NL9400836A - Stable ADP-dependent kinases, and preparation and use thereof - Google Patents

Abstract

The invention relates to an enzyme preparation which catalyzes the phosphorylation and/or dephosphorylation of a substrate other than adenosine diphosphate (ADP) or polyphosphate, wherein ADP is converted into adenosine monophosphate (AMP). In particular, the substrate is a sugar or a sugar derivative. The enzyme of the preparation is novel and is active at a temperature above 70 degree C. The enzyme can be prepared from hyperthermophilic microorganisms such as those of the genus Pyrococcus.

Description

Stable ADP-dependent kinases, their preparation, as well as their use

The invention relates to enzymes and enzyme preparations which catalyze the phosphorylation of substrates in which ADP is converted into AMP. The invention also relates to expression systems and microorganisms capable of producing these enzymes. The invention also relates to methods for the preparation and use of such ADP-dependent kinases with high stability.

Background

Enzymes are capable of reversibly catalyzing biochemical conversion and are widely used in various branches of industry such as the pharmaceutical and food industries. In addition, enzymes are essential for many diagnostic determinations, such as those used in health care or research and development laboratories. The enzymes used here often come from micro-organisms. Micro-organisms generally lend themselves well to the efficient and large-scale production of enzymes. In addition, microorganisms can be used as a source of DNA fragments encoding useful enzymes. It is well known that by using genetic engineering techniques, introducing such DNA fragments into suitable hosts, new microorganisms can be obtained that can efficiently produce the desired enzymes.

An important factor determining the utility of enzymes is their activity. The temperature at which the conversion is carried out plays a very important role here [for an overview, see P.W. Hochachka and G.N. Somero (1973) Strategies of Biochemical Adaptation, W.B. Saunders Company, Philadelphia]. It is well known that enzymatic reactions proceed faster as the temperature is raised. In addition, the temperature influences the substrate affinity of some enzymes. Furthermore, by choosing an appropriate temperature, the solubility of one or more reactants can be changed, thereby usefully influencing the equilibrium of the reactions. Finally, there are conversions that can only be carried out at high temperatures, such as, for example, the polymerase chain reaction.

There is therefore a great need for enzymes that have a wide temperature range and are active at high temperatures. Enzymes from their microorganisms have generally been found to have high activity at high temperatures and are therefore very suitable for a number of applications [H. Rossi (1986) European Conference on Biotechnology, European Institute of Technology & Conzorzio ZAI, Verona, pp. 46-50, Verona; R. Jaenicke and P. Zavodsky (1990) FEBS Lett. 268: 344-349; D. Cowan (1992) Trends Biotechnol. 10: 315 * 323; R.A. Herbert (1992) Trends Biotechnol. 10: 395 * 402].

The utility of enzymes is determined not only by their activity but also by their stability. This stability is necessary not only during the conversion but also in the stage before the enzymes are used. In many cases, special precautions must be taken to improve the stability of enzymes during preparation, transport and storage. In a number of cases this can be achieved by drying enzymes. However, this is an expensive procedure. In addition, many enzymes lose their activity during drying. Finally, enzymes are often used in conversions where water is used as a solvent and the dried enzyme has to be dissolved again. Another way in which stability can be increased is by storing aqueous solutions of enzymes in cooled or frozen form. However, this entails large cooling, transport and storage costs. Therefore, there is a great need for enzymes that have inherently high stability. Enzymes from thermophilic microorganisms are known to have high physical and chemical stability, thereby increasing the scope of the enzyme [Rossi (1988); Jaenicke and Zavodsky (1990); Cowan (1992); Herbert (1992); vide supra]. The use of such enzymes for many applications therefore offers great advantages.

The enzymatic activation of substrates by means of phosphorylation plays a crucial role in the metabolism of living things and phosphate transfer is a basic reaction in biochemistry [L. Stryer, Biochemistry (1988) W.H. Freeman & Company, New York]. Not surprisingly, enzymes have been isolated from numerous organisms to catalyze this phosphorylation in a reaction that converts the energy carrier adenosine triphosphate (ATP) into adenosine diphosphate (ADP) according to the reaction below where X is the substrate and XP is the substrate with an additional phosphate represents:

