EP0153943A1 - Methanol dehydrogenase enzyme; a process for culturing micro-organisms; chemical products; epoxidations or hydroxylations catalyzed by micro-organisms; methylotrophic micro-organisms - Google Patents

Methanol dehydrogenase enzyme; a process for culturing micro-organisms; chemical products; epoxidations or hydroxylations catalyzed by micro-organisms; methylotrophic micro-organisms

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Publication number
EP0153943A1
EP0153943A1 EP84903204A EP84903204A EP0153943A1 EP 0153943 A1 EP0153943 A1 EP 0153943A1 EP 84903204 A EP84903204 A EP 84903204A EP 84903204 A EP84903204 A EP 84903204A EP 0153943 A1 EP0153943 A1 EP 0153943A1
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Prior art keywords
enzyme
micro
organisms
producing
nad
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German (de)
French (fr)
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Johannis Adriaan Duine
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Technische Universiteit Delft
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Technische Universiteit Delft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • 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

Definitions

  • Methanol dehydrogenase enzyme a process for culturing micro-organisms; chemical products; epoxidations or hydroxylations catalyzed by micro-organisms; methylotrophic micro-organisms. -----
  • This invention relates to a methanol dehydrogenase enzyme which consists of a quinoprotein.
  • NAD nicotinamide adenine dinucleotide
  • suitable substrates are, for example, methanol, ethanol, formaldehyde
  • the holo-enzyme comprises the apo-enzyme (generally a dimer) and two prosthetic groups consisting of pyrrolo-quinoline quinone (PQQ; complete name 2,7,9-tricarboxy-1H pyrrolo[2,3 f]-quinolin -4,5-dione).
  • PQQ pyrrolo-quinoline quinone
  • the enzyme functions as a catalyst in the reaction: CH 3 OH ⁇ CH 2 O-2H, and thus enables the micro-organisms to grow on a nutrient containing methanol as a source of carbon.
  • methanol is an inexpensive and expiosion-safe material, there is a great interest in micro-organisms which, using this substrate, can be cultured for producing microbial protein or other chemical products. Examples are the production of cattle food, various enzymes, including detergent enzymes (proteases and upases), antibiotics, vitamins, such as vitamin B12, and biopolymers, such as polyhydroxybutyric acid.
  • the known MAD-independent methanol dehydrogenase does not function optimally, however, because it is coupled to the respiratory chain (sometimes referred to as the electron-transport chain) at an advanced level (cytochrome c); this implies that little ATP (adenosine triphosphata) is formed and that the energy required must be supplied by a substantial degree of combustion, so that less formaldehyde product is available for biosyntheses.
  • ATP adenosine triphosphata
  • MDH methanol dehydrogenase enzyme
  • a methanol dehydrogenase enzyme consisting of a quinoprotein
  • the enzyme is NAD-dependent and, in vivo, forms part of a complexwith NADH dehydrogenase enzyme and formaldehyde dehydrogenase enzyme, which complex is coupled to the respiratory chain via the NADH dehydrogenase enzyme.
  • the enzyme occurs in, and can be isolated from, methylotrophic micro-organisms whose cell-free extracts exhibit methanol oxidizing activity in the presence of both NAD and an anionic dye, such as 2,6-dichlorophenol-indophenol.
  • an anionic dye is indicated to enable selective detection. If a cationic dye is used, methanol oxidizing activity need not necessarily indicate the presence of the enzyme according to the invention. The reason is that the known NAD-independent methanol dehydrogenase can be detected by means of a cationic dye, but not with an anionic dye.
  • the NAD-dependent methanol dehydrogenase according to the invention does not have this restriction with regard to the nature of the dye.
  • the invention accordingly relates to a naturally-occurring enzyme whose existence and characteristics were unknown and which has not been isolated and manipulated before.
  • new MDH occurs in all gram-positive bacteria growing on methanol.
  • the new MDH found requires not only PQQ, but also NAD as a co-enzyme.
  • the need of NAD is specific: nicotinamide adenine dinucleotide phosphate (NADP) is not a suitable co-enzyme.
  • NADP nicotinamide adenine dinucleotide phosphate
  • the new MDH is found to be deactivated by oxygen, like classical MDH, in the case of in-vitro manipulations. The deactivation can be prevented or eliminated by the presence of ammonium ions.
  • the substrate specificity is narrower than in the case of classical MDH: the new MDH specifically uses methanol as a substrate; formaldehyde and ethanol being unsuitable substrates.
  • NADH dehydrogenase and formaldehyde dehydrogenase which complex is tightly coupled, via the NADH dehydrogenase, to the respiratory chain at the beginning thereof, and does not produce free NADH.
  • Fig. 1 of the accompanying drawings This is shown diagrammatically in Fig. 1 of the accompanying drawings.
