CS272660B1 - Immobilized biocatalyst for nicotinamidadenindinucleotide's enzymic reduction and method of its preparation - Google Patents
Immobilized biocatalyst for nicotinamidadenindinucleotide's enzymic reduction and method of its preparation Download PDFInfo
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- CS272660B1 CS272660B1 CS455387A CS455387A CS272660B1 CS 272660 B1 CS272660 B1 CS 272660B1 CS 455387 A CS455387 A CS 455387A CS 455387 A CS455387 A CS 455387A CS 272660 B1 CS272660 B1 CS 272660B1
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- cells
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- arthrobacter ureafaciens
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 21
- 239000011942 biocatalyst Substances 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title abstract description 9
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 241001524178 Paenarthrobacter ureafaciens Species 0.000 claims abstract description 9
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 239000000725 suspension Substances 0.000 claims abstract description 4
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229920001817 Agar Polymers 0.000 claims abstract description 3
- 239000008272 agar Substances 0.000 claims abstract description 3
- 229940072056 alginate Drugs 0.000 claims abstract description 3
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 3
- 229920000615 alginic acid Polymers 0.000 claims abstract description 3
- 238000001816 cooling Methods 0.000 claims abstract description 3
- 239000000499 gel Substances 0.000 claims abstract description 3
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 13
- 229950006238 nadide Drugs 0.000 claims description 10
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 210000001822 immobilized cell Anatomy 0.000 claims description 4
- 229920001525 carrageenan Polymers 0.000 claims description 3
- 239000000679 carrageenan Substances 0.000 claims description 2
- 229940113118 carrageenan Drugs 0.000 claims description 2
- 239000003349 gelling agent Substances 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 239000000463 material Substances 0.000 abstract 2
- 241000894006 Bacteria Species 0.000 abstract 1
- 230000015271 coagulation Effects 0.000 abstract 1
- 238000005345 coagulation Methods 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 15
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 15
- 239000011541 reaction mixture Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 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
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000000852 hydrogen donor Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000590020 Achromobacter Species 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- ZNOZWUKQPJXOIG-XSBHQQIPSA-L [(2r,3s,4r,5r,6s)-6-[[(1r,3s,4r,5r,8s)-3,4-dihydroxy-2,6-dioxabicyclo[3.2.1]octan-8-yl]oxy]-4-[[(1r,3r,4r,5r,8s)-8-[(2s,3r,4r,5r,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-sulfonatooxyoxan-2-yl]oxy-4-hydroxy-2,6-dioxabicyclo[3.2.1]octan-3-yl]oxy]-5-hydroxy-2-( Chemical compound O[C@@H]1[C@@H](O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H]2OC[C@H]1O[C@H](O[C@H]1[C@H]([C@@H](CO)O[C@@H](O[C@@H]3[C@@H]4OC[C@H]3O[C@H](O)[C@@H]4O)[C@@H]1O)OS([O-])(=O)=O)[C@@H]2O ZNOZWUKQPJXOIG-XSBHQQIPSA-L 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 229940043264 dodecyl sulfate Drugs 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
Vynález se týká zmobilizovaného biokatalyzátoru pro enzymovou redukci nikotinamidadenindinukleotidu (NAD+) v jeho redukovanou formu (NADH) a způsobu jeho přípravy.The invention relates to a mobilized biocatalyst for the enzymatic reduction of nicotinamide adenine dinucleotide (NAD + ) in its reduced form (NADH) and a process for its preparation.
