C1=C(O)C(OC)=CC(\C=C\C=2C=C(OC)C(O)=CC=2)=C1 KQPXJFAYGYIGRU-ONEGZZNKSA-N 0.000 description 1
- HAQLHRYUDBKTJG-UHFFFAOYSA-N 3,5-dihydroxybenzaldehyde Chemical compound OC1=CC(O)=CC(C=O)=C1 HAQLHRYUDBKTJG-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 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
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- BJIOGJUNALELMI-ONEGZZNKSA-N Isoeugenol Natural products COC1=CC(\C=C\C)=CC=C1O BJIOGJUNALELMI-ONEGZZNKSA-N 0.000 description 1
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- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
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- 239000004472 Lysine Substances 0.000 description 1
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- 230000009467 reduction Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000019086 sulfide ion homeostasis Effects 0.000 description 1
- BJIOGJUNALELMI-UHFFFAOYSA-N trans-isoeugenol Natural products COC1=CC(C=CC)=CC=C1O BJIOGJUNALELMI-UHFFFAOYSA-N 0.000 description 1
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 description 1
- PTNLHDGQWUGONS-UHFFFAOYSA-N trans-p-coumaric alcohol Natural products OCC=CC1=CC=C(O)C=C1 PTNLHDGQWUGONS-UHFFFAOYSA-N 0.000 description 1
- PTNLHDGQWUGONS-OWOJBTEDSA-N trans-p-coumaryl alcohol Chemical compound OC\C=C\C1=CC=C(O)C=C1 PTNLHDGQWUGONS-OWOJBTEDSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
【発明の詳細な説明】 〈産業上の利用分野〉 本発明は、アルデヒド類の製造方法に関する。[Detailed description of the invention] <Industrial application field> The present invention relates to a method for producing aldehydes.
更に詳しくは、本発明は、スチレン誘導体をシュードモ
ナス（Ｐ ｓｅｕｄｏｍｏｎａｓ）属に属する成る特定
の菌の産生ずる酵素を用いて、そのスチレン誘導体中の
ベンゼン環に共役するエチレン結合を選択的且つ酸化的
に分解し、アルデヒド類を製造する方法に関する。More specifically, the present invention uses an enzyme produced by a specific bacterium belonging to the genus Pseudomonas to selectively and oxidatively oxidize the ethylene bond conjugated to the benzene ring in the styrene derivative. This invention relates to a method for decomposing aldehydes to produce aldehydes.
〈従来技術〉
従来酵素を用いてスチレン誘導体中のベンゼン環に共役
するエチレン結合を選択的且つ酸化的に分解し、アルデ
ヒド類を製造することは本発明者の知る限り、知られて
いない。<Prior Art> To the best of the present inventor's knowledge, it has not been known to selectively and oxidatively decompose the ethylene bond conjugated to the benzene ring in a styrene derivative using an enzyme to produce aldehydes.
〈発明の目的〉
本発明の目的は、スチレン誘導体中のベンゼン環に共役
するエチレン結合を酵素を用いて選択的且つ酸化的に分
解し、アルデヒド類を製造し得る方法を提供することに
ある。<Objective of the Invention> An object of the present invention is to provide a method for producing aldehydes by selectively and oxidatively decomposing the ethylene bond conjugated to the benzene ring in a styrene derivative using an enzyme.
本発明の他の目的は、シュードモナス属に属する菌株が
産生する酵素を用いて、スチレン誘導体中のベンゼン環
に共役するエチレン結合を酸化的に分解し、アルデヒド
類を製造し得る方法を提供することにある。Another object of the present invention is to provide a method for producing aldehydes by oxidatively decomposing an ethylene bond conjugated to a benzene ring in a styrene derivative using an enzyme produced by a strain belonging to the genus Pseudomonas. It is in.
本発明者の研究によれば、上記本発明の目的は、下記一
般式［Ｉ］
式中Ｒ，は炭素数１〜１０の直鎖もしくは分岐状アルキ
ル基、炭素数６〜１０のアリール基、炭素数５〜１５の
シクロアルキル基、基−ＣＯＯＲ’ （ここでＲ′は水
素原子または炭素数１−１０のアルキル基）、ホルミル
基また基、シクロアルキル基およびアリール基は置換基
を有していてもよい。According to the research of the present inventors, the object of the present invention is to provide the following general formula [I], where R is a linear or branched alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, A cycloalkyl group having 5 to 15 carbon atoms, a group -COOR' (where R' is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms), a formyl group, a cycloalkyl group and an aryl group have a substituent. You can leave it there.
Ｒ３、Ｒ８、Ｒ４，Ｒ１およびＲ６は、互いに同一もし
くは異なり、水素原子、水酸基もしくはその配糖体、炭
素数１−１０の直鎖もしくは分岐状アルキル基、または
炭素数１〜１０のアルコキシ基を示すか或いはこれら互
いに隣接する２つの基は、（ＣＨｚ′＋−１−ｏ ＋
ＣＨ！ ） または −０＋ＣＨ，）Ｏ−ｎ
ｎであって
もよい（ここでｎは１〜４の整数を示す）。R3, R8, R4, R1 and R6 are the same or different and each represents a hydrogen atom, a hydroxyl group or a glycoside thereof, a linear or branched alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. The two groups shown or adjacent to each other are (CHz'+-1-o +
CH! ) or -0+CH,)O-n
It may be n (here, n represents an integer of 1 to 4).
