JPH0227992A - Production of polyester copolymer - Google Patents
Production of polyester copolymerInfo
- Publication number
- JPH0227992A JPH0227992A JP63178448A JP17844888A JPH0227992A JP H0227992 A JPH0227992 A JP H0227992A JP 63178448 A JP63178448 A JP 63178448A JP 17844888 A JP17844888 A JP 17844888A JP H0227992 A JPH0227992 A JP H0227992A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- culture
- copolymer
- hydroxybutylate
- component
- 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
- 229920000728 polyester Polymers 0.000 title claims abstract description 7
- 238000004519 manufacturing process Methods 0.000 title claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 20
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 10
- 241000588986 Alcaligenes Species 0.000 claims abstract description 9
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000011574 phosphorus Substances 0.000 claims abstract description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 8
- 229920000070 poly-3-hydroxybutyrate Polymers 0.000 claims description 8
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical group CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 claims description 3
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical group OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 claims description 3
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 3
- 239000003960 organic solvent Substances 0.000 abstract description 2
- 239000000306 component Substances 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 23
- 229920001577 copolymer Polymers 0.000 description 22
- 230000001580 bacterial effect Effects 0.000 description 16
- 244000005700 microbiome Species 0.000 description 15
- 229910052799 carbon Inorganic materials 0.000 description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 241000252867 Cupriavidus metallidurans Species 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002844 melting Methods 0.000 description 4
- 230000008018 melting Effects 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 3
- 150000001342 alkaline earth metals Chemical class 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000588813 Alcaligenes faecalis Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 208000010392 Bone Fractures Diseases 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 239000005715 Fructose Substances 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
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 229940005347 alcaligenes faecalis Drugs 0.000 description 2
- 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 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000007334 copolymerization reaction Methods 0.000 description 2
- 238000012136 culture method Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012567 medical material Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 2
- HASWNCHYBAFUSB-PMACEKPBSA-N (3s,6s)-3,6-bis[(5-hydroxy-1h-indol-3-yl)methyl]piperazine-2,5-dione Chemical compound C1=C(O)C=C2C(C[C@H]3C(=O)N[C@H](C(N3)=O)CC3=CNC4=CC=C(C=C43)O)=CNC2=C1 HASWNCHYBAFUSB-PMACEKPBSA-N 0.000 description 1
- REKYPYSUBKSCAT-UHFFFAOYSA-N 3-hydroxypentanoic acid Chemical group CCC(O)CC(O)=O REKYPYSUBKSCAT-UHFFFAOYSA-N 0.000 description 1
- 241000193033 Azohydromonas lata Species 0.000 description 1
- 229920001634 Copolyester Polymers 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-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 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
- 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
- 101100424709 Danio rerio tbxta gene Proteins 0.000 description 1
- 241001299659 Halomonas aquamarina Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000003905 agrochemical Substances 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
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003698 anagen phase Effects 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
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001844 chromium Chemical class 0.000 description 1
- 239000011651 chromium Substances 0.000 description 1
- 150000001868 cobalt Chemical class 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 150000001879 copper Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 108010087681 cyclo(5-hydroxytryptophyl-5-hydroxytryptophyl) Proteins 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- -1 for example Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 229960004275 glycolic acid Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 150000002497 iodine compounds Chemical class 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 150000002696 manganese Chemical class 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 150000002751 molybdenum Chemical class 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 150000002815 nickel Chemical class 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polyesters Or Polycarbonates (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、3−ヒドロキシブチレート単位(以下3HB
成分と記す)と4−ヒドロキシブチレート単位(以下4
HB成分と記す)を含有する共重合体の製造法に関し、
更に詳しくは、ポリエステルを蓄積できる微生物を用い
て製造される3HB成分、48B成分からなる新規の共
重合ポリエステルの製造法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a 3-hydroxybutyrate unit (hereinafter referred to as 3HB
component) and 4-hydroxybutyrate unit (hereinafter referred to as 4
Regarding the method for producing a copolymer containing HB component),
More specifically, the present invention relates to a method for producing a novel copolyester comprising a 3HB component and a 48B component, which is produced using a microorganism capable of accumulating polyester.
