JP2000078969A - Alcohol dehydrogenase and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new alcohol dehydrogenase derived from a microorganism belonging to the genus Sphingobacterium, having action oxidizing a primary or secondary alcohol, etc., to an aldehyde in the presence of NAD+, having high heat stability and high industrial utilization value. SOLUTION: This new alcohol dehydrogenase has the following properties. Action; catalyzing a reaction producing an aldehyde and a reduction type nicotinamide adenine dinucleotide (NADH) from an alcohol and nicotinamide dinucleotide (NAD+). Substrate specificity: widely acting on a 2-10C primary alcohol or secondary alcohol; Optimum pH: 7.0; stable pH range: 6.0-9.0; stable temperature area: 0-60 deg.C; molecular structure: a tetramer of polypeptide having about 168,000 molecular weight. The enzyme is obtained by culturing a microorganism belonging to the genus Sphingobacterium and having above enzyme- producing ability [Sphingobacterium spiritivorum P1 (FERM P-16974)].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel alcohol dehydrogenase and a method for producing the same.

[0002]

2. Description of the Related Art Existing alcohol dehydrogenases (hereinafter referred to as A
DH) are coenzyme-independent and nicotinamide adenine dinucleotide (hereinafter NA) as a coenzyme.
D ⁺ ) or reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH), or those requiring pyrroloquinolinoquinone (PQQ). Many of these enzymes are derived from microorganisms and have limited substrate specificity, and work well for certain alcohols such as ethanol and methanol, but not enough for other alcohols. Many did not work. As a microorganism that produces ADH, Escherichia coli (Esche
richia coli), Clostridium,
Pseudomonas, Acinetobacter, Mycobacteriu
m), a thermophilic bacterium of the genus Bacillus (Bacillus stearotherm)
ophilus), all of which were normal-temperature or thermophilic bacteria. Recently, however, a paper on ADH produced by psychrophilic microorganisms has been published (Eu
r. J Biochem., 254, 356-362, 1998).
It was shown that H is not always produced only by a normal-temperature bacterium or a thermophilic bacterium, and that ADH derived from a psychrophilic bacterium can also exist.

[0003] Generally, enzymes obtained from thermophilic bacteria have higher thermostability than those derived from normal-temperature bacteria. On the other hand, although enzymes derived from psychrophilic microorganisms have the property of being able to act even at low temperatures, in most cases they are inferior in thermostability as compared with those derived from room-temperature bacteria or thermophilic bacteria. For example, A derived from the psychrophilic bacterium (Maraxella sp. TAE123)
In the case of DH, the residual activity after incubation for 30 minutes was about 20% at 37 ° C and 5% or less at 50 ° C. in this way,
Enzymes derived from psychrophilic bacteria often have low stability at room temperature or high temperature, and can be used only for specific applications. Therefore, their industrial utility value is lower than enzymes derived from ordinary temperature bacteria or thermophilic bacteria.

[0004]

As described above, most of the ADHs derived from microorganisms which have been reported so far are derived from room temperature or thermophilic microorganisms, and ADH derived from chilled microorganisms. There were very few reports on this. Also, no enzyme having high thermostability has been reported, even if it is derived from a psychrophilic microorganism. An object of the present invention is to provide ADH having high ADH activity and high thermal stability, and a method for producing the same.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, an enzyme obtained from a microorganism belonging to the genus Sphingobacterium, which is a psychrophilic microorganism, has been obtained. The present inventors have found that the enzyme has high ADH activity, high heat stability, and that the above-mentioned enzyme can be produced by culturing the above-mentioned microorganism.

That is, the present invention provides a method for producing ADH, comprising culturing ADH having the following properties and a microorganism capable of producing the ADH, and collecting the ADH according to claim 1 from the culture. It is the gist. (A) Action: catalyzes the following reaction. Alcohol + NAD ⁺ → aldehyde + NA
DH (b) Substrate specificity: widely acts on primary or secondary alcohols having 2 to 10 carbon atoms. (C) Optimum pH: 7.0 (d) Stable pH range: 6.0 to 9.0 (e) Optimum temperature: 60 ° C. or higher (f) Stable temperature range: 0 to 60 ° C. (g) Molecular structure : 4 of polypeptide with a molecular weight of about 168,000
Monomer

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention may be any microorganism as long as it can produce ADH having the above-mentioned properties, but a psychrophilic microorganism is preferred. As the psychrophilic microorganism, a microorganism belonging to the genus Sphingobacterium is preferable. Spigotobacterium spiritivorum P1 is particularly preferred. The bacteriological properties of the above strains are shown below.

