JP2003180343A - Membrane bound cyclic alcohol dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To elucidate that a membrane bound cyclic alcohol dehydrogenase presenting in cell membranes of acetic acid bacteria, especially those belonging to the genus Gluconobacter, and requiring PQQ as a coenzyme is different from already known quinoprotein ADH and NAD dependent CCAD and suitable for producing oxidation products such as a cyclic alcohol, a secondary alcohol and the like. <P>SOLUTION: The presence of oxidation activity by PQQ as a coenzyme in cell membranes of aerobic bacteria is pointed out for the first time and screening of a MCAD-producing strain is carried out for ≥100 cell strains including aerobic bacteria such as acetic acid bacteria and the like. A bacterium strain presenting in the cell membranes of acetic acid bacteria of the genus Gluconobacter and having high MCAD activity is found. The MCAD is solubilized and purified and the properties of the same are elucidated for the first time. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a membrane-bound cyclic alcohol dehydrogenase (MCAD) having PQQ as a coenzyme, which is present in the cell membrane of acetic acid bacteria, particularly acetic acid bacteria of the genus Gluconobacter.

[0002]

BACKGROUND ART It is known that NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) exists in the cytoplasm of acetic acid bacteria and other Psuedmonas spp.
The cyclic alcohol dehydrogenase activity of D is very low. C existing in the cytoplasm of acetic acid bacteria of the genus Gluconobacter
Although CAD also has a cyclic alcohol dehydrogenase activity, a membrane-bound cyclic alcohol dehydrogenase (MC having pyrroloquinoline quinone (PQQ) of the present invention as a coenzyme is used.
The activity was only 100 times lower than that of AD). Therefore, using quinoprotein alcohol dehydrogenase (ADH) or CCAD in the oxidative fermentation process for industrially producing an oxidation product of a cyclic alcohol has a problem in terms of production efficiency.

[0003]

[Problems to be Solved by the Invention] MCAD, which is present in the cell membrane of acetic acid bacteria, particularly acetic acid bacteria of the genus Gluconobacter, and which has PQQ as a coenzyme, is an enzyme different from conventional ADH and CCAD, and is a cyclic alcohol. The challenge is to clarify that it is suitable for the production of oxidation products such as alcohols and secondary alcohols.

[0004]

[Means for Solving the Problems] A large number of studies on microbial and enzymatic oxidation of cyclic alcohols have been carried out using various bacteria. Most of them are related to NAD-dependent cyclohexanol dehydrogenase (EC 1.1.1.245) derived from Psuedomonas and Acinetobacter. Cyclohexanol oxidation has also been studied in the course of cyclohexane metabolism. Genetic analysis of the gene cluster for cyclohexanol oxidation was studied in Acinetobacter.

The reduction of cyclohexane to cyclohexanol by NAD-dependent cyclohexanol dehydrogenase has been proposed to produce cyclohexanol by coupling with a hydrogenase. This is a case similar to cyclic ketone reductase or cyclic aldehyde reductase. Early studies reported a NADPH-dependent acetone reductase that produces isopropanol. Similar ketone reductases have been reported in mammals. Numerous aldo-keto reductases that catalyze the asymmetric reduction of carbonyl compounds have proven useful in the synthesis of optically active compounds.

Cyclopentanol oxidation by microorganisms has also been studied for Pseudomonas, and NAD-dependent dehydrogenase (EC 1.1.1.163) is the enzyme acting in the first step of oxidative degradation of cyclic alcohols. Has been shown to be. Cyclohexanol oxidation was also observed in secondary alcohol oxidase derived from Pseudomonas, and the flavin-dependent enzyme that produces hydrogen peroxide functions.

Enzymes that catalyze the oxidation of myo-inositol and related cyclitols have also been reported in many references to NA.
It is known as a D-dependent enzyme. However MCA
Since nothing has been reported on D, there is no information on the function, localization, catalytic, physicochemical and physiological functions of MCAD.

