Mutant creatine amidinohydrolase

Abstract

By modifying the protein using protein engineering techniques, we provide a mutant creatine amidinohydrolase with improved affinity for the substrate. [Solution] A mutant creatine amidinohydrolase having an amino acid at position 105 or an equivalent position in an amino acid sequence that is 90% or more identical to SEQ ID NO: 1, in which the amino acid is substituted with another amino acid.

Description

Technical Field

[0001] The present invention relates to a mutant creatine amidinohydrolase having improved affinity for creatine by introducing mutations in the amino acid sequence of creatine amidinohydrolase, preferably derived from a microorganism. The present invention also relates to a gene encoding the mutant creatine amidinohydrolase, a recombinant vector containing the gene, a transformant transformed with the recombinant vector, a method for producing the mutant creatine amidinohydrolase using the transformant, and various applications of the mutant creatine amidinohydrolase to a reagent for measuring creatine or creatinine.

Background Art

[0002] Creatine and creatinine are present in blood or urine and serve as indices for diagnosing diseases such as uremia, chronic nephritis, acute nephritis, gigantism, and myotonic dystrophy. In order to perform these diagnoses, it is very important to quickly and accurately quantify the amount of creatine in blood or urine.

[0003] Conventionally, creatine amidinohydrolase (EC 3.5.3.3) has been used as an enzyme for measuring creatine in body fluids together with other enzymes such as creatinine amidinohydrolase, sarcosine oxidase, and peroxidase. Creatine amidinohydrolase is an enzyme that catalyzes an irreversible reaction that acts on creatine in the presence of water to produce sarcosine. Such creatine amidinohydrolase is widely found in the microbial world, has already been industrially produced, and is used as a clinical test reagent.

[0004] However, conventional creatine amidinohydrolases produced from various bacterial cells had high Km values ​​relative to creatine. For example, enzymes derived from the genera Corynebacterium, Micrococcus, Actinobacillus, and Bacillus (Patent Document 1) had high Km values ​​of approximately 20 mM relative to creatine. Therefore, they could not be said to possess sufficient characteristics for use as diagnostic enzymes.

[0005] To solve the above problems, the present inventors have already found that bacteria belonging to the genus Alcaligenes produce creatine amidinohydrolase that is highly thermally stable and has a relatively low Km value relative to creatine (Km value: approximately 15.2) (Patent Document 2). Furthermore, they have isolated a creatine amidinohydrolase gene with a relatively low Km value relative to creatine from this strain and have established a technique for producing this enzyme in large quantities using Gram-negative bacteria as a host (Patent Document 3). In addition, a creatine amidinohydrolase that is stable in the high pH range and has a low Km value (13 mM) has also been reported from bacteria of the same genus Alcaligenes (Patent Document 4). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 3-76915 [Patent Document 2] Japanese Patent Application Publication No. 7-265074 [Patent Document 3] Japanese Patent Application Publication No. 8-308579 [Patent Document 4] Japanese Patent Application Publication No. 7-170979 [Overview of the project] [Problems that the invention aims to solve]

[0007] However, the aforementioned creatine amidinohydrolase does not yet have a sufficiently low Km value for use in general clinical diagnostic reagents, and there has been a demand for the development of creatine amidinohydrolase with an even lower Km value. [Means for solving the problem]

[0008] The object of the present invention is to provide an enzyme that can be used in creatine measurement reagents and is practically advantageous compared to creatine amidinohydrolase having the amino acid sequence described in Sequence ID No. 1 of the sequence listing. More specifically, the object is to provide a creatine amidinohydrolase with improved affinity for creatine, obtained by substituting the amino acid sequence of creatine amidinohydrolase with other amino acids using protein engineering techniques.

[0009] Creatine amidinohydrolase, which has the amino acid listed in Sequence ID No. 1 of the sequence listing, is expected to be used as an enzyme for creatine measurement reagents due to its excellent stability. However, this creatine amidinohydrolase has a drawback in clinical diagnostics because it has low affinity for creatine, resulting in a time-consuming creatine quantification process.

