JPH0665299B2 - Novel aldehyde dehydrogenase complex and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention provides 4,5-dihydro-4,5-dioxo-1H.
-A new aldehyde containing pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid (hereinafter abbreviated as PQQ), capable of oxidizing an aldehyde having 2 or more carbon atoms and stable in a wide range of pH. The present invention relates to a dehydrogenase complex and a method for producing the same.

[Prior art and its problems] Aldehydes, especially acetaldehyde, are known as intermediate metabolites of ethanol metabolism in the animal body and are attracting attention as a causative agent for hangovers and also during fermentation of various brews. It is a toxic substance. This substance can be measured relatively easily by an enzyme quantification method using aldehyde dehydrogenase.Because the optimum pH of the reaction is near neutral, and the reaction is reversible in the presence of high concentration of acetic acid, it is large. It is not suitable for the quantification of aldehydes in brewed foods and sake fermentation which have a strong buffering capacity in the acidic range due to the error.

The above-mentioned aldehyde dehydrogenase is an enzyme that uses nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme. And Glu
(Agric.Biol.Chem., 4)
4,503-515 (1980); ibid. 45,1889-1890 (1981)). This enzyme has many advantages as a quantifying enzyme, such as being active only in the acidic range and not being inhibited by coexisting acids in the aldehyde oxidation direction only, but the stability of the enzyme at each pH Problem remains. Especially at pH 3 5
After left at 4 ° C for 4 days, the residual activity decreases to about 30%. Moreover, the enzyme is normally inactivated to the same level as pH 3 even at the stable pH 7.

The present inventors pay attention to the fact that the above-mentioned aldehyde dehydrogenase is obtained from acetic acid bacteria that are not used in actual vinegar brewing, and if the acetic acid bacteria exhibit high acetic acid fermentation ability even in the presence of high concentration acetic acid. It was thought that it would produce aldehyde dehydrogenase, which is much more acid resistant than conventional enzymes. Therefore, for the purpose of searching for an aldehyde dehydrogenase which is active even in a strongly acidic region, an aldehyde hydrogenase of an acetic acid bacterium which shows good growth even in the presence of a relatively high concentration of acetic acid and has a high aldehyde oxidation activity was examined. As a result, Acetobacter altoacetigenes MH
It was found that a group of acetic acid bacteria belonging to the genus Acetobacter represented by -24 (FERM BP-491) produces a novel aldehyde dehydrogenase which is stable not only at pH 3 but also in the alkaline range.

In addition, there has been a problem that when a membrane enzyme is conventionally solubilized from a cytoplasmic membrane, it is easily deactivated, and the activity recovery rate is low even if purification is performed. The present inventors noted that the conventional enzyme solubilization often uses only one kind of surfactant, and by combining two or more kinds of surfactants, aldehyde dehydrogenase can be more efficiently used than a cytoplasmic membrane. Moreover, it was studied to solubilize it in a stabilized state.

As a result, it has been found that the aldehyde dehydrogenase of the present invention can be efficiently solubilized in a stabilized state by using Triton X-100 in combination with sodium N-lauroyl sarcosinate. The present invention has been completed based on these findings.

[Means for Solving Problems] The present invention provides a novel aldehyde dehydrogenase complex having the following properties: (1) having a molecular weight of about 75,000 and about 20,000 measured by SDS-polyacrylamide gel electrophoresis. An enzyme complex containing a protein (2) containing 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid, ) Action This enzyme acts on an aldehyde having 2 or more carbon atoms to oxidize to a corresponding carboxylic acid.

(4) Substrate specificity This enzyme acts as a good substrate for linear primary aldehydes, that is, acetaldehyde, propionaldehyde, n-butyraldehyde and the like. However, it does not act on glyceraldehyde, glyoxal, benzaldehyde, nor on alcohols, various sugars and organic acids.

(5) Optimum pH The optimum pH of this enzyme is
It is around pH 7.0.