Redukovaný nikotinamidadenindinukleotid se používá při enzymovém stanovení řady organických sloučenin, v biochemickém výzkumu a pří enzymové přípravě některých organických sloučenin. Zatímco nikotinamidadenindinukleotid je většinou připravován izolací z buněk kvasinek, jeho redukovaná forma se získává redukcí izolovaného nikotinamidadenindinukleotidu. Nejčastěji je používána redukce pomocí imobilizováných čistých enzymů, např. alkoholdehydrogenasy (E.C.1.1.1.1.), malátdehydrogenasy (E.C.1.1.1.37, E.C.I.1.1.38, nebo E.C.1.1,1. 39) nebo formátdehydrogenasy (E.C.1.2.1.2.) za použití příslušných enzymových substrátů, jako donoru vodíku. Použití imobilízovaných enzymů pro tento proces vyžaduje technicky i ekonomicky náročnou izolaci příslušných enzymů, jejichž stabilita je značně omezená. Proto bylo navrženo použití celých, permeabilizovaných nebo rozdrcených buněk mikroorganismů jako zdroje příslušných dehydrogenas, případně hydrogenas pro enzymovou redukci nikotinamidadenindinukleotidu. Isuzumi Y. a sp. (J. Ferment. Technol. 61, 135, 1983) používají k tomuto účelu permeabilizované buňky mikroorganismů s vysokou aktivitou formátdehydrogenasy, avšak k proběhnutí redukce je zapotřebí poměrně dlouhá doba (3 hod. pro redukcí 60 X přítomného nikotinamidadenindínukleotídu), což vede k částečnému rozkladu vzniklého produktu ve sloučeniny, jež inhibují reakce dehydrogenas, čímž se redukovaný nikotinamidadenindinukleotid znehodnocuje. Z přidaného mravenčanu, sloužícího zde jaké donor vodíku, se při použitém pH uvolňuje těkavá mravenčí kyselina, působící korozivně na zařízení a dráždivě na sliznice pracovníků. II rozdrcených.buněk Achromobacter parvulus (Yokoyama S. a Suye S., franc. patent 2562089) je pro redukci 98 % přidaného nikotinamidadenindínukleotídu zapotřebí dokonce 6 h. Také enzymová hydrogenace nikotinamidadenindínukleotídu v jeho redukovanou formu plynným vodíkem za součinnosti zmobilizovaných bakteriálních buněk (Klibanov A.M. a sp ., Biotechnol. Letters 2, 445, 1980, Matsunaga T. a sp., Biotechnol. Bioeng.27, 1277, 1985) vyžaduje 5 až 6 h při velmi nízkých koncentracích produktu 0,8 mM. Přitom je u tohoto postupu velmi náročná manipulace s plynným vodíkem, jenž se v modifikaci popsané Matsunagem a sp.Reduced nicotinamide adenine dinucleotide is used in the enzymatic determination of a number of organic compounds, in biochemical research, and in the enzymatic preparation of some organic compounds. While nicotinamide adenine dinucleotide is mostly prepared by isolation from yeast cells, its reduced form is obtained by reduction of isolated nicotinamide adenine dinucleotide. Reduction using immobilized pure enzymes, such as alcohol dehydrogenase (EC1.1.1.1.), Malate dehydrogenase (EC1.1.1.37, ECI1.1.38, or EC1.1.1.39) or format dehydrogenase (EC1.2.1.2), is most commonly used. using appropriate enzyme substrates as a hydrogen donor. The use of immobilized enzymes for this process requires a technically and economically demanding isolation of the respective enzymes, the stability of which is very limited. Therefore, it has been suggested to use whole, permeabilized or crushed cells of microorganisms as a source of the respective dehydrogenases or hydrogenases for the enzymatic reduction of nicotinamide adenine dinucleotide. Isuzumi Y. et al. (J. Ferment. Technol. 61, 135 (1983)) use permeabilized microorganisms with high format dehydrogenase activity for this purpose, but a relatively long time (3 hours to reduce 60% of the nicotinamide adenine dinucleotide present) is required for reduction, resulting in partial decomposition of the resulting product into compounds that inhibit dehydrogenase reactions, thereby degrading the reduced nicotinamide adenine dinucleotide. The added formate, which serves as a hydrogen donor, releases volatile formic acid at the pH used, which acts corrosively on the device and irritates the workers' mucous membranes. Even crushed Achromobacter parvulus cells (Yokoyama S. and Suye S., French patent 2562089) even 6 hours are required to reduce 98% of the added nicotinamide adenine dinucleotide. et al., Biotechnol. Letters 2, 445 (1980), Matsunaga T. et al., Biotechnol. Bioeng. 27, 1277, 1985) requires 5-6 h at very low product concentrations of 0.8 mM. In this process, the handling of hydrogen gas, which in the modification described by Matsunag et al.