[0010] The inventors diligently studied to overcome the above-mentioned drawbacks and succeeded in creating a mutant creatine amidinohydrolase with a lower Km value for creatine using protein engineering techniques with the above-mentioned creatine amidinohydrolase gene derived from the genus Alcaligenes. Furthermore, they found that by reducing the Km value of creatine amidinohydrolase, the time required for the enzymatic reaction to reach the endpoint in creatine quantification can be shortened, thus completing the present invention.

[0011] In other words, the present invention consists of, for example, the following configuration. Section 1. A mutant creatine amidinohydrolase in which the amino acid at position 105 or an equivalent position in the amino acid sequence, which is 90% or more identical to SEQ ID NO: 1, is substituted with another amino acid. Section 2. A variant of creatine amidinohydrolase according to item 1, wherein the arginine at position 105 or equivalent is substituted with lysine or histidine. Section 3. This gene consists of a nucleotide sequence that is more than 90% identical to Sequence ID No. 2, and encodes a mutant creatine amidinohydrolase of item 1. Section 4. A vector containing the gene described in item 3. Section 5. A transformant obtained by transforming with the vector of item 4. Section 6. A method for producing mutant creatine amidinohydrolase, comprising the steps of culturing the transformant described in item 5 and collecting creatine amidinohydrolase from the resulting culture. Section 7. A reagent for creatine measurement containing the mutant creatine amidinohydrolase described in item 1. Section 8. A method for measuring creatine in a sample using the mutant creatine amidinohydrolase described in item 1. [Effects of the Invention]

[0012] This invention creates a novel creatine amidinohydrolase with a lower Km value, which is useful as a clinical diagnostic enzyme, and enables the industrial mass production of this creatine amidinohydrolase. This invention also allows for a reduction in the amount of enzyme used in creatine quantification, and, if the amount used is the same as before, the measurement time can be shortened, thereby increasing the number of samples processed. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows an example of the principle of creatinine measurement using the mutant creatine amidinohydrolase of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] As one embodiment of the present invention, there is provided a protein in which at least one amino acid of an amino acid sequence constituting a protein having creatine amidinohydrolase activity is mutated by addition, deletion, insertion or substitution, the protein having creatine amidinohydrolase activity and having improved affinity for creatine as compared to that before introduction of the mutation. This is a mutant creatine amidinohydrolase.

[0015] Alternatively, the mutant creatine amidohydrolase of the present invention preferably has 50% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 85% or more, still more preferably 90% or more. Particularly preferably, it has 95% or more identity, still more preferably 98% or more, still more preferably 99% or more identity.

[0016] ​​​​​​​​​​​​The creatineamide hydrolase used as the basis for the modification of the modified creatineamide hydrolase of the present invention is not particularly limited, but is derived from Alcaligenes faecalis TE3581 (FERMP-14237), and its amino acid sequence is shown in SEQ ID NO: 1. These are all as described in Patent Document 3. In SEQ ID NO: 1, the amino acids are numbered starting with methionine as 1.

[0019] The creatineamide hydrolase used as the basis for modifying the mutant creatineamide hydrolase of the present invention is not particularly limited as long as it has creatineamide hydrolase activity. Modifications include, but are not particularly limited to, those in which amino acids are deleted, substituted or added, those in which intermolecular or intramolecular crosslinks are formed, or those that are chemically modified with sugar chains or other functional groups.

[0020] Methods for modifying the amino acid sequence that constitutes creatinamide hydrolase typically involve genetic modification techniques. Specifically, DNA containing the genetic information of the modified protein is created by altering specific bases in the DNA containing the protein's genetic information, or by inserting or deleting specific bases. Specific methods for altering the base sequence in DNA include the use of commercially available kits (such as the Transformer Mutagenesis Kit (Clonetech), EXOIII / Mung Bean Deletion Kit (Stratagene), and Quick Change Site Directed Mutagenesis Kit (Stratagene)), or the use of polymerase chain reaction (PCR).

[0021] In the present invention, the gene encoding the mutant creatinamide hydrolase can be, for example, a nucleic acid consisting of a nucleotide sequence having 90% or more identity with SEQ ID NO: 2. More preferably, it is a nucleic acid consisting of a nucleotide sequence having 95% or more identity with SEQ ID NO: 2, and even more preferably, a nucleotide sequence having 98% or more identity with SEQ ID NO: 2. SEQ ID NO: 2 can be obtained by substituting the codon sequence of the site encoding the amino acid at position 105 or an equivalent position in the amino acid sequence described in SEQ ID NO: 1 with a codon sequence corresponding to lysine or histidine.