(6) Stable pH range This enzyme has almost all the activity after being left at pH 3 to 10 at 5 ° C for 4 days. PH even after standing at 5 ℃ for 7 days
3 to 10 has an activity of 80% or more.

(7) Optimum temperature range for action The temperature range for action is 20 to 80 ° C, and the optimum temperature is around 55 ° C.

(8) Conditions of inactivation due to pH and temperature When treated at pH 6.0 for 10 minutes, inactivation does not occur up to 60 ° C.
Approximately 50% deactivation at 65 ° C.

(9) Inhibition It is inhibited by heavy metal ions such as mercury, copper and cadmium at a concentration of 1 mM.

On the other hand, it is not inhibited by sodium azide, sodium arsenite and ethylenediaminetetraacetic acid.

(10) Color This enzyme solution has a light pink color.

(11) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻² M when acetaldehyde is used as a substrate.

(12) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP, molecular enzyme are electron acceptors. Cannot be

The present invention also provides a method for producing the above enzyme complex, which comprises culturing a microorganism belonging to the genus Acetobacter and having the ability to produce the above enzyme complex, and collecting the complex from the culture. It is a thing.

Next, the properties of the aldehyde dehydrogenase complex of the present invention will be shown.

(1) Action This enzyme acts on an aldehyde having 2 or more carbon atoms and oxidizes it to the corresponding carboxylic acid.

(2) Substrate specificity Table 1 shows examples of the substrate specificity of this enzyme. This enzyme acts on a linear primary aldehyde, that is, acetaldehyde, propionaldehyde, n-butyraldehyde, etc. as a good substrate. However, it does not act on glyceraldehyde, glyoxal, and benzaldehyde, and also does not act on alcohols, sugars, and organic acids.

(3) Optimum pH The optimum pH of this enzyme is when acetaldehyde is used as the substrate.
As shown in Fig. 1, the pH is around 7.0.

(4) Stable pH range As shown in Fig. 2, this enzyme has almost all the activity after being left at pH 3 to 10 at 5 ° C for 4 days. 5 ° C,
It is an extremely stable enzyme having an activity of 80% or more at pH 3 to 10 even after standing for 7 days. The stable pH curve in FIG. 2 is obtained by measuring the residual activity after being left at each pH at pH 6.0.

An example of a known enzyme is Acetobacter aceti (A.ace
ti) IFO 3284 and Gluconobacter suboxydans IFO 12528 can be mentioned as aldehyde dehydrogenase (A
gric.Biol.Chem., 44,503-515 (1980); ibid.45,1889-189
0 (1981)), this aldehyde dehydrogenase has a residual activity of about 30% after being left at pH 3.0 at 5 ° C. for 4 days,
Similar results were obtained under neutral conditions (pH 7.0).

Therefore, this enzyme is remarkably superior in pH stability to these conventional aldehyde dehydrogenases of acetic acid bacteria.

(5) Range of suitable working temperature As shown in FIG. 3, the working temperature range is 20 to 80 ° C,
Especially, it has the optimum temperature around 55 ℃.

(6) Conditions of deactivation by pH and temperature As shown in Fig. 4, when treated at pH 6.0 for 10 minutes at each temperature, it does not deactivate up to 60 ° C, but deactivates by about 50% at 65 ° C.

(7) Inhibition, activation and stabilization It is inhibited by heavy metal ions such as mercury, copper and cadmium at a concentration of 1 mM. On the other hand, it is not inhibited by sodium azide, sodium arsenite and ethylenediaminetetraacetic acid.

(8) Molecular weight The molecular weight of the aldehyde dehydrogenase complex estimated by gel filtration with Sephacryl S-300 in the presence of 0.1% Triton X-100 is about 95,000.

