JPH05115281A - New sarcosine oxidase m, its gene and new recombinant dna and production of new sarcosine oxidase m - Google Patents

Abstract

PURPOSE:To obtain a new sarcosine oxidase M suitable as an enzyme for diagnosis of kidney diseases, etc. CONSTITUTION:Sarcosine oxidase M having an amino acid sequence of the formula. The sarcosine oxidase M is obtained by culture of a microorganism [e.g. Escherichia coli JM109 (FERM P-3604)] having sarcosine oxidase producing ability and belonging to the genus Escherichia, and has high substrate specificity and thermal stability.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel sarcosine oxidase M, its gene, a novel recombinant DNA and a sarcosine oxidase M, which are more suitable for use as a diagnostic enzyme for renal diseases in the field of clinical medicine.
Manufacturing method.

[0002]

2. Description of the Related Art Conventionally, in the field of clinical medicine, creatinine or creatine in serum or urine is measured by an enzyme assay method to diagnose diseases of renal function, muscle diseases and the like. The quantification method is as follows, and sarcosine oxidase is used for this. In the formulas below, the parentheses represent the enzyme used.

[0003]

[Chemical 2]

Conventionally, sarcosine oxidase has been classified into the genus Corynebacterium [J. Biochem., 89 , 599 (1981)], the genus Bacillus (JP-A-54-52789, JP-A-61-162174), Cylindrocarbon genus (JP-A-56-92790)
It is known to be produced by strains of the genus Pseudomonas (JP-A-60-43379), the genus Arthrobacter (JP-A-2-265478), and the like. However, it was an enzyme with poor specificity, such as poor thermostability, a large Km value for sarcosine, and a large effect on ethyl glycine and proline. The ethyl glycine and proline are derived from the medication during treatment, and when sarcosine oxidase also acts on this, it gives a positive measurement error,
It is a therapeutic problem.

[0005]

As described above, the method of enzymatically quantifying creatinine or creatine is used, but the amount of creatinine or creatine contained in serum or urine is extremely small. As such, acting on the substance from the medication will render the measurement unreliable.

[0006] Therefore, in its quantification, with high sensitivity,
Moreover, enzymatic assay methods with high specificity are expected. In addition, as the enzyme used for the quantification, a highly pure enzyme that does not contain a contaminating enzyme is required, and in order to obtain it, a contaminating enzyme (for example, catalase,
Uricase etc.) should be completely inactivated by heat treatment to obtain a highly pure enzyme with high specific activity.

[0007]

[Means for Solving the Problems] Based on the background described above,
One of the inventors of the present invention is suitable for highly sensitive and specific enzymatic assay of creatinine or creatine,
As a result of extensive studies to provide a novel sarcosine oxidase that is heat-stable and simultaneously possesses an enzymatic property highly specific to sarcosine, a strain newly belonging to the genus Bacillus isolated from soil {Bacillus sp. KS
It was found that a new sarcosine oxidase M having high substrate specificity and thermostability at the same time can be obtained by culturing -11A [Microtechnical Laboratory Article 3605]}.

Further, the present inventors have made a DNA containing a gene encoding this novel sarcosine oxidase M.
Addition of creatinine, creatine and / or sarcosine to the medium by creating a strain having the ability to produce sarcosine oxidase M, which is obtained by including a recombinant having the vector inserted into vector DNA in a strain belonging to the genus Escherichia. , It was found that sarcosine oxidase M can be efficiently produced, and as a result of further studies on the sarcosine oxidase M gene, the present inventors succeeded in isolating and determining the structure of this novel sarcosine oxidase M gene for the first time. Was completed.

That is, the first aspect of the present invention is a novel sarcosine oxidase M represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and the second aspect of the present invention is derived from a microorganism belonging to the genus Bacillus. Sarcosine oxidase M gene defined by restriction enzyme cleavage map of Escherichia coli

[0010]

[Chemical 3]

( Where Ps is Pst I, Pv is Pvu II, and H is Hin dI
II and N represent Nru I. The third of the present invention is a novel sarcosine oxidase M gene which encodes the amino acid sequence of SEQ ID NO: 1 in the sequence listing and is represented by the base sequence of SEQ ID NO: 2 in the sequence listing. 4 is a novel recombinant DNA characterized by inserting the second novel sarcosine oxidase M gene of the present invention into a vector DNA, and 5th of the present invention is SEQ ID NO: 1 in the sequence listing. A novel sarcosine oxidase M which encodes an amino acid sequence and is represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing
A novel recombinant DNA characterized by inserting a gene into vector DNA. The sixth aspect of the present invention is a novel recombinant DNA obtained by inserting the second novel sarcosine oxidase M gene of the present invention into vector DNA. A method for producing sarcosine oxidase M, which comprises culturing a microorganism belonging to the genus Escherichia having a sarcosine oxidase M-producing ability in a medium, and collecting sarcosine oxidase M from the culture. A seventh aspect of the invention is a novel recombinant DNA in which the novel sarcosine oxidase M gene encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing and represented by the nucleotide sequence of SEQ ID NO: 2 is inserted into vector DNA. Including, sarcosine
A method for producing sarcosine oxidase M, which comprises culturing a microorganism belonging to the genus Escherichia capable of producing oxidase M in a medium and collecting sarcosine oxidase M from the culture.

The present invention will be described in detail below. First, the physicochemical properties of the enzyme of the present invention are shown below. a. Action This enzyme catalyzes the reaction of oxidatively decomposing sarcosine to produce glycine, formaldehyde and hydrogen peroxide.