ポリ−3−ヒドロキシブチレート(PHB)は、エネル
ギー貯蔵物質として数多(の微生物の菌体内に蓄積され
、優れた生物分解性と生体適合性を示す熱可塑性高分子
であることから、環境を保全する“クリーン”プラスチ
ックとして注目され、手術糸や骨折固定用材などの医用
材料および医薬や農薬を徐々に放出する徐放性システム
などの多方面への応用が長年にわたり期待されてきた。Poly-3-hydroxybutyrate (PHB) is a thermoplastic polymer that accumulates in the bodies of many microorganisms as an energy storage substance and exhibits excellent biodegradability and biocompatibility, making it environmentally friendly. It has been attracting attention as a "clean" plastic for conservation, and has long been expected to be used in a variety of fields, including medical materials such as surgical threads and fracture fixation materials, and sustained-release systems that gradually release pharmaceuticals and agricultural chemicals.
特に近年、合成プラスチックが環境汚染や資源循環の観
点から深刻な社会問題となるに至り、PHBは石油に依
存しないバイオポリマーとして注目されている。Particularly in recent years, synthetic plastics have become a serious social problem from the viewpoint of environmental pollution and resource recycling, and PHB is attracting attention as a biopolymer that does not depend on petroleum.
しかしながら、PHBは耐衝撃性に劣るとゆう物性上の
問題とともに、生産コストが高いことから工業的生産が
見送られてきた。However, industrial production of PHB has been postponed due to physical property problems such as poor impact resistance and high production costs.
近時、3HB成分および3−ヒドロキシバリレ−ト単位
(以下3HV成分と記す)を含有する共重合体およびそ
の製造法について、研究、開発がなされ、たとえば、特
開昭57−150393号公報および特開昭59−22
0192号公報にそれぞれ記載されている。Recently, research and development have been conducted on copolymers containing a 3HB component and 3-hydroxyvalerate units (hereinafter referred to as 3HV component) and methods for producing the same. Japanese Patent Publication No. 59-22
Each of these is described in Publication No. 0192.
しかしながら、共重合体の3HV成分がOから33モル
%まで増大するとこの増大に伴って融解温度(Tm)が
180℃から85℃まで急激に低下することが知られて
おり(T、L、Bluhmet al、Macrom
olecules。However, it is known that when the 3HV component of the copolymer increases from O to 33 mol%, the melting temperature (Tm) decreases rapidly from 180°C to 85°C (T, L, Bluhmet al, Macrom
olecules.
■、2871 (1986))そのため、3HV成分
含有率の高い共重合体は耐熱性に劣っていた。2, 2871 (1986)) Therefore, copolymers with a high 3HV component content were inferior in heat resistance.
一方、本発明者は、3HB成分および4HB成分を含有
する共重合体およびその製造法について研究、開発を行
ない、先に出願したく特願昭62−204538)。か
かる共重合体は4HB成分の共重合成分含有率が高い場
合でも、高い融点を有することから工業的な価値は高い
。しかしながら、この方法では炭素源として高価な試薬
を使う必要があったため、工業的に容易に入手できる汎
用の炭素源を見い出すことに対する極めて高い要請があ
った。On the other hand, the present inventor has conducted research and development on a copolymer containing a 3HB component and a 4HB component and a method for producing the same, and has previously filed a patent application (Japanese Patent Application No. 62-204538). Such a copolymer has a high melting point even when the copolymerization component content of the 4HB component is high, and therefore has high industrial value. However, since this method requires the use of expensive reagents as a carbon source, there has been an extremely high demand for finding a general-purpose carbon source that can be easily obtained industrially.
本発明者は、以上の点を鑑み、3HB成分および4HB
成分からなる共重合体を工業的に有利にかつ容易に製造
すべく鋭意検討した結果、後段の窒素もしくはリンを制
限する培養においてT−ブチロラクトンの存在下でPH
B生産能を有する微生物を培養するとこの菌体中に所望
の共重合体が生成・蓄積されるとの新知見を得て、本発
明に到達した。In view of the above points, the present inventor has determined that the 3HB component and the 4HB component
As a result of intensive studies to industrially advantageously and easily produce a copolymer consisting of these components, we found that in the presence of T-butyrolactone in the subsequent nitrogen or phosphorus limiting culture,
The present invention was achieved based on the new finding that when a microorganism capable of producing B is cultured, a desired copolymer is produced and accumulated in the microorganism.