The present inventors have found that P on the cell membrane of aerobic bacteria.
The existence of oxidative activity with QQ as a coenzyme was pointed out for the first time.
Therefore, as a result of screening of MCAD-producing strains for 100 or more cell lines containing acetic acid bacteria, Pseudomonas, Acinetobacter and various other aerobic bacteria, acetic acid bacteria, particularly acetic acid of the genus Gluconobacter. For the first time, MCAD, which has PQQ as a coenzyme, present in the cell membrane of the bacterium was found and solubilized and purified.

MCA of Gluconobacter frateurii CHM 9
The property of D will be described below as a typical example of the MCAD of the present invention. MCAD exists in the cell membrane of acetic acid bacteria, PQQ
It is named as a cyclic alcohol dehydrogenase having a coenzyme as a coenzyme. It is an enzyme that widely oxidizes cyclic alcohols as well as secondary alcohols, and has wide substrate specificity. C existing in already known cytoplasm
It has been found that CAD is a novel enzyme that is clearly different from the viewpoint of intracellular site, substrate specificity, kinetic properties and the like. The cyclic alcohol oxidation activity of MCAD is
It was 100 times higher than CCAD. The high enzymatic activity of MCAD is a great advantage in industrially producing the corresponding cyclic ketone by oxidative fermentation of cyclic alcohols.

As an interesting fact, it was also revealed that the MCAD of the present invention allows resolution of optical isomers. For example, MCAD is (S, S)-(+)-2,3-bu
It did not oxidize tanediol at all, whereas (2R, 3R)-(-)
It was found that -2,3- butanediol was oxidized (Table 3). Utilizing this property, racemic 2,3- butanedi
When ol is oxidized by MCAD to convert only the R form into the corresponding ketone, only the unreacted S form is concentrated. After that, it is concentrated (S, S)-(+) by the usual purification method.
Purification of -2,3- butanediol has the advantage of facilitating the separation of optical isomers.

In the production of the compound by the fermentation method, there may be a problem in that the induction time of the reaction is long. In the case of MCAD of the present invention, it was shown that the reaction occurs in proportion to both the reaction time and the population of cells used, without any time lag. In most cases of oxidative fermentation, no reaction was observed to occur without a time lag. This is because the substrate oxidation is performed on the outer surface of the cell. Compared with the case where the substrate has to be taken up into the cell and reacted, and then the product must be released to the outside of the cell again, the production method using MCAD of the present invention has an advantage that energy loss can be reduced. Have

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described based on examples with reference to the drawings and tables.

[0013]

Example 1 Screening of MCAD-producing strains was carried out on 100 or more cell lines containing acetic acid bacteria, Pseudomonas, Acinetobacter and various other aerobic bacteria. The composition of the screening medium is 0.03%
Glycerol, 0.01% glucose, 0.1% polypeptone, 0.2% cyclopentanol or 0.2
% Cyclohexanol for more than 48 hours,
The culture was performed under aeration conditions. The culture temperature was 30 ° C in all cases.

The majority of strains isolated in Thailand grew well with both cyclopentanol and cyclohexanol as cyclic alcohols. However, cyclohexanol appeared to be more toxic, or rather bacteriostatic, than cyclopentanol. Instit
ute For Fermentation (IFO) or American Type
Most of the strains obtained from the Culture Collection (ATCC) required a long culture time of 48 hours or longer, judging from the turbidity of the culture solution, but most of the strains isolated in Thailand had 24 hours. Equivalent turbidity was reached by culturing for a period of time, indicating high resistance to cyclohexanol and cyclopentanol.

As a result of judging by the turbidity of the culture solution after 24 hours of culture, Gluconobacter frateurii CHM 9 and Psuedmon were representatively selected from the 100 or more strains tested.
as sp. CHM 32, Acetobacter rancens SKU 1111, Acine
Four strains of tobacter calcoaceticus LMD79.41 were selected.
Table 1 shows a comparison of the relative growth rate (Growth) of these strains, the final pH of the culture solution, and the enzyme activity of the cell membrane fraction with respect to cyclopentanol.

[0016]

[Table 1]

Regarding the final pH of the culture medium, Gluconobac
Only ter frateurii CHM 9 showed acidic values. On the other hand, Psuedmonas sp. CHM 32 and Acinetobacter calcoac
eticusLMD 79.41 was a weakly alkaline value. In the case of Acetobacter rancens SKU 1111, it was the same as the initial pH value.