Sarcosine + H ₂ O + O ₂ → Glycine + Formaldehyde + H ₂ O ₂ b. Substrate specificity Each substrate (0.01 M) 0.5 ml, 0.3 M phosphate buffer (pH 7.7)
0.1 ml, 0.2% (W / V) of 2,4-dichlorophenol
Sulfonate 0.1 ml, 70 mg / dl 4-aminoantipyrine 0.1 ml and 70 units / ml peroxidase 0.1
To the mixture of 1 ml, 0.1 unit of the enzyme solution of 17 units / ml and the thermostable sarcosine oxidase N described in the example of Japanese Examined Patent Publication No. 1-34035 were added, and the mixture was reacted at 37 ° C. for 20 minutes to give 1. The reaction was stopped by adding 2 ml of 0N acetic acid, and then the absorbance at a wavelength of 510 nm was measured using the reaction solution having a reaction time of zero as a control. The relative activity (%) is the comparative value (%) when the absorbance at a wavelength of 510 nm obtained by enzymatic reaction with N-methyl glycine (sarcosine) as a substrate is 100% and other substances are used as substrates. Indicated. Relative activity (%) substrate Sarcosine oxidase N N-methyl-glycine (sarcosine) 100 100 N-ethyl-glycine 2.5 4.4 N-methyl-valine 1.9 7.7 7.7 N-methyl- Leucine 6.0 25.4 N-methyl-isoleucine 3.2 7.2 N-methyl-phenylalanine 0 0 N-methyl-serine 0 0 N-methyl-glutamic acid 0 0 N-methyl-lysine 0 0 L-alanine 0 0 L-serine 0 0 L- Glutamic acid 0 N, N-dimethyl-glycine 0 0 Betaine 0 0 Choline 0 0 Proline 0.3 0.5 c. Optimum pH The optimum pH of this enzyme is shown in Fig. 1 when sarcosine is used as a substrate.
The pH is 8.0 to 9.0 as shown in. In FIG. 1, × − ×: Na
₂ HPO ₄ -Citric acid, ○-○: Phosphate buffer, ●-
●: Tris-HCl, △-△: TES-NaOH, ▲-▲: PO
PSO-NaOH, □-□: Gly ・ Gly-NaOH, ■ − ■: Gly ・ N
aCl-NaOH. d. Determination of titer 0.3 ml of 0.2 M sarcosine solution, 0.1 ml of 0.3 M Tris-HCl buffer (pH 8.8) and 0.1 ml of this enzyme solution of appropriate concentration.
After adding 10 ml of the reaction mixture at 37 ° C. for 10 minutes, the reaction was stopped by adding 0.5 ml of 1.0 N acetic acid solution, and then the acetyl-acetone coloring solution [acetyl-acetone. 2% (V /
V), diammonium hydrogen phosphate 10% (W / V)] (pH 6.5)
Was added for 3 minutes at 37 ° C., and the absorbance value at a wavelength of 410 nm was measured by a photoelectric colorimeter.

The amount of formaldehyde produced was checked from a previously prepared formaldehyde calibration curve, and the amount of enzyme producing 1 μM formaldehyde per minute at 37 ° C. was defined as 1 unit. e. Optimum temperature range of action As shown in Fig. 2, the optimum temperature range of action of this enzyme is 50 to 60 ° C.
It is in. f. Thermostability 0.5 ml of enzyme solution containing 3 units of the purified enzyme [0.
3M Tris-HCl buffer (pH 8.0)] was allowed to stand at each temperature for 20 minutes, and the residual enzyme activity was examined. As a result, as shown in FIG. 3, 100% at 50 ° C. and 64% at 60 ° C. The residual activity of g. pH stability 0.5 ml of each buffer solution containing 0.2 unit of the enzyme at 5 ° C
After being left at 48 ° C. for 48 hours, the remaining enzyme activity was examined. The result is pH 7.0 to 10.0 as shown in FIG. The buffer solution used is the same as that used to determine the optimum pH. h. Inhibition, Activation and Stabilization The present enzyme is capable of reacting with ions such as copper, zinc, silver and mercury and SDS.
It is strongly inhibited by (sodium dodecyl sulfate). There is no particular activator or stabilizer. i. Molecular weight The molecular weight of the polypeptide translated from the nucleotide sequence of the gene of this enzyme is 43072.

Next, the preparation of a DNA containing a gene encoding this novel sarcosine oxidase M (hereinafter referred to as "SOM") will be described. Examples of the donor microorganism of the gene encoding SOM include Bacillus sp. (Bacillus sp.) KS-11A. Bacillus sp. (Bacilluc sp.) KS-11A is a strain newly isolated from the soil by the present inventor, and its mycological properties are as follows.

(A) Morphology Microscopic observation (cultured on broth agar medium at 30 ° C. for 1 to 3 days) (1) Shape and size of cells: 0.8 to 1.1 × 3.0 to 4.5 micron Bacilli (2) Polymorphism of cells: Not observed. (3) Motility: Exercise with periflagellates.

(4) Presence of spores: Endospores are extremely difficult to form. (5) Gram stainability: Positive. (6) Anti-acidity: Negative. (b) Growth condition in each medium (1) Meat broth agar plate culture: Light gray brown colonies at 30 ° C. for 24 hours, surface smooth with dull luster, and opaque. No dye formation.

(2) Meat agar slope culture: Growth is good
Same as (1). (3) Liquid culture of broth: The static culture does not grow well and precipitates viable cells. (4) Meat broth gelatin stab culture: Culture at 25 ° C to liquefy. (5) Litmus milk: No change.

(C) Physiological properties (1) Reduction of nitrate: Negative (2) Denitrification reaction: Negative (3) MR test: Negative (4) VP test: Negative (5) Indole formation: Negative (6) Generation of hydrogen sulfide: Negative (7) Hydrolysis of starch: Positive (8) Hydrolysis of casein: Negative (9) Degradation of uric acid: Negative (10) Degradation of tyrosine: Negative (11) Hydrolysis of cellulose: Negative ( 12) Use of citrate: Negative (13) Use of inorganic nitrogen source: Use of ammonia is weak, nitrate is not used.

