CS233318B1 - Method of 3-hydroxybutyratedehydrogenase preparation - Google Patents
Method of 3-hydroxybutyratedehydrogenase preparation Download PDFInfo
- Publication number
- CS233318B1 CS233318B1 CS8981A CS8981A CS233318B1 CS 233318 B1 CS233318 B1 CS 233318B1 CS 8981 A CS8981 A CS 8981A CS 8981 A CS8981 A CS 8981A CS 233318 B1 CS233318 B1 CS 233318B1
- Authority
- CS
- Czechoslovakia
- Prior art keywords
- hydroxybutyrate
- acetoacetate
- ferment
- cultivation
- cultured
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims 8
- 102000034279 3-hydroxybutyrate dehydrogenases Human genes 0.000 title claims 2
- 108090000124 3-hydroxybutyrate dehydrogenases Proteins 0.000 title claims 2
- 238000002360 preparation method Methods 0.000 title claims 2
- 241000589776 Pseudomonas putida Species 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 2
- QIMLADMAMHXKHL-UHFFFAOYSA-N 2-hydroxybutanoic acid;3-oxobutanoic acid Chemical group CCC(O)C(O)=O.CC(=O)CC(O)=O QIMLADMAMHXKHL-UHFFFAOYSA-N 0.000 claims 1
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 claims 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims 1
- 206010021143 Hypoxia Diseases 0.000 claims 1
- 239000004472 Lysine Substances 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 241000589516 Pseudomonas Species 0.000 claims 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims 1
- WDJHALXBUFZDSR-UHFFFAOYSA-M acetoacetate Chemical compound CC(=O)CC([O-])=O WDJHALXBUFZDSR-UHFFFAOYSA-M 0.000 claims 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims 1
- 239000001166 ammonium sulphate Substances 0.000 claims 1
- 235000011130 ammonium sulphate Nutrition 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 claims 1
- PHIQHXFUZVPYII-UHFFFAOYSA-N carnitine Chemical compound C[N+](C)(C)CC(O)CC([O-])=O PHIQHXFUZVPYII-UHFFFAOYSA-N 0.000 claims 1
- 230000003247 decreasing effect Effects 0.000 claims 1
- 235000014113 dietary fatty acids Nutrition 0.000 claims 1
- 230000000694 effects Effects 0.000 claims 1
- 239000000194 fatty acid Substances 0.000 claims 1
- 229930195729 fatty acid Natural products 0.000 claims 1
- 150000004665 fatty acids Chemical class 0.000 claims 1
- 238000000855 fermentation Methods 0.000 claims 1
- 230000004151 fermentation Effects 0.000 claims 1
- 238000005194 fractionation Methods 0.000 claims 1
- 229930195733 hydrocarbon Natural products 0.000 claims 1
- 150000002430 hydrocarbons Chemical class 0.000 claims 1
- 239000012051 hydrophobic carrier Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims 1
