JPH0632608B2 - Process for producing thermostable sarcosine oxidase N - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing thermostable sarcosine oxidase N.

Sarcosine oxidase is an enzyme that catalyzes the reaction of sarcosine + H ₂ O + O ₂ → glycine + formaldehyde + H ₂ O ₂ , and is disclosed in, for example, JP-A-54-52789.
Bulletin, Journal of Biochemistry, 89th
Volume, p. 599, 1981 Japan [J. Biochem. 89 , 599 (198
1) JAPAN] and is publicly known.

Meanwhile, a method for enzymatically quantifying creatinine or creatine has been reported [Clinical Chemistry Symposium, Vol. 19,
196 (1979)], sarcosine oxidase is used in the assay, and the enzyme is a useful enzyme. The quantification method is as follows, and in the following formula, parentheses represent the enzyme used.

Creatinine + H ₂ O Creatinine (creatinine amidohydrolase) Creatinine + H ₂ O → sarcosine + urea (creatine aminohydrolase) Sarcosine + H ₂ O + O ₂ → Glycine + formaldehyde + H ₂ O ₂ (sarcosine oxidase) [Prior art] First , One of the inventors of the present invention showed a new enzyme having a sufficient enzyme activity even in a slightly acidic condition, a thermostability, and a low km value for sarcosine, as compared with the case of culturing a strain belonging to the genus Bacillus newly isolated from soil. Knowing that sarcosine oxidase can be obtained, a patent application for a heat-resistant sarcosine oxidase N and a method for producing the same was filed (JP-A-61-162174).

[Problems to be solved by the invention]

However, in order to maximize the yield of the enzyme by the above thermostable sarcosine oxidase N production method,
It is necessary to add expensive creatinine, creatine and / or sarcosine to the medium, which is economically disadvantageous, and there is a problem that the manufacturing operation becomes complicated.

[Means for solving problems]

Therefore, as a result of various studies to solve the above problems, the present inventors obtained a recombinant DNA in which a DNA containing a gene encoding thermostable sarcosine oxidase N was inserted into a vector DNA. When a microorganism having a thermostable sarcosine oxidase N-producing ability contained in a microorganism belonging to the genus Escherichia is cultured in a medium, creatinine, creatine and / or sarcosine are not added to the medium at all,
The present invention has been completed based on the findings that the thermostable sarcosine oxidase N is efficiently produced.

That is, the present invention provides a recombinant D in which a DNA containing a gene encoding a thermostable sarcosine oxidase N derived from Bacillus sp.
A thermostable sarcosine oxidase N is characterized in that a microorganism belonging to the genus Escherichia containing NA and capable of producing a thermostable sarcosine oxidase N is cultured in a medium, and the thermostable sarcosine oxidase N is collected from the culture. It is a manufacturing method.

Hereinafter, the present invention will be described in detail.

First, thermostable sarcosine oxidase N (hereinafter referred to as SO
It is abbreviated as N. DNA containing a gene encoding
The preparation will be described.

Bacillus SP NS-129 [Micromachinery Research Institute of Microbiology No. 2922 (FERM
The BP-671)] strain is cultured in exactly the same manner as described in JP-A-61-162174 to obtain a culture. This culture is centrifuged at, for example, 3000 rpm or more, preferably 8000 to 10000 rpm, for 5 minutes or more, preferably 10 to 15 minutes to obtain Bacillus sp. NS-129 strain cells.

From this cell, for example, the method of Saito and Miura [Biochem.
Biofizz. Actor. (Biochem.Biophys.Acta), 72nd
Vol. 6, page 619 (1963)], etc. to obtain chromosomal DNA.

Then, a restriction enzyme, such as Sau3AI (manufactured by Toyobo Co., Ltd.), which produces a protruding end is added to the chromosomal DNA at a temperature of 30 ° C.
Above, preferably 37 ℃, enzyme concentration 1-10 units / ml
The mixture is digested for 20 minutes or longer, preferably for 0.5 to 2 hours to obtain various chromosomal DNA fragment mixtures.

From the DNA fragment mixture thus obtained, a DNA fragment mixture is obtained by, for example, an ordinary agarose gel electrophoresis method, further purified by a purification means such as phenol extraction, and further concentrated by a concentration means such as ethanol precipitation method. And purified DNA fragment mixture (in which SO
DNA fragments containing the gene encoding N are included).

