JPS6312279A - Novel aldehyde dehydrogenase complex and production thereof - Google Patents

Abstract

NEW MATERIAL:An aldehyde dehydrogenase complex having the following characteristics. (A) An enzyme complex containing proteins having molecular weights of about 75,000 and about 20,000 determined by SDS-polyacrylamide electrophoresis, (B) containing 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3- f]quinoline-2,7,9-tricarboxylic acid (C) capable of oxidizing >=2C aldehyde as substrate, (D) solubilizable from cytoplasm membrane with a surfactant and (E) exhibiting residual activity of >=80% even after being left for 1 week at 3-10pH and 4 deg.C. USE:Determination of aldehyde in brewed food or during fermentation of Japanese rice wine. PREPARATION:A microbial strain belonging to Acetobacter genus such as Acetobacter altoacetigenes MH-24 (FERM BP-491) is cultured and the objective substance is separated from the cultured product.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to 4,5-dihydro-4,5-dioxo-1H-
A novel aldehyde dehydrogenase containing pyrrolo[2,3-fl quinoline-2,7,9-)licarpon m (hereinafter abbreviated as PQQ), capable of oxidizing aldehydes having 2 or more carbon atoms, and stable over a wide range of pH. The present invention relates to a composite and a method for producing the same.

[Prior art and its problems] Aldehydes, especially acetaldehyde, are known as intermediate metabolites of ethanol metabolism in animal bodies, and are attracting attention as a causative agent of hangovers, etc., and are also generated during the fermentation of various brews. Although this substance, which is a toxic substance, can be measured relatively easily by the enzymatic determination method using aldehyde dehydrogenase, the optimum pH for the reaction is around neutrality,
Since the reaction is reversible when a high concentration of acetic acid coexists,
It is not suitable for quantifying aldehydes in brewed foods or during sake fermentation, which have a strong buffering capacity in acidic regions, as large errors occur.

The above-mentioned aldehyde dehydrogenase is an enzyme that uses nicotinamide adenine dinucleotide (MAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a coenzyme, but in recent years, a different and beautiful type of aldehyde dehydrogenase has been developed in Acetobacter bacterium.
Acter spp. and Gluconobacter spp.) (Agric.

Bio'1. Chem, *su, 503-515 (
1980); 45.1889-1890 (1981
)) Although this enzyme has many advantages as a quantitative enzyme, such as being active only in the acidic range and not being inhibited by coexisting acids, the reaction only occurs in the direction of oxidation of aldehydes. There are still problems with the stability of # to P
After being left at 5°C for 4 days in H3, residual activity is approximately 30%.
decreases to Moreover, even at pH 7, where enzymes are normally stable, they are inactivated to the same level as at pH 3.

The present inventors focused on the fact that the above-mentioned aldehyde dehydrogenase is obtained from acetic acid bacteria that are not used in actual vinegar brewing, and found that if the aldehyde dehydrogenase is an acetic acid bacterium that exhibits high acetic acid fermentation ability even in the presence of high concentrations of acetic acid. For example, they thought that they could produce aldehyde dehydrogenase, which is far more acid-resistant than conventional enzymes. Therefore, with the aim of searching for aldehyde dehydrogenases that are active even in strongly acidic regions, we investigated aldehyde dehydrogenases of Acetobacter aldehyde bacteria, which grow well even in the presence of relatively high concentrations of acetic acid and have high aldehyde oxidation activity. As a result, Acetobacter altoacetigenes (Acetobacter a
ltoacetigenes) MH-24 (FERMB
We have discovered that a group of acetic acid bacteria belonging to the genus Acetobacter, represented by P-491), produce a novel aldehyde dehydrogenase that is stable not only at pH 3 but also in an alkaline range.

In addition, conventionally, when enzymes are solubilized from the cytoplasmic membrane, they are easily deactivated, and even when purified, the recovery rate of activity is low. Noting that there were many cases in which only one type of surfactant was used, we investigated the possibility of solubilizing aldehyde dehydrogenase in a more efficient and stable state than in the cytoplasmic membrane by using two or more types of surfactants in combination.

