JP2007228925A - New oxidase and method for producing glyoxal by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an oxidase or a microorganism, converting glycolaldehyde to glyoxal, and to provide a method for efficiently producing glyoxal. <P>SOLUTION: The oxidase acting on glycolaldehyde to produce glyoxal and hydrogen peroxide in the presence of oxygen is provided, wherein, as substitute specificity, the oxidase exhibits activity on glycolaldehyde and glyceraldehyde but substantially does not exhibit activity on methanol, ethanol, 1,2-propanediol, 1,3-propanediol and glyoxal. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a novel oxidase having the ability to convert glycolaldehyde to glyoxal and a method for producing glyoxal using the enzyme. Glyoxal is a compound useful as a raw material for pharmaceuticals such as fiber processing agents, paper processing agents, soil hardening agents, agricultural chemicals and glyoxylic acid.

At present, glyoxal is produced by a chemical method, but the production method is complicated, and a large amount of by-products are produced, so that the yield of glyoxal is low. In order to solve the disadvantages of this chemical production method, methods using microorganisms and enzymes have been studied, and alcohol oxidase derived from yeast and glycerol oxidase derived from Aspergillus Japanicas have ethylene glycol via glycolaldehyde. It was clarified that it can be converted to glyoxal (Patent Document 1, Non-Patent Documents 1, 2, and 3). On the other hand, an enzyme that converts glycolaldehyde to glyoxal has been found by microorganisms belonging to the genus Rhizobiales and Arthrobacter, and it has been reported that glyoxal can be synthesized by this enzyme (patent). Reference 2).

Conventional alcohol oxidase derived from yeast or glycerol oxidase derived from Aspergillus japanicas accumulates glycol aldehyde in the presence of high ethylene glycol concentration, and it is difficult to convert ethylene glycol to glyoxal. There has been a demand for a microorganism that efficiently converts glycolaldehyde to glyoxal, has high thermal stability, and produces a stable enzyme in a wide range of pH, and acquisition of the enzyme.
JP-A-8-84592 JP 2005-245235 A Biosci. Biotech. Biochem. 58, 170-173 (1994) Biosci. Biotech. Biochem. 59, 576-581 (1995) J. et al. Mol. Catal. B: Enzymatic, 1, 37-43 (1995)

Accordingly, an object of the present invention is to provide a novel oxidase that converts glycolaldehyde to glyoxal, a microorganism that produces the oxidase, a method for producing the oxidase, and the efficiency of glyoxal using the oxidase or the microorganism. It is in providing the method of manufacturing automatically.

As a result of intensive studies to solve the above-mentioned problems, the present inventor has found a novel oxidase that converts glycolaldehyde to glyoxal in the genus Paenibacillus. And this oxidase was isolated and refine | purified from the said microorganism, and this invention was completed.
That is, the present invention provides a novel oxidase having the following properties (1) and (2).
(1) Action:
Acts on glycolaldehyde in the presence of oxygen to produce glyoxal and hydrogen peroxide.
(2) Substrate specificity:
It shows activity against glycolaldehyde and glyceraldehyde, and has substantially no activity against methanol, ethanol, 1,2-propanediol, 1,3-propanediol and glyoxal. (In the present specification, “substantially no activity” means that the activity for the compound is less than 5% of the activity for glycolaldehyde.)
Preferably, in addition to the above properties, an oxidase having a property that the activity with respect to ethylene glycol is 10% or less of the activity with respect to glycol aldehyde.
Preferably, it is an oxidase having the following properties (3) and (4) in addition to the above properties.
(3) Optimum pH of action: 6.0-7.0.
(4) Optimum temperature of action: 45-55 ° C.
More preferably, it is an oxidase having the following properties (5) and (6) in addition to the above properties.
(5) Thermal stability: After pretreatment at 40 ° C. for 1 hour at pH 6.5, 85% or more activity remains.
(6) pH stability: After pretreatment of heating at 40 ° C. for 1 hour at pH 5.0 to pH 8.5, 80% or more activity remains.
More preferably, it is an oxidase having the following properties (7) and (8) in addition to the above properties.
(7) Molecular weight: 4.9 × 10 ^{4 in} gel filtration analysis, 2.4 × 10 ⁴ in SDS-polyacrylamide electrophoresis analysis.
(8) Inhibitor: Inhibited by phenylhydrazine, hydrazine, semicarbazide, α, α′-dipyridyl, 8-oxyquinoline, monoiodoacetic acid, NiCl ₂ and CoCl ₂ .