(9) Subunit This enzyme becomes a single band by disc gel electrophoresis. This enzyme is divided into two components by treatment with sodium dodecyl sulfate (hereinafter abbreviated as SDS). Phosphorylase b (molecular weight 94,000), albumin (molecular weight as standard molecular weight protein by SDS-polyacrylamide gel electrophoresis)
67,000), ovalbumin (molecular weight 43,000), carbonic anhydrase (molecular weight 30,000), trypsin inhibitor (molecular weight 20,100) and α-lactalbumin (molecular weight 14,400) The two components had a molecular weight of about 75,000 and about 20,000. Since this enzyme does not show the absorption spectrum peculiar to cytochrome, cytochrome is not included as a component.

As mentioned above, the enzyme of the present invention is considered to be a complex enzyme because it is composed of two subunits having molecular weights of about 75,000 and about 20,000. Therefore, it is appropriate to call this enzyme an aldehyde dehydrogenase complex.

An example of a known enzyme is Acetobacter aceti IFO 32.
84 and Gluconobacter suboxidans IFO 1252
Aldehyde dehydrogenase obtained from 8 can be mentioned (Agric.Biol.Chem., 44, 503-515 (1980);
45,1889-1890 (1980)), the former aldehyde dehydrogenase is composed of three subunits with molecular weights of 78,000, 45,000 and 14,000, and the latter aldehyde dehydrogenase is composed of two subunits with molecular weights of 86,000 and 55,000. It is configured. From the above points, this enzyme is a novel enzyme that is clearly different from the conventional aldehyde dehydrogenase of acetic acid bacteria.

(10) Color This enzyme solution has a light pink color.

(11) Fluorescence spectrum of hot water extract fraction The fluorescence spectrum of the hot water extract of this enzyme is shown in FIG.
By this spectrum and bioassay, 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-
It was shown to contain f] quinoline-2,7,9-tricarboxylic acid (PQQ).

(12) Km value The reaction rate constant Km of this enzyme was about 1.2 × 10 ^-2 M when ataldehyde was used as a substrate.

(13) Electron acceptor Phenazine mesosulfate (PMS), 2,6-dichlorophenol indophenol (DCIP), nitroblue tetrazolium chloride (NBT), potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP and molecular enzymes cannot serve as electron acceptors.

From the above properties, the aldehyde dehydrogenase complex of the present invention is a novel enzyme which is different from any aldehyde dehydrogenase known so far.

(15) Purification method The purification method is described in the manufacturing method.

Next, a method for producing the present enzyme will be described.

The bacterium used for producing the aldehyde dehydrogenase complex of the present invention may be any bacterium that produces the present enzyme, and examples thereof include bacteria of the genus Acetobacter. As a specific example, Acetobacter altoacetigenes MH-24
(FERM BP-491) can be mentioned.

The medium used for producing the aldehyde dehydrogenase complex of the present invention may be any medium as long as the above bacteria grow to produce the present enzyme. As the carbon source, for example, glucose, glycerol, fructose, mannitol, acetic acid and the like can be used, and as the nitrogen source, for example, yeast extract, casein hydrolyzate,
Natural products such as corn steep liquor and peptone, and ammonium salts can be used. If necessary, inorganic salts, various organic substances, vitamins, nucleic acid-related compounds, etc. can be added. Preferably, if ethanol is further added to the medium to carry out the acetic acid fermentation, the enzyme content is increased, and a large amount of enzyme can be easily obtained.

A solid medium can be used for the culture, but in order to obtain a large amount of cells, it is preferable to carry out aeration stirring culture or shaking culture using a liquid medium. In liquid culture, controlling the added ethanol so that it is not consumed completely and supplying the enzyme sufficiently may be effective for efficiently accumulating the enzyme. In the culture, the main culture can be performed from the beginning, but in the case of performing the large-scale culture, it is preferable to first perform a small-scale preculture and inoculate the obtained culture into the main medium. The culturing temperature, culturing period, composition of the medium and the like may be appropriately selected and adjusted so that the production amount of the enzyme of the present invention is maximized, but usually 1 to 3 at 25 to 35 ° C under aerobic conditions. Day culture is preferred.