X + ATP - X-P + ADP

These enzymes belong to the group of kinases and well-known examples are the enzymes hexokinase, glucokinase and phosphofructokinaee. Hexokinases and glucokinases catalyze the conversion of glucose to glucose-6-phosphate with different efficiency while phosphofructokinae converts fructose-6-phosphate to fructose-1,6-diphosphate. These and other kinases are widely used in diagnostic tests where the concentration of the starting materials can be determined [J. Keesey (1987) Biochemica Information (1st Edition) Boehringer Mannheim Bio-chemicals, Germany]. Since all enzymatic reactions are reversible, use is also made of the reverse reaction in which ATP is formed from a phosphorylated substrate and ADP as indicated above. In particular, there is interest in the determination of sugars and their degradation products that are formed in living cells. A well-known example is the determination of glucose using hexokinase. The yeast hexokinase, which has a molecular weight of 96 kDa [H.R. Mahler and E.H. Cordes (1975) Biological Chemistry, Harper & Row Publishers, New York].

A problem with the above phosphorylation is the dependence on ATP. ATP is not always available in a conversion, for example in the case of a coupled reaction in which ATP is converted into ADP. In addition, ATP is unstable at high temperatures in the presence of divalent ions and hydrolyses faster than, for example, ADP [M. Tetas and J.M. Lowenstein (1963) Biochemistry 2, pp. 350-357] The presence of divalent cations in phosphotransferase reactions is often necessary. Finally, ATP is a costly connection. These problems are nonexistent or greatly reduced for ADP-dependent kinases that form adenosine monophosphate (AMP) according to the following reaction in which X is the substrate and X-P represents the substrate with an additional phosphate:

X + ADP - X-P + AMP

A very limited number of ADP-dependent phosphorylations are known. For example, adenylate kinase catalyzes the conversion of 2 ADP to ATP and AMP. In addition, there is a polyphosphate: AMP phosphotransferase that dephosphorylates polyphosphate while AMP is phosphorylated. Although these enzymes have potential applications, they are unable to phosphorylate substrates other than ADP or polyphosphate.

In particular, these enzymes are unable to phosphorylate sugars and their breakdown products. In addition, in the case of adenylate kinase, ATP is again formed which, as indicated above, is not stable at high temperature, and the equilibrium of the reaction of the polyphosphate: AMP phosphotransferase strongly depends on the formation of ADP and dephosphorylation of polyphosphate.

In recent years, a large number of thermophilic and hyperthermophilic microorganisms have been described [K.O. Stetter, G. Fiala, G. Huber, R. Huber, and G. Segerer (1990) FEMS Microbiol. Rev. 75: 117-124]. Various thermostable and thermoactive enzymes have already been described from these organisms. An excellent example is the Archaeon Pyrococcus furiosus, belonging to the Thermococcales family, which is one of the most extremely thermophilic micro-organisms with an optimal growth temperature of 100 aC [G. Fiala and K.O. Stetter (1986) Arch. Microbiol. 145: 56-61; Stetter et al. (1990) vide supra]. It is known that enzymes produced by P. furiosus can be counted among the most thermostable and thermoactive. For example, the B-glucosidase of P. furiosus still has about half of its activity after heating in aqueous solutions at a temperature of 100 ° C for 85 hours [S.W.M. Kengen, E.J. Luesink, A.J.M. Stams, A.J.B. Zehnder (1993) Eur. J. Biochem. 213: 305-312].

From the above, it is clear that there is a need for ADP-dependent kinases that catalyze a reaction in which the substrate is not ADP or polyphosphate. However, such ADP-dependent kinases have not been described to date.

Description of the invention

The invention relates to a new group of ADP-dependent kinases that have high thermoactivity and high thermal stability and catalyze a reaction in which the substrate is not ADP or polyphosphate. In particular, the invention relates to an ADP-dependent hexokinase and phosphofructokinase from the hyperthermophilic microorganism Pyrococcus furiosus.

Surprisingly, it has been found that ADP-dependent kinases exist in the hyperthermophilic organism Pyrococcus furiosus, which has a maximum growth temperature of 103 * C, but still shows growth at 70 * C [Fiala and Stetter (1986) vide supra]. In particular, considerable activity has been found of an ADP-dependent hexokinase subsequently obtained in purified form and consisting of polypeptides with a molecular weight of about 50 kDa. The ADP-dependent kinase also appears to be in slide form. In addition, considerable activity of an ADP-dependent phosphofructokinase has also been found in P. furiosus. These discoveries indicate that there is a new family of ADP-dependent kinases, the existence of which was previously unknown or even suspected. In view of the higher thermal stability of ADP over ATP [Tetas and Lowenstein (1963) vide supra], it is believed that there are more ADP-dependent enzymes in P. furiosus, the existence of which is not yet known.