で表わされるスチレン誘導体を、シュードモナス属に属
し且つスチレン誘導体に対するジオキシゲナーゼ活性酵
素を産生し得る菌株が生産した酵素と好気的条件下に接
触せしめることを特徴とするアルデヒド類の製造方法に
より達成される。A method for producing aldehydes characterized by bringing a styrene derivative represented by the following into contact with an enzyme produced by a strain belonging to the genus Pseudomonas and capable of producing an enzyme with dioxygenase activity against styrene derivatives under aerobic conditions. Ru.
本発明によれば、上記スチレン誘導体［Ｉ］にシュード
モナス属し且つスチレン誘導体中のベンゼン環の共役エ
チレン結合に対するジオキシゲナーゼ活性を有する酵素
を産生じ得る菌株が生産した酵素を作用せしめることに
より、該スチレン誘導体［１１におけるエチレン結合が
酸化的に分解されアルデヒド基に転換される。これを模
式的に反応式で表わすと下記の通りとなる。According to the present invention, the styrene derivative [I] is treated with an enzyme produced by a strain of Pseudomonas that is capable of producing an enzyme having dioxygenase activity against the conjugated ethylene bond of the benzene ring in the styrene derivative. The ethylene bond in derivative [11] is oxidatively decomposed and converted to an aldehyde group. This is schematically represented by the reaction formula as follows.
Ｏ！ ・′−・ＣＨＯ＋ＯＨＣ〜
・−・ＣＨ−ＣＨ〜−〉
かくして本発明において、ジオキシゲナーゼ活性とは、
スチレン誘導体中のベンゼン環に共役するエチレン結合
が、上記反応式の如く選択的に酸素添加反応によって分
解され２つのアルデヒド基に転換される作用を意味する
。O!・'-・CHO+OHC~ ・-・CH-CH~-> Thus, in the present invention, dioxygenase activity is
This refers to the action in which the ethylene bond conjugated to the benzene ring in the styrene derivative is selectively decomposed by an oxygen addition reaction and converted into two aldehyde groups as shown in the above reaction formula.
本発明者が知る限り、スチレン誘導体におけるエチレン
結合が、シュードモナス属の成る菌株の産生ずる酵素に
よって上記ジオキシゲナーゼ活性を受けることは全く新
しい知見である。As far as the present inventors know, it is a completely new finding that ethylene bonds in styrene derivatives undergo the dioxygenase activity described above by enzymes produced by strains of the genus Pseudomonas.
かくして本発明によれば上記スチレン誘導体［Ｉ］は上
記した酵素のジオキシゲナーゼ活性の作用を受け、アル
デヒドが得られる。Thus, according to the present invention, the above-mentioned styrene derivative [I] is subjected to the action of the dioxygenase activity of the above-mentioned enzyme to obtain an aldehyde.
その反応例のいくつかを下記Ｉこ示す。Some of the reaction examples are shown below.
本発明の上記一般式［Ｉ］で表わされるスチレン誘導体
として、好ましいＲ，、Ｒ，、Ｒ３、ＲいＲ，およびＲ
，は以下に説明する通りである。Preferred examples of the styrene derivative of the present invention represented by the above general formula [I] include R,, R,, R3, R, and R.
, is as explained below.
Ｒ１としては、炭素数１〜６の直鎖もしくは分岐状アル
キル基；炭素数６〜８のアリール基、特にフェニル基；
炭素数５〜ｌＯのシクロアルキル基、特にシクロヘキシ
ル基；基−ＧＯＯＲ’ （ここでＲＩは水素原子または
炭素数１〜４のアルキ［バニリン］
なお本発明方法によれば前記反応例（２）に示されるよ
うに、原料スチレン誘導体中に基−０Ｃ１ｕが含まれて
いる場合、生成したアルデヒド類中の基−０ＧＱｕは、
菌株の培養生成物から分離された酵素中にグリコシダー
ゼが含まれているとこの基０ＧＱｕはその作用に起因す
ると思われる水酸基に転換された形として得られる場合
がある。R1 is a straight chain or branched alkyl group having 1 to 6 carbon atoms; an aryl group having 6 to 8 carbon atoms, especially a phenyl group;
A cycloalkyl group having 5 to 10 carbon atoms, especially a cyclohexyl group; -GOOR' (where RI is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms [vanillin]) According to the method of the present invention, in the reaction example (2), As shown, when the raw material styrene derivative contains the group -0C1u, the group -0GQu in the generated aldehydes is
When glycosidase is contained in the enzyme isolated from the culture product of the bacterial strain, this group 0GQu may be obtained in the form converted to a hydroxyl group, which is believed to be due to its action.
好ましい。また上記アルキル基、およびアリール基には
、水酸基もしくはその配糖体または炭素数１〜４のアル
コキシ基で置換されていてもよい。preferable. Further, the above alkyl group and aryl group may be substituted with a hydroxyl group or a glycoside thereof, or an alkoxy group having 1 to 4 carbon atoms.