The present invention also provides a microorganism that produces the oxidase. Furthermore, this invention also provides the manufacturing method of the said oxidase.
On the other hand, the present invention also provides a method for producing glyoxal, wherein the oxidase or the microorganism is allowed to act on glycolaldehyde to oxidize an alcohol hydroxyl group present in glycolaldehyde and convert it to glyoxal.
Furthermore, by reacting ethylene glycol with an oxidase having the ability to convert ethylene glycol into glycol aldehyde or a microorganism producing the oxidase to produce glycol aldehyde, the resulting glyceraldehyde is subjected to the oxidase of the present invention. Alternatively, the present invention also relates to a method for producing glyoxal, wherein the microorganism of the present invention is allowed to act to convert to glyoxal.
The present invention is described in detail below.

The novel oxidase of the present invention is a highly specific oxidase that oxidizes glycolaldehyde to glyoxal but does not act on the reaction product glyoxal. If the oxidase of the present invention is used, It can be manufactured efficiently.

The present invention has found a microorganism that produces a novel oxidase that does not act on glyoxal by acting on glycol aldehyde that has not been reported so far, reveals various properties of the enzyme, and is effective in the production of glyoxal. It was completed by clarifying. For such microorganisms, soil samples are added to a medium containing ethylene glycol, glycol aldehyde, glycolic acid or the like as a carbon source and cultured, and then the microorganisms that have grown are examined for oxidation activity against glycol aldehyde and glyoxal. Can be obtained.

The microorganism that is the origin of the enzyme of the present invention is not particularly limited, but bacteria and the like are preferable, preferably a microorganism belonging to the genus Paenibacillus, particularly preferably Paenibacillus species isolated from the soil of Lake Notori, Abashiri, Hokkaido. (Paenibacillus sp.) AIU AL311 strain. The AIU AL311 strain has been deposited as FERM P-20786 at the National Institute of Advanced Industrial Science and Technology Patent Microorganisms Depositary Center (1-6 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 305-8586). Table 1 shows the mycological properties of AIU AL311 strain.

In addition, for identification of AIU AL311 strain, a region of about 500 bp on the 5 ′ end side of the 16S ribosomal RNA gene (16SrDNA) of the strain is amplified by PCR, the nucleotide sequence is determined, and the international nucleotide sequence database (GenBank / DDBJ / EMBL) was carried out by homology search and a method of preparing a molecular phylogenetic tree.

The oxidase of the present invention can be obtained from the microorganism producing the enzyme as follows. For example, the microorganism having this activity is cultured under suitable conditions, and after culturing, the cells are collected from the culture solution by centrifugation, etc., and disrupted by a method such as ultrasonic disruption or disruption using glass beads. A crude enzyme solution is obtained. Further, the enzyme of the present invention can be obtained by purifying the crude enzyme solution by a method such as salting out or various chromatography.

The oxidase of the present invention, for example, a bacterium belonging to the genus Paenibacillus, and representatively, the oxidase produced by Paenibacillus sp. AIU AL311 (FERM P-20786) is glycol aldehyde, It works well with aldehyde alcohols such as seraldehyde, but has little effect on aliphatic aldehydes and alcohols, and is clearly different in substrate specificity from alcohol oxidase and aldehyde oxidase reported so far. In addition, the oxidase of the present invention is highly stable, has a pH of 6.5, retains almost 100% of activity after heating at 30 ° C. for 1 hour, and is about 90% even at 40 ° C. for 1 hour. Activity remains. The oxidase of the present invention also has high pH stability, such as maintaining 80% activity after heating at 40 ° C. for 1 hour at pH 5.0 to 8.5.

The optimum pH or temperature of action of the oxidase of the present invention is determined by measuring the activity by changing the pH or temperature of the reaction conditions.

The molecular weight of the oxidase of the present invention can be calculated from the relative elution time with a standard protein by gel filtration analysis using, for example, a TSK-G3000SW (7.8 mm × 30 cm) (manufactured by Tosoh Corporation) column. The molecular weight can be calculated from the relative mobility with the standard protein by SDS-polyacrylamide electrophoresis.

Further, the N-terminal amino acid sequence can be determined by subjecting the enzyme purified by the above method or further purified by reverse phase HPLC to a protein sequencer.