The apparent cyclopentanol dehydrogenase activity of the cell membrane fraction was determined by phenazine methosulfate (PMS).
And the PMS-DCIP method using 2,6-dichlorophenol indophenol (DCIP). As the substrate, 25 μmol of cyclopentanol was used. McIlvaine buffer (pH 5.0) was used as the measurement solution. One unit of enzyme activity was defined as the activity to catalyze 1 μmol of substrate oxidation per minute under reaction conditions. As a result of the measurement, Gluconobacter frateurii CHM
In the case of 9, activity was observed in two pH regions. One was the major activity peak at pH 5.0 and the other was the minor activity peak at pH 9.0. On the other hand, no cyclopentanol dehydrogenase activity was observed in the cell membrane fractions of the other three strains.

Psuedmonas sp. CHM 32 and Acinetoba
In the case of cter calcoaceticus LMD 79.41, it is known that the NAD-dependent CCAD activity existing in the cytoplasm is predominant. It is known that CCAD performs two reactions, depending on the reaction conditions, oxidation of cyclopentanol to cyclopentanone and reduction of cyclopentanone to cyclopentanol. Therefore, the cyclopentanol oxidation activity is 100 μmol cyclopentanol, 50
μmol glycine-NaOH (pH9.5), 0.1μmol NAD
The enzyme was added to a 1 ml solution consisting of and the rate of increase of NADH at 25 ° C was recorded by the absorbance at 340 nm. One unit of enzyme activity was defined as the activity of producing 1 μmol NADH per minute under the reaction conditions. As a result of the measurement, in the case of Psuedmonas sp. CHM 32, it is considered that cyclopentanol was consumed in the cytoplasm. Further, in the case of Acetobacter rancens SKU 1111, it is considered that it is difficult to grow in the presence of cyclopentanol, and it seems that it survived after utilizing a very small amount of cyclopentanol. Next, regarding the reducing activity of cyclopentanone, 100 μmol cyclopentanone, 50
μmol potassium phosphate (pH 6.0), 0.1 μmol
Add the enzyme to a 1 ml solution consisting of NADH,
The rate of decrease of NADH at 25 ° C was recorded by the absorbance at 40 nm. One unit of enzyme activity was defined as the activity of reducing 1 μmol NADH per minute under the reaction conditions. Furthermore, the amount of enzyme produced was compared under various culture conditions. As a result, Acetobacter ra
The enzyme of ncens SKU 1111 was found to be a constitutive enzyme.

Gluconobacter frateurii CHM 9 resting cells reacted with cyclopentanol. The reaction was then analyzed to see if the direct oxidation product, cyclopentanone, accumulated during the reaction.
Pure cyclopentanone is 2,4-dinitrophenylhydrazi
Since it is known that a derivative can be formed by reacting with ne, the reaction mixture was reacted with 2,4-dinitrophenylhydrazine and then analyzed by thin layer chromatography (TLC). 2,4-dinitrophenylhy produced by the reaction
The drazone derivative is pure cyclopentanone 2,4-din
The same Rf value as itrophenylhydrazone derivative 0.8
A value of 2 was shown. Therefore, as expected, the oxidation of cyclopentanol to cyclopentanone was shown to occur without any time lag, proportional to both the reaction time and the number of cells used. In most cases of oxidative fermentation, no reaction was observed to occur without a time lag. This is because in the cells of the present invention, substrate oxidation is performed on the outer surface of the cells. Therefore, the MCAD of the present invention is the same as the conventional CCAD or quinoprotein alcohol dehydrogenase (AD) of acetic acid bacterium cell membrane.
H) is a result showing that it is a novel enzyme which is clearly different.