[0022] The DNA encoding the mutant creatine amidinohydrolase described above is transferred into a host microorganism in a ligated state with a plasmid, resulting in a transformant that produces the modified protein. For example, when using Escherichia coli as the host microorganism, pBluescript and pUC18 can be used as plasmids. Suitable host microorganisms include Escherichia coli W3110, Escherichia coli C600, Escherichia coli JM109, and Escherichia coli DH5α. Methods for transferring the recombinant vector into the host microorganism include, for example, transferring the recombinant DNA in the presence of calcium ions when the host microorganism belongs to the Escherichia genus, and electroporation may also be used. Furthermore, commercially available competent cells (e.g., Competent Hi JM109; manufactured by Toyobo) may be used.

[0023] The transformed microorganisms obtained in this way can stably produce large quantities of modified proteins by being cultured in a nutrient medium. The culture form of the transformed host microorganism should be selected considering the nutritional and physiological properties of the host, and although liquid culture is usually used in most cases, aerated and stirred culture is advantageous industrially. As nutrients for the medium, those commonly used for culturing microorganisms can be widely used. As a carbon source, any assimilated carbon compound is acceptable, such as glucose, sucrose, lactose, maltose, fructose, molasses, and pyruvic acid. As a nitrogen source, any available nitrogen compound is acceptable, such as peptone, meat extract, yeast extract, casein hydrolysate, and soybean meal alkali extract. In addition, phosphates, carbonates, sulfates, salts such as magnesium, calcium, potassium, iron, manganese, and zinc, as well as specific amino acids and specific vitamins, can be used as needed. The culture temperature can be appropriately changed within the range in which the bacteria grow and produce modified proteins, but in the case of Escherichia collie, it is preferably around 20-42°C. The incubation time varies slightly depending on the conditions, but it is sufficient to terminate the incubation at an appropriate time, timing it to coincide with the point when the modified protein reaches its highest yield; this is usually around 6 to 48 hours. The pH of the culture medium can be adjusted as needed within the range in which the bacteria grow and produce the modified protein, but a pH of around 6.0 to 9.0 is particularly preferred.

[0024] While it is possible to directly collect and use the culture medium containing the bacterial cells that produce the modified protein, generally, if the modified protein is present in the culture medium, it is separated from the microbial cells by filtration, centrifugation, etc., according to conventional methods, before use. If the modified protein is present within the bacterial cells, the bacterial cells are collected from the obtained culture by means of filtration or centrifugation, and then these cells are destroyed by mechanical methods or enzymatic methods such as lysozyme. If necessary, chelating agents such as EDTA and / or surfactants are added to solubilize the modified protein, and it is then separated and collected as an aqueous solution.

[0025] The modified protein-containing solution obtained in this manner can be precipitated by, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate or sodium sulfate, or fractional precipitation using hydrophilic organic solvents such as methanol, ethanol, or acetone. Heat treatment and isoelectric focusing are also effective purification methods. The purified modified protein can be obtained by gel filtration using adsorbents or gel filters, adsorption chromatography, ion exchange chromatography, or affinity chromatography.

[0026] In the examples, the activity of creatine amidinohydrolase was measured as follows. In this invention, the enzyme activity is defined as 1 unit (U) of enzyme activity that produces 1 μmol of sarcosine per minute under the following conditions.

[0027] Composition of reaction mixture 0.3M HEPES (pH 7.6) 0.005% 4-aminoantipyrine 0.015% phenol 1.8% Creatine 6 U / ml sarcosin oxidase 6U / ml peroxidase

[0028] Place 3 ml of the above reaction mixture into a cuvette (d=1 cm) and preheat at 37°C for approximately 3 minutes. Next, add 0.1 ml of enzyme solution and gently mix. Then, using water as a control, record the absorbance change at 500 nm for 5 minutes using a spectrophotometer controlled at 37°C, and determine the absorbance change per minute from the linear portion between 2 and 5 minutes (ΔOD test).