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

[Means for Solving the Problems] The present invention provides a novel aldehyde dehydrogenase complex having the following properties: (1) The molecular weight measured by 5OS-polyacrylamide electrophoresis is about 75,000 and about 20,000.
(2) 4,5-dihydro-4,5-dioxo-1H-
Contains pyrrolo[2,3-flquinoline-2,7,9-tricarboxylic acid, (3) oxidizes using an aldehyde having 2 or more carbon atoms as a substrate, (4) solubilizes from the cytoplasmic membrane with a surfactant, (5
) 4°C in the range from p) 13 to p) +10,
It shows residual activity of 80% or more even after being left for one week.

and further provides a method for producing the enzyme complex, which comprises culturing a microorganism belonging to the genus Acetobacter and having the ability to produce the enzyme complex, and collecting the complex from the culture. It is something.

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

(1) Action This enzyme acts on aldehydes having two or more carbon atoms and oxidizes them to the corresponding carboxylic acids.

(2) Qualitative specificity of this enzyme (Examples of qualitative isomerism are shown in Table 1). Acts as a substrate.However, glyceraldehyde,
It does not act on glyoxal or benzaldehyde, nor does it act on alcohols, sugars, or organic acids.

Table 1 (3) Optimal p.

The optimum pH of this enzyme is around pH 7.0, as shown in FIG. 1, when acetaldehyde is used as a substrate.

(4) Stable pH range As shown in Figure 2, this enzyme is stable at pH 3 to IO at 5°C.
After standing for 4 days, it has almost all its activity. 5
It is an extremely stable enzyme with an activity of more than 80% at p)+3 to 10 even after being left at 7 days at ℃. In addition, the stable PH curve in Figure 2 shows the residual activity after being left at each PH as pne,
It was measured at o.

Examples of known enzymes include Acetobacter aceti (ETI) IFO3284 and Gluconobacter-9 suboxidans (Gluconobacter 5ub).
Aldehyde dehydrogenase obtained from IFO12528 (Agr oxydans) can be mentioned.
ic, Rial.

Cbem, 44.503-515 (1980);
45.1889-1890 (1981)), this aldehyde dehydrogenase reacts at 5°C at pH 3.0.
After standing for 4 days, the residual activity was about 30%, and under neutral conditions (
Similar results were obtained at pH 7.0).

Therefore, this enzyme has significantly better pH stability than these conventional aldehyde dehydrogenases of acetic acid bacteria.

(5) Range of suitable operating temperature As shown in FIG. 3, the operating temperature range is 20 to 80°C, with the optimum temperature particularly around 55°C.

(6) Conditions for deactivation by pH and temperature As shown in Figure 4, when treated at pH [i, 0 for 10 minutes at each temperature, it does not deactivate up to 60°C, and about 50% deactivates at 65°C. .

(7) Inhibition, activation and stabilization Inhibited by heavy metal ions such as mercury, copper and cadmium at 1a+M concentrations. 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 from the gel filtration method using heptaphryl S-300 in the presence of 0.1% Triton X-100 is about 95,000.

(9) The subunit Motoichi 2-stroke yields a single band by disk gel electrophoresis. The enzyme is separated into two components by treatment with sodium dodecyl sulfate (hereinafter abbreviated as SOS). 5O9
- Phosphorylase B (molecular weight 94,000), albumin (molecular weight Ei 7,000), ovalbumin (molecular i 43,000), carbonic anhydrase (molecular weight 130,000), )lipsin as molecular weight standard proteins in polyacrylamide electrophoresis When using a Pharmacia calibration kit containing the inhibitor (molecule 1420,100) and α-lactalbumin (molecule ff 114.400), the molecular weights of the two components of this enzyme were approximately 75,000 and approximately 20,000.
It was 00. Since this enzyme does not exhibit an absorption spectrum specific to cytochrome, cytochrome is not included as a component.

As mentioned above, the tfI element of the present invention has a molecular weight of about 75,
This enzyme is considered a complex enzyme because it is composed of two types of subunits: 1,000 and 20,000 subunits. Therefore, it is appropriate to call this enzyme an aldehyde dehydrogenase complex.