[0021]

Example 2 A cell membrane fraction having a novel enzyme MCAD was treated with E
It was investigated whether or not the treatment with DTA had some influence on the oxidation activity of cyclopentanol. The method for preparing the cell membrane fraction and the cytoplasmic fraction is as follows.
The cells after culture of Gluconobacter frateurii CHM 9 were collected, and a cell suspension was prepared so that the wet cells were about 10 g per 10 ml of 10 mM Tris-HCl (pH 7.5) buffer. Rannie high pressure laboratory homogenize the cell suspension at a pressure of 1,000 psi.
passed through r. After removing unbroken cells by ordinary low speed centrifugation, the resulting crude cell-free extract solution was adjusted to 68.0%.
The precipitated cell membrane fraction was separated by further centrifugation at 00 × g for 90 minutes. The cell supernatant was used as the cytosolic fraction.
The cell membrane fraction was 10 mM Tris-HCl (pH 7.
5) After resuspending in a buffer and washing the precipitate by homogenizing, it was centrifuged at 68,000 xg for 90 minutes. This washing operation was repeated twice to minimize contamination of the cytoplasmic fraction. Obtained cell membrane fraction (Native
MCAD activity was increased by adding PQQ and CaCl ₂ to the membrane (FIG. 1). From this result, it was shown that MCAD partially exists as an apoenzyme when the cell membrane fraction was prepared.

EDTA treatment was performed with 10 mM Tris-H.
10 mg / ml with Cl (pH 7.5) buffer
After preparing the cell membrane suspension so that it becomes a protein, 2
Incubation was carried out for 30 minutes in an ice bath under the condition of 0 mM EDTA. After performing ultracentrifugation at 90,000 xg for 60 minutes to remove the supernatant, precipitate was washed with 10 mM Tr.
It was resuspended in an is-HCl (pH 7.5) buffer and subjected to ultracentrifugation under the same conditions to remove EDTA. EDT
The cell membrane after treatment with A showed only a slight MCAD activity, and most of MCAD was found to be an apoenzyme (data of EDTA treated membrane in FIG. 1). Cell membrane fraction containing apoenzyme (EDTA treated membrane)
rane), 5 μM PQQ and 5 mM CaC
When l ₂ was added and the mixture was incubated at 25 ° C. for 30 minutes and the activity was measured again, the enzyme activity was recovered.

For the apoenzyme of the cell membrane fraction, PQQ
5 μM or CaCl ₂ 5 mM was added alone, 25
When the activity was measured again after incubation at 30 ° C. for 30 minutes, recovery of the enzyme activity was observed in both cases, but the effect was greater with CaCl ₂ (FIG. 1).
From this result, it is speculated that the main action of EDTA is to remove Ca ²⁺ ions from the enzyme by the chelating effect. Therefore, by adding both PQQ and CaCl _2, the apoenzyme was converted to the holoenzyme, and MCAD
It was shown that the activity could be restored to its original level. Therefore, the MCAD of the present invention has a typical quinoprotein (qu
It was concluded that it is a cyclic alcohol dehydrogenase.

[0024]

Example 3 MCAD was separately solubilized from the cell membrane fraction. Prior to solubilization of the enzyme, PQQ (5 μM) and CaCl _{2 (} 5 mM) were added to the cell membrane suspension to change the apoenzyme into a holoenzyme. In the cell membrane fraction used for solubilization, the total protein was 1.4 g, and the cyclopentanol dehydrogenase activity was 3,120 units (Membrane black bar on the left side of FIG. 2). Also, quinoprotein A
The DH activity was 1,440 units (M on the left side of Fig. 2).
White bar of embrane). 10 mM Tris-HC
After adjusting the protein concentration of the cell membrane fraction to 10 mg / ml with 1 (pH 7.0) buffer, Na-cholate
Was added to 0.5% and the mixture was stirred slowly for 1 hour. If you centrifuge at 68,000 xg for 60 minutes,
Most of the quinoprotein ADH activity is due to centrifugation supernatant (Supe
rnatant). However, 70% or more of the original cyclopentanol dehydrogenase activity remained in the precipitated cell membrane fraction (Precipitate) (Fig. 2).
Na-cholate data).

The precipitated cell membrane fraction was treated with 10 mM Tri
It was suspended in s-HCl (pH 7.0) buffer and the protein concentration was adjusted to 10 mg / ml. To this suspension of cell membrane fraction, Mydol 10 ^™ was added at 1.0% and gently stirred for 1 hour. 68,000 ×
After centrifugation at 60 g for 60 min, about one-third of the cyclopentanol dehydrogenase activity in the original cell membrane fraction appeared in the supernatant (Fig. 2, right side, black bar of Mydol 10 ^™) . ). On the other hand, the quinoprotein ADH activity was so low that it could not be detected under the usual activity test conditions. These results indicate that MCAD activity is not due to quinoprotein ADH, and it was shown for the first time that the enzyme MCAD that specifically oxidizes cyclic alcohol is present in the cell membrane fraction of acetic acid bacteria.

