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  • 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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• 1983
  • 1983-09-05 NL NL8303082A patent/NL8303082A/nl not_active Application Discontinuation
• 1984
  • 1984-08-28 EP EP84903204A patent/EP0153943A1/de not_active Withdrawn
  • 1984-08-28 AU AU33175/84A patent/AU3317584A/en not_active Abandoned
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