  • the advantage of this coupling to the beginning of the respiratory chain will be clear to those skilled in the art: as a consequence much ATP is produced, and hence the energy stored in the substrate is optimally utilized.
  • Fig. 2 of the drawings diagrammatically shows the structure and hypothetical action of the complex (the FAD shown therein stands for flavine adenine dinucleotide).
  • the NADH formed during the oxidation of methanol is not liberated from the complex, but is probably oxidized by the NADH dehydrogenase coupled to the respiratory chain (in vitro the NADH dehydrogenase dependent on a dye, such as DCPIP) .
  • the exact mechanism is not as yet clear.
  • the complex has been demonstrated by HPLC gel filtration in a sorbitol containing buffer: all three activities were found to be present; the molecular weight was found to be approximately 200,000.
  • the complex comprises an NAD-dependent formaldehyde dehydrogenase (an enzyme which catalyzes the dehydrogenation of hydrated formaldehyde to form formate), which does specifically require NAD, but does not have formaldehyde as the only substrate. Acetaldehyde even turns out to be a better substrate, while propionaldehyde is also suitable.
  • the complex can easily be isolated and dissociated into its components. The isolation of the complex can be realized, for example, by breaking up the harvested cells (centrifugation), suspended in a buffer, (e.g. under pressure), and transferring the supernatant, obtained after centrifugation, to a column of ion exchange material
  • the complex can be eluted with a buffer solution containing a compound having hydroxyl groups, such as sorbitol, glycerol,polyethylene glycol.
  • a buffer solution containing a compound having hydroxyl groups, such as sorbitol, glycerol,polyethylene glycol.
  • the components of the complex can be isolated by again transferring the cell-free extract to a column of ion exchange material (e.g. DEAE-Sephacel) and eluting the NADH-dehydrogenase component from it (e.g. with 0.5 M potassium phosphate butter; pH 6.8), whereafter the new MDH component together with the formaldehyde dehydrogenase component is eluted with a buffer solution which, again, contains a compound having hydroxyl groups (e.g. 0.02 M potassium phosphate buffer; pH 7.2; containing 1M KCl and 2% sorbitol).
  • the new MDH component and formaldehyde dehydrogenase component can subsequently be separated and isolated by gel filtration (e.g. on a TSK-G 3000 SW column in 0.2 M potassium phosphate buffer; pH 7.0) and/or further standard biochemical separation and purification techniques.
  • bioenergetically favourable properties of the new MDH are expressed in a process for cu ⁇ ring micro-organisms, using methanol as C source, which process, according to the invention, is characterize by
  • methylotrophic micro-organisms capable of producing both NAD-independent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by using a selective blocking agent for said former type of enzyme; or
  • genetic manipulation is used to denote all processes whereby the genetic information of a micro-organism can be modified.
  • genetic manipulation In the case of introducing specific genetic information, one should in this connection be thinking of using DNA recombinant techniques.
  • destroying genetic information that is present one may be thinking of mutations, for example, by a treatment with mutagenics or radiation, or alternatively of interference using DNA recombinant techniques.
  • a cyclopropane derivative may be used for this purpose, for example, cyclopropanol or cyclopropanone.
  • Cyclopropanol has been found to be very suitable as it did block the classical enzyme, but not the new enzyme. This possibility of selective blocking is of great importance as it affords a suitable selection method for transformants (non-transformed micro-organism which only contain classical MDH perish).
  • the process according to the invention makes it possible to improve the growth yield of bacteria or other micro-organism, such as fungi and yeasts, growing on methanol and used for the production of microbial protein or other interesting chemical products, such as enzymes (e.g. detergent enzymes, such as proteases or upases), antibiotics, vitamins , biopolymers, etc.
  • enzymes e.g. detergent enzymes, such as proteases or upases
  • antibiotics e.g. antibiotics, vitamins , biopolymers, etc.
  • the present process also makes it possible to create from micro-organisms not naturally growing on methanol, by genetic manipulation, new micro-organisms which do grow on the inexpensive and explosion-safe methanol and have a high production efficiency.
  • the genetic manipulation with recombinant DNA techniques should also comprise the introduction into the micro-organism of the genetic information for a suitable formaldehyde dehydrogenase and/or a suitable NADH dehydrogenase.
  • Another utility is for the new NDH to be used as a reduction equivalents producing system in methane mono-oxygenase (MMO) containing bacteria capable of serving as biocatalysts for epoxidation and hydroxylation reactions .