一方Ｒ３、Ｒ８、Ｒ１、Ｒ１およびＲ６としては、同一
もしくは異なり、水素原子；水酸基もしくはその配糖体
；炭素数ｌ〜６、特に１〜３のアルキル基：炭素数ｌ〜
６、特に１〜３のアルコキシ基；Ｒ１とＲ，またはＲ４
とＲ，は、互いに結合して一０ＣＲ１Ｏ−を形成する環
であるものが好ましい。On the other hand, R3, R8, R1, R1 and R6 are the same or different, hydrogen atom; hydroxyl group or glycoside thereof; alkyl group having 1 to 6 carbon atoms, especially 1 to 3 carbon atoms; carbon number 1 to 6;
6, especially 1 to 3 alkoxy groups; R1 and R, or R4
and R, are preferably rings that combine with each other to form -0CR1O-.
上記式［Ｉ］の化合物はＲ３−Ｒ１のうち、１〜２個が
水酸基もしくはその配糖体または炭素数１〜３のアルコ
キシ基で置換されたものが特に好ましい。In the compound of the above formula [I], one in which 1 to 2 of R3-R1 are substituted with a hydroxyl group, a glycoside thereof, or an alkoxy group having 1 to 3 carbon atoms is particularly preferable.
以下本発明において前記ジオキシゲナーゼ活性を有する
酵素を用いて分解することができる前記スチレン誘導体
［Ｉ］の具体例としては下記のものを例示することがで
きる。In the present invention, the following can be exemplified as specific examples of the styrene derivative [I] that can be decomposed using the enzyme having dioxygenase activity.
なお下記式においてＭｅはメチル基、基０Ｃ１ｕはグル
コシル基を表わすものとする。In the following formula, Me represents a methyl group, and the group 0C1u represents a glucosyl group.
イソオイゲノール
コニ７エリルアルコール
インサ７０−ル
インカビコール
アネトール
０Ｍｅ
シナビルアルコール
（Ｐ−プロペニルカテコール）
Ｐ−クマリルアルコール
Ｍａ
ＭｅＯ
アサロン
Ｍｅ
シリンゲン
ＯＭｅ
Ｍｅ
ＨＯ”
インラボテン
インアビオール
ジフェニル）エチレン
シルアビオール
ゝ’ｃ−ｃ
次に本発明方法において、前記一般式［Ｉ］で表わされ
るスチレン誘導の酸化分解に用いられる前記ジオキシゲ
ナーゼ活性を有する酵素、その酵素を産生ずるシュード
モナス属の菌株および該酵素の取得方法について説明す
る。Isoeugenol Coni 7Eryl Alcohol Insa 70-Ruin Cavicol Anethole 0Me Cinavir Alcohol (P-Propenylcatechol) P-Coumaryl Alcohol Ma MeO Asalon Me Syringen OMe Me HO” Inlabotenin Abiol Diphenyl) Ethylene Sylaviolゝ'c-c Next, in the method of the present invention, the enzyme having the dioxygenase activity used for the oxidative decomposition of styrene derivatives represented by the general formula [I], the Pseudomonas strain that produces the enzyme, and the acquisition of the enzyme Explain the method.
本発明方法において用いられるジオキシゲナーゼ活性を
有する酵素は、シュードモナス属に属する細菌より産生
され且つ下記理化学性質を有している。The enzyme having dioxygenase activity used in the method of the present invention is produced by bacteria belonging to the genus Pseudomonas and has the following physical and chemical properties.
（ａ） 作用および基質特異性；
本ジオキシゲナーゼは上記一般式［Ｉ］で表わされるス
チレン誘導体中のベンゼン環に共役しｔ；エチレン結合
に選択的に作用しアルデヒド類に転換せしめる作用を有
する。(a) Action and substrate specificity: This dioxygenase is conjugated to the benzene ring in the styrene derivative represented by the above general formula [I] and has the action of selectively acting on the ethylene bond and converting it into aldehydes.
０１１）至適ｐＨおよび安定ｐＨの範囲；至適ｐＨ６〜
１０
安定ｐＨの範囲６．５〜９．５
（ｃ）作用適温の範囲；
ＰＨ８，０で２０〜５０℃
（ｄ） 失活条件；
ｐＨ；５以下或いは１２以上
温度−６０℃以上
（ｅ） 力価の測定
後述する実施例２のａに記載
（ｆ） 分子量；
５ＤＳ−ＰＡＧＥ電気泳動によって測定された分子量は
約５２，０００であった。またゲル濾過で求められた分
子１約９４．０００であった。従って本ジオキシゲナー
ゼは分子量が約５２．０００の同一サブユニットからな
る２量体の酵素であると考えられる。011) Optimal pH and stable pH range; optimal pH 6~
10 Stable pH range 6.5-9.5 (c) Suitable temperature range for action; 20-50°C at pH 8.0 (d) Deactivation conditions; pH: 5 or less or 12 or more, temperature -60°C or more (e) (f) Molecular weight: The molecular weight measured by 5DS-PAGE electrophoresis was about 52,000. Moreover, the number of molecules 1 determined by gel filtration was about 94,000. Therefore, this dioxygenase is considered to be a dimeric enzyme consisting of identical subunits with a molecular weight of about 52,000.
伝） 精製方法；
粗酵素液をヒドロキシルアパタイト処理し、イオン交換
クロマトグラフィー（ＤＥＡＥ、 トヨバール）およ
び疎水クロマトグラフィー（プチルトヨバール）を組合
せることにより精製される。Purification method: Crude enzyme solution is treated with hydroxylapatite and purified by a combination of ion exchange chromatography (DEAE, Toyovar) and hydrophobic chromatography (Putyl Toyovar).
