JP2010088303A - Modified glycerol kinase having improved thermal stability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glycerol kinase having high thermal stability in addition to high durability to preservatives, and to provide a method for measuring neutral fat with the enzyme. <P>SOLUTION: There are provided the modified glycerol kinase having more improved thermal stability than that of a wild type glycerol kinase originated from a bacterium in genus Cellulomomas, and having an amino acid sequence having amino acids substituted at specific positions, a gene for the enzyme, a transformant having the same transduced therein, a neutral fat sensor using the enzyme, and a method for measuring a neutral fat. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a modified glycerol kinase having improved thermal stability compared to wild-type glycerol kinase, and more particularly to a modified glycerol kinase useful as a clinical test agent.

  Glycerol kinase (EC 2.7.1.30) is an enzyme that catalyzes a reaction for converting glycerol to glycerol-3-phosphate by phosphorylation dependent on magnesium and ATP. Glycerol kinase has been discovered in the liver by Kalkkar in 1937 (see, for example, Non-Patent Document 1). Purification from thermus flavus has been reported (for example, see Non-Patent Documents 2 to 5 and Patent Document 1), and is known to exist widely in all living organisms. In addition, cloning of genes from humans, Bacillus subtilis, Saccharomyces cerevisiae, Thermus flavus, etc. has been reported (see, for example, Non-Patent Documents 6 to 9). In particular, in Escherichia coli, the enzyme has been studied in detail. It was purified by Hayashi et al. In 1967 (see, for example, Non-Patent Document 10), and its cloning was reported in 1988. (For example, refer nonpatent literature 11). In addition, studies on gene regulation and inhibition by allosteric inhibitors have already been conducted in a wide range.

  On the other hand, glycerol kinase is used as a raw material enzyme for clinical test drugs in industrial applications. For example, in the measurement of neutral fat in blood, glycerol kinase is used to convert glycerol produced by hydrolyzing neutral fat (triglyceride) in a sample with lipase to glycerol-3-phosphate. .

  The current clinical chemistry tests for biochemical tests, including those for measuring triglycerides, are mainly in the state of solutions, and as a necessary characteristic of the enzyme of the raw material, high stability in the test solution Sex is required. It is particularly important that the resistance to the preservative is high as a characteristic that contributes to the stability of the enzyme in the test solution. This is because a liquid test agent is generally added with a preservative to enable long-term storage, and this preservative may destabilize the enzyme.

  Enzymes with high thermostability have been considered to have high stability in liquid diagnostics, and thermophilic bacteria such as Bacillus stearothermophilus and Thermus flavus. The derived glycerol kinase has been widely used as a glycerol kinase for liquid diagnostic agents. However, these glycerol kinases have the problem of relatively low resistance to preservatives.

The present inventors isolated a gene encoding glycerol kinase highly resistant to preservatives from bacteria of the genus Cellulomonas, established a method for producing the enzyme by gene recombination technology, and neutralized fat and glycerol of the enzyme However, the glycerol kinase has a weakness that it is less thermostable than the glycerol kinase derived from the thermophilic bacterium (patent) Reference 2). Therefore, as another approach, glycerol kinase derived from thermophilic bacteria has been modified by protein engineering to try to achieve both high thermal stability and antiseptic resistance (see Patent Document 3). However, due to changes in the required specifications due to the recent spread of clinical diagnostic drugs overseas, glycerol kinase has also been required to have higher heat resistance and preservative resistance, and further improvements have been desired.
JP-A-56-121484 JP 2004-121234 A JP 2007-195453 A H. Kalkkar, “Enzymologia”, 1937, Vol. 2, p47 C. Bublitz et al., “J. Biol. Chem.”, 1954, 211, p951. E. P. Kennedy, “Methods Enzymol.”, 1962, Vol. 5, p476 H. U. Bergmeyer et al., “Biochem.”, 1961, No. 333, p471. H. S. Huang et al., “J. Ferment. Bioeng.”, 1997, No. 83, p328. C. A. Sargent et al., “Hum. Mol. Genet.”, 1994, Volume 3, p1317. C. Holberg et al., “J. Gen. Microbiol.”, 1990, 136, p2367. P. Pavlik et al., “Curr. Genet.”, 1993, 24, p21. H. S. Huang et al., “Biochim. Biophys. Acta”, 1998, 1382, p186. S. Hayashi et al., “J. Biol. Chem.” 1967, Vol. 242, p1030. D. W. Pettigrew et al., “J. Biol. Chem.” 1988, 263, p135.

