JP2768473B2 - Method for producing NADH oxidase - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a method for oxidizing reduced nicotinamide adenine dinucleotide (hereinafter, abbreviated as NADH) to produce a hydrogen peroxide or water by using NADH oxidase which is involved in an enzymatic reaction. The present invention relates to a method for producing NADH oxidase having a stable activity according to the method of the present invention.

Nicotinamide adenine dinucleotide (hereinafter, NAD
Are abbreviated as coenzymes for various dehydrogenase reactions performed in vivo, and among these enzymes, those which can be suitably used for the production of industrially useful optically active compounds, reaction intermediates, etc. Not a few. When the target enzyme is used as a reaction element in an enzyme reactor, using a reaction equivalent of NAD disposes of expensive NAD and cannot be a practical method. Therefore, it is necessary to reduce the amount of NAD fed into the reaction system from the reaction equivalent, and to regenerate the generated NADH in the system. Enzymatic and chemical methods are known as regeneration methods, but have various disadvantages [Journa of American Chemical Society (Journa).
l of American Chemical Society), 107, 6999-7
008, 1985). For example, there are problems such as a low rate of the regeneration reaction, separation of the substrate-product used in the regeneration application from the product of the target reaction, and the price of the regeneration reaction substrate. Some of these problems can be solved by oxidizing NADH using molecular oxygen as a direct electron acceptor. NA
DH oxidase oxidizes NADH using molecular oxygen to produce NAD
It is a useful enzyme to regenerate.

2. Description of the Related Art Conventionally, NADH oxidases that oxidize NADH using a molecular enzyme as an electron acceptor include those derived from Lactobacillus plantarum [Agrical tural and Biological chemistry]. Chemistry) 25 volumes,
876, 1961], derived from Streptococcus faecalis [Journal of Joyall
of Biological Chemistry), 237, 2647, 19
62 years], Acholeplasma Ride
laidlawii) [European journal
European Jouranl of Bioc
hemistry), volume 120, page 329, 1981], derived from Bacillus megaterium [Journal of Biochemistry (Journal of Biochemistry)].
Biochemistry, 98, 1433, 1985], Leuconostoc mese
nterojdes) [Jouranl of Biochemistry, 97, 127
9 pages, 1985].

However, the NAs known in these technologies
DH oxidase is difficult to cultivate the microorganism and is difficult to prepare in large quantities (such as Acoreplasma Ridelaui),
Since it is present in the intracellular membrane fraction, large-scale preparation is difficult, and it is difficult to increase the intracellular content (Bacillus megaterium, etc.). Nostock, Mecenteroides, etc.).

The present inventors have searched extensively for NADH oxidase suitable for industrial production, and found that optimal growth in the alkaline region was achieved.
NA with excellent temperature stability, facultative anaerobic bacteria with pH
The inventors have found that DH oxidase is produced, and have completed the present invention. In the present invention, "facultative anaerobic bacteria" refers to anaerobic bacteria capable of growing in the presence or absence of oxygen.

SUMMARY OF THE INVENTIONAccording to the present invention, a facultatively anaerobic NADH oxidase-producing bacterium having an optimum growth pH in an alkaline region is anaerobically or aerobically cultured in a medium, and NADH oxidase is collected from the culture. A process for producing NADH oxidase is provided.

DETAILED DESCRIPTION OF THE INVENTION The NADH oxidase-producing strain used in the present invention belongs to an alkalophilic facultative anaerobic bacterium and may be any strain as long as it has NADH oxidase-producing ability. Is also good. Preferred examples of strains having these properties include, for example, Agricaltural and Biological Chemistry, Vol.
Ep01 strain described on pages 2271-2275 (1987) can be mentioned. This strain is accumulated and separated in compost whose pH has been adjusted to alkaline, and according to the literature, has the following mycological properties.

(1) Morphology (a) Cell shape and size: Bacillus, (0.3-0.6) ×
(1.5-2.5) μm (b) Presence or absence of polymorphism: no (c) Motility: no (d) Spores: with heat-resistant spores (e) Gram stain: positive early in culture (f) Acid-fast : Negative (2) Growth in each medium (a) Gravy agar medium: does not grow (b) Gelatin: does not liquefy (3) Physiological properties (a) Range of growth: Does not grow at pH 7.0, pH 8. 5-10.0
Is optimized. Optimum around 40 ° C.

(B) Oxidase: negative (c) Catalase: negative (d) Fermentable sugar: D-xylose, D-arabinose, D-ribose, D-glycose, D-mannose, D-fructose, amygdalin, esculin, salicin, Maltose, saccharose, cellobiose, trehalose, soluble starch, pectin, xylan (e) Growing in the presence of 3% salt (4) Other properties (a) Cell wall component: containing mesodiaminopimelic acid (b) Cytochrome: cytochrome a, b, c, d
Does not contain.

 (C) Quinone: does not contain menaquinone or ubiquinone.

