JP2009082132A - Stabilized sarcosine oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sarcosine oxidase improved in stability in its liquid state, obtained by modifying a protein having sarcosine oxidase activity by a protein engineering means, to provide a method for producing the same and uses of the same. <P>SOLUTION: This modified type sarcosine oxidase is obtained by substituting an amino acid at a specific position of the sarcosine oxidase originated from Arthrobacter, etc., and having the specific amino acid sequence. The creatine and creatinine-measuring reagent containing the above enzyme are also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a sarcosine oxidase with improved liquid stability obtained by modifying a protein having sarcosine oxidase activity by a protein engineering technique, a method for producing the same, and use thereof.

Sarcosine oxidase (EC 1.5.3.1) is an enzyme for measuring creatine and creatinine in body fluids, which is clinically an indicator for diagnosis of muscle and kidney diseases, and other enzymes such as creatininase. , Creatinase and peroxidase. Sarcosine oxidase acts on the substrate sarcosine in the presence of water and oxygen to produce glycine, formaldehyde and hydrogen peroxide.

Such sarcosine oxidase is known to be produced by bacteria such as Bacillus genus, Corynebacterium genus, Cylindrocarpon genus, Pseudomonas genus, Arthrobacter genus (for example, Patent Documents 1 to 5 and (Refer nonpatent literature 1.). In addition, a technique for mass-producing sarcosine oxidase genes of these producing bacteria using a host such as Escherichia coli by genetic engineering techniques has also been reported (see, for example, Patent Documents 6 to 8).
JP-A-54-52789 JP 61-162174 A JP 56-92790 A JP-A-60-43379 JP-A-2-265478 Japanese Patent Laid-Open No. 5-115281 JP-A-6-113840 JP-A-8-238087 “J. Biochem.”, 1981, 89, p. 599

With the recent liquefaction of clinical diagnostic reagents, various methods for stabilizing reagent components in liquid form have been studied. However, sarcosine oxidase used in creatinine and creatine measurement reagents also has excellent liquid stability. Is desired. Our group previously modified a wild-type sarcosine oxidase by protein engineering and reported a mutant sarcosine oxidase with improved stability against metal ions (see, for example, Patent Document 9). Further improvements in long-term storage stability in pharmaceutical reagents are expected.
An object of the present invention is to provide a modified sarcosine oxidase with improved liquid stability.
JP-A-7-163341

As a result of intensive studies to achieve the above object, the present inventors have found that a modified sarcosine oxidase with improved stability in liquid form can be produced without impairing the action on sarcosine, and the present invention has been completed. I came to let you.

That is, the present invention has the following configuration.
[Item 1] The 89th lysine of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is arginine, the 204th methionine is alanine, the 213th serine is proline, the 233rd cysteine is serine, 240 A modified sarcosine oxidase, wherein the asparagine of the 2nd is substituted with tyrosine and the glutamic acid of the 250th is substituted with glutamine.
[Item 2] A gene encoding the modified sarcosine oxidase according to Item 1.
[Claim 3] A vector comprising the gene according to item 2.
[Item 4] A transformant transformed with the vector according to item 3.
[Item 5] A method for producing a modified sarcosine oxidase, comprising culturing the transformant according to item 4 and collecting sarcosine oxidase from the culture.
[Item 6] A reagent for measuring creatine containing the sarcosine oxidase according to item 1.
[Item 7] A reagent for measuring creatinine containing the sarcosine oxidase according to item 1.

According to the present invention, it has become possible to supply a modified sarcosine oxidase with improved liquid stability by modifying a protein having sarcosine oxidase activity by a protein engineering technique. By using the modified sarcosine oxidase of the present invention as an enzyme for measuring creatine and creatinine in a body fluid that is clinically an indicator for diagnosis of muscle disease and kidney disease, the liquid stability of the reagent is improved. Can be made.

The modified sarcosine oxidase of the present invention is useful for the analysis of creatine and creatinine in the clinical laboratory field.

One embodiment of the present invention is a protein obtained by converting at least one amino acid of an amino acid sequence constituting a protein having sarcosine oxidase activity by addition, deletion insertion or substitution, and having sarcosine oxidase activity, It is a modified sarcosine oxidase characterized in that its stability in liquid form is improved as compared with that before conversion.