In the present invention, the medium for culturing a microorganism that produces an oxidase converting glycol aldehyde to glyoxal is not particularly limited as long as the microorganism can grow. For example, as a carbon source, sugars such as glucose and sucrose, alcohols such as ethanol, glycerin, ethylene glycol and propylene glycol, organic acids such as glycolic acid, fatty acids such as oleic acid and stearic acid and esters thereof, rapeseed oil and Oils such as soybean oil; ammonium sources such as ammonium sulfate, ammonium nitrate, peptone, casamino acid, yeast extract, meat extract and corn steep liquor; inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium hydrogen phosphate, phosphorus A normal liquid medium having malt extract, meat extract and the like may be used.

The method for producing glyoxal according to the present invention is characterized in that the oxidase of the present invention or the microorganism of the present invention producing the oxidase is allowed to act on glycolaldehyde to be converted and accumulated into glyoxal.

Furthermore, the method for producing glyoxal according to the present invention was produced by producing glycol aldehyde by allowing an oxidase having the ability to convert ethylene glycol to glycol aldehyde or a microorganism producing the oxidase to act on ethylene glycol. It is characterized in that it is converted to glyoxal by allowing the oxidase of the present invention or the microorganism of the present invention to act on glycolaldehyde.

The oxidase having the ability to convert ethylene glycol to glycol aldehyde is not particularly limited as long as it has the ability to convert ethylene glycol to glycol aldehyde, and examples thereof include alcohol oxidase and glycerol oxidase. The origin of alcohol oxidase is not particularly limited, and examples include alcohol oxidase produced by yeast, mold, and bacteria. Examples include Pichia, Candida, Hansenula, Toluropsis, and Ogataair. Examples include alcohol oxidase produced by yeast belonging to the genus (Ogataea). Examples of glycerol oxidase include glycerol oxidase produced by yeast, mold, and bacteria, and examples include glycerol oxidase produced by the genus Aspergillus. For enzymes other than the above, microorganisms belonging to the genus Aspergillus, such as Aspergillus ochraceus (Aspergillus ochraceus), for example, Aspergillus ochraceus (Aspergillus ochraceus) AIU 031 strain (FERM P-20785) is converted to glycol aldehyde. An oxidase having the ability to The AIU 031 strain was isolated and identified from the soil by the present inventors, and was registered with the Patent Microorganism Deposit Center of the National Institute of Advanced Industrial Science and Technology (1st Higashi 1-chome, Tsukuba City, Ibaraki 305-8586, Central No. 6). ).

The oxidase used in the present invention may be a single or partially purified enzyme. As the microorganism used in the present invention, a culture of the microorganism or a processed product thereof can be used. Here, “microorganism culture” means a culture solution or culture containing bacterial cells, and “the processed product” means, for example, a crude enzyme solution, freeze-dried cells, acetone-dried cells, or These crushed materials and their mixtures are meant. Furthermore, the oxidase or the microorganism can be used after being immobilized by a known means (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.). The microorganism of the present invention or the microorganism that produces an oxidase having the ability to convert ethylene glycol to glycol aldehyde may be a wild strain or a mutant strain, or may encode the oxidase as long as it has the ability to produce the oxidase. It may be a transformant (recombinant) in which the DNA to be incorporated is incorporated into a vector and introduced into the host. The transformant having the ability to produce the oxidase used in the present invention can also be produced by stably integrating DNA encoding the oxidase into the host genome.

The conditions for reacting the oxidase of the present invention with glycol aldehyde or the conditions for reacting oxidase with the ability to convert ethylene glycol to glycol aldehyde with ethylene glycol vary depending on the enzyme used, but the temperature ranges from 5 ° C to 5 ° C. 80 degreeC, Preferably it is the range of 5-60 degreeC, pH is 4-12, Preferably it is the range of pH 5-9. An oxidase having the ability to convert ethylene glycol into glycolaldehyde, or a microorganism that produces the oxidase is allowed to act to produce glycolaldehyde, and the resulting glycolaldehyde is subjected to the oxidase of the present invention or the microorganism of the present invention. When converting to glyoxal by acting, the concentration of ethylene glycol is preferably set high so that an enzyme that converts ethylene glycol to glycol aldehyde works efficiently, preferably 100 mM or more, and more preferably 1 M or more. Further, since glycol aldehyde has a property of inactivating the enzyme, it is not preferable to accumulate it at a high concentration during the reaction. Therefore, it is effective to control the amount of glycolaldehyde accumulated by adjusting the amount of enzyme having the ability to convert ethylene glycol to glycolaldehyde and the amount of enzyme having the ability to convert glycolaldehyde to glyoxal. . On the other hand, when glycolaldehyde is converted to glyoxal using the enzyme of the present invention as the initial substrate, it is not preferable to add glycolaldehyde to the reaction system at a high concentration from the viewpoint of enzyme stability. Preferably it is 1 M or less, more preferably 100 mM or less. In this case, it is preferable to add glycolaldehyde intermittently or continuously with the progress of the reaction, which leads to improvement in productivity. The reaction is preferably performed under oxygen conditions. In order to promote dissolution of oxygen in the reaction solution, the reaction is preferably performed under shaking and stirring conditions. Furthermore, by performing the reaction under a pressure higher than atmospheric pressure, the solubility of oxygen in the reaction solution is improved, and the reaction may further progress.