- 239000010452 phosphate Substances 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 claims 1
- 101100366940 Mus musculus Stom gene Proteins 0.000 description 3
- 229920002943 EPDM rubber Polymers 0.000 description 2
- 101100283604 Caenorhabditis elegans pigk-1 gene Proteins 0.000 description 1
- 101150096418 Mepe gene Proteins 0.000 description 1
- 241001553014 Myrsine salicina Species 0.000 description 1
- 101150104466 NOCT gene Proteins 0.000 description 1
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium on carbon Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 1
- 241001441724 Tetraodontidae Species 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Compounds Of Unknown Constitution (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
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Cnocod nonynemiH 3-rHnpoKCHdyTHpaT.nerHnporeHa3n yne H3-BecTOH. JBlh stoh ne jih BnpaníHBaioT daKTepnantHEH ιιιτθμμRhodopseudomonas spheroides Ha 3-rH.npOKCHdyTHpaTe, K8Khctohhhk yrjiepo.ua, 3θτθμ οτββτεηιοτ daKTepKH nocpencTBOM neHT-pH$yrHpoBaHHH, odpadaTHBaioT κχ h nonsepraioT cBodonHHH otKJieTOK deJEKOBHH 3KCTpaKT, ΗΜΘΚΙΙξΗΗ HanaJIBHyiO 8KTHBH0CTL no- - 2 - ρπηκθ 0,1 mojib/mhh/μγ denna, B03.neiicTBHio npoTaMHHcyjiB$a-Ta ujih ocax:.neHHH h nocne.nyioinero y.naneHHH HyKJieííHOBHX kkc-JIOT. IlOCJie φρδΚΠΚΟΗΧρΟΒΘΗΙΜ nOCpe.UCTBOM aMMOHHllcyJIL^iaTHOrOooaKneHEH, cooTBSTCTByiomaH $paKiw οτ.ηθηΗθτοΗ, προμηβθθτοη,pacTBopaeTcn b tíygpepe κ OHHmaeTcn Hnu se odoramaeTCH mho-roKpaTHO xpoMaTorparaHHecKlí no θκτηβηοοτη nopnnKa 17 mojib/μηη/μγ denna (toepaTypa, HanpHMep, H.U.Bergmeyer et al.,Biochem. J. (1967),102, 423). npsncTaBJíeHHHH cnocod odna.naeT τθμ Ηθ.ηοστθτκοΜ, ητο κοηθη-hhh npo.nyKT oneHB nopor, ocodeHHO ecnn neno xacaeTCH cnnB-ho odorameHHOH 3-rH.npoKCHtíyTHpaT.nerHnporeHa3H. IIpHHHHa sto-po He tojibko b Tpy.noeMKOCTH onepaiiHH ohhctkh, ho h b bhco-KOÍi CTOHMOCTH CydCTpSTa w KyJILTilBIípOBaHEH. JtaJIBHeilIIIHHΗθ,ηοοτετοκ coctoht b tom, ητο βκτηβηοοτβ nonynaeMoro φθρ-μθητθ He npeBKiiiaeT 3Η3Ηθηηη b 17 - 20 mojil/mhh/μγ dejma.HpHHHHa b tom, hto πρημθηηθμηη ιιιτθμμ Rhodopseudomongsspheroides He B COCTOHHHH np0H3B0.HHTB φθρΜβΗΤ C dOJiee BH-C0K0Í1 HanaJIBHOK aKTHBHOCTBIO, ΗΘΜ BHHieonHCaHHOH.
UenB H3odpeTeHiiH
UejiB. ' H3odpeTeHKH coctoht b tom, HTodn naTB cnocod nonyne-hiih cpepMeHTa, κοτορκη TpedyeT 3HannTenBH0 μθηβιπηχ 3aTpaT,HeM no chx nop H3BecTHHe h no3BOJMeT CBepx Toro nonynaTBφβρΜθκτ co 3HaHHTenBH0 donee bhcokoh οπθΐίΗφΗΗθοκοΗ θκτηβ-HOCTBK).
