DE10139958A1 - Process for the enzymatic reduction of substrates with molecular hydrogen - Google Patents

Abstract

The invention relates to a method for the enzymatic reduction of substrates with molecular hydrogen. According to the invention, a mediator is reacted with hydrogen using a hydrogenase. DOLLAR A In particular, this process can be used to reduce chemical mediators or cofactors of enzymes. The main application of this process is the reduction of NADP · + · to NADPH or the use of the enzyme described in the patent for the cofactor regeneration of NADPH with molecular hydrogen. The advantage of this process, compared to all established processes for the regeneration of NADPH, lies in the use of the very inexpensive molecular hydrogen for the generation or regeneration of the cofactor and therefore not in the formation of a by-product which can be separated still further. In this case, water is formed as a by-product. Various purifications and preparations of the enzyme Pyrococcus furiosus can be used as the catalyst.

Description

The invention relates to a method for enzymatic Reduction of substrates with molecular hydrogen.

More recently, more and more 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 still very expensive today, especially NADPH, there is a need for a process with which it is possible to produce NADPH in particular in a particularly inexpensive manner. Furthermore, a process that enables NADPH to be regenerated is of the greatest 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 because these cofactors are far too expensive to use stoichiometrically. The current method of chemical reduction of NADP ⁺ uses inexpensive and commercially available reagents, such as. B. sodium dithionite (Peters J. Dehydrogenases - characteristics, design of reaction conditions, and applications. In: Biotechnology, biotransformations I. Kelly Dr, Editor, ed. 8a, Weinheim: Wiley-VCH, 1998. p. 391-473), however, the complex disadvantages here are the elaborate product work-up, the formation of by-products and low yields (Chenault H, Whitesides G. Regeneration of nicotinamide cofactors for use in organic synthesis. Appl Biochem Biotech 1987, 14: 147-197). The enzymatic production of NADPH has also been described in various publications. The glucose dehydrogenase from Gluconobacter 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. Appl Microbiol Biotechnol 1989, 30: 337 -342), the alcohol dehydrogenase from Thermoanaerobium brockii (Lamed RJ, Keinan E, Zeikus JG. Potential applications of an alcoholaldehyde / ketone oxidoreductase from thermophilic bacteria. Enzyme Microb Technol 1981, 3: 144-148) and the malate dehydrogenase from Acromobacter parvulus (Suye S, Yokohama S. NADPH production from NADP ⁺ using malic enzyme of Achromobacter parvulus IFO-13182. Enz Microbial Technol 1985, 7: 418-424). The use of various hydrogenases is also already to be found in the scientific literature: for example the hydrogenase from Alkanigines eutrophus (Klibanov A, Puglisi A. The regeneration of coenzymes using immobilized hydrogenase. Biotech Lett 1980, 2: 445-450) and the hydrogenase Methanobacterium thermoautotrophicum (Wong CH, Daniels L, Orme-Johnson WH, Whitesides GM. Enzyme-catalyzed organic synthesis: NAD (P) H regeneration using dihydrogen and the hydrogenase from Methanobacterium thermoautotrophicum. J Am Chem Soc 1981, 103: 6227-6228) , All publications that describe hydrogenases to date for the use of cofactor regeneration do this either in relation to the direct reduction of NAD ⁺ , or to the indirect reduction of NADP ⁺ by means of a further mediator. The enzymes used according to the prior art are relatively unstable, go through a few cycles and have a low thermodynamic driving force for the reactions.

It is the object of the invention to provide a simple method for the direct reduction of mediators, in particular of NADP ^+, without the use of an additional, other mediator, which is used for the production of NADPH or the hydrogenated form of the mediators and the cofactor regeneration of NADPH, and the regeneration of other mediators can be used.

Starting from the preamble of claim 1 Object achieved in accordance with the characterizing Features specified in claim 1.

With the method according to the invention it is now possible to hydrogenate and thus regenerate mediators, in particular NADP ⁺ . The process is particularly inexpensive because of the inexpensive hydrogen as an agent. The removal of the reducing agent presents no difficulties 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 become.

Advantageous developments of the invention are in the Subclaims specified.

The invention is to be explained below.

According to the invention, a mediator is operated using a Hydrogenase reacted with molecular hydrogen.

The hydrogenase is preferably from the hyperthermophilic strain of the archaebacterium Pyrococcus furiosus (DSM 3638).

The hydrogenases can be used in situ.

According to the invention, a substance is used as a mediator referred to, the hydride ions or electrons Can transfer substances. These connections will, if they occur naturally in organisms as cofactors designated. Other substances that are functional with are equivalent to these natural cofactors referred to as mediators. For the purposes of the invention the term mediators all known mediators including natural cofactors mean that the term covers both equally acting types should.