Since the aldehyde dehydrogenase complex of the present invention accumulates in bacterial cells, the bacterial cells are first collected and crushed for recovery and purification of the enzyme. Since this enzyme is localized in the cytoplasmic membrane fraction, it is solubilized by adding a suitable concentration of a surfactant, for example, a nonionic surfactant. Preferably, Triton X-100 and sodium N-lauroyl sarcosinate (Sodium N-Lauroyl sarcosinate) are used in combination.
By this combined use, the present enzyme can be efficiently stabilized and solubilized. The solubilized enzyme was diethylaminoethyl-sepharose (hereinafter DEAE-
It is called Sepharose. ) And other various ion-exchange agents, adsorption chromatography such as hydroxyapatite, or gel filtration using Sephadex or the like, and a conventional enzyme purification method singly or in combination is applied, and optionally purified. Moreover, the enzyme of the present invention can be obtained.

Next, the method for measuring the enzyme activity of the enzyme of the present invention will be described.

The activity of the present enzyme can be measured using any of the above-mentioned electric receptors, and one example is shown below. 0.
Add 0.1 ml of 1M potassium ferricyanide solution, 0.7 ml of McIlvaine's buffer (pH 6.0) and an appropriate amount of this enzyme, and add with water.
Adjust to 0.9 ml. Furthermore, add 1M acetaldehyde solution to 0.1
Start the reaction by adding ml, and after 5-15 minutes at 30 ℃, ferric s
ulfate-Dupanol reagent (WA Wood et al., “Method in enzymol
0.5 g (see ogy "Vol.V.287 (1962)) to stop the reaction, and then immediately add water to make 5 ml. After standing at 37 ° C for 30 minutes, measure the absorbance at 660 nm. The difference in the absorbance with the sample reacted in the same manner as above without the addition of acetaldehyde means the activity of the enzyme, and the amount of the enzyme that oxidizes 1 μmole of acetaldehyde in 1 minute is defined as 1 unit.

[Examples] Next, the present invention will be described in detail with reference to Examples.

Example 1 Glucose 3%, yeast extract 0.5% and polypeptone
100 ml of 0.2% liquid medium was dispensed into a 500 ml shake flask and sterilized in an autoclave at 120 ° C for 15 minutes. After cooling, ethanol and acetic acid were added so that ethanol was 5% and acetic acid was 6%. This medium was inoculated with 5 platinum loops of Acetobacter altoacetylene MH-24 (FERM BP-491), and cultured on a reciprocal shaker at 30 ° C for 4 days.
A medium 20 having the same composition was prepared with 30 jar fermenters, inoculated with the above-mentioned culture solution 2 and aerated at 20 / min,
It was cultured at 30 ° C. for 2 days with stirring. After completion of the culture, the culture solution was centrifuged at 5 ° C. to obtain a microbial cell having a wet weight of about 2 g.

The cells were suspended in 0.01 M potassium phosphate buffer (pH 6.0) and passed through a French press at 20,000 psi to crush the cells, followed by ultracentrifugation at 68,000 xg for 60 minutes to precipitate the membrane fraction. Got as. This precipitate was added to 0.01M potassium phosphate buffer (pH
6.0) and 20% Triton X-100 and 10% sodium N-lauroyl sarcosinate to a final concentration of 1.5 each.
% And 1%, and stirred for 3 hours. This solution was again centrifuged at 68,000 × g for 60 minutes to obtain a supernatant in which the enzyme was solubilized. Ammonium sulphate was added to this supernatant to 60% saturation and completely dissolved, then 10,000 xg
It was centrifuged for 10 minutes. 0.6% Trito was added to the obtained precipitate.
0.01M potassium phosphate buffer containing n X-100, 20mM benzaldehyde and 0.1% cetylpyridinium chloride (pH
After 6) was added and dissolved, it was dialyzed against the same buffer. The dialysate was subjected to ultracentrifugation at 68,000 × g for 60 minutes to remove insoluble substances as a precipitate.

