JP2593481B2 - DNA having genetic information of sarcosine oxidase and use thereof - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a polydeoxyribonucleic acid having genetic information of a novel sarcosine oxidase. The present invention also provides
The present invention relates to a transformant carrying the polydeoxyribonucleic acid.

[Conventional technology]

Sarcosine oxidase is obtained from one molecule of sarcosine, one molecule of glycine, formaldehyde and one molecule of oxygen, respectively, as shown by the following reaction formula: sarcosine + O ₂ + H ₂ O → glycine + formaldehyde + H ₂ O ₂ An enzyme that catalyzes a reaction that produces hydrogen oxide, which has long been known to exist in nature, particularly in animal organs.
cillium), Pseudomonas (Fr.
isell, WR & Mackenzie, CG (1970) Meth.Enzymol.17
A.976-981], Arthrobacter
[JP-A-54-28893], Bacillus
[JP-A-54-52789, JP-A-61-162174], Cylindrocarpon (JP-A-56-92790), Pseudomonas
s) It has been reported that it is present in microorganisms belonging to the family Streptomycetaceae (Japanese Unexamined Patent Publication No. Sho 61-280271).

Since sarcosine oxidase is an oxidase using sarcosine as a substrate, it is used for quantification of sarcosine present in body fluids such as serum, but this enzyme is not limited to sarcosine, and is used for creatinine or choline metabolism. Creatinine, creatine, by producing formaldehyde and hydrogen peroxide, and quantifying one or both of them, in particular by causing a conjugation reaction with other enzymes of the former metabolic system, namely creatininase and creatinase. Can be simply and specifically quantified. Therefore, it is a major enzyme in biochemical quantification methods that replace conventional non-specific quantification methods of creatinine or creatine, and is extremely useful in the field of clinical diagnosis and research.

[Problems to be Solved by the Invention] Conventionally reported sarcosine oxidase-producing bacteria have low productivity of sarcosine oxidase, and not only have the disadvantage that the production cost is originally high, but also
During production, the addition of substances that induce sarcosine oxidase production, such as choline, sarcosine or creatine, was indispensable, resulting in higher production costs, and the removal of other coexisting enzymes after culture was extremely difficult. Is difficult, there is a problem in terms of purification process and cost to obtain high-quality sarcosine oxidase of high purity,
It was not always satisfactory for easy and wide use as a research reagent and a clinical diagnostic reagent.

However, recently, the heat-resistant sarcosine oxidase obtained from a heat-resistant bacterium can be completely deactivated by heat treatment to completely inactivate the activities of contaminating enzymes (eg, catalase, uricase, etc.) [Japanese Patent Laid-Open No. Sho 61]. -162174)
However, contaminating enzymes derived from thermotolerant bacteria (for example, catalase, uricase, etc.) have some heat resistance in any case, and in fact, as described in the above-mentioned publication, the contaminating enzymes are completely inactivated. It was very difficult to do.

Further, no detailed primary structure of sarcosine oxidase having the following physicochemical properties has been reported.

(A) Action It is an enzyme that catalyzes the following enzymatic reaction that produces glycine, formaldehyde and one molecule of hydrogen peroxide from sarcosine, one molecule of enzyme and one molecule of water.

Sarcosine + O ₂ + H ₂ O → glycine + formaldehyde + H ₂ O ₂ (b) Substrate specificity Sarcosine has substrate specificity.

(C) Extraction pH 8.-9.5 (d) Isoelectric point 4.7 (e) Molecular weight 40,000 (measured by gel filtration method) (f) Thermal stability Stable at 40 ° C for 10 minutes.

[Means for solving the problem]

The present inventors have made intensive studies to improve the productivity of sarcosine oxidase, reduce the content of coexisting other enzymes (contaminating enzymes), and reduce the production cost, and found a novel sarcosine oxidase gene from microorganisms. As described below, a method for producing sarcosine oxidase that is highly productive and does not require an inducing substance in the production medium by applying genetic engineering techniques to the extraction of sarcosine oxidase and the analysis of its primary structure. Established.

That is, the present invention comprises a polydeoxyribonucleic acid comprising a base sequence encoding the amino acid sequence described below of a polypeptide comprising sarcosine oxidase having the following physicochemical properties, and the polydeoxyribonucleic acid is retained. A transformant characterized in that:

(A) Action It is an enzyme that catalyzes the following enzymatic reaction that produces glycine, formaldehyde and one molecule of hydrogen peroxide from sarcosine, one molecule of enzyme and one molecule of water.

Sarcosine + O ₂ + H ₂ O → glycine + formaldehyde + H ₂ O ₂ (b) Substrate specificity Sarcosine has substrate specificity.

(C) Extraction pH 8.0-9.5 (d) Isoelectric point 4.7 (e) Molecular weight 40,000 (measured by gel filtration method) (f) Thermal stability Stable at 40 ° C for 10 minutes.