  • MMO methane mono-oxygenase
  • the present invention provides a process for epoxidizing or hydroxylating saturated or unsaturated aliphatic, alicylic and aromatic hydrocarbons, organic multi-ring compounds, chlorinated hydrocarbons, and phenols, using methane mono-oxygenase (MMO) containing micro-organisms as catalyst, said process being characterized by
  • methylotrophic micro-organisms capable of using both NADindependent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by a selective blocking agent for said former type of enzyme; or
  • MDH (either by adding a selective blocking agent such as cyclopropanol, or by genetic manipulation, such as a deliberate mutation).
  • a selective blocking agent such as cyclopropanol, or by genetic manipulation, such as a deliberate mutation.
  • RH 1/3 mole methanol per mole RH
  • RH fully converted into ROH.
  • the chance of breakdown of the ROH formed is less (which is a .result of the blocking of classical MDH, whose wide substrate specificity will often permit further oxidation of the ROH formed) .
  • a highly specific use of this process is the preparation of methanol from methane, using biocatalysts.
  • the invention is also embodied in methylotrophic microorganisms, characterized by having the capacity of producing NADdependent methanol dehydrogenase according to claim 1 or 2, produced from
  • micro-organisms which are capable of producing both NAD-independent as NAD-dependent methanol dehydrogenase, by genetic manipulations such as to destroy the capacity of producing NAD-independent methanol dehydrogenase; or
  • the supernatant was transferred to a DEAE-Sephacel column equilibrated with 0.02 M potassium phosphate buffer; pH 7.2, whereafter the column was washed with the same buffer/
  • the complex of new MDH/formaldehyde dehydrogenase/NADH dehydrogenase was eluted with 0.02 M potassium phosphate buffer; pH 7.2, containing 1 M KCl and 2% sorbitol.
  • the cell-free extract was transferred to the DEAE-Sephacel column as described above. After washing the column,the NADH dehydrogenase component was eluted with 0.5 M potassium phosphate buffer; pH 6.8.
  • the activities of the complex were measured in 0.1M tetrasodium pyrophosphate in the presence or absence of 0.12 M NH 4 Cl; pH 0.9, the reduction of 2,6-dichlorophenol-indophenol (40 ⁇ M) being followed at 600 nm.
  • methanol oxidizing activity 2.5 mM NAD and 2 mM methanol were used.
  • the formaldehyde oxidizing activity vas measured in the presence of 2.5 mM NAD and 1 mM formaldehyde (hydrolysed paraformaldehyde).
  • the NADH dehydrogenase activity was measured with 280 ⁇ M NADH in the assay mixture.
  • the activity of the isolated NADH dehydrogenase was measured in the same way as for the complex.
  • the cell-free extract only showed dye (DCPIP) -dependent methanol oxidation, if NAD was present. Formaldehyde oxidizing activity could be measured both via NADH formation and via dye reduction, if NADH was present.
  • the cell-free extract also showed dye-dependent NADH oxidizing activity.
  • the three activities were readily adsorbed on DEAE ion exchangers.
  • the methanol oxidizing activity could only be eluted with buffers containing hydroxyl compounds, such as sorbitol, glycerol or polyethylene glycol.
  • the eluate then obtained exhibited all three activities as specified above for the cell-free extract. The activities found are summarized in the following Table A.
  • the methanol oxidizing activity was lost after some time, evidently as a result of the presenceof oxygen.
  • Such a situation has been observed in the case of classical MDH, where, when O 2 is admitted to an anaerobic preparation, a rapid transformation into an NH 3 -dependent enzyme form takes place (see Duine et al., J.Gen.Microbiol. 115, pp. 523-526 (1979)) .
  • the methanol oxidizing acitivity was increased by adding ammonium salts, as shown by the following Table 3 for a partially aged preparation.
  • HPLC gel filtration in a sorbitol containing buffer solution demonstrated the existence of a complex having a molecular weight of about 200,000, which complex contains all three activities.
  • NAD-dependent formaldehyde dehydrogenase was eluted, which no longer could be demonstrated via dye reduction and exhibited a K m value for formaldehyde thirty times as high as did the enzyme in the complex.
  • the complex By recombination, i.e. adding the individual components in buffer solutions, the complex could be re-constituted, which was apparent from the re-occurence of a dye-linked, NAD-dependent methanol oxidation.

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Abstract

Enzyme méthanol-déshydrogénase NAD-dépendante qui, in vivo, fait partie d'un complexe avec l'enzyme NADH-déshydrogénase et l'enzyme formaldéhyde déshydrogénase, complexe qui est couplé à la chaîne respiratoire par l'enzyme NADH-déshydrogénase. Procédé de culture de micro-organismes capables de produire cette enzyme, où le méthanol est utilisé comme source de C; produit chimique récupéré et réactions d'époxydations et d'hydroxylations exécutées grâce auxdits micro-organismes; micro-organismes méthylotrophes per se.NAD-dependent methanol-dehydrogenase enzyme which, in vivo, is part of a complex with the NADH-dehydrogenase enzyme and the formaldehyde dehydrogenase enzyme, a complex which is coupled to the respiratory chain by the NADH-dehydrogenase enzyme. A method of growing microorganisms capable of producing this enzyme, where methanol is used as the source of C; chemical recovered and reactions of epoxidation and hydroxylation carried out by said microorganisms; methylotrophic microorganisms per se.