H3JI0SeHHe CyiItHOCTH H30dpeTeHHH B ocHO3y H3odpeTeHHH nononeHa 3a.nana Η3μθηητβ cymecTBeHHBieοτοροΗΗ y^e ηθβθοτηηχ cnooodoB BH.neJieHHn h, HcnonB3OBaB npyrae iuTaMMti, a Taiose .npyrne cydcTpaTH nnn KynBTHBHpoBaHHHb coneTaHHH o hoboh TexHonornen, nonymiTB ^enaeMHH ΦθρμθητnpOCTEM CnOCOdOM H C BHCOKOH ΟΠΘΗΗφΗΗΘΟΚΟΖ aKTHBHOCTBIO. - 3 -
CorjiacHO n3oópeTemro pememie 3a,Hanu dnjio nocTurHyTo nyTeMΒΗΑθΛΘΗΚΗ (pepweHTa 3-rn.npoKCíidyTKpaTnernnporeHa3H H3 uiTaM-Ma Pseudomonas putida. llhraMM coxpaHHJin npenjviynecTBeHHo HadyjiLOHe c 2 % arapoM npn 4° C b τθμηοτθ. KyjiLTHBHpoBaHnePseudomonas putida ocyinecTBJíHeTcn b cydMepcHOHHon xy jíl Ty-pe Ha SH.HKOH MHHHMaJILHOH ΟρθΠθ nOCJie ΠβρβΟβΒΘΗΗΗ H3 ΠθρΒΗΗ-Hofí KyjiLTypti npH TewnepaTypax ot 20 .no 40° C (npeHMynecTBenho npn 30° C). BtipanHíBaHne corjiacHO Μ3θόρβτθΗΗΐο npoBO.nHJiocLHa τβχ HGTOHHHKax yrjiepo.na, npn pacna.ne κοτορκχ odpa3yKTcn'aueToaueTaT h m.npoKcnóyTHpaT. Tamní ηοτοηηιικθμη yrjiepo.naHBJIHIOTCH, HanpHMep, D,L -JIH3HH, D,L-JieHUHH, D,L-KapHHTHHH T.,n. H ΚρΟΜθ ΤΟΓΟ, Hepa3B6TBJieHHHe yrJieBO.nopO.HBI C HJIIIHOHnenH Cg no Cjq SHpHLie khcjioth ot C4 .no Cjg. B KanecTBeHCTOHHHKOB a30Ta npHMeHHJIHCL NH^Cl, H Τ.Π. B ΤΘΧ cjiyHanx, b κοτορπχ hctohhhk yrjiepona ne μοιςθτ óhtl o.nnoBpe-ΜΘΗΗΟ HCnOJILSOBaH H K3K HCTOHHHK a30Ta. IlOCJie BHpaiIIHBaHHH Bτθηθηηθ βρθμθηη ot 5 no 40 nacoB - b 3θβηοημοοτη οτ φΗ3Η0-jiornnecKoro cogtohhhh h KOJiinecTBa βηοθβθθμογο MaTepnaJia -. óaKTepHH coónpaJincL, HanpHMep, nyTeM neHTpii$yrapoBaHHH npo-MHBaJIHCL φΟΟψθΤΗΗΜ Óy^epOM Η B 3aKJH0HeHHe npeBpanaJIHCL BdecKJieTOHHHH rpydtni SKCTpaKT,· HanpHMep, nyTeM odpadoTKHyjrLTpa3ByxoM npn TewnepaTypax dJiH3KHX κ τοηκθ 3aMep3aHHH cnocJie.nyioiniíM neHTpHžyrHpoBaHHe m. Ηθχο.κηιιιηηοη b rpydoM skct-paKTe φβρΜθΗτ npoHBjmeT b 3aBHciLM0CTH ot ηοτοηηηκθ yrjieponanpHBe.neHHiie b Tadjinue I οπθιιηΦηηθοκηθ θκτηβηοοτη. C nOMOUfLK) mpaKHHOHHOrO OCazneHHH HJIH se 3KCTpawm cojlhmh,HanpHMep c cyjn^aTOM θμμοηηή, ocyinecTBJíneTCH 10 - 15 KpaT—Hoe odoraneHne φθρΜθΗΤθ. Hjih stopo n,ejiecoodpa3HHM HBJíHeTcnpa3Be.neHHe dejnwBoro SKCTpaKTa c nocJienywiM ocasneHiieM no-naBJíHKinero KOJinnecTBa dejina 70 % cyjn^aTOM θμμοηηη. nojman θκτηβηοοτβ ocraeTcn npn stom b οθ.κημθητθ. Azih HaJiB-Hefiiiiero odoraneHKa 3-rHnpoKCHdyTHpaT.nerK.nporeKa3H oca.noKnocJienoBaTejiLHo odpadaTHBaeTCH npn 4° C pacTBopaMH cyjiBčpaTa - 4 - 3MM0HHH, KOTOpHe COflepjKST 0,05 MOJIb/jI TpHG, npií nOHOSIOIUeíí-ch KOHueHTpaiw (NH4)2soZj. H B ksehom GJiynae nyTe;i πητημη-HyTHoro nepeMeuiHBaHHH. Πρκ stom φθρΜθΗΤ Bce dojiBine pacTBopn-eTCH. TjiaBHaH wacca φθρΜθΗΤθ pacTBopneTCH b pauoHe. KOHupHTpa-Uiffl Mejxny 35 h 20 % HacwHHH pacTBopa cyjiL$aTa θμμοηηη.Φθρμθητ nepeocasnaeTCH nyTeM nodaBJíeHHH cynBroaTa θμμοηηη .noκοηθηηοη KOWHTpaiw b 50 %.
