JPS6155946B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel formaldehyde dehydrogenase and a method for producing the same. Formaldehyde dehydrogenase, which has been known for a long time, is produced by yeast and bovine liver.
NAD) produces enzymatic action only in the presence of reduced glutathione (Enzyme Handbook,
(Refer to Asakura Shoten, p. 97, 1977). Also, although the addition of NAD and/or reduced glutatin is not required, dichlorophenol indophenol (hereinafter referred to as
Formaldehyde dehydrogenase (Antonie van Leeuwenh
-ock Vol. 41, pp. 89-95, 1975), and furthermore
Formaldehyde dehydrogenase is known that does not require the addition of NAD or reduced glutathione, but requires the addition of pteridine or phenazine methosulfate, and also has an oxidizing effect on methanol. (Agr.Biol.Chem.Volume 41 No.
467, 1977). In addition, microorganisms belonging to the genus Pseudomonas are NAD.
It is known that it has the ability to produce formaldehyde dehydrogenase, which uses as an electron acceptor and does not require the addition of reduced glutathione to exhibit its activity (see Biochem. J., Vol. 116, pp. 357-365, 1970). The present inventors discovered that the mechanism of action is completely different from that of formaldehyde dehydrogenase, which acts on formaldehyde, acetaldehyde, propionaldehyde, methylglyoxal, and glyoxal using only NAD as an electron acceptor; We have discovered a novel formaldehyde dehydrogenase that does not act on methanol, ethanol, propanol, ethylene glycol, glycerol, fatty acids, and amino acids (substrate concentration 50mM), and does not require the addition of reduced glutathione to express its activity, and has achieved the present invention. Reached. That is, the present invention provides (1) at least the following properties ~
This is a novel formaldehyde dehydrogenase comprising the following. HCHO+NAD+ _H2O →HCOOH+NADH _2As mentioned above, formaldehyde is oxidized to form formic acid via nicotinamide adenine dinucleotide, and it does not have the opposite effect. Nicotinamide adenine dinucleotide is the only electron acceptor. It does not utilize other electron acceptors such as nicotinamide adenine dinucleotide phosphate. Addition of reduced glutathione is not required for activity expression. Km value for formaldehyde is 8×10 ^-5 M
(PH7.5), and the Km value for NAD is 12×
10 ^-5 M (PH7.5). In addition to formaldehyde, acetaldehyde,
propionaldehyde, methylglyoxal,
Although it acts on glyoxal, it does not act on aldehydes with 4 or more carbon atoms, alcohols such as methanol, ethanol, propanol, ethylene glycol, glycerol, fatty acids, and amines (substrate concentration 50 mM). The molecular weight is 150000±1000 and the isoelectric point is PI=
5.2±0.1, and the optimum pH is 7.0 to 8.0. Stable against nonionic surfactants, but susceptible to anionic, cationic and amphoteric surfactants. The formaldehyde dehydrogenase of the present invention does not require the addition of reduced glutathione, and has the advantage that it does not require the addition of reduced glutathione.
(see p. 1977). In addition, conventional formaldehyde dehydrogenase (Antonie van Leeuwenh
(see ock Vol. 41, pp. 89-95, 1975). Furthermore, in that it does not have an oxidizing effect on methanol, conventional formaldehyde dehydrogenase (Agr. Biol. Chem. Vol. 41, p. 467)
1977), and methylamine,
Conventional formaldehyde dehydrogenase (Biochem.J.
116, pp. 357-865, 1970). Next, the novel formaldehyde dehydrogenase of the present invention will be described in detail. (1) Action As shown below, formaldehyde is oxidized to form formic acid via NAD, and it does not have the opposite action. HCHO + NAD + H ₂ O → HCOOH + NADH ₂ Utilizing only NAD as the electron acceptor, other substances such as nicotinamide adenine dinucleotide phosphate (NADP), dichlorophenol indophenol (DCPIP), pteridine, phenazine methosulfate (PMS) Does not utilize electron acceptors. Addition of reduced glutathione is not required for activity expression. (2) Substrate specificity In addition to formaldehyde, it also acts on acetaldehyde, propionaldehyde, methylglyoxal, and glyoxal, but aldehydes with 4 or more carbon atoms, alcohols such as methanol, ethanol, propanol, ethylene glycol, glycerol, fatty acids, It does not act on amines (substrate concentration 50mM). The table below shows the relative activity of the novel formaldehyde dehydrogenase of the present invention against various substrates.