Description

Methanol dehydrogenase enzyme; a process for culturing micro-organisms; chemical products; epoxidations or hydroxylations catalyzed by micro-organisms; methylotrophic micro-organisms. -----
This invention relates to a methanol dehydrogenase enzyme which consists of a quinoprotein.
Such an enzyme is known from C. Anthony, "The Biochemistry of Methylotrophs", pp. 167-186 (1982), Academic Press, New York. The known enzyme (EC 1.1.99.8) is MAD-independent (NAD means nicotinamide adenine dinucleotide, which has a positive charge and is an electron or hydrogen acceptor; by accepting 2 electrons and 1 proton the electron or hydrogen donor NADH is formed); has a wide but well-defined substrate specificity (suitable substrates are, for example, methanol, ethanol, formaldehyde); uses as an electron acceptor in vivo cytochrome c, in in-vitro detections an artificial cationic dye, such as phenazine methosulphate, or the cation radical of tetramethγl-p-phenylene diamine; and under aerobic conditions in vitro requires ammonium ions. The holo-enzyme comprises the apo-enzyme (generally a dimer) and two prosthetic groups consisting of pyrrolo-quinoline quinone (PQQ; complete name 2,7,9-tricarboxy-1H pyrrolo[2,3 f]-quinolin -4,5-dione).
The enzyme functions as a catalyst in the reaction: CH3OH →CH2 O-2H, and thus enables the micro-organisms to grow on a nutrient containing methanol as a source of carbon. As methanol is an inexpensive and expiosion-safe material, there is a great interest in micro-organisms which, using this substrate, can be cultured for producing microbial protein or other chemical products. Examples are the production of cattle food, various enzymes, including detergent enzymes (proteases and upases), antibiotics, vitamins, such as vitamin B12, and biopolymers, such as polyhydroxybutyric acid.
From a bioenergetic viewpoint, the known MAD-independent methanol dehydrogenase does not function optimally, however, because it is coupled to the respiratory chain (sometimes referred to as the electron-transport chain) at an advanced level (cytochrome c); this implies that little ATP (adenosine triphosphata) is formed and that the energy required must be supplied by a substantial degree of combustion, so that less formaldehyde product is available for biosyntheses. Loginova and Trotsenko (FEMS Microbiology Letters 5, pp. 239-243 (1979)), observed that, in celifree extracts of some grampositive methylotrophic bacteria, neither NAD-independent, nor NADdependent methanol dehydrogenase, nor methanol oxidase (a flavoprotein occurring in methylotrophic yeast) could be demonstrated. Ttey speculated that the enzyme responsible for the primary methanol oxidation in these micro-organisms is particularly unstable or requires particular detection conditions.
It has now been found that a methanol dehydrogenase enzyme (MDH) , consisting of a quinoprotein, occurs in such micro-organisms, which is characterized, according to the invention, in that the enzyme is NAD-dependent and, in vivo, forms part of a complexwith NADH dehydrogenase enzyme and formaldehyde dehydrogenase enzyme, which complex is coupled to the respiratory chain via the NADH dehydrogenase enzyme. It has also been found that the enzyme occurs in, and can be isolated from, methylotrophic micro-organisms whose cell-free extracts exhibit methanol oxidizing activity in the presence of both NAD and an anionic dye, such as 2,6-dichlorophenol-indophenol.
It is noted that the use of an anionic dye is indicated to enable selective detection. If a cationic dye is used, methanol oxidizing activity need not necessarily indicate the presence of the enzyme according to the invention. The reason is that the known NAD-independent methanol dehydrogenase can be detected by means of a cationic dye, but not with an anionic dye. The NAD-dependent methanol dehydrogenase according to the invention does not have this restriction with regard to the nature of the dye.
The invention accordingly relates to a naturally-occurring enzyme whose existence and characteristics were unknown and which has not been isolated and manipulated before.
Cell-free extract assays in the presence of both NAD and the anionic dye 2,6-dichorophenol-indophenol (DCPIP), besides methanol as the substrate, showed that the new enzyme occurs in the gram-positive bacteria Nocardia spec .239; Eubacterium limosum and Clostrididium thermoautotrophicum, and also in the gram-negative bacterium Methylococcus capsulatus, strain Bath. On the other hand, the new enzyme turned out not to occur in the gram-negative Kyphomicrobium X and Pseudomonas spec. In view of the presence of the new enzyme in all gram-positive bacteria investigated, and in view of the fact that NAD-independent MDH (hereinafter sometimes referred to as classical MDH) has never been found in gram-positive bacteria, it would appear to be plausible that the new enzyme (hereinafter sometimes referred to as new MDH) occurs in all gram-positive bacteria growing on methanol.