The modified sarcosine oxidase of the present invention is characterized in that the stability in liquid form is improved as compared with that before modification. The liquid stability in the present invention means, for example, the ratio of the remaining enzyme activity after the modified enzyme is dissolved in an appropriate buffer and stored at an appropriate temperature for a certain period.
The “appropriate buffer” is not particularly limited as long as the type and concentration thereof are selected so as to have sufficient buffering capacity around pH 7 to 8, which is the optimum pH of sarcosine oxidase, but preferably 50 mM potassium phosphate buffer. Solution (pH 7.5) or 50 mM PIPES-NaOH buffer (pH 7.5) is selected. The buffer may further contain a surfactant, salts, chelating agents, preservatives and the like as necessary.
The conditions for “storing at a suitable temperature for a certain period of time” are not particularly limited, but preferably, accelerated (severe) test conditions are selected in consideration of long-term storage stability in a liquid diagnostic reagent. Specifically, “storage at 40 ° C. for 3 days”, “storage at 60 ° C. for 30 minutes” or the like can be mentioned. If time permits, storage for 6 months or more may be selected under refrigerated conditions of 2 ° C. to 10 ° C., which is generally used as a temperature at which the liquid diagnostic agent is actually stored for a long time.
The concentration of sarcosine oxidase in storage is not particularly limited, but 1 to 30 U / ml assuming a concentration used for a normal diagnostic reagent is preferably selected. More preferably, it is 5-20 U / ml.
“Stability is improved compared to before modification” means that the activity retention after storage for a certain period is higher than the activity retention of the enzyme before modification measured under the same conditions.

As one embodiment of the present invention, a modified sarcosine oxidase in which the residual enzyme activity rate after storage at 60 ° C. for 30 minutes in 50 mM potassium phosphate buffer (pH 7.5) is improved as compared with that before modification. It is. In another embodiment, 50 mM PIPES- containing 2 mM EDTA, 50 mM NaCl, 0.1% (W / V) 2-methylisothiazolone, 0.1% (W / V) Triton X-100. It is a modified sarcosine oxidase in which the residual enzyme activity rate after storage for 3 days in NaOH buffer (pH 7.5) at 40 ° C. is improved as compared with that before modification.

The sarcosine oxidase used for the modification of the present invention is not particularly limited, and for example, sarcosine oxidase derived from the genus Bacillus, Pseudomonas, Corynebacterium and the like can be used.

In the present invention, as an example, sarcosine oxidase of Arthrobacter sp. TE1826 (Microtechnical Laboratories No. 10737) was used (see, for example, Patent Document 10). The group of the present inventors has already succeeded in isolating the sarcosine oxidase gene from the chromosomal DNA extracted from Arthrobacter sp. TE1826, and determined the entire structure of the DNA (see, for example, Non-Patent Document 2). ) And succeeded in producing a high yield of the present sarcosine oxidase in a transformant by a genetic engineering technique, and it is possible to supply a large amount of high-purity sarcosine oxidase at low cost (see, for example, Patent Document 11). ). The amino acid sequence of sarcosine oxidase of Arthrobacter sp. TE1826 is shown in SEQ ID NO: 1 in the sequence listing. The DNA sequence encoding these amino sequences is shown in SEQ ID NO: 2 in the sequence listing.
However, the present invention is not limited to the modified sarcosine oxidase having the amino acid sequence described in SEQ ID NO: 1, but may be modified other proteins having sarcosine oxidase activity. As a suitable example of another protein having sarcosine oxidase activity, sarcosine oxidase having a three-dimensional structure similar to that of sarcosine oxidase having the amino acid sequence shown in SEQ ID NO: 1, specifically, the homology of the amino acid sequence is 50 %, More preferably, other sarcosine oxidase activity proteins having amino acid sequence homology of 80% or more. In the case of enzyme proteins having 50% to 80% homology in the amino acid sequence and exhibiting the same catalytic activity, the three-dimensional structure is usually highly similar, and amino acid residues involved in substrate specificity This is based on the fact that the reaction mechanism is often the same.
Further, the modified sarcosine oxidase of the present invention is one in which one or several amino acids are further deleted, substituted or added as long as the stability that is the essence of the enzyme characteristics of the present invention is not impaired. Also good. Specific examples include those in which a histidine tag is added to the N-terminal side or C-terminal side of the amino acid sequence in order to simplify the purification of sarcosine oxidase (see, for example, Non-Patent Document 3).
The homology of amino acid sequences in the present invention can be determined using known gene analysis software. Here, the homology refers to the percentage of amino acid residues that match within a range having similarity to the amino acid sequence to be compared.