  The present invention has been made in view of the current state of the prior art, and an object thereof is to provide a glycerol kinase having high thermal stability in addition to high resistance to preservatives.

  As a result of intensive studies to achieve the above-described object, the present inventors have modified wild-type glycerol kinase having excellent antiseptic resistance by protein engineering, thereby providing a modified glycerol having high heat stability and antiseptic resistance. The present invention was completed by successfully obtaining a kinase.

  That is, according to the present invention, a modified glycerol kinase having improved thermal stability as compared with a wild-type glycerol kinase derived from a bacterium of the genus Cellulomonas, which is located at position 50 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and / or Or a modified glycerol kinase having an amino acid sequence introduced with amino acid substitutions at positions 137 and 138, and / or 163 and 164, and / or 274, and / or 386 and 388 Is provided.

  According to a preferred embodiment of the present invention, the amino acid substitution is selected from Q50E, (G137A + P138S), (N163T + T164I), L274M, (T386I + F388Y), (Q50E + L274M), (Q50E + T386I + F388Y), and (Q50E + L274M + T38F + T38F + T38 +).

  According to the present invention, a gene encoding the modified glycerol kinase according to any one of the above, a vector containing the gene, a transformant transformed with the vector, and culturing the transformant A method for producing the modified glycerol kinase is also provided.

  In addition, according to the present invention, there is also provided a neutral fat assay kit, a neutral fat sensor, or a neutral fat measurement method using any of the modified glycerol kinases described above.

  The modified glycerol kinase of the present invention is derived from a bacterium belonging to the genus Cellulomonas, and has an amino acid substitution introduced at a specific position, and thus exhibits high thermal stability in addition to high preservative resistance. Therefore, the modified glycerol kinase of the present invention can be stably stored for a long time in a solution state, and can be effectively used as a clinical test agent having excellent storage stability.

  The modified glycerol kinase of the present invention has improved stability by modifying protein-engineered wild-type glycerol kinase derived from a bacterium of the genus Cellulomonas that has high antiseptic resistance but poor thermal stability, Specifically, the thermal stability is improved by introducing an amino acid substitution at a specific position of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

  In the modified glycerol kinase of the present invention, amino acid substitutions are introduced at positions 50 and / or 137 and 138, and / or 163 and 164, and / or Or 274 and / or 386 and 388. The amino acid substitution to be introduced may be appropriately determined by experiment so that the thermal stability of the glycerol kinase after the introduction of the amino acid substitution is higher than that of the wild-type glycerol kinase. For example, Q50E, (G137A + P138S), (N163T + T164I) ), L274M, (T386I + F388Y), (Q50E + L274M), (Q50E + T386I + F388Y), and (Q50E + L274M + T386I + F388Y).

The state in which the thermal stability is improved in the present invention is, for example, after dissolving the modified protein (a) and the unmodified protein (b) in an appropriate buffer and storing them for a certain period at an appropriate temperature. Is a state in which the residual activity ratio of (a)> (b).
Here, the conditions for storing at an appropriate temperature for a certain period of time are, for example, accelerated (severe) test conditions such as “store at 60 ° C. for 15 minutes”. In addition, storage for 6 months or more may be selected under refrigerated conditions of 2 ° C. to 10 ° C., which are widely used as temperatures for long-term storage.
The appropriate buffer solution is not particularly limited as long as the type and concentration thereof are selected so as to have sufficient buffering ability in the pH range where glycerol kinase acts, but for example, 50 mM Tris buffer solution (pH 8.0), or , 50 mM PIPES-NaOH buffer (pH 7.0) and the like are selected. Assuming diagnostic use, the buffer may further contain a surfactant, salts, chelating agents, preservatives and the like.
The concentration of glycerol kinase in storage is not particularly limited, but is preferably selected from 0.2 to 30 U / ml assuming a concentration used for a normal diagnostic reagent. More preferably, it is 1-10 U / ml.

