EP1419262A2 - Procede de reduction enzymatique de substrats au moyen d'hydrogene moleculaire - Google Patents

Procede de reduction enzymatique de substrats au moyen d'hydrogene moleculaire

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Publication number
EP1419262A2
EP1419262A2 EP02767065A EP02767065A EP1419262A2 EP 1419262 A2 EP1419262 A2 EP 1419262A2 EP 02767065 A EP02767065 A EP 02767065A EP 02767065 A EP02767065 A EP 02767065A EP 1419262 A2 EP1419262 A2 EP 1419262A2
Authority
EP
European Patent Office
Prior art keywords
reaction
hydrogenase
hydrogen
nadph
enzyme
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.)
Withdrawn
Application number
EP02767065A
Other languages
German (de)
English (en)
Inventor
Andreas Liese
Rita Mertens
Lasse Greiner
Eyke Van Den Ban
Huub Haaker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP1419262A2 publication Critical patent/EP1419262A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/36—Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the invention relates to a method for the enzymatic reduction of substrates with molecular hydrogen.
  • oxidoreductases have found their way into the production of fine chemicals and pharmaceuticals. If these are enzymatic, stereoselective reductions, a cofactor (hydrogen donor) in the form of NADH or NADPH is usually required. Since these cofactors are also now very high price, especially NADPH, s there is a need for a method by which it is possible to produce NADPH especially particularly inexpensive. Furthermore, a process that enables the regeneration of NADPH is of the highest interest, whereby the individual NADPH molecule should be able to go through as many cycles as possible (high total turn-over number). The latter is particularly important since these cofactors are far too expensive to be used stoichiometrically.
  • the glucose dehydrogenase from Gl conobacter suboxidans (Izumi Y, Nath PK, Yamamoto H, Yamada H. NADPH production from NADP + with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans were used here.
  • the process is particularly inexpensive because of the inexpensive hydrogen as an agent.
  • the removal of the reducing agent is not difficult since hydrogen is gaseous.
  • the enzymes used according to the invention are less labile and go through more reaction cycles. Quantitative conversions can be achieved with the method according to the invention.
  • the process can preferably be carried out in situ.
  • a mediator is implemented with molecular hydrogen by means of a hydrogenesis.
  • the hydrogenase is preferably from the hyperthermophilic strain of the archaebacterium Pyrococcus furiosus (DSM 3638).
  • the hydrogenases can be used in situ.
  • a mediator is a substance that can transfer hydride ions or electrons to substances. These compounds, when naturally occurring in organisms, are called cofactors. Other substances that are functionally equivalent to these natural cofactors are called mediators.
  • mediators is intended to mean all known mediators, including the natural cofactors, so that both types of action having the same effect should be included in the term. Examples of mediators are flavin mononucleotide (FMN), phenazine methosulfate (PMS), 2,6 dichlorophenolinophenol (DCPIP), Janus Grün, methylene blue, flavin adenine dinucleotide (FAD) but especially NADP + . However, NAD + is excluded from the invention.
  • the mediators are converted into their reduced form by means of the hydrogenase in the presence of hydrogen. The products are known to the person skilled in the art.
  • the reaction preferably takes place in an aqueous medium.
  • the reaction can be carried out in a pH range from 6-10, but preferably takes place in a pH range from 7 to 9, preferably from 7.5 to 8.5, since the enzyme activity and the stability of NADPH are greatest here is.
  • the reaction can be carried out in a temperature range from -10 ° C. to 150 ° C., preferably in a range from 0 ° C. to 100 ° C., particularly preferably from 20 ° C. to 80 ° C.