používá dokonce za tlaku 10 MPa.even at a pressure of 10 MPa.
Uvedené nevýhody odstraňuje biokatalyzátor pro enzymovou redukci nikotinamidadenindinukleotidu podle vynálezu, který obsahuje 1 až 60 hmot. dílů vlhkých permeabilizovaných buněk bakterie Arthrobacter ureafaciens Krebs a Eggleston, s výhodou Arthrobacter urěafaciens DBM 1076, uložené ve sbírce katedry biochemie a mikrobiologie Vysoké školy chemicko-technologické v Praze pod číslem 1076, na 1 hmot. díl gelotvorné látky například alginátu, akrylamidu, agaru, kapa-carrageenanu nebo genu-carrageenanu.These disadvantages are overcome by the biocatalyst for the enzymatic reduction of the nicotinamide adenine dinucleotide according to the invention, which contains 1 to 60 wt. parts of damp permeabilized cells of Arthrobacter ureafaciens Krebs and Eggleston, preferably Arthrobacter ureafaciens DBM 1076, deposited in the collection of the Department of Biochemistry and Microbiology of the Institute of Chemical Technology in Prague under number 1076, per 1 wt. a portion of a gelling agent, for example, alginate, acrylamide, agar, kappa-carrageenan or gene-carrageenan.
Způsob přípravy biokatalyzátoru podle vynálezu spočívá v tom, že se vlhké, popřípadě permeabilizované buňky Arthrobacter ureafaciens suspendují do roztoku gelotvorné látky, načež se suspenze převede v gel vysrážením, polymerací a/nebo ochlazením, popřípadě se pak provede permeabílizace buněk. Permeabilizaci je vhodné provádět třepáním s acetonem nebo jiným rozpouštědlem, působením povrchově aktivní látky, například dodecylsulfátem nebo opakovaným zmražováním buněk. Takto připravený biokatalyzátor se použije v přítomnosti fosfátu a metabolizovatelných sacharidů, jakožto jediného' zdroje vodíku k enzymové redukci rozpuštěného nikotinamidadenindinukleotidu v jeho redukovanou formu. Biokatalyzátor může být použit vsádkově v reaktoru za mírného míchání nebo v koloně s průtokem reakčni směsi. Může být používán opakovaně, neboť si zachovává aktivitu po dobu 7 až 14 dnů.The process for the preparation of the biocatalyst according to the invention consists in suspending wet or possibly permeabilized Arthrobacter ureafaciens cells into a gel-forming solution, whereupon the suspension is converted into a gel by precipitation, polymerization and / or cooling, optionally by permeabilizing the cells. Permeabilization is preferably carried out by shaking with acetone or other solvent, by a surface-active agent, for example dodecyl sulfate, or by repeated freezing of the cells. The biocatalyst thus prepared is used in the presence of phosphate and metabolizable carbohydrates as the only hydrogen source to enzymatically reduce dissolved nicotinamide adenine dinucleotide in its reduced form. The biocatalyst can be used batchwise in the reactor with gentle stirring or in a column with a flow of reaction mixture. It can be reused because it retains activity for 7 to 14 days.