(14) Urease: Negative (15) Oxidase: Positive (16) Catalase: Positive (17) Pigment formation: Negative (18) Salt tolerance: Does not grow in 5% NaCl.

(19) Attitude toward oxygen: Aerobic (20) Range of growth: Growth pH range is 6 to 9, growth temperature is 22 ° C to 37 ° C and 85 ° C. (21) OF test: Oxidation (glucose) (22) Production of acid and gas from saccharides Production of sugar acid Production of gas (1) L-arabinose --- (2) D-xylose --- (3) D-Glucose +-(4) D-Mannose --- (5) D-Fructose --- (6) D-Galactose --- (7) Maltose --- (8) Sucrose --- (9) Lactose --- (10 ) Trehalose- (11) D-sorbit- (12) D-mannite- (13) inosit- (14) glycerin- (15) starch- (d) Other properties meso- in cell wall The presence of diaminopimelic acid was positive, the quinone type was MK-7, and the GC content (%) of DNA was 37%.
Is.

Catalase-positive Gram-positive bacilli are considered to be strains belonging to the genus Bacillus based on the measurement of cell wall diamino acids and quinone strains. They are strains close to Bacillus circulans, but they are not considered to be the same, and Bacillus sp.
(Bacillus sp.) KS-11A strain was named. In addition, Bacillus sp. (Bacillus sp.) KS-11A strain was transferred to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology at the Micro Engineering Research Center No. 3605 (FERM BP
-3605) has been deposited.

For example, Bacillus sp. The KS-11A strain is cultured in exactly the same manner as described in JP-A No. 61-162174 to obtain a culture. This culture, for example 3000r.
pm or more, preferably 8000 to 10000r.pm for 5 minutes or more,
Bacillus sp. Preferably after centrifugation for 10 to 15 minutes. Obtain KS-11A cells. From this bacterium, for example, Current Protocols in Molecular Biology, No. 1
Vol. 2, No. 2 (1987)] and the like.
A can be obtained.

Then, a restriction enzyme having no cleavage site on the SOM gene, such as Bam HI (Takara Shuzo), is added to the chromosomal DNA at a temperature of 30 ° C. or higher, preferably 37 ° C., and the enzyme concentration is 10 to 1000 units / ml. After digestion for 3 hours or longer, preferably 16 hours, various chromosomal DNA fragment mixtures are obtained. From the DNA fragment mixture thus obtained, for example, a DNA having a size of 15 to 20 Kb (kilobase pair) is preferably prepared by a conventional agarose gel electrophoresis method.
After obtaining a fragment mixture and subjecting it to an alkaline phosphatase treatment and the like, the mixture is further purified by a purification means such as phenol extraction, and further concentrated by means such as ethanol precipitation to obtain a purified DNA fragment mixture ( DNA containing a gene encoding SOM therein
Fragments are included. ) Get.

On the other hand, the vector DNA that can be used in the present invention includes, for example, bacteriophage vector DNA, plasmid vector DNA and the like. Specifically, for example, λEMBL4 DNA (manufactured by Stratagene) and the like. preferable. In order to make the above-mentioned phage vector DNA ready for Bam HI fragment incorporation, for example, Bam HI and Sal I (each manufactured by Takara Shuzo)
At a temperature of 30 ° C or higher, preferably 37 ° C, an enzyme concentration of 10 to 1000
The phage vector DNA capable of incorporating the Bam HI fragment is obtained by digesting with 1 unit / ml for 1 hour or more, preferably 1 to 6 hours, and further performing isopropanol precipitation treatment and the like.

Then, the Bacillus.
A mixture of DNA fragments derived from SPS KS-11A and containing a gene encoding SOM, and a phage vector DNA capable of incorporating the Bam HI fragment obtained as described above.
And mixed with, for example, T4 DNA ligase (manufactured by Boehringer Mannheim).
Temperature 4-37 ° C, preferably 4-16 ° C, enzyme concentration 1-100
Recombinant phage DNA is obtained by reacting with unit / ml for 1 hour or more, preferably 6 to 24 hours.

The recombinant phage DNA thus obtained can be used to form phage particles by an ordinary in vitro packaging method. Further, the phage can be obtained by, for example, Escherichia coli P2 392 (obtained from Toyobo). Plaques of recombinant phages carrying various DNA fragments can be obtained. Among the plaques thus obtained, DN containing the SOM gene
To search for a recombinant phage carrying the A fragment,
For example, [α- ³² P] dCTP [Amersham Japan
(Procedure (Amersham Japan)] using the sarcosine oxidase N gene (Japanese Patent Laid-Open No. 1-668287) as a probe, the current protocols in molecular biology [Current Protocols in Molecular
Biology, Volume 1, No. 6 (1987)] can be used to perform plaque hybridization.

To obtain a purified phage DNA from the recombinant phage thus obtained (containing a DNA fragment containing the SOM gene therein),
For example, molecular cloning [Molecular Clon
ing, pp. 371-372, Cold Spring Harbor Labolatory (198
2 years)] can be obtained by the method described.

In the recombinant phage DNA thus obtained and purified, the DNA containing the gene encoding SOM contains a large amount of unnecessary DNA in addition to the gene encoding SOM. Therefore, the unnecessary DNA is removed by the following operation. Then, the recombinant DNA thus obtained and purified is treated with a restriction enzyme having no cleavage site on the SOM gene, such as Bgl II or Eco RI, at a temperature of 30 ° C. or higher,
Preferably, the enzyme is allowed to act at 37 ° C. and an enzyme concentration of 10 to 1000 units / ml for 1 hour or more, preferably 1 to 6 hours for digestion to give DN.
A mixture of A fragments is obtained.

