JPH0665298B2 - Novel alcohol dehydrogenase complex and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to 4,5-dihydro-4,5-dioxo-1H-
Stability that contains pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid (hereinafter abbreviated as PQQ) and can oxidize alcohols having 2 or more carbon atoms, formaldehyde and acetaldehyde over a wide range of pH And a novel alcohol dehydrogenase complex having a significantly high activity and a method for producing the same.

[Prior art and its problems]

Alcohol is a substance with a long history of contact with humankind,
There are many methods for quantifying this substance. Although methods using gas chromatographs and enzymes have been put to practical use, enzyme sensors have begun to be researched in recent years due to advances in bioelectronics, and methods for using sensors with immobilized alcohol dehydrogenase have been proposed for the determination of ethanol. . However, the conventional ones have poor stability, and this point has been left as the biggest problem for practical use of enzyme sensors.

On the other hand, conventionally widely known animal, plant, and yeast-derived alcohol dehydrogenases use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme. Is on the alkaline side, (b) the reaction is reversible, and the coexisting aldehyde causes a large error in the measurement. (C) Relatively effective NAD, NAD
Since P was used, it was unsuitable for the determination of ethanol in an acetic acid fermentation process showing a strong acidity, or in a brewed food having a strong buffering ability in an acidic region or during a fermentation of sake.

In recent years, although a new alcohol dehydrogenase which is active only in the acidic range and whose reaction is biased only to the oxidation method of alcohol has been obtained from acetic acid bacteria (Acetobacter bacterium and Gluconobacter bacterium) (Agric.Biol.Chem., 42 , 2
045-2056 (1978); the 42, 2063 to 2069 (1978); the 42, 2331
~ 2340 (1978)), there is a problem in the stability of the enzyme in the strongly acidic region. When the above-mentioned already known alcohol dehydrogenase of acetic acid bacterium is allowed to stand at pH 3, temperature of 4 ° C. for 4 days, the residual activity is reduced to 0 to 10%. Therefore, it cannot be used for continuous long-term alcohol determination in acetic acid fermentation, which is usually performed at a very low pH of around pH 3.

The present inventors have focused on the fact that the alcohol dehydrogenase described above 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. For example, it was expected that it would produce alcohol dehydrogenase, which is much more acid resistant than conventional enzymes. Therefore, for the purpose of searching for alcohol dehydrogenase that acts on alcohol in a wide pH range and has high stability, it shows good growth even in the presence of relatively high concentration of acetic acid, and has high acetic acid fermentation ability (alcohol oxidation ability). Alcohol dehydrogenase of acetic acid bacterium having a As a result, Acetobacter altoacetigenes MH-24 (FERM
BP-491) and Acetobacter xylinum (Acetobacter xy
linum) (IFO3288), a group of acetobacters belonging to the genus Acetobacter exhibit unchanged activity in a wide pH range and produce a novel alcohol dehydrogenase that stably retains the activity in a wide pH range including pH3. I found the fact to do. The present invention has been completed based on these findings.

[Means for solving problems]

The present invention is a novel alcohol dehydrogenase complex having the following properties, (1) an enzyme complex containing proteins having molecular weights of about 72,000 and about 44,000 as 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, and (3) Action This enzyme has 2 or more carbon atoms. It acts on alcohol and oxidizes to the corresponding aldehyde. It also acts on formaldehyde and acetaldehyde and oxidizes to the corresponding carboxylic acid.

(4) Substrate specificity This enzyme is a linear primary alcohol, namely ethanol,
n-Propanol, n-butanol, etc. act as good substrates. In addition, formaldehyde, acetaldehyde and glyceraldehyde can also be oxidized. However, it does not act on alcohols and aldehydes such as methanol, 3-butanol, propionaldehyde, and benzaldehyde, and also does not act on various sugars and organic acids.

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

(6) Stable pH range The enzyme is left at pH 3.0 to 8.0 at 5 ℃ for 15 hours,
Has almost all activity. After being left at 5 ° C for 100 hours, it will be inactivated by about 15% at pH 3.0, and after being left at 5 ° C for 240 hours, it will be inactivated by about 50% at pH3.0.
Almost no deactivation occurs above 4.0.