The supernatant containing the solubilized enzyme was equilibrated with 0.01 M potassium phosphate buffer (pH 6) containing 0.6% Triton X-100.
It was injected into a column filled with DEAE-Sepharose CL-6B, and after adsorbing the present enzyme, gradient elution was performed with 0.01M to 0.1M potassium phosphate buffer. The active fractions eluted with a potassium phosphate buffer solution around 0.05 M were collected and collected in 0.1% Triton X-
It was dialyzed against 0.01 M potassium phosphate buffer (pH 6) containing 100%. This dialysate was mixed with 0.2% Triton X-100, 5 mM benzaldehyde in 0.01 M potassium phosphate buffer (pH
It was injected into a column filled with hydroxyapatite equilibrated in 6) to adsorb the present enzyme. Then 0.01M
Gradient elution with potassium phosphate buffer from 0.1 to 0.1M. Active fractions around 0.05 M were collected and concentrated to obtain 1.2 mg of a purified sample of this enzyme in a yield of 50%. The titer of this purified enzyme preparation was 800 units / mg protein. Further, this enzyme had the above-mentioned properties.

Example 2 Acetobacter altoacetigenes MH-24 (FERM BP-491) was cultured under the same conditions as in Example 1 to obtain bacterial cells having a wet weight of about 2 g. The cells were disrupted and subjected to ultracentrifugation in the same manner as in Example 1 to obtain a membrane fraction. After suspending the membrane fraction in 0.01 M potassium phosphate buffer (pH 6.0), 20% Triton X-100 was added to a final concentration of 1.5% and the mixture was stirred for 3 hours. This solution was again subjected to ultracentrifugation at 68,000 × g for 60 minutes to obtain a supernatant in which the enzyme of the present invention was solubilized. 0.6% Triton X-10
The above supernatant was injected into a column packed with DEAE-Sepharose CL-6B equilibrated with 0.01 M potassium phosphate buffer (pH 6) containing 0,20 mM benzaldehyde and 0.1% cetylpyridinium chloride to adsorb the enzyme. I'm sorry. Hereinafter, the present enzyme was purified in the same manner as in Example 1 to obtain 0.15 mg of the purified enzyme preparation with a yield of 7%. The titer of this purified enzyme preparation was 750 units / mg protein. Further, this enzyme had the above-mentioned properties.

[Effect of the Invention] The aldehyde dehydrogenase complex of the present invention is adjusted to pH 3 or higher.
It is superior to conventional aldehyde dehydrogenases in that it can stably retain activity in a wide range up to pH 10, and it is more predominantly used as a reagent for clinical diagnosis. Further, it can be used more favorably than the conventional aldehyde dehydrogenase for an enzyme sensor which is strongly required to have high stability. Further, in solubilizing the enzyme from the cytoplasmic membrane, by using Triton X-100 and sodium N-lauroyl sarcosinate as a surfactant together, the enzyme can be efficiently and stably solubilized. This method is remarkably excellent in the purification of the present enzyme since the activity recovery rate of the purified enzyme preparation is remarkably higher than that when Triton X-100 is used alone.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the reaction pH and the relative activity of the enzyme of the present invention, FIG. 2 is a graph showing the pH stability of the enzyme of the present invention, and FIG. 3 is the reaction temperature of the enzyme of the present invention. Fig. 4 is a graph showing the relationship with relative activity, Fig. 4 is a graph showing the temperature stability of the enzyme of the present invention, and Fig. 5 is a graph showing the fluorescence spectrum of the hot water extract of the enzyme of the present invention.