JP-A-2-265478 JP-A-6-113840 “Journal of Fermentation and Bioengineering”, 1993, Vol. 75, No. 4, p. 239-244 "Experimental Medicine", 2002, 20, p479-482

Another embodiment of the present invention is characterized in that at least one amino acid of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with another amino acid, and the stability in liquid state is before conversion. It is a modified sarcosine oxidase which is improved compared to

Another embodiment of the present invention is arranged at positions 155 to 250 of the amino acid sequence of Arthrobacter sp. TE1826 described in SEQ ID NO: 1 in the sequence listing, or sarcosine oxidase other than Arthrobacter sp. TE1826. Stability in liquid form before conversion, characterized in that at least one amino acid at a position corresponding to positions 155 to 250 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence table is substituted with another amino acid It is a modified sarcosine oxidase that is improved compared to the above.

Another embodiment of the present invention is arranged at positions 82 to 92 of the amino acid sequence of Arthrobacter sp. TE1826 described in SEQ ID NO: 1 in the sequence listing, or sarcosine oxidase other than Arthrobacter sp. TE1826. Stability in liquid form before conversion, characterized in that at least one amino acid at a position corresponding to positions 82 to 92 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence table is substituted with another amino acid It is a modified sarcosine oxidase that is improved compared to the above.
There is a report of sarcosine oxidase whose steric structure has been clarified by X-ray crystallography (for example, see Non-Patent Document 4). According to the report, the sarcosine oxidase is homologous to the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing, and is the amino acid sequence of Sarcosine oxidase of Arthrobacter sp. TE1826. The site | part corresponding to the 82nd-92th position of the amino acid sequence described in 1, or the 82nd-92th positions of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing in sarcosine oxidase other than Arthrobacter sp. TE1826 Is considered to constitute a linking site between the catalytic domain of sarcosine oxidase and the FAD binding domain.

In another embodiment of the present invention, positions 354 to 366, which are presumed to constitute an α helix containing the position 364 of the amino acid sequence of Arthrobacter sp. In sarcosine oxidase other than Bacter sp. TE1826, at least one amino acid between sites corresponding to positions 354 to 366 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing is substituted with another amino acid. It is a modified sarcosine oxidase whose characteristic stability in liquid form is improved compared to that before conversion.
“Structure”, 1999, Vol. 7, No. 3, p. 331-345

Preferably, position 89, position 155, position 166, position 204, position 213, position 233, position 240, position 250, position 364 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, or other sarcosine oxidase A modified sarcosine oxidase in which at least one amino acid selected from the group consisting of corresponding sites is substituted with another amino acid.

More preferably, it is a modified sarcosine oxidase in which at least one amino acid selected from the following group is substituted with another amino acid. In the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, lysine at position 89 is substituted with arginine, cysteine at position 155 is substituted with isoleucine, asparagine at position 166 is substituted with lysine, methionine at position 204 is substituted with alanine, 213 Serine at position is replaced with proline, cysteine at position 233 is replaced with serine, asparagine at position 240 is replaced with tyrosine, glutamic acid at position 250 is replaced with glutamine, and alanine at position 364 is replaced with valine.

Another embodiment of the present invention includes a gene encoding the above modified sarcosine oxidase, a vector containing the gene, a transformant transformed with the vector, and further culturing the transformant, A method for producing a modified sarcosine oxidase, which comprises collecting sarcosine oxidase from a culture.

The method for producing the modified sarcosine oxidase of the present invention is not particularly limited, but it can be produced by the following procedure. As a method for modifying the amino acid sequence constituting the protein having sarcosine oxidase activity, a commonly used technique for modifying genetic information is used. That is, a DNA having genetic information of a modified protein is created by converting a specific base of DNA having genetic information of the protein, or by inserting or deleting a specific base. Specific methods for converting bases in DNA include, for example, commercially available kits (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, QuickChange SiteDirectedMetStainedMistageStained Kit, etc.) Use of the chain reaction method (PCR) is mentioned.

