JPH0358782A - Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereof - Google Patents
Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereofInfo
- Publication number
- JPH0358782A JPH0358782A JP1193835A JP19383589A JPH0358782A JP H0358782 A JPH0358782 A JP H0358782A JP 1193835 A JP1193835 A JP 1193835A JP 19383589 A JP19383589 A JP 19383589A JP H0358782 A JPH0358782 A JP H0358782A
- Authority
- JP
- Japan
- Prior art keywords
- butanol
- mutant
- clostridium
- clostridium bacterium
- bacterium
- 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.)
- Pending
Links
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 title claims abstract description 91
- 241000193403 Clostridium Species 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 9
- WCASXYBKJHWFMY-NSCUHMNNSA-N 2-Buten-1-ol Chemical compound C\C=C\CO WCASXYBKJHWFMY-NSCUHMNNSA-N 0.000 claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims description 17
- 230000004151 fermentation Effects 0.000 claims description 17
- 239000000543 intermediate Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 8
- XXROGKLTLUQVRX-UHFFFAOYSA-N allyl alcohol Chemical compound OCC=C XXROGKLTLUQVRX-UHFFFAOYSA-N 0.000 claims description 6
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 230000007886 mutagenicity Effects 0.000 claims 1
- 231100000299 mutagenicity Toxicity 0.000 claims 1
- 230000035772 mutation Effects 0.000 abstract description 6
- 239000003471 mutagenic agent Substances 0.000 abstract description 3
- 239000012299 nitrogen atmosphere Substances 0.000 abstract description 2
- 241000193469 Clostridium pasteurianum Species 0.000 abstract 1
- 241001483585 Clostridium pasteurianum DSM 525 = ATCC 6013 Species 0.000 abstract 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 231100000707 mutagenic chemical Toxicity 0.000 abstract 1
- 230000003505 mutagenic effect Effects 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 12
- 239000002609 medium Substances 0.000 description 8
- 239000002994 raw material Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 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 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 229930091371 Fructose Natural products 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
- 239000005715 Fructose Substances 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
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 2
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- -1 combinations Substances 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000000126 substance Substances 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
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 1
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193401 Clostridium acetobutylicum Species 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
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-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
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 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
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 229940023020 acriflavine Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 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
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 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
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- DLRVVLDZNNYCBX-ZZFZYMBESA-N beta-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000025938 carbohydrate utilization Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- VAYGXNSJCAHWJZ-UHFFFAOYSA-N dimethyl sulfate Chemical compound COS(=O)(=O)OC VAYGXNSJCAHWJZ-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- 229940099607 manganese chloride Drugs 0.000 description 1
- 235000002867 manganese chloride Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
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- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、クロストリジウム属細菌変異株の取得方法,
該変異株および該変異株を用いるブタノールの製造法に
関する。本発明の変異株はブタノールを著量に生産する
能力を有する突然変異株であり、本発明の製造法により
生産されるブタノールは溶剤,合或原料.燃料等として
溶剤工業,有機化学工業等において利用される。[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for obtaining a mutant strain of a Clostridium bacterium,
The present invention relates to the mutant strain and a method for producing butanol using the mutant strain. The mutant strain of the present invention is a mutant strain that has the ability to produce a significant amount of butanol, and the butanol produced by the production method of the present invention can be obtained from solvents, combinations, or raw materials. It is used as fuel in the solvent industry, organic chemical industry, etc.
〔従来の技術および発明が解決しようとする課題〕ブタ
ノールは、溶剤,合戒原料,燃料等として有用なもので
あり、石油化学により生産することが可能であるが、石
油資源の減少等の問題から、1)[を原料とする発酵法
による安価な製造法が望まれている。従来、発酵法によ
るブタノールの製造にはクロストリジウム・アセトブチ
リクム等を王に用いていたが、ブタノールの生産性を向
上すべく種々の変異株分離が試みられている。ブタノ一
ル生産変異株によるブタノールの発酵生産法としては、
ブタノール耐性変異株を用いた特開昭58165786
号公報に記載の方法、BiotechnolLett.
+ 9, 175(1987),同7, 711(1.
985)に記載の方法、Appl. Environ.