In order to determine which of the DNA fragments in the thus obtained mixture of DNA fragments contains the gene encoding SOM, the DNA labeling and detection by Boehringer Mannheim Co. Kit (DNA Labeling and Detection Kit)
By using the sarcosine oxidase N gene labeled with non-radioactive digoxigenin (JP-A-1-168287) as a probe, Southern hybridization and the like can be carried out by a conventional method.

Then, D obtained by digesting as described above
The NA fragment mixture is subjected to ordinary agarose electrophoresis, and, for example, only the DNA fragment determined to contain the gene encoding SOM as described above is used as a gene clean II kit (GENECLEAN II K manufactured by Funakoshi Co., Ltd.).
(IT) to obtain a purified BglII- digested fragment (in which the gene encoding SOM is contained).

On the other hand, any vector DNA can be used in the present invention, and examples thereof include plasmid vector DNA and bacteriophage vector DNA. Specific examples include plasmid pUC118 and pUC119 DNA [respectively, Takara Shuzo] and the like are preferable. A restriction enzyme such as Ba is added to the vector DNA.
m HI, Eco RI, etc. (respectively, Takara Shuzo Co., Ltd.) Temperature 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 1 to 6 hours to obtain a digested vector DNA.

Then, the Bacillus.
A mixture of DNA fragments derived from SPS KS-11A and containing a gene encoding SOM and cleaved vector DNA are mixed, and for example, T4 DNA ligase (Boehringer Mannheim) is added at a temperature of 4 to 37 ° C, preferably Is 4
~ 16 ℃, enzyme temperature 1 ~ 100 units / ml for 1 hour or more,
Preferably, the reaction is carried out for 6 to 24 hours to obtain a recombinant DNA.

Using this recombinant DNA, for example, E. coli K-12, preferably E. coli JM109 (obtained from Takara Shuzo), E. coli HB101 (ATCC 33694), E. coli DH1 (A
Each strain is obtained by transforming or transducing TCC 33849), Escherichia coli x-1776 (ATCC 31244) and the like. This transformation is done by DM Morrison (Methods in Enzymology).
ology), 68, 326-331 (1979)]. For transduction, B. Hoh
Method n) [Methods in Enzymology, Volume 68,
Pp. 299-309 (1979)].

By screening a strain having sarcosine oxidase activity from the above strains, a DNA containing the full length gene encoding SOM is obtained.
It is possible to obtain a strain belonging to the genus Escherichia having the ability to produce SOM, which contains the recombinant DNA in which the fragment is inserted into the vector DNA. In order to obtain a purified recombinant DNA from the strain thus obtained, for example, the method of P. Guerry, etc. [J. Bacteriology, Vol. 116, Vol. 1064 ~ 10
66 (1973)], D.B.Crewell (DB C
Lewell) method (J. Bacteriology, Volume 110,
Pp. 667-676 (1972)] and the like.

Using the DNA containing the above SOM gene, SO was prepared by the method as described in item (7) of the Example.
The entire base sequence of the M gene is analyzed (see SEQ ID NO: 2 in the sequence listing), and then the amino acid sequence of the polypeptide translated by the gene having the base sequence is determined (see SEQ ID NO: 1 in the sequence listing). The gene encoding the amino acid sequence thus determined is the SOM gene of the present invention.

Next, to produce SOM using a strain belonging to the genus Escherichia containing the recombinant DNA in which the DNA containing the SOM gene obtained as described above is inserted into vector DNA, This strain may be cultivated by an ordinary solid culturing method, but it is preferable to cultivate by using a liquid culturing method as much as possible. Also,
Examples of the medium for culturing the above-mentioned strain include, for example, yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat koji leaching solution, and the like, one or more nitrogen sources, potassium dihydrogen phosphate, dipotassium hydrogen phosphate. , One containing at least one inorganic salt such as magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate or manganese sulfate, and optionally a sugar raw material, vitamins and the like are used.

The initial pH of the medium is appropriately adjusted to 7-9. Also, the culture is 30 to 42 ° C, preferably 37 ° C.
It is preferable to carry out for 4 to 24 hours, preferably 6 to 8 hours before and after aeration and agitation deep culture, shaking culture, static culture and the like. After the culture is completed, the SOM can be collected from the culture by using an ordinary enzyme collecting means.

For example, the cells are sonicated by a conventional method,
This enzyme may be subjected to grinding treatment, extracted with a lysing enzyme such as lysozyme, or shaken or allowed to stand in the presence of toluene or the like for autolysis to discharge the enzyme out of the cells. .. The solution is filtered, the solid portion is removed by centrifugation, etc., and if necessary, nucleic acid is removed by 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 vacuum dried to obtain a crude enzyme preparation.

The enzyme of the present invention is optionally subjected to a conventional method for isolation and purification of the enzyme, for example, (1) DEAE-cellulose column chromatography, (2) ammonium sulfate fractionation, (3) QA.
E-Sephadex column chromatography, (4)
TSK-GEL Butyl-Toyopearl 650 C [Toyo Soda Co., Ltd.] hydrophobic chromatography, (5) Sephadex gel filtration, etc. A purified SOM standard can be obtained.

The physicochemical properties of the recombinant-derived purified SOM obtained by the above-mentioned purification means are as follows.
The physicochemical properties of SOM derived from SPK-11A are exactly the same.

[0042]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the examples. Example (1) Preparation of chromosomal DNA of Bacillus sp. KS-11A strain Bacillus sp. KS-11A strain was mixed with TY medium {1% (W /
V) Bacto-trypton (Difco
co), 0.5% (W / V) Bacto-yeast extract (Difco), 0.5% (W / V) NaCl} (pH 7. 2) 200 ml was inoculated and shake-cultured at a temperature of 30 ° C for 16 hours to obtain a culture.