The prepared DNA having genetic information of the modified protein is transferred into a host microorganism in a state of being linked to a plasmid, and becomes a transformant that produces the modified protein. As the plasmid at this time, for example, pBluescript, pUC18, etc. can be used when Escherichia coli is used as a host microorganism. Examples of host microorganisms that can be used include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. An electroporation method may be used. Furthermore, a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

Microorganisms which are transformants thus obtained can stably produce a large amount of modified protein by being cultured in a nutrient medium. 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. Usually, the culture is performed in liquid culture, but industrially, aeration and agitation culture is performed. Is advantageous. As a nutrient source of the medium, those commonly used for culturing microorganisms can be widely used. Any carbon compound that can be assimilated may be used as the carbon source. For example, glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid and the like are used. The nitrogen source may be any nitrogen compound that can be used. For example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkaline extract, 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. The culture temperature can be appropriately changed within the range in which the fungus grows and produces the modified protein. In the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culturing time varies somewhat depending on conditions, the culturing may be terminated at an appropriate time in consideration of the time when the modified protein reaches the maximum yield, and is usually about 6 to 48 hours. The medium pH can be appropriately changed within the range in which the bacteria grow and produce the modified protein, but is particularly preferably about pH 6.0 to 9.0.

Although it is possible to collect and use the culture solution containing the bacterial cells producing the modified protein in the culture as it is, in general, when the modified protein is present in the culture solution according to a conventional method, filtration, centrifugation, etc. It is used after separating the modified protein-containing solution and the microbial cells. When the modified protein is present in the microbial cells, the microbial cells are collected from the obtained culture by means of filtration or centrifugation, and then the microbial 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 is added to solubilize the modified protein, and it is separated and collected as an aqueous solution.

The modified protein-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting-out treatment such as ammonium sulfate or sodium sulfate, or fractional precipitation using a hydrophilic organic solvent such as methanol, ethanol, acetone or the like. It may be allowed to settle. Heat treatment and isoelectric point treatment are also effective purification means. A purified modified protein can be obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

Another embodiment of the present invention is a creatine measurement reagent containing the modified sarcosine oxidase and a creatinine measurement reagent. The reagent for measuring creatine and the reagent for measuring creatinine of the present invention can extend the effective period of the reagent or improve the measurement accuracy by using a modified sarcosine oxidase with improved liquid stability.

The creatine measurement reagent of the present invention includes the modified sarcosine oxidase, creatine amidinohydrolase, peroxidase, and hydrogen peroxide detection reagent having improved liquid stability. The creatinine measurement reagent includes the modified sarcosine oxidase, creatinine amide hydrolase, creatine amidinohydrolase, peroxidase, and hydrogen peroxide detection reagent with improved liquid stability. The hydrogen peroxide detection reagent is a reagent for measuring hydrogen peroxide generated by the modified sarcosine oxidase as a generated dye in the presence of peroxidase, and includes an oxidative coloring reagent and 4-aminoantipyrine or 3 as required. -Couplers such as methyl-2-benzothiazolinone. Various commercially available reagents can be used as the hydrogen peroxide measuring reagent of the present invention, but the reagent is not particularly limited. Furthermore, the above creatine or creatinine measuring reagent can also use a metal salt, protein, amino acid, saccharide, organic acid or the like as a stabilizer. Usually, a preservative and a surfactant are added to the extent that they do not adversely affect the reagent performance, and they are used together with an appropriate buffer. One or a plurality of types, concentrations, and pH of the buffer solution are selected depending on purposes such as storage of each reagent component and enzyme reaction, but the pH at the time of enzyme reaction is 5. It is preferably used in the range of 0 to 10.0.

In the present invention, sarcosine oxidase activity is measured under the following conditions.
<Reagent>
100 mM Tris-HCl buffer (pH 8.0) (containing 200 mM sarcosine and 0.1% Triton X-100)
0.1% 4-aminoantipyrine 0.1% phenol 25 U / ml peroxidase <Measurement conditions>
The above-mentioned Tris-HCl buffer, 4-aminoantipyrine solution, phenol solution, and peroxidase solution are mixed at a ratio of 5: 1: 2: 2 to prepare a reaction mixture. Take 1 ml of the reaction mixture in a test tube, preheat at 37 ° C. for about 5 minutes, and then add 0.05 ml of the enzyme solution to start the reaction. After making it react exactly at 37 degreeC for 10 minutes, 2.0 ml of 0.25% SDS aqueous solution is added, reaction is stopped, and the light absorbency of 500 nm of this liquid is measured. In the blind test, distilled water is added to the reagent mixture instead of the enzyme solution, and the absorbance is measured in the same manner. The amount of enzyme that produces 1 micromole hydrogen peroxide per minute under the above conditions is defined as 1 unit. Moreover, the reactivity with respect to proline was measured as a relative ratio of activity when sarcosine in the reagent was replaced with L-proline at the same concentration.

