EP1419262A2

EP1419262A2 - Method used for the enzymatic reduction of substrates by molecular hydrogen

Info

Publication number: EP1419262A2
Application number: EP02767065A
Authority: EP
Inventors: Andreas Liese; Rita Mertens; Lasse Greiner; Eyke Van Den Ban; Huub Haaker
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2001-08-21
Filing date: 2002-07-27
Publication date: 2004-05-19
Also published as: DE10139958A1; WO2003018824A2; WO2003018824A3

Abstract

The invention relates to a method for the enzymatic reduction of substrates by molecular hydrogen, according to which a mediator is reacted with hydrogen using hydrogenase. The method is especially useful for reducing chemical mediators or cofactors of enzymes. The method is mainly utilized to reduce NADP<+> to NADPH or to use the enzyme described in the application to regenerate the cofactor of NADPH by molecular hydrogen. The advantage of the inventive method over all the methods for regenerating NADPH so far described is the use of inexpensive molecular hydrogen for generating or regenerating the cofactor, and thereby avoiding an additional by-product to be removed. In this case, water is formed as the by-product. As a catalyst, various purifications and preparations of the enzyme Pyrococcus furiosus can be used.

Description

Process for the enzymatic reduction of substrates with molecular hydrogen

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

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 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 current method of chemical reduction of NADP ⁺ uses inexpensive and commercially available reagents such as 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), but here, in particular, the elaborate product processing is the Formation of by-products and low yields have the major disadvantages (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 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.

Appl. Microbiol. Biotechnol. 1989, 30: 337-342)

Alcohol dehydrogenase from Thermoanaerobium brockii

(Lamed RJ, Keinan E, Zeikus JG. Potential applications of an alcohol-aldehyde / ketone oxidoreductase from thermophilic bacteria. Enzyme Microb. Technol. 1981, 3: 144-148) and the malate dehydrogenase from Äcromobacter parvulus (Suye S, Yokohama S. NADPH production from NADP ⁺ using malic enzyme of Achromobacter parvulus IFO- 13182. Enz. Microbiol. 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 from 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 thermoautotrophicu. J. Am. Chem. Soc. 1981, 103: 6227-6228). All publications that have been using hydrogenases for the use of cofactor describe generation, do this either related to the direct reduction of NAD ⁺ , or to the indirect reduction of NADP ⁺ using another 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 process 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, the object is achieved according to the invention with the features specified in the characterizing part of 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 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.

Advantageous developments of the invention are specified in the subclaims.

The invention is to be explained below.

According to the invention, 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.

According to the invention, 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. For the purposes of the invention, the term 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. According to 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. In the high pressure ranges> 20 bar, the solubility of hydrogen is higher, so that more molecules are available for the hydrogenation in the solution. In the pressure ranges below 20 bar, reactors are required which cannot withstand such high pressures, so that the technical outlay here is lower. However, the boundaries of the specified areas are fluid. At the pressures mentioned, 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.

In a further development of the invention, a further enzyme is added to the reaction solution, which converts a substrate. For this second reaction with this enzyme 2, 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. 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. Further enzymes E2 are available, for example, from Thermoanaerobium brockii (ADH), (Pyrococcus furiosus (GDH). In principle, 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.

In an advantageous embodiment of the method, 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. In the experiment shown in Example 6, a maximum space-time yield of 10 gL ^"1 d ^{" 1 was} achieved. In the experiment given 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 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 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 since higher sales are achieved than in the case of regeneration systems coupled with cosubstrates.

The figures show test results: It shows:

Fig.l: Sales-time curve for the reaction acetophenone - phenylethanol examples

4 and 5. Fig. 2: Repetitive batch tests for the reaction from Fig. 1 (Example 6). 3: Yield of NADPH for repetitive batch tests according to Example 7.

Fig. 4: Dependence of the NADPH formation on the temperature.

Description of the catalyst preparation

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-moisturizer were added 400 mL 50 mM Tris-HCl pH 8.0 suspended, and 10 μg / 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):

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) up to 30-50 mg protein / mL.

Two different 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). Hydrogenase 1 (90% of the total activity) elutes from the ion exchange chromatography column at 0.25 M NaCl and 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, 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.

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 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.

Example 2:

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 . This was demonstrated by carrying out the following test batch: 3.9 units of hydrogenase were incubated under a hydrogen atmosphere in a volume of 3 ml 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 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.

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 (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 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. 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. 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. Quantitative conversion was achieved after 3 hours with an enantiomeric excess greater than 99.5%. GC: Agilent 6890GC with a Chirasil-Dex capillary column (Chrompack-Varian, Germany) Figure 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 reduced 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 introduced 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. When filling, only alcohol dehydrogenase (ADHM) was replenished at the times which were identified in FIG. 2.

Starting with the sixth cycle, the substrate was changed from acetophenone to (S) -2-hydroxy-l-phenyl-propanone. 1, 2-Dihydroxyphenylpropanon with high enantiomeric excess was obtained as product.

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

The cycle was repeated eight times. The pressure was increased to 7.5 bar hydrogen from the 7th cycle. No hydrogenase preparation was added. The permeate was examined by 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 shown.

Claims

Patent claims

1. Process for the enzymatic reduction of substrates with molecular hydrogen, characterized in that a hydrogenase as the enzyme and as a substrate a mediator except NAD ⁺ without the addition of a second

Mediators is used.

2. The method according to claim 1, characterized in 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 in that the reaction is carried out in an aqueous medium.

5. The method according to any one of claims 1 to 4, characterized in that the reaction takes place in a pH range between 6 and 10.

6. The method according to any one of claims 1 to 5, characterized in that the reaction is carried out in a temperature range between -10 ° C and 150 ° C.

7. The method according to any one of claims 1 to 6, characterized in that the reaction is carried out in a pressure range between 0 and 300 bar.

8. The method according to claim 7, characterized in that the reaction is carried out in a pressure range between 0 and 20 bar.

9. The method according to any one of claims 1 to 8, characterized in that the hydrogen in pure form or in one

Mixture with a gas which is inert for the process is present and the liquid phase is covered.

10. The method according to claim 9, characterized in that the inert gas at least one component from the

Group consisting of C0 ₂ , He, Ar, Kr or N ₂ .

11. The method according to claim 9 or 10, characterized in that the hydrogen concentration in the gas phase is 50 to 100%.

12. The method according to any one of claims 1 to 11, characterized in that a second enzyme and a second substrate is added to the reaction mixture, which is converted into a product, so that a coupling takes place with another chemical process.

13. The method according to claim 12, characterized in that an alcohol dehydrogenase is added as the second enzyme.

14. The method according to claim 12 or 13, characterized in that a ketone is used as the second substrate.

15. The method according to any one of claims 1 to 14, characterized in that the reaction is carried out in a membrane reactor which is equipped with an ultrafiltration membrane which effects the retention of enzymes.