すなわち本発明は、ポリ−3−ヒドロキシブチレート生
産能を有するアルカリ土類金属菌を前段で菌体を増殖さ
せ、後段で該菌体を窒素あるいはリンの制限下で培養し
て該菌体内にポリ−3−ヒドロキシブチレートを生成・
蓄積させるに際して、後段の培養をT−ブチロラクトン
の存在下で行なうことを特徴とする3−ヒドロキシブチ
レート単位および4−ヒドロキシブチレート単位からな
るポリエステル共重合体の製造方法に存する。That is, in the present invention, cells of alkaline earth metal bacteria capable of producing poly-3-hydroxybutyrate are grown in the first step, and in the second step, the cells are cultured under limited nitrogen or phosphorus to inject into the cells. Produces poly-3-hydroxybutyrate.
The present invention provides a method for producing a polyester copolymer comprising 3-hydroxybutyrate units and 4-hydroxybutyrate units, characterized in that during accumulation, the latter stage of cultivation is carried out in the presence of T-butyrolactone.
以下、本発明の詳細な説明する。The present invention will be explained in detail below.
本発明において、共重合体に含有される3HB成分およ
び4HB成分はそれぞれ次式であられされる。In the present invention, the 3HB component and 4HB component contained in the copolymer are expressed by the following formulas.
3HB成分; −0CH(CH3)CH,C−4HB成
分; OCH! CHt CHt C−本発明で使
用される微生物は、PHB生産能を有する微生物であれ
ば特に制限はないが、実用上は、たとえば、アルカリゲ
ネス フェカリス(AIcaligenes fae
calts)、アルカリゲネス ルーランディイ (A
lcaligenes ruhlandti)、
アルカリゲネスラタス(Alcaligenes I
atuS)。3HB component; -0CH(CH3)CH, C-4HB component; OCH! CHt CHt C - The microorganism used in the present invention is not particularly limited as long as it has the ability to produce PHB, but in practice, for example, AIcaligenes fae
calts), Alcaligenes roulandii (A
lcaligenes ruhlandti),
Alcaligenes latus (Alcaligenes I)
atuS).
アルカリゲネス アクアマリヌス(Alcaligen
es aquamarinus)およびアルカリゲネ
ス ユウトロフス(Alcaligenes eut
rophs)等のアルカリ土類金属などがある。Alcaligenes aquamarinus (Alcaligen)
es aquamarinus) and Alcaligenes eutophus (Alcaligenes eut
rophs) and other alkaline earth metals.
これらの菌種に属する菌株の代表例として、アルカリゲ
ネス フェカリスATCC8750,アルカリゲネス
ルーランディイATCC15749、アルカリゲネス
ラタスATCC29712゜アルカリゲネス アクアマ
リヌス ATCC14400ならびにアルカリゲネス
ユウトロフスH−16ATCC17699およびこのH
−16株の突然変異株であるアルカリゲネス ユウトロ
フスNClB11597.同NClB11598゜同N
ClB11599.同NClB11600などを挙げる
ことができる。これらのうち、実用上、アルカリゲネス
ユウトロフスH−16ATCC17699およびアル
カリゲネス ユウトロフスNClB11599が特に好
ましい。Representative examples of strains belonging to these bacterial species include Alcaligenes faecalis ATCC8750 and Alcaligenes faecalis ATCC8750.
Rulandii ATCC15749, Alcaligenes
Rattus ATCC29712゜Alcaligenes aquamarinus ATCC14400 and Alcaligenes
Eutrophus H-16ATCC17699 and this H
-16 strain, Alcaligenes eutrophus NClB11597. Same NClB11598゜ Same N
ClB11599. Examples include the same NClB11600. Among these, Alcaligenes eutrophus H-16ATCC17699 and Alcaligenes eutrophus NClB11599 are particularly preferred for practical purposes.