[0026]

Example 4 Next, MCAD was purified. Example 3
As described above, the stepwise solubilized MCAD solution was added to
5 mM CaCl₂And Mydol 10^TM10m including 0.3%
DEA equilibrated with M acetate buffer (pH 5.0)
It was applied to an E-Toyopearl column (2.6 x 9 cm). M
CAD is not adsorbed, and some impurities are
Was removed. The solution that has passed through the column is collected and 5 mM
 CaCl₂And Mydol 10^{T M}10 mM acetic acid containing 0.3%
It was dialyzed against a buffer (pH 4.0).

Next, the solution after dialysis was treated with 5 mM CaCl ₂ .
When applied to a CM-Toyopearl column (1.6 × 6 cm) equilibrated with 10 mM acetate buffer (pH 4.0) containing Mydol 10 ^™ 0.3%, MCAD and remaining quinoprotein ADH were loaded on the column. Adsorbed.
10 mM acetate buffer containing 5 mM CaCl ₂ (p
After washing with H4.0), elution was performed with a 5-40 mM CaCl ₂ linear concentration gradient. As shown in FIG.
A peak having MCAD activity (● in FIG. 5) was detected in the peak fraction detected by the absorbance at 280 nm (circle in FIG. 5).
Mark) was detected. The peak fraction having MCAD activity was collected and analyzed by SDS-PAGE. As a result, it was found to be a single peak with a molecular weight of 83 kDa (lane 2 in FIG. 4). Lane 1 in FIG. 4 is the size marker band, phosphorylase b (94 kDa), bovine.
serum albumin (68 kDa), ovalbumin (43 kDa), ca
rbonicanhydrase (31 kDa) and lysozyme (14.4 kDa) were used.

The absorbance at 420 nm (marked by □ in FIG. 5)
The rear peak of the two peaks detected by E. coli was the elution peak of quinoprotein ADH. MCAD was completely separated from quinoprotein ADH, and it was simultaneously proved that they are not the same. By such a two-step purification operation, MCAD was purified 25 times (Table 2).

[0029]

[Table 2]

[0030]

Example 5 For both the cell membrane fraction and the purified enzyme, M
The substrate specificity of CAD was investigated (Table 3).

[0031]

[Table 3]

MCAD was highly specific for cyclic alcohols, especially cyclopentanol and cyclohexanol. The apparent Michaelis constant for cyclopentanol was 1 mM when tested under optimal conditions.
Was measured. By MCAD, cyclooctanol,
cis-1,2-cyclopentanediol and 2,3-butanediol
However, it was an interesting result that it was well oxidized. MCAD also oxidized some aliphatic secondary alcohols. MCAD, in addition to the oxidation of cyclic alcohols and secondary alcohols, glycerol, meso-erythritol, ribitol, D-
Sugar alcohols such as arabitol, D-sorbitol and D-mannitol were also oxidized. MCAD appears to recognize these sugar alcohols as secondary alcohols.

As in the case of cyclopentanone, the individual oxidation products accumulated extracellularly. Therefore MCAD
Is considered to be a useful enzyme in chiral chemistry. The localization of MCAD on the outer surface of the cell membrane has the advantage of facilitating the oxidative fermentation of cyclic alcohols. Since cyclic alcohols have some biological toxicity to living cells due to the mechanism of oxidative fermentation, it is not preferred to incorporate such toxic substances into the cytoplasm for oxidation. Also, extra bioenergy is required to pump the oxidation products out of the cell through the cell membrane. PQ as a coenzyme
Only Q is valid, NAD (P), NAD (P) H,
FAD and FMN were not effective as coenzymes.