Ha ΓΗηροφοόΗΟΜ HocHTejie, τθκομ κθκ, Hanpnwep, IO-KapdoKcn.ne-UHJK^apo3a, bo3mokho nanBHeninee odorameHHe φβρΜθΗτθ. lípustom Ha KOJioHKy, 3anojmeHHyío ΙΟ-κβρόοκοΗΟθφθροΒΟΗ, cajKaeT-gh φθρΜβΗΤ b 20 % HaowHHH pacTBopa cyjiB$aTa θμμοκιιη. IIOGJie npOMHBKH paCTBOpOM Ογ,ΗΒφθΤθ 3MM0HHH 3TOH ΗΘ ΚΟΗΒζθΗΤ-paiw npOH3BO.nHTCH φρθΚΙίΗΟΗΗΟβ SJHOKpOBaHUe G 17 % HaCWHHHpacTBopa cyjii^aTa θμμοηηη. B nepBoS mpaKiw HaóJnonaeTCH 4-xKpaTHoe h bo BTopoS - 7-mh xpaTHoe ποβηπιθηηθ cneHiKtaecKoSaKTHBHOCTH. Ancopdiw cnocoócTByioT cTpyKTyj^opMtfpyioiHHehohh, Tamie κοκ φοοφβτ, iwpaT H cyjiB$aT. IIonyHeHHHfi corjiac-HO H30ÓpGT6HHK) φβρΜβΗΤ XOpOUlO COXpaHHeTCH. HpH ΟΠΘίχηφΗΗΘΟΚΚΧaKTHBHOGTHX b 10 — 20 mojib/mhh/μγ denna noKa3HBaioTCH cjie-nywie MaKCHMaJiBHHe nyxepo.nHHe βκτηβηοοτη: an£KorojiB.nerHHporeHa3a ( I %
ManaT.nerH,nporeHa3a < 0,02 % jiaKTaTnerH.nporeHa3a <0,001%
Ha maře BapamíBaHaa daKTepnajiBHOH KyjiBTypu bbho.uho padoTaTBco cTyneHHaTHM ηθφιαιιιτοΜ KHCJiopo.na. H3odpeTeHHe. no.npodHee ποηοηηθτοη Ha cjie.nynueM npKMepe npo-BS.nSHKH BH.neJieHHH.
ΠρκΜθρ ocymecTBJíeHHH H3odpeTemiH
Pseudomonas putida (PpG 6) BHpaiííHBaeTCH b cydMepcKOHHoSKyjiBType b bgi.hkoh MHHHManBHOĚ cpe.ne. Cpe.na conepmiT Ha ηπτρ:6,97 K2HP04, 1,5 r NaH2P04, 0,2 Γ MgS04 . 7 H20, 7,5 ΜΓ PeS047 H20, 0,75 ΜΓ I'*mS04 . 4 H20, 0,75 ΜΓ ZnSO4 . 7 H2°» - 5 - 0,15 ΜΓ CuSO4 . 5 HgO, 0,15 ΜΓ CoC12 . 6 HgO H 0,15 ΜΓdopHOM KHCJIOTH. B nanecTBe πρμβηβοηηηχ nyjiLTyp cjiy^ma npe.nBapHTejiLHue ny-jn>Typtj, y κοτορκχ κ mí.bkoh mhhilmoJilhoh cpe.ne nodaBJíHjm 3kct-paKT .upereš (0,4 r/ji) h rJH0KO3y (10 γ/ji) . ITpH KyJIBTHBHpOBaHBH Ha D,L -KapHHTIÍHe nOCJie.HHHM .HOtíaBJIHJICHb KOHiieHTpaiíHE 2 γ/ji, κοκ θ.βηηοτβθηηβιη ηοτοηηηκ yrjie.po.ua na30Ta. Πρη KyjiLTUBiipoBaHun Ha npyrnx ηοτοήηκκθχ yrjiepo.ua(10 γ/ji) b KanecTBe ηοίόηηηκβ a30Ta npHMeHHJicn NH^Cl(3 γ/ji) . nocjie BHpamHBaHHH b τθηθηηθ 15 nacos óaKTepnH codn-pajiií HeHTpií$yriípoBaHiíeM, npoMBiBaJin MHoroKpaTHo φοοφβτΗΐΐΜóy$epoM (0,067 mojil/ji, pH = 7,5) n pa3pyuiajin b 3a'KJnoHeHHenocpe.ncTBOM oópadoTKH yjiLTpa3ByK0M npn 4° 0. PpytíHii decKJie-tohhhh θκοτρβκτ nojiynajin c noMOintio iíeHTpn$yrnpoBaHHH.
