JPH0665303B2 - Novel recombinant DNA - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel recombinant DNA useful for producing thermostable sarcosine oxidase N.

Sarcosine oxidase is an enzyme that catalyzes the reaction of rucosin + 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 (19
81) 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) Creatine + 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 newly developed a strain of Bacillus genus isolated from soil, which shows sufficient enzyme activity even in slightly acidic conditions, is thermostable, and has a low Km value against sarcosine. 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 strain having a thermostable sarcosine oxidase N-producing ability contained in a strain belonging to the genus Escherichia is cultured in a medium, the efficiency can be improved without adding creatinine, creatine and / or sarcosine to the medium. The present invention has been completed based on the finding that thermostable sarcosine oxidase N is often produced.

That is, the present invention is characterized by including a gene encoding a thermostable sarcosine oxidase N derived from the genus Bacillus, having the following enzyme cleavage map and having a base length of 1700 bp inserted into a vector DNA. It is a novel recombinant DNA.

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
For example, 3000r.pm or more, preferably 8000-10,000r.pm
The cells of Bacillus sp. NS-129 strain are obtained by centrifugation for 5 minutes or longer, preferably 10 to 15 minutes.

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 DA that can be used in the present invention
Any N may be used, and examples thereof include plasmid vector DNA and bacteriophage vector DAN. 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, Vol. 68, pp. 326-331 (1979)]. The transduction can be carried out by the method of B. Hohn [Methods in Enzymology Vol. 68, 299-309 (1979)].

Then, a strain having the SON-producing ability is screened from the above-mentioned strain to obtain a strain belonging to the genus Escherichia having the SON-producing ability and containing the recombinant DNA in which the DNA containing the gene encoding the SON is inserted into the vector DNA. be able to.

To obtain a purified recombinant DNA from the strain thus obtained, for example, the method of P. Guerry et al. [J. J. Bacteriology
116, 1064-1066 (1973)], the method of DB Clewell [J. Bacteriology 110, 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 Bam HI and Hi are added to the vector DNA.
nc 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, a strain having the SON-producing ability is screened from the above-mentioned strain to obtain a strain belonging to the genus Escherichia having the SON-producing ability and containing the recombinant DNA in which the DNA containing the gene encoding the SON is inserted into the vector DNA. be able to.

To obtain a purified novel recombinant DNA from the strain thus obtained, for example, P. Gue (P. Gue
rry) etc. [J. Bacteriology (J. Bacteriol
ogy) 116, 1064-1066 (1973)], and the method of DB Clewell [J. Bacteriology 110, 667-676 (1972)] and the like. You can

To produce SON using a strain belonging to the genus Escherichia containing the recombinant DNA in which the DNA containing the gene encoding SON obtained as described above is inserted into the vector DNA, The strain may be cultivated by an ordinary solid culturing method, but it is preferable to cultivate by adopting a liquid culturing method as much as possible.

Examples of the medium for culturing the above-described strain include yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat malt exudate, and the like, one or more nitrogen sources, potassium potassium phosphate, and phosphate. Second potassium,
One or more inorganic salts such as magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, a sugar raw material, vitamins and the like are appropriately added.

It is appropriate to adjust the initial pH of the medium to 7-9. The culture is carried out at 30 to 42 ° C, preferably around 37 ° C for 4 to 24 ° C.
It is preferably carried out for a time, 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 then vacuum dried to obtain a crude enzyme product.

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), gel filtration with (5) Cedex, etc., or other methods in combination as necessary.

The physicochemical properties of the purified SON obtained by the above purification means are exactly the same as those of the SON described in JP-A No. 61-162174.

〔The invention's effect〕

As is apparent from the above, when a strain belonging to the genus Escherichia containing the novel recombinant DNA of the present invention is cultivated 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 (pH7.2) )) 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 )
The mixture was mixed with (pH7.4) and reacted at a temperature of 37 ° C. for 2 hours to obtain a digestion solution, which was subjected to phenol extraction and ethanol precipitation by a conventional method, and the plasmid pBR32 digested with BamHI.
2 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 (Boehrikger Mannheim)) 6.6 mM MgC
Contains l ₂ , 10 mM dithiothreitol and 10 mM ATP
Add to 66mM Tris-HCl buffer (pH7.5) and
After reacting for 6 hours, DNA was ligated.

Then the method of DM Morrison (Medthods in Enz
ymology), 68, 326-331 (1979)], calcium chloride-treated Escherichia coli DHI strain (ATCC 33849).
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
A solution obtained by adding μl toluene and reacting at 37 ° C. for 1 hour was centrifuged at 3500 rpm for 10 minutes to obtain a reaction solution, and 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)
V) Add 0.15 ml each of sodium metaperiodate,
The transformant having a purple color in the reaction solution was a transformant having SON activity, and an E. coli DHI (pKLS 601) strain which was a transformant having SON activity was obtained.