山）阻害、活性化および安定化；
酵素反応液に下記各阻害剤を加えて酵素活性に与える影
響を調べた。酵素反応条件はｐＨ８，０，５０°Ｃで１
０分間。阻害剤を加えない場合を１００とした相対活性
で阻害を評価した。M) Inhibition, activation, and stabilization; The following inhibitors were added to the enzyme reaction solution to examine their effects on enzyme activity. Enzyme reaction conditions are pH 8, 0, 1 at 50°C.
0 minutes. Inhibition was evaluated by relative activity, with the case where no inhibitor was added being set as 100.
ＫＣＮ ｌ
９３Ｎａ−ａｚｉｄｅ ｌ
９７Ｄｉｐｙｒｉｄｉｌｅ
ｌ ９７Ａｓｃｏｒｂａｔｅ
８ ７７本発明において、上記
ジオキシゲナーゼは、上記活性を有し且つシュードモナ
ス属に属する菌株より産生されるが、特に好ましくは、
シュードモナス属に属する細菌ＴＭＹ Ｉ Ｏ０９株が
産生する酵素である。KCN l
93Na-azide l
97 Dipyridile
l 97 Ascorbate
8 77 In the present invention, the dioxygenase has the above activity and is produced by a strain belonging to the genus Pseudomonas, and particularly preferably,
This enzyme is produced by the TMY I O09 strain of bacteria belonging to the genus Pseudomonas.
このシュードモナス属の細菌ＴＭＹ１００９株は、微工
研へ微工研菌寄第１０３６２号として寄託されている。This bacterial strain TMY1009 of the genus Pseudomonas has been deposited with the National Institute of Fine Arts and Technology under the title No. 10362.
か−る細菌ＴＭＹＩＯ０９株は、新規であり下記菌学的
性質を有している。The bacterium TMYIO09 strain is new and has the following mycological properties.
ω−亙厘
■、細胞の形と大きさ：桿菌、０．８Ｘ１．ｌｐｍ２、
運動性の有無と鞭毛の着生状態有り、極鞭毛１本
３、ダラム染色性：陰性
各種培地における生育状態
■ ＬＢ培地平板培養
円形でなめらかなコロニー形成、黄褐色■ 肉汁液体培
養
コロニーは円形、表面はスムーズ、色はうすい黄色、光
沢あり
■ 肉汁寒天斜面培養
表面はスムーズ、色はうすい黄色、光沢あり、色素拡散
は見られなかった。ω-亙厘■, Cell shape and size: Bacillus, 0.8X1. lpm2,
Presence of motility and epiphytic status of flagella, 1 polar flagellum 3, Durham staining: negative Growth status on various media ■ LB medium plate culture round, smooth colony formation, yellowish brown■ Flesh liquid culture Colonies are round, The surface was smooth, the color was pale yellow, and there was a gloss ■ The surface of the broth agar slant culture was smooth, the color was a pale yellow, and there was a gloss, and no pigment diffusion was observed.
■ 肉汁液体培養
液表面での生育は認められなかった。液全体かや−濁っ
ていた。底部に菌体が沈降していた。■ No growth was observed on the surface of the meat juice liquid culture solution. The entire liquid was slightly cloudy. Bacterial cells had settled at the bottom.
■ 肉汁ゼラチン穿刺培養 全体に小さな画境、ゼラチン液化は認められなかった。■ Meat juice gelatin puncture culture Small borders and no gelatin liquefaction were observed throughout.
■ リドマスミルク
底部に菌体沈降、ミルクの凝固、液化、ｐＨの変化なし
。■ There was no sedimentation of bacterial cells at the bottom of the lidmus milk, no coagulation or liquefaction of the milk, and no change in pH.
鉤 生理学的性質
（１）硝酸塩の還元：陰性
（２）色素の生成二カロチノイド極大吸収４７９．４５
０ （４２５）ｎｍ
（３）オキシダーゼ：陽性
（４）カタラーゼ：陽性
（５）酸素に対する態度：好気的
（６）Ｏ−Ｆテスト二酸化的条件で有機酸生成ω 脱窒
反応：陰性
（８）ＭＲテスト：陰性
（９）ＶＰテスト：陰性
（１０）インドールの生成：陰性
（１１）硫化水素の生成：陰性
（１２）デンプンの加水分解：陽性
（１３）クエン酸の利用
Ｋｏｓｅｒの培地：陰性
Ｃｈｖｉｓｔｅｎｓｅｎの培地：陽性
（１４）無機窒素源の利用
硝酸塩：陰性
アンモニウム塩：陽性
（１５）ウレアーゼ：陰性
（１６）糖類から酸及びガスの生成
酸生成
■Ｌ−アラビノース ＋
■Ｄ−キシロース ＋
■Ｄ−グルコース ＋
■Ｄ−マンノース ＋
■Ｄ−フラクトース ＋
■Ｄ−ガラクトース ＋
■麦芽糖 十
■ショ糖 十
■乳 糖 ＋
［相］トレハロース ＋
ガス生成
■Ｄ−ソルビット
■Ｄ−マンニット
＠イノジット
■グリセリン
■デンプン
（１７）その他の特徴
■グルコン酸の酸化：陰性
■アルギニンの分会：陰性
■リジンの脱炭酸反応：陰性
■オルニチンの脱炭酸反応：陰性
■プロトカテキン酸の分解二メタ開裂
（１８）その他
Ｔｗｅｅｎの分解
Ｔｖｅｅｎ ４０ ：陰性
丁ｗａｅｎ ６０ ：陰性
Ｔｗｅｅｎ ８０ ：陰性
ＤＮａｓｅ ：陽性
３−ケト乳酸の生成：陰性
鏝−ｉｓ町！
温度 ２０〜３３℃
ｐＨ５，５〜ＩＯ
Ｄｏｕｂｌｉｎｇ Ｔｉｍｅ ２７℃ＬＢ培地（ｐＨ
７，４）で２ 、５ ｈｒｓ
Ｖ 炭素化合物の資化性
カテコール
ベンゼン
安息香酸
フェルラ酸 十
ｐ−フマル酸 十
プロトカテキン酸 十
バラハイドロキシ安息香酸 十
バニリン酸 十
グルタミン酸ナトリウム ＋
本発明におけるシュードモナス属に属し且つ前記ジオキ
シゲナーゼ活性を有する酵素を産生じ得る菌株、殊に菌