Hlavní výhoda zmobilizovaného biokatalyzátoru je jeho mimořádně vysoká aktivita, jež umožňuje například redukci trojnásobného množství (počítáno na objem hydratovaného katalyzátoru) 20 mM nikotinamidadenindinukleotidu během 1 h s výtěžkem 90 až 95% redukovaného nikotinamidadenindinukleotidu. Další výhodou tohoto biokatalyzátoru je jeho vysoká stabilita, takže může být používán kontinuálně nebo opakovaně po dobu 14 dnů i více, což vede k úsporám na přípravě mikrobiálních buněk i biokatalyzátoru. Mimořádně výhodné je, že biokatalyzátor podle vynálezu umožňuje použiti metabolizovatelných sacharidů jako donoru vodíku λThe main advantage of the mobilized biocatalyst is its extremely high activity, which makes it possible, for example, to reduce the triple amount (calculated on the volume of hydrated catalyst) of 20 mM nicotinamide adenine dinucleotide over 1 hour with 90-95% yield of reduced nicotinamide adenine dinucleotide. Another advantage of this biocatalyst is its high stability, so that it can be used continuously or repeatedly for 14 days or more, resulting in savings in both microbial cell and biocatalyst preparation. It is particularly preferred that the biocatalyst of the invention allows the use of metabolizable carbohydrates as a hydrogen donor λ
CS 272 660 Bl ά při enzymové redukci, což předčí ostatní postupy po ekonomické a v některých případech také hygienické stránce.CS 272 660 Bl ά in enzyme reduction, which outperforms other processes in economic and, in some cases, hygiene terms.
Vynález je v dalším blíže objasněn v příkladech provedení, aniž by se jimi omezoval. Příklad 1The invention is illustrated by the following non-limiting examples. Example 1
Arthrobacter ureafaciens DBM 1076 se aerobně kultivuje při 30° C v prostředí o pH 7,2 obahujícím v 1 litru: 20 g glukosy, 1,0 g (NH^^SO^, 0,1 g MgSO^.7 I^O·, 8,7 g KgHPO^, 10 g sušeného kvasničného autolyzátu a 10 g peptonu. Po 18 h kultivace se buňky odstředí při 800 g a promyjí vodou. 15 g vlhkých buněk se rozmíchá v 30 ml v 1,5 % (hmot./obj.) vodného roztoku alginátu sodného a takto vzniklá suspenze se kape pomocí stříkačky do 0,05 M CaC^ obsahujícího 10 % (hmot./obj.) glukosy. Pak se srážecí roztok odstraní filtrací přes skleněnou fritu a imobilizované buňky se na fritě promyjí 0,05 M roztokem CaC^v Tris-HCl pufru o pH 9,0. Promyté imobilizované buňky se míchají 15 min v dvojnásobném objemu acetonu, načež se aceton slije a jeho zbytky se odstraní proudem vzduchu. Takto připravený biokatalyzátor se přidá k trojnásobnému množství (hmot./obj.) reakční směsi obsahující (v konečných koncentrátech) 20 mM nikotinamidadenindinukleotid, 40 mM MgCIz, 0,05 M CaC^, 1 mM NaNj, % (hmot./obj.) kyseliny metařosforečné zneutralizované pomocí IN NaOH a 2 % (hmot./obj.) glukosy v Tris-HCl pufru o pH 9,0 a inkubuje se za mírného míchání 1 h při 30 C. Pak se reakční kapalina oddělí od biokatalyzátoru filtrací přes fritu a z filtrátu se některým ze známých postupů izoluje redukovaná forma nikotinamidadenindinukleotidu. Biokatalyzátor se použije pro další podíl reakční směsi. Jeho stabilita zůstává zachována po dobu 7 až 14 dnůArthrobacter ureafaciens DBM 1076 is aerobically cultured at 30 ° C in an atmosphere of pH 7.2 containing 1 liter: 20 g glucose, 1.0 g (NH 4 O 2 SO 4, 0.1 g MgSO 4 .7 I O 2 O). , 8.7 g of KgHPO4, 10 g of dried yeast autolysate and 10 g of peptone After 18 h of cultivation, the cells are centrifuged at 800 g and washed with water, 15 g of wet cells are stirred in 30 ml at 1.5% (w / v). The aqueous solution of sodium alginate and the suspension thus obtained are dripped