Incidentally, hydrogen peroxide is produced by an oxidation reaction by the oxidase or the oxidase having the ability to convert ethylene glycol into glycolaldehyde, but this hydrogen peroxide may deactivate the enzyme. However, by adding catalase to the reaction system, the generated hydrogen peroxide can be decomposed and removed. From the viewpoint of rapidly decomposing and removing peroxidase, the catalase used may be used in an amount of activity that is 10 times or more, preferably 100 times or more, more preferably 1000 times or more the activity of the oxidase used. desirable. In addition, hydrogen peroxide can be efficiently decomposed by allowing the above-mentioned transformant to recombinantly express catalase together with an oxidase. On the other hand, it is preferable to use a microorganism originally having an ability to produce catalase as a host of a transformant.

The novel oxidase of the present invention is a highly specific oxidase that oxidizes glycolaldehyde to glyoxal but does not act on the reaction products glyoxal and ethylene glycol at all. If the oxidase of the present invention is used, Glyoxal can be produced efficiently.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
(Example 1) Separation of microorganisms capable of producing an oxidase which acts on glycolaldehyde but does not act on glyoxal As a medium for screening, a medium containing 1% glycolic acid shown in Table 2 is 120C. And a small amount of soil was added to the test tube into which 5 ml of this glycolic acid-containing medium was dispensed, and cultured with shaking at 30 ° C. for 1 to 7 days. About the culture solution with which the growth of microorganisms was confirmed, the culture solution 0.1ml was inoculated to the culture medium of the same composition as the above, and was similarly shake-cultured at 30 degreeC for 1 to 7 days. After repeating this operation once more, the culture solution was plated out on an agar plate medium prepared by adding 2% agar to the same composition as the liquid medium, and cultured at 30 ° C. And after the growth of microorganisms was confirmed, the coloring solution shown in Table 3 was added to this agar plate medium to which glycolaldehyde for confirming the production of glycolaldehyde and hydrogen peroxide was added to 50 mM. The reaction was performed at 30 ° C. By this method, microorganisms in which the production of hydrogen peroxide was observed were inoculated into a slant medium having the same composition as the plate medium, cultured at 30 ° C. and stored. Next, the microorganisms separated into the slant medium were cultured with shaking in a liquid medium containing 5% ethylene glycol shown in Table 2.

Then, the cells were collected from the culture by centrifugation, the collected cells were suspended in 0.1 M phosphate buffer (pH 7.0), and glycolaldehyde or glyoxal was added to a concentration of 50 mM. The reaction was added to the color developing solution for measurement. In this reaction, the reaction solution was colored purple in the presence of glycolaldehyde (hydrogen peroxide was generated), and in the presence of glyoxal, the color of the reaction solution was hardly changed (hydrogen peroxide was not generated). . Subsequently, the selected microorganisms were subjected to shaking culture at 30 ° C. using a Sakaguchi flask containing 150 ml of a liquid medium having the above composition containing 5% ethylene glycol shown in Table 2. The cells of 900 ml obtained by this culture were collected by centrifugation, suspended in 0.1 M phosphate buffer (pH 7.0) and disrupted using glass beads. The cell disruption residue was removed by centrifugation, and the resulting supernatant was subjected to DEAE-Toyopearl Karakum chromatography. That is, 20 ml of DEAE equilibrated with 10 mM phosphate buffer (pH 7.0) after desalting the supernatant collected after removing the cell disruption residue to the same salt concentration as 10 mM phosphate buffer. -The solution was passed through a Toyopearl column, and this column was washed with 10 mM phosphate buffer (pH 7.0). The adsorbed enzyme was eluted by a linear concentration gradient method using 10 mM phosphate buffer (pH 7.0) and 10 mM phosphate buffer (pH 7.0) containing 0.5 M NaCl. The oxidase activity for glyoxal was examined. Then, microorganisms that produce an enzyme that acts well on glycolaldehyde and does not exhibit oxidase activity on glyoxal were selected.