The new MDH found requires not only PQQ, but also NAD as a co-enzyme. The need of NAD is specific: nicotinamide adenine dinucleotide phosphate (NADP) is not a suitable co-enzyme. The new MDH is found to be deactivated by oxygen, like classical MDH, in the case of in-vitro manipulations. The deactivation can be prevented or eliminated by the presence of ammonium ions. The substrate specificity is narrower than in the case of classical MDH: the new MDH specifically uses methanol as a substrate; formaldehyde and ethanol being unsuitable substrates.
The most remarkable and, at the same time, most favourable aspect of the new MDH is that, in vivo, it forms part of a complex with
NADH dehydrogenase and formaldehyde dehydrogenase, which complex is tightly coupled, via the NADH dehydrogenase, to the respiratory chain at the beginning thereof, and does not produce free NADH. This is shown diagrammatically in Fig. 1 of the accompanying drawings. The advantage of this coupling to the beginning of the respiratory chain will be clear to those skilled in the art: as a consequence much ATP is produced, and hence the energy stored in the substrate is optimally utilized.
Fig. 2 of the drawings diagrammatically shows the structure and hypothetical action of the complex (the FAD shown therein stands for flavine adenine dinucleotide). The NADH formed during the oxidation of methanol is not liberated from the complex, but is probably oxidized by the NADH dehydrogenase coupled to the respiratory chain (in vitro the NADH dehydrogenase dependent on a dye, such as DCPIP) . The exact mechanism is not as yet clear. The complex has been demonstrated by HPLC gel filtration in a sorbitol containing buffer: all three activities were found to be present; the molecular weight was found to be approximately 200,000.
The complex comprises an NAD-dependent formaldehyde dehydrogenase (an enzyme which catalyzes the dehydrogenation of hydrated formaldehyde to form formate), which does specifically require NAD, but does not have formaldehyde as the only substrate. Acetaldehyde even turns out to be a better substrate, while propionaldehyde is also suitable. The complex can easily be isolated and dissociated into its components. The isolation of the complex can be realized, for example, by breaking up the harvested cells (centrifugation), suspended in a buffer, (e.g. under pressure), and transferring the supernatant, obtained after centrifugation, to a column of ion exchange material
(e.g. DEAE-Sephacel). The complex can be eluted with a buffer solution containing a compound having hydroxyl groups, such as sorbitol, glycerol,polyethylene glycol.
The components of the complex can be isolated by again transferring the cell-free extract to a column of ion exchange material (e.g. DEAE-Sephacel) and eluting the NADH-dehydrogenase component from it (e.g. with 0.5 M potassium phosphate butter; pH 6.8), whereafter the new MDH component together with the formaldehyde dehydrogenase component is eluted with a buffer solution which, again, contains a compound having hydroxyl groups (e.g. 0.02 M potassium phosphate buffer; pH 7.2; containing 1M KCl and 2% sorbitol). The new MDH component and formaldehyde dehydrogenase component can subsequently be separated and isolated by gel filtration (e.g. on a TSK-G 3000 SW column in 0.2 M potassium phosphate buffer; pH 7.0) and/or further standard biochemical separation and purification techniques.
Furthermore it has proved possible for the complex to be reconstituted from the components by combining the components in buffer solutions.
The bioenergetically favourable properties of the new MDH are expressed in a process for cuϋπring micro-organisms, using methanol as C source, which process, according to the invention, is characterize by
A.using methylotrophic micro-organisms which are capable of producing NAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase, according to claim 1 or 2, after carrying out genetic manipulations such as to introduce into said micro-organisms the capacity of producing the latter type of enzyme and, if so desired, destroying the capacity of producing the former type of enzyme by genetic manipulation, or blocking the action of the former type of enzyme by using a selective blocking agent for said former type of enzyme; or B. using methylotrophic micro-organisms capable of producing both NAD-independent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by using a selective blocking agent for said former type of enzyme; or
C. using micro-organisms incapable of producing methanol dehydrogenase enzyme after carrying out genetic manipulations such as to introduce into said micro-organisms the capacity of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2.
The term "genetic manipulation" is used to denote all processes whereby the genetic information of a micro-organism can be modified. In the case of introducing specific genetic information, one should in this connection be thinking of using DNA recombinant techniques. In the case of destroying genetic information that is present, one may be thinking of mutations, for example, by a treatment with mutagenics or radiation, or alternatively of interference using DNA recombinant techniques.