The present invention also relates to a transformant characterized in that the host microorganism retains the polydeoxyribonucleic acid. Further, the polypeptide produced by the transformant may contain one unit of sarcosine oxidase activity and a contaminating enzyme. The activity of certain catalase, creatinase, N-ethyl-glycine oxidase is sarcosine oxidase having a catalase activity of 0.05 units or less, a creatinase activity of 0.0004 units or less, and an N-ethyl-glycine oxidase activity of 0.03 units or less. Expression from end (In the formula, A represents the amino acid residue, a hydrogen atom or an acetyl group, B denotes an amino acid residue, -OH or -NH ₃₎
Is a polypeptide constituting sarcosine oxidase which catalyzes a reaction that produces glycine, formaldehyde and one molecule of hydrogen peroxide from one molecule of enzyme and one molecule of water represented by the following formula: The object is achieved by transferring a recombinant DNA obtained by incorporating the polydeoxyribonucleic acid into an expression vector into a host microorganism, further culturing the transformant to express the genetic information of the polydeoxyribonucleic acid, and then The present invention relates to the production of sarcosine oxidase obtained by collecting a polypeptide as a constituent component of sarcosine oxidase having the above physicochemical properties.

In the polypeptide (I), the amino acid residue represented by A is composed of one or more amino acid residues, and A preferably includes a hydrogen atom, Met, or a signal peptide. The compound represented by B may be an acid amide or one or more amino acid residues.

The polydeoxyribonucleic acid which is the sarcosine oxidase gene having the above-mentioned physicochemical properties may be a polydeoxyribonucleic acid containing at least the sarcosine oxidase gene itself having the above-mentioned physicochemical properties, such as the nuclear sarcosine oxidase gene itself. From the N-terminal side And polydeoxyribonucleic acid, which is a nucleotide sequence encoding the amino acid sequence of a polypeptide as a component of sarcosine oxidase represented by

In the amino acid sequence of the polypeptide that is sarcosine oxidase represented by the formula (II), any polydeoxyribonucleic acid consisting of any one of a series of codons corresponding to each amino acid may be used. It may be a polydeoxyribonucleic acid having one or more codons other than one or more nonsense codons at the 5 'end of the deoxyribonucleic acid and / or one or more codons at the 3' end. For example, as a typical example, the formula (Wherein, X represents a codon or hydrogen atom other than TAA, TAG and TGA, and Y represents a codon or hydrogen atom).

Regarding the base sequence represented by the formula (III), the codon represented by X may be any codon that encodes an amino acid, and further has one or more codons encoding an amino acid at the 5′-terminal thereof. However, a polydeoxyribonucleic acid corresponding to ATG or a signal peptide can be preferably used.

The codon represented by Y may be any translation termination codon or a codon encoding an amino acid.
One or more codons encoding an amino acid may be present on the 3'-terminal side. In this case, it is preferable that the plurality of codons further have a translation termination codon at the 3'-terminal.

A polydeoxyribonucleic acid having a sarcosine oxidase gene as a component or a polydeoxyribonucleic acid having a base sequence encoding an amino acid sequence represented by the formula (II) or a polydeoxyribonucleic acid represented by the formula (III) For example, after separating and purifying the DNA of a microorganism that is a donor of a sarcosine oxidase gene that produces sarcosine oxidase, the ultrasound,
The DNA cleaved using a restriction enzyme or the like and the expression vector cleaved and linearized are ligated and closed with a DNA ligase or the like at the blunt or cohesive ends of both DNAs, and the thus obtained recombinant DNA vector is replicated. After transferring to a possible host microorganism, culturing the microorganism holding the recombinant DNA vector obtained by screening using the marker of the vector and the activity of sarcosine oxidase as an index,
The recombinant DNA vector may be separated and purified from the cultured cells, and then a polydeoxyribonucleic acid that is a sarcosine oxidase gene may be collected from the recombinant DNA vector.

The microorganism that is a donor of the sarcosine oxidase gene may be any microorganism that has sarcosine oxidase-producing ability, such as JP-A-54-28893, JP-A-54-52789, and JP-A-56-92790. No.
JP-A-60-43379, JP-A-61-162174 and JP-A-61-61174
No. 280271, Bacillus sp. B-0618 strain, Bacillus sp.
Bacillus sarcosine oxidase-producing bacteria such as illus sp NS-129 strain, Arthrobacter sarcosine oxidase-producing bacteria such as Arthrobacter ureafashiense IFO 12140 strain, and Cylindrocarpon deidayum (Hardig). ) Wollen webe (Cylindrocarpon didymum (Hartig) Wollenwebe
r) A sarcosine oxidase-producing bacterium of the genus Cylindrocarpon such as M-1 strain, Pseudomonas maltophylia (Ps
eudomonas maltophilia) BMTU254 strain, Pseudomonas
Pseudomonas sarcosine oxidase-producing bacteria such as Pseudomonas maltophilia strain BMTU288 and Chainia purpurogena D
SM43156 strain, Chainia ochrace
ae) DSM43155 strain, Streptomyces flocculus DSM40327 strain, Streptoverticilliun sp
DSM40237 strain, Kitasatoa purp
ures) It is appropriately selected from the group of sarcosine oxidase-producing bacteria such as Streptomyces family such as DSM43362 strain.