On the other hand, the vector DN that can be used in the present invention
Any A may be used, and examples thereof include plasmid vector DNA and bacteriophage vector DNA. Specifically, for example, plasmid pBR322D
NA (Bethesda Research Laboratories
Research Laboratories)] and the like are preferable.

A restriction enzyme, such as BamHI (manufactured by Takara Shuzo Co., Ltd.), which produces a protruding end, is added to the vector DNA at a temperature of 30 ° C or higher,
The digested vector DNA is obtained by digesting at 37 ° C. at an enzyme concentration of 10 to 1000 units / ml for 1 hour or more, preferably for 2 to 3 hours to obtain a cleaved vector DNA.

Then Bacillus sp. NS-1 obtained as described above
DNA derived from 29 and containing a gene encoding SON
The fragment mixture and the cleaved vector DNA are mixed, and for example E. coli DNA ligase (New England Biolabs), T4 DNA ligase (Boehringer Mannheim), etc., preferably T4DN.
A ligase is allowed to act at a temperature of 4 to 37 ° C., preferably 4 to 16 ° C., and an enzyme concentration of 1 to 100 units / ml for 1 hour or more, preferably 6 to 24 hours to obtain a recombinant DNA.

Using this recombinant DNA, for example, E. coli K-12,
Preferably Escherichia coli HB101 (ATCC33694), E. coli D
HI (ATCC33849), E. coli X-1776 (ATCC312
44), etc. are transformed or transduced to obtain the respective strains. This transformation is DM Morrison
(DM Morrison) [Methods in Enzymology], 68, 326-331 (1
979)]. The transduction can be carried out by the method of B. Hohn [Methods in Enzymology Vol. 68, 299-309 (1979)].

Then, by screening a microorganism capable of producing SON from the above strain, a microorganism belonging to the genus Escherichia containing the recombinant DNA in which the DNA containing the gene encoding SON is inserted into the vector DNA and having the ability of producing SON is obtained. be able to.

To obtain a recombinant DNA purified from the microorganism thus obtained, for example, P. Guerry
Etc. [J. Bacteriology
116, 1064-1066 (1973)], Day. Bee
It can be obtained by the method of DB Clewell [J. Bacteriology Vol. 110, pp. 667-676 (1972)] and the like.

Then, D containing the gene encoding SON in the purified recombinant DNA obtained as described above
In NA, unnecessary DN other than the gene encoding SON
Since A is present, the unnecessary DNA is removed by the following operation.

Then, the recombinant DNA thus obtained and purified is treated with a restriction enzyme, for example, Hpa I and Bgl II (each manufactured by Takara Shuzo) at a temperature of 30 ° C. or higher, preferably 37 ° C.
The mixture is digested at an enzyme concentration of 10 to 1000 units / ml for 20 minutes or more, preferably 1 to 6 hours to obtain a DNA fragment mixture.

From the DNA fragment mixture thus obtained, a DNA fragment mixture is obtained by, for example, an ordinary agarose gel electrophoresis method, further purified by a purification means such as phenol extraction, and further concentrated by a concentration means such as ethanol precipitation method. And purified DNA fragment mixture (in which SO
DNA fragments containing the gene encoding N are included).

On the other hand, the vector DN that can be used in the present invention
Any A may be used, and examples thereof include plasmid vector DNA and bacteriophage vector DNA. Specific examples include plasmids pUC18 and pUC18.
UC12, pUC8 DNA (Pharmacia) and the like are preferable.

Restriction enzymes such as BamHI and H are added to the vector DNA.
inc II (manufactured by Takara Shuzo Co., Ltd.) was digested and digested by allowing it to act at a temperature of 30 ° C or higher, preferably 37 ° C, and an enzyme concentration of 10 to 1000 units / ml for 1 hour or longer, preferably 1 to 3 hours. Obtain vector DNA.

Then Bacillus sp. NS-1 obtained as described above
DNA derived from 29 and containing a gene encoding SON
The fragment mixture and the cleaved vector DNA are mixed, and for example E. coli DNA ligase (New England Biolabs), T4 DNA ligase (Boehringer Mannheim), etc., preferably T4DN.
A ligase at a temperature of 4 to 37 ° C., preferably 4 to 16 ° C., and an enzyme concentration of 1 to 100 units for 1 hour or more, preferably 6 to 2
React for 4 hours to obtain recombinant DNA.

