EP0584104A1 - Procede d'obtention de dimethylamine-deshydrogenase ou de trimethylamine-deshydrogenase, deshydrogenases ainsi obtenues et leur utilisation - Google Patents

Procede d'obtention de dimethylamine-deshydrogenase ou de trimethylamine-deshydrogenase, deshydrogenases ainsi obtenues et leur utilisation

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Publication number
EP0584104A1
EP0584104A1 EP92909154A EP92909154A EP0584104A1 EP 0584104 A1 EP0584104 A1 EP 0584104A1 EP 92909154 A EP92909154 A EP 92909154A EP 92909154 A EP92909154 A EP 92909154A EP 0584104 A1 EP0584104 A1 EP 0584104A1
Authority
EP
European Patent Office
Prior art keywords
dehydrogenase
tma
trimethylamine
dma
dimethylamine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92909154A
Other languages
German (de)
English (en)
Inventor
Werner Hummel
Udo Wendel
Susanne Sting
Georg Krix
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Juelich GmbH
Original Assignee
Forschungszentrum Juelich GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Juelich GmbH filed Critical Forschungszentrum Juelich GmbH
Publication of EP0584104A1 publication Critical patent/EP0584104A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A23B2/70—Preservation of foods or foodstuffs, in general by treatment with chemicals
    • A23B2/725—Preservation of foods or foodstuffs, in general by treatment with chemicals in the form of liquids or solids
    • A23B2/729—Organic compounds; Microorganisms; Enzymes
    • A23B2/783—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B4/00—Preservation of meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase

Definitions

  • the invention relates to the production of analytically useful dimethylamine or trimethylamine dehydrogenase (DMA or TMA dehydrogenase) and it encompasses the enzymes obtained thereafter and their use.
  • DMA or TMA dehydrogenase analytically useful dimethylamine or trimethylamine dehydrogenase
  • DMA or TMA dehydrogenase are suitable enzymes for the selective detection of dimethylamine or trimethylamine.
  • dimethylamine or trimethylamine is important in the food sector (e.g. fish spoilage) and in clinical diagnostics (trimethylaminuria, betaine analysis).
  • the trimethylamine detection is particularly important for determining the freshness of fish in fresh fish.
  • the fish muscle contains high levels of trimethylamine N-oxide, which is broken down postmortem by bacterial decomposition to trimethylamine (TMA).
  • TMA trimethylamine
  • the striking pungent smell of spoiling sea fish is mainly caused by TMA.
  • a number of different, rather unspecific parameters have already been proposed in the analytical literature for determining fish freshness, for example the nucleotide content, the ammonia concentration, volatile acids, catalase activity or the pH value. As the primary breakdown product, however, TMA is of particular diagnostic value.
  • a chemical TMA determination method using picric acid has found its way into practice (Dyer, W., J. Fish Res. Vol. Can. 6.
  • Trimethylaminuria is a disorder of the choline metabolism.
  • the defective enzyme is trimethylamine N-oxidase, which catalyzes the formation of trimethylamine N-oxide.
  • a specific trimethylamine detection is required for the diagnosis.
  • mass spectrometry King GS et al. Biomed. Mass Spectrom.
  • the selective determination of dimethylamine is particularly suitable for the detection of the fresh state of frozen fish, in which other degradation routes predominate, which is why the DMA content can provide information about a consumption limit.
  • the object of the invention is therefore to provide an enzyme which is suitable for specific DMA or TMA detection.
  • This object is achieved according to the invention essentially by multiplying and harvesting a Paracoccus strain on a culture medium containing dimethylamine or trimethylamine, mechanically disrupting the harvested cells for enzyme release, removing liquid fragments from the liquid obtained and possibly cleaning up the enzyme contained in the crude extract
  • Paracoccus strains isolated from soil samples can be used to obtain two dehydrogenases that are similar per se and which either - grown on DMA-containing medium - for selective DMA conversion or - grown on TMA-containing medium - are capable of selective TMA implementation.
  • the new enzymes are capable of selectively converting dimethylamine (DMA-DH) or trimethylamine (TMA-DH), which can be used for analytical purposes.
  • DMA-DH dimethylamine
  • TMA-DH trimethylamine
  • Soil samples are suitable as the starting material for the production of the TMA or DMA dehydrogenase-forming microorganisms, from which in particular microorganisms of the genus Paracoccus are isolated which, when grown on a nutrient medium containing DMA or TMA, contain a suitable DMA or Form TMA dehydrogenase and specifically the Paracoccus strain DSM 6512.
  • TMA dehydrogenase isolated from this Paracoccus strain shows the following essential parameters in purified form:
  • the DMA dehydrogenase isolated from this Paracoccus strain shows the following essential parameters in purified form:
  • dehydrogenases according to the invention show a high specificity with regard to the conversion of DMA or TMA in the presence of other alkylamines, so that they are used for the analytical determination of dimethylamine (DMA dehydrogenase) or trimethylamine (TMA dehydrogenase) in a mixture with others Alkylamines are particularly suitable in the food and diagnostic fields.
  • dimethylamine or trimethylamine are formed as degradation products of a chemical or enzymatic reaction, in particular for determining the freshness of fresh (with TMA-DH) or frozen (with DMA-DH) fish.
  • betaine in clinical samples after its reaction with lyase with elimination of trimethylamine, which is reacted with TMA-DH in the presence of phenazineethosulfate or phenazinemethosulfate and dichlorophenolindophenol and evaluated photometrically.
  • Fig. 1 Growth and TMA-DH formation of Paracoccus DSM 6512 as a function of time;
  • Example 1 Isolation of microorganisms which contain the enzyme TMA dehydrogenase
  • a bioreactor contains 1 L of medium A with the following
  • the pH is adjusted to 7.0 and the solution is sterilized at 121 ° C. for 30 minutes.
  • the bioreactor is inoculated with approx. 5 g of a soil sample (compost). Suitable microorganisms of the soil sample grow in the closed vessel, the temperature is kept at 22 ° C. By supplying sterile-filtered air, the organisms grow largely under oxygen saturation conditions.
  • sterilized medium A is pumped in from a storage bottle (0.1 l / h), and reactor contents are removed at the same time with the same flow rate.
  • a sample is taken daily and tested for TMA dehydrogenase activity.
  • TMA-1 Taxonomic determination of the isolate TMA-1: The microbiological characterization showed that the strain TMA-1 belongs to the genus Paracoccus. The following properties were identified: Gram-negative cocci, immobile, strictly aerobic, no acid formation, not chemolithoautotrophic. Good utilization of methylamine, dimethylamine, trimethylamine, trimethylamine-N-oxide, N-methylformamide, N, N-dimethylformamide; weak growth on form amide. The TMA-1 strain differs from the two Paracoccus species denitrificans and halodenitrificans in that it is not chemolithoautotrophic. The strain was deposited on May 2, 1991 with the German Collection of Microorganisms, Braunschweig, under the number DSM 6512.