In case a selective blocking agent for classical MDH is used, a cyclopropane derivative may be used for this purpose, for example, cyclopropanol or cyclopropanone. Cyclopropanol has been found to be very suitable as it did block the classical enzyme, but not the new enzyme. This possibility of selective blocking is of great importance as it affords a suitable selection method for transformants (non-transformed micro-organism which only contain classical MDH perish).
The process according to the invention makes it possible to improve the growth yield of bacteria or other micro-organism, such as fungi and yeasts, growing on methanol and used for the production of microbial protein or other interesting chemical products, such as enzymes (e.g. detergent enzymes, such as proteases or upases), antibiotics, vitamins , biopolymers, etc.
The present process also makes it possible to create from micro-organisms not naturally growing on methanol, by genetic manipulation, new micro-organisms which do grow on the inexpensive and explosion-safe methanol and have a high production efficiency. If necessary, the genetic manipulation with recombinant DNA techniques should also comprise the introduction into the micro-organism of the genetic information for a suitable formaldehyde dehydrogenase and/or a suitable NADH dehydrogenase. Another utility is for the new NDH to be used as a reduction equivalents producing system in methane mono-oxygenase (MMO) containing bacteria capable of serving as biocatalysts for epoxidation and hydroxylation reactions . For this purpose, the present invention provides a process for epoxidizing or hydroxylating saturated or unsaturated aliphatic, alicylic and aromatic hydrocarbons, organic multi-ring compounds, chlorinated hydrocarbons, and phenols, using methane mono-oxygenase (MMO) containing micro-organisms as catalyst, said process being characterized by
A. using methylotrophic micro-organisms which are capable of producingNAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2, after carrying out genetic manipulations such as to introduce into the micro-organisms the capacityofprcducLngthe latter type of enzyme, and, if desired, destroying the capacity of producing the former type of enzyme by genetic manipulation, or blocking the action of the former type of enzyme by using a selective blocking agent for the former type of enzyme; or B. using methylotrophic micro-organisms capable of using both NADindependent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by a selective blocking agent for said former type of enzyme; or
C. using micro-organisms incapable of producing methanol dehydrogenase, after carrying out genetic manipulations, such as to introduce into the micro-organisms the capacity of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2. Such epoxidation or hydroxylation reactions often produce only one type of product. Conversions of propylene into epoxypropane, cyclohexane into cyclohexanol, benzene into phenol, m-chlorotoluene into a mixture of benzyl alcohol and benzylepoxide, are good examples. The reaction which takes place, for example, when a compound RH is hydroxylated is as follows: In some cases,NADH functions as compound XH2, but it is assumed that there mustbeanunknown XH2. Now it seems very probable that this unknown reductionequivalents producing material is the new MDH, which has also been found, for example, in the methane-grower Methylococcus capsulatus, strain Bath. Now an improved biocatalyst can be produced by blocking the route via classical
MDH (either by adding a selective blocking agent such as cyclopropanol, or by genetic manipulation, such as a deliberate mutation). When the reaction is carried out in the presence of 1/3 mole methanol per mole RH, an optimum amount of reduction equivalents will be formed, and RH will be fully converted into ROH. Moreover, the chance of breakdown of the ROH formed is less (which is a .result of the blocking of classical MDH, whose wide substrate specificity will often permit further oxidation of the ROH formed) . A highly specific use of this process is the preparation of methanol from methane, using biocatalysts.
The invention is also embodied in methylotrophic microorganisms, characterized by having the capacity of producing NADdependent methanol dehydrogenase according to claim 1 or 2, produced from
A. micro-organisms which are capable of producing NAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase, by genetic manipulations such as to introduce the capacity of producing NAD-dependent methanol dehydrogenase and, if desired, to destroy the capacity of producing NAD-independent methanol dehydrogenase; or
B. micro-organisms which are capable of producing both NAD-independent as NAD-dependent methanol dehydrogenase, by genetic manipulations such as to destroy the capacity of producing NAD-independent methanol dehydrogenase; or
C. micro-organisms incapable of producing methanol dehydrogenase enzyme by genetic manipulations such as to introduce the capacity of producing NAD-dependent methanol dehydrogenase.
For such artificially created micro-organisms, exclusive rights are claimed too. The invention is illustrated in and by the following experimental section.
Cultivation of Nocardia spec. 239 bacteria. Use was made of a bacterium described by Kato et al. in Microbial Growth on C -Compounds, pp. 91-98 (1975). In it the bacterium is designated as Streptomyces spec. 239, but further investigation showed that the bacterium belongs to the Nocardia. The bacterium was grown at 37°C on 0.5% ethanol + 0.5% methanol with good aeration in a medium containing per litre 2 g NH4Cl;
1 g K2HPO4; 1 g NaH2PO4.2H2O; 0.2 g MgSO4.7H2O; 1 mg ZnSO4.7H2O; 1 mg CuSO4.5H2O; 1 mg MnSO4.7H2O; 0.5 mg MoO3.H2O; 0.5 mg CoCl2.6H2O; 10 mg FeCl3.H2O; and 10 mg CaCl2. 2H2O. During the cultivation, the pH of the culture was maintained at 7.0 by means of a concentrated ammonia solution, and the conversion of the alcohol was monitored by gas chromatography on a Porapak Q column. The ethanol was first consumed. When the methanol was consumed, the cells were harvested.