Examples of known enzymes include Acetobacter acetiIF0
32B4 and Gluconobacter suboxidans I
Mention may be made of the aldehyde dehydrogenase obtained from FO 12528 (Agric.

Biol, Cheta, 44.503-515
(1980); Docho.

1889-1HO (+980),), the former aldehyde dehydrogenase has a molecular weight of 78,000 and 45,00
It is composed of three subunits: 0 and 14,000, and the latter aldehyde dehydrogenase is composed of molecule B
a6. It is composed of two subunits: ooo and 55,000. 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 exhibits a pale pink color.

(11) Fluorescence spectrum of hot water extract fraction The fluorescence spectrum of the hot water extract of the present enzyme is shown in FIG. This spectrum and bioassay show that the hot water extract contains 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-moquinoline-2.7.9-tricarvone # (PQQ). It was shown that

(12) Km value The reaction rate constant of this enzyme was approximately 1.2 Xl0-2M when ethanol was used as a substrate.

(13) Electron acceptor phenazine mesosulfate (PMS), 2. G
-dichlorophenolindophenol (DCIP)
, nitro blue tetrazolium chloride (NBT)
, potassium ferricyanide, etc. can serve as electron acceptors for this enzyme, but HA, NADP, and molecular oxygen cannot serve as electron acceptors.

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

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

Next, the method for producing this enzyme will be explained.

The bacteria used in the production of the aldehyde dehydrogenase complex of the present invention may be any species as long as it produces the present enzyme, such as Acetobacter
r) genus Bacteria. A specific example is Acetobacter altoacetigenes (Acetobacter altoacetigenes).
r altoacetigenes) MH-24 (F
ERN BP-491).

The medium used for producing the aldehyde dehydrogenase complex of the present invention may be any medium as long as the above-mentioned bacteria can grow and produce the present enzyme. As a carbon source, for example, glucose, glycerol, fructose, mannitol, acetic acid, etc. can be used, and as a nitrogen source, for example, yeast FFJ extract, casein hydrolyzate.

Natural products such as cornstarch liquor, peptone, and ammonium salts can be used. Inorganic salts, various organic substances, vitamins, nucleic acid-related compounds, etc. can be added as necessary. Preferably, if ethanol is further added to the medium and acetic acid fermentation is performed, the content of the enzyme increases and a large amount of enzyme can be easily obtained.

18 Although solid media can be used for cultivation,
In order to obtain a large number of bacterial cells, it is preferable to perform aeration agitation culture or shaking culture using a liquid medium.When performing liquid culture, it is necessary to control the added ethanol so that it is not consumed completely. Supplying this enzyme upwards may be effective in killing this enzyme efficiently;)': In culture, main culture can be performed from the beginning, but when performing large-scale culture, it is preferable to first perform small-scale pre-culture and inoculate the obtained culture into the main medium.Culture temperature, The culture period, liquid properties of the medium, etc. may be appropriately selected and adjusted to maximize the production amount of the enzyme of the present invention, but usually culture is carried out for 1 to 3 days at 25 to 35°C under aerobic conditions. It is preferable to do so.

Since the aldehyde dehydrogenase complex of the present invention is accumulated within bacterial cells, in order to recover and purify the enzyme, the cells are first collected and disrupted. Since this enzyme is localized in both cytoplasmic membranes, it can be solubilized by adding an appropriate concentration of surfactant 1, such as a nonionic surfactant. Preferably Triton X-100 and Sodium N-Laur sarcosinate.
oil 5arcosinate). This combination allows the enzyme to be efficiently stabilized and solubilized. The solubilized enzyme is subjected to chromatography using various ion exchange agents such as diethylaminoethyl-Sepharose (hereinafter referred to as DEAE-Sepharose), adsorption chromatography using hydroxyapatite, or Sephadex in the presence of a surfactant. The enzyme of the present invention purified as desired can be obtained by applying conventional enzyme purification methods such as gel filtration methods alone or in appropriate combinations.