Microbiol., 53, 2854(1987
)トj]斤Q−. 1.238(1985).同塁−j
), 477(1985)に記載の方法等が知られてい
る。また、ク[′:Iストリジウム・バストリアヌムに
よるブタノールの発酵生産法としては、特開昭63
1.57989弓公報やRiotechnolLett
.,旦, p889(1986)等に記載の方法が知ら
れている。しかしながら、いずれの力法もブタノールの
収率が不]一分であるとい・う問題があった。[Prior art and problems to be solved by the invention] Butanol is useful as a solvent, raw material, fuel, etc., and can be produced by petrochemistry, but there are problems such as a decrease in petroleum resources. Therefore, there is a desire for an inexpensive production method using a fermentation method using 1) [ as a raw material. Conventionally, Clostridium acetobutylicum and the like have been used to produce butanol by fermentation, but attempts have been made to isolate various mutant strains in order to improve the productivity of butanol. The fermentation production method for butanol using a butanol-producing mutant strain is as follows:
JP-A-58165786 using a butanol-resistant mutant strain
The method described in Biotechnol Lett.
+ 9, 175 (1987), 7, 711 (1.
985), the method described in Appl. Environ.
Microbiol. , 53, 2854 (1987
) Toj] Catty Q-. 1.238 (1985). Same base-j
), 477 (1985) and the like are known. In addition, as a fermentation production method of butanol using Ku[':I Stridium bastrianum, JP-A-63
1.57989 Bow Bulletin and Riotechnol Lett
.. , Dan, p. 889 (1986) and the like are known. However, both force methods have the problem that the yield of butanol is insignificant.
〔課題を解決するための手段]
そこで、本発明者らはブタノールを発酵法6こより著搦
に生産ずることを目的として、これに適ずる菌株の検索
を行なったところ、クロス1・リジウl、属細菌を変異
させて得られる変異株が適することを見出し、本発明を
完威した。[Means for Solving the Problems] Therefore, the present inventors conducted a search for suitable bacterial strains for the purpose of producing butanol effectively using fermentation methods, and found that cross 1. We have found that mutant strains obtained by mutating genus bacteria are suitable, and have completed the present invention.
すなわち、本発明はクロスI・リジウム属細菌を突然変
異処理後、ブタノール発酵中間体のアナログ耐性により
クロスI・リジウl、属細菌の変異株を行ることを特徴
とするクロス1〜リジウム属細菌の変異株の取得力法、
ブタノール発酵中間体のアナログ耐111,をイjし、
ブクノール生産能を有ずるクロスI・リソウl、属突然
変異株および該変異株を培養してブタノールを産1.1
せしめ、これを採取するこどを特徴とするブタノールの
製造法を提供するものである。That is, the present invention is characterized in that after mutation treatment of Cross I.Rhydium bacteria, a mutant strain of Cross I.Rhydium bacteria is generated by analog resistance of a butanol fermentation intermediate. acquisition power method of mutant strains,
Analog resistance of butanol fermentation intermediate 111,
Cross I. risou l, genus mutant strain having the ability to produce bukunol, and culturing the mutant strain to produce butanol 1.1
The present invention provides a method for producing butanol, which involves collecting the same.
本発明のクロス1・リジウム属細菌変異株は、クロスト
リジウム属に属する細菌を親株として、これを変異処理
し、ブタノール発酵中間体のアナログに対する剛性によ
り分離するこどにより得られる。The Clostridium genus bacterial mutant strain of the present invention is obtained by mutating a parent strain of Clostridium genus bacteria and separating it based on the stiffness of the butanol fermentation intermediate relative to the analogue.
ここで、クロスI・リジウム属細菌としては、クロス1
−リソウム・バストリアヌム(Clostridium
pasteur.ianum)DSM 525株が挙げ
られる。また、親株の変異処理は既知の方沃によって行
なえばよく、たどえば、紫外線照則,X線照射,γ線な
どの放躬線照射,突然変異誘起剤による処理などの方法
を適用することができる。なお、突然変異誘起剤としで
は、エチルメタンスルホ不−1−N−メチル−N”−ニ
トロ−N−二I・ロソグアニジン ジメチルサルフェー
ト 2−アミノブリン,アクリフラビン,アクリジンオ
レンジ,ヒドラジン 4ニトロキノリンーN−オキシト
,塩化マンガンなどがある。Here, as Cross I and Rhydium bacteria, Cross 1
- Clostridium
pasteur. ianum) DSM 525 strain. In addition, mutation treatment of the parent strain can be carried out using known methods, such as ultraviolet irradiation, X-ray irradiation, radioactive irradiation such as gamma rays, treatment with mutagenic agents, etc. I can do it. In addition, as a mutagenic agent, ethylmethanesulfoun-1-N-methyl-N''-nitro-N-2I-rosoguanidine dimethyl sulfate, 2-aminobuline, acriflavine, acridine orange, hydrazine 4-nitroquinoline-N-oxyto , manganese chloride, etc.