Isolation of the complex. A frozen paste of bacterial cells was defrosted and mixed with an equal volume of 0.02 M potassium phosphate buffer; pH 7.2.The bacteria were disrupted in a French pressure cell at a pressure of 110 MPa. DNA-ase was added to decrease the viscosity. The suspension was centrifuged at 48000 x g at 4°C for 20 minutes. The supernatant was transferred to a DEAE-Sephacel column equilibrated with 0.02 M potassium phosphate buffer; pH 7.2, whereafter the column was washed with the same buffer/ The complex of new MDH/formaldehyde dehydrogenase/NADH dehydrogenase was eluted with 0.02 M potassium phosphate buffer; pH 7.2, containing 1 M KCl and 2% sorbitol.
Isolation of the components of the complex.
The cell-free extract was transferred to the DEAE-Sephacel column as described above. After washing the column,the NADH dehydrogenase component was eluted with 0.5 M potassium phosphate buffer; pH 6.8.
Thereafter the new MDH and the formaldehyde dehydrogenase were togeher eluted with 0.02 M potassium phosphate buffer; pH 7.2, containing
1 M KCl and 2% sorbitol. The new methanol dehydrogenase was separated from the formaldehyde dehvdrogenase bv gel filtration on a TSK-G 3000 SW columnin 0.2 M potassium phosphate buffer; pH 7.0.
Enzyme assays.
The activities of the complex were measured in 0.1M tetrasodium pyrophosphate in the presence or absence of 0.12 M NH4Cl; pH 0.9, the reduction of 2,6-dichlorophenol-indophenol (40 μM) being followed at 600 nm.
For the methanol oxidizing activity, 2.5 mM NAD and 2 mM methanol were used. The formaldehyde oxidizing activity vas measured in the presence of 2.5 mM NAD and 1 mM formaldehyde (hydrolysed paraformaldehyde).
The NADH dehydrogenase activity was measured with 280 μM NADH in the assay mixture.
The activities of the new MDH and formaldehyde dehydrogena components were measured in the same NAD and substrate concentrations as for the complex, but followed at 340 nm (indication for NADH production)
The activity of the isolated NADH dehydrogenase was measured in the same way as for the complex.
Results. The cell-free extract only showed dye (DCPIP) -dependent methanol oxidation, if NAD was present. Formaldehyde oxidizing activity could be measured both via NADH formation and via dye reduction, if NADH was present. The cell-free extract also showed dye-dependent NADH oxidizing activity. The three activities were readily adsorbed on DEAE ion exchangers. The methanol oxidizing activity, however, could only be eluted with buffers containing hydroxyl compounds, such as sorbitol, glycerol or polyethylene glycol. The eluate then obtained exhibited all three activities as specified above for the cell-free extract. The activities found are summarized in the following Table A.
Unlike the NADH and formaldehyde oxidizing activity, the methanol oxidizing activity was lost after some time, evidently as a result of the presenceof oxygen. Such a situation has been observed in the case of classical MDH, where, when O2 is admitted to an anaerobic preparation, a rapid transformation into an NH3-dependent enzyme form takes place (see Duine et al., J.Gen.Microbiol. 115, pp. 523-526 (1979)) . Here, too, the methanol oxidizing acitivity was increased by adding ammonium salts, as shown by the following Table 3 for a partially aged preparation.
HPLC gel filtration in a sorbitol containing buffer solution demonstrated the existence of a complex having a molecular weight of about 200,000, which complex contains all three activities.
HPLC gel filtration in a buffer without surbitol indicated dissociation of the complex,because NADH dehydrogenase was found at a retention time of 21 minutes instead of 14 minutes. Elution of the NADH dehydrogenase from the DEAE Sephacel indeed proved to be possible with a sorbitol-free buffer (0.5 M potassium phosphate; pH 6.8; K NADH = 50 yM), with the remaining two activities continuing to be adsorbed. With a 1 M KCl, 2% sorbitol containing 0.02 M potassium phosphate buffer; pH 7.2,
NAD-dependent formaldehyde dehydrogenase was eluted, which no longer could be demonstrated via dye reduction and exhibited a Km value for formaldehyde thirty times as high as did the enzyme in the complex.