[0029] For the blind test, 0.1 ml of enzyme diluent (50 mM potassium phosphate buffer, pH 7.5) is added instead of the enzyme solution, and the procedure is performed as described above to determine the change in absorbance per minute (△OD blank).

[0030]

number

[0031] 13.3: Millimolecular extinction coefficient (cm²) of quinone imine pigments under the above measurement conditions. 2 / μM) 1 / 2: The coefficient indicating that the quinone imine pigment formed from one molecule of hydrogen peroxide produced by an enzymatic reaction is 1 / 2 the size of a molecule. · 0: Optical path length (cm) 0.1: Amount of added enzyme solution (ml)

[0032] In this invention, the Km value is the Km value for creatine when activity is measured using sarcosine oxidase and peroxidase as conjugated enzymes.

[0033] The mutant creatinamide hydrolase of the present invention has improved affinity for the substrate, making it useful for use in reagents for creatine measurement. An example of the principle of measuring creatinine in a sample is shown below (Figure 1). Examples of samples to be measured include plasma, serum, and urine.

[0034] Creatinine in the sample is reacted with creatinine amidohydrolase to convert it to creatine, and then creatine amidinohydrolase is reacted with creatine to produce sarcosine. Sarcosine oxidase is reacted with the produced sarcosine to produce hydrogen peroxide, and this hydrogen peroxide is oxidatively condensed with ESPMT (N-ethyl-N-(3-sulfopropyl)-3-methylaniline) and 4-AA (4-aminoantipyrine) in the presence of peroxidase to produce a quinone dye. The absorbance of the produced quinone dye can be measured to determine the creatinine content in the sample. The reagent consists of, for example, enzyme reagent A containing creatine amidinohydrolase, sarcosine oxidase, N-ethyl-N-(3-sulfopropyl)-3-methylaniline, and ascorbic acid oxidase, and enzyme reagent B containing 4-aminoantipyrine, creatinine amidohydrolase, and peroxidase.

[0035] The creatinine measurement reagent, comprising the mutant creatinamide hydrolase of the present invention and having the above configuration, can reduce the amount of enzyme used in creatinine quantification. Furthermore, if the same amount of enzyme is used as in conventional methods, the reaction time to reach the endpoint is shorter, thus shortening the measurement time and increasing the number of samples that can be processed. [Examples]

[0036] The present invention will be described in detail below with reference to examples. The present invention is not particularly limited by the examples.

[0037] fruit Example 1: Preparation of a mutant creatine amidinohydrolase expression plasmid PCR was performed using the recombinant plasmid pCRH273 described in Patent Document 3 as a template, with complementary synthetic oligonucleotide pairs designed to substitute specific amino acids with different amino acids, as shown in Table 1, as primers for each mutant creatinine amidinohydrolase. Subsequently, commercially available E. coli competent cells (E. coli JM109; Toyobo Co., Ltd.) were transformed with the PCR product and cultured on ampicillin-containing LB agar medium at 37°C for 16 hours. After that, single colonies were inoculated into ampicillin-containing LB liquid medium and cultured overnight with shaking at 37°C. Then, 1 mL of the culture medium was inoculated and the plasmid was extracted by a conventional method. The substituted sites of the extracted plasmid were identified using a DNA sequencer (ABI PRISM® 3700 DNA Analyzer; Perkin-Elmer). Plasmids containing the base sequence encoding a modified creatine amidinohydrolase in which specific amino acids in SEQ ID NO: 1 were substituted with different amino acids were obtained, as shown in Table 1.

[0038] [Table 1]

[0039] [Table 2]

[0040] Example 2: Expression of a mutant creatine amidinohydrolase expression plasmid Competent cells of Escherichia coli (JM109; Toyobo Co., Ltd.) were transformed with the recombinant plasmid prepared in Example 1 and cultured on LB agar medium containing ampicillin at 37°C for 16 hours. Subsequently, single colonies were inoculated into LB liquid medium containing ampicillin and cultured overnight with shaking at 37°C. The above bacterial cells were collected by centrifugation, suspended in 50 mM potassium phosphate buffer (pH 7.5), then disrupted by sonication, and further centrifugation was performed to obtain the supernatant as a crude enzyme solution.