Preferably, Bacillus sp. B
Sarcosine oxidase-producing bacteria of the strain -0618, and in particular, microorganisms having a sarcosine oxidase-producing ability having the above-mentioned physicochemical properties.
Bacillus sp. B-0618 strain described in JP-A-54-28893. Also,
The transformed microorganism to which the sarcosine oxidase-producing ability has been imparted by making full use of gene recombination technology may be used as a sarcosine oxidase gene donor.

DNA derived from a microorganism as a gene donor is collected as follows. That is, any of the above-described microorganisms that are the donor microorganisms is, for example, cultured under aeration and stirring for about 1 to 3 days in a liquid medium, and the obtained culture is collected by centrifugation,
Then, the lysate is lysed to prepare a lysate containing the sarcosine oxidase gene.
As a lysis method, for example, treatment with a cell wall lysing enzyme such as lysozyme or β-glucanase is performed, and other enzymes such as a protease or a surfactant such as sodium lauryl sulfate are used in combination, if necessary. May be performed in combination with the lysis method described above.

In order to separate and purify DNA from the lysate obtained in this manner, a method such as protein removal by phenol extraction, protease treatment, ribonuclease treatment, alcohol precipitation, or centrifugation is appropriately combined in accordance with a conventional method. It can be carried out.

The method of cutting the separated and purified microbial DNA can be performed, for example, by sonication, restriction enzyme treatment, and the like.However, in order to facilitate the binding of the obtained DNA fragment to the vector, restriction enzymes, especially specific nucleotides, are used. Suitable are type II restriction enzymes which act on the sequence, such as, for example, EcoRI, HindIII, BamHI.

As a vector, a vector constructed for gene recombination from a phage or a plasmid capable of autonomous propagation in a host microorganism is suitable.

Examples of the phage include Escherichia coli (Es
cherichia coli) is used as the host microorganism.
λC, λgt and λB can be used.

As plasmids, for example, Escherichia
When using E. coli as the host microorganism, pBR322, pBR325, pACY
When C184, pUC12, pUC13, pUC18, pUC19 and the like are used as Bacillus subtilis as a host microorganism, pUB110 and pC194 can be used. Furthermore, a shuttle vector capable of autonomous propagation in two or more kinds of host microorganisms spanning both gram and amphoteric amphoteric such as Escherichia coli and Saccharomyces cerevisiae can be used. Preferably, such a vector is cleaved with the same restriction enzyme used for cutting the microorganism DNA that is the sarcosine oxidase gene donor described above to obtain a vector fragment.

The method of binding the microbial DNA fragment to the vector fragment may be a method using a known DNA ligase.For example, after annealing the cohesive end of the microbial DNA fragment and the cohesive end of the vector fragment, the action of an appropriate DNA ligase is performed. To prepare a recombinant DNA of the microorganism DNA fragment and the vector fragment. If necessary, after annealing, the DNA can be transferred to a host microorganism, and recombinant DNA can be prepared using in-vivo DNA ligase.

The host microorganism may be any microorganism as long as the recombinant DNA can stably and autonomously proliferate and can express the trait of the exogenous DNA.For example, when the host microorganism is Escherichia coli, Escherichia coli DH1, Escherichia coli・ Kori H
B101, Escherichia coli W3110, Escherichia coli C
600 can be used.

As a method for transferring the recombinant DNA to the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, transfer the recombinant DNA in the presence of calcium iono, and in the case of a microorganism belonging to the genus Bacillus In
A competent cell method, a method for electrically transferring a liposome recombinant DNA to a protoplast host cell, or the like may be employed, and a microinjection method may be used. It has been found that the microorganism as a transformant thus obtained can stably produce a large amount of sarcosine oxidase by being cultured in a nutrient medium.

Selection of the presence or absence of transfer of the target recombinant DNA to the host microorganism may be performed by examining a microorganism capable of simultaneously expressing the drug resistance marker of the vector, which is a recombinant DNA holding the target DNA fragment, and the sarcosine oxidase, For example, a microorganism that grows on a selective medium based on a drug resistance marker and that produces the sarcosine oxidase may be selected.

The recombinant DNA having the sarcosine oxidase gene once selected in this manner can be easily removed from a transformed microorganism and transferred to another host microorganism. Further, the sarcosine oxidase gene is cleaved with a restriction enzyme or the like from the recombinant DNA holding the sarcosine oxidase gene to cut out the sarcosine oxidase gene DNA,
It can also be easily carried out to prepare a recombinant DNA having novel characteristics by ligating it to another vector end obtained by cleavage by the same method as described above, and to transfer it to another host microorganism.