The method for producing the modified glycerol kinase of the present invention is not particularly limited, but it can be produced by the following procedure.
First, in accordance with the teachings of JP-A No. 2004-121234, chromosomal DNA is isolated from Cellulomonas sp. JCM2471 strain (available for purchase from the Research Institute for Biological Systems, RIKEN Biological Research Institute), and the nucleotide sequence of a known glycerol kinase is determined. The glycerol kinase gene is amplified by PCR using PCR primers designed based on the base sequence, and the base sequence shown in SEQ ID NO: 1 in the sequence listing (the sequence of the gene encoding the amino acid sequence shown in SEQ ID NO: 2) is obtained.

  Next, among the bases in this base sequence, the amino acid at the specific position of the amino acid sequence shown in SEQ ID NO: 2 is modified by modifying the base corresponding to the amino acid at the specific position in the amino acid sequence shown in SEQ ID NO: 2. A gene encoding the amino acid sequence into which the substitution is introduced is prepared. This base modification can be performed by, for example, using a commercially available kit (Transformer Mutagenesis Kit; manufactured by Clonetech, EXOIII / Mung Bean Deletion Kit; manufactured by Stratagene, Quick Change Site Mutated Kit, using Quick Change Site Mutation Kit). It can be carried out.

Next, the gene thus prepared is ligated with a suitable plasmid to prepare a recombinant vector, and a host microorganism is transformed with this vector to obtain a transformant that produces modified glycerol kinase. .
As a plasmid at this time, for example, when Escherichia coli is used as a host microorganism, pBluescript, pUC18 and the like can be used. Examples of host microorganisms that can be used include Escherichia coli DH5, Escherichia coli W3110, and Escherichia coli C600. To avoid contamination with host-derived glycerol kinase, it is disclosed in Japanese Patent Application Laid-Open No. 2007-195453. It is preferable to use Escherichia coli KM1 strain which is a glycerol kinase deficient strain. As a method for transferring the recombinant vector into the host microorganism, for example, when the host microorganism is a microorganism belonging to Escherichia coli, a method of transferring the recombinant DNA in the presence of calcium ions can be employed. . Instead, an electroporation method or a commercially available competent cell (for example, competent high JM109; manufactured by Toyobo) may be used.

  The microorganism, which is the transformant thus obtained, can stably produce a large amount of modified glycerol kinase 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 physiological properties of the host. Usually, liquid culture is performed in many cases, but industrially, aeration and agitation culture are performed. Is advantageous. As a nutrient source of the medium, those commonly used for culturing microorganisms are widely used. The carbon source is not particularly limited as long as it is an assimitable carbon compound, and for example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid and the like are used. The nitrogen source is not particularly limited as long as it is a nitrogen compound that can be used, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean cake alkaline decomposition product, 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 bacteria grow and produce modified glycerol kinase. In the case of Escherichia coli, it is preferably about 20 to 42 ° C. Although the culture time varies somewhat depending on conditions, the culture may be terminated at an appropriate time in consideration of the time when the modified glycerol kinase 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.

  After completion of the culture, the culture solution containing the cells producing the modified glycerol kinase in the culture can be collected as it is and subjected to purification treatment. Generally, however, the modified protein is present in the culture solution according to a conventional method. In that case, the modified glycerol kinase-containing solution may be separated from the microbial cells by filtration, centrifugation, etc., and then subjected to a purification treatment. When the modified protein is present in the microbial cells, the microbial cells are collected from the obtained culture by a technique such as 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 may be added to solubilize the modified glycerol kinase, and then separated and collected as an aqueous solution.