株ＴＭＹ Ｉ ＯＯ９株から、目的とする前記酵素を得
るには、それ自体公知のシュードモナス属の菌株が生育
する培地中で菌体を培養し、得られた培養物から酵素を
分離すればよい。Hook Physiological properties (1) Nitrate reduction: negative (2) Pigment production Dicarotenoid maximum absorption 479.45
0 (425) nm (3) Oxidase: Positive (4) Catalase: Positive (5) Attitude towards oxygen: Aerobic (6) O-F test Organic acid production under oxidative conditions ω Denitrification reaction: Negative (8) MR test: Negative (9) VP test: Negative (10) Indole production: Negative (11) Hydrogen sulfide production: Negative (12) Starch hydrolysis: Positive (13) Utilization of citric acid Koser's medium: Negative Chvistensen Medium: Positive (14) Utilization of inorganic nitrogen sources Nitrate: Negative Ammonium salt: Positive (15) Urease: Negative (16) Generation of acid and gas from sugars Acid production ■L-arabinose + ■D-xylose + ■D- Glucose + ■D-mannose + ■D-fructose + ■D-galactose + ■maltose 10■sucrose 10■lactose + [phase] Trehalose + gas production ■D-sorbitol ■D-mannite@inozit ■glycerin ■starch (17) Other characteristics ■ Oxidation of gluconic acid: negative ■ Subdivision of arginine: negative ■ Decarboxylation of lysine: negative ■ Decarboxylation of ornithine: negative ■ Decomposition of protocatechuic acid Dimeta cleavage (18) Other Tween Decomposition Tween 40: Negative Tween 60: Negative Tween 80: Negative DNase: Positive 3-keto-lactic acid production: Negative trowel-is town! Temperature 20-33°C pH 5,5-IO Doubling Time 27°C LB medium (pH
7,4) 2,5 hrs V Assimilation of carbon compounds Catecholbenzenebenzoic acid ferulic acid Tenp-fumaric acid Tenprotocatechinic acid Tenbarahydroxybenzoic acid Tenvanillic acid Sodium tenglutamate + Belongs to the genus Pseudomonas in the present invention In order to obtain the desired enzyme from a strain capable of producing an enzyme having dioxygenase activity, particularly the strain TMY I OO9, the bacterial cells are grown in a medium in which a known strain of the Pseudomonas genus grows. The enzyme may be separated from the culture obtained by culturing.
以下、上記菌株を生育および酵素を産生するために使用
しうる培地の組成と酵素の抽出法について説明するがこ
れは単に説明のためであって、本発明はこの組成の培地
および酵素の抽出法に限定されるわけではない。Hereinafter, the composition of a medium that can be used to grow the above-mentioned bacterial strain and the enzyme extraction method will be described, but this is merely for the sake of explanation, and the present invention describes the medium with this composition and the enzyme extraction method. It is not limited to.
炭素源としては、たとえばグルコース、フラクトースな
どの炭水化物、エタノールのごとき有機化合物、コハク
酸などのごとき有機酸があげられ、これらは本発明の菌
株が利用できる１種または２種以上の炭素化合物を任意
に炭素源として利用できる。Examples of carbon sources include carbohydrates such as glucose and fructose, organic compounds such as ethanol, and organic acids such as succinic acid. can be used as a carbon source.
また、窒素源としては、特に限定されないが例えば、硫
酸アンモニウム、硫酸アンモニウムなどの無機窒素化合
物、およびペプトンなどの有機窒素源が利用できる。Further, as the nitrogen source, although not particularly limited, for example, inorganic nitrogen compounds such as ammonium sulfate and ammonium sulfate, and organic nitrogen sources such as peptone can be used.
また、無機塩類としては、各種のリン酸塩、硫酸マグネ
シウムなどが使用できる。さらに、微量の金属（鉄塩、
カルシウム塩など）を培地に含有させてもよい。Moreover, various phosphates, magnesium sulfate, etc. can be used as inorganic salts. In addition, trace amounts of metals (iron salts,
Calcium salts, etc.) may be included in the medium.