Further, the DNA of sarcosine oxidase mutein, which is essentially sarcosine oxidase activity, means an artificially mutated gene produced by a genetic engineering technique from the sarcosine oxidase gene of the present invention. Sarcosine oxidase mutein DNA obtained by using various genetic engineering methods such as replacing a specific DNA fragment of the target gene with an artificially mutated DNA fragment and thus having particularly excellent properties among the thus obtained artificially mutated genes Finally, this mutein D
A sarcosine oxidase mutein can be produced by inserting a NA into a vector to prepare a recombinant DNA and transferring it to a host microorganism.

Furthermore, the nucleotide sequence of the sarcosine oxidase gene obtained by the above-described method is Science 214 1205-1210.
(1981), and the amino acid sequence of the sarcosine oxidase was predicted and determined from the salting-out sequence. On the other hand, the partial amino acid sequence constituting the N-terminal of the sarcosine oxidase peptide was determined as follows.

That is, a sarcosine oxidase gene-donating microorganism having sarcosine oxidase-producing ability is cultured in a nutrient medium to produce and accumulate the sarcosine in the cells, and after completion of the culture, the resulting culture is filtered or centrifuged to remove the cells. , And then the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme, and sarcosine oxidase is solubilized by adding EDTA and / or a suitable surfactant, if necessary, to obtain an aqueous solution. Was isolated and collected.

The aqueous solution of the sarcosine oxidase thus obtained is concentrated, or the ammonium sulfate fraction without concentration.
Gel filtration, adsorption chromatography, and ion exchange chromatography were performed to obtain the high-purity sarcosine oxidase. Using the high-purity sarcosine oxidase, a liquid-phase protein sequencer (Beckman: BECKMAN System 890ME) was used. A partial amino acid sequence constituting the N-terminal portion of a certain peptide is determined, and at least the partial amino acid sequence is
It was confirmed that the amino acid sequence was identical to the N-terminal partial amino acid sequence of the sarcosine oxidase obtained by genetic manipulation.

The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional and physiological properties of the host.In most cases, the culture is usually performed in liquid culture or industrially by deep aeration and stirring culture. It is advantageous to do so. As the nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose,
Maltose, fructose, molasses and the like are used.

Any available nitrogen compound may be used as the nitrogen source. For example, peptone, meat extract, yeast extract, casein hydrolyzate and the like are used. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, specific amino acids, specific vitamins, and the like are used as necessary, and the usual sarcosine oxidase-producing bacteria are used. The sarcosine oxidase production inducer such as choline, sarcosine or creatine required in the production of the sarcosine oxidase is not required in the production method.

The culture temperature can be appropriately changed within a range in which the bacteria grow and produce the sarcosine oxidase. In the case of Escherichia coli, the temperature is preferably about 20 to 42 ° C. The culturing time varies somewhat depending on the conditions, but the culturing may be terminated at an appropriate time in consideration of the time when the sarcosine oxidase reaches the maximum yield, and is usually about 12 to 48 hours. Medium pH
The pH of the sarcosine oxidase can be appropriately changed within a range where the bacterium grows and the sarcosine oxidase is produced, and the pH is particularly preferably about 6 to 8.

When sarcosine oxidase in the culture is present in the culture, the culture solution containing the bacterial cells can be directly collected and used.However, in general, filtration, centrifugation, etc., are performed in the culture solution according to a conventional method. The sarcosine oxidase solution obtained after separating the sarcosine oxidase from the microbial cells is used.

If sarcosine oxidase is present in the cells, the obtained culture is collected by filtration or centrifugation to collect the cells, and then the cells are destroyed by a mechanical method or an enzymatic method such as lysozyme. If necessary, a chelating agent such as EDTA and / or a surfactant are added to solubilize the sarcosine oxidase, and the sarcosine oxidase is separated and collected as an aqueous solution.

The sarcosine oxidase-containing solution thus obtained is subjected to, for example, concentration under reduced pressure, membrane concentration, salting-out treatment with ammonium sulfate, sodium sulfate or the like, or a fractional precipitation method using a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. May be allowed to precipitate. The precipitate can then be dissolved in water and dialyzed through a semi-permeable membrane to remove lower molecular weight impurities.

Gel filtration using an adsorbent or gel filtration agent,
The sarcosine oxidase-containing solution obtained by purification by adsorption chromatography or ion-exchange chromatography and using these means can be purified to a more purified sarcosine oxidase by treatment such as freeze-drying under reduced pressure. The sarcosine oxidase thus obtained is measured by the following activity measurement method. That is, first, the next reaction solution is prepared.

0.2 M Tris-HCl buffer (pH 8.0) 0.5 ml 15 mM 4-aminoantipyrine 0.5 ml 0.2% (w / v) phenol 0.5 ml Peroxidase (50 U / ml) 0.5 ml 1.0 M sarcosine aqueous solution 1.0 ml water 2.0 ml 0.5 ml is placed in a test tube, preliminarily heated at 37 ° C. for 3 minutes, 10 μl of an enzyme solution containing sarcosine oxidase (SOX) is added, and the mixture is incubated at 37 ° C. for exactly 5 minutes. 2.5ml
Of ethanol was added to stop the reaction, and colorimetric quantification was performed at 480 nm. The activity to produce 1 μmole of hydrogen peroxide per minute was defined as 1 unit (U). Therefore, the sarcosine oxidase activity value (SOX activity value) (U / ml) is determined by the following equation.