(7) Optimum temperature range for action The temperature range for action is 10 to 50 ° C, and the optimum temperature is 40 ° C.

(8) Deactivation condition depending on pH and temperature When treated at pH 6.0 for 10 minutes at each temperature, it does not deactivate up to 40 ° C, but deactivates at about 80% at 50 ° 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 red color.

(11) Visible absorption spectrum Reduced cytochrome components are 553nm, 522nm,
At 417 nm, the oxidized cytochrome component is 409 nm
Shows the maximum absorption.

(12) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻³ M when ethanol is used as a substrate.

(13) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP and molecular oxygen 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 alcohol dehydrogenase complex of the present invention will be shown.

1) Action This enzyme acts on an alcohol having 2 or more carbon atoms and oxidizes it to a corresponding aldehyde. It also acts on formaldehyde and acetaldehyde and oxidizes to the corresponding carboxylic acid.

2) Substrate specificity An example of the substrate specificity of the present enzyme is shown in Table 1. The present enzyme acts as a good substrate with a linear primary alcohol, that is, ethanol, n-propanol, n-butanol and the like. In addition, formaldehyde, acetaldehyde and glyceraldehyde can also be oxidized. However, it does not act on alcohols and aldehydes such as methanol, 3-butanol, propionaldehyde, and benzaldehyde, and also does not act on various sugars and organic acids.

3) Optimum pH The optimum pH of this enzyme is around pH 5.5 when ethanol is used as the substrate, as shown in FIG.

4) Stable pH range As shown in Fig. 2, this enzyme has a pH of 5 from 3.0 to 8.0.
After being left for 15 hours at ℃, it has almost all activity. Approximately 15% deactivated at pH 3.0 after standing at 5 ° C for 100 hours, and about 50% at pH 3.0 after standing at 5 ° C for 240 hours
Although it is deactivated, it hardly deactivates above pH 4.0,
It is a very stable enzyme. 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 Aceto.
bacter aceti) IFO 3284 and gluconolactone (can be exemplified an alcohol dehydrogenase derived from Gluconobacter suboxydans) IFO 12528 (Agric.Biol.Chem, 42, 2045~2056 (1978.) suboxydans; same 4
2 , 2063-2069 (1978); 42 , 2331-2340 (1978)), these alcohol dehydrogenases are almost inactivated when left to stand at pH 3, 4 ° C for 4 days (about 100 hours), and the residual activity is 0-10. %. From the above points, this enzyme is clearly superior in acid resistance to the conventional alcohol dehydrogenase of acetic acid bacteria.

5) Range of suitable working temperature As shown in FIG. 3, the working temperature range is 10 to 50 ° C, and the optimum temperature is 40 ° C.

6) Deactivation condition depending on pH and temperature As shown in Fig. 4, when it is treated at pH 6.0 for 10 minutes at each temperature, it does not deactivate up to 40 ° C, but deactivates by about 80% at 50 ° C.

7) Inhibition, activation and stabilization It is inhibited by heavy metal ions such as mercury, copper and cadmium at 1 mM concentration.

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

8) Molecular weight 0.1% Triton X-100 in the presence of Sephacryl S-3
The molecular weight of the alcohol dehydrogenase complex deduced from the gel filtration method with 00 is about 340,000.

9) Subunit This enzyme becomes a single band by disc gel electrophoresis. This enzyme is sodium dodecyl sulfate (hereinafter abbreviated as SDS. It is divided into two components by treatment. SDS-
Phosphorylase b (molecular weight 94,000), albumin (molecular weight 67,000), ovalbumin (molecular weight 43,000), carbonic anhydrase (molecular weight 30,000), trypsin inhibitor (molecular weight 20,100) and α-lactalbumin as molecular weight standard proteins by polyacrylamide gel electrophoresis. When using a calibration kit manufactured by Pharmacia containing (molecular weight 14,400), the molecular weights of the two components of this enzyme were about 72,000 and about 44,000. Further, since this enzyme shows an absorption spectrum peculiar to cytochrome, it is also clear that it contains cytochrome as a constituent component.