Using this recombinant DNA, for example, E. coli K-12,
Preferably Escherichia coli JM101 (ATCC33876), E. coli H
B101 (ATCC33694), E. coli DHI (ATCC3384
9), Escherichia coli X-1776 (ATCC31244), etc. are transformed or transduced to obtain respective strains. This transformation is done by DM Morrison [Methods in Enzymology].
mology), 68, 326-331 (1979)]. Transduction is also by B. Hohn
Method [Methods in Enzymology Volume 68, 29
9-309 (1979)].

Then, by screening a microorganism capable of producing SON from the above strain, a microorganism belonging to the genus Escherichia containing the recombinant DNA in which the DNA containing the gene encoding SON is inserted into the vector DNA and having the ability of producing SON is obtained. be able to.

To obtain a novel recombinant DNA purified from the microorganism thus obtained, for example, PG
uerry) etc. [J. Bacteriology (J. Bacteri
ology) 116, 1064-1066 (1973)], Day
It can be obtained by the method of DB Clewell [J. Bacteriology Vol. 110, pp. 667-676 (1972)] and the like.

To produce SON using a microorganism belonging to the genus Escherichia containing the recombinant DNA obtained by inserting the DNA containing the gene encoding SON obtained above into a vector DNA, The microorganism may be cultivated by an ordinary solid culturing method, but it is preferable to adopt a liquid culturing method as much as possible.

As the medium for culturing the above-mentioned microorganism, for example, yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat fusuma leachate, and the like, one or more kinds of nitrogen sources, potassium potassium phosphate, and potassium phosphate second One in which one or more inorganic salts such as potassium, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate is added, and if necessary, a sugar raw material, vitamins and the like are appropriately added is used.

The initial pH of the medium is appropriately adjusted to 7-9. Culturing is performed at 30 to 42 ° C, preferably around 37 ° C for 4 to
It is preferably carried out for 24 hours, preferably 6 to 8 hours, by aeration and agitation deep culture, shaking culture, static culture and the like.

After completion of the culture, to collect SON from the culture, it can be obtained using a usual enzyme collection means.

For example, the cells are subjected to ultrasonic disruption treatment, grinding treatment, etc. by a conventional method, or the enzyme is extracted using a lysing enzyme such as lysozyme, or shaken or left in the presence of toluene or the like. The enzyme is self-digested and the enzyme is excreted outside the cells. The solution is filtered, the solid portion is removed by centrifugation, etc., and if necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate or manganese sulfate, and then ammonium sulfate, alcohol, acetone or the like is added to fractionate the solution. The precipitate is collected, dialyzed against water, and dried under vacuum to obtain a crude enzyme preparation.

And this enzyme is, if necessary, according to a conventional method of isolation and purification of the enzyme, for example, (1) DEAE-cellulose column chromatography, (2) ammonium sulfate fractionation, (3) QAE-Sephadex column chromatography, ( 4) TSK-GEL
Butyl-Toyopearl 650C [Toyo Soda Co., Ltd.]
A highly purified SON standard product can be obtained by using the hydrophobic chromatography described in 1. above, (5) gel filtration using Sephadex, or the like, or any other method in combination as necessary.

The physicochemical substances of the purified SON obtained by the above-mentioned purification means are exactly the same as the physicochemical properties of the SON described in JP-A-61-162174.

〔The invention's effect〕

As is clear from the above, when a microorganism belonging to the genus Escherichia containing the novel recombinant DNA of the present invention is cultured in a medium, SON can be efficiently obtained without adding and using creatine, creatinine, sarcosine and the like. Therefore, the present invention is industrially useful.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples.

Example (1) Preparation of chromosomal DNA of Bacillus sp. NS-129 strain Bacillus sp. NS-129 (FERM BP-671) strain was used as TY
Medium [1% (W / V) Bacto-tryptone
on (manufactured by Difco), 0.5% (W / V)
Bacto-yeast extract (manufactured by Difco), 0.5% (W / V) NaCl (pH 7.2)] 2
00 ml was inoculated and shake-cultured at a temperature of 30 ° C. for 16 hours to obtain a culture.

This culture was centrifuged at 10,000 rpm for 10 minutes by a conventional method to obtain 0.5 g of wet cells. Saito cells, Saito,
Miura's method [Biochem, Biofizz. Actor. (Bio
Chem. Biophys. Acta.), 72, 619 (1963)].