A ₀ : Colorimetric value at 480 nm obtained by adding a buffer instead of the enzyme solution and performing the same operation as above. The content of the impurity enzyme is determined by the enzyme activity measurement method described below.

1. Catalase activity measurement method 0.5 enzyme solution diluted with 0.1 M phosphate buffer (pH 7.0)
ml in a test tube and preheated at 30 ° C for 5 minutes, then 0.4%
Add 0.5 ml of hydrogen peroxide solution and keep at 30 ° C for exactly 5 minutes. The reaction was stopped by adding 2.5 ml of 0.1 M perchloric acid solution, and 240
The colorimetric determination is performed in nm, and the value is set to B. Also, 0.5 ml of a 0.4% hydrogen peroxide solution was placed in a test tube, preliminarily heated at 30 ° C. for 5 minutes, and then 2.5 ml of a 0.1 M perchloric acid solution was added.
M phosphate buffer enzyme solution 0.5ml diluted with (pH 7.0) was added, and colorimetry at 240 nm, its value as B _0, 1 unit of activity resulting hydrogen peroxide 1μmole per minute ( If U) is determined, the catalase activity value (CL activity value) (U / ml) is obtained by the following equation.

2. Method for measuring creatinase activity The method for measuring the activity of creatinase used in the present invention is as follows. First, the following solutions are prepared.

1st liquid 0.2M phosphate buffer (pH7.5) 0.5ml 50mM creatine aqueous solution 4.5ml 2nd liquid 0.2M Tris-HCl buffer (pH8.0) 0.5ml 15mM 4-aminoantipyrine aqueous solution 0.5ml 0.2% phenol 0.5ml par Oxidase (50 U / ml) 0.5 ml Sarcosine oxidase (30 U / ml) 1.0 ml 0.5 mM p-chloromercury benzene 2.0 ml Third liquid 0.5 mM p-chloromercury benzene 0.5 ml Take the first liquid into a test tube and pre-add at 37 ° C for 3 minutes. After warming,
Add 10 μl of enzyme solution and incubate for exactly 5 minutes. 0.5 ml of the third solution was added, then 0.5 ml of the second solution was added, and the mixture was further reacted at 37 ° C. for 20 minutes. Then, 1.5 ml of distilled water was added, and colorimetric determination was performed at 500 nm. The activity to produce hydrogen was defined as one unit (U).

3. Method for measuring N-ethylglycine oxidase activity First, prepare the following reaction solution.

0.2 M Tris-HCl buffer (pH 8.0) 0.5 ml 15 mM 4-aminoantipyrine 0.5 ml 0.2% (w / v) phenol 0.5 ml Peroxidase (50 U / ml) 0.5 ml 1.0 M N-ethylglycine aqueous solution 1.0 ml water 2.0 ml 0.5 ml of the reaction solution is placed in a test tube and preliminarily heated at 37 ° C. for 3 minutes. Then, 10 μl of the enzyme solution is added and the temperature is kept at 37 ° C. for exactly 5 minutes. The reaction was stopped by adding 2.5 ml of ethanol, and 480 nm
, And the value is set to A. The activity producing 1 μmole of hydrogen peroxide per minute was defined as 1 unit (U).

Using the enzyme activity measurement method described above, the sarcosine oxidase standard of the present invention was prepared based on one unit of sarcosine oxidase with respect to catalase, creatinase, N-
Ethyl-glycine oxidase activity, catalase activity 0.05 units or less, creatinase activity 0.0004 units or less,
It is a sarcosine oxidase having an N-ethyl-glycine oxidase activity of 0.03 units or less, and has extremely excellent properties as an enzyme used in clinical diagnosis with very few contaminating enzymes.

The sarcosine oxidase thus obtained has the following physicochemical properties.

(A) Action This is an enzyme for culturing the following enzymatic reaction that produces glycine, formaldehyde and one molecule of hydrogen peroxide from sarcosine, one molecule of oxygen and one molecule of water.

Sarcosine + O ₂ + H ₂ O → glycine + formaldehyde + H ₂ O ₂ (b) Substrate specificity 0.05 ml of 0.2 M Tris-HCl buffer (pH 8.0), 0.05 ml of 0.2% phenol, 0.05 ml of 0.5 mg / ml peroxidase , 0.2 ml of distilled water and 0.5 M substrate solution using the following compounds as substrates
To 0.5 ml of a reaction solution consisting of 0.20 ml, 0.5 u of the sarcosine oxidase was added, and reacted at 37 ° C. for 5 minutes.
After stopping the reaction by adding 2.5 ml of ethanol, colorimetry was performed at 480 nm.