次に、ブタノール発酵中間体のアナログとは、例えばク
ロトニルアルコール、アリルアルコールアクリル酸をい
い、好ましくはクロトニルアルコールである。また、ブ
タノール発酵中間体のアナログ耐性とは、該アナログを
含む培地で生育団害を受けることなくブタノールを生産
できる性質をいう。Next, analogs of butanol fermentation intermediates refer to, for example, crotonyl alcohol and allyl alcohol acrylic acid, preferably crotonyl alcohol. Furthermore, the analog resistance of a butanol fermentation intermediate refers to the property that it is possible to produce butanol in a medium containing the analog without suffering growth damage.
さらに、変異株の分離方法について具体的6こ説明する
。まず、クロストリジウム・パスl・リアヌムDSM5
25株を第1表に示すCM3培地で37゜Cにて窒素雰
囲気下で嫌気的に12時間培養する。培養終S後、遠心
分離を行なって集菌し、50mM1・リス−マレー1−
(Tris−Malate)緩衝液(p.+1 5.
5にて2回洗菌する。次いで、N−メチルーN)
ニ1−口 N−二1・ロソグアニシン0.2■/ mp
を添力11Lた同緩衝液にこの菌休を懸濁した後、37
゛Cで15分間振とう処理することによって変異処理を
行なう。変異処理終了後、集菌して1・リスマl/−1
−緩衝液にて3回洗菌する。Furthermore, six specific methods for isolating mutant strains will be explained. First, Clostridium passi Lianum DSM5
The 25 strains were cultured anaerobically in the CM3 medium shown in Table 1 at 37°C under a nitrogen atmosphere for 12 hours. After the completion of culture, centrifugation was performed to collect the bacteria, and 50mM 1.
(Tris-Malate) buffer (p.+1 5.
Wash twice at step 5. Then, N-methyl-N) N-21 Rosoguanicine 0.2 / mp
After suspending this bacterial suspension in the same buffer solution containing 11 L of
Mutation treatment is carried out by shaking at °C for 15 minutes. After the mutation process is completed, collect the bacteria and obtain 1・lisma l/−1
- Wash 3 times with buffer.
数L表
グルコース
ポリペブトン
酵母エキス
(Ni+−)2SO4
K 2 I+ 11 0.
MgcN z.6H20
門ops”
CaCε2
FeSO,
レリスリンナl・リウム (酸化還元色素)t.−cy
s++・Ic℃(還元剤)
10g
5g
2g
1,3 g
5g
1g
5g
150mg
1.3mg
2mg
0.5g
合計1.. O p.(pl+ 7. 0 )*モルホ
リノプロパンスルホン酸
次に、変異処理して得られる菌体を第1表に示したCM
3培地にクロトニルアルコール4g/j!を添加した培
地に植菌し、37゜Cで3日間集積培養を行なう。培養
終了後、集菌してクロトニルアルコール4g/Qを添加
したCM3培地の寒天平板上に塗布し、37゜Cで3日
間培養したのち、寒天平板上のクロ1・ニルアルコール
耐性株のコロニーを分離する。このようにして、本発明
のブタノール発酵中間体のアナログに耐性を有し、ブタ
ノール生産能を有するクロストリジウム属細菌が得られ
る。本発明者らは、この細菌をクロス1・リジウム・バ
ストリアヌム(Clostridium 7CA101
株と命名した。本菌は、工業技術院微生物工業技術研究
所にFERM P−10817として寄託されている。Number L Table Glucose Polypebutone Yeast Extract (Ni+-)2SO4 K 2 I+ 11 0. MgcNz. 6H20 Ops” CaCε2 FeSO, Relisulinalium (redox pigment) t.-cy
s++・Ic°C (reducing agent) 10g 5g 2g 1.3 g 5g 1g 5g 150mg 1.3mg 2mg 0.5g Total 1. .. Op. (pl+ 7.0) * Morpholinopropanesulfonic acid Next, the bacterial cells obtained by mutation treatment are CM shown in Table 1.
4g/j of crotonyl alcohol in 3 medium! The cells were inoculated into a medium supplemented with the following, and enrichment culture was carried out at 37°C for 3 days. After culturing, the bacteria were collected and spread on an agar plate of CM3 medium supplemented with 4 g/Q of crotonyl alcohol, and cultured at 37°C for 3 days. Separate. In this way, Clostridium bacteria that are resistant to the analog of the butanol fermentation intermediate of the present invention and capable of producing butanol can be obtained. The present inventors identified this bacterium as Clostridium 7CA101.
It was named a stock. This bacterium has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10817.
本菌の菌学的性質を以下に示す。The mycological properties of this bacterium are shown below.