アルカリ土類金属に属するこれらの微生物の菌学的性質
は、たとえば、”BERGEY’ SMANUAL
OF DETERMINATIVE BACTE
RIOLOGY:EighthEdition、The
Williams &Wilkins Com
pany/Baltimore”に、また、アルカリゲ
ネス ユウトロフスH−16の菌学的性質は、たとえば
、“J。The mycological properties of these microorganisms belonging to alkaline earth metals are, for example, “BERGEY” SMANUAL
OF DETERMINATIVE BACTE
RIOLOGY: Eighth Edition, The
Williams & Wilkins Com
Pany/Baltimore", and the mycological properties of Alcaligenes eutrophus H-16 are described, for example, in "J.
Qen、 Miclobiol、、115,185〜1
92 (1979)にそれぞれ記載されている。Qen, Microbiol, 115, 185-1
92 (1979).
これらの微生物は、従来の方法と同様に、主として菌体
を増殖させる前段の培養と、窒素もしくはりんを制限し
て菌体内に共重合体を生成、蓄積させる後段の培養との
2段で培養される。Similar to conventional methods, these microorganisms are cultured in two stages: a first stage culture in which the bacterial cells are mainly grown, and a second stage culture in which nitrogen or phosphorus is restricted to produce and accumulate copolymers within the bacterial cells. be done.
前段の培養は、微生物を増殖させる為の通常の培養法を
適用することができる。すなわち、使用する微生物が増
殖し得る培地および培養条件を採用すればよい。For the first-stage culture, a normal culture method for propagating microorganisms can be applied. That is, it is sufficient to adopt a medium and culture conditions that allow the microorganisms used to proliferate.
培地成分は、使用する微生物が責化し得る物質であれば
特に制限はないが、実用上は、炭素源としては、たとえ
ば、メタノール、エタノールおよび酢酸などの合成炭素
源、二酸化炭素などの無機炭素源、酵母エキス、糖蜜、
ペプトンおよび肉エキスなどの天然物、アラビノース、
グルコース、マンノース、フラクトースおよびガラクト
ースなどのtieならびにソルビトール、マンニトール
およびイノシトールなど、窒素源としては、たとえば、
アンモニア、アンモニウム塩、硝酸塩などの無機窒素化
合物および/または、たとえば、尿素、コーン・ステイ
ープ・リカー、カゼイン、ペプトン、酵母エキス、肉エ
キスなどの有機窒素含有物ならびに無機成分としては、
たとえば、カルシウム塩、マグネシウム塩、カリウム塩
、ナトリウム塩、りん酸塩、マンガン塩、亜鉛塩、鉄塩
、銅塩、モリブデン塩、コバルト塩、ニッケル塩、クロ
ム塩、はう素化合物およびよう素化合物などからそれぞ
れ選択される。There are no particular restrictions on the medium components as long as they can be used by the microorganisms used, but in practical terms carbon sources include synthetic carbon sources such as methanol, ethanol and acetic acid, and inorganic carbon sources such as carbon dioxide. , yeast extract, molasses,
Natural products such as peptone and meat extract, arabinose,
Nitrogen sources include, for example, ties such as glucose, mannose, fructose and galactose, and sorbitol, mannitol and inositol.
Inorganic nitrogen compounds such as ammonia, ammonium salts, nitrates and/or organic nitrogen-containing substances and inorganic components such as, for example, urea, corn steep liquor, casein, peptone, yeast extract, meat extract, etc.
For example, calcium salts, magnesium salts, potassium salts, sodium salts, phosphates, manganese salts, zinc salts, iron salts, copper salts, molybdenum salts, cobalt salts, nickel salts, chromium salts, boromine and iodine compounds. Each is selected from the following.
また、必要に応じて、ビタミン類なども使用することが
できる。Additionally, vitamins and the like can be used as needed.
培養条件としては、温度は、たとえば、20〜40℃程
度、好ましくは25〜35℃程度とされ、また、pHば
、たとえば、6〜10程度、好ましくは6.5〜9.5
程度とされる。このような条件で好気的に培養する。As for the culture conditions, the temperature is, for example, about 20 to 40°C, preferably about 25 to 35°C, and the pH is, for example, about 6 to 10, preferably 6.5 to 9.5.