Hereinafter, the present invention will be specifically described by way of examples.
However, the present invention is not limited to these. For example, among the 13 types of modified sarcosine oxidase shown in Example 3 described later, SAOM1 is a mutant in which the lysine at position 89 in the amino acid sequence described in the sequence listing / SEQ ID NO: 1 is substituted with arginine However, one or several amino acids may be further deleted, substituted or added as long as the performance of the mutant is not substantially affected. The same applies to mutants other than SAOM1.
Example 1 Construction of an expression plasmid for sarcosine oxidase The sarcosine oxidase expression plasmid pSAOEP3 derived from Arthrobacter sp. TE1826 was constructed according to the method described in JP-A-7-163341. This expression plasmid contains an inserted DNA fragment of about 1.7 Kbp containing a gene encoding sarcosine oxidase of TE1826 at the multicloning site of PUC18. The base sequence is shown in SEQ ID NO: 2 in the sequence listing, and the sarcosine oxidase amino acid sequence deduced from the base sequence is shown in SEQ ID NO: 1 in the sequence listing.

Example 2 Production of Modified Sarcosine Oxidase Gene Using the expression plasmid pSAOEP3 containing the sarcosine oxidase gene, the synthetic oligonucleotide described in SEQ ID NO: 3 in the sequence listing and a synthetic oligonucleotide complementary thereto, QuickChangeTM Site-Directed Mutagenesis Using Kit (manufactured by STRATAGENE), a mutation treatment operation was performed according to the protocol, and the nucleotide sequence was further determined. An encoding recombinant plasmid (pSAOM1) was obtained.
By using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 4 of the Sequence Listing and a synthetic oligonucleotide complementary thereto, by the same operation as described above, SEQ ID NO: 1 A recombinant plasmid (pSAOM2) encoding a mutant sarcosine oxidase in which the 155th cysteine of the amino acid sequence described was replaced with isoleucine was obtained.
Using pSAOEP3, a synthetic oligonucleotide shown in SEQ ID NO: 5 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 166th asparagine of the amino acid sequence shown in SEQ ID NO: 1 was replaced with lysine by the same operation as above. A recombinant plasmid (pSAOM3) encoding the mutated sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide shown in SEQ ID NO: 6 in the sequence listing and a synthetic oligonucleotide complementary thereto, the methionine at position 204 in the amino acid sequence shown in SEQ ID NO: 1 was substituted with alanine by the same operation as above. A recombinant plasmid (pSAOM4) encoding the obtained mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 7 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 213rd serine of the amino acid sequence described in SEQ ID NO: 1 is replaced with proline by the same operation as above. A recombinant plasmid (pSAOM5) encoding the obtained mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 8 in the sequence listing and a synthetic oligonucleotide complementary thereto, the cysteine at position 233 in the amino acid sequence described in SEQ ID NO: 1 is replaced with serine by the same operation as described above. A recombinant plasmid (pSAOM6) encoding the obtained mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide shown in SEQ ID NO: 9 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 240th asparagine of the amino acid sequence shown in SEQ ID NO: 1 was replaced with tyrosine by the same operation as above. A recombinant plasmid (pSAOM7) encoding the obtained mutant sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide shown in SEQ ID NO: 10 in the sequence listing and a synthetic oligonucleotide complementary thereto, the glutamic acid at position 250 in the amino acid sequence shown in SEQ ID NO: 1 was replaced with glutamine by the same operation as above. A recombinant plasmid (pSAOM8) encoding the mutated sarcosine oxidase was obtained.
Using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 11 of the Sequence Listing and a synthetic oligonucleotide complementary thereto, the 364th alanine in the amino acid sequence described in SEQ ID NO: 1 is replaced with valine by the same operation as described above. A recombinant plasmid (pSAOM9) encoding the obtained mutant sarcosine oxidase was obtained.
Using pSAOM1, a synthetic oligonucleotide described in SEQ ID NO: 7 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 89th lysine of the amino acid sequence described in SEQ ID NO: 1 is converted into arginine and 213 by the same operation as described above. A recombinant plasmid (pSAOM10) encoding a mutant sarcosine oxidase in which the second serine was replaced with proline was obtained.
By using pSAOM10, a synthetic oligonucleotide described in SEQ ID NO: 10 of the Sequence Listing and a synthetic oligonucleotide complementary thereto, and the same operation as described above, the 89th lysine of the amino acid sequence described in SEQ ID NO: 1 is arginine, 213 A recombinant plasmid (pSAOM11) encoding a mutant sarcosine oxidase in which the first serine was replaced with proline and the 250th glutamic acid was replaced with glutamine was obtained.
Using pSAOM11, each of the synthetic oligonucleotides described in SEQ ID NOs: 4, 5, and 11 in the sequence listing and each of the synthetic oligonucleotides complementary thereto, the same operation as described above was repeated, and the amino acid sequence described in SEQ ID NO: 1 89th lysine is arginine, 155th cysteine is isoleucine, 166th asparagine is lysine, 213th serine is proline, 250th glutamic acid is glutamine, 364th alanine is substituted with valine. A recombinant plasmid (pSAOM12) encoding was obtained.
Using pSAOM11, each synthetic oligonucleotide described in SEQ ID NOs: 6, 8, and 9 in the sequence listing and each synthetic oligonucleotide complementary thereto, the same operation as described above was repeated, and the amino acid sequence described in SEQ ID NO: 1 89th lysine is arginine, 204th methionine is alanine, 213th serine is proline, 233rd cysteine is serine, 240th asparagine is tyrosine, 250th glutamic acid is replaced with glutamine A recombinant plasmid (pSAOM13) encoding was obtained.