(1)0.8X3〜10μmの桿菌
(2)周鞭毛で運動性あり
(3)内生胞子あり
(4)ダラム染色陽性
(5)生育温度30〜4
(6)糖の利用性
アミクタリン − マルトースアラビノー
ス 士 マンニトール七■ヒ才−ス
− マンノースフラクトース +
メレシトースカラクトース 士 メリヒ
才一スクリコーケン − ラフィノースイ
ヌリン − ラムノースラクトース
− リネース」二記のようにクロストリ
ジウム・バストリアヌム(;A 101株(FERM
P−1.0817)の菌学的性質は、クロトニルアルコ
ールの他にアリルアルコール,アクリル酸に耐性を示す
こと、およびブタノールを著量に発酵生産する能力を有
すること以外は親株であるクロストリジウム・バストリ
アヌムDSM 525株と全く同一の菌学的性質を示す
。(1) Bacillus of 0.8 x 3-10 μm (2) Motile with periflagellate (3) Endospores present (4) Positive Durham staining (5) Growth temperature 30-4 (6) Sugar utilization amictalline - maltose Arabinose mannitol
− Mannose fructose +
Melecitose Charactose - Raffinose Inulin - Rhamnose Lactose
- Clostridium bastrianum (; A 101 strain (FERM
The mycological properties of Clostridium P-1.0817) are similar to the parent strain, Clostridium P-1.0817, except that it is resistant to allyl alcohol and acrylic acid in addition to crotonyl alcohol, and has the ability to ferment and produce a significant amount of butanol. It shows completely the same mycological properties as DSM 525 strain.
本発明では、ブタノール発酵中間体のアナログに耐性を
有し、ブタノール生産能を有するクロストリジウム属細
菌突然変異株、例えばクロストリサリンン
ソルヒトール
スターチ
ンユクロース
トレハυ−ヌ
キンロース
O′C
ジウム・バストリアヌムCA 101株(FERM P
−10817)を培養してブタノールを産生せしめ、こ
れを採取することによってブタノールを製造する。In the present invention, mutant bacterial strains of the genus Clostridium that are resistant to analogues of butanol fermentation intermediates and have the ability to produce butanol, such as clostrisalin sorchitol startin uclostreha υ-nuquinulose O'C Dium bastrianum CA 101 shares (FERM P
-10817) to produce butanol, which is collected to produce butanol.
培養に用いる培地は、炭素源,窒素源,無機イオン.有
機栄養源などを含有する通常の培地でよく、炭素源とし
てはグルコース,シュクロースフラクトース,マルトー
ス.マンノース,メリビオース,ソルビトール,マンニ
トール,メレジトース,ラフィノース トレハロース,
デンプン等のII!類が使用される。The culture medium used includes a carbon source, nitrogen source, and inorganic ions. A conventional medium containing organic nutrients may be used, and carbon sources include glucose, sucrose, fructose, and maltose. Mannose, melibiose, sorbitol, mannitol, melezitose, raffinose trehalose,
II of starch etc. class is used.
培養は、温度20〜40゜C,pH5〜8、好ましくは
6〜7に調節し、嫌気的条件下で静置または弱く攪拌し
て行なう。また、本菌の植菌量は0.1〜10%、好ま
しくは1〜5%である。培養期間は、ブタノールが十分
量生産されるまで行なえばよく、通常は1〜10日間、
好ましくは2〜5日間である。培養液中に生威したブタ
ノール等のアルコール類および有機酸類は、培養液をリ
ン酸で酸性にした後、ガスクロマトグラフィーにより確
認定量する。The culture is carried out under anaerobic conditions at a temperature of 20 to 40°C, a pH of 5 to 8, preferably 6 to 7, and left standing or with gentle stirring. Moreover, the amount of inoculation of this bacterium is 0.1 to 10%, preferably 1 to 5%. The cultivation period may be continued until a sufficient amount of butanol is produced, usually for 1 to 10 days.
Preferably it is 2 to 5 days. Alcohols such as butanol and organic acids that have survived in the culture solution are confirmed and quantified by gas chromatography after making the culture solution acidic with phosphoric acid.
次に、本発明を実施例により説明する。 Next, the present invention will be explained by examples.
実施例
前記第1表に示したCM3培地にクロストリジウム・バ
ストリアヌムCA 101株(FERM P−1081
7)を接種し、37゜Cで18時間嫌気的に培養を行な
った。この培養液を CM3培地5成に2%の割合で植
菌し、10mRネジロ試験管中で37゜Cにて2日間嫌
気的に静置培養した。Example Clostridium bastrianum strain CA 101 (FERM P-1081) was added to the CM3 medium shown in Table 1 above.