  • the pressure range in which the reaction can be carried out is preferably between 0 to 300 bar, particularly preferably between 0 and 20 bar.
  • the solubility of hydrogen is higher, so that more molecules are available for the hydrogenation in the solution.
  • the pressure ranges below 20 bar reactors are required which cannot withstand such high pressures, so that the technical outlay here is lower.
  • the boundaries of the specified areas are fluid.
  • the gas phase can be composed differently over the reaction solution. There should be no oxygen in the gas phase or at least so little that there is no explosive mixture.
  • the hydrogen can be present in pure form or in a mixture of a gas inert to the process, for example with at least one component from the group consisting of N 2 , CO 2 , Ar, He and Kr.
  • the hydrogen content can be between> 0 to 100%.
  • a hydrogen content of between 50 and 100% is preferred.
  • a further enzyme is added to the reaction solution, which converts a substrate.
  • the mediator converted by the hydrogenase can serve as a cofactor or more generally as a mediator, which is regenerated by the subsequent repeated reaction with the hydrogenase by being hydrogenated with hydrogen.
  • the enzyme 2 can be, for example, an alcohol dehydrogenase, which converts a ketone to an alcohol.
  • NADP + is converted by means of the hydrogenase to NADPH, which serves as an H - donor for the reduction of the ketone to an alcohol. This in turn produces NADP + , which is regenerated again by the hydrogenase.
  • acetophenone or acetone can be mentioned as substrates for the second reduction.
  • Further enzymes E2 are available, for example, from Thermoanaerobium brockii (ADH), (Pyrococcus furiosus (GDH).
  • ADH Thermoanaerobium brockii
  • GDH Panococcus furiosus
  • the process of mediator regeneration according to the invention can be carried out with any further enzyme reaction tion that the regenerated mediator needs.
  • the hydrogenase reaction is thus coupled with a further enzymatic reaction.
  • Typical examples of reactions of enzyme 2, which can be coupled with the regeneration of the mediator are described in “Wong, C.-H .; Whitesides, G.M. ; 1994 Enzymes in synthetic organic chemistry "Baldwin, J.E.; Magnus, P.D .; Tetrahedron organic chemistry series; Oxford; Elsevier Science Ltd.; Vol 12; 370 pages”.
  • the method according to the invention can therefore be used both for the production of mediators and for their in situ regeneration in coupled systems.
  • the coupled enzyme reaction with enzyme 2 is not disturbed by the hydrogen, rather the hydrogen even represents an inert atmosphere for the reaction.
  • the process control can take place in a batch reactor. Furthermore, the reaction according to the invention can be carried out in a membrane reactor, as described, for example, in German Patent 44 36 149, which is equipped with an ultrafiltration membrane which enables the retention of the hydrogenase.
  • the membrane reactor can be used as a repetitive batch or as a continuous reactor. The process control in the continuously operated membrane reactor is particularly preferred.
  • the hydrogenase from Pyrococcus furiosus is used repeatedly in a 'repetitive batch' process to obtain NADPH to produce. This results in low catalyst consumption rates and high productivity.
  • a maximum space-time yield of 10 gL "1 d " 1 was achieved.
  • a catalyst consumption of less than 0.1 mg protein / gNADPH and a maximum space-time yield of 74 g / L "1 d ⁇ 1 is achieved.
  • the hydrogenase from Pyrococcus furiosus for the cofactor regeneration of NADPH in the asymmetrical reduction of prochiral ketones by means of a dehydrogenase In the experiment shown in example 6, a cycle number of 100, in example 1 of 320, is achieved.
  • the hydrogenase from Pyrococcus furiosus used here is a particularly stable enzyme which, as shown by the repetitive batch experiments, can be reused many times.
  • the catalyst can be used both in coupled and alone.