Otot rpyóHH dejiKosaifí οκοτρθκτ pa3BO.nnJiH 0,067 mojil/ji φοοφθτ-hhm dyφepoM (pH 8) no ηοοτη&θΗΗΗ KOHiieHTpaiiHH d.ejiKa b 5 -20 μγ/mji. no.naBjmwtf HacTL dejma ocajKuaJm npn 4° C H3 θτογο pacTBopacpe.ncTBOM nodaBJíeHHH cyjiLŽaTa θμμοηηη no 70 % HacLnneHHH. IIojiHaH aKTHBHOGTL 3-rHnpoKCHdyTHpaT.nerH.nporeHasH Haxo.niiTCHnpn 3tom b ocanne. IlocJie neHTpngĎyrnpoBaHKH ce.nHMeHT cycneH-nupoBajin npn 0° C b pacTBope cyjiL$aTa ομμοηηη (45 % Hacnme-hhh b 0,05 mojil/ji Tpnc) n nocjie θτογο chob3 neHTpnctjyrnpoBa-jih. B ce.niiMeHTe ocTaeTcn nojiHan θκτηβηοοτβ. Jpin naJiLHeíimeroodorameHHH ocTaroK nepeMeumBajiii no 5 miih. npn 4° C c paoTBO-paMH cyjiLcoaTa θμμοηηη co.nepHainne κθκηημ 0,05 mojil/ji Tpuc-HCI,pH 8 noHimaioinnxoH KOHiieKTpanitíi.
QepneHT pacTBopneTcn npn stom b B03pacTaK)iqefi Mepe, npeiiMy-mecTBeHHO b pafione KOHpeHTpannn Mesny 35 n 20 / HacwHiiHcyjiLcpaTOM θμμοηι-ιη (cm. Tadjinny 2). - 6 - Φθρ^θητ nepe oca^na jih CHOBa nocpe.ncTBOM .nodaBJíeHHH cyjiLinaTa3MM0HHH .no ΚΟΗΘΗΗΟΜ ΚΟΗΙξθΗΤρθΙΧΗΗ B 50 % HaCHlUeHHH.