Next, 0.1 mg of this chromosomal DNA and the restriction enzyme Sau3AI
Two units (manufactured by Toyobo Co., Ltd.) were mixed with 10 mM Tris-hydrochloric acid buffer solution (containing 50 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH 7.4) and reacted at a temperature of 37 ° C. for 45 minutes. A DNA fragment having a size of 4 to 8 Kb (kilobase pair) was obtained from the reaction-completed solution subjected to 0.7% (W / V) agarose gel (Takara Shuzo Co., Ltd.) electrophoresis by a conventional method. (RCA Yang) and other methods (Methods in Enzymo
logy), 68, 176-182 (1979)] to obtain an eluate, which is then subjected to phenol extraction by a conventional method, and then ethanol precipitation, followed by Sau3AI-digested Bacillus sp. NS. -129 strain chromosomal DNA fragment 10
μg was obtained.

(2) Preparation of recombinant plasmid pKLS601DNA Plasmid pBR322DNA [Bethesda Research Laboratories] 10 μ
g and 100 units of BamHI restriction enzyme (Takara Shuzo) at 50 mM
Tris-HCl buffer (containing 100 mM NaCl and 10 mM MgSO ₄ )
(pH7.4) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digested liquid, which was subjected to phenol extraction and ethanol precipitation by a conventional method, and the plasmid pBR322 digested with BamHI.
DNA was obtained.

Then, this BamHI-digested plasmid pBR322D
NA 10 μg, Sau3AI-digested Bacillus sp. NS-129 strain chromosomal DNA fragment 10 μg obtained in (1) above.
And 5 units of T4 DNA ligase [Boehringer
Mannheim (Boehriger Mannheim)) 6.6 mM MgC
Contains l ₂ , 10 mM dithiothreitol and 10 mM ATP
Add to 66 mM Tris-HCl buffer (pH 7.5),
After reacting for a time, DNA was ligated.

Then, DM Morrison's Method in Enzy
Mology), 68, 326-331 (1979)], calcium chloride-treated Escherichia coli DHI strain (ATCC33849).
Was transformed with the DNA ligated as described above, and a transformant resistant to ampicillin and sensitive to tetracycline was obtained.
I got 1000 shares.

Whether or not the transformant thus obtained had SON activity was tested as follows.

Each strain was inoculated into 5 ml of TY medium containing 0.5% (W / V) sarcosine (manufactured by Tokyo Chemical Industry Co., Ltd.) and 50 μg / ml ampicillin, and cultured at 30 ° C. for 24 hours to obtain a culture solution. . This was centrifuged at 3500 rpm for 10 minutes to obtain wet cells, which contained 1% (W / V) sarcosine.
Suspend in 1 ml of 10 mM phosphate buffer (pH 7.0) and add 20 μm to it.
1 Toluene was added, and the reaction solution was allowed to react at 37 ° C. for 1 hour by centrifugation at 3500 rpm for 10 minutes to obtain a reaction solution. 1% (W / V) 4-amino was added to the reaction solution. -3-hydrazino-5-mercapto-1,2,4-triazole, 23
% (W / V) potassium hydroxide and 0.08% (W / V) sodium metaperiodate were added in an amount of 0.15 ml each, and the reaction solution turned purple was a transformant having SON activity. E. coli (Ec
oli) DHI (pKLS 601) strain was obtained.

(3) Isolation of recombinant plasmid pKLS601 DNA Tryptone 1% (W / V), yeast extract 0.5% (W / V)
V) and 0.5% NaCl (W / V) in medium 1,
20 ml of a culture solution of E. coli DHI (pKLS 601) obtained by pre-culturing at 37 ° C. for 16 to 24 hours using the medium was inoculated, shake-cultured at 37 ° C. for 3 hours, and then the culture solution. Chloramphenicol (0.2 g) was added thereto, and the mixture was further cultured at the same temperature for 20 hours to obtain a culture solution.

Then, this culture solution was subjected to centrifugation at 10,000 rpm for 10 minutes by a conventional method to obtain wet cells, which were added to 20 ml of 25%.
(W / V) 50 mM Tris-HCl buffer containing sucrose (pH
8.0), and then 10 mg of lysozyme,
8 ml of 0.25 M EDTA solution (pH 8.0) and 8 ml of 20% (W / V) sodium dodecyl sulfate solution were added, and the temperature was adjusted to 60
Lysis was performed by incubating at 30 ° C for 30 minutes to obtain a lysate.

To this lysate, 13 ml of 5M NaCl solution was added, and the temperature was 4 ° C.
What was treated for 16 hours was centrifuged at 15000 rpm for 30 minutes by an ordinary method to obtain an extract, which was subjected to phenol extraction by an ordinary method and then ethanol precipitation was performed by an ordinary method to obtain a precipitate.