Continuation of front page (56) References J. Ferment. Techno l. 59 (4) (1981) P. 247-255

Claims (5)

[Claims] 1. A novel aldehyde dehydrogenase complex having the following properties. (1) Includes proteins having molecular weights of about 75,000 and about 20,000 measured by SDS-polyacrylamide gel electrophoresis. (2) Contains 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid. (3) Action This enzyme acts on an aldehyde having 2 or more carbon atoms and oxidizes it to the corresponding carboxylic acid. (4) Substrate specificity This enzyme acts as a good substrate for linear primary aldehydes, that is, acetaldehyde, propionaldehyde, n-butyraldehyde and the like. However, it does not act on glyceraldehyde, glyoxal, benzaldehyde, nor on alcohols, various sugars and organic acids. (5) Optimum pH The optimum pH of this enzyme is
It is around pH 7.0. (6) Stable pH range This enzyme has almost all the activity after being left at pH 3 to 10 at 5 ° C for 4 days. PH even after standing at 5 ℃ for 7 days
3 to 10 has an activity of 80% or more. (7) Optimum temperature range for action The temperature range for action is 20 to 80 ° C, and the optimum temperature is around 55 ° C. (8) Conditions of inactivation due to pH and temperature When treated at pH 6.0 for 10 minutes, inactivation does not occur up to 60 ° C.
Approximately 50% deactivation at 65 ° C. (9) Inhibition It is inhibited by heavy metal ions such as mercury, copper and cadmium at a concentration of 1 mM. On the other hand, it is not inhibited by sodium azide, sodium arsenite and ethylenediaminetetraacetic acid. (10) Color This enzyme solution has a light pink color. (11) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻² M when acetaldehyde is used as a substrate. (12) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP, molecular enzyme are electron acceptors. Cannot be 2. A novel aldehyde dehydrogenase complex which belongs to the genus Acetobacter and has the following properties: (1) It contains proteins having molecular weights of about 75,000 and about 20,000 measured by SDS-polyacrylamide gel electrophoresis. . (2) Contains 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid. (3) Action This enzyme acts on an aldehyde having 2 or more carbon atoms and oxidizes it to the corresponding carboxylic acid. (4) Substrate specificity This enzyme acts as a good substrate for linear primary aldehydes, that is, acetaldehyde, propionaldehyde, n-butyraldehyde and the like. However, it does not act on glyceraldehyde, glyoxal, benzaldehyde, nor on alcohols, various sugars and organic acids. (5) Optimum pH The optimum pH of this enzyme is
It is around pH 7.0. (6) Stable pH range This enzyme has almost all the activity after being left at pH 3 to 10 at 5 ° C for 4 days. PH even after standing at 5 ℃ for 7 days
3 to 10 has an activity of 80% or more. (7) Optimum temperature range for action The temperature range for action is 20 to 80 ° C, and the optimum temperature is around 55 ° C. (8) Conditions of inactivation due to pH and temperature When treated at pH 6.0 for 10 minutes, inactivation does not occur up to 60 ° C.
Approximately 50% deactivation at 65 ° C. (9) Inhibition It is inhibited by heavy metal ions such as mercury, copper and cadmium at a concentration of 1 mM. On the other hand, it is not inhibited by sodium azide, sodium arsenite and ethylenediaminetetraacetic acid. (10) Color This enzyme solution has a light pink color. (11) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻² M when acetaldehyde is used as a substrate. (12) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP, molecular enzyme are electron acceptors. Cannot be A method for producing a novel aldehyde dehydrogenase complex, which comprises culturing a microorganism having the ability to produce aldehyde and collecting the complex from the culture. 3. A novel aldehyde dehydrogenase complex-producing bacterium belonging to the genus Acetobacter is Acetobacter altoacetylenes MH-24 (FERM BP-491).
The method of claim 2 wherein: 4. A patent for collecting a novel aldehyde dehydrogenase complex from a culture by crushing the bacterial cells separated from the culture, adding a surfactant to solubilize it, and then applying a usual enzyme purification means. The method according to claim 2. 5. The surfactants are Triton X-100 and N.
A method according to claim 4 which is a combination of sodium lauroyl sarcosinate.