JP2009095348A - Modified sarcosine oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sarcosine oxidase with reduced activity to proline obtained by modifying a protein having sarcosine oxidase activity by a protein engineering technology, and to provide a method for producing the same and use of the same. <P>SOLUTION: Provided are modified sarcosine oxidase in which two amino acids on a specific position in a specific amino acid sequence are substituted, and the method for producing the same. Provided also are creatinine using the enzyme, and measuring reagent for the creatine. The activity of the enzyme to proline is reduced and the measuring accuracy for the creatine and creatinine is improved. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sarcosine oxidase with reduced activity against proline, 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 used as an enzyme for measuring creatine and creatinine in body fluid, which is clinically an indicator for diagnosis of muscle disease and kidney disease. It is used with nuclease, 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

However, it is known that conventional sarcosine oxidase also acts on proline, which is one of the amino acids present in blood, and may cause a positive error when measuring creatinine and creatine. (For example, refer nonpatent literature 2-3.). In order to solve this problem, our group reported sarcosine oxidase in which wild-type sarcosine oxidase was modified by protein engineering to reduce its activity on proline (see, for example, Patent Document 9). The action on sarcosine, which is the original substrate, is unknown, and further improvements have been desired.
An object of the present invention is to provide a sarcosine oxidase with reduced activity against proline.
JP-A-10-248572 “Clinical Chemistry”, 1991, Volume 20, p. 144-152 “Biological Sample Analysis”, 1994, Vol. 17, p. 332-337

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that a modified sarcosine oxidase having a reduced activity against proline can be produced without impairing the activity against sarcosine, and the present invention is completed. It came to.

That is, the present invention has the following configuration.
[Item 1] A modified sarcosine oxidase in which the 94th valine in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with glycine and the 250th glutamic acid 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 having a reduced activity against proline 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, Creatine and creatinine can be measured.

  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, And it is a modified sarcosine oxidase characterized in that its activity on L-proline is reduced as compared with that before conversion.

  The modified sarcosine oxidase of the present invention is characterized in that the reactivity to proline is reduced as compared with that before modification. Here, the reactivity to proline means the relative ratio of the enzyme activity when proline is used as a substrate to the enzyme activity when sarcosine, which is the original substrate, is used as a substrate. And if the reactivity with respect to proline is falling, even if the specific activity at the time of using sarcosine as a substrate is changing, it will be included in the modified sarcosine oxidase of the present invention. Further, the Km value of sarcosine of the modified sarcosine oxidase of the present invention may change, but when applied to a creatinine or creatine measurement reagent, it may cause a decrease in reactivity, Within 3 times before modification, more preferably within 1.5 times.

  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). Our group 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 4). ) 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, and may be modified other proteins having sarcosine oxidase activity. . Preferable examples of other sarcosine oxidase protein having sarcosine oxidase activity include 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. Examples thereof include other sarcosine oxidase activity proteins having 50% or more, more preferably amino acid sequence homology of 80% or more. This is based on the fact that amino acid residues and reaction mechanisms involved in substrate specificity are often the same in the case of enzyme proteins having 50% or 80% homology in the amino acid sequence and exhibiting the same catalytic activity. And The modified sarcosine oxidase of the present invention may be one in which at least one amino acid is further added, deleted or inserted as long as the enzyme activity is not impaired. 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 5).

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 a modified sarcosine oxidase, wherein the protein having sarcosine oxidase activity has the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing.

  Another embodiment of the present invention is a modified sarcosine oxidase in which at least one amino acid in the catalytic domain of sarcosine oxidase and the region constituting the linking site of the catalytic domain and the FAD binding domain is substituted with another amino acid. is there. From the sarcosine oxidase which has homology with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing and whose steric structure has been clarified by X-ray crystallography, It is presumed that positions 82 to 152, 216 to 328 constitute the catalytic domain of sarcosine oxidase and the linking site between the catalytic domain and the FAD binding domain (see, for example, Non-Patent Document 6).

  Preferably, a modified form in which at least one amino acid in the region constituting the β-sheet structure of the catalytic domain adjacent to the catalytic domain of sarcosine oxidase and the FAD-binding domain and the catalytic domain thereof is substituted with another amino acid Sarcosine oxidase. 82 to 97, 313 to 328 of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing are the linking sites of the sarcosine oxidase catalytic domain and the FAD binding domain, and the β sheet structure of the catalytic domain adjacent thereto (For example, refer nonpatent literature 6).

“Structure”, 1999, Vol. 7, No. 3, p. 331-345

  Another embodiment of the present invention is a modification in which at least one amino acid selected from the group consisting of positions 89, 94, and 322 of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing is substituted with another amino acid. Type sarcosine oxidase.