IlocJie iieHTpnroyriipoBaHiw cpepMeHT cycneHnupoBajm b 3,2 mojib/jipacTBopa cyjiB$aTa θμμοηηη, pH 7, h coxpaHHJiH npn 4° C. JUfl najiLHéHiiiero odorainemra I mji pacTBopa $epweHTa b cyjiLcpaTeθμμοηηη 20 % HacHineHKH caJKajm Ha 2-x mji kojiohky c IO-napdo-Kcnce$apo3oií; cneiyičpKHecKaH θκτηβηοοτβ οοοτοβλήθτ II mojib/μηη/μγ dejiKa; oóiuhh dejioK - 1,5 μγ. HojmaH θκτηβηοοτβ οοτθθτch Ha HOGHTejie. Ilocjie προμηβθηηη 10 mji pacTBopa cyjitčpaTa aM-mohhh (20 % HactimeHEH) Ha KOJiOHKy no.naBaJHi nccJienoBaTejiLHOno 2 mji pacTBopa cyjrBípaTa θμμοηηη (17 % ηθοητιιθηηη) . B noJiy-ΗΘΗΗΗΧ ,HByX φρθΚΗΗΗΧ HaXO.nHTCH BCH 3KTIÍBHOCTB, HO TOJILKO25 % dejina. ΟπβΗΗφΗΗΘΟΚΘΗ aKTHBHOCTB COCTaBJIHOT B I φρθΚΙξΗΗ 41 MOJIB/μηη/μγ dejma (0,3 μγ dejma) h bo 2 φρθκκΗΗ - 80 mojib/mhh/μγ dsjína (0,1 μγ dejma).
CycneH3HH ^epweHTa coxpaHseMU b HacwHHOM pacTBope cyjiBfóaTaθμμοηηη. Ilocjie 6 μθοηιϊθβ coxpaHeHHH npn TeMnepaTypax okojio4° C noTepn aKTHBHOCTH 3-rHnpoKCHtíyTHpaT.nerHnporeHa3H HeHadJHonajiact. - 7 -
Tatíjixua I
ΟπβΙίΚφΚΉΘΟΚΘΗ aKTHBHOCTB δ-ΓΗΙίρΟΚΟΗόγΤΗρεΤ,ΙίθΓΗ.ΙίρΟΓΘΗΘδΗ B rpydhix SKGTpaKTax H3 Pseudomonas putida nocuie pocTa Ha aHeToaueTaTodpa3ynwx cydcTpaTax .ujih KyjiL- THBHpOBaHHfl
Mctohhhk yrjiepo.na S-rHupoKCHdyTHpaT.nerHuporeHa 3H mojil/mhh/μγ dejiKa D,L- rKupoKCHdyTHpaT 3,5 D,L- KapHHTHH 2,0 D,L- jieínw 3,6 D,L- JUÍ3HH 0,8 - 8 -
TaÓJinua 2 Οήηοτκθ 3-rH.npoKCHdyTHpaT.nerHi];poreHa3H (cydcTpaT .wKyjiBTHBiípoBaHiiH D,L -κθρβήτεη) H3 Pseudomonas putidanojiyHeHiie npoBo.wiH H3 17 mji rpydoro sxcTpaKTa (7,5 μγ/mjidejixa).
Oca.noK nocJie odpadoTKH 45 % cyjn>$aTOM θμμοηκη, odpadaTHBaJica:noojie.noBaTejiBHo 5-k> mji pacTBopa cyjn>$aTa θμιλοηηη nomuKawix-CH KOHUeHTpanHH. 0 = ocaupK,’ H = Ha.iíoca.iíOHHaH wekoctl
Odiwi dejioK Odiw aKTHBHOGTB CneiíH$ircecKafl aKTZBHOCTB OniicTKa(-pa3) Bhxou w (μγ) ( mojil/meh) ( μολβ/μηη/μγ) rpydtm 3KCTpaKT 228 470 2,06 - 0 70 % 202 490 2,3 1,1 H 45 % (I) 2,6 H 45 % (2) 3,2 H 35 $ 2,6 H 33 $ 4,2 H 30 % 58 H 27 % 7 136 19 9,2 29 H 25 % 8 160 20 9,7 34 - 9 -
Tataua H 22 %7,5 H 19 % l ' (npO.HOJWHHe) 102 34 14 6,8 . 22 -Μ-
