METHANOL DEHYDROGENASE ENZYME; A PROCESS FOR CULTURING MICRO-ORGANISMS; CHEMICAL PRODUCTS; EPOXIDATIONS OR HYDROXYLATIONS CATALYZED BY MICRO-ORGANISMS; METHYLOTROPHIC MICRO-ORGANISMS

Abstract: An NAD-dependent methanol dehydrogenase enzyme which, 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. The invention further relates to a process for culturing micro-organisms capable of producing this enzyme, in which methanol is used as a C source; to chemical products recovered, and epoxidation and hydroxylation reactions carried out, by means of such micro-organisms; and also to such methylotrophic micro-organisms per se.

Description: Methanol dehydrogenase enzyme; a process for culturing micro-organisms; chemical products; epoxidations or hy- droxylations catalyzed by micro-organisms; methylotroph- ic micro-organisms. This invention relates to a methanol dehydrogenase enzyme which consists of a quinoprotei . Such an enzyme is known from C. Anthony, "The 3iochemist of Methylotrophs" , pp. 167-186 (1982), Academic Press, New York. The kn enzyme (EC 1.1.99.8) is MAD-independent (NAD means nicotinamide adenine dinucleotide, which has a positive charge and is an electron or hydroge acceptor; by accepting 2 electrons and 1 proton the electron or hydroge donor NADH is formed) ; has a wide but well-defined substrate specificit (suitable substrates are, for example, methanol, ethanol, formaldehyde); uses as an electron acceptor in vivo cytochrome c, in in-vitro detecti an artificial cationic dye, such as phenazine methosulphate, or the cat radical of tetramethγl-p-phenylene diamine; and under aerobic conditions in vitro requires ammonium ions. The holo-enzyme comprises the apo-enzy (generally a dimer) and two prosthetic groups consisting of pyrrolo-quin line quinone (POO; complete name 2,7,9-tricarboxy-ljϊ pyrrolo[2,3 f -quin lin -4,5-dione). The enzyme functions as a catalyst in the reaction: CH 3 OH→-CH- 0-2H, and thus enables the micro-organisms to grow on a nutrie containing methanol as a source of carbon. As methanol is an inexpensive and expiosion-safe material, there is a great interest in micro-organism which, using this substrate, can be cultured for producing microbial pro or other chemical products. Examples are the production of cattle food, various enzymes, including detergent enzymes (proteases and upases), an biotics, vitamins, such as vitamin B12, and biopolymers, such as poly- hydroxybutyric 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 elec¬ tron-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. OMPI Loginova and Trotsenko (FΞMS Microbiology Letters 5, pp. 239-243 (1979)), observed that, in celifree extracts of some gram- positive methylotrophic bacteria, neither NAD-independent, nor NAD- dependent methanol dehydrogenase, nor methanol oxidase (a flavoprotein occurring in methylotrophic yeast) could be demonstrated. Tteyspeculate that the enzyme responsible for the primary methanol oxidation in these micro-organisms is particularly unstable or requires particular detecti conditions. It has now been found that a methanol dehydrogenase enzy (MDH) , consisting of a quinoprotei , 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 dehydro genase 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 ca be isolated from, methylotrophic micro-organisms whose cell-free extrac 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 oxid activity need not necessarily indicate the presence of the enzyme accor to the invention. The reason is that the known NAD-independent methanol hydrogenase can be detected by means of a cationic dye, but not with an anionic dye. The NAD-dependent methanol dehydrogenase according to the vention does not have this restriction with regard to the nature of the The invention accordingly relates to a naturally-occurri enzyme whose existence and characteristics were unknown and which has n been isolated and manipulated before. Cell-free extract assays in the presence of both NAD an the anionic dye 2,δ-dichorophenol-indophenol (DCPIP) , besides methanol as the substrate, showed that the new enzyme occurs in the gram-positiv bacteria Nocardia spec.239? Ξubacterium limosum and Clostrididium therm autotrophicum, and also in the gram-negative bacterium Methylococcus ca sulatus, strain Bath. On the other hand, the new enzyme turned out not occur in the gram-negative Kyphomicrobium X and Pseudomonas spec. In vi of the presence of the new enzyme in all gram-positive bacteria investi < gτOT_ OMPI and in view of the fact that NAD-independent MDH (hereinafter sometimes referred to as classical MDH) has never been found in gram-positive bac¬ teria, 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 P , but also NAD as a co-enzyme. The need of NAD is specific: nicotinamide adenine dinucleo tide 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-vitr manipulations. The deactivation can be prevented or eliminated by the presence of ammonium ions. The substrate specificity is narrower than i 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 favourab aspect of the new MDH is that, in vivo, it forms part of a complex with NADH dehydrogenase and formaldehyde dehydrogenase, which complex is tig coupled, via the NADH dehydrogenase, to the respiratory chain at the beginning thereof, and does not produce free NADH. This is shown diagra matically in Fig. 1 of the accompanying drawings. The advantage of this coupling to the beginning of the respiratory