 (D) DNA containing guanine and cytosine (GC content) 35.9% (e) Cell fatty acid composition: antein 23.7% (during anaerobic culture) Isobrand 35.4% linear 37.2% (g) Xylanase is generated during anaerobic and aerobic culture.

These properties are described in "Bergey's Manual of Systematic Bacteriology"
f Systematic Bacteriology) ", Volume 2 (1986). What is alkalophilic Bacillus?
Many have the same properties such as positive Gram stain, sporulation, aerobic growth, and growth on various carbohydrates, but differ in properties such as catalase negative, motility negative, and no growth on gravy agar. To The absence of cytochromes and quinones does not indicate an assignment to the genus Bacillus. There is a point that the bacterial cell fatty acid composition, aerobic growth and the like do not match that of the genus Clostridium, and there is a possibility that the strain may not be a new strain belonging to these existing genera. Therefore, in the present invention, only the unidentified bacterium Ep01 strain will be referred to. This strain has been deposited with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology under Microorganism Accession No. 10096 (FERMP-10096).

As a nitrogen source that is a nutrient source of a medium that can be used for culturing the microbial cells used in the present invention, for example, ammonia,
Inorganic nitrogen compounds such as ammonium sulfate, ammonium chloride and ammonium nitrate can be used alone or in combination. Examples of the inorganic metal salt include potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, manganese sulfate, and ferrous sulfate. In addition to the above inorganic nutrients, natural nutrients such as corn steep liquor, yeast extract and peptone may be added. Further, as a carbon source, for example, glucose, xylose, mannose, fructose, artose, sucrose, cellobiose, xylan and the like can be added to the medium.

In culturing the aforementioned NADH oxidase-producing bacterium in a medium having the above composition, the culturing temperature is usually about 20 to about 50.
C., preferably in the range of about 35 to about 45.degree. Further, the pH during culturing is not strictly limited as long as it is in an alkaline region of about pH 7.5 or more, but is generally 8 to 10.
5, preferably in the range of 8.5 to 10.0 is advantageous. It is convenient to adjust the pH during the cultivation to be within the optimum range by adding an alkali such as sodium hydroxide, potassium hydroxide, sodium ammonium carbonate or the like to the medium. The culture time is usually 3 to 24 hours, preferably 5 to 24 hours.
It can be about 20 hours. The cultivation can be performed under aerobic conditions such as aeration stirring and shaking, or may be performed under anoxic anaerobic conditions.

When culturing under anaerobic conditions, reduce the oxygen-free gas atmosphere such as nitrogen gas, helium gas, argon gas, or a mixture thereof, from which oxygen has been removed with a reduced copper column, or reduce the redox units of the medium. Techniques commonly used for cultivating obligate anaerobic bacteria, such as adding a reducing agent such as titanium (III) citric acid or dithionite to facilitate the growth of anaerobic bacteria [for example, Yamasato, Udagawa, Kodama, Morichi , "Separation of microorganisms" R & D planning (1986) 246-274
Page, etc.] can be applied as long as the condition is effective in an alkaline region where growth of the present bacterium is optimal and does not adversely affect the growth of the present application.

Furthermore, it is not necessary to perform all the stages of culturing up to obtaining the cells under only one condition under anaerobic conditions or aerobic conditions, and from anaerobic conditions to aerobic conditions during culturing, or from aerobic conditions. It is also a preferable method to carry out cultivation by combining both conditions by changing to anaerobic conditions.

Since the cells cultured in this way contain NADH oxidase in the cells, the obtained cells are disrupted by commonly used means such as sonication, enzyme treatment, homogenization, etc., and the resulting cell-free extraction is performed. The liquid is subjected to separation and purification methods known per se such as salting out, ion exchange chromatography, and gel filtration [for example, Biochemical Experiment Lecture, edited by the Biochemical Society of Japan, Vol. Pp. 1-334; Takeichi Kudio, J. Yamashita, "Basic Experimental Methods for Proteins and Enzymes," Nankodo, pp. 1-379, etc.], and NAD.
H oxidase can be collected.

The enzymatic and physicochemical properties of NADH oxidase obtained according to the present invention are as follows.

(1) Action: This enzyme is NA as shown in the following reaction formula.
Oxidizes DH to produce NAD and hydrogen peroxide.

NADH + H ⁺ + O ₂ → NAD ⁺ + H ₂ O ₂ (2) Optimum pH: When measured using a phosphate buffer, Tris buffer, or carbonate buffer, this enzyme has an optimum pH around 7.0.
(See FIG. 1 attached).

(3) Optimum temperature and thermal stability: phosphate buffer (pH 7.
0), the enzyme had an optimum temperature of about 37 ° C. (see FIG. 2 attached).

(4) Temperature stability: The enzyme was suspended in the same buffer as used in (3) above, stored at 37 ° C., and the temperature stability of the activity was examined. Each had the same activity. On the other hand, the already known Leuconostoc Mesert
eroides), Journal of Biochemistry, 97, 1285 (1985)
As shown in FIG. 7B, the activity is maintained at 30 ° C. even after 60 minutes, but the activity is reduced to about 60% at 40 ° C. and 0% at 50 ° C., resulting in poor stability.