[Table] (3) Optimal PH and stable PH range The PH action curve of the enzyme of the present invention is shown in Figure 1.
Figure 2 shows the PH stability curve for 16 hours. Optimal PH is around PH7.5, stable PH range is 25
℃, pH 8.5-9.5 after 16 hours treatment. (4) Potency measurement method (i) 340nm absorption increase method in NAD reduction 10mM formaldehyde solution (PH7.5) 1
ml and 0.5 ml of 10 mM NAD solution are added to a 1 cm wide quartz cell and prewarmed at 37°C. Immediately after adding 0.5 ml of an appropriately diluted enzyme solution, record the absorbance at 340 nm using a spectrophotometer, read the change in absorbance over 1 minute from the linear portion, and calculate the enzyme activity using the following formula. U/ml=△E340×2.0(ml)/6.22×0
．． 5 (ml) (ii) Diformazan method 0.5ml of 10mM formaldehyde solution (PH7.5) containing 0.5% Triton X-100, 0.01% PMS and
Mix 0.1 ml of a solution containing 0.1% NTB and 0.1 ml of an aqueous solution containing 3 mM NAD and prewarm to 37 °C. Pour 0.5ml of enzyme solution to allow enzyme reaction.
After 15 minutes, the reaction was stopped with 3 ml of 0.3NHCl.
Read the absorbance at 570nm. Using the case where 0.3NHCl is added and then the enzyme solution is added as a control, the enzyme activity is calculated using the following formula. U/ml=△E570×4.2ml/20.1×15×
0.5 In either case, the enzyme action and color change follow the following formula, and the enzyme activity display values in both cases match. One unit is defined as the amount that decomposes 1 micromole of formaldehyde per minute at 37°C and pH 7.5. (5) Range of action temperature The temperature activity curve of the enzyme of the present invention is shown in Figure 3.
7.5, the temperature stability curve for 30 minutes treatment is shown in Figure 4. The suitable temperature for action is around 40℃, PH7.5,
Stable up to 40°C for 30 minutes. (6) Effects of various metals and surfactants The enzyme is inhibited by Ni〓Zn〓Mn〓Hg〓Cd〓, but is not affected by other metals. It is not affected by nonionic surfactants, but is inhibited by anionic and cationic surfactants.