Another species of the genus Pyrocoeeus, P. woesei [W. Zillig, et al. (1987) Syst. Appl. Microbiol. 9: 62-70] has now been found to contain ADP-dependent hexokinase and phosphofructokinase. These and other ADP-dependent kinases will also be present in other species of the genus Pyrococcus and other representatives of the Thermococcales. Because these microorganisms belong to the Archaea [Stetter et al. (1990) vide supra], ADP-dependent hexokinase, phosphofructokinase and other kinases may also exist in other representatives of this super kingdom or domain that occupy a separate phylogenetic position [ CR Woese, 0. Kandier and M.L. Wheelis (1990) Proc. Natl. Acad. Sci. USA 87: 4576-579] In addition, it is also believed that other thermophilic microorganisms, which have the ability to grow above a temperature of 70 ° C, have ADP-dependent hexokinase, phosphofructokinase and other kinases. Finally, since the existence of ADP-dependent hexokinase and phosphofructokinase was not yet known, it is quite possible that, after targeted searches, such ADP-dependent kinases can also be found in other, non-thermophilic microorganisms.

The use of ATP-dependent hexokinase and ATP-dependent phosphofructokinase in diagnostic assays is known [Keesey (1987) vide supra]. Similar applications can be realized in the family of stable ADP-dependent kinases described here. In addition, as indicated above, other, for example linked, reactions can also be caused by such ADP-dependent kinases. The activity of ADP-dependent kinases can be easily determined in the cell-free extract of P. furiosus. This shows that the use of such enzymes is not limited to purified enzymes. Despite the fact that P. furiosus can only grow under anaerobic conditions, the ADP-dependent hexokinase and phosphofructokinase also appear to function well under aerobic conditions, which is consistent with the activity of many other enzymes from this organism [see Kengen et al (1993) vide supra]. As a result, these ADP-dependent enzymes have excellent applications.

The ADP-dependent hexokinase has also been purified and can be used to determine the N-terminal amino acid sequence. This sequence can then be used to isolate DNA fragments encoding the ADP-dependent hexokinase from P. furiosus. These DNA fragments can then be used for the production of this enzyme in suitable hosts such as Escherichia colt. An analogous example for another enzyme from P. furiosus has recently been described [H.I.L. Eggen, A.C.M. Geerling, K. Waldkötter, G. Antranikian & W.M. de Vos (1993) Gene 132: 143-1 ^ 8]. Then, the gene encoding the ADP-dependent hexokinase can be used to detect similar homologous genes from other microorganisms. This can be done by DNA-DNA hybridization under different conditions or, if the nucleotide sequence of the gene has been determined, by amplification via the polymerase chain reaction (PCR) [see, inter alia, J. Sambrook, E.F. Fritsch, & T. Maniatis (1989) Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.]. Given that the hexokinase and also other enzymes such as the S-glucosidase (vide supra) can be purified from known P. furiosus purification methods, such a genetic approach could also be applied to the ADP-dependent phosphofructokinase and other ADP-dependent kinases. The genes found can then be expressed in suitable hosts and the obtained enzyme activity can be used in ADP-dependent conversions.

The enzyme preparations according to the invention may, in addition to the new ADP-dependent enzyme, contain other enzymes, optionally other finely divided cell residues, diluents such as water, stabilizers and the like. Preferably, the preparations are homogeneous. They can also have a dried, for example spray-dried or freeze-dried, form.

The invention relates to methods for the preparation of ADP-dependent enzymes and enzyme preparations, both by culturing microorganisms containing these enzymes and separating them from the enzyme or enzyme mixture, and by culturing recombinant cells containing the genetic code for this enzyme.

The invention also relates to enzymatic assays in which the above-described enzymes and enzyme preparations are used. The invention also covers diagnostic kits for performing such determinations. These kits contain the enzyme or enzyme preparation in suitable forms, and preferably further reactants, such as ADP in the case of a sugar determination or glucose in the case of an ADP determination, and further the necessary aids such as diluents, detecting agents, manuals for the performing diagnostic determinations.

V & orbfifill.l

This example describes the method for the determination of hexo * kinase activity in cell extracts of the hyperthermophilic microorganism Pyrococcus furiosus. P. furiosus was obtained from the Deutsche Sammlung ftlr Microorganisms (DSM) of Braunschweig, Germany, under number DSM 3638, and was grown as described [Kengen et al. (1993). vide supra]. Standard procedures such as enzyme and protein assays were performed as described [Kengen et al. (1993) * vide supra].