  In order to purify the modified glycerol kinase of the present invention from the thus obtained modified glycerol kinase-containing solution, the solution is concentrated, for example, under reduced pressure, membrane concentration, and further, salting out treatment such as ammonium sulfate, sodium sulfate, etc. Alternatively, the modified glycerol kinase in the solution may be precipitated by subjecting it to a fractional precipitation method using a hydrophilic organic solvent (eg, methanol, ethanol, acetone, etc.). Heating treatment, isoelectric point treatment, gel filtration with an adsorbent or gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography are also effective purification means.

  The modified glycerol kinase of the present invention purified as described above can be used as an alternative to the conventional glycerol kinase in the conventionally known neutral fat assay kit, neutral fat sensor, or neutral fat measurement method. The storability of these kits and sensors can be increased.

  Hereinafter, examples of the present invention will be described in detail. Of the reagents used in the examples, ATP was purchased from Roche Diagnostics, bovine serum albumin was purchased from Sigma-Aldrich, and 4 aminoantipyrine was purchased from Daiichi Chemicals. Glycerol-3-phosphate oxidase (code number G3O-301) and peroxidase (code number PEO-301) manufactured by Toyobo were used, and other reagents purchased from Nacalai Tesque were used. used.

In the examples, the activity of glycerol kinase was measured as follows. The activity of glycerol kinase was measured by the amount of glycerol-3-phosphate produced using glycerol as a substrate. 0.23 mM 4 aminoantipyrine, 1.5 mM phenol, 2 mM MgCl ₂ , 0.15 mM EDTA · 2Na, 0.2% bovine serum albumin, 4 mM ATP, 7 U / ml glycerol-3-phosphate oxidase, 5 U / ml A 100 mM HEPES buffer solution (pH 7.9) containing peroxidase was prepared and used as a stock solution for measurement. To 3 ml of this measurement stock solution, 0.1 ml of glycerol kinase enzyme solution was added and mixed. After preheating at 37 ° C. for about 5 minutes, 0.45 ml of 33 mM glycerin aqueous solution was added thereto, and after mixing, the absorbance at 500 nm was recorded with a spectrophotometer controlled at 37 ° C. for 3 to 4 minutes. The change in absorbance per minute was determined from the portion (ΔODtest). In the blind test, 0.1 ml of an enzyme diluent (20 mM potassium phosphate buffer solution containing 0.2% bovine serum albumin, pH 7.5) was used instead of the enzyme solution, and the same operation was carried out as described above. Absorbance change was determined (ΔOD blank). From the obtained change in absorbance, the enzyme activity of glycerol kinase was calculated based on the following formula. The amount of enzyme that phosphorylates 1 micromole of glycerol per minute under the above conditions was defined as 1 unit (U).
a formula:
Activity value (U / ml) = {(ΔODtest−ΔODblank) × 3.55 (ml) × dilution ratio} / {13.3 × 1/2 × 1.0 (cm) × 0.1 (ml)}
3.55 (ml): reaction liquid mixture amount 13.3: millimolar extinction coefficient when quinone dye is measured under the above measurement conditions 1/2: quinone dye formed from one molecule of hydrogen peroxide generated by enzyme reaction Is a factor of 1.0 molecule: cell optical path length 0.1 (ml): enzyme sample liquid volume

Example 1: Construction of expression plasmid pCGK14 of glycerol kinase The same amino acid sequence as that deduced from the DNA sequence encoding glycerol kinase derived from Cellulomonas sp. JCM2471 disclosed in Japanese Patent Application Laid-Open No. 2004-121234 A coding DNA sequence, which is modified so that the codon usage is optimal for expression in E. coli, and the recognition sequences for restriction enzymes NcoI and NotI are added to the 5 ′ end side and 3 ′ side, respectively. Synthesized artificially. A DNA fragment obtained by cleaving this synthetic DNA with NcoI and NotI and a plasmid pSE380 (manufactured by Invitrogen) cleaved with NcoI and NotI were reacted with a Ligation High kit at 16 ° C. for 1 hour and ligated. A recombinant plasmid pCGK14 was obtained, and a competent cell (manufactured by Toyobo Co., Ltd.) of Escherichia coli JM109 strain was transformed with this recombinant vector, and an LB agar medium containing 100 μg / ml ampicillin And transformed to