The strain obtained by such a method is a microorganism exhibiting the above-mentioned mycological properties, and Paenibacillus sp. It was named AIU AL311.
In addition, the color developing solution shown in Table 3 was used for the oxidase activity with respect to glycolaldehyde on the agar plate medium, and the color developing solution shown in Table 4 was used for the oxidase activity with respect to glycolaldehyde in the solution.

(Example 2) Production of oxidase which acts on glycolaldehyde but does not act on glyoxal The microorganisms separated in Example 1 were used in a test tube using a liquid medium containing 5% ethylene glycol shown in Table 2. A seed culture solution was prepared by shaking culture at 30 ° C. for 2 days. Next, 1.5 ml of this seed culture solution is inoculated into a 500 ml flask containing 150 ml of a liquid medium having the same composition as that of the seed culture, and cultured at 30 ° C. with shaking. Examined. As a result, the production amount of the oxidase acting on glycolaldehyde showed the highest value in the 1-day culture, and the activity of the oxidase maintained a high value even around the 6th day of the culture.

(Example 3) Purification of oxidase which acts on glycolaldehyde but does not act on glyoxal Based on the result of Example 2, using a liquid medium containing 5% ethylene glycol shown in Table 2 in a test tube A primary seed medium was prepared by shaking culture at 30 ° C. for 2 days. 1.5 ml of this primary seed culture solution was inoculated into a 500 ml flask in which 150 ml of a liquid medium having the same composition was dispensed, and shake-cultured at 30 ° C. for 2 days to prepare a secondary seed culture solution. Subsequently, 20 ml of this secondary seed culture was inoculated into a 3 L flask containing 2 L of liquid medium having the same composition as the primary seed culture, and cultured with shaking at 30 ° C. for 1 day. Bacteria were collected from 9 L of the culture medium thus cultured, and the enzyme was purified by the following method. In addition, a pH 7.0 phosphate buffer was used for the purification of the enzyme.

1) Preparation of crude enzyme solution Cell bodies (wet weight: 7.5 g) collected from a 9 L culture solution are suspended in 10 mM phosphate buffer, and 0.5 mm glass beads are used at a temperature of 10 ° C. or less for 12 minutes. Cells were disrupted (2 minutes x 6 times). The cell lysate was centrifuged to remove cell residue, and the supernatant fraction was used as a crude enzyme solution for enzyme purification.
2) DEAE-Toyopearl column chromatography The crude enzyme enzyme solution was desalted and concentrated to 2 ms / cm with an ultrafiltration membrane, and then equilibrated with a 10 mM phosphate buffer (2.2 × 17. 5 cm). The column was washed with 650 ml of 10 mM phosphate buffer, and then the enzyme was eluted by a linear concentration gradient method using 10 mM phosphate buffer (340 ml) and 10 mM phosphate buffer (340 ml) containing 0.5 M NaCl.
3) Hydroxyapatite column chromatography The eluate from the DEAE-Toyopearl column was desalted and concentrated with an ultrafiltration membrane, and then passed through a hydroxyapatite column (1 x 6 cm) buffered with 5 mM phosphate buffer. Subsequently, this column was washed with 100 ml of 5 mM phosphate buffer to elute the target enzyme.
The enzyme preparation of 3) obtained by the above method was analyzed by Native and SDS-polyacrylamide electrophoresis. As a result, the sample was electrophoretically single. The purification yield of the enzyme by this purification method was as shown in Table 5.

(Example 4) Properties of oxidase that acts on glycolaldehyde but not on glyoxal The enzyme preparation purified by the method of Example 3 was analyzed using Native and SDS-polyacrylamide electrophoresis. As a result, the protein was single in any analysis. Therefore, various properties were examined using this purified enzyme preparation.
(Function)
0.05 ml of the purified enzyme solution was added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 to which glycolaldehyde was added to 21 mM, and the reaction was performed at 30 ° C., and the increase in absorbance at 555 nm was observed over time. It was measured. As a result, it was revealed that the absorbance at 555 nm increased with the passage of time, and hydrogen peroxide was produced by the reaction of ethylene glycol and this enzyme.