  • the implementation of the pyrodine nucleotides according to the invention is not subject to any thermodynamic limitation. This is surprising and particularly advantageous since higher sales are achieved than in the case of regeneration systems coupled with cosubstrates.
  • Fig.l Sales-time curve for the reaction acetophenone - phenylethanol examples
  • Fig. 2 Repetitive batch tests for the reaction from Fig. 1 (Example 6).
  • Fig. 3 Yield of NADPH for repetitive batch tests according to Example 7.
  • Fig. 4 Dependence of the NADPH formation on the temperature.
  • bio-moisturizer 100 g of bio-moisturizer were added 400 mL 50 mM Tris-HCl pH 8.0 suspended, and 10 ⁇ g / mL DNase and RNase added. After 4 h at room temperature, the cell extract was centrifuged for 1 h at 30,000 g. 3 different preparations were prepared from the cell supernatant obtained in this way (diluted, cell-free extract, 5 - 10 mg protein / mL):
  • the hydrogenase activity (H 2 formation) is measured at 40 ° C (Silva PJ; Van den Ban ECD; Wassink H.; Haaker H .; De Castro B .; Robb FT; Hagen WR Enzymes of hydrogen metabolism in Pyrococcus furiosus, Eur. J. Biochem., 2000, 267, 6541-6551). If one of the enzyme preparations described above is to be used for the preparation or the cofactor regeneration of NADPH, any phosphatase activity still present which catalyzes the dephosphorilation of NADP + to NAD + must first be removed. The corresponding activities on hydrogenase and phosphatase are given in Table 1 for the three enzyme preparations described above.
  • the rate of dephosphorilation of NADP + was determined under the following conditions: 50 mM EPPS pH 8.0, 0.5 mM NADPH, protein sample, 40 ° C., total volume 1.5 ml. No phosphodiesterase activity could be detected.
  • Table 1 Specific activities of hydrogenase and phosphatase. All activities were determined at 40 ° C.
  • the hydrogenase is only highly stable if anaerobic conditions are present.
  • the reduction of NADP + is only carried out in the presence of hydrogen. Appropriate precautions have been taken to exclude disruptive components.
  • the reaction stability was tested using a fed-batch experiment. 1.6 units of hydrogenase are incubated in 3 ml of 200 mM EPPS, pH 8.0 at 80 ° C. for 5 minutes. After the addition of 8 mM (24 ⁇ mol), the solution was incubated at 40 ° C. under a hydrogen atmosphere for 24 h. After each addition of NADP + , quantitative conversion with respect to the fresh substrate was achieved after one hour. However, it was found that the NADPH produced was more unstable than the metered te NADP + is. The decay of NADPH is not enzyme-catalyzed. The half-life of NADPH in the different experiments was on average 20 h. Similar half-lives for NADPH at 41 ° C are described in the literature. An HPLC analysis after a reaction time of 2 and 48 h showed no decay products which could be caused by phosphatase or by phosphodiesterase activity.
  • the standard oxidation and reduction potentials of H + -H 2 and a NADP + -NADPH at pH 8 differ by 133 mV. Assuming that there is no inhibition of the hydrogenase by one of its starting materials or products, the enzyme must be capable of completely reducing the pyrodine nucleotide cofactors. A potential difference of 133 mV at pH 8.0 and a hydrogen atmosphere shifts the balance from NADPH / NADP + to 24,000 / 1 (Clark, WM, etc.). This means that there is no thermodynamic limitation for the reduction of pyrodine nucleotides by H 2 .
  • GDH glutamate dehydrogenase
  • the hydrogenase from Pyrococcus furiosus was used for regeneration.
  • the reaction solution consisted of 50 mM Tris / HCL buffer, pH 7.5, 50 mM NH 4 CL, 0.2 units GDH and 0.06 units hydrogenase from a cell-free extract.
  • Purified Pyrococcus furiosus glutamate dehydrogenase was used as a test system for reductive cofactor regeneration. As can be seen in Table 2, sufficient NADPH could be regenerated at 50 C within 16 h in order to achieve 100% conversion of 2-keto-gluterate to glutamate, which had a cycle number (TTN) of 100 under the aforementioned reaction conditions equivalent.