Then, this precipitate was subjected to normal vacuum drying treatment,
6 ml of 10 mM Tris-HCl buffer containing 1 mM EDTA (pH
7.5), and further cesium chloride 6g and 10mg
/ Ml Ethidium bromide 0.2 ml was added and subjected to equilibrium density gradient centrifugation using an ultracentrifuge at 39,000 rpm for 42 hours by the conventional method. Recombinant plasmid pKLS
601 DNA was isolated and ethidium bromide was further removed using normal butanol.
Recombinant plasmid pKLS purified by dialysis against 10 mM Tris-HCl buffer (pH 7.5) containing EDTA
2700 μg of 601 DNA (size is 9.1 Kb) was obtained.

(4) Construction of new recombinant pKLS612 DNA Plasmid pU
0.2 μg of C18 DNA (Pharmacia) and restriction enzymes
10 units each of Hinc II (Takara Shuzo) and BamHI (Takara Shuzo) each containing 10 mM Tris-HCl buffer (50 mM NaC)
l, containing 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH 7.
4) and react for 1 hour at 37 ℃ to obtain digestive juice,
The solution was subjected to phenol extraction and ethanol precipitation by a conventional method, and the plasmid pU digested with Hinc II and BamHI was obtained.
C18 DNA was obtained.

Then, the recombinant pKLS601DNA12 obtained in (3) above.
μg, 12 units HpaI (Takara Shuzo) and Bgl I
30 units of I (manufactured by Takara Shuzo Co., Ltd.) were added to 10 mM Tris-HCl buffer (5
It was mixed with 0 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4) and reacted at a temperature of 37 ° C. for 5 hours to obtain a digestive juice.

A 1.7 Kb DNA fragment was obtained by subjecting the digested solution to electrophoresis on 0.7% (W / V) agarose gel by a method such as RCA Yang (Methods in Enzymology). ), Vol 08, 176-
182 (1979)] to obtain an eluate, which is subjected to phenol extraction and ethanol precipitation by a conventional method to obtain DNA.
A fragment of 1.3 μg was obtained.

The fragment is a DNA fragment containing a gene encoding SON.

Then, the above-mentioned DNA fragment was added to Bgl II, Dra I, Pvu II, H
ind III, Hpa I (both manufactured by Takara Shuzo, restriction enzymes), As
u II (Promega Biotech, restriction enzyme) and Bst
The mobility patterns of the DNA fragments obtained by single-digestion and double-digestion using E II (Nippon Gene Co., Ltd., restriction enzyme) were analyzed by agarose gel electrophoresis. Was compared with the standard mobility pattern of the DNA fragment obtained by digestion with Hind III, and the molecular weight obtained was 1,700 bp (base pair) [Journal of Molecular Biology (Journal of Molecular Biology). Molecular Biology) Vol. 98, 55
1-564, 1975], and a restriction map of the above DNA fragment with a restriction enzyme is shown in FIG.

0.15 μg of the DNA fragment thus obtained, Hinc II and B
0.2 μg of plasmid pUC18DNA digested with amHI and T4 DNA ligase (Boehringer Mannheim) 1
The unit was replaced with 66 mM Tris-HCl buffer (6.6 mM MgCl ₂ , 10 mM
It was mixed with dithiothreitol and 10 mM ATP (pH 7.5) and reacted at a temperature of 4 ° C. for 16 hours to ligate the DNA.

Then, DM Morrison (DM Morriso)
E. coli JM101 treated with calcium chloride by the method of n)
(ATCC33876) was transformed with the DNA ligated as described above, and the obtained transformant was transformed with 50 μg / ml ampicillin, 0.5 mM isopropyl-β-D-thiogalactoside and 0.004% (W / −) 5-. Bromo-4-chloro-3-
T containing indolyl-β-D-galactoside, respectively
E. coli JM101 (pKLS) that forms a white colony, since a strain that produces a white colony by culturing for 24 hours at 37 ° C. using Y agar plate medium forms a white colony.
612) was obtained.

Thus obtained E. coli JM101 (pKLS612), which is a transformant having SON-producing ability, was obtained from the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Institute No. 1146 (FE
It has been deposited as RM BP-1146).