As described 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 72,000 and about 44,000. Therefore, it is appropriate to call this enzyme an alcohol dehydrogenase complex.

An example of a known enzyme is Acetobacter aceti IFO 32.
84 and Gluconobacter suboxidans IFO 1252
Alcohol dehydrogenase obtained from 8 (Agric.Biol.Chem., 42 , 2045-2056 (197
8); 42 , 2063-2069 (1978); 42 , 2331-2340 (197)
8)), the former alcohol dehydrogenase has a molecular weight of 63,0.
It is composed of four subunits of 00,44,000,29,000 and 13,500, and the latter alcohol dehydrogenase is composed of three subunits of molecular weights 85,000,49,000 and 14,400. From the above points, this enzyme is a novel enzyme which is clearly different from the conventional alcohol dehydrogenase of acetic acid bacteria.

10) Color This enzyme solution has a red color.

11) Visible part absorption spectrum Fig. 5 shows the visible part absorption spectrum of this enzyme.

Reduced cytochrome components are 553nm, 522nm,
At 417 nm, the oxidized cytochrome component is 409 nm
Shows the maximum absorption.

12) Fluorescence spectrum of hot water extraction fraction Figure 6 shows the fluorescence spectrum of the hot water extract of this enzyme.
By this spectrum and bioassay, the hot water extract contained 4,5-dihydro-4,5-dioxo-1H-pyrrolo [2,3-f] quinoline-2,7,9-tricarboxylic acid (PQQ). It was shown that.

13) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻³ M when ethanol is used as a substrate.

14) Electron acceptor Phenazine mesosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), nitroblue tetrazolium chloride (NBT), potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP ， Molecular oxygen cannot be an electron acceptor.

From the above properties, the alcohol dehydrogenase complex of the present invention is a novel enzyme that is different from any of the alcohol dehydrogenases 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 alcohol dehydrogenase complex of the present invention may be any bacterium that produces the present enzyme, and examples thereof include bacterium of the genus Acetobacter. As a specific example, Acetobacter altoacetigenes MH-24
(FERM BP-491), Acetobacter xylinum (Acetobac
ter xylinum) (IFO 3288) and the like.

The medium used for producing the alcohol 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, corn steep liquor, natural products such as peptone and ammonium salts, etc. 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 up and supplying oxygen sufficiently may be effective in efficiently accumulating the present 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 alcohol dehydrogenase complex of the present invention is accumulated in bacterial cells, bacterial cells are first collected and crushed for the purpose of recovering and purifying the enzyme. Since this enzyme is localized in the cytoplasmic membrane fraction, it is solubilized by adding an appropriate concentration of a surfactant, for example, a nonionic surfactant. This solubilized enzyme is diethylaminoethyl-sepharose (hereinafter referred to as DEAE-sepharose) in the presence of a surfactant.
, Etc. were applied to various ion exchange agents such as chromatography, adsorption chromatography of hydroxyapatite or gel filtration using Sephadex, etc., and the conventional enzyme purification means singly or in appropriate combination was applied to obtain an arbitrary purification. 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 this enzyme can be measured using any of the electron acceptors described above, and one example is shown below.
0.1 M potassium ferricyanide solution 0.1 ml. McIl
Add 0.7 ml of vaine buffer (pH 6.0) and an appropriate amount of this enzyme, and make up to 0.9 ml with water. Furthermore, 0.1 ml of 1M ethanol solution was added to start the reaction, and the reaction was performed at 30 ° C for 5 minutes.
~ 15 minutes later, Ferricsulfate-Dupanol reagent (WAWood
(See "Method in enzymology" Vol.V.287 (1962))
After adding 0.5 ml to stop the reaction, immediately add water to make 5 ml. After leaving at 37 ° C. for 30 minutes, the absorbance at 660 nm is measured. The difference in the absorbance with the sample reacted in the same manner as above without adding the substrate ethanol, means the activity of the enzyme. One unit is the amount of enzyme that oxidizes 1 μmole of ethanol in 1 minute.