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

The activity of this enzyme can be measured using any of the electron acceptors mentioned above; one example is shown below.
0.1M potassium ferricyanide solution 0.1ral
! , NcIl MAINE buffer (PH8,0) 0.
Add 7mj7 and an appropriate amount of this enzyme, and dilute with water to 0.9tj.
). Furthermore, add 1M acetaldehyde solution to 0.1
Cabinet! In addition, start the reaction and after 5 to 15 minutes at 30'C,
Ferric 5ulfate-Dupanol reagent (
W., A., Wood et al.

“Method in enzyso1ogy”Va1
．． V, 287 (19 ft2)) After adding 0.5 mA+ to stop the reaction, immediately add water to make 5 tal. After standing at 37°C for 30 minutes, absorbance at 880 nm is measured. The difference in absorbance from a sample reacted in the same manner as above without adding the substrate acetaldehyde indicates the activity of the enzyme. 1 JLmole of acetaldehyde
One ton of enzyme is oxidized per minute.

[Example] Next, the present invention will be explained in detail with reference to examples.

Example 1 A liquid medium containing 3% glucose, 0.5% yeast extract, and 0.2% polypeptone at 500+sI! Dispense 100mf into each volume shaker flask and place in an autoclave for 12
Sterilized at 0°C for 15 minutes. After cooling, ethanol and acetic acid were added such that ethanol was 5% and acetic acid was 6%. Acetobacter altoacetigenes MH was added to this medium.
-24 (FERN BP-491) was inoculated into 5 platinum loops,
Cultures were incubated for 4 days at 30°C on a reciprocating shaker. A medium 205 with the same composition was prepared using a 30β jar fermenter, inoculated with the above culture solution 2I2, and 2Of! The cells were cultured at 30° C. for 2 days with aeration and stirring at a rate of 1/min. After the culture was completed, the culture solution was centrifuged at 5°C to obtain bacterial cells with a wet weight of about 2 g.

The bacterial cells were dissolved in 0.01M potassium phosphate buffer (pH 6,
0), the cells were crushed by passing through a French press at 20,000 psi, and then ultracentrifuged at 88,0 OOX g for 60 minutes to obtain a membrane fraction as a precipitate. After suspending this precipitate in 0.01M potassium phosphate buffer (pH 6,0), 20% triton X-100 and 1O%
Sodium N-lauroyl sarcosinate was added to final concentrations of 1.5% and 1%, respectively, and stirred for 3 hours. This solution was centrifuged again at 68.0 OOX g for 60 minutes to obtain a supernatant in which the enzyme was solubilized. Ammonium sulfate was added to the supernatant to achieve 60% saturation and after complete dissolution, centrifugation was performed at 10,000X g for 10 minutes. Add 0.6% triton X-100 to the resulting precipitate.
．． A 0.01M potassium phosphate buffer (p)+6) containing 20mM benzaldehyde and 0.1% cetylpyridinium chloride was added and dissolved, followed by dialysis against the same buffer. This dialysate was subjected to ultracentrifugation for 60 minutes at ea, ooox g, and insoluble substances were removed as precipitates.

The supernatant containing the solubilized enzyme was 0.6% Triton.
0.01M potassium phosphate buffer containing X-100 (
l1EAE-Sepharose CL- equilibrated at pH 6
After injecting into a column filled with 6B and adsorbing this enzyme, 0
．． Gradient elution was performed with potassium phosphate buffer from 0.1M to 0.1M. The active fraction eluted with around 0.05M potassium phosphate buffer was collected and added with 0.1% Triton
-0.01M potassium phosphate buffer (pH
6). This dialysate was treated with 0.2% Trit.
on X-100, 0.0.
Equilibrated with 01M potassium phosphate buffer (pH 6)/
＼Inject into a column filled with hydroxyapatite 1
0 to which this enzyme was adsorbed, then 0. 0.1M from OIM
gradient elution with potassium phosphate buffer up to 0.05
The active fractions near M were collected to obtain 1.2 sg of purified sample of this enzyme in eml with a yield of 50%. The titer of the wood purified enzyme preparation was 800 units/mg protein. Moreover, this enzyme had the above-mentioned properties.