Gel filtration on an HPLC column confirmed the idea that two enzymes were involved, as a partial separation of the two enzymes was achieved.
New MDH containing fractions were analyzed for PQQ by denaturation for 2 minutes in a boiling water bath, cooling, centrifugation, and inverted phase HPLC of the supernatants by the procedure described by Duine et al. in Analytical Biochemistry 133 (1983). Amounts of PQQ proportional to the original methanol dehydrogenase activity were found
Isolated new methanol dehydrogenase proved to be incapable of catalyzing a methanol oxidation. A change in the absorption spectrum did occur in the presence of methanol and NAD, but this was located at about 325 nm (NADH production should be visible at 340 nm) and proved to be insensitive to further additions of methanol and NAD.
By recombination, i.e. adding the individual components in buffer solutions, the complex could be re-constituted, which was apparent from the re-occurence of a dye-linked, NAD-dependent methanol oxidation.

Claims

CLAIMS .
1. A methanol dehydrogenase enzyme consisting of a quinoprotein, characterized in that the enzyme is NAD-dependent and, in vivo, forms part of a complex with NADH dehydrogenase enzyme and formaldehyde dehydrogenase enzyme, which complex is coupled to the respiratory chain via the NADH dehydrogenase enzyme.
2. A methanol dehydrogenase enzyme according to claim 1, characterized in that the enzyme occurs in, and can be isolated from, methylotropic micro-organisms whose cell-free extracts exhibit methanol oxidizing activity in the presence of both NAD and an anionic dye, such as 2,6-dichlorophenol-indophenol.
3. A process for culturing micro-organisms, using methanol as a C source, characterized by
A. using methylotrophic micro-organismswhich are capable of producing NAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2, after carrying out genetic manipulations such as to introduce into said micro-organism the capacity of producing the latter type of enzyme and, if so desired, destroying the capacity of producing the former type of enzyme by genetic manipulation, or blocking the action of the former type of enzyme by using a selective blocking agent for said former type of enzyme; or
3. using methylotrophic micro-organisms capable of producing both NAD-independent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by using a selective blocking agent for said former type of enzyme; or
C. using micro-organisms incapable of producing methanol dehydrogenase enzyme after carrying out genetic manipulations such as to introduce into said micro-organisms the capacity of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2.
4. A process according to claim 3 , characterized by using cyclopropanol as the selective blocking agent for NAD-independent methanol dehydrogenase.
5. Chemical products produced by, and recovered from, micro-organisms grown by the process as claimed in claim 3 or 4.
6. A process for epoxidizing or hydroxylating saturated or unsaturated aliphatic, alicylic and aromatic hydrocarbons, organic multi-ring compounds, chlorinated hydrocarbons, and phenols, using methane mono-oxygenase (MMO) containing micro-organisms as catalyst, said process being characterized by
A. using methylotrophoc micro-organisms which are capable of producing NAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2, after carrying out genetic manipulations such as to introduce into the micro-organisms the capacity of producing the latter type of enzyme, and, if desired, destroying the capacity of producing the former type of enzyme by genetic manipulation, or blocking the action of the former type of enzyme by using a selective blocking agent for the former type of enzyme; or
B. using methylotrophic micro-organisms capable of using both NADindependent methanol dehydrogenase and NAD-dependent methanol dehydrogenase according to claim 1 or 2, with the capacity of producing the former type of enzyme being destroyed by genetic manipulation, or the action of the former type of enzyme being blocked by a sleective blocking agent for said former type of enzyme; or
C. using micro-organisms incapable of producing methanol dehydrogenase, after carrying out genetic manipulations such as to introduce into the micro-organisms the capacity of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2.
7. Methylotrophic micro-organisms, characterized by having the capacity of producing NAD-dependent methanol dehydrogenase according to claim 1 or 2 , produced from A. micro-organisms which are capable of producing NAD-independent methanol dehydrogenase, but are not capable of producing NAD-dependent methanol dehydrogenase, as a result of genetic manipulations such as to introduce the capacity of producing NAD-dependent methanol dehydrogenase and, if desired, to destroy the capacity of producing NAD-independent methanol dehydrogenase; or
B. micro-organisms which are capable of producing both NAD-independent as NAD-dependent methanoldehydrogenase as a result of genetic manipulations such as to destroy the capacity of producing NAD-independent methanol dehydrogenase; or
C. micro-organisms incapable of producing methanol dehydrogenase enzyme as a result of genetic manipulations such as to introduce the capacity of producing NAD-dependent methanol dehydrogenase.
EP84903204A 1983-09-05 1984-08-28 Methanol dehydrogenase enzyme; a process for culturing micro-organisms; chemical products; epoxidations or hydroxylations catalyzed by micro-organisms; methylotrophic micro-organisms Withdrawn EP0153943A1 (en)

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