Substrate Relative activity (%) Sarcosine 100.0 Choline 0 Serine 0 Threonine 0 Alanine 0 Valine 0 N-Methylethanolamine 0 N-Dimethylethanolamine 0 (c) Effect on chromogenic system of 4-aminoantipyrine-phenol-peroxidase As a result of quantifying the formaldehyde produced by the acetylacetone method to avoid the influence, it is recognized that the sarcosine oxidase has a maximum pH of around 8.0 to 9.5. As the buffer used, pH: 4.7: dimethylglutarate buffer, pH: 6 to 8: phosphate buffer, pH 7.5 to
9: Tris-HCl buffer, pH 9-10: glycine-sodium hydroxide buffer, pH 10-11: Sodium carbonate-sodium borate buffer.

(D) Isoelectric point around 4.7 (isoelectric point fractionation method using carrier ampholite) (e) Molecular weight about 40,000 (measured by gel filtration method) (f) Thermostability Contains 20 μg / ml enzyme protein 0.5 ml of the resulting 10 mM Tris-HCl buffer (pH 8.0) was allowed to stand at each temperature for 10 minutes, and then the enzyme activity against sarcosine was measured.
Even at 40 ° C., it had 100% residual activity.

(G) Optimal temperature Using the above-described method for measuring enzyme activity for sarcosine, an enzyme reaction was performed by changing the temperature conditions, and the enzyme activity for sarcosine was measured. As a result, the optimum temperature was found to be around 50 ° C. .

(H) pH stability As a buffer, pH 4-7: dimethylglutarate buffer, p
H: 6-8: phosphate buffer, pH 7.5-9: Tris-HCl buffer,
pH 9 to 10: Glucine-sodium hydroxide buffer, pH 10 to 1
1: each using sodium carbonate-sodium borate buffer
In 0.1 ml of pH buffer, add enzyme solution (enzyme protein concentration 100 μg / m
l) Add 100 μl, leave at 37 ° C for 60 minutes, then add
After adding 0.3 ml of 1.0 M Tris-HCl buffer (pH 8.0) to adjust the pH, 20 μl was taken, added to the reaction solution, and the enzyme activity was measured according to the enzyme activity measurement method for sarcosine described above. , Its pH stability is found to be around 6.0 to 10.0.

The abbreviations for amino acids, peptides, nucleic acids, nucleic acid-related substances, and the like described herein are based on the commonly used abbreviations in the relevant field, and examples thereof are listed below. In addition, all amino acids show L-form.

DNA: deoxyribonucleic acid RNA: ribonucleic acid A: adenine T: thymine G: guanine C: cytosine Ala: alanine Arg: arginine Asn: asparagine Asp: aspartic acid Cys: cysteine Gln: glutamine Glu: glutamic acid Gly: glutamic acid Isoleucine Leu: Leucine Lys: Lysine Met: Methionine Phe: Phenylalanine Pro: Proline Ser: Serine Thr: Threonine Trp: Tryptophan Tyr: Tyrosine Val: Valine [Example] Hereinafter, the present invention will be described in detail with reference to Examples. The invention is not limited in any way by these.

Example 1 Isolation of Chromosomal DNA Chromosomal DNA was isolated by the following method using Bacillus sp. Strain B-0618 [Microorganism Deposit No. 0750 (FERM BP-0750)]. 150 ml of the same strain was cultured in a normal broth medium (containing 0.5% sodium thiosulfate) at 37 ° C. overnight after shaking, and then collected by centrifugation (3000 rpm for 10 minutes). Ten
% Sucrose, 50mM Tris-HCl (pH8.0), 50mM EDTA
Is suspended in 5 ml of a solution containing lysozyme solution (10 ml).
mg / ml) and incubated at 37 ° C for 15 minutes, then 1 ml of 10% SD
An S (sodium dodecyl sulfate) solution was added. An equal volume of a mixed solution of chloroform and phenol (1: 1) was added to the suspension, mixed with stirring, centrifuged at 10,000 rpm for 3 minutes to separate an aqueous layer and a solvent layer, and the aqueous layer was separated.

Twice the amount of ethanol was gently overlaid on this aqueous layer, and the DNA was sprinkled on the glass rod while slowly stirring with a glass rod to separate the DNA. This was added to 10 ml of 10 mM Tris-HCl (pH 8.
0), and dissolved in a solution containing 1 mM EDTA (hereinafter abbreviated as TE). This was mixed with an equal volume of a chloroform-phenol mixture (1:
1) was added and the same treatment as above was carried out. The aqueous layer was separated, twice the amount of ethanol was added, the DNA was separated again by the above method, and dissolved in 2 ml of TE.

Example 2 Isolation of pACYC184 Plasmid DNA Escherichia coli pM191 [J. Bac
teriol., 134, 1141 (1981); ATCC37033] was shake-cultured in 11 BHI media (manufactured by Difco). When the turbidity grew to OD ₆₆₀ = 1.0, spectinomycin (final concentration 300 μg / m
l) was added and shaking was continued at 37 ° C. for more than 16 hours. 300
The cells were collected by centrifugation at 0 rpm for 10 minutes, and the lysozyme-SDS method and the cesium chloride-ethidium bromide method (Maniat
s et al., Molecular, Cloning, 86-94 Cold Spring Harbor (19
82)] to prepare a plasmid DNA.