(3) Isolation of recombinant plasmid pKLS601 DNA Tryptone 1% (W / V), yeast extract 0.5% (W / V)
V) and a medium 1 consisting of 0.5% NaCl (W / V),
20 ml of a culture broth of E. coli DHI (pKLS 601) obtained by preculturing the medium at 37 ° C. for 16 to 24 hours was inoculated, cultivated with shaking at 37 ° C. for 3 hours, and then cultivated. 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 centrifuged at 1000 rpm for 10 minutes by a conventional method to obtain wet bacterial cells, which were added to 20 ml of 25% (W
/ V) 50 mM Tris calculation buffer containing sucrose (pH 8.0)
Lysozyme 10mg, 0.25
8 ml of M EDTA solution (pH 8.0) and 8 ml of 20% (W / V) sodium dodecyl sulfate solution were added, and the temperature was 60 ° C.
The mixture was kept warm for 30 minutes to lyse and a lysate was obtained.

To this lysate, add 13 ml of 5M NaCl solution and
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 DTA (pH 7.
5) dissolved in cesium chloride 6g and 10mg /
After adding 0.2 ml of ethidium bromide, the recombinant plasmid pKLS60 was subjected to equilibrium density gradient centrifugation using an ultracentrifuge at 39,000 rpm for 42 hours in a conventional manner.
1 DNA was isolated and ethidium bromide was further removed using normal butanol.
Recombinant plasmid pKLS60 purified by dialysis against 10 mM Tris-HCl buffer (pH 7.5) containing DTA
2700 μg of 1 DNA (size is 9.1 Kb) was obtained.

(4) Preparation of a novel recombinant pKLS612DAN Pyramid pUC18 DNA (Pharmacia) 0.2 μg and restriction enzymes HincII (Takara Shuzo) and BamHI (Takara Shuzo) 10 units each in 10 mM Tris-HCl buffer (50 mM NaCl, 50 mM NaCl, It was mixed with 10 mM MgSO ₄ and 1 mM dithiothreitol (pH 7.4) and reacted at a temperature of 37 ° C. for 1 hour to obtain a digestive solution, which was subjected to phenol extraction and ethanol precipitation by a conventional method to obtain HincII and A plasmid pUC18DAN digested with BamHI was obtained.

Then, the recombinant pKLS601DNA12 obtained in (3) above.
μg, 12 units HpaI (Takara Shuzo) and BalII
(Takara Shuzo) 30 units of 10 mM Tris-HCl buffer (50
The mixture was mixed with 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 0.7% (W / V) agarose gel electrophoresis, and then using 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 EII (Nippon Gene, restriction enzyme) and double digestion were analyzed by agarose gel electrophoresis, and the obtained mobility patterns and λDNA were analyzed. The base length obtained by comparing with the standard mobility pattern of the DNA fragment obtained by digestion with Hind III is 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 m
The mixture was mixed with M 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) ligated as described above
The obtained transformant was transformed with 50 μg / ml ampicillin, 0.5 mM isopropyl-β-D-thiogalactoside and 0.004% (W / V) 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, Microtechnology Research Institute 1146 (FERM).
It has been deposited as BP-1146).

(5) Isolation of recombinant plasmid pKLS612DNA Tryptone 1% (W / V), yeast extract 0.5% (W / V)
V) and a medium 1 consisting of 0.5% NaCl (W / V),
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, add 13 ml of 5M NaCl solution and
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 crude SON enzyme solution (2
90 units / ml) was obtained.

The crude enzyme solution thus obtained was subjected to nucleic acid removal treatment using streptomycin sulfate and heated at 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 0.01 M phosphate buffer containing potassium chloride, elution was performed with 0.01 M phosphate buffer containing 0.37 M 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.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12N 9/06 C12R 1:19) (72) Inventor Hideko Yamamoto 50-Shimoyasumatsu, Tokorozawa, Saitama Prefecture 217 (56) Reference JP-A-61-162174 (JP, A)

Claims (2)

[Claims] 1. A vector DNA containing a gene encoding a thermostable sarcosine oxidase N derived from the genus Bacillus and having the following restriction enzyme cleavage map and having a base length of 1700 bp is inserted into a vector DNA. A novel recombinant DNA that does. 2. The vector DNA is the plasmid pUC18DN.
The novel recombinant DNA according to claim 1, which is A.