In addition, the reaction product was subjected to the method of Isobe and Nishise [Biosci. Biotech. Biochem. , 58, 170-173, (1994)], and reacted with N-methyl-2-benzothiazolinone hydrazone (MBTH), and analyzed its absorption spectrum and elution time from C18 reverse phase column. First, the reaction product was dissolved in 0.75 ml of 0.2 M glycine-HCl buffer (pH 4.0), and 0.3 ml of 1.0% (w / v) MBTH solution was added thereto (reaction 1). Thereafter, the absorption spectrum of the reaction solution obtained in Reaction 1 was measured. As a result, similar to the absorption spectrum of the reaction solution obtained in the reaction 1 of glyoxal, a spectrum having a maximum absorption near 410 nm was obtained in the reaction solution of the reaction product 1 described above. When the reaction solution of reaction 1 was analyzed with a C18 reverse phase column, a peak was obtained at the same elution position as the product obtained in reaction 1 of glyoxal. Therefore, it became clear that this enzyme oxidizes glycol aldehyde to glyoxal according to the following formula.
OHCCHO + O ₂ + H ₂ O → OHCCOOH + H ₂ O ₂

Next, 0.05 ml of the purified enzyme solution was added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 to which glyoxal was added to 21 mM, and the reaction was carried out at 30 ° C. to increase the absorbance at 555 nm over time. Measured. As a result, no increase in absorbance at 555 nm was observed. Therefore, it was clarified that this enzyme oxidizes glycolaldehyde to glyoxal but does not act on glyoxal which is a reaction product.

(Substrate specificity)
0.05 ml of purified enzyme solution is added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 to which aldehydes, organic acids or alcohols shown in Table 6 are added to 53 mM, and the reaction is carried out at 30 ° C. The increase in absorbance at 555 nm was continuously measured with a spectrophotometer, the increase in 555 nm per hour was calculated, and the activity against each compound was examined.
As a result, as shown in Table 6, this enzyme works well on glycolaldehyde and glyceraldehyde, and shows a slight action on formaldehyde, acetaldehyde, ethylene glycol, propionaldehyde, but isobutyraldehyde, valeraldehyde, isoforms. It did not substantially act on valeraldehyde, glyoxal and glyoxylic acid. Moreover, it did not act on primary alcohols such as methanol, ethanol, 1-propanol, 1-butanol, 1,2-propanediol, 1,3-propanediol and the like. In addition, each numerical value in Table 6 is a relative value when the activity with respect to glycolaldehyde is 100.

(Km value for glycolaldehyde)
Add 0.05 ml of purified enzyme solution to 0.95 ml of coloring solution for enzyme activity measurement shown in Table 4 to which glycol aldehyde has been added to a concentration of 10 mM to 45 mM, and react at 30 ° C. to increase the absorbance at 555 nm. Was continuously measured with a spectrophotometer, an increase of 555 nm per hour was calculated, and the activity against glycolaldehyde at each concentration was measured. Furthermore, Km value was computed by Lineweaver-Burk plot created from the obtained activity value and substrate concentration. As a result, the Km value for glycolaldehyde was about 13 mM.

(Molecular weight)
Gel filtration using TSK-G3000SW (7.8 mm × 30 cm) column (manufactured by Tosoh Corporation) by high performance liquid chromatography using the purified enzyme, and 10% SDS-polyacrylamide electrophoresis in the presence of 1% 2-mercaptoethanol The molecular weight was estimated from the relative mobility with the standard protein. As a result, the molecular weight was about 49,000 by gel filtration and about 24,000 by SDS-polyacrylamide electrophoresis [molecular weight measurement conditions by gel filtration].
Column: TSK-G3000SW (7.8 mm × 30 cm) (manufactured by Tosoh Corporation)
Eluent: 0.1 M phosphate buffer + 0.3 M sodium chloride (pH 7.0)
Flow velocity: 1.0ml / min
Temperature: Room temperature detection: 220nm

(Influence of various compounds)
0.05 ml of purified enzyme solution was added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 in which glycolaldehyde was added to 53 mM and the compound shown in Table 7 to a concentration of 1.05 mM, and the reaction was performed at 30 ° C. The increase in absorbance at 555 nm was continuously measured with a spectrophotometer, the increase in 555 nm per hour was calculated, and the activity was examined.
As a result, as shown in Table 7, the enzyme, phenylhydrazine, hydrazine, semicarbazide, alpha,. Alpha .'- Jipirijiru, 8-oxyquinoline, monoiodoacetic _acid, inhibited with NiCl 2, CoCl _2, activated with MnCl ₂ It became clear that

(Optimum pH)
Glycolaldehyde was added to 53 mM, and the kind of buffer solution was changed, and 0.05 ml of purified enzyme solution was added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 prepared at pH 5.5 to pH 8.5. Then, the reaction was carried out at 30 ° C., and the increase in absorbance at 555 nm was continuously measured with a spectrophotometer, the increase in 555 nm per hour was calculated, and the activity was examined.
As a result, as shown in FIG. 1, the enzyme showed activity in a wide range from an acidic region to a weakly alkaline region, and the optimum pH of the reaction was 6.0 to 7.0.