  Particularly preferred is a modified sarcosine in which the lysine at position 89 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with arginine, valine at position 94 with glycine, or lysine at position 322 with arginine. Oxidase.

  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 use a modified sarcosine oxidase having a reduced action on L-proline, thereby suppressing the influence of proline to less than 7%, preferably less than 5%. It has been.

  The creatine measurement reagent of the present invention includes a modified sarcosine oxidase, creatine amidinohydrolase, peroxidase, and a hydrogen peroxide detection reagent that have a decreased response to proline. The creatinine measurement reagent includes a modified sarcosine oxidase, a creatinine amide hydrolase, a creatine amidinohydrolase, a peroxidase, and a hydrogen peroxide detection reagent in which the reaction to proline is reduced. 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. Preservatives and surfactants may be added as long as reagent performance is not adversely affected. Usually, it is 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 nine 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 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 QuickChange ™ Site-Directed 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. Mutagenesis Kit (manufactured by STRATAGENE) is used to perform a mutation treatment operation according to the protocol, further determine the base sequence, and mutated sarcosine oxidase in which the 89th lysine of the amino acid sequence described in SEQ ID NO: 1 is substituted with arginine A recombinant plasmid (pSAOM1) encoding was obtained.
By using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 4 in the Sequence Listing and a synthetic oligonucleotide complementary thereto, by the same operation as above, SEQ ID NO: A recombinant plasmid (pSAOM2) encoding a mutant sarcosine oxidase in which the 94th valine in the amino acid sequence described in 1 was replaced with glycine was obtained.
Using pSAOEP3, a synthetic oligonucleotide described in SEQ ID NO: 5 in the sequence listing and a synthetic oligonucleotide complementary thereto, 322 lysine of the amino acid sequence described in SEQ ID NO: 1 was replaced with arginine by the same operation as above. A recombinant plasmid (pSAOM3) encoding the mutated sarcosine oxidase was obtained.
Using pSAOM2, a synthetic oligonucleotide described in SEQ ID NO: 6 in the sequence listing and a synthetic oligonucleotide complementary thereto, the 94th valine in the amino acid sequence described in SEQ ID NO: 1 is glycine, 250 by the same operation as described above. A recombinant plasmid (pSAOM4) encoding a mutant sarcosine oxidase in which the first glutamic acid was replaced with glutamine was obtained.
By using pSAOM4, a synthetic oligonucleotide described in SEQ ID NO: 3 in the sequence listing and a synthetic oligonucleotide complementary thereto, by the same operation as described above, the 89th lysine of the amino acid sequence described in SEQ ID NO: 1 is arginine, 94 A recombinant plasmid (pSAOM5) encoding a mutant sarcosine oxidase in which the first valine was replaced with glycine and the 250th glutamic acid with glutamine was obtained.
Using pSAOM5, 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 arginine and 94 by the same operation as described above. A recombinant plasmid (pSAOM6) encoding a mutant sarcosine oxidase in which the first valine is glycine, the 213th serine is proline, and the 250th glutamic acid is replaced by glutamine was obtained.
Using pSAOM6, a synthetic oligonucleotide described in SEQ ID NO: 8 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 arginine and 94 A recombinant plasmid (pSAOM7) encoding a mutant sarcosine oxidase in which the first valine was replaced with glycine, the 204th methionine with alanine, the 213th serine with proline, and the 250th glutamic acid with glutamine was obtained.
Using pSAOM7, a synthetic oligonucleotide described in SEQ ID NO: 9 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 arginine and 94 Recombinant plasmid (pSAOM8) encoding a mutant sarcosine oxidase in which the valine is glycine, the 166th asparagine is lysine, the 204th methionine is alanine, the 213th serine is proline, and the 250th glutamic acid is replaced with glutamine ).
Using pSAOM8, a synthetic oligonucleotide described in SEQ ID NO: 5 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 arginine and 94 The second valine is glycine, the 166th asparagine is lysine, the 204th methionine is alanine, the 213th serine is proline, the 250th glutamic acid is glutamine, and the 322nd lysine is substituted with arginine. An encoding recombinant plasmid (pSAOM9) was obtained.