[0041] Competent E. coli cells (E. coli JM109; Toyobo Co., Ltd.) were transformed with a DNA sequence encoding the artificial CRH sequence designed in Example 1 inserted into the multi-cloning region of the recombinant plasmid pBluescript SK(+). The transformed cells were spread on LB agar medium containing ampicillin (1.0% polypeptone, 0.5% yeast extract, 1.0% NaCl, 1.5% agar; pH 7.3) and cultured overnight at 37°C. The transformed cells that formed colonies on the agar medium were inoculated into LB liquid medium containing ampicillin (50 mg / ml; Nacalai Tesque Co., Ltd.) (1% polypeptone, 0.5% yeast extract, 1.0% NaCl; pH 6.5) and cultured overnight with shaking at 37°C. Plasmids were extracted and purified from the obtained cells using miniprep extraction to obtain a plasmid in which the nucleotide sequence of the pCRH273 mutant was inserted into the multi-cloning region of pBluescript SK(+).

[0042] Example 3: Preparation of mutant creatine amidinohydrolase E. coli (Tuner DE3) transformed with the artificially synthesized CRH expression plasmid obtained in Example 2 was cultured on LB agar medium containing kanamycin at 37°C for 16 hours to obtain single colonies. Subsequently, the single colonies of the transformants were inoculated into 5 ml LB liquid medium containing kanamycin and cultured with shaking at 25°C for 16 hours. A portion of the culture medium was inoculated into 100 ml LB liquid medium containing kanamycin and cultured with shaking at 25°C for 72 hours. Cells obtained from a portion of the culture medium by centrifugation were collected, and a crude enzyme solution was prepared by disrupting the cells using an ultrasonic disruptor in 50 mM phosphate buffer (pH 6.0).

[0043] Example 4: Screening of mutant creatine amidinohydrolase The creatine amidinohydrolase activity of the obtained crude enzyme solution was measured using the activity assay method described above. Creatine amidinohydrolase activity was measured at two creatine concentrations: 1.8% and 0.18%. The affinity to the substrate was evaluated by the ratio of the value calculated as (activity at 0.18% creatine concentration) / (activity at 1.8% creatine concentration) to the unmodified creatine amidinohydrolase. The results are shown in Table 3.

[0044] [Table 3]

[0045] Example 5: Evaluation of mutant creatine amidinohydrolase From the crude enzyme solution prepared in Example 3, four enzymes were purified using HisTrap® HP (Cytiva) according to its protocol: the original creatine amidinohydrolase and mutant creatine amidinohydrolase mutants 9, 11, and 18, which showed high affinity for the substrate in Example 4. Then, the creatine amidinohydrolase activity was measured using the activity assay method described above, and the Km value was evaluated. The results are shown in Table 4. In particular, mutant 11, in which the arginine at position 105 was modified to lysine, had a Km value of 1.5 mM, which was confirmed to be significantly reduced compared to the original mutant.

[0046] [Table 4] [Industrial applicability]

[0047] The mutant creatine amidinohydrolase of the present invention is expected to be widely used, particularly in the medical and diagnostic fields, by being applied to reagents and sensors for measuring creatinine in samples.

Claims

1. A mutant creatine amidinohydrolase in which the amino acid at position 105 or an equivalent position in the amino acid sequence, which is 90% or more identical to SEQ ID NO: 1, is substituted with another amino acid.

2. The mutant creatine amidinohydrolase according to claim 1, wherein the arginine at position 105 or an equivalent position is substituted with lysine or histidine.

3. A gene comprising a nucleotide sequence having 90% or more identity with SEQ ID NO: 2, which encodes the mutant creatine amidinohydrolase described in claim 1.

4. A vector comprising the gene described in claim 3.

5. A transformant obtained by transforming with the vector described in claim 4.

6. A method for producing mutant creatine amidinohydrolase, comprising the steps of culturing the transformant described in claim 5 and collecting creatine amidinohydrolase from the obtained culture.

7. A reagent for measuring creatine, comprising the mutant creatine amidinohydrolase described in claim 1.

8. A method for measuring creatine in a sample using the mutant creatine amidinohydrolase described in claim 1.

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