(Optimum temperature)
0.05 ml of purified enzyme solution was added to 0.95 ml of the coloring solution for enzyme activity measurement shown in Table 4 to which glycolaldehyde was added to 53 mM, and the reaction was carried out in the range of 30 ° C. to 60 ° C. The increase was continuously measured with a spectrophotometer, the increase amount of 555 nm per hour was calculated, and the activity was examined.
As a result, as shown in FIG. 2, this enzyme showed activity at any measured temperature, and the optimal temperature for the reaction was 45-55 ° C.

(PH stability)
The enzyme solution was pretreated at 40 ° C. for 1 hour at pH 5.0 to 8.5. Thereafter, 0.05 ml of the pretreated enzyme solution was added to 0.95 ml of the color developing solution for enzyme activity measurement shown in Table 4 to which glycolaldehyde was added to 53 mM, and the reaction was carried out at 30 ° C., and the absorbance at 555 nm. Was continuously measured with a spectrophotometer, the amount of increase at 555 nm per hour was calculated, and the remaining enzyme activity was examined. FIG. 3 shows the residual activity after each treatment (relative activity after each treatment relative to the activity before pretreatment: the activity before pretreatment is taken as 100).
As a result, as shown in FIG. 3, the enzyme activity remained in a wide range from the acidic region to the alkaline region, and particularly in the range of pH 5.0 to pH 8.5, 80% or more of the activity remained. Therefore, it was revealed that this enzyme is stable in a wide range from the acidic region to the alkaline region.

(Thermal stability)
After pretreatment of heating the enzyme solution at 0 to 70 ° C. for 1 hour at pH 6.5, the residual enzyme activity was measured by the same method as the above pH stability. As a result, as shown in FIG. 4, 60% or more of the enzyme activity remained even after heating at 70 ° C. for 1 hour. Therefore, it became clear that this enzyme is heat stable.

(N-terminal amino acid sequence)
Using purified enzyme, the N-terminal amino acid sequence of this enzyme was examined. The N-terminus of this purified enzyme is shown in SEQ ID NO: 1.

(Example 5) Synthesis of glyoxal using crude enzyme solution The cells collected from 250 ml of AIU AL311 strain culture solution obtained by the method described in Example 2 were suspended in 5 ml of 10 mM phosphate buffer, The cells were disrupted for 10 minutes (2 minutes × 5 times) using 0.5 mm glass beads at a temperature of 10 ° C. or lower. The cell lysate was centrifuged to remove cell residue, and a supernatant fraction was obtained as a crude enzyme solution. To 0.2 ml of the obtained crude enzyme solution, 0.2 ml of 250 mM glycol aldehyde aqueous solution and 0.1 ml of 50,000 U / ml catalase (derived from Bovive Liver; manufactured by Nakarai Co., Ltd.) solution were added, and 28 ° C. for 10 hours in a test tube The reaction was carried out by shaking. Thereafter, the obtained reaction solution was analyzed by high performance liquid chromatography (HPLC). As a result, 13 mM glyoxal was generated in the reaction solution.

[HPLC analysis conditions]
Column: Aminex HPX-87H (7.8 mm × 300 mm) manufactured by Bio-Rad
Detection: Differential refractive index (RI)
Column temperature: 25 ° C
Eluent: 5 mM H ₂ SO ₄ aqueous solution Flow rate: 0.4 ml / min Elution time: Glyoxal-16 minutes

(Example 6) Purification purified enzyme 0.1U of enzyme obtained in Synthesis <br/> Example 3 of glyoxal with, glycolaldehyde 50 mM, catalase (Bovive Liver derived; manufactured by Nacalai Tesque, Inc.) 500 U 100 mM phosphate buffer containing 1 ml of the solution (pH 6.5) was added to the test tube, and the reaction was performed by shaking at 30 ° C. for 24 hours. Thereafter, the obtained reaction solution was analyzed by HPLC. As a result, 20 mM glyoxal was generated in the reaction solution.