[Table] (7) Molecular weight by gel filtration method using Sephadex G-200
150000±1000, and 72000±2000 by DISC electrophoresis using SDS polyacrylamide. (8) Isoelectric point PI=5.2±0.1 by ampholine electrophoresis. (9) Km value From the Lineweber-Burk plot, it is 8×10 ^-5 M (PH7.5) for formaldehyde and 12×10 ^-5 M (PH7.5) for NAD. Also, from the action diagram, it is estimated that the reaction is due to the ping-pong BiBi mechanism. The enzyme of the present invention is produced by culturing microorganisms, and microorganisms such as Pseudomonas, Achromobacter, Arthrobacter, Proteus, Serratia, Agrobacterium, Ethscherichia, or Micrococcoccus are used to produce the enzyme. have the ability. Microorganisms of the genus Pseudomonas are particularly desirable. Next, the method for producing the enzyme of the present invention will be described. The medium used in the present invention may be either a synthetic medium or a natural medium as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Particularly preferred is a liquid medium. As carbon sources, various carbohydrates such as glucose, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate, and molasses, or various sugar alcohols such as glycerol, sorbitol, and mannitol can be used, and acetic acid,
Various organic acids such as lactic acid, pyruvic acid, fumaric acid, and citric acid, various alcohols such as methanol and ethanol, various glycols such as ethylene glycol and propylene glycol, various amino acids,
Alternatively, hydrocarbons such as n-hexadecane can also be used. Nitrogen sources include ammonia, ammonium chloride, ammonium carbonate, ammonium phosphate,
Various inorganic and organic ammonium salts such as ammonium nitrate, ammonium acetate, or urea,
Amino acids and other nitrogenous compounds, as well as nitrogenous organic substances such as peptone, NZ-amine, meat extract, corn staple liquor, casein hydrolyzate, pupa hydrolyzate, fruit meal or its digested product, defatted soybean or its digested product, etc. Can be used. Examples of inorganic substances that can be used include primary potassium phosphate, secondary potassium phosphate, potassium chloride, magnesium sulfate, manganese sulfate, ferrous sulfate, sodium chloride, and calcium carbonate. As other nutrients, yeast extract or malt extract is preferable. The medium used in the present invention contains sarcosine, betaine, dimethylglycine,
A larger amount of formaldehyde dehydrogenase can be produced by adding metabolic precursors such as choline, betaine aldehyde, creatine, creatinine, methylamine, dimethylamine, trimethylamine, formaldehyde, or mixtures thereof, or substances containing these. The amount of the metabolic precursor added is preferably 0.05 to 1.0% by weight based on the medium. When a very small amount of formaldehyde is added to the medium at the final stage of culture, enzyme productivity increases. The culture temperature is usually in the range of 15-40℃, preferably 28℃.
~33℃. The pH during culture is usually in the range of 6.0 to 8.5, preferably 6.5 to 8.5. If cultured under such conditions for 1 to 2 days, a large amount of formaldehyde dehydrogenase will be produced in the culture solution and/or the bacterial cells. The bacterial cells are crushed to obtain a bacterial cell extract by a known method such as freezing and thawing, ultrasonic crushing, glass bead grinding, mechanical compression, and autolysis. The enzyme is extracted from the culture solution and the above bacterial cell extract by salting out ammonium sulfate, desalting, and ion exchange chromatography. The novel formaldehyde dehydrogenase of the present invention is also highly significant in industrial applications. For example, hydrogen peroxide is generated from uric acid in the blood by the action of uricase, which is converted to formaldehyde by catalase in the presence of methanol.
When formaldehyde is measured using formaldehyde dehydrogenase, (i) reduced glutathione interferes with quantitative determination using conventional enzymes, but the enzyme of the present invention is not affected by reduced glutathione. (ii) Although enzymes that act on methanol cannot be used in the measurement of formaldehyde, the enzyme of the present invention is useful because it does not act on methanol. (iii) When measuring DCPIP as an electron acceptor,
DCPIP is colored in its oxidized state, so there is a limit to the amount that can be added, but the enzyme of the present invention
Since it does not act as an electron acceptor, there is no limit to the amount added. Hereinafter, a specific method for producing enzymes from microbial cells and the effects of the obtained enzymes on electron acceptors and coenzymes will be explained with reference to Examples. Example 1 Betaine 1%, polypeptone 1%, yeast extract
0.4%, malt extract 0.1%, ammonium chloride 0.2
%, dibasic potassium phosphate 1.4%, and primary potassium phosphate 0.3%. 500 ml of a culture medium prepared by dissolving various nutrient sources in tap water was placed in a 2-capacity shoulder flask and sterilized at 120° C. for 15 minutes. Pseudomonas aeruginosa IFO
3445 was grown in the same medium in advance, and this inoculum solution 10
ml was inoculated into the above medium, cultured with shaking at 30° C. at a speed of 144 rpm, and after 40 hours, the bacterial cells were collected by centrifugation. Suspend the above bacteria in 100ml of 50mM phosphate buffer (PH7.5), centrifuge again, and wash.
The cells were suspended in 50 ml of the same buffer. This suspension was crushed using an ultrasonic crusher for 10 minutes, and then centrifuged to obtain a crude enzyme solution. Ammonium sulfate was then added to 50% saturation, and the resulting precipitate was collected by centrifugation.
After redissolving in 25mM phosphate buffer, dialysis and desalting were performed using the same buffer as an external solution. Pour the obtained liquid in advance
It was loaded and adsorbed onto a DEAE-cellulose column equilibrated with 25mM phosphate buffer. After thorough washing with a buffer solution of the same concentration, the crude enzyme was eluted by flowing a phosphate buffer solution containing 0.3M potassium chloride. The eluate contained 110 units of formaldehyde dehydrogenase and 44 mg of protein. Example 2 The eluate obtained in Example 1 was desalted, and to 1 ml of the obtained solution (0.03 units/ml) was added 1 ml of 10 mM formaldehyde solution, 3 mM NAD, and 0.2 ml of 50 mM phosphate buffer. , reacted at 37°C.
Immediately after the addition of NAD, changes in absorbance at 340 nm were measured. Increase in absorbance per minute is 0.07
It was hot. Similarly, substrates, coenzymes, and reduced glutathione were added as shown in Table 1 below, and changes in absorbance were measured.