〔Example〕

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

Example 1 A liquid medium containing 3% glucose, 0.5% yeast extract and 0.2% polypeptone was dispensed in 100 ml aliquots into a 500 ml shake flask and placed in an autoclave at 120 ° C. for 1 hour.
Sterilized for 5 minutes. After cooling, ethanol and acetic acid were added so that ethanol was 5% and acetic acid was 6%. Acetobacter alto acetigenes MH-24 (FERM BP-
491) was inoculated with 5 platinum loops, and 4 times at 30 ° C on a reciprocal shaker.
Cultured for a day. A medium 20 having the same composition was prepared with 30 jar fermenters, inoculated with the above culture solution 2, aerated at 20 / min, and cultured at 30 ° C. for 2 days while 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 bacterial cells were mixed with 0.01M potassium phosphate buffer (pH 6.
The cells were suspended in 0) and passed through a French press at 20,000 psi to disrupt the cells, followed by ultracentrifugation at 68,000 × g for 60 minutes to obtain a membrane fraction as a precipitate. This precipitate was suspended in 0.01M potassium phosphate buffer (pH 6.0) and then suspended in 20% Triton.
X-100 was added to a final concentration of 1.5% and 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 present enzyme was solubilized. This solution was injected into a column filled with DEAE-Sepharose CL-6B equilibrated with 0.01 M potassium phosphate buffer containing 0.1% Triton X-100, and after adsorbing this enzyme, 0.0
Gradient elution was performed with 1M to 0.1M potassium phosphate buffer. Active fractions eluted with a potassium phosphate buffer solution around 0.03 M were collected, and the active fraction containing 0.1% Triton X-100 was added.
It was dialyzed against 01M potassium phosphate buffer. This dialysate was injected into and adsorbed on a column filled with hydroxyapatite equilibrated with 0.01 M potassium phosphate buffer containing 0.1% Triton X-100. Then 0.01
Gradient elution was performed with potassium phosphate buffer from M to 0.1M. Active fractions eluted with a potassium phosphate buffer solution around 0.05 M were collected and concentrated to obtain 2 mg of a purified sample of this enzyme in a yield of 22%. The titer of this purified enzyme preparation is 200.
Units / mg protein. Further, this enzyme had the above-mentioned properties.

Example 2 In Example 1, Acetobacter altoacetylenes
Acetobacter xylinum (IFO 3288) was used instead of MH-24 (FERM BP-491), and the acetic acid concentration of the medium was adjusted to 0.
The same procedure was performed except that the amount was 1%, and 0.7 mg of a purified standard of the enzyme of the present invention was obtained with a yield of 7%. The titer of this purified enzyme preparation was 190 units / mg protein. Further, this enzyme had the above-mentioned properties.

〔The invention's effect〕

Since the alcohol dehydrogenase complex of the present invention exhibits high activity in a wide pH range and can stably maintain the activity in a high pH range including pH 3, it is superior to conventional alcohol dehydrogenase as a reagent for clinical diagnosis. In addition, the enzyme of the present invention can be used as an advantage over conventional alcohol dehydrogenases in enzyme sensors that are highly required to have high stability.

[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, FIG. 5 is a diagram showing a visible region absorption spectrum of the enzyme of the present invention, and FIG. It is a figure which shows the fluorescence spectrum of the hot water extract of the enzyme of this invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References J. Ferment. Techno l. , 60 (1) (1982) P. 41-50

Claims (3)