It is considered to be a degree. Cultivate aerobically under these conditions.
これらの条件をはずして培養した場合には、微生物の増
殖は比較的悪くなるが、これらの条件をはずして培養す
ることを妨げない。If culture is performed under these conditions, the growth of microorganisms will be relatively poor, but this does not preclude cultivation under these conditions.
培養方式は、回分培養または連続培養のいずれでもよい
。The culture method may be either batch culture or continuous culture.
前段の培養によって得られた菌体を、さらに窒素および
/またはりん制限条件下で培養する。The bacterial cells obtained in the first stage of culture are further cultured under nitrogen and/or phosphorus-limited conditions.
すなわち、前段の培養で得られた培養液から微生物の菌
体を、濾過および遠心分離のような通常の固液分離手段
により分離回収し、この菌体を後段の培養に付するか、
または、前段の培養において、窒素および/またはりん
を実質的に枯渇させて、菌体を分離回収することなく、
この培養液を後段の培養に移行させることによってもで
きる。That is, either the microbial cells are separated and recovered from the culture solution obtained in the first-stage culture by ordinary solid-liquid separation means such as filtration and centrifugation, and the microbial cells are subjected to the second-stage culture, or
Alternatively, in the first stage of culture, nitrogen and/or phosphorus are substantially depleted, without separating and collecting the bacterial cells.
This can also be done by transferring this culture solution to the subsequent stage of culture.
この後段の培養においては、培地または培養液に窒素お
よび/またはりんを実質的に含有させず、γ−ブチロラ
クトンを炭素源として含有させること以外は前段の培養
と異なるところはない。This second-stage culture is similar to the first-stage culture except that the medium or culture solution does not substantially contain nitrogen and/or phosphorus and contains γ-butyrolactone as a carbon source.
尚、培養液にγ−ブチロラクトンを含有させる場合は、
培養の初期ないし後期のどの時点でもよいが、培養の初
期が好ましい。In addition, when containing γ-butyrolactone in the culture solution,
It may be carried out at any time from the early stage to the late stage of the culture, but the early stage of the culture is preferable.
本発明に用いられるγ−ブチロラクトンは、共重合体を
生成させることができ、かつ微生物の生育を阻害しない
ような量であればよく使用した微生物の菌株および所望
の共重合割合(モル比)などによって異なるが、一般的
には培地もしくは培養液Ilに3〜100g程度が適当
である。γ-Butyrolactone used in the present invention may be used in an amount that can produce a copolymer and does not inhibit the growth of microorganisms, such as the strain of the microorganism used and the desired copolymerization ratio (molar ratio). Although it varies depending on the situation, in general, about 3 to 100 g is appropriate for the medium or culture solution Il.
この後段の培養においてはT−ブチロラクトンを唯一の
炭素源としてもよいが、使用した微生物が責化し得る他
の炭素源、たとえば、グルコース、フラクトース、メタ
ノール、エタノール、酢酸、プロピオン酸、n−醋酸、
乳酸および吉草酸、4−ヒドロキシ醋酸およびそれらの
塩、1.4−ブタンジオールなどのジオール類などを共
存させることもできる。たとえば、グルコースを使用す
る場合には、多くても1.5g/l程度とされる。Although T-butyrolactone may be used as the sole carbon source in this subsequent culture, other carbon sources that can be used by the microorganisms used may be used, such as glucose, fructose, methanol, ethanol, acetic acid, propionic acid, n-acetic acid,
Diols such as lactic acid and valeric acid, 4-hydroxyacetic acid and salts thereof, and 1,4-butanediol can also be present. For example, when glucose is used, the amount is about 1.5 g/l at most.
このように培養して得られた培養液から、濾過および遠
心分離などの通常の固液分離手段によって菌体を分離回
収し、この菌体を洗浄、乾燥して乾燥菌体を得、この乾
燥菌体から、常法により、たとえば、クロロホルムのよ
うな有機溶媒で生成された共重合体を抽出し、この抽出
液に、たとえば、ヘキサンのような貧溶媒を加えて、共
重合体を沈澱させる。From the culture solution obtained by culturing in this way, the bacterial cells are separated and recovered by ordinary solid-liquid separation means such as filtration and centrifugation, and the bacterial cells are washed and dried to obtain dry bacterial cells. A copolymer produced from the bacterial cells is extracted using an organic solvent such as chloroform by a conventional method, and a poor solvent such as hexane is added to the extract to precipitate the copolymer. .