[0034]

Example 6 NAD-dependent CCAD present in the cytoplasm of Gluconobacter frateurii CHM 9 is the MCA of the present invention.
It was proved to be an enzyme different from D. The above cytoplasmic fraction was added to buffer A (5 mM β-mercaptoethanol
DEA equilibrated with 2 mM KPB pH 7.2) containing
It was applied to an E-cellulose column (2.5 x 20 cm).
After washing the column with buffer A, CCAD was eluted with buffer A containing 0.2 M KCl. CCA recovered
Fraction D was dialyzed overnight against buffer A and then applied to a blue dextran Sepharose column (2 x 20 cm) equilibrated with buffer A. CCAD is
The NAD (P) -dependent enzyme, which was not adsorbed on the Blue Dextran Sepharose column, was removed at this step.

Next, DEA equilibrated with buffer A
E-Sephadex A-50 column (1.5 x 20c
m). After washing the column with buffer A containing 75 mM KCl, the column was eluted with buffer A containing 100 mM KCl and then with buffer A containing 125 mM KCl. The main part of CCAD is 125 mM KC
Elution with buffer A containing 1 was performed. CCAD recovered
Fractions were applied to a hydroxyapatite column (5 x 7 cm) equilibrated with buffer A. 15 mMphosp
After washing with buffer A containing hate, CCAD was 30
Elution was performed with buffer A containing mM phosphate. Finally, CCAD was crystallized with ammonium sulphate (Fig. 5).

The outline of CCAD purification is shown in Table 4. C
CAD was purified 38 times from the crude extract, and the total yield was 2
Obtained with a recovery of 8%. Hitachi analytical ultracentrifuge SCP85H
At 20 ° C. and 60,000 rpm,
As a result of measuring the sedimentation pattern of CCAD every 10 minutes,
It was shown that the apparent sedimentation rate was a symmetrical peak at 3.0 s, and roughly, the molecular weight of CCAD was equivalent to 60 kDa ((A) of FIG. 6). The results of SDS-PAGE revealed that the protein was composed of two identical subunits having a molecular weight of 29,000 ((B) of FIG. 6).

[0037]

[Table 4]

Lane 2 in FIG. 6 is a cell-free extract (20
μg protein), lane 3 after purification with DEAE-cellulose (15 μg protein), lane 4 after purification with DEAE-Sephadex A-50 (10 μg protei).
n), lane 5 after purification with hydroxyapatite (5 μg protein), lane 6 after the first crystallization (5 μg p
rotein) result. Lane 1 is a size marker band, phosphorylase b (110 kDa), bovi
ne serum albumin (77 kDa), ovalbumin (50.5 kD
a), carbonic anhydrase (35.2 kDa), soybean tryps
in inhibitor (29.1 kDa) and lysozyme (14.4 kDa) were used.

[0039]

Example 7 The substrate specificity of CCAD is shown in Table 3.
CCAD oxidised cyclic alcohols under alkaline pH conditions of 8.5-10.0 when tested in 50 mM glycine-NaOH. CCAD oxidised cyclic alcohols but was narrower than MCAD in terms of substrate specificity in the oxidation of cyclic alcohols (Table 3).
CCAD is considered to be a secondary alcohol dehydrogenase since it oxidizes 2-butanol well. The crystallized CCAD showed a specific activity of 0.45 units / mg protein for cyclopentanol, which was compared with the specific activity of membrane-bound MCAD for cyclopentanol (44.3 units / mg protein). Then, the value was about 100 times lower.

It is believed that the cytoplasmic NAD (P) -dependent enzyme, which has a low Michaelis constant, reflects the rapid elimination system of toxic compounds that happens to occur in the cytoplasm. Of course, the cyclopentanol and cyclohexanol used in the present invention are not naturally present in high concentrations. Therefore, cyclopentanol and cyclohexanol should be considered as true and real substrates of CCAD.

The apparent Michaelis constant of CCAD for cyclohexanol and NAD is 7 each.
Measured as mM and 73 μM. In addition, CC for cyclohexanone and NADH
Apparent Michaelis constants for AD were measured at 18 mM and 0.4 mM, respectively.

On the other hand, the reduction of cyclic ketones by CCAD, when tested in 50 mM sodium acetate, resulted in a pH of 5.
It showed the highest activity under the acidic condition of 0. Thus, CCAD has a high reactivity with cyclohexanone and was easily reduced to the corresponding cyclic alcohol, cyclohexanol. From the above results,
It was proved that MCAD of the present invention and conventional CCAD are completely different enzymes.