This culture was centrifuged at 5000 rpm for 10 minutes by a conventional method to obtain 0.5 g of wet cells, and the current cells were inoculated with the Current Protocols in Molecular Biology [Current Protocols]. in Molecular Biolog
y, Vol. 1, No. 2 (1987)] to obtain a chromosomal DNA. Next, 50 μg of this chromosomal DNA and 50 units of restriction enzyme Bam HI (Takara Shuzo) were mixed with 20 mM Tris-HCl buffer (containing 100 mM KCl, 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH 8.5), and the temperature was 37 ° C. The reaction was allowed for 16 hours. DN of about 15 to 20 Kb (kilo base pair) is obtained by subjecting the reaction-completed solution to electrophoresis of 0.7% (W / V) agarose gel (manufactured by Takara Shuzo) by a conventional method.
Fragment A is a method such as ARC Young (Methods in Enz
ymology), Vol. 68, pp. 176-182 (1979)] to obtain an eluate, which is then subjected to phenol extraction by a conventional method and further ethanol precipitation to purify Ba.
An mHI digested fragment was obtained. This was added to 50 mM Tris-HCl buffer (pH 8.0) together with 1 unit of alkaline phosphatase (Takara Shuzo Co., Ltd.) and reacted at a temperature of 37 ° C. for 45 minutes,
The phosphate at the 5'end was removed. The reaction-terminated solution was subjected to phenol extraction treatment by a conventional method, and then subjected to ethanol precipitation treatment to obtain Bacillus sp. KS-11A chromosomal DNA.
5 μg of a mixture of Bam HI digested fragments (15 to 20 Kb) (containing a DNA fragment containing a gene encoding SOM) was obtained. (2) Preparation of recombinant phage λSOM1 λEMBL4 phage DNA [STRATAGEN
E)] 2 μg and 100 units of restriction enzymes Bam HI and Sal I (respectively Takara Shuzo Co., Ltd.) each in 50 mM Tris-HCl buffer (containing 100 mM NaCl, 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH 7.5). ) And reacted at a temperature of 37 ° C. for 2 hours, then 15 mM ethylenediamine tetraacetic acid (EDT
A) (pH8.0) was added, and the enzymatic reaction was stopped by treating at 70 ° C. for 10 minutes. The reaction solution by a conventional method with phenol extraction, after removing the small DNA fragment further by isopropanol precipitation treatment, and ethanol precipitation treatment to obtain a λEMBL4 phage DNA has become possible incorporation of Bam HI fragment.

Then, λ capable of incorporating this Bam HI fragment
EMBL4 phage DNA 1 μg, obtained in the above item (1)
Bacillus sp. KS-11A chromosomal DNA fragment mixture (15-20Kb) 3 µg and 1 unit of T4 DNA ligase (Boehringer Mannheim) digested with Bam HI
6.6 mM MgCl ₂ , 10 mM dithiothreitol and 10 mM AT
It was added to 66 mM Tris-HCl buffer (pH 7.5) containing P and reacted at a temperature of 16 ° C. for 16 hours to ligate DNA.

Then, this recombinant phage DNA1
μg by standard in vitro packaging method
Firstly taken into the phage particles were infected into E. coli as a phage, dissolved bacteria was added chloroform, 50 mM Tris-HCl buffer _{(100mM NaCl, 8mM MgSO 4 ·} 7
H ₂ O and 0.01% (W / V) gelatin). Among the recombinant phages thus obtained, S
The sarcosine oxidase N gene labeled with [α- ³² P] dCTP [obtained from Amersham Japan] to search for recombinant phage carrying a Bam HI fragment containing the OM gene (JP-A-1-168287).
Current Protocols in Molecular Biology [Current Protoc]
ols in Molecular Biology, Volume 1, Issue 6 (1987)
] Plaque hybridization was performed by the method described. DN hybridized with this probe
The phage having A was a recombinant phage carrying the SOM gene, whereby a recombinant λEMBL4 phage carrying the SOM gene (λSOM1) was obtained.

(3) Isolation of recombinant phage λSOM1 DNA 1 ml of a culture solution of E. coli LE392 (obtained from Toyobo) was added to
TY supplemented with 1% (W / V) glucose and 8 mM MgSO _4.
Medium [1% (W / V) Bacto-tryptone, 0.5% (W / V) Bacto-Yeast Extract, 0.5% (W / V) NaCl}
(pH7.2) 30 ml was inoculated, shake-cultured at a temperature of 37 ° C. for 2-3 hours, and the lysate obtained by plating the recombinant phage λSOM 1 carrying the SOM gene prepared in (2) (2.
0 to 2.5 x 10 ⁹ phage) and LE3
Shake at 37 ℃ until 92 lysates (3-5 hours),
Further, 0.3 ml of chloroform was added and shaken for 15 minutes. This lysate was centrifuged at 8000 rpm for 15 minutes by a conventional method, 30 μg of RNase and 30 μg of DNase were added to the supernatant, and the mixture was reacted at 37 ° C. for 30 minutes, and then the lysate and the like were mixed. 20% of the amount (W / V) polyethylene glycol (PEG)
/2.5M NaCl was added, and PEG precipitation was performed at 0 ° C for 1 hour. This precipitate was added to 50 mM Tris-HCl buffer (100 mM NaCl,
Eluted 8mM MgSO ₄ · 7H ₂ O and 0.01% (W / V) gelatin containing) conventional manner phenol extraction, and further isopropanol precipitation process, recombinant phage λSOM1
DNA was obtained.