Cell extracts were obtained by treating cell suspensions of Pyrococcus furiosus (1 gram / ml Tris / Cl 'buffer; 10 mM, pH 7.8) with the French press (138 MPa). Cell debris was removed by centrifugation (60 min, 100,000xg). The supernatant was used as a cell-free extract. The protein content is between 23 and 35 mg / ml. The cell extracts were not treated anaerobically, since the hexokinase is oxygen stable.

Hexokinase activity was measured in a coupled assay, converting the glucose-6-phosphate formed into 6-phosphogluconate using yeast glucose-6-phosphate dehydrogenase (Boehringer Mannheim, Germany). The associated reduction of NADP to NADPH2 is used as a measure of activity. Since the yeast enzyme is not stable at high temperature, the assay was performed at 50 ° C. The hexokinase of P. furiosus is still sufficiently active at this temperature to be measured. Thus, the complete reaction mixture for determining hexokinase activity contains; Tris / Cl 'buffer (100 mM; pH 7.8). glucose (15 mM), ADP (2 mM). Mg2 * (10 mM). NADP (0.5 mM), glucose-6-phosphate dehydrogenase (1.75 units) and 10 μΐ cell-free extract of P. furiosus. When ADP was replaced by ATP, low hexokinase activity in the cell-free extract could be demonstrated. However, this was due to contamination of ATP with trace ADP.

In this way, high activities ADP-dependent hexokinase could be demonstrated in cell-free extracts of P. furiosus. Depending on the growth substrate used, values up to 1.4 μαοί glucose were converted to glucose-6-phosphate per min per mg protein (50 * C). Assuming that the Q10 of the reaction rate is 2 [Hochachika and Somera (1973) vide supra], a specific activity can be calculated of 45 µl / min.mg protein at 100 ° C.

Example 2.

This example describes the enzyme assay for detecting phosphofructokinase in cell-free extracts of P. furiosus. Like the hexokinase, this enzyme also depends on ADP instead of ATP.

Phosphofructokinase was measured in an assay, in which the ADP-dependent formation of fructose-1,6-bisphosphate from fructose-6-phosphate was coupled to the oxidation of NADK using aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase (rabbit auxiliary enzymes supplied by Boerhinger Mannheim, Germany). The complete reaction mixture for the determination of phosphofructokinase contained: Tris / Cl "buffer (100 mM; pH 7.8), dithiothreitol (1 mM), ADP (2 mM), Mg24 (10 mM), NADH (0.4 mM), fructose -6-phosphate (4 mM), aldolase (0.22 units), triose phosphate isomerase (11.4 units), glycerol-3 ~ phosphate dehydrogenase (3.7 units) and 20 μΐ cell-free extract of P. furiosus.

In this way, specific activities were achieved up to 0.05 μπιοί fructose-6-phosphate converted to fructose-1,6-bisphosphate per minute per mg of protein (50 * C). At 100 * 0, the specific activity is then 1.6 lanol / min.mg protein.

Example 3

In this example, it is described that the ADP-dependent hexokinase and phosphofructokinase also occur in cell extracts of Pyrococcus uoesei. P. uoesei DSM 3773 was obtained, grown and treated as described above for P. furiosus. In a cell-free extract, an ADP-dependent hexokinase activity of 1 pmol / min.mg protein and an ADP-dependent phosphofructokinase activity of 0.05 µmol / min.mg protein were found at 50 ° C.

Example 4

This example describes the purification and characterization of the ADP-dependent hexokinase from P. furiosus. Standard biochemical operations were performed as previously described [Kengen et al. (1993). vide supra].

Cell-free extract was prepared by suspending 25 g of cells of P. furiosus in 25 ml of 100 aM Tris / Cl "buffer (pH 7-8). The cell suspension was treated once with the French Press at 138 MPa and cell debris removed by centrifugation for 30 min at 100,000 x g The supernatant with a protein content of 29 µg / ml was used in further purifications.