[Table] As is clear from Table 1, the enzyme of the present invention does not require the addition of reduced glutathione, and it is clear that it does not act on formic acid. Further, the eluate obtained in Example 1 was desalted, and an equal amount of 5 mM formaldehyde solution was mixed with the obtained solution, and the mixture was heated to 37° C. in advance. 2ml of this mixture
0.2 ml of the following electron acceptors were added to the solution, and the change in absorbance for each specific absorber was measured. The result is the second
Shown in the table.

[Table] As is clear from Table 2, the enzyme of the present invention uses only NAD as an electron acceptor. Furthermore, the eluate obtained in Example 1 was desalted, and the molecular weight of formaldehyde dehydrogenase was measured using the gel filtration method using Sephadex G-200 using the obtained solution. The isoelectric point of formaldehyde dehydrogenase was measured using PI=5.2±0.1. Further, the PH action curve and PH stability curve at 25° C. for 16 hours of formaldehyde dehydrogenase obtained in Example 1 are shown in FIGS. 1 and 2. Furthermore, the temperature activity curve is shown in Fig. 3, and the temperature stability line after treatment at pH 7.5 for 30 minutes is shown in Fig. 4. When the Km value of formaldehyde dehydrogenase obtained in Example 1 was measured from a Lineweber-Burk plot, it was found to be 8×10 ^-5 M (PH7.5) for formaldehyde and 12× for NAD.
It was 10 ^-5 M (PH7.5). The relative activity of formaldehyde dehydrogenase obtained in Example 1 against various substrates was determined using the first
The effects of various metals and surfactants are shown in the table above.

[Brief explanation of the drawing]

FIG. 1 shows the PH action curve of the enzyme of the invention. FIG. 2 shows the PH stability curve of the enzyme of the invention. FIG. 3 shows the temperature activity curve of the enzyme of the invention. FIG. 4 shows the temperature stability curve of the enzyme of the invention.

Claims (1)

[Scope of Claims] 1. A novel formaldehyde dehydrogenase having at least the following properties. HCHO+NAD+ _H2O →HCOOH+NADH _2As mentioned above, formaldehyde is oxidized to form formic acid via nicotinamide adenine dinucleotide, and it does not have the opposite effect. Nicotinamide adenine dinucleotide is the only electron acceptor. Addition of reduced glutathione is not required for activity expression. Km value for formaldehyde is 8×10 ^-5 M
(PH7.5), and the Km value for NAD is 12×
10 ^-5 M (PH7.5). In addition to formaldehyde, acetaldehyde,
propionaldehyde, methylglyoxal,
Although it acts on glyoxal, it does not act on aldehydes with 4 or more carbon atoms, alcohols such as methanol, ethanol, propanol, ethylene glycol, glycerol, fatty acids, and amines. (Substrate concentration 50mM) The molecular weight (gel filtration method using Cephadex G-200) is 15000±1000, and the isoelectric point is PI=
5.2±0.1, and the optimum pH is 7.0 to 8.0. It is stable to nonionic surfactants, but unstable to anionic, cationic, and amphoteric surfactants.