Example 2 Acetobacter altoacetigenes MH-24 (FERN BP-491) was cultured under the same conditions as in Example 1 to obtain bacterial cells with a wet weight of about 2 g. This bacterial cell was crushed and ultracentrifuged in the same manner as in Example 1 to obtain a membrane fraction. After suspending the membrane fraction in 0.01M potassium phosphate buffer (pH 6.0), 20% Triton

This solution was again subjected to ultracentrifugation for 60 minutes at ee and ooox g to obtain a supernatant in which the enzyme of the present invention was solubilized.

0.6% Triton X-100, 20mM benzaldehyde.

0.01 containing 0.1% cetylpyridinium chloride
DEA equilibrated with M potassium phosphate buffer (pH 6)
The above supernatant was injected into a column filled with E-Sepharose CL-8B to adsorb the enzyme. Hereinafter, the present enzyme was purified in the same manner as in Example 1, purified enzyme preparation Q,
15 mg was obtained with a yield of 7%. The titer of the wood-purified enzyme preparation was 750 tons/mg protein. Moreover, this enzyme had the above-mentioned properties.

〔Effect of the invention〕

The aldehyde dehydrogenase complex of the present invention is superior to conventional aldehyde dehydrogenases in that it can stably maintain its activity over a wide desired range of pH 3 to pH 10, and is more advantageously used as a clinical diagnostic reagent. Furthermore, it can be used more advantageously than conventional aldehyde dehydrogenases for enzyme sensors that strongly require high stability. Furthermore, Triton X-10 was used as a surfactant to solubilize the enzyme from the cytoplasmic membrane.
By using sodium 0 and N-lauroyl sarcosinate in combination, the present enzyme can be efficiently and stably solubilized. This method has a significantly higher activity recovery rate for the purified enzyme preparation than when Triton X-100 is used alone, and is an extremely excellent method for purifying the present enzyme.

[Brief explanation of the drawing]

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 a graph showing the relationship between the reaction temperature and the relative activity of the enzyme of the present invention. 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 a hot water extract of the enzyme of the present invention. Figure 1 pH Figure 2 pH Figure 3 Figure 4 Kan-r/'Cノ Figure 5 2υ Xυ 4oo5oo
αυjl length (nm)

Claims (1)

[Claims] 1. A novel aldehyde dehydrogenase complex having the following properties. (1) It is an enzyme complex containing proteins with molecular weights of about 75,000 and about 20,000 as measured by SDS-polyacrylamide electrophoresis, (2) 4,5-dihydro-4,5-dioxo-1H - Contains pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid, (3) oxidizes using an aldehyde with 2 or more carbon atoms as a substrate, (4) solubilizes from the cytoplasmic membrane with a surfactant. (5) 4°C, 1°C in the range from pH 3 to pH 10.
It shows residual activity of 80% or more even after being left for a week. 2. A novel aldehyde dehydrogenase complex belonging to the genus Acetobacter and having the following properties: (1) An enzyme complex containing proteins with molecular weights of about 75,000 and about 20,000 as measured by SDS-polyacrylamide electrophoresis. (2) contains 4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid, and (3) has 2 or more carbon atoms. oxidizes aldehyde as a substrate, (4) is solubilized from the cytoplasmic membrane by a surfactant, and (5) is oxidized at 4°C in the pH range of 3 to 10.
1. A method for producing a novel altehyde dehydrogenase complex, which comprises culturing a microorganism capable of producing an altehyde dehydrogenase that exhibits residual activity of 80% or more even after being left for a week, and collecting the complex from the culture. 3. The method according to claim 2, wherein the novel aldehyde dehydrogenase complex-producing bacterium belonging to the genus Acetobacter is Acetobacter altoacetigenes MH-24 (FERMBP-491). 4. Claims in which the novel aldehyde dehydrogenase complex is collected from the culture by crushing the bacterial cells isolated from the culture, solubilizing them by adding a surfactant, and then applying ordinary enzyme purification means. The method described in Section 2. 5. The method according to claim 4, wherein the surfactant is a combination of Triton X-100 and sodium N-lauroylsarcosinate.