  • TTN cycle number
  • Table 2 Conversion of 50 mM 2-ketoglutarate to glutamate by P. furiosus GDH with NADPH regeneration by hydrogenase in a cell-free extract from P. furiosus.
  • a solution of 10 mM acetophenone in 50 mM aqueous potassium phosphate buffer (pH8) with 0.5 mM oxidized cofactor NADP + and a NADP + -dependent alcohol dehydrogenase (ADHM) is degassed by flowing the solution through with water-saturated helium.
  • 20 ⁇ L of an activated enzyme preparation (5 minutes at 80 ° C. under a hydrogen atmosphere) from Pyrococcus furiosus are added to this solution, so that the protein concentration corresponds to 0. lmg / mL. It is added through a gas-tight septum using a suitable syringe.
  • the solution is kept at 40 ° C.
  • the inert argon atmosphere is replaced by hydrogen.
  • GC gas chromatography
  • the substrate was changed from acetophenone to (S) -2-hydroxy-l-phenyl-propanone. 1, 2-Dihydroxyphenylpropanon with high enantiomeric excess was obtained as product.
  • a modified ultrafiltration cell (Amicon, Germany), which is equipped with an ultrafiltration membrane (YM10, Amicon), 3 ml of 200 mM EPPS, pH 8.0, 12 mM NADP + are filled in under an argon atmosphere.
  • the cell outlet is opened periodically and the permeate of the ultrafiltration cell is analyzed for conversion and yield by capillary electrophoresis. The outlet was checked for protein content with a negative result. After more than 95% of the conversion had been achieved (with respect to NADP + ), the cell contents were filtered to a minimal residual volume (approx. 0.2 ml) and again filled with the same solution.
  • Various hydrogen pressures were applied.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé de réduction enzymatique de substrats au moyen d'hydrogène moléculaire, selon lequel un médiateur est mis à réagir avec de l'hydrogène par le biais d'une hydrogénase. Ce procédé peut être appliqué en particulier pour réduire des médiateurs chimiques ou des cofacteurs d'enzymes. Le domaine principal d'application dudit procédé est la réduction de NADP+ en NADPH et l'utilisation de l'enzyme décrite dans ce brevet pour régénérer le cofacteur de NADPH au moyen d'hydrogène moléculaire. L'avantage du procédé par rapport à tous les autres procédés établis pour la régénération de NADPH, réside en ce qu'il fait appel à l'hydrogène moléculaire, qui est très bon marché, pour la génération ou la régénération du cofacteur, ne donnant ainsi naissance à aucun produit secondaire à séparer. Dans ce cas, le produit secondaire est l'eau. Différentes purifications ou préparations de l'enzyme Pyrococcus furiosus peuvent être employées comme catalyseur.
EP02767065A 2001-08-21 2002-07-27 Procede de reduction enzymatique de substrats au moyen d'hydrogene moleculaire Withdrawn EP1419262A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10139958 2001-08-21
DE2001139958 DE10139958A1 (de) 2001-08-21 2001-08-21 Verfahren zur enzymatischen Reduktion von Substraten mit molekularen Wasserstoff
PCT/DE2002/002775 WO2003018824A2 (fr) 2001-08-21 2002-07-27 Procede de reduction enzymatique de substrats au moyen d'hydrogene moleculaire

Publications (1)

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EP1419262A2 true EP1419262A2 (fr) 2004-05-19

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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004007029A1 (de) * 2004-02-12 2005-09-08 Consortium für elektrochemische Industrie GmbH Verfahren zur enantioselektiven Reduktion von Ketoverbindungen durch Enzyme
US20070190596A1 (en) * 2006-01-20 2007-08-16 Jones Gerald S Jr Synthesis of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid
JP2013511973A (ja) * 2009-11-30 2013-04-11 ファルマツェル、ゲーエムベーハー 新規７β−ヒドロキシステロイドデヒドロゲナーゼおよびその使用

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WO2003018824A2 (fr) 2003-03-06
DE10139958A1 (de) 2003-03-20

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