Example 3 Construction of Plasmid pOXI101 Having Sarcosine Oxidase (SOX) Gene (i) Bacillus Espi-B-0618 Prepared in Example 1
2 μl (about 0.5 μg) of chromosomal DNA of the strain and 10 times concentration of EcoRI
Cutting buffer (500 mM Tris-HCl (pH 7.5), 70 mM M
gCl ₂ , 1 M NaCl, 70 mM mercaptoethanol) 1 μl, E
1 μl of coRI (Takara Shuzo 10 unit / μl) and 6 μl of water were mixed and cut at 37 ° C. for 1 hour. Plasmid pACY prepared separately
Approximately 0.3 μg of C184 DNA was digested with EcoRI using the same method, and 0.6 unit of alkaline phosphatase (hereinafter sometimes abbreviated as BAP, Takara Shuzo) was added, followed by treatment at 65 ° C. for 1 hour.

Mix these two DNA solutions cut with EcoRI,
One-tenth the volume of 3M sodium acetate was added, and the mixture was treated with a chloroform-phenol mixed solution in the same amount as the whole amount, and the aqueous layer was separated by centrifugation. Two volumes of ethanol was added to the aqueous layer, DNA was precipitated by centrifugation, and dried under reduced pressure.
After dissolving the obtained DNA in 89 μl of water, ligation buffer [0.5 M Tris-HCl buffer (pH 7.6), 0.1
M MgCl ₂ , 0.1 M dithiothreitol, 10 mM spermidine, 10 mM ATP] 10 μl and T4 DNA ligase 1 μl (Takara Shuzo)
175 units / μl), mixed, and allowed to stand at 4 ° C. overnight. After the DNA solution was treated with chloroform-phenol and precipitated with ethanol, the resulting precipitate was collected by centrifugation, dried under reduced pressure, and dissolved in 10 µl TE.

(Ii) 100 ml of BHI medium (Brein Heart Infusion medium, Dif
E. coli DH in logarithmic growth phase
One strain (stock number ME8569, distributed by the National Institute of Genetics) is collected by centrifugation (10000 rpm, 2 minutes)
40 ml of ice-cold 30 mM potassium acetate, 100 mM RbCl, 10 mM Ca
A solution containing Cl ₂ , 50 mM MnCl ₂ and 15% glycerin (p
H5.8). After standing at 0 ° C. 5 min, discarded by centrifugation supernatant (manufactured by Dotite Co.) Further 10 mM MOPS buffer 4 ml, in 75 mM CaCl _2, 10 mM RbCl and 15% containing glycerol solution (pH 6.5) The cells were suspended and left at 0 ° C. for 15 minutes to obtain competent cells.

(Iii) DN prepared in (i) was added to 200 μl of this E. coli suspension.
A solution (10 μl) was added and left at 0 ° C. for 30 minutes. BHI medium 1m
After adding l, and keeping the mixture at 37 ° C for 90 minutes, 100 µl of the mixture was spread on a BHI agar plate containing tetracycline (15 µg / ml), and cultured at 37 ° C overnight to obtain a transformant. This transformant was added to a detection medium (composition: peptone 5 g, meat extract 2 g, yeast extract 5 g, NaCl 1 g, K ₂ HPO ₄ 1 g, MgSO ₄ 0.5 g, peroxidase 500 IU, dianidin 0.1 g, sarcosine 9 g, agar 15 g, distilled water
(1 l, pH 7.0) and replicated overnight at 37 ° C.

When the transformants of about 6000 colonies were examined, four strains having brownish brown around the colonies were obtained.
Strain Escherichia coli DH1 pOXI10
It was named 1 strain. After pure isolation, the strain was cultured overnight in a BHI medium at 37 ° C., and the plasmid was separated in the same manner as in Example 2 to obtain a plasmid containing the sarcosine oxidase gene and the pACYC184 gene, which was named pOXI101.

Example 4 Mapping and Subcloning of pOXI101 Restriction enzymes Cla I, Hpa I, Sal
Cut maps were prepared using I, Sma I, and Xho I (all manufactured by Takara Shuzo). The results are shown in FIG. Example 3
In the same manner as in (i), each fragment of an EcoRI-ClaI5.3kb fragment containing two XhoI cleavage sites obtained from pOXI101 and an EcoRI-CioI4.3kb fragment obtained from pBR322 was obtained, and these fragments were obtained. And subcloning operations such as conjugation, transformation of Escherichia coli DH1 and screening after transformation were attempted by the same method as in Example 3, and one clone having sarcosine oxidase productivity was obtained. The strain was named Escherichia coli DH1 pOXI103 strain [Microcosms Deposit No. 9494 (FERM P-949).
Four)〕.