[Claims] 1. A novel alcohol dehydrogenase complex having the following properties. (1) Includes proteins having molecular weights of about 72,000 and about 44,000 as 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 alcohol having 2 or more carbon atoms and oxidizes it to the corresponding aldehyde. It also acts on formaldehyde and acetaldehyde and oxidizes to the corresponding carboxylic acid. (4) Substrate specificity This enzyme is a linear primary alcohol, namely ethanol,
n-Propanol, n-butanol, etc. act as good substrates. In addition, formaldehyde, acetaldehyde and glyceraldehyde can also be oxidized. However, it does not act on alcohols and aldehydes such as methanol, 3-butanol, propionaldehyde, and benzaldehyde, and also does not act on various sugars and organic acids. (5) Optimum pH The optimum pH of this enzyme is pH 5.
It is near 5. (6) Stable pH range The enzyme is left at pH 3.0 to 8.0 at 5 ℃ for 15 hours,
Has almost all activity. After being left at 5 ° C for 100 hours, it will be inactivated by about 15% at pH 3.0, and after being left at 5 ° C for 240 hours, it will be inactivated by about 50% at pH3.0.
Almost no deactivation occurs above 4.0. (7) Optimum temperature range for action The temperature range for action is 10 to 50 ° C, and the optimum temperature is 40 ° C. (8) Deactivation condition depending on pH and temperature When treated at pH 6.0 for 10 minutes at each temperature, it does not deactivate up to 40 ° C, but deactivates at about 80% at 50 ° 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 red color. (11) Visible absorption spectrum Reduced cytochrome components are 553nm, 522nm,
At 417 nm, the oxidized cytochrome component is 409 nm
Shows the maximum absorption. (12) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻³ M when ethanol is used as a substrate. (13) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP and molecular oxygen are electron acceptors. Cannot be 2. A novel alcohol dehydrogenase complex having the following properties belonging to the genus Acetobacter (1) An enzyme containing a protein having a molecular weight of about 72,000 and about 44,000 as measured by SDS-polyacrylamide gel electrophoresis. It is a complex. (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 alcohol having 2 or more carbon atoms and oxidizes it to the corresponding aldehyde. It also acts on formaldehyde and acetaldehyde and oxidizes to the corresponding carboxylic acid. (4) Substrate specificity This enzyme is a linear primary alcohol, namely ethanol,
n-Propanol, n-butanol, etc. act as good substrates. In addition, formaldehyde, acetaldehyde and glyceraldehyde can also be oxidized. However, it does not act on alcohols and aldehydes such as methanol, 3-butanol, propionaldehyde, and benzaldehyde, and also does not act on various sugars and organic acids. (5) Optimum pH The optimum pH of this enzyme is pH 5.
It is near 5. (6) Stable pH range The enzyme is left at pH 3.0 to 8.0 at 5 ℃ for 15 hours,
Has almost all activity. After being left at 5 ° C for 100 hours, it will be inactivated by about 15% at pH 3.0, and after being left at 5 ° C for 240 hours, it will be inactivated by about 50% at pH3.0.
Almost no deactivation occurs above 4.0. (7) Optimum temperature range for action The temperature range for action is 10 to 50 ° C, and the optimum temperature is 40 ° C. (8) Deactivation condition depending on pH and temperature When treated at pH 6.0 for 10 minutes at each temperature, it does not deactivate up to 40 ° C, but deactivates at about 80% at 50 ° 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 red color. (11) Visible absorption spectrum Reduced cytochrome components are 553nm, 522nm,
At 417 nm, the oxidized cytochrome component is 409 nm
Shows the maximum absorption. (12) Km value The reaction rate constant Km of this enzyme is about 1.2 × 10 ⁻³ M when ethanol is used as a substrate. (13) Electron acceptor Phenazine mesosulfate, 2,6-dichlorophenolindophenol, nitroblue tetrazolium chloride, potassium ferricyanide, etc. can be electron acceptors of this enzyme, but NAD, NADP and molecular oxygen are electron acceptors. Cannot be A method for producing a novel alcohol dehydrogenase complex, which comprises culturing a microorganism having the ability to produce and then collecting the complex from the culture. 3. A novel alcohol dehydrogenase complex-producing bacterium belonging to the genus Acetobacter is Acetobacter altoacetylenes MH-24 (FERM BP-491).
Or Acetobacter xylinum (IFO 328
The method according to claim 2, which is 8).