(5) Requirement of monovalent cation: 50 mM cell-free cell extract
Assuming that the NADH oxidase activity of the enzyme solution obtained by dialysis with Tris buffer was 100, the activity when K ⁺ , Na ⁺ , and NH ₄ ⁺ were added to the enzyme solution to a concentration of 20 mM, respectively, Three
10, 180 and 310.

(6) NADH oxidase is 100,000 ×
g in the supernatant fraction centrifuged for 1 hour.

The qualitative analysis of the present enzyme was performed by observing the decrease in the absorbance of NADH at 340 nm or the absorption of oxygen in the presence of NADH using an oxygen electrode or a manometer. Also,
The activity of the present enzyme can be measured as follows. That is, after pre-holding the reaction solution consisting of 50 mM buffer 1.9 ml and enzyme solution 0.05 ml at the designated temperature, 5 mM NADH 0.05 ml
To start the reaction, and the decrease in absorbance at 340 nm is measured. Enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol of NADH per minute as one unit.

According to the present invention described above, the use of NADH oxidase-producing bacteria having an optimal growth pH in the alkaline region, the use of NADH oxidase-producing bacteria with a low risk of contamination during culturing, such as temperature stability. Excellent NADH oxidase can be easily produced.

EXAMPLES Next, the method for producing NADH oxidase of the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

Example 1 K ₂ HPO ₄ 1 g, ammonium nitrate 2 g, MgSO ₄ .7H ₂ O ₂₀₀ mg,
_{MnSO 4 · 7H 2 O5mg, FeSO} 4 · 7H 2 O5mg, CaCl 2 · 2H 2 O700mg,
3 g of yeast extract, 300 mg of polypeptone, 10 g of xylan, and 1 mg of resazurin were dissolved in 900 ml of distilled water, and 90 ml was dispensed into 100 ml serum bottles. After boiled and degassed, oxygen-free nitrogen gas prepared by passing through a reduced copper column was aerated, covered with a butyl rubber stopper, sealed with an aluminum cap, and sterilized at 120 ° C for 15 minutes. To this medium, 10 ml of a 10% aqueous sodium carbonate solution (pH 10.6) sterilized at 120 ° C. for 15 minutes was added to adjust the pH of the medium to 10. Furthermore, Science No. 194 is used as a reducing agent.
AJB Zehnder & K. Wuhrmann on pages 1165-1166
A titanium (III) citric acid solution prepared according to these methods was added to a final concentration of 0.13 mM. This medium was inoculated with an alkalophilic bacterium Ep01 strain (Microtechnical Laboratories No. 10096), and allowed to stand at 40 ° C. for 20 hours to obtain a seed culture solution.

Except that 10 g of xylan was changed to 10 g of glucose, 450 ml of a medium of the same composition was dispensed into a 500 ml serum bottle, and the same operation was performed.
l, Titanium (III) citric acid solution was added to a final concentration of 0.13 mM. Inoculate 10 ml of the seed culture, 40
The culture was allowed to stand at 24 ° C. for 24 hours. After the culture is completed, 100 ml of the culture solution
The cells obtained by centrifugation at 1,000 × g for 20 minutes were suspended in 50 mM Tris-HCl buffer, and collected by centrifugation to obtain washed cells. After suspending the cells in 5 ml of the same buffer as above, the cells were disrupted by sonication, and the supernatant obtained by centrifugation was used as a crude enzyme solution, and the NADH oxidase activity was measured. (1 unit indicates the amount of enzyme that oxidizes 1 μmole of NADH per minute).

Example 2 Same medium as described in Example 1, except that 10 g of glucose is used instead of 10 g of xylan, and no resazurin is contained.
Dispense 90 ml into a 500 ml Erlenmeyer flask and sterilize at 120 ° C for 15 minutes. A 10% aqueous sodium carbonate solution, which was separately heat-sterilized, was added to adjust the pH to 10, and then 5 ml of the seed culture described in Example 1 was added.
l, and cultured with shaking at 30 ° C. for 20 hours. After culturing, 5 ml of cells obtained by centrifugation from 100 ml of culture solution
Was suspended in 50 mM Tris-HCl buffer, sonicated, and centrifuged to obtain a supernatant, which was used as a crude oxygen solution. Its activity is 0.
18 units 1 ml.

[Brief description of the drawings]

FIG. 1 shows the optimum NADH oxidase obtained by the present invention.
FIG. 2 is a graph showing pH, and FIG. 2 is a graph showing an optimum temperature of NADH oxidase obtained by the present invention.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C12N 9/02 BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. A process for producing NADH oxidase, comprising culturing a facultatively anaerobic NADH oxidase-producing bacterium (Ep01 strain) having an optimum growth pH in an alkaline region in a medium, and collecting NADH oxidase from the culture. Law.