培養方法としては、振とう培養法、深部通気撹拌培養法
などの方法により行うことができる。培養温度は、例え
ば２０″〜３３℃、ｐＨは６〜ＩＯ程度の範囲が好まし
くあげられる。また培養日数は、特に限定されないがた
とえば、通常は１〜７日の範囲で行われる。The culturing method may be a shaking culture method, a deep aeration agitation culture method, or the like. The culture temperature is preferably in the range of, for example, 20'' to 33°C, and the pH is preferably in the range of about 6 to IO.Although the number of days of culture is not particularly limited, it is usually carried out, for example, in the range of 1 to 7 days.
このようにして酵素が生産蓄積された培養物中の菌体を
超音波処理により破壊して得られる無細胞抽出液を分離
し、得られた無細胞抽出液中の低分子物を除くためにリ
ン酸ナトリウム緩衝液で透析処理するこ七により酵素液
が得られる。In order to separate the cell-free extract obtained by destroying the bacterial cells in the culture in which enzymes have been produced and accumulated by ultrasonication, and to remove low-molecular substances from the obtained cell-free extract. An enzyme solution is obtained by dialysis with a sodium phosphate buffer.
かくして本発明方法においては、前記酵素液自体或いは
、その酵素液から精製された酵素を前記一般式［１］の
スチレン誘導体と好気的条件下で接触せしめ、かくして
目的とするアルデヒド類を得ることが可能となる。Thus, in the method of the present invention, the enzyme solution itself or the enzyme purified from the enzyme solution is brought into contact with the styrene derivative of the general formula [1] under aerobic conditions, thereby obtaining the target aldehyde. becomes possible.
その際の温度は２０〜５０℃、好ましくは３０〜４０℃
の範囲が有利でありｐＨは６〜ｌＯ１好ましくは６．５
〜９．５範囲が望ましい。The temperature at that time is 20-50℃, preferably 30-40℃
Advantageously, the pH ranges from 6 to 1O1, preferably 6.5.
A range of ~9.5 is desirable.
また反応は、バッチ方式または連続方式いずれで行なっ
てもよく、さらに酵素は、固定化して使用しても差支え
ない。Further, the reaction may be carried out either batchwise or continuously, and the enzyme may be used in an immobilized state.
以下実施例を掲げて本発明を詳述する。The present invention will be described in detail below with reference to Examples.
実施例（１）酵素液の調製
Ｉｆｆ（７）栄養培地（ＬＢ） ”ｃ’ＴＭｙ ｌ Ｏ
Ｏ９株を約２２時間好気的に２７℃で振とう培養するこ
とにより湿菌体約３．５ｇが得られた。菌体を集菌、洗
浄後１５ｍ１２の５０ｍＭリン酸ナトリウム緩衝液に懸
濁した後、超音波処理することにより菌体を破壊し、遠
心分離処理しｆ： （１０，ｏｏＯＸｇ、２０ｍ１ｎ）
。得られた無細胞抽出液中の低分子物を除くために２０
ｗ＋Ｍリン酸ナトリウム緩衝液（ｐＨ７，５）で透析を
行ない粗酵素液とした（タンパク濃度： ｌ ５．４
ｍｇ／ｍ（１）。Example (1) Preparation of enzyme solution If (7) Nutrient medium (LB) "c'TMy l O
About 3.5 g of wet bacterial cells were obtained by culturing O9 strain aerobically at 27° C. with shaking for about 22 hours. The bacterial cells were collected, washed, suspended in 15 ml of 50 mM sodium phosphate buffer, destroyed by ultrasonication, and centrifuged. f: (10,ooOXg, 20mln)
. 20 to remove low molecular weight substances in the obtained cell-free extract.
Dialysis was performed with w+M sodium phosphate buffer (pH 7.5) to obtain a crude enzyme solution (protein concentration: l 5.4
mg/m(1).
実施例（２）バニリンの製法
ａ、イソオイゲノール１ｐｒａｏＱを含む４５０μαの
緩衝液（ｐＨ７，５）に酵素液（１５，４ｍｇ／ｒａＱ
）を５０μａ加え２６℃で３０分間インキュベートする
。５０μａのＩＮ Ｈ（ｌを加え酸性にしたのち、５
００μａの酢酸エチルで抽出する。抽出液をＨＰＬＣで
定量分析した結果、０，１６μｍｏａのバニリンを認め
Ｉ；。Example (2) Vanillin production method a, enzyme solution (15.4 mg/raQ) in 450 μα buffer (pH 7.5) containing isoeugenol 1 praoQ
) and incubate at 26°C for 30 minutes. After adding 50 μa of IN H (l) to make it acidic,
Extract with 00 μa ethyl acetate. As a result of quantitative HPLC analysis of the extract, 0.16 μmoa of vanillin was detected.
ＨＰＬＣの測定条件
機 種； ５ｈｉｎ＋ａｄｚｕ Ｌ Ｃ−６Ａカラム
；ＬｉＣｈｒｏｓｏｒｂ ＲＰ−１８（４，６Ｘ２５
Ｏｒａｍ）
溶 出；０．０５％リン酸水（Ａ）／アセトニトリル（
Ｂ）−７０／３０−−− （５分間”） −−−（Ａ）
／ （Ｂ） −７０／３０−−−（ｉｎｃｒｅａｓｉ
ｎｇ ５％ｐｅｒ ｍ１ｎ）−（Ａ）／ （Ｂ）−０／
１００
上記におけるバニリンの保持時間は６．１分す、実施例
ａと同じ条件下に基質としてインラボテン１ｐ１１０＜
２を用いて行ッｔ；結果、Ｏ，１６＊ｍｏｄのバニリン
を認めた。また３、５−ジヒドロキシベンズアルデヒド
も認められた。HPLC measurement conditions Model; 5hin+adzu L C-6A column; LiChrosorb RP-18 (4,6X25
Oram) Elution; 0.05% phosphoric acid water (A)/acetonitrile (
B) -70/30 --- (5 minutes") --- (A)
/ (B) -70/30---(increasi
ng 5% per m1n)-(A)/(B)-0/
100 The retention time of vanillin in the above is 6.1 minutes.
As a result, O, 16*mod vanillin was observed. 3,5-dihydroxybenzaldehyde was also observed.