(A) Morphology: non-motile bacilli (b) Stainability: negative for Gram stain (c) Physiological properties (1) Nitrate reduction: negative (2) FO test: O (oxidation) (3) Pigment production: water-insoluble yellow pigment production

[0010] The mycological properties were identified by Union Vitec (1-17-10 Hoshin, Higashiyodogawa-ku, Osaka-shi). From the mycological properties, the above strain was found to be a bacterium belonging to Sphingobacterium spilitivolim, named sphingobacterium spiritivorum P1, and was contacted by the Ministry of International Trade and Industry, National Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology on August 31, 1998. Deposited. The deposit number is FERM P-1
6974.

The ADH of the present invention can be produced, for example, by culturing the ADH-producing bacterium in a nutrient medium and collecting from the culture.

[0012] The medium used for the culture of ADH may be a natural medium as long as it contains a carbon source, a nitrogen source, inorganic salts, and other necessary nutrients which can be used by the used strain in an appropriate amount.
Any of the synthetic media can be used. For example, glucose, lactose, maltose and the like can be used as a carbon source, and nitrogen-containing natural products such as polypeptone and corn steep liquor, and inorganic nitrogen compounds such as ammonium salts and urea can be used as a nitrogen source. Potassium, sodium, calcium, magnesium, iron, manganese, cobalt, copper, phosphoric acid and the like are used as inorganic substances. In addition, alcohols such as ethanol, propanol, and butanol may be added to the medium as an ADH inducer.

The culture of ADH-producing bacteria is carried out by shaking culture or aeration-agitation culture. The culture temperature at this time is 4-25.
℃, preferably 10-20 ℃, the medium p
H is controlled in the range of 5 to 9, preferably 6 to 8. However, implementation is possible even under conditions other than these as long as the strain to be used grows. The culture period of the strain is usually 1 to 4 days, preferably 36 to 72 hours. The strain produces ADH during the growth process and accumulates in the cells.

For the purification of ADH of the present invention, a commonly used purification method can be used. For example, as a method for extracting an enzyme from cells, ultrasonic, glass beads, a physical crushing method using a French press, a chemical method using a surfactant, a biochemical method using lysodium. And the like.

The target enzyme can be purified to a high purity by treating the extract obtained by the above method in combination with a commonly used enzyme purification method. Examples include methods such as ammonium sulfate precipitation, ion exchange chromatography, molecular weight fractionation by gel filtration, and affinity chromatography.

[0016]

Next, the present invention will be described specifically with reference to examples. In addition,% represents weight%.

Embodiment 1

(1) Cultivation Sphingobacterium spiritibolum
terium spiritivorum) P1 (FERM P-1697
4) One loopful of the agar slant culture in the nutrient medium (1.0%
Yeast extract, 2.0% polypeptone: pH 7.0) 20
The cells were inoculated into 0 ml and cultured at 15 ° C. with shaking for two days. The cultured cells were subcultured in a 10 L jar fermenter containing a 7 L medium, and cultured with stirring for 48 hours. The obtained cultured cells were separated into a medium component and the cells by centrifugation.
g of wet cells were obtained.

(2) Cell disruption 20 g of wet cells were immersed in a 10 mM phosphate buffer (pH 7.0).
After suspending in 40 ml, it was crushed by ultrasonic waves (treatment time: 3 minutes x 5 times). The cell lysate was used to precipitate solids by centrifugation, and the resulting supernatant was used as a crude enzyme extract for purification.

(3) Purification 3-1 DEAE-Toyopearl column chromatograph The crude enzyme extract obtained by the above method was applied to a DEAE-Toyopearl column equilibrated with 10 mM phosphate buffer (pH 7.0). The non-adsorbed ADH (flow-through fraction) was collected.