Example 3 Preparation of modified sarcosine oxidase Then, each of the transformants was obtained.
400 ml of terrific broth was dispensed into a 2 liter Sakaguchi flask, autoclaved at 121 ° C. for 20 minutes, allowed to cool, and then sterile filtered ampicillin was added to 100 μl / ml. This medium was inoculated with 5 ml of a culture solution of Escherichia coli JM109 (pSAOM1) previously cultured at 30 ° C. for 16 hours in an LB medium containing 100 μl / ml ampicillin, and cultured with aeration and stirring at 30 ° C. for 20 hours. The sarcosine oxidase activity at the end of the culture was about 9.5 U / ml per 1 ml of the culture solution in the activity measurement.
The cells were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.5), crushed by sonication, and further centrifuged to obtain a supernatant as a crude enzyme solution. . The obtained crude enzyme solution was subjected to denucleic acid removal with polyethyleneimine and ammonium sulfate fractionation, dialyzed with 20 mM phosphate buffer (pH 7.5), DEAE Sepharose CL-6B (Amersham Biosciences), and further for 1 hour. Separation and purification by heat treatment gave a purified enzyme preparation. The sample obtained by this method showed an almost single band by SDS-PAGE. This mutant was named SAOM1.
pSAOM2, pSAOM3, pSAOM4, pSAOM5, pSAOM6, pSAOM7, pSAOM8, pSAOM9, pSAOM10, pSAOM11, pSAOM12, and pSAOM13 were obtained in the same manner as in the above-mentioned method. The obtained enzyme preparations were named SAOM2, SAOM3, SAOM4, SAOM5, SAOM6, SAOM7, SAOM8, SAOM9, SAOM10, SAOM11, SAOM12, and SAOM13, respectively.

Comparative Example 1 Preparation of wild type sarcosine oxidase As a comparative example, Escherichia coli JM109 transformant with pSAOEP3 was obtained in the same manner as above to obtain a purified enzyme preparation before modification.

Example 4 Evaluation 1 of Modified Sarcosine Oxidase
The mutant sarcosine oxidase (SAOM1, SAOM2, SAOM3, SAOM4, SAOM5, SAOM7, SAOM8, SAOM10) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 were each 50 mM potassium phosphate buffer (pH 7). 5) was added to 5 U / ml, and the residual enzyme activity rate (%) after storage at 60 ° C. for 30 minutes was measured. The results are shown in Table 1. As can be seen from Table 1, it was confirmed that the modified sarcosine oxidase of the present invention has improved liquid stability compared to before modification.