by syringe into 0.05 M CaCl 2 containing 10% (w / v) glucose, and then the precipitating solution is removed by filtration through a glass frit and the immobilized cells are washed on the frit. 0.05 M CaCl 2 solution in Tris-HCl buffer, pH 9.0 The washed immobilized cells are stirred for 15 min in 2-fold volume of acetone, then acetone is decanted and its residues are removed by air flow. (w / v) reaction mixture containing (in final concentrates) 20 mM nicotinamidade nindinucleotide, 40 mM MgCl 2, 0.05 M CaCl 2, 1 mM NaN 3,% (w / v) metaphosphoric acid neutralized with 1N NaOH and 2% (w / v) glucose in Tris-HCl buffer pH 9 The reaction liquid is separated from the biocatalyst by filtration through a frit and a reduced form of nicotinamide adenine dinucleotide is isolated from the filtrate by any known method. The biocatalyst was used to further charge the reaction mixture. Its stability is maintained for 7 to 14 days
Příklad 2Example 2
Buňky Arthrobacter ureafaciens DBM 1076 se získají postupem uvedeným v příkladu 1 a promyjí vodou. K 10 g vlhkých buněk se přidá 20 ml destilované vody, 4 g monomeru akrylamidu a 0,4 g Ν,Ν’-methylen-bis-akrylamidu a po rozmíchání se nechá ztuhnout při teplotě 7 až 12° C. Z této hmoty se nařežou krychličky o hraně 2 až 3 mm, jež se pak míchají 15 min s dvojnásobným objemem acetonu, aby se imobilizované buňky permeabilizovaly. Pak se aceton slije, získaný biokatalyzátor se promyje vodou a naplní do kolony, kterou se poté nechá pro těkat reakční směs stejného složení jako v příkladu 1. Rychlost průtoku reakční směsi se volí tak, aby se veškerá reakční směs v koloně vyměnila během 45 až 70 min. Tento kontinuální způsob může probíhat až 14 dnů a může být také kdykoliv přerušen a znovu obnoven. Výtěžnost redukovaného nikotinamidadenindinukleotidu je kolem 95 % vneseného nikodinamidadenindinukleotidu. Z eluátu se izoluje redukovaný nikotinamidadenindinukleotid některým ze známých postupů.Arthrobacter ureafaciens DBM 1076 cells were obtained as described in Example 1 and washed with water. 20 ml of distilled water, 4 g of acrylamide monomer and 0.4 g of Ν, Ν'-methylene-bis-acrylamide are added to 10 g of wet cells and, after stirring, solidified at 7 to 12 ° C. cubes with an edge of 2-3 mm, which are then mixed for 15 min with twice the volume of acetone to permeabilize the immobilized cells. Then the acetone is decanted, the biocatalyst obtained is washed with water and packed into a column, which is then allowed to flow through the reaction mixture of the same composition as in Example 1. The flow rate of the reaction mixture is chosen so that all the reaction mixture in the column is changed within 45 to 70 min. This continuous process can take up to 14 days and can also be interrupted and resumed at any time. The yield of reduced nicotinamide adenine dinucleotide is about 95% of the nicodinamide adenine dinucleotide loaded. The reduced nicotinamide adenine dinucleotide is isolated from the eluate by any of the known methods.
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| CS455387A CS272660B1 (en) | 1987-06-22 | 1987-06-22 | Immobilized biocatalyst for nicotinamidadenindinucleotide's enzymic reduction and method of its preparation |
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| CS455387A CS272660B1 (en) | 1987-06-22 | 1987-06-22 | Immobilized biocatalyst for nicotinamidadenindinucleotide's enzymic reduction and method of its preparation |
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| CS272660B1 true CS272660B1 (en) | 1991-02-12 |
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