3-2 Phenyl-Toyopearl column chromatograph Ammonium sulfate was added to the flow-through fraction collected by the DEAE-Toyopearl column to a final concentration of 1.5 M, and the formed precipitate was removed. 10m containing 0.5M ammonium sulfate
Phenyl-equilibrated with M phosphate buffer (pH 7.0)
It was applied to a Toyopearl column. The enzyme adsorbed on the column
Eluted with a linear gradient of 1.5-0 M ammonium sulfate.

3-3 Affinity Column Chromatography The active fraction of the enzyme eluted by the above method is dialyzed against a 10 mM phosphate buffer (pH 7.0), and then adsorbed on a 5-AMP Sepharose 4B column to give 0-2 mM. NAD
^The enzyme was eluted with a linear gradient of ⁺ .

(4) Purity Assay The purity of the alcohol dehydrogenase purified by the above three-step method was confirmed by non-denaturing acrylamide gel electrophoresis, and the obtained ADH had an Rm value of 0.24.
A single band was shown. Inspection of the subunit structure revealed that the enzyme was a tetramer of a polypeptide having a molecular weight of 168,000.

The properties of the enzyme obtained by the above method were examined as follows.

[Measurement of Activity] The activity of ADH was measured by using ethanol as a substrate at 15 ° C.
The change in absorbance at 340 nm due to DH was measured. The reaction solution has a final composition of 100 mM CHES (pH 9.
0) It was prepared to be 1 mM NAD ⁺ and 10 mM ethanol. 2.95 ml of the reaction solution was placed in a cuvette and kept warm for 5 minutes, the reaction was started by adding 50 μl of the crude enzyme extract, and the increasing absorbance at 340 nm was measured. In addition, this enzyme activity is 1 under the above conditions.
The amount of enzyme capable of purifying 1 μmol of NADH per minute was defined as one unit.

[Substrate specificity] Of the above reaction solutions, various alcohols are added as substrates instead of ethanol,
The relative activity when the activity in ethanol was set to 100 was examined. Table 1 shows the results. From Table 1, the carbon number is 2 to 10
It has been found to work widely on primary alcohols or secondary alcohols.

[0027]

[Table 1]

[Inhibitor] Various metal ions and the like were added to a reaction solution using ethanol as a substrate, and the inhibitory activity against ADH of the present invention was examined. Table 2 shows the results. as a result,
The enzyme activity of the present invention is HgCl ₂ , NiCl ₂ , CuCl
₂ significantly inhibited. Also, monoiodoacetic acid, N
Since the activity was lost by -ethylmaleimide, it was suggested that the enzyme of the present invention was an SH enzyme having an SH group at the active center.

[0029]

[Table 2]

[Optimal pH and pH Stability] To determine the optimal pH of the enzyme of the present invention, the pH of the buffer of the reaction solution was changed from 2.0 to 11.0 (pH 2.0-3.5).
: Gly-HCl, pH 3.0-6.0: Citrat
e, pH 6.0-8.0: KPB, pH 8.0-9.0
: TAPS, pH 9.0-10.0: CHES, pH1
0.0-11.0: CAPS), and the enzyme activity at each pH was measured. The results are shown in FIG. as a result,
The optimum pH of this enzyme was shown to be 7.0. Next, in order to examine the pH stability of the present enzyme, the present enzyme was incubated at 30 ° C. in the presence of a 10 mM buffer (pH 2.0 to 11.0).
For 60 minutes, and then the residual activity was measured at pH 7.0. The results are shown in FIG. As a result, the enzyme had a pH of 6.
It was shown to be stable between 0 and 10.0.

[Optimal Temperature and Thermal Stability] In order to determine the optimal temperature of the present enzyme, the reaction temperature during the activity measurement reaction was varied from 0 ° C. to 75 ° C., and the reactivity at that time was examined. The results are shown in FIG. As a result, the activity of this enzyme increased as the reaction temperature increased, and no remarkable decrease in the activity was observed until 75 ° C. Next, in order to examine the thermostability of the enzyme, the enzyme was incubated at 0 to 80 ° C for 60 minutes (pH 7.0).
Then, the remaining activity was measured at 15 ° C. Fig. 4 shows the results.
Shown in The results showed that this enzyme was stable up to 60 ° C.