Example 4 Evaluation 2 of Modified Sarcosine Oxidase
Mutant sarcosine oxidase (SAOM1, SAOM2, SAOM5, SAOM6, SAOM7, SAOM8, SAOM9) obtained in Example 3 and the various sarcosine oxidases obtained in Comparative Example 1 were respectively 2 mM ethylenediaminetetraacetate disodium dihydrogen tetraacetate and 50 mM NaCl. In PIPES-NaOH buffer (pH 7.5) containing 0.1% (W / V) 2-methylisothiazolone (Roche Diagnostics), 0.1% (W / V) Triton X-100 The residual enzyme activity rate (%) after storage at 40 ° C. for 3 days was measured. The results are shown in Table 2. As can be seen from Table 2, it was confirmed that the modified sarcosine oxidase of the present invention has improved liquid stability compared to before modification.

Example 5 Evaluation 3 of Modified Sarcosine Oxidase
The stability of the mutant sarcosine oxidase (SAOM1, SAOM10, SAOM11, SAOM12, SAOM13) obtained in Example 3 and various sarcosine oxidases obtained in Comparative Example 1 in the creatinine measurement reagent was measured. 1 mM disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 0.1% (W / V) 2-methylisothiazolone (manufactured by Roche Diagnostics), 0.1% (W / V) Triton X-100, 0.02% (W / V) 4-aminoantipyrine, 0.02% (W / V) TOOS (manufactured by Dojindo Laboratories), 100 U / ml creatinine amide hydrolase (manufactured by Toyobo; CNH-211), 50 U / ml The above sarcosine oxidase is adjusted to 10 U / ml in 50 mMPIPES-NaOH buffer (pH 7.5) containing creatine amidinohydrolase (Toyobo; CRH-221), 10 U / ml peroxidase (Toyobo; PEO-301). In addition, the remaining rate of sarcosine oxidase activity after 2 weeks storage at 35 ° C It was boss. The results are shown in Table 3. As can be seen from Table 3, it was confirmed that the modified sarcosine oxidase of the present invention has improved liquid stability in the creatinine measuring reagent as compared with that before modification.

Example 6 Evaluation 4 of Modified Sarcosine Oxidase
The stability in the creatine measuring reagent of the mutant sarcosine oxidase (SAOM1, SAOM10, SAOM11, SAOM12, SAOM13) obtained in Example 3 and various sarcosine oxidases obtained in Comparative Example 1 was measured. 1 mM disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 0.1% (W / V) 2-methylisothiazolone (manufactured by Roche Diagnostics), 0.1% (W / V) Triton X-100, 0.02% (W / V) 4-aminoantipyrine, 0.02% (W / V) TOOS (manufactured by Dojindo Laboratories), 50 U / ml creatine amidinohydrolase (manufactured by Toyobo; CRH-221), 10 U / ml The above sarcosine oxidase was added to 50mMPIPES-NaOH buffer (pH 7.5) containing peroxidase (Toyobo; PEO-301) to a concentration of 10 U / ml and stored at 35 ° C for 2 weeks. The residual rate was measured. The results are shown in Table 3. As can be seen from Table 3, it was confirmed that the modified sarcosine oxidase of the present invention has improved liquid stability in the creatine measuring reagent as compared with that before modification.

According to the present invention, it has become possible to supply a modified sarcosine oxidase with improved liquid stability by modifying a protein having sarcosine oxidase activity by a protein engineering technique. By using the modified sarcosine oxidase of the present invention as an enzyme for measuring creatine and creatinine in a body fluid that is clinically an indicator for diagnosis of muscle disease and kidney disease, the liquid stability of the reagent is improved. Can be made.

Claims (7)

The 89th lysine of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is arginine, the 204th methionine is alanine, the 213th serine is proline, the 233rd cysteine is serine, and the 240th asparagine is A modified sarcosine oxidase, wherein the 250th glutamic acid is substituted with glutamine for tyrosine. A gene encoding the modified sarcosine oxidase according to claim 1. A vector comprising the gene according to claim 2. A transformant transformed with the vector according to claim 3. A method for producing a modified sarcosine oxidase, comprising culturing the transformant according to claim 4 and collecting sarcosine oxidase from the culture. A reagent for measuring creatine comprising the sarcosine oxidase according to claim 1. A reagent for measuring creatinine comprising the sarcosine oxidase according to claim 1.