SOBOTA H30EPETEHMH 1. Cnocod nojiyzeHzn 3-rznpoKCzdyTHpaTnerznporeHa3H xa-OTnzzaJomzfícH τθμ, ζτο BHpamzBaeTcn ιιιτθμμ Psuedomonas putidaz πρκΜθΗΗίοτοίΐ ts zctozhzkz yrjiepo.na, npz pacna.ne κοτορζχ od-pa3yioTGB aneToaneTaT z rznpozczdyTZpaT. 2. Cnocod no nyHKTy I . oTJiEzawzcH τθμ, zto ιιιτθμμPseudomonas putida BLipaiiíZBacTcn npz TeMnepsTypax ot 20 .no40° C b cydMepczoHHofi zyjiBType b ztz.nzofi mzhzmsjibhoz cpene.Hocjie BpeweHZ BbipaiuzBaHZH b 5 - 40 zscob, tíazTepzz OT.nejiH-íotch, nepeBO.UHTCH e deozjieTozmiz denzoBtiz θκοτρθκτ, Z3 koto-poro φθρΜθΗΤ BÍJHeJIHeTOH nOCpe.nCTBOM φρθΚΙίΚΟΗΗΟΓΟ OCaJKUeHHHcyjiB$aTa ομμοηζη z b cjiyzae Heodxo.nzMOCTZ nonsepraeTCH ozzct-tze zjiz odorameHZio. 3. Cnocod no nyHKTaM I z 2 o Tanzanii ech tom, zto b zazecTBe zctozhzkob yrjiepo.na, npz pacna.ne κοτορπχ odpa3yioT-cn aneToaneTaT z rznpoKczdyTzpaT, npzMeHEJizcB D»L -jreznZH,.D,L-jiz3HH,D,L - KapHZTZH, Hepa3BeTBJieHHtie yrjieBO.nopo.ZH cZjixhoz nenz ot Cg no CjQ z JKZpHtie kzczoth - ot C^ no Cjg. 4. Cnocod no nyHKTaM I z 3 OTHHzaramziícH τθμ, zto Z3rpydoro deczjieTOZHoro SKCTpazTa tojibko nocpencT3OM φρβκηζοΗ-ηογο ocasnenzn cyjiB$aTa aMMOHzn nojiyzeH φθρΜβκτ co cnenz^z-zecKOH θκτζβηοοτβ b 10 - 20 mojib/μζη/μγ dejiza. 5. Cnocod no nyHKTaM I z 4 OTJizzaioinHzcH τβΜ, ζτο odo-raiueHze φβρΜθΗτθ ocyiyecTBjLHeTCH xpoMaTorpa$zzecKH Ha rznpo-$odHOM HoczTejie, ískom Kaz I0-zapdozczneiiHJice$apo3a zjhino.nodHOM. 6. Cnocod no nyHKTy 5 . othzzsioiukzch Ter.i, zto $epMeHTnpz bhcokzx KOHneHTpanzHX cyzB^aTa, cóoc$aTa zjiz nzTpaTaancopdzpyeTCH, a npz cHZ&aiaitHXCH KOHneHTpanzHX stzx zohob' - sjnonpyeTCH. -441 - 7. Cnocoó no nyHKTSM I h 3 OTJiKnaiomKHCfl τθμ, ητο bnpoiíecce BNpamHBaHHH óaKTepmí πρΗΜβΗηθτοη nocTyneiraaTHfi .neφηΐίΗτ KHCJiopo.ua.
AHHOTMW I430ÓpeTeHne KacaeTcn cnocoóa nojiyveiwi 3~rHnpoKCHóyTHpaT.neri<[nporeHa3H.
HeJIL H30ÓpSTSHHH GOGTOHT B Πρθ,ηΠΟ&ΘΗΙΙΗ HH3KOCTOHMOCTHOIO MG—To.ua, KOTopuii ηθθτ bosmojkiioctl nojiynaTB φΘρΜβΗΤ g bhcokoécner^BHecKOĚ θκτηβηοοτβιο. 3a.naHa ooGTosuia b nojiyneHHH acejiaeMoro $epweHTa nocpe.ncTBOMBbipaimiBaHiíH ηοβογο ÓaKTepjaaJiLHoro niTawa Ha cyócTpaTax nn&KyjILTHBHpOBaHHH, .no CHX ΠΟρ Ηθ ΠρΗΜΘΗΗβΜΗΧ.