When the resting cell or cell membrane fraction was mixed with the substrate cyclic alcohol, the cyclic alcohol was converted to the corresponding cyclic ketone in a short time. The conversion rate was so large that it could not be compared with the reaction rate due to NAD-dependent CCAD. Therefore, it is shown that CCAD does not make any contribution to oxidative fermentation,
It was possible to clarify that only the membrane-bound MCAD was functioning.

[0044]

EFFECTS OF THE INVENTION The present inventors have found that in the cell membrane of aerobic bacteria,
For the first time, we pointed out the existence of oxidative activity with PQQ as a coenzyme,
Screening of 100 or more cell lines containing aerobic bacteria such as acetic acid bacteria was carried out for a membrane-bound cyclic alcohol dehydrogenase producing strain. As a result, 1 of the conventional CCAD, which is present in the cell membrane of Gluconobacter acetic acid bacteria
For the first time, a membrane-bound cyclic alcohol dehydrogenase (MCAD) having activity as high as 00 times was found and solubilized and purified.

[Brief description of drawings]

FIG. 1 Gluconobacter frateurii C after EDTA treatment
FIG. 6 shows that MCAD from HM 9 strain is reactivated by PQQ and CaCl ₂ .

Fig. 2 M from Gluconobacter frateurii CHM 9 strain
It is a figure which shows fractional solubilization of CAD.

Figure 3: M from Gluconobacter frateurii CHM 9 strain
It is a figure which shows the elution profile from CM-Toyopearl column chromatography of CAD.

FIG. 4 M from Gluconobacter frateurii CHM 9 strain
It is a figure which shows the result of SDS-PAGE of CAD.

Fig. 5 C from Gluconobacter frateurii CHM 9 strain
It is a figure which shows the crystal form of CAD.

FIG. 6: C from Gluconobacter frateurii CHM 9 strain
It is a figure which shows the purity of CAD.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Hirohide Toyama             1677-1 Yoshida, Yamaguchi City, Yamaguchi Prefecture Yamaguchi University             Faculty of Agriculture (72) Inventor Ganjana Teragur             Kingdom of Thailand Bangkok 10900 Kasetsart Large             Study (72) Inventor Zuangtip Moonman Me             Kingdom of Thailand Bangkok 10140             Institute of Technology Thonburi F-term (reference) 4B050 CC01 CC07 DD02 FF09 FF11                       FF12 LL05                 4B064 AC09 AC31 CA21 CB13 CB28                       DA16

Claims (9)

[Claims] 1. A membrane-bound cyclic alcohol dehydrogenase of acetic acid bacteria and other aerobic Gram-negative bacteria. 2. The membrane-bound cyclic alcohol dehydrogenase according to claim 1, wherein the acetic acid bacterium is an acetic acid bacterium of the genus Gluconobacter. 3. An acetic acid bacterium of the genus Gluconobacter is Glucono.
The membrane-bound cyclic alcohol dehydrogenase according to claim 1 or 2, which is bacter frateurii CHM 9. 4. PQQ is used as a coenzyme, and Ca is used for the expression of activity.
The membrane-bound cyclic alcohol dehydrogenase according to any one of claims 1 to 3, which requires ²⁺ ions. 5. A method for producing a cyclic ketone from a cyclic alcohol, which uses the membrane-bound cyclic alcohol dehydrogenase according to any one of claims 1 to 4. 6. A method for producing a secondary ketone from a secondary alcohol, which uses the membrane-bound cyclic alcohol dehydrogenase according to any one of claims 1 to 4. 7. The cell membrane fraction or cell having a membrane-bound cyclic alcohol dehydrogenase is used, according to claim 5 or 6.
The manufacturing method described in. 8. A membrane-bound cyclic alcohol dehydrogenase is used to oxidize only one optical isomer of a racemate,
A method for separating optical isomers, which comprises separating only non-oxidized optical isomers. 9. The membrane-bound cyclic alcohol dehydrogenase is G
An enzyme derived from luconobacter frateurii CHM 9,
The method for separating optical isomers according to claim 8, wherein the racemic compound is 2,3-butanediol.