(4) Construction of recombinant plasmid pSOM 1 2.5 μg of plasmid pUC119 DNA (Takara Shuzo) and 10 units of restriction enzyme Bam HI (Takara Shuzo) were added to 20 mM Tris-HCl buffer (100 mM KCl, 10 mM MgSO _4). And 1 mM dithiothreitol) (pH 8.5) and mix at 37 ℃.
The reaction mixture was allowed to react for a period of time to obtain a digestive solution, which was subjected to phenol extraction and ethanol precipitation by a conventional method, and then added to 50 mM Tris-HCl buffer (pH 8.0) together with 1 unit of alkaline phosphatase (Takara Shuzo) at a temperature of The reaction was carried out at 37 ° C for 45 minutes to remove the phosphate at the 5'end. The reaction-terminated solution was subjected to a phenol extraction treatment by a conventional method, and then an ethanol precipitation treatment to obtain a plasmid digested with Bam HI.
pUC119 DNA was obtained.

[0048] Then, the above-mentioned items (Takara Shuzo Co., Ltd.) resulting recombinant phage λSOM1DNA5μg and Bgl II (3) to 10 units, 50 mM Tris-HCl buffer (100 mM NaC
l, containing 10 mM MgSO ₄ and 1 mM dithiothreitol) (pH
7.5), and reacted at a temperature of 37 ° C for 16 hours to obtain a digestive juice. The digested solution was electrophoresed on a 0.7% (W / V) agarose gel, and only a DNA fragment of about 1.5 Kb was purified and purified using the GeneCLEAN II KIT manufactured by Funakoshi. 0.1 μg of the BglII digested fragment was obtained.
The fragment is a DNA fragment of about 1.5 kb containing a gene encoding SOM.

0.1 μg of the DNA fragment thus obtained,
Plasmid pUC119 DNA 0.1 μg digested with Bam HI
And 1 unit of T4 DNA ligase (Boehringer Mannheim) were added to 66 mM Tris-HCl buffer (6.6 mM MgCl 2).
₂ , containing 10 mM dithiothreitol and 10 mM ATP) (p
H7.5) and mixed at a temperature of 16 ° C. for 16 hours to ligate the DNA.

Next, DM Maurison (D.
M. Morrison) [Methods in Enzymology, 68, 326-331.
(1979)], E. coli JM109 treated with calcium chloride
(Obtained from Takara Shuzo Co., Ltd.) was transformed with the recombinant plasmid DNA obtained as described above, and the obtained transformant was treated with 50 μg / ml ampicillin, 0.5 mM isopropyl-β-D-thiogalactoside and 0.004 % (W / V) 5-
Bromo-4-chloro-3-indolyl-β-D-galactoside is cultured for 24 hours at 37 ° C. using TY agar plate containing each of them, and those forming white colonies produce SOM. As a strain, Escherichia coli JM109 (pSOM1) forming a white colony was obtained.

The thus obtained SOM-producing transformant E. coli JM109 (pSOM)
1) has been deposited at the Institute of Microbial Engineering, Institute of Industrial Science and Technology, as Micro Engineering Research Article No. 3604 (FERM BP-3604). (5) Isolation of recombinant plasmid pSOM1 DNA T-consisting of Bact-tryptone 1% (W / V), Bact-yeast extract 0.5% (W / V), and NaCl 0.5% (W / V). 12.5 mg of ampicillin was added to 250 ml of Y medium (pH 7.2), and E. coli JM109 (pSOM1) was added to this.
Was inoculated and cultured at 37 ° C. for 20 to 24 hours with shaking to obtain a culture solution.

Then, this culture solution was subjected to 5000 r.
Centrifuge at 10 pm for 10 minutes to obtain moist bacterial cells.
After suspending in 5 ml of 25 mM Tris-HCl buffer (pH 8.0) containing mM glucose and 10 mM EDTA,
Lysozyme (25 mg) was added and lysed at room temperature for 5 minutes to obtain a lysate. To this lysate, 10 ml of 0.2N-NaOH solution containing 1% (W / V) sodium dodecyl sulfate was added, and the mixture was left at 0 ° C. for 10 minutes to denature the DNA. Furthermore, 7.5 ml of 5M potassium acetate-acetic acid buffer (pH 4.8) was added to this, and the mixture was added at 0 ° C.
After allowing to stand for 10 to 30 minutes at room temperature to regenerate only the plasmid DNA, centrifugation was performed at 9000 rpm for 20 minutes to obtain an extract, which was subjected to chloroform extraction according to a conventional method, followed by ethanol precipitation according to a conventional method. A precipitate was obtained.

Then, this precipitate was dried under normal vacuum, and dissolved in 6 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, and 6 g of cesium chloride and 10 mg / ml of ethidium bromide were added thereto. Equilibrium density gradient centrifugation using an ultracentrifuge was carried out for 20 hours at 50000 rpm with 3 ml added, the recombinant plasmid pSOM1DNA was isolated, and n-butanol was used to ethidium.・ After extracting and removing bromide,
10 mM Tris-HCl buffer containing 1 mM EDTA (pH 7.5)
After dialysis, 100 μg of purified recombinant plasmid pSOM1DNA (size: about 4.7 Kb) was obtained.

(6) Production of SOM by E. coli JM109 (pSOM1) and separation and purification of the enzyme 1% (W / V) bacto-tryptone, 0.5% (W / V) bacto-
Yeast extract, 1 mM isopropyl-β-D
-A stirring type small culture device (made by Iwashiya) using 2 L (pH 7.2) of a medium containing thiogalactoside and 0.5% (W / V) NaCl.
E. coli JM109 (pSOM1) culture broth, which had been aliquoted in a high-pressure sterilized solution by a conventional method and shake-cultured in a medium having the same composition as described above for 24 hours at 37 ° C.
The procedure of inoculating ml of the mixture and culturing with aeration and stirring at 37 ° C. for 8 hours was repeated 5 times to obtain 37 g of wet cells.