As the first purification step, an ammonium sulfate precipitation was performed and it was found that the hexokinase precipitated between 60% and 70% ammonium sulfate. Then the supernatant from a 58% ammonium sulfate precipitation was used for hydrophobic interaction chromatography using a Phenylsepharose CL-B column (Pharmacia. Uppsala, Sweden). The column was eluted with a decreasing gradient of ammonium sulfate (2.5 M to 0 M) in 100 mH Tris / Cl 'buffer (pH 7.8). The hexokinase was found to elute at 2 N ammonium sulfate. The active fractions were then concentrated and desalted by ultrafiltration with an Amicon YM-5 filter (5 kDa cut-off). To the active concentrate was added 5M of the detergent 3 * ([3'Cholamidopropyl] -dimethylammonio) -1-propanesulfonate (CHAPS; Sigma Chemie, Axel, The Netherlands). Subsequently purification was further carried out by chromatography on a Mono-Q HR anion exchanger (Pharmacia). Active fractions eluted at 0.18 M NaCl in 100 mM Tris / Cl 'buffer (pH 7.8) and 1 mM CHAPS. After desalting by Amicon YM-5 ultrafiltration, the active fractions were further purified by chromatography on a hydroxylapatite column eluting with a gradient of 1-50 mM sodium phosphate (pH 6.8; 1 mM CHAPS). Active fractions eluted at 0.33 mM phosphate. The protein was then bound to a Phenyl-Superose HR column and eluted via a decreasing gradient of ammonium sulfate in 50 mM sodium phosphate (pH 7.0). Active fractions eluted at 0.58 M ammonium sulfate. After desalting, a second Mono-Q HR purification was performed in 50 mM PIPES buffer (pH 6.2). The hexokinase was found to elute at 0.2 M NaCl.

The thus purified hexokinase gave a band with an apparent molecular weight of about 46 kDa on an SDS polyacrylamide gel electrophoresis. Native polyacrylamide gel electrophoresis revealed a molecular weight of about 92 kDa. From this it can be concluded that the hexokinase ie consists of two Identical subunits.

The purified protein has a specific activity of 224 /anol / min.mg protein (50'C). The cell-free extract that served as the starting material in this purification had a specific activity of 0.228 janol / min.mg protein. Thus, a purification factor of 982 times was achieved.

For glucose and ADP, a K 2 of 0.8 mM and 0.042 mM was determined, respectively.

With regard to specificity, it has been found that ADP could not be replaced by possible other phosphoryl group donors, namely ATP, GDP, phosphoenol pyruvate (PEP) or pyrophosphate (PPj).

The hexokinase is oxygen stable, i.e. the enzyme is not inactivated under normal atmospheric conditions. The purified protein was also not oxygen sensitive.

Claims (17)

1. Enzyme preparation which catalyzes the phosphorylation and / or dephosphorylation of a substrate, is characterized in that it contains an enzyme that catalyzes the phosphorylation of a substrate other than ADP or polyphosphate, converting ADP to ANP. Enzyme preparation that catalyzes the phosphorylation and / or dephosphorylation of a substrate, characterized in that it contains an enzyme that catalyzes the phosphorylation of a substrate, converting ADP into AMP, and is active at a temperature above 70 ° C . Enzyme preparation according to claim 1 or 2, characterized in that the substrate is a sugar or a degradation product of a sugar. Enzyme preparation according to any one of claims 1 to 3, characterized in that it is a cell extract, or a purified fraction thereof, from a micro-organism. Enzyme preparation according to claim 4, characterized in that the microorganism can grow at a temperature of 75 ° C or higher. Enzyme preparation according to claim 4 or 5. characterized in that the microorganism belongs to the Archaea. Enzyme preparation according to any one of claims 4-6, characterized in that the microorganism belongs to the Thermococcales. Enzyme preparation according to claim 7, characterized in that the microorganism belongs to the genus Pyrococcus. Enzyme preparation according to claim 8, characterized in that the microorganism is Pyrococcus furiosus DSM 3636 or Pyrococcus voesei DSM 3773. Enzyme preparation according to any one of claims 1-9. characterized in that the enzyme is a protein with a subunit molecular weight between 40 and 60 kDa. Purified enzyme according to any one of claims 1-10. A process for the preparation of an enzyme preparation according to any one of claims 1-10 or an enzyme according to claim 11. wherein a microorganism of the Archaea group, in particular of the Thermococcales family, more particularly of the genus Pyrococcus culture and separates an enzyme fraction from the culture medium, and optionally isolates the enzyme therefrom. DNA fragment encoding an enzyme according to claim 11. A recombinant microorganism capable of expressing the DNA fragment of claim 12. A method for the enzyme-catalyzed phosphorylation or dephosphorylation of a substrate, wherein an enzyme preparation according to any one of claims 1-10 or an enzyme according to claim 11 is used. The method of claim 11, wherein the substrate is glucose or fructose 6-phosphate. Diagnostic kit containing an enzyme preparation according to any one of claims 1-10 or an enzyme according to claim 11 and optionally ADP or glucose.