本発明の製造法によれば、共重合体中の3HB成分、4
HB成分の割合は任意に調節することができる。According to the production method of the present invention, the 3HB component in the copolymer, the 4
The proportion of the HB component can be adjusted as desired.
本発明を、実施例によりさらに具体的に説明する。なお
、本発明は、これらの実施例に限定されるものではない
。The present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to these examples.
実施例1〜5及び比較例1〜2
アルカリゲネス ユウトロフスH16(ATCC176
99)を使用して共重合体を製造した。Examples 1 to 5 and Comparative Examples 1 to 2 Alcaligenes eutrophus H16 (ATCC176
99) was used to produce the copolymer.
すなわち、
前段培養:
つぎの組成を有する培地で前記の微生物を30℃で24
時間培養し、対数増殖期終期の培養液から遠心分離によ
り菌体を分離した。That is, preliminary culture: The above microorganisms were incubated at 30°C for 24 hours in a medium having the following composition.
The cells were cultured for several hours, and the bacterial cells were separated from the culture solution at the end of the logarithmic growth phase by centrifugation.
前段培養用培地の組成
酵母エキス 10g ポリペプトン 10g肉エキ
ス 5 g (Ntla)zs045 gこれらを脱
イすン水1zに溶解し、pH7,0に調整した。Composition of culture medium for first stage: Yeast extract 10g Polypeptone 10g Meat extract 5g (Ntla)zs045g These were dissolved in 1z of demineralized water and adjusted to pH 7.0.
後段培養:
前記培養で得られた菌体を、つぎの組成を有する培地に
、11あたり5gの割合で懸濁させ30℃で48時間培
養し、得られた培養液から遠心分離により菌体を分離し
て、菌体を得た。後段培養用培地の組成
0、5 M りん酸水素カリウム水溶液39.0 閤
i
0、5 M りん酸水素二カリウム水溶液53.6惰
1
20wt/V% 硫酸マグネシウム水溶液1.0++1
1
炭素源0
ミネラル溶液” 1.0s+t!
* 炭素源として後記第1表に記した様な種々の化合物
を用いた。(単位g/l培地)**ミネラル溶液
c o c J z 119. O
wzFeCls 9.7gCa
CIt z 7.8 gN
量 ct、 11B、
0 寡■−蘭【Cr C41z
62.2111Ca S Oa 1
56.4 wを0.lN−HCl IIlに溶解
これらを脱イオン水11に溶解し、pH7,0に調整し
た。Post-stage culture: The bacterial cells obtained in the above culture were suspended in a medium having the following composition at a ratio of 5 g per 11 cells, cultured at 30°C for 48 hours, and the bacterial cells were removed from the resulting culture solution by centrifugation. The cells were separated to obtain bacterial cells. Composition of medium for second stage culture 0,5 M Potassium hydrogen phosphate aqueous solution 39.0 0,5 M Dipotassium hydrogen phosphate aqueous solution 53.6 1 20wt/V% Magnesium sulfate aqueous solution 1.0++1
1 Carbon source 0 Mineral solution 1.0s+t! * Various compounds as listed in Table 1 below were used as carbon sources. (Unit: g/l medium) ** Mineral solution co c J z 119. O
wzFeCls 9.7gCa
CIt z 7.8 gN
Amount ct, 11B,
0 small ■-ran [Cr C41z
62.2111Ca S Oa 1
56.4 w to 0. Dissolved in 1N HCl II1 These were dissolved in deionized water 11 and adjusted to pH 7.0.
菌体の処理:
後段培養で得られた菌体を蒸溜水で洗浄し、引続きアセ
トンで洗浄し、これを減圧乾燥(20℃。Treatment of bacterial cells: The bacterial cells obtained in the second-stage culture were washed with distilled water, followed by acetone, and dried under reduced pressure (20°C).