This bacterial cell was treated with 0.01 M phosphate buffer (pH 8.0)
After suspending in 120 ml and sonicating by a conventional method,
Normal centrifugation treatment was performed at 000 rpm for 30 minutes to obtain 130 ml (3.2 unit / ml) of a crude SOM enzyme solution. The crude enzyme solution thus obtained was subjected to nucleic acid removal treatment using streptomycin sulfate, heat-treated at a temperature of 50 ° C. for 1 hour, and then the insoluble substance was centrifuged at 10,000 rpm for 10 minutes. Removed.

Then, this crude enzyme solution was equilibrated with 0.01M phosphate buffer to a QAE-Sephadex A-50 column.
(1.4 × 40 cm), washed with 0.01M phosphate buffer containing 0.27M potassium chloride, and then eluted with 0.01M phosphate buffer containing 0.37M potassium chloride.
An OM-containing eluate was obtained. The SOM-containing eluate was concentrated by a conventional method and then freeze-dried to obtain a purified SOM powder.

[0057] (7) using various restriction enzyme cleavage sites within Bgl II fragment incorporated in the resulting plasmid pSOM1DNA the analysis the item (5) of the base sequence of DNA containing the SOM gene,
The nucleotide sequence of the SOM gene was analyzed by the procedure shown in FIG. Various DNA fragments cut out from pSOM1 with various restriction enzymes were used as plasmids pUC118 or pUC119D.
Recombinant plasmid DN obtained by cloning into the multiple cloning site of NA (Takara Shuzo Co., Ltd.)
E. coli JM109 (obtained from Takara Shuzo Co., Ltd.) was transformed with A to obtain each transformant strain.

Cleavage of DNA by restriction enzyme, T4
Method for ligation and transformation with DNA ligase, and D
The isolation and purification method of the NA fragment by agarose gel electrophoresis was based on the method described in the above item (4). The transformant thus obtained was added to the helper phage M13K07.
(Takara Shuzo Co., Ltd.) infection method [Methods in Enzymology (Methods in Enzymo
logy), 101, 20-78 (1983)].

Sequencing with the obtained single-stranded DNA was carried out using the Dye Primer Taq Sequencing Kit [Applied
Biosystems (manufactured by Applied Biosystems)] according to the above meshing method. In addition, gel electrophoresis for the analysis of nucleotide sequences was performed using 6% (W / V) polyacrylamide gel (Nation containing 50% (W / V) urea).
al Diagnostics Company), DNA sequencer 370A
(Manufactured by Applied Biosystems).

The entire nucleotide sequence of the obtained SOM gene is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the polypeptide translated from the gene is shown in SEQ ID NO: 1.

[0061]

INDUSTRIAL APPLICABILITY The novel SOM of the present invention has high substrate specificity for sarcosine and thermostability at the same time, and is suitable for a highly sensitive and highly specific enzymatic assay of creatinine or creatine. Furthermore, when a strain belonging to the genus Escherichia containing the novel recombinant DNA into which the SOM gene of the present invention is cultivated in a medium, SOM can be efficiently obtained without adding and using creatinine, creatine, sarcosine, etc. The present invention is extremely useful industrially.