(5) Isolation of recombinant plasmid pKLS612DNA Tryptone 1% (W / V), yeast extract 0.5% (W / V)
V) and 0.5% NaCl (W / V) in medium 1,
20 ml of a culture solution of E. coli JM101 (pKLS612) obtained by pre-culturing at 37 ° C. for 16 to 24 hours using the medium was inoculated and shake-cultured at a temperature of 37 ° C. for 3 hours. Chloramphenicol (0.2 g) was added, and the mixture was further cultured at the same temperature for 20 hours to obtain a culture solution.

Then, this culture solution was subjected to centrifugation at 10,000 rpm for 10 minutes by a conventional method to obtain wet cells, which were added to 20 ml of 25%.
(W / V) 50 mM Tris-HCl buffer containing sucrose (pH
8.0), and then 10 mg of lysozyme,
8 ml of 0.25 M EDTA solution (pH 8.0) and 8 ml of 20% (W / V) sodium dodecyl sulfate solution were added, and the temperature was adjusted to 60
Lysis was performed by incubating at 30 ° C for 30 minutes to obtain a lysate.

To this lysate, 13 ml of 5M NaCl solution was added, and the temperature was 4 ° C.
What was treated for 16 hours was centrifuged at 15000 rpm for 30 minutes by an ordinary method to obtain an extract, which was subjected to phenol extraction by an ordinary method and then ethanol precipitation was performed by an ordinary method to obtain a precipitate.

Then, this precipitate was subjected to normal vacuum drying treatment,
6 ml of 10 mM Tris-HCl buffer containing 1 mM EDTA (pH
7.5), and further cesium chloride 6g and 10mg
/ Ml Ethidium bromide 0.2 ml was added and subjected to equilibrium density gradient centrifugation using an ultracentrifuge at 39,000 rpm for 42 hours by the conventional method. Recombinant plasmid pKLS
612 DNA was isolated and ethidium bromide was further removed using normal butanol.
Recombinant plasmid pKLS purified by dialysis against 10 mM Tris-HCl buffer (pH 7.5) containing EDTA
612 DNA (size is 4.4 Kb) 2300 μg was obtained.

(6) Production of SON by E. coli JM101 (pKLS612) and separation and purification of the enzyme Tryptone 1% (W / V), yeast extract 0.5% (W /
V), isopropyl-β-D-thiogalactoside / mM,
And a medium 2 consisting of 0.5% salt (W / V) were dispensed into a culture tank of a stirring type small culture device (made by Iwashiya Co., Ltd.) and subjected to high-pressure sterilization by a conventional method. E. coli JM that had been shake-cultured at 37 ° C for 24 hours in advance
20 ml of a culture solution of 101 (pKLS612) was inoculated, and the operation of culturing with aeration and stirring at 37 ° C. for 8 hours was repeated 5 times to obtain 35 g of wet cells.

The cells were suspended in 100 ml of 0.01 M phosphate buffer (pH 8.0),
After ultrasonic destruction by a conventional method, 30 at 15000r.pm
Centrifugal centrifugation for 120 minutes, 120 ml of SON crude enzyme solution (29
0 unit / ml) was obtained.

The crude enzyme solution thus obtained was subjected to nucleic acid removal treatment with streptomycin sulfate and then heat-treated at a temperature of 50 ° C. for 1 hour.
It was removed by centrifugation for 0 minutes.

After adsorbing on a QAE-Sephadex A-50 column (1.4 × 40 cm) that has been equilibrated with 0.01 M phosphate buffer, 0.27
After washing with 0.01M phosphate buffer containing potassium chloride, elution was performed with 0.01M phosphate buffer containing 0.37M potassium chloride to obtain a SON-containing eluate.

This SON-containing eluate was concentrated by a conventional method and then freeze-dried to obtain 20,800 units of purified SON powder. The yield was 60%.

[Brief description of drawings]

FIG. 1 is a DNA containing a gene encoding SON
It is a figure which shows the cutting | disconnection map by the restriction enzyme of a fragment.

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Claims (2)

[Claims] 1. A recombinant DN in which a DNA containing a gene encoding a thermostable sarcosine oxidase N derived from Bacillus sp. NS-129 strain is inserted into a vector DNA.
A thermostable sarcosine oxidase N is characterized in that a microorganism belonging to the genus Escherichia containing A and having the ability to produce a thermostable sarcosine oxidase N is cultured in a medium, and the thermostable sarcosine oxidase N is collected from the culture. Manufacturing method. 2. The vector DNA is the plasmid pUC18DN.
The method for producing thermostable sarcosine oxidase N according to claim 1, which is A.