7) and cultured anaerobically at 37°C for 18 hours. This culture solution was inoculated into CM3 medium at a ratio of 2% and statically cultured anaerobically for 2 days at 37°C in a 10 mR screw test tube.
得られた培養液から菌体を遠心分離して除去した後、ガ
スクロマトグラフィーによりブタノールの生威量を測定
したところ、2.1g/f(対糖収率21%)であり、
その他第2表に示す量の生戒物が得られた。After removing the bacterial cells from the obtained culture solution by centrifugation, the yield of butanol was measured by gas chromatography and found to be 2.1 g/f (yield based on sugar: 21%).
Other raw materials were obtained in the amounts shown in Table 2.
比較例
実施例において、クロストリジウム・バストリアヌムC
A 101株(FERM P−10817)の代わりに
クロストリジウム・バストリアヌムDSM 525株を
用いたこと以外は実施例と同様の操作を行なった。フタ
ノールの生或量は0.6g/EC対糖収率6%)であり
、その他第2表に示す量の生威物が得られた。Comparative Example In the example, Clostridium bastrianum C
The same operation as in Example was performed except that Clostridium bastrianum DSM 525 strain was used instead of A 101 strain (FERM P-10817). The raw amount of phthanol was 0.6 g/EC (sugar yield: 6%), and other raw materials were obtained in the amounts shown in Table 2.
鏡!L表
生威量(g/L)
エタノール フクノー}1実施例
0.1 0.5 2.1.. 0.2 0
.10.2 1.2 0.6
0.7 0.2〔発明の効果〕
本発明のクIコス1−リジウ1、属細菌変異株は、ブタ
ノール発酵中間体のアナログに剛性を有し、ブタノール
を安価かつ著量に生産する能力を有ずる。mirror! L surface yield (g/L) Ethanol Fukuno}1 example
0.1 0.5 2.1. .. 0.2 0
.. 10.2 1.2 0.6
0.7 0.2 [Effects of the Invention] The bacterial mutant strain of the genus Cuicos 1-Rhijiu 1 of the present invention has rigidity in analogs of butanol fermentation intermediates, and has the ability to produce butanol at low cost and in significant quantities. have.
本発明により得られるブタノールは、有機化学工業,溶
剤工業等の分野で有用なものである。The butanol obtained by the present invention is useful in fields such as organic chemical industry and solvent industry.
Claims (4)
ノール発酵中間体のアナログ耐性によりクロストリジウ
ム属細菌の変異株を得ることを特徴とするクロストリジ
ウム属細菌変異株の取得方法。(1) A method for obtaining a Clostridium bacterium mutant, which is characterized by obtaining a Clostridium bacterium mutant strain by mutagenicity of Clostridium bacterium and analog resistance of a butanol fermentation intermediate.
タノール生産能を有するクロストリジウム属細菌突然変
異株。(2) A Clostridium bacterial mutant strain that has analog resistance to butanol fermentation intermediates and has the ability to produce butanol.
ルコール、アリルアルコールまたはアクリル酸である請
求項2記載のクロストリジウム属細菌突然変異株。(3) The mutant strain of Clostridium bacteria according to claim 2, wherein the analog of the butanol fermentation intermediate is crotonyl alcohol, allyl alcohol, or acrylic acid.
ブタノール生産能を有するクロストリジウム属細菌突然
変異株を培養してブタノールを産生せしめ、これを採取
することを特徴とするブタノールの製造法。(4) resistant to analogs of butanol fermentation intermediates;
1. A method for producing butanol, which comprises culturing a mutant strain of a Clostridium bacterium capable of producing butanol to produce butanol, and collecting the same.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1193835A JPH0358782A (en) | 1989-07-28 | 1989-07-28 | Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1193835A JPH0358782A (en) | 1989-07-28 | 1989-07-28 | Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0358782A true JPH0358782A (en) | 1991-03-13 |
Family
ID=16314530
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP1193835A Pending JPH0358782A (en) | 1989-07-28 | 1989-07-28 | Obtaining of clostridium bacterium mutant, same mutant and production of butanol using thereof |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0358782A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9260731B2 (en) | 2011-08-01 | 2016-02-16 | Reliance Industries Limited | Butanol fermentation using acid pretreated biomass |
-
1989
- 1989-07-28 JP JP1193835A patent/JPH0358782A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9260731B2 (en) | 2011-08-01 | 2016-02-16 | Reliance Industries Limited | Butanol fermentation using acid pretreated biomass |
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