Example 3 Production of Modified Sarcosine Oxidase pSAOM1, pSAOM2, pSAOM3, pSAOM4, pSAOM5, pSAOM6, pSAOM7, pSAOM8, pSAOM9 Recombinant plasmids were transformed into Escherichia coli JM109 competent cells, respectively. I got it.
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 nucleic acid removal with polyethyleneimine and ammonium sulfate fractionation, dialyzed with 20 mM phosphate buffer (pH 7.5), then DEAE Sepharose CL-6B (Amersham Biosciences), and further separated by heat treatment. -Purified to obtain a purified enzyme preparation. The sample obtained by this method showed an almost single band by SDS-PAGE. This mutant was named SAOM1.
Purified enzyme preparations were also obtained for Escherichia coli JM109 transformants using recombinant plasmids of pSAOM2, pSAOM3, pSAOM4, pSAOM5, pSAOM6, pSAOM7, pSAOM8, and pSAOM9 in the same manner as described above. The obtained enzyme preparations were named SAOM2, SAOM3, SAOM4, SAOM5, SAOM6, SAOM7, SAOM8, and SAOM9, respectively.

Comparative Example 1 Production of Wild Type Sarcosine Oxidase As a comparative example, a purified enzyme preparation before modification was obtained in the same manner as described above for Escherichia coli JM109 transformant by pSAOEP3.

Example 4 Evaluation of Modified Sarcosine Oxidase Various sarcosine oxidases obtained in Example 3 and Comparative Example 1 were evaluated.
The proline activity was determined from the relative ratio (%) of the enzyme activity when L-proline was used as a substrate to the enzyme activity when sarcosine was used as a substrate by the above-described activity measurement method. The Km values for sarcosine and L-proline were measured by changing the substrate concentration in the above activity measurement method. The results are shown in Table 1. As can be seen from Table 1, it was confirmed that the reactivity of the modified sarcosine oxidase of the present invention to proline was lower than that before the modification. In addition, the Km value of the modified sarcosine oxidase with respect to sarcosine was almost the same as that of the sarcosine oxidase before modification, or within 1.5 times.

Example 6 Effect of Proline in Measurement Agent for Creatinine The degree of influence of proline when the various sarcosine oxidases obtained in Example 3 and Comparative Example 1 were applied to the creatinine measurement reagent was evaluated. 10 U / ml sarcosine oxidase (prepared in Example 3 and Comparative Example 1), 1 mM disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 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 (Toyobo; CNH-211), 50 U / ml creatine amidinohydrolase (Toyobo; CRH-221) 10 μl of a 5 mg / dl creatinine aqueous solution was added to 300 μl of 50 mMPIPES-NaOH buffer solution (pH 7.5) containing 10 U / ml peroxidase (Toyobo; PEO-301). The reaction is allowed to proceed for 5 minutes, and the change in absorbance at 546 nm is measured by HITACHI 7060. Measurement was performed using a type automatic analyzer. Further, a change in absorbance was measured by the same operation as described above using a 100 mg / dl L-proline aqueous solution instead of the creatinine aqueous solution. The degree of influence of proline was determined from the relative ratio (%) of the increase in absorbance for 5 minutes produced when L-proline was used as a substrate to the increase in absorbance for 5 minutes produced when creatinine was used as a substrate. The results are shown in Table 2. As can be seen from Table 2, it was confirmed that by using the modified sarcosine oxidase of the present invention as a reagent for measuring creatinine, the influence of proline in the reagent is reduced.

Example 7 Influence of Proline in Reagent for Measuring Creatine The degree of influence of proline when the various sarcosine oxidases obtained in Example 3 and Comparative Example 1 were applied to the reagent for measuring creatine was evaluated. 10 U / ml sarcosine oxidase (prepared in Example 3 and Comparative Example 1), 1 mM disodium ethylenediaminetetraacetate, 50 mM sodium chloride, 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 peroxidase (Toyobo) 10 μl of 5 mg / dl creatine aqueous solution is added to 300 μl of 50mMPIPES-NaOH buffer solution (pH 7.5) containing PEO-301) and reacted at 37 ° C. for 5 minutes, and the change in absorbance at 546 nm is measured with an HITACHI 7060 type automatic analyzer. And measured. Moreover, the change in absorbance was measured by the same operation as described above using a 100 mg / dl L-proline aqueous solution instead of the creatine aqueous solution. The degree of influence of proline was determined from the relative ratio (%) of the increase in absorbance for 5 minutes produced when L-proline was used as a substrate to the increase in absorbance for 5 minutes produced when creatine was used as a substrate. The results are shown in Table 3. As can be seen from Table 3, by using the modified sarcosine oxidase of the present invention as a creatine measuring reagent, it was confirmed that the degree of influence of proline in the reagent was reduced.

  According to the present invention, it has become possible to supply a modified sarcosine oxidase having a reduced activity against proline 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, Creatine and creatinine can be measured.

Claims (7)

A modified sarcosine oxidase, wherein the 94th valine in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is substituted with glycine and the 250th glutamic acid with glutamine. 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.