Examples of mediators that can be mentioned are flavin mononucleotide (FMN), phenazine methosulfate (PMS), 2,6 dichlorophenolindophenol (DCPIP), Janus Grün, methylene blue, flavin adenine dinucleotide (FAD), but in particular NADP ⁺ . However, NAD ⁺ is excluded from the invention.

The mediators are inventively by means of Hydrogenase in the presence of hydrogen in your reduced form transferred. The products are the specialist known.

The reaction preferably takes place in an aqueous medium instead of.

The reaction can be in a pH range of 6-10 be carried out, but preferably takes place in a pH Range from 7 to 9, preferably from 7.5 to 8.5 instead because of the enzyme activity and stability of NADPH is largest here.

The reaction can take place in a temperature range of -10 ° C to 150 ° C, preferably in a range from 0 ° C to 100 ° C particularly preferably from 20 ° C to 80 ° C be performed.

The pressure range in which the reaction is carried out is preferably between 0 to 300 bar, particularly preferably between 0 and 20 bar. In the high Pressure ranges> 20 bar is the solubility of Hydrogen higher, so that more molecules for the solution Hydrogenation are available. In the printing areas Reactors below 20 bar are required, which not have to withstand such high pressures, so that the technical effort is less here. The However, the boundaries of the specified ranges are fluid.

At the pressures mentioned, the gas phase over the reaction solution can have different compositions. 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 which is inert for 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.

In a further development of the invention Reaction solution added another enzyme, which is a Implement substrate. For this second implementation with this Enzyme 2 can be the hydrogenase-converted Mediator as a cofactor or more generally as a mediator serve by the subsequent repeated implementation with the hydrogenase, is regenerated again by is hydrogenated with hydrogen.

The enzyme 2 can be, for example, an alcohol dehydrogenase, which converts a ketone to an alcohol. In such a cycle, for example, 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. In this example, acetophenone or acetone can be mentioned as substrates for the second reduction.

Other enzymes E2 are, for example Thermoanaerobium brockii (ADH), (Pyrococcus furiosus (GDH) available. Basically, the process of the invention the mediator regeneration with every further one Enzyme reaction coupled to the regenerated mediator needed.

Thus the hydrogenase reaction with another enzymatic reaction coupled. Typical examples for reactions of the enzyme 2, which with the Regeneration of the mediator can be coupled 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 both Production of mediators, as well as their in situ Regeneration used in coupled systems become. The coupled enzyme reaction with enzyme 2 is not disturbed by the hydrogen, but rather the hydrogen even provides an inert atmosphere for the reaction.

The process can be carried out in a batch reactor. Furthermore, the reaction according to the invention can be carried out in one Membrane reactor, as exemplified in the German patent 44 36 149 is carried out, which is equipped with an ultrafiltration membrane that allows the retention of the hydrogenase. The Membrane reactor can be used as a repetitive batch or as continuous reactor can be used. Litigation in continuously operated membrane reactor is special prefers.

In an advantageous embodiment of the method, the hydrogenase from Pyrococcus furiosus is used repeatedly in a "repetitive batch" process to produce NADPH. This results in low catalyst consumption rates and high productivity. In the experiment shown in Example 6, a maximum space-time yield of 10 gL ^-1 d ^{-1 was} achieved. In the experiment shown in Example 7, 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. In a further advantageous embodiment of the method, the hydrogenase from Pyrococcus furiosus is used for the cofactor regeneration of NADPH in the asymmetrical reduction of prochiral ketones by means of a dehydrogenase. In the experiment listed in Example 6, a cycle number of 100, in Example 1 of 320, is achieved.

• - Bei der hier verwendeten Hydrogenase aus Pyrococcus furiosus handelt es sich um ein besonders stabiles Enzym, welches wie durch die repetitiv batch Experimente gezeigt, vielfach wiederverwendet werden kann.
• - Der Katalysator kann sowohl in gekoppelten als auch alleinig eingesetzt werden.
• - Da die Hydrogenase aus einem thermophilen Stamm kommt, kann sie wie in den Ansprüchen beschrieben, über einen weiten Temperaturbereich eingesetzt werden.

The following advantages result from the procedure presented here:

• - 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.
• - Since the hydrogenase comes from a thermophilic strain, it can be used over a wide temperature range as described in the claims.

The implementation of the pyrodine nucleotides according to the invention is not subject to any thermodynamic limitation. This is surprising and particularly advantageous because higher Turnover than for those coupled with cosubstrates Regeneration systems can be achieved.

The figures show test results:

It shows:

Fig. 1 sales-time curve for the reaction acetophenone → phenylethanol Examples 4 and 5.

Fig. 2 Repetitive batch experiments for the reaction of Fig. 1 (Example 6).

Fig. 3 yield of NADPH for repetitive batch experiments according to Example 7.

Fig. 4 dependence of the NADPH formation on the temperature.