chain will be clear to thos skilled in the art: as a consequence much ATP is produced, and hence th energy stored in the substrate is optimally utilized. Fig. 2 of the drawings diagrammatically shows the struct and hypothetical action of the complex (the FAD shown therein stands fo 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 mechani is not as yet clear. The complex has been demonstrated by HPLC gel filtr tion 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 dehyd genase (an enzyme which catalyzes the dehydrogenation of hydrated formal dehyde to form formate) , which does specifically require NAD, but does n have formaldehyde as the only substrate. Acetaldehyde even turns out to a better substrate, while propionaldehyde is also suitable. "ξUZ T, OMPI far 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 componentsof 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 KC1 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 producin NAD-dependent methanol dehydrogenase, according to claim 1 or 2, after carrying out genetic manipulations such as to introduce into sai micro-organisms the capacity of producing the latter type of enzyme an 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 presen 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 importanc as it affords a suitable selection method for transformants (non-transfo 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 enzyme (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 biocatalystsfor 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 producing NAD-independent methanol dehydrogenase, but are not capable of producin 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 ty of enzyme by using a selective blocking agent for the former type of enzyme; or B. using methylotrophic micro-organisms capable of using both NAD- independent methanol dehydrogenase and NAD-dependent methanol dehydroge according to claim 1 or 2, with the capacity of producing the former ty 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-organismsincapable 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 hydrox lated is as follows: MMO RH + O + XH ■ * ■ ROH + H O - X O ι In some cases, ADH functions as compound XH , but it is assumed that there mus beanunknown XH . Now it seems very 2 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 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, butare 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 foDowing experimental section. Cultivation of Nocardia spec. 239 bacteria. Use was made of a bacteriumdescribed by Kato et al. in Microbial Growth on C -Compounds, pp. 91-98 (1975). In it the bacterium is designated as Strepto yces 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 NH Cl; 1 g K HPO ; 1 g NaH PO .2Ξ.O; 0.2 g MgSO .7H O; 1 mg ZnSO .7H 0; 1 mg CuSO .5H 0; 1 mg MnSO .7H 0; 0.5 mg MoO .H 0; 0.5 mg CoCl .αH 0; 10 mg FeCl .H 0; and 10 mg CaCl_ . 2H O. 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 gaschrom tography on a Porapak 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 dehydrogena 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 bvgel filtration on a TSK-G 3000 ... - tTR 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 NH.Cl; pH 0.9, the reduction of 2,6-dichlorophenol-indophenol (40 yM) 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 inthepresence 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 methanoloxidation, 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 DEAΞ 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. OMPI v TABLE A Dehydrogenase activities of fraction eluted from DEAE-Sepharose. (DCPIP + NAD) assay NADH production substrate presence of ymo-ssDCPIP reduced molesNADH per (mM) NAD per in. and per min. and per mg protein mg protein methanol (2) _. 0 0 methanol (2) + 0.032 0 formal¬ dehyde (2) - 0 0 formal¬ dehyde (2) + 0.110 0.080 NADH (0.3) 0.318 Unlike the NADH and formaldehyde oxidizing activity, the methanol oxidizing activity was lost after some time, evidently as a result of the " resenceof oxygen. Such a situation has been observe in the case of classical MDH, where, when 0 is admitted to an anaerobi preparation, a rapid transformation into an NH -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 preparatio . TABLE B Dialysis of the fraction eluted from DEAE-Sepharose. 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-fre 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 K value for m formaldehyde thirty times as high as did the enzyme in the complex. Gel filtration on an HPLC column confirmed the idea that two enzymes we 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, centrifuga 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 oxidatio .

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 produci 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 producin 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, an 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 NAD- independent methanol dehydrogenase and NAD-dependent methanol dehydrogen according to claim 1 or 2, with the capacity of producing the former typ 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 manipulat 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. OMP fa b WIP

Inventors: DUINE JOHANNIS ADRIAAN, DUINE, JOHANNIS, ADRIAAN
Applicants: DELFT TECH HOGESCHOOL, TECHNISCHE HOGESCHOOL DELFT
Application year: 1984
Main classification: C12N9
IPC codes: C12N1, C12N9, C12P7, C12P17