[0062]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 387 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence: Met Ser Thr His Phe Asp Val Ile ValVal 10 Gly Ala Gly Ser Met Gly Met Ala AlaGly 20 Tyr Tyr Leu Ala Lys Gln Gly Val LysThr 30 Leu Leu Val Asp Ala Phe Asp Pro ProHis 40 Thr Glu Gly Ser His His Gly Asp ThrArg 50 Ile Ile Arg His Ala Tyr Gly Glu GlyArg 60 Glu Tyr Val Pro Phe Ala Leu Arg AlaGln 70 Glu Leu Trp Tyr Glu Leu Glu Asn GluThr 80 His Asn Lys Ile Phe Thr Lys Thr GlyVal 90 Leu Val Phe Gly Pro Lys Gly Glu SerAsp 100 Phe Val Ala Glu Thr Met Glu Ala AlaAla 110 Glu His Ser Leu Thr Val Asp Leu LeuGlu 120 Gly Asp Glu Ile Asn Thr Arg Trp ProGly 130 Ile Thr Val Pro Glu Asn Tyr Asn AlaIle 140 Phe Glu Pro Asn Ser Gly Val Leu PheSer 150 Glu Asn Cys Ile Arg Ser Tyr Arg GluLeu 160 Ala Val Ala Lys Gly Ala Lys Ile LeuThr 170 Tyr Thr Arg Val Glu Asp Phe Glu ValSer 180 Gln Asp Gln Val Lys Ile Gln Thr AlaAsn 190 Gly Ser Tyr Thr Ala Asp Lys Leu IleVal 200 Ser Met Gly Ala Trp Asn Ser Lys LeuLeu 210 Ser Lys Leu Asn Leu Asp Ile Pro LeuGln 220 Pro Tyr Arg Gln Val Val Gly Phe PheAsp 230 Ser Asn Glu Ala Lys Tyr Ser Asn AspVal 240 Asp Tyr Pro Ala Phe Met Val Glu ValPro 250 Lys Gly Ile Tyr Tyr Gly Phe Pro SerPhe 260 Gly Gly Cys Gly Leu Lys Ile Gly TyrHis 270 Thr Tyr Gly Gln Gln Ile Asp Pro AspThr 280 Ile Asn Arg Glu Phe Gly Ala Tyr GlnGlu 290 Asp Glu Ser Asn Leu Arg Asp Phe Leu Leu Met Pro Glu Ala Asn Gly GluLeu 310 Lys Arg Gly Ala Val Cys Met Tyr ThrLys 320 Thr Pro Asp Glu His Phe Val Ile AspThr 330 His Pro Glu His Ser Asn Val Phe ValAla 340 Ala Gly Phe Ser Gly HIs Gly Phe LysPhe 350 Ser Ser Val Val Gly Glu Val Leu SerGln 360 Leu Ala Thr Thr Gly Lys Thr Glu HisAsp 370 Ile Ser Ile Phe Ser Ile Asn Arg ProAla 380 Leu Lys Gln Lys Thr Thr Ile SEQ ID NO: 2 Sequence length: 1164 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: genomic DNA Origin organism name: Bacillus sp. lus sp.) Strain name: KS-11A strain Sequence: ATG AGT ACA CAT TTT GAT GTG ATT GTTGTT 30 GGA GCA GGA TCA ATG GGA ATG GCT GCAGGG 60 TAC TAT TTA GCA AAA CAA GGA GTC AAAACA 90 TTA TTG GTG GAT GCA TTC GAT CCG CCGCAT 120 ACA GAA GGA AGC CAT CAC GGT GAT ACTCGC 150 ATT ATC CGC CAT GCT TAC GGT GAA GGAAGA 180 GAA TAT GTT CCA TTT GCA CTA AGA GCACAA 210 GAA TTA TGG TAT GAA CTT GAA AAT GAAACA 240 CAC AAT AAG AAAATT TTT ATA ACA GGCGTT 270 CTA GTT TTT GGT CCG AAA GGT GAA TCCGAT 300 TTC GTT GCC GAA ACA ATG GAG GCA GCTGCA 330 GAA CAT TCA TTG ACT GTG GAT TTA CTTGAG 360 GGT GAT GAA ATC AAT ACG CGC TGG CCCGGC 390 ATA ACG ATT CCT GA AAT GCAATT 420 TTT GAA CCA AAT TCA GGC GTA TTG TTCAGT 450 GAG AAT TGT ATT CGT TCA TAC CGT GAGCTG 480 GCT GTA GCA AAA GGA GCA AAA ATT TTAACA 510 TAT ACT CGT GTT GAG GAT TTT GAA GTTTCT 540 CAA GTC CAA GTT AAA ACG GCAAAT 570 GGA TCG TAC ACA GCT GAT AAA TTA ATCGTA 600 AGT ATG GGT GCT TGG AAT AGT AAA CTACTT 630 TCT AAA TTA AAT CTT GAC ATC CCA TTACAG 660 CCA TAC CGC CAA GT T GTA GGA TTT TTTGAT 690 TCT AAT GAA GCA AAG TAC AGC AAT GATGTG 720 GAT TAT CCA GCA TTC ATG GTA GAA GTACCA 750 AAA GGT ATT TAT TAC GGA TTC CCA AGCTTC 780 GGT GGC TGC GGT TTG AAA ATA GGG TATCATGT 810 ACG CAA ATC GAC CCT GATACG 840 ATT AAC CGT GAA TTT GGT GCT TAT CAAGAG 870 GAT GAA AGT AAT CTT CGC GAT TTC TTGGAA 900 AAA TAT ATG CCA GAA GCA AAT GGC GAGTTA 930 AAA CGA GGC GCA GTC TGT ATG TAC ACGACAAT 960 ACA CAT TTC GTG ATT GATACT 990 CAT CCA GAA CAT TCC AAT GTT TTC GTAGCA 1020 GCT GGT TTC TCT GGA CAC GGC TTT AAATTT 1050 TCA AGT GTA GTC GGT GAA GTG TTA AGTCAA 1080 TTA GCG ACA ACA GGT AAA ACA GAA CATGAT 1110 TATT TCA ATA AAT CGT CCTGCT 1140 TTA AAA CAG AAA ACA ACG ATT TAA

[Brief description of drawings]

FIG. 1 is a graph showing the optimum pH of this enzyme.

FIG. 2 is a diagram showing a range of suitable temperature for the action of the present enzyme.

FIG. 3 is a view showing the thermostability of the present enzyme.

FIG. 4 is a diagram showing the pH stability of this enzyme.

FIG. 5: D containing a gene encoding this enzyme SOM
The figure which shows the cutting | disconnection map by the main restriction enzymes of NA fragment (Additional procedure of a base sequence analysis).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12R 1: 185) (C12N 1/21 C12R 1: 185) (72) Inventor Taiji Koyama Chiba Prefecture Kita Korman Co., Ltd., 339 Noda, Noda City (72) Inventor Eiichi Nakano 339 Noda, Noda, Chiba Prefecture

Claims (10)

[Claims] 1. A novel sarcosine oxidase M represented by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. Derived from a microorganism belonging to the genus Bacillus,
A novel sarcosine oxidase M gene defined by the following restriction enzyme cleavage map. [Chemical 1] (Wherein, Ps is Pst I, Pv is Pvu II, H is Hin dIII, N is Nru
Represents I. ) 3. The novel sarcosine oxidase M gene according to claim 2, which encodes the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 4. The novel sarcosine oxidase M gene according to claim 2 or 3, which is represented by the base sequence of SEQ ID NO: 2 in the sequence listing. 5. A novel recombinant DNA comprising the novel sarcosine oxidase M gene according to claim 2 inserted into vector DNA. 6. The novel recombinant DNA according to claim 5, wherein the sarcosine oxidase M gene encodes the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 7. The novel recombinant DNA according to claim 5 or 6, wherein the sarcosine oxidase M gene is represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 8. The novel recombinant DN according to claim 5.
A method for producing sarcosine oxidase M, which comprises culturing a microorganism containing A and having the ability to produce sarcosine oxidase M and belonging to the genus Escherichia in a medium, and collecting sarcosine oxidase M from the culture. 9. The sarcosine oxidase M according to claim 8, wherein the sarcosine oxidase M gene encodes the amino acid sequence of SEQ ID NO: 1 in the sequence listing.
Manufacturing method. 10. The sarcosine oxidase M according to claim 8 or 9, wherein the sarcosine oxidase M gene is represented by the nucleotide sequence of SEQ ID NO: 2 in the sequence listing.
Manufacturing method.