(Example 7) Synthesis of glyoxal from ethylene glycol 1 M ethylene glycol, 2 U / ml (methanol oxidation activity) Pichia pastris-derived alcohol oxidase (Sigma), 0.2 U / ml (glycolaldehyde oxidation activity) ) 0.5 ml of 300 mM phosphate buffer (pH 8.0) containing the purified oxidase obtained in Example 3 of 10000 U / ml of catalase (derived from Bovive River; manufactured by Nacalai) in a test tube at 28 ° C. The reaction was carried out with shaking for 4 hours. Thereafter, the obtained reaction solution was analyzed by HPLC. As a result, 5 mM glycolaldehyde and 18 mM glyoxal were produced.
[HPLC analysis conditions]
Column: Shodex Rspak KC-811 (manufactured by Showa Denko KK), connected detection: differential refractive index (RI)
Column temperature: 40 ° C
Eluent: 50 mM perchloric acid aqueous solution Flow rate: 1 ml / min
Elution time: Glycolaldehyde; 17.9 minutes, Glyoxal; 15.7 minutes

The graph which shows the action | operation optimal pH of the oxidase acquired in Example 3 The graph which shows the action | operation optimal temperature of the oxidase acquired in Example 3 Graph showing the pH stability of the oxidase obtained in Example 3 The graph which shows the thermostability of the oxidase acquired in Example 3

Claims (16)

An oxidase having the following properties (1) and (2).
(1) Action:
Acts on glycolaldehyde in the presence of oxygen to produce glyoxal and hydrogen peroxide.
(2) Substrate specificity:
It shows activity against glycolaldehyde and glyceraldehyde, and has substantially no activity against methanol, ethanol, 1,2-propanediol, 1,3-propanediol and glyoxal. The oxidase of Claim 1 whose activity with respect to ethylene glycol is 10% or less of the activity with respect to glycol aldehyde. Furthermore, the oxidase of Claim 1 or 2 which has the property of following (3) and (4).
(3) Optimum pH of action: 6.0-7.0.
(4) Optimum temperature of action: 45-55 ° C. Furthermore, the oxidase in any one of Claims 1-3 which has the property of following (5) and (6).
(5) Thermal stability: After pretreatment at 40 ° C. for 1 hour at pH 6.5, 85% or more activity remains.
(6) pH stability: 80% or more of the activity remains after pretreatment of heating at 40 ° C. for 1 hour at pH 5.0 to 8.5. Furthermore, the oxidase in any one of Claims 1-4 which has the property of following (7) and (8).
(7) Molecular weight: 4.9 × 10 ^{4 in} gel filtration analysis, 2.4 × 10 ⁴ in SDS-polyacrylamide electrophoresis analysis.
(8) Inhibitor: Inhibited by phenylhydrazine, hydrazine, semicarbazide, α, α′-dipyridyl, 8-oxyquinoline, monoiodoacetic acid, NiCl ₂ and CoCl ₂ . The oxidase according to any one of claims 1 to 5, which has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing at the N-terminus. The oxidase according to any one of claims 1 to 6, which is produced by a bacterium belonging to the genus Paenibacillus. The oxidase according to claim 7, wherein the bacterium belonging to the genus Paenibacillus is Paenibacillus sp. AIU AL311 strain (FERM P-20786). A microorganism that produces the oxidase according to any one of claims 1 to 8. A method for producing the oxidase according to any one of claims 1 to 8, wherein the microorganism according to claim 9 is cultured. A method for producing glyoxal, which comprises converting the oxidase according to any one of claims 1 to 8, or the microorganism culture according to claim 9 or a processed product thereof to glycolaldehyde to convert it into glyoxal. Glycolaldehyde is produced by reacting ethylene glycol with an oxidase having the ability to convert ethylene glycol to glycolaldehyde, or a culture of microorganisms that produce the oxidase or its treated product. A method for producing glyoxal, wherein the oxidase according to any one of claims 1 to 8, or the microorganism culture according to claim 9 or a treated product thereof is allowed to act to convert to glyoxal. The oxidase having the ability to convert ethylene glycol into glycolaldehyde is at least one oxidase selected from the group consisting of alcohol oxidase, glycerol oxidase, and an oxidase obtained from Aspergillus ochraceus. 12. A method for producing glyoxal according to 12. The alcohol oxidase is an alcohol obtained from at least one microorganism selected from the group consisting of Pichia genus, Candida genus, Hansenula genus, Toluropsis genus, and Ogataea genus The method for producing glyoxal according to claim 13, which is an oxidase, and the glycerol oxidase is a glycerol oxidase obtained from a microorganism belonging to the genus Aspergillus. The method for producing glyoxal according to any one of claims 11 to 14, wherein catalase is allowed to coexist during the reaction. The method for producing glyoxal according to claim 11, wherein the reaction is carried out in the presence of oxygen.

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