[Half-life of activity at 75 ° C.] In order to examine the thermostability of the present enzyme, the half-life of the activity of the present enzyme at 75 ° C. was examined. Keep the enzyme at 75 ° C for 0-60 minutes (p
H7.0), and the remaining activity thereafter was measured at 15 ° C.
The residual activity of the enzyme (relative activity when the enzyme activity at the time of 0 minute incubation was taken as 100%) was plotted against the incubation time.
FIG. 5 shows the results. The results showed that the half-life of the activity of this enzyme at 75 ° C. was 26 minutes.

Comparative Example 1 Yeast [Saccharomyce
s. cerevisiae)], the optimal temperature, thermal stability, and half-life of the activity at 75 ° C. of ADH (purchased from Sigma) were measured in the same manner as in Example 1.

[Activity measurement] AD derived from yeast [Saccharomyces cerevisiae]
H (purchased from Sigma) was measured for enzyme activity in the same manner as in Example 1 (reaction solution composition: 100 mM CHES (pH 9.
0) ¹ mM NAD ⁺ , 10 mM ethanol: However, the reaction temperature was 25 ° C.). The enzymatic activity was defined as the amount of enzyme capable of producing 1 μmol of NADH per minute under the above conditions as one unit.

[Optimal temperature] The optimal temperature of the above enzyme was determined in the same manner as in Example 1. The results are shown in FIG.
As a result, the optimum temperature of yeast-derived ADH was 25 to 30 ° C.

[Thermal Stability] The thermal stability of the above enzyme was determined in the same manner as in Example 1. FIG. 4 shows the results.
As a result, the heat resistance temperature of yeast-derived ADH was 40 ° C.

[Half-life of activity at 75 ° C.]
The half-life of the activity of the above enzyme at 75 ° C. was examined in the same manner as described above. FIG. 5 shows the results. As a result, the yeast-derived ADH was completely inactivated at 75 ° C. within several minutes.

[0038]

The ADH of the present invention acts widely on substrates,
It has excellent pH stability, high ADH activity, and high thermal stability. In addition, the production method of the present invention relates to the above ADH.
Can be manufactured.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of ADH of the present invention.

FIG. 2 shows the pH stability of ADH of the present invention.

FIG. 3 shows the optimum temperatures of ADH of the present invention and yeast-derived ADH.

FIG. 4 shows the thermal stability of ADH of the present invention and yeast-derived ADH.

FIG. 5 shows the half-life of activity at 75 ° C. of ADH of the present invention and yeast-derived ADH.

Claims (4)

[Claims] 1. An alcohol dehydrogenase having the following properties: (A) Action: catalyzes the following reaction. Alcohol + nicotinamide adenine dinucleotide (NAD ⁺ ) → aldehyde + reduced nicotinamide adenine dinucleotide (NADH) (b) Substrate specificity: acts widely on primary or secondary alcohols having 2 to 10 carbon atoms. (C) Optimum pH: 7.0 (d) Stable pH range: 6.0 to 9.0 (e) Optimum temperature: 60 ° C. or higher (f) Stable temperature range: 0 to 60 ° C. (g) Molecular structure : 4 of polypeptide with a molecular weight of about 168,000
Monomer 2. A method for producing an alcohol dehydrogenase, comprising culturing a microorganism capable of producing alcohol dehydrogenase according to claim 1 and collecting the alcohol dehydrogenase according to claim 1 from the culture. Method. 3. Sphingobacterium
A microorganism belonging to the genus rium), which is capable of producing the alcohol dehydrogenase according to claim 1, is cultured, and the microorganism is cultured.
A method for producing an alcohol dehydrogenase, comprising collecting the alcohol dehydrogenase according to any one of the preceding claims. 4. Sphingobacterium
rium), a microorganism capable of producing alcohol dehydrogenase is Sphingobacterium spiritivorum P1 (FERM).
The method for producing an alcohol dehydrogenase according to claim 3, which is P-16974).