This strain has been transferred to a deposit based on the Budapest Treaty and deposited as a microfabricated article No. 1828 (FERM BP-1828). Plasmid DNA was isolated from this strain as in Example 2 and named pOXI103. When a cut map was created for the DNA in pOXI103 using restriction enzymes Cla I, Hpa I, Sal I, Sma I, and Xhol I (all manufactured by Takara Shuzo), Sm
It was found that there was a clone lacking from EcoR I to the vicinity of Xho I containing the aI cleavage site, and is shown in FIG.

This Escherichia coli DH1 pOXI103 strain was
After culturing overnight at ℃ C, the sarcosine oxidase productivity was about 2 u / ml sarcosine oxidase activity according to the sarcosine oxidase activity measurement method described above. The nucleotide sequence of the DNA containing the sarcosine oxidase gene was determined by the dideoxy method using phage M13 [Science, 2
14, 1206-1210, (1981)]. The nucleotide sequence and amino acid sequence of the sarcosine oxidase gene are shown in FIG. 3 (shown in FIGS. 3-1 and 3-2).

Example 5 Method for producing sarcosine oxidase Escherichia coli DH1 pOXI103 strain was cultured in 20 l of BHI medium for 3 hours.
The cells were cultured by aeration and agitation using a 30 l Jaffer meter at 7 ° C. for 18 hours, and the cells were collected by centrifugation at 5000 rpm for 10 minutes. At this time, the productivity of sarcosine oxidase was 4.6 u / ml. After washing with physiological saline 21, 21 of 10 mM phosphate buffer (pH 7.
5) Suspended. Lysozyme chloride 0.1%, EDTA-2Na 2m
M, and stir at 37 ° C for 60 minutes.
A supernatant liquid 1.81 was obtained by centrifugation at 00 rpm for 10 minutes.

To this solution was added 1.8 l of saturated ammonium sulfate, and the resulting precipitate was 12000 rpm,
Collected by centrifugation for 10 minutes and dissolved in 300 ml of 10 mM phosphate buffer (pH 7.5). 10 mM phosphate buffer (pH 7.
After desalting with Sephadex G-25 (trade name) equilibrated in 5), ion exchange chromatography using DEAE-Sepharose CL-6B was performed to collect an active fraction. After desalination,
By freeze-drying, 0.807 g of a powder sample was obtained. At this time, the recovery yield was 36%, and the specific activity was 41 U / mg. When the molecular weight of the obtained sarcosine oxidase was measured by a gel filtration method, it was found to be about 40,000, which was almost the same as the molecular weight calculated from the amino acid sequence.

〔The invention's effect〕

According to the present invention, the sarcosine oxidase gene and the amino acid sequence of sarcosine oxidase have been elucidated, and a method for efficiently producing sarcosine oxidase by genetic engineering has been provided. The present invention also provides a more efficient method for producing sarcosine oxidase by using the sarcosine oxidase gene of the present invention and various genetic engineering techniques.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the structure of the pOXI101 vector, FIG. 2 is a schematic diagram showing the structure of the pOXI103 vector, and FIG.
-1 and 3-2) show the coding strand of sarcosine oxidase gene DNA (5 '→ 3') and the respective sequences of the resulting translation products.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical indication (C12N 1/21 C12R 1:19) (C12N 9/06 C12R 1:19)

Claims (7)

(57) [Claims] (1) a polydeoxyribonucleic acid having the formula (Wherein A represents an amino acid residue or a hydrogen atom, and B represents an amino acid residue or -OH), which comprises a base sequence encoding an amino acid sequence of a polypeptide as a component of sarcosine oxidase. A polydeoxyribonucleic acid, characterized in that: 2. The polydeoxyribonucleic acid which is exogenous to the host is 2. The polydeoxyribonucleic acid according to claim 1, wherein the polydeoxyribonucleic acid is a base sequence represented by the formula: wherein X represents a codon or hydrogen atom other than TAA, TAG and TGA, and Y represents a codon or hydrogen atom. 3. The compound is exogenous to a host microorganism and has the following formula (Wherein, A represents an amino acid residue or a hydrogen atom, and B represents an amino acid residue or -OH), a polydeoxyribonucleotide containing a base sequence encoding the amino acid sequence of a polypeptide as a component of sarcosine oxidase represented by A transformant characterized by holding a nucleic acid. 4. The method according to claim 1, wherein the host microorganism is of the formula 4. A polydeoxyribonucleic acid having a base sequence represented by the formula: wherein X represents a codon or hydrogen atom other than TAA, TAG and TGA, and Y represents a codon or hydrogen atom. Transformants. 5. The transformant according to claim 3, wherein the host microorganism belongs to the genus Escherichia. 6. The transformant according to claim 4, wherein the host microorganism belonging to the genus Escherichia is a microorganism belonging to Escherichia coli. The transformant may be Escherichia coli (Esch).
The transformant according to claim 6, which is E. coli DH1 pOXI101 strain (FERM BP-1828).