実施例（３）バニリンの製法
８．５− ［２’−（４″−ヒドロキシ−３１−メトキ
シフェニル）ビニル］７エルラ酸１．２μａＩＯＱヲ含
む９５０μａのリン酸ナトリウム緩衝液（ｐＨ７，５）
に酵素液（１５，４＋＋＋ｇ／ｗＱ）５０ ｔｔＱ加え
２７℃で３０分間インキュベートした。酸素１ｐｔｍｏ
（ｌが消費された時点で反応を止めた。この反応生成物
を１０００μａの酢酸エチルで抽出し、ＨＰＬＣで定量
分析した結果、ｌμ＋１０Ｑのバニリンと１μ−Ｏａの
５−ホルミルフェルラ酸を認めた。ＨＰＬＣの測定条件
は、実施例（２）のａと同条件で測定。Example (3) Preparation of vanillin 8.5-[2'-(4''-Hydroxy-31-methoxyphenyl)vinyl]7 erulic acid 1.2 μa IOQ containing 950 μa sodium phosphate buffer (pH 7.5)
Enzyme solution (15,4+++g/wQ) 50 ttQ was added to the mixture and incubated at 27°C for 30 minutes. oxygen 1ptmo
The reaction product was extracted with 1000 μa of ethyl acetate and quantitatively analyzed by HPLC. As a result, 1 μ+10Q of vanillin and 1 μ-Oa of 5-formylferulic acid were observed. The HPLC measurement conditions were the same as in Example (2) a.
ｂ、実施例（２）のａと同じ条件下に１．２−ビス−（
４−ヒドロキシ−３−メトキシフェニル）エチレンｌｐ
ｍｏＱを用いて反応し、酸素０．５４．ｕｍｏＩ２が消
費された時点で反応を止めた。この反応生成物を１００
０μｇの酢酸エチルで抽出し、ＨＰＬＣで定量分析した
結果、１．１μｍ〇αのバニリンを認めた。b, 1,2-bis-(
4-hydroxy-3-methoxyphenyl)ethylene lp
react using moQ, oxygen 0.54. The reaction was stopped when umoI2 was consumed. This reaction product is 100
As a result of extraction with 0 μg of ethyl acetate and quantitative analysis by HPLC, vanillin of 1.1 μm〇α was observed.
実施例（４） ｐ−ヒドロキシベンズアルデヒドの製造
実施例（２）のａと同じ条件下に基質としてピッイドｌ
μｌ１ＯＱを用いて行った。ＨＰＬＣで定量分析した結
果、０．１６μｍＯ＋２のｐ−ヒドロキシベンズアルデ
ヒドを認めた。Example (4) Production of p-hydroxybenzaldehyde Under the same conditions as in Example (2) a, p-hydroxybenzaldehyde was used as a substrate.
It was performed using μl1OQ. As a result of quantitative analysis by HPLC, p-hydroxybenzaldehyde of 0.16 μm O+2 was observed.
ＨＰＬＣの測定条件は、実施例（２）のａと同じ条件で
行った。The HPLC measurement conditions were the same as in Example (2) a.
実施例（５） ジヒドロキシベンズアルデヒドの製造
実施例（２）のａと同じ条件下に基質としてアストリジ
ン１μｍｏＱを用いて行った。ＨＰＬＣで定量分析した
結果、０．０２６μｒａｏＱのジヒドロキシベンズアル
デヒドを認めた。ＨＰＬＣの測定条件は、実施例（２）
のａと同じ条件で行った。Example (5) Production of dihydroxybenzaldehyde This was carried out under the same conditions as in Example (2) a using astridine 1 μmoQ as a substrate. As a result of quantitative HPLC analysis, 0.026 μraoQ of dihydroxybenzaldehyde was observed. The HPLC measurement conditions are as in Example (2).
It was carried out under the same conditions as in a.