Description of the catalyst preparation

• 1. Zellfreies Extrakt wurde mittels Ultrafiltration (Amicon YM 10 Membran) auf 30 mg/mL aufkonzentriert und über Nacht mit 50 mM Tris-HCl pH 8,0 Puffer dialysiert.
• 2. Zu dem zellfreien Extrakt wurde Ammoniumsulfat hinzugesetzt, bis zu 10%iger Sättigung. Die so erhaltene Suspension wird für 15 min. bei 30 000 g und 4°C zentrifugiert und der Überstand mit Ammoniumsulfat bis zu 60% gesättigt. Nach einem weiteren Zentrifugationsschritt, wird das Pellet resuspendiert und gegen 50 mM Tris-HCl-Puffer pH 8 dialysiert. Die Proteinkonzentration beträgt 30 mg/mL.
• 3. Das zellfreie Extrakt wird auf eine Source 15Q- Säule (Pharmacia) aufgetragen und mit 250 mM NaCl eluiert. Die Fraktionen, welche Hydrogenaseaktivität enthalten, werden mittels Ultrafiltration (Amicon YM 30 Membran) bis 30-50 mg Protein/mL aufkonzentriert.

In the invention presented here, an in vitro method is described which enables molecular hydrogen as a reducing agent for the preparation of pyridine nucleotides and dyes by means of a soluble hydrogenase. Pyrococcus furiosus (DSM 3638) was cultivated according to the instructions given in the literature, with the addition of table salt and potato starch (Hagedoorn, PL., MCPF Driessen, M. van den Bosch, I. Landa, and WR Hagen. 1998. Hyperthermophilic redox chemistry : a re-evaluation. FEBS Lett. 440: 311-314). The cells were disrupted by autolysis. For this purpose, 100 g of bio-moist mass were suspended in 400 mL of 50 mM Tris-HCl pH 8.0, and 10 µg / mL DNase and RNase were added. After 4 h at room temperature, the cell extract was centrifuged for 1 h at 30,000 g. 3 different preparations were made from the cell supernatant (diluted, cell-free extract, 5-10 mg protein / mL):

• 1. Cell-free extract was concentrated to 30 mg / mL by means of ultrafiltration (Amicon YM 10 membrane) and dialyzed overnight with 50 mM Tris-HCl pH 8.0 buffer.
• 2. Ammonium sulfate was added to the cell-free extract, up to 10% saturation. The suspension thus obtained is for 15 min. Centrifuged at 30,000 g and 4 ° C and the supernatant saturated with ammonium sulfate up to 60%. After a further centrifugation step, the pellet is resuspended and dialyzed against 50 mM Tris-HCl buffer pH 8. The protein concentration is 30 mg / mL.
• 3. The cell-free extract is applied to a Source 15Q column (Pharmacia) and eluted with 250 mM NaCl. The fractions, which contain hydrogenase activity, are concentrated by ultrafiltration (Amicon YM 30 membrane) to 30-50 mg protein / mL.

Two soluble hydrogenases are present in the soluble cell-free fraction of the hypothermophilic archae bacterium Pyrococcus furiosus (Ma, K., Weiss, R. and Adams, MWW Characterization of hydrogenase II from the hyperthermophilc archaeon Pyrococcus furiosus and assessment of its role in sulfur reduction. J. Bacteriol., 2000, 1864-1871). The hydrogenase 1 (90% of the total activity) elutes from the ion exchange chromatography column at 0.25 M NaCl and the hydrogenase 2 at 0.4 M NaCl (10% of the total activity). The fractionation was carried out under an argon atmosphere and the fractions are stored at -80 ° C. 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, a phosphatase activity which is still present and which catalyzes the dephosphorilation of NADP ⁺ to NAD ^{+ must first be} removed. The corresponding activities in 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 phophodiesterase 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.

example 1

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 μmol), 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 is more unstable than the added NADP ⁺ . 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.

Example 2

The standard oxidation and reduction potentials of H ⁺ -H ₂ and an 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 equilibrium of 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 . This was demonstrated by carrying out the following test approach: 3.9 units of hydrogenase were incubated under a hydrogen atmosphere in a volume of 3 ml of 1 M EPPS buffer, pH 8.0, 110 mM NADP ⁺ . Quantitative conversion was achieved after 240 minutes.

Example 3

The reductive amination of two ketogluterates to glutamate is catalyzed by glutamate dehydrogenase (GDH), which is dependent on NADPH. 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 to achieve 100% conversion of 2-ketogluterate to glutamate, which corresponds to a cycle number (TTN) of 100 under the aforementioned reaction conditions. 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

Example 4

A solution of 10 mM acetophenone in 50 mM aqueous potassium phosphate buffer (pH 8) with 0.5 mM oxidized cofactor NADP ⁺ and a NADP ⁺ -dependent alcohol dehydrogenase (ADHM) is degassed by flowing the solution with water-saturated helium. 20 μL 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 0.1 mg / 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. Samples are taken from the stirred reaction solution and analyzed without further processing by gas chromatography (GC) with regard to conversion and enantiomeric excess or ratio.