CyuíHOCTL H3oópeTeHHH goctoht b tom, hto hitsmm Pseudomonaaputida BHpaumBajm Ha τθχ cyócTpaTax, npn pacna.ne κοτορπχB03HHKaioT aneToaneTaT hjih rH.npoKCHÓyTHpaT, hjih sce Ha cyócT-paTax H3 Hepa3BeTBJieHHHX - οτ Οθ no Cjq - yrjieBO.nopo.noB.
Claims (4)
- 4$ Předmět vynálezu L. Způsob přípravy 3-hydroxybutyrátdehydrogenázy,vyznačující se tlm, že se kultivuje kmen Pseudomonas putidaa používají se takové zdroje uhlíku, při jejichž rozpadu setvoří acetoacetát a hydroxybutyrát.
- 2. Způsob podle bodu 1, vyznačující se tím, že se kmenPseudomonas putida kultivuje při teplotách od 20 do 40 °Cv submerzní kultuře v kapalném minimálním mediu, po kultivacitrvající 5 až 40 hodin se bakterie oddělí, převedou na bezbu-něčný bilkovinový extkakt, ze kterého se ferment oddělí frak-čním srážením sulfátem amonným a v případě potřeby se čistí nebo obohacuje· ' ' ' ' ' .·' 'AS 3· Způsob podle bodů 1 a 2, vyznačující se tím, že sejako zdroje uhlíku, při jejichž rozpadu se tvoří acetoacetáta hydroxybutyrát se používají D,L-leucia, D,l-lysin, D,L-kar-nitin, nerozvětvené uhlovodíky s délkou řetězce 6 až 10 atomůuhlíku a mastné kyseliny od čtyč do šestnácti atomů uhlíku.' A*·
- 4. Způsob podle bodů 1 a 3, vyznačující se tím, že se zhrubého bezbuněčného extraktu pouhým frakčním srážením sulfátemamonným získá ferment se specifickou aktivitou 10 až 20 mol//min/ mg bílkoviny. 5· Způsob podle bodů 1 a 4, vyznačující se tím, že seobohacení fermentu provádí chromatograficky na hydrofobnímnosiči jako je 10-karboxydecylcefaroza.- Λ* &. Způsob podle bodu 5, vyznačující se tím, že se fermentpři vysokých koncentracích sulfátu, fosfátu nebo citrátuadsorbuje a při poklesu těchto koncentrací se evuuje. ,'x
- 7. Způsob podle bodů 1 až 3, vyznačující se tím, že sepři kultivaci bakterií používá stupňovitého deficitu kyslíku, Uznáno vynálezem na základě výsledků expertizy, provedenéÚřadem pro vynálezectví a patentnictví, Berlín, DD
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DD22006080A DD149874A3 (de) | 1980-03-31 | 1980-03-31 | Verfahren zur herstellung von 3-hydroxybutyratdehydrogenase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CS233318B1 true CS233318B1 (en) | 1985-02-14 |
Family
ID=5523436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS8981A CS233318B1 (en) | 1980-03-31 | 1981-01-05 | Method of 3-hydroxybutyratedehydrogenase preparation |
Country Status (5)
| Country | Link |
|---|---|
| BG (1) | BG41060A1 (cs) |
| CS (1) | CS233318B1 (cs) |
| DD (1) | DD149874A3 (cs) |
| DE (1) | DE3109748A1 (cs) |
| SU (1) | SU1731813A1 (cs) |
-
1980
- 1980-03-31 DD DD22006080A patent/DD149874A3/de not_active IP Right Cessation
- 1980-12-31 SU SU807771547A patent/SU1731813A1/ru active
-
1981
- 1981-01-03 BG BG5029081A patent/BG41060A1/xx unknown
- 1981-01-05 CS CS8981A patent/CS233318B1/cs unknown
- 1981-03-13 DE DE19813109748 patent/DE3109748A1/de not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| SU1731813A1 (ru) | 1992-05-07 |
| DD149874A3 (de) | 1981-08-05 |
| BG41060A1 (en) | 1987-04-15 |
| DE3109748A1 (de) | 1982-05-06 |
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