Claims (1)
キル基、炭素数６〜１０のアリール基、炭素数５〜１５
のシクロアルキル基、基 −ＣＯＯＲ＾１（ここでＲ＾１は水素原子または炭素数
１〜１０のアルキル基）、ホルミル基または基▲数式、
化学式、表等があります▼を示す。但し、上記アルキル 基、シクロアルキル基およびアリール基は置換基を有し
ていてもよい。 Ｒ＿３、Ｒ＿３、Ｒ＿４、Ｒ＿５およびＲ＿６は、互い
に同一もしくは異なり、水素原子、水酸基もしくはその
配糖体、炭素数１〜１０の直鎖もしくは分岐状アルキル
基、または炭素数１〜１０のアルコキシ基を示すか或い
はこれら互いに隣接する２つの基は、▲数式、化学式、
表等があります▼、 ▲数式、化学式、表等があります▼または▲数式、化学
式、表等があります▼ であってもよい（ここでｎは１〜４の整数を示す）。 で表わされるスチレン誘導体を、シュードモナス属に属
し且つスチレン誘導体に対するジオキシゲナーゼ活性酵
素を産生し得る菌株が生産した酵素と好気的条件下に接
触せしめることを特徴とするアルデヒド類の製造方法。 ２、該菌株がＴＭＹ１００９株である特許請求の範囲第
１項記載の製造方法。[Claims] 1. The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼[I] In the formula, R_1 is a straight chain or branched alkyl group having 1 to 10 carbon atoms, or a straight chain or branched alkyl group having 6 to 10 carbon atoms. aryl group, carbon number 5-15
cycloalkyl group, group -COOR^1 (where R^1 is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms), formyl group or group ▲ formula,
There are chemical formulas, tables, etc. Showing ▼. However, the above alkyl group, cycloalkyl group and aryl group may have a substituent. R_3, R_3, R_4, R_5 and R_6 are the same or different and each represents a hydrogen atom, a hydroxyl group or a glycoside thereof, a linear or branched alkyl group having 1 to 10 carbon atoms, or an alkoxy group having 1 to 10 carbon atoms. The two groups shown or adjacent to each other are ▲mathematical formula, chemical formula,
There are tables, etc. ▼, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (Here, n indicates an integer from 1 to 4). A method for producing aldehydes, which comprises bringing a styrene derivative represented by the following formula into contact with an enzyme produced by a strain belonging to the genus Pseudomonas and capable of producing an enzyme with dioxygenase activity against styrene derivatives under aerobic conditions. 2. The production method according to claim 1, wherein the bacterial strain is TMY1009 strain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5511289A JP2679739B2 (en) | 1988-10-25 | 1989-03-09 | Method for producing aldehydes |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63-267285 | 1988-10-25 | ||
| JP26728588 | 1988-10-25 | ||
| JP5511289A JP2679739B2 (en) | 1988-10-25 | 1989-03-09 | Method for producing aldehydes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02200192A true JPH02200192A (en) | 1990-08-08 |
| JP2679739B2 JP2679739B2 (en) | 1997-11-19 |
Family
ID=26395965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5511289A Expired - Fee Related JP2679739B2 (en) | 1988-10-25 | 1989-03-09 | Method for producing aldehydes |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2679739B2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0583687A2 (en) * | 1992-08-17 | 1994-02-23 | Haarmann & Reimer Gmbh | Method for the preparation of substituted methoxyphenols and microorganisms therefore |
| WO1996022381A1 (en) * | 1995-01-19 | 1996-07-25 | V. Mane Fils | Biochemical process for preparing aromatic substances |
| WO1997035999A3 (en) * | 1996-03-23 | 1998-01-08 | Inst Of Food Research | Production of vanillin |
| DE102010039833A1 (en) * | 2010-08-26 | 2012-03-01 | Symrise Ag | Whole-cell biotransformation of fatty acids to the fatty aldehydes truncated by one carbon atom |
| US9131648B2 (en) | 2006-07-07 | 2015-09-15 | Washington State University | Genes encoding chavicol/eugenol synthase from the creosote bush Larrea tridentata |
| CN113767173A (en) * | 2019-04-29 | 2021-12-07 | 科纳根公司 | Biosynthesis of vanillin from isoeugenol |
-
1989
- 1989-03-09 JP JP5511289A patent/JP2679739B2/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0583687A2 (en) * | 1992-08-17 | 1994-02-23 | Haarmann & Reimer Gmbh | Method for the preparation of substituted methoxyphenols and microorganisms therefore |
| EP0583687A3 (en) * | 1992-08-17 | 1994-06-01 | Haarmann & Reimer Gmbh | Method for the preparation of substituted methoxyphenols and microorganisms therefore |
| WO1996022381A1 (en) * | 1995-01-19 | 1996-07-25 | V. Mane Fils | Biochemical process for preparing aromatic substances |
| FR2729661A1 (en) * | 1995-01-19 | 1996-07-26 | Mane Fils V | PROCESS FOR THE PREPARATION OF AROMATIC SUBSTANCES BY BIOCHEMICAL |
| WO1997035999A3 (en) * | 1996-03-23 | 1998-01-08 | Inst Of Food Research | Production of vanillin |
| US6323011B1 (en) | 1996-03-23 | 2001-11-27 | Institute Of Food Research | Production of vanillin |
| US6664088B2 (en) | 1996-03-23 | 2003-12-16 | Plant Bioscience Limited | Production of vanillin |
| US9131648B2 (en) | 2006-07-07 | 2015-09-15 | Washington State University | Genes encoding chavicol/eugenol synthase from the creosote bush Larrea tridentata |
| DE102010039833A1 (en) * | 2010-08-26 | 2012-03-01 | Symrise Ag | Whole-cell biotransformation of fatty acids to the fatty aldehydes truncated by one carbon atom |
| US10017790B2 (en) | 2010-08-26 | 2018-07-10 | Symrise Ag | Whole-cell biotransformation of fatty acids to obtain fatty aldehydes shortened by one carbon atom |
| CN113767173A (en) * | 2019-04-29 | 2021-12-07 | 科纳根公司 | Biosynthesis of vanillin from isoeugenol |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2679739B2 (en) | 1997-11-19 |
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