0、1 mmHg) L/て乾燥菌体を得た。0.1 mmHg)/L/dry cells were obtained.
共重合体の分離回収:
このようにして得られた乾燥菌体から熱クロロホルムで
共重合体を抽出し、この抽出液にヘキサンを加えて共重
合体を沈澱させ、この沈澱を濾取、乾燥して共重合体を
得た。Separation and recovery of copolymer: Extract the copolymer from the dried bacterial cells thus obtained with hot chloroform, add hexane to this extract to precipitate the copolymer, collect this precipitate by filtration, and dry. A copolymer was obtained.
共重合体の特性:
このようにして得られた共重合体の組成、固有粘度、融
解温度および融解熱を、つぎにようにして測定した。す
なわち、
組 成 :500MHz の ’H−NM
Rスペクトルによる。Properties of copolymer: The composition, intrinsic viscosity, melting temperature and heat of fusion of the copolymer thus obtained were measured as follows. That is, composition: 'H-NM of 500MHz
According to R spectrum.
固有粘度〔η〕 =30℃、クロロホルム中。Intrinsic viscosity [η] = 30°C, in chloroform.
融解温度および融解熱: DSC測定(10℃/aki
n)による。Melting temperature and heat of fusion: DSC measurement (10°C/aki
According to n).
測定結果などを第1表に示す。The measurement results are shown in Table 1.
本発明によれば、3HB成分、4HB成分を含有する新
規のポリエステル共重合体を容易に得ることができる。According to the present invention, a novel polyester copolymer containing a 3HB component and a 4HB component can be easily obtained.
さらに本発明で得られた共重合体は、優れた種々の特性
を有しているので、手術糸および骨折固定用材などの医
用材料の原料として極めて好適であり、また徐放性シス
テムへの利用などの多方面への応用が期待される。Furthermore, the copolymer obtained by the present invention has various excellent properties, so it is extremely suitable as a raw material for medical materials such as surgical threads and materials for fixing bone fractures, and can also be used for sustained release systems. It is expected to be applied in many fields such as.
Claims (1)
アルカリゲネス属菌を前段で菌体を増殖させ、後段で該
菌体を窒素あるいはリンの制限下で培養して該菌体内に
ポリ−3−ヒドロキシブチレートを生成・蓄積させるに
際して、後段の培養をγ−ブチロラクトンの存在下で行
なうことを特徴とする、3−ヒドロキシブチレート単位
および4−ヒドロキシブチレート単位からなるポリエス
テル共重合体の製造方法。(1) In the first stage, cells of the Alcaligenes genus having the ability to produce poly-3-hydroxybutyrate are grown, and in the second stage, the cells are cultured under nitrogen or phosphorus limitation, and poly-3- A method for producing a polyester copolymer consisting of 3-hydroxybutyrate units and 4-hydroxybutyrate units, which comprises performing the subsequent cultivation in the presence of γ-butyrolactone when producing and accumulating hydroxybutyrate. .
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63178448A JPH0714354B2 (en) | 1988-07-18 | 1988-07-18 | Method for producing polyester copolymer |
US07/230,461 US4876331A (en) | 1987-08-18 | 1988-08-10 | Copolyester and process for producing the same |
EP88307635A EP0304293B1 (en) | 1987-08-18 | 1988-08-17 | Copolyester and process for producing the same |
DE8888307635T DE3879320T2 (en) | 1987-08-18 | 1988-08-17 | COPOLYESTER AND METHOD FOR THE PRODUCTION THEREOF. |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63178448A JPH0714354B2 (en) | 1988-07-18 | 1988-07-18 | Method for producing polyester copolymer |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0227992A true JPH0227992A (en) | 1990-01-30 |
JPH0714354B2 JPH0714354B2 (en) | 1995-02-22 |
Family
ID=16048696
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP63178448A Expired - Lifetime JPH0714354B2 (en) | 1987-08-18 | 1988-07-18 | Method for producing polyester copolymer |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0714354B2 (en) |
-
1988
- 1988-07-18 JP JP63178448A patent/JPH0714354B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
JPH0714354B2 (en) | 1995-02-22 |
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