There was quantitative turnover after 3 hours at one Enantiomeric excess greater than 99.5% reached.

GC: Agilent 6890GC with a Chirasil-Dex capillary column (Chrompack-Varian, Germany) Fig. 1 (circles) shows the turnover-time curve.

The number of cycles (TTN) with respect to NADP ⁺ corresponds to 20.

Example 5

Concentrations as in Example 4, the Cofactor concentration was decreased to 0.1 mM.

The turnover-time curves for both cofactor concentrations are shown in FIG. 1 (triangles).

Quantitative conversion was achieved after 3.5 hours with an enantiomeric excess greater than 99.5%. The number of cycles (TTN) with respect to NADP ⁺ corresponds to 100.

Example 6

In a modified ultrafiltration cell (Amicon, Germany), which is equipped with an ultrafiltration membrane (YM10, Amicon), the solutions described above are filled in under an argon atmosphere. The cell outlet is opened periodically and the permeate of the ultrafiltration cell is analyzed by GC for conversion and yield. The outlet was checked for protein content with a negative result. After more than 95% of the conversion had been reached, the cell contents were filtered to a minimal residual volume and again filled with the same solution. During the filling, only alcohol dehydrogenase (ADHM) was replenished at the times that were identified in FIG. 2.

Starting with the sixth cycle, the substrate from acetophenone to (S) -2-hydroxy-1-phenyl-propanone changed. As a product Obtained 1,2-dihydroxyphenylpropanone with high enantiomeric excess.

The cycle was repeated eight times. The results of the first six cycles are shown in FIG. 2.

Example 7

In 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 introduced} under an argon atmosphere. The cell outlet is opened periodically and the permeate of the ultrafiltration cell is analyzed for conversion and yield by means of capillary electrophoresis. The outlet was checked for protein content with a negative result. After more than 95% of the conversion was achieved (with respect to NADP ⁺ ), the cell contents were filtered down to a minimal residual volume (approx. 0.2 mL) and filled again with the same solution. Various hydrogen pressures were applied.

The cycle was repeated eight times. It was from 7th cycle the pressure increased to 7.5 bar hydrogen. It no hydrogenase preparation was added. The Permeate was examined using capillary electrophresis. (Beckmann PACE MDQ, capillary: fused silica, 24 ° C, U = 20 kV, c (total) = 100 mM, 50% potassium phosphate (50 mM Potassium phosphate + 50 mM borate), pH = 6)

The results are shown in FIG. 3.

Claims (15)

1. A process for the enzymatic reduction of substrates with molecular hydrogen, characterized in that a hydrogenase is used as the enzyme and a mediator except NAD ^{+ is used} as the substrate without the addition of a second mediator. 2. The method according to claim 1, characterized, that the hydrogenase from Pyrococcus furiosus is used. 3. The method according to any one of claims 1 or 2, characterized in that the mediator is a component from the group NADP ⁺ , FMN, PMS, DCPIP, Janus Green, methylene blue and FAD. 4. The method according to any one of claims 1 to 3, characterized, that the reaction is carried out in aqueous medium becomes. 5. The method according to any one of claims 1 to 4, characterized, that the reaction in a pH range between 6 and 10 takes place. 6. The method according to any one of claims 1 to 5, characterized, that the implementation in a temperature range between -10 ° C and 150 ° C is carried out. 7. The method according to any one of claims 1 to 6, characterized, that the reaction in a pressure range between 0 and 300 bar is carried out. 8. The method according to claim 7, characterized, that the reaction in a pressure range between 0 and 20 bar is carried out. 9. The method according to any one of claims 1 to 8, characterized, that the hydrogen in pure form or in one Mixture with a gas inert to the process is present and the liquid phase is covered. 10. The method according to claim 9, characterized in that the inert gas is at least one component from the group consisting of CO ₂ , He, Ar, Kr or N ₂ . 11. The method according to claim 9 or 10, characterized, that the hydrogen concentration in the gas phase is 50 is up to 100%. 12. The method according to any one of claims 1 to 11, characterized, that the reaction mixture a second enzyme and a second substrate is added, which to a Product is implemented so that a coupling with another chemical process takes place. 13. The method according to claim 12, characterized, that as a second enzyme an alcohol dehydrogenase is added. 14. The method according to claim 12 or 13, characterized, that a ketone is used as the second substrate. 15. The method according to any one of claims 1 to 14, characterized, that the reaction in a membrane reactor is carried out, which with a Ultrafiltration membrane is equipped, which the retention of Enzymes causes.