  • the strain TMA-1 was grown in the following medium B (per 1 1):
  • the medium was inoculated with 300 ml of a 24-hour-old preculture after the brood temperature had reached 30 ° C.
  • the course of the enzyme activity over time could be determined by taking samples at different times and the activity of the TMA dehydrogenase after cell disruption in the cell-free Supernatant was determined.
  • FIG. 1 shows that maximum enzyme activity can only be obtained within a short period of time towards the end of the growth phase.
  • the 8 1 fermenter was after 21 hours at an OD 660 of 4 - 35 cropped by a centrifuge, to obtain 160 g of wet biomass.
  • the biomass can be stored frozen at -20 ° C, whereby no loss of activity can be seen over several months.
  • the enzyme can be released from the cells by methods known per se (ultrasound, high-pressure homogenization, wet grinding, etc.).
  • the cells were disrupted by wet grinding with glass beads.
  • the biomass was suspended in 0.1 M Tris-HCl buffer (pH 7.0) so that the concentration of the cell wet mass was 40%.
  • the cell contents were obtained from the suspension by mechanical disruption using a vibrating mill (type MM2; Retsch, Haan, FRG). The digestion took place in 1.5 ml Eppendorf tubes.
  • the Eppendorf tubes were filled with 1.2 g of glass beads (0.5 mm in diameter) and 0.6 ml of 40% cell suspension and shaken for 15 min at a frequency of 1800 / sec. After centrifugation (3 min at 12000 rpm) in a table centrifuge, a viscous supernatant was obtained which was used for the further enzyme extraction.
  • the supernatant after PEI precipitation was subjected to ion exchange chromatography on a Mono-Q column (Fast Protein Liquid Chromatography (FPLC); Pharmacia, Freiburg FRG). 10 ml of the with a 0.2 ⁇ m Filters filtered crude extract were applied to the column in 2 ml portions and eluted with a concentration gradient of 0.05 M - 0.2 M sodium pyrophosphate buffer pH 9.0. The enzyme was detached from the column at a buffer concentration of approximately 0.09 M. The fractions containing the enzyme were then combined and diluted 1: 2 with 0.05 M sodium pyrophosphate buffer.
  • FPLC Fast Protein Liquid Chromatography
  • the solution was again applied in 2 ml portions to the Mono-Q column and eluted with a NaCl gradient (0-1 M); the enzyme was detached at a NaCl concentration of 400 mM.
  • a specific activity of 4 U / mg was measured in fractions with high enzyme activity.
  • An SDS gel prepared with these fractions showed on the basis of the protein staining that the TMA dehydrogenase was here at least 90% purified.
  • TMA dehydrogenase The activity of the TMA dehydrogenase was determined using the following test approach (C):
  • the initial reaction rate was measured at 595 nm on a spectrophotometer at 30 ° C.
  • Fig. 2 shows the pH profile of this reaction, maximum activity is obtained at a pH of 9.
  • the TMA dehydrogenase has its stability maximum at pH 9.0.
  • the pure enzyme solution has a residual activity of 80% after 6 days at 4 ° C. With the addition of 50% glycerol, a residual activity of 70% could be measured after 4 weeks at - 20 ° C.
  • reaction rate was measured at different temperatures. After sufficient preheating of the test components without enzyme, the reaction was started by adding enzyme solution.
  • the TMA dehydrogenase shows maximum activity at 45 ° C, at 60 ° C no activity can be measured.
  • Table 2 Influence of various metal cations and inhibitors. As a control, the compounds were used in parallel tests without TMA, in order to show a possible influence of the compounds on the enzyme test.
  • the test (see Example 2.D.) was replaced by TMA on the one hand with a number of other methylamine compounds (1 mM), and on the other hand the electron acceptor phenazinethosulfate (including the redox dye dichlorophenolindophenol) was replaced by other electron acceptors and then the Enzyme activity determined.
  • the results showed that only TMA is apparently reacted with respect to the amine component, and were inactive: dimethylamine, methylamine, dimethylethylamine, trimethylamine-N-oxide, choline chloride, betaine, carnitine and D, L-carnitine amide.
  • TMA is not converted if PES / DCPIP is replaced by the following compounds: methylene blue, K 3 (Fe (CN) 6 ), Wurster's Blau, PES, DCPIP, NAD + or NADP + ; only with the structurally analogous compound phenazine methosulfate does a reaction take place (98% conversion compared to PES).
  • some of the amine compounds mentioned above were tested as to whether they inhibit the conversion of TMA.
  • the activity of the TMA dehydrogenase was measured as a function of the substrate concentration.
  • the Lineweaver-Burke evaluation shows a K M value of 6.6-10 -7 M. Substrate saturation occurs above 0.03 mM.
  • the reaction rate is proportional to the Konzentra ⁇ tion of TMA (see Fig. 3).
  • This photometric reaction can thus be used to determine TMA of unknown concentration in samples.
  • This detection method can also be used to detect other TMA-containing compounds, provided that these compounds are broken down in a chemical or enzymatic pretreatment in such a way that TMA is released and this is then determined enzymatically by means of TMA dehydrogenase.
  • the strain DSM 6512 was grown in the following medium B (per 1 1):
  • the medium was inoculated with 300 ml of a 24-hour-old preculture after reaching the gross temperature of 30 ° C. and harvested after about 21 hours with an OD 660 of> 4 using a centrifuge.
  • the biomass can be stored frozen at -20 ° C., no loss of activity being discernible over several months.
  • the enzyme can be released from the cells as in Example 2B.
  • the cells are suspended in 0.1 M Tris-HCl buffer (pH 7.0) with the addition of a protease inhibitor (Pefabloc; Pentapharm, Basel, Switzerland) and disrupted by wet grinding with glass beads. After centrifugation (15 minutes at 16000 rpm), the supernatant was filtered (0.45 ⁇ m pores) and then subjected to a further chromatographic separation.
  • C Purification using ion exchange chromatography
  • the supernatant was subjected to ion exchange chromatography on a Q-Sepharose (Pharmacia, Freiburg FRG).
  • the column was equilibrated with 50 mM Na 4 P 2 0 7 buffer pH 9.
  • elution was carried out first with two bed volumes of the same buffer, to which 200 mM NaCl had been added, and then with a linear gradient from 200 to 500 mM NaCl (dissolved in the above sodium phosphate buffer).
  • the active fractions are combined and concentrated with an ultrafiltration membrane (cut-off 30 kDalton; Amicon).
  • the concentrated active fractions after the ion exchange chromatography were subjected to gel filtration.
  • the separation takes place via a Sephadex G-200 gel (Pharmacia), which was equilibrated with 50 mM KPi buffer, pH 8 with 150 mM NaCl. Elution is also carried out with the same buffer.
  • the active fractions can be stored at -20 ° C with 50% glycerol added.
  • the activity of the DMA dehydrogenase was determined analogously to the TMA dehydrogenase with a test approach as for the TMA dehydrogenase, except that 50 ⁇ l of a 100 M dimethylamine solution (in buffer) were provided instead of the TMA solution.
  • Fig. 4 shows the pH profile of this reaction, maximum activity is obtained at a pH of 8.6.
  • reaction rate was measured at various temperatures (25-60 ° C.). After the test components had been adequately tempered without enzyme, the reaction was started by adding enzyme solution. The DMA dehydrogenase shows maximum activity at 40-45 ° C, at 60 ° C no activity can be measured.
  • TMA dehydrogenase 100 ⁇ l enzyme solution were incubated for 30 min in a water bath at the desired temperature. The enzyme solution was then briefly cooled, the remaining test components were added and the remaining activity was determined by measurement at 30 ° C. After preincubation at 63.degree. C., 63% residual activity is obtained, at 37.degree. C. still 45%, at 56.degree. C. no activity was detectable.
  • Table 4 Influence of various metal cations and inhibitors. As a control, the compounds were used in parallel tests without TMA, in order to show a possible influence of the compounds on the enzyme test.
  • the substrate spectrum was determined as indicated in Example 3F.
  • the results showed that the enzyme shows a high specificity for DMA with regard to the amine component, trimethylamine is still reacted with 6% side activity and was inactive: methylamine, 2-dimethylaminoethylamine, dimethylformamide, formaldehyde and methanol.
  • the enzyme is also apparently highly specific with regard to the electron acceptor, TMA is not converted if PES / DCPIP is caused by - -
  • the reaction of DMA in the presence of PES / DCPIP is independent of ⁇ ; If a test is carried out under anaerobic conditions (buffer and test components gassed with argon for 15 min), an identical conversion rate is obtained as in the test with 0 2 gassed buffer. This distinguishes the enzyme from those in the group of oxidases in which 0 2 is the co-substrate of the reaction. Biochemically, the enzyme belongs to the group of dehydrogenases.
  • the activity of the DMA dehydrogenase was measured as a function of the substrate concentration.
  • the Lineweaver-Burke evaluation gives a K M value of 2.6-10 -5 M.
  • the isoelectric point of the DMA dehydrogenase is 4.7 (measured by means of isoelectric focusing (Pharmacia, Freiburg)).
  • the sequence of the first amino acids of the N-terminus was determined using a protein sequencer (from Applied to the first amino acids of the N-terminus).
  • dimethylamine dehydrogenase can be used especially where dimethylamine is the product of a chemical or enzymatic reaction.
  • the determination method for TMA described in Example 4 can be modified in a simple manner by replacing the trimethylamine dehydrogenase with dimethylamine dehydrogenase. Such a test is useful, for example, for determining the freshness of frozen fish, since the DMA content increases with increasing storage.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Des souches de Paracoccus provenant d'échantillons du sol fournissent, par culture sur un milieu de culture à base de diméthylamine ou de triméthylamine, respectivement, une diméthylamine-déshydrogénase et une triméthylamine-déshydrogénase hautement sélectives, c'est-à-dire deux enzymes qui sont particulièrement appropriées pour l'estimation de la fraîcheur des poissons chez des poissons frais ou congelés. La réaction s'effectue en présence d'étho- ou de méthosulfate de phénazine comme accepteur d'électrons, et peut avoir lieu en présence de dichlorophénolindophénol et être contrôlée par photométrie. Le dosage sélectif de DMA ou de TMA convient pour l'analyse dans le domaine des produits alimentaires et du diagnostic. Une souche de Paracoccus particulièrement efficace a été déposée sous le numéro DSM 6512.
EP92909154A 1991-05-10 1992-05-06 Procede d'obtention de dimethylamine-deshydrogenase ou de trimethylamine-deshydrogenase, deshydrogenases ainsi obtenues et leur utilisation Withdrawn EP0584104A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4115340 1991-05-10
DE4115340A DE4115340A1 (de) 1991-05-10 1991-05-10 Mikrobiologisch hergestellte trimethylamin-dehydrogenase, verfahren zu ihrer gewinnung und ihre verwendung

Publications (1)

Publication Number Publication Date
EP0584104A1 true EP0584104A1 (fr) 1994-03-02

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Application Number Title Priority Date Filing Date
EP92909154A Withdrawn EP0584104A1 (fr) 1991-05-10 1992-05-06 Procede d'obtention de dimethylamine-deshydrogenase ou de trimethylamine-deshydrogenase, deshydrogenases ainsi obtenues et leur utilisation

Country Status (6)

Country Link
EP (1) EP0584104A1 (fr)
JP (1) JPH06507073A (fr)
AU (1) AU1650292A (fr)
DE (1) DE4115340A1 (fr)
NO (1) NO934055D0 (fr)
WO (1) WO1992020789A1 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9617709D0 (en) * 1996-08-22 1996-10-02 Univ Leicester Enzyme
GB2331100A (en) * 1996-08-22 1999-05-12 Univ Leicester Modified forms of trimethylamine dehydrogenase
JP2006081481A (ja) * 2004-09-17 2006-03-30 Kikkoman Corp 非対称ジメチルアルギニン測定法及び非対称ジメチルアルギニン測定キット
FR3003169B1 (fr) * 2013-03-13 2018-08-31 Universite D'auvergne Clermont I Utilisation de microorganismes pour diminuer le taux de trimethylamine dans une cavite du corps humain, notamment pour le traitement de la trimethylaminurie ou d'une vaginose bacterienne et la prevention des maladies cardiovasculaires
FR3008317B1 (fr) * 2013-07-15 2017-04-28 Univ D'auvergne Clermont I Utilisation de microorganismes pour diminuer le taux de trimethylamine dans une cavite du corps humain, notamment pour le traitement de la trimethylaminurie ou d'une vaginose bacterienne et la prevention des maladies cardiovasculaires
EP2925346A1 (fr) 2012-11-30 2015-10-07 Universite d'Auvergne Clermont I Utilisation de microorganismes pour diminuer le taux de triméthylamine dans une cavité du corps humain, notamment pour le traitement de la triméthylaminurie ou d'une vaginose bactérienne et la prévention des maladies cardiovasculaires
FR2998799B1 (fr) * 2012-11-30 2015-09-04 Univ Dauvergne Clermont I Utilisation de microorganismes pour diminuer le taux de trimethylamine dans l'intestin, traitement de la trimethylaminuremie et prevention de la formation des plaques d'atherome
CN105540869B (zh) * 2015-12-17 2018-04-03 苏州大学 一种负载脱氮副球菌的改性氧化石墨烯复合材料及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9220789A1 *

Also Published As

Publication number Publication date
NO934055L (no) 1993-11-09
AU1650292A (en) 1992-12-30
NO934055D0 (no) 1993-11-09
DE4115340A1 (de) 1992-11-12
WO1992020789A1 (fr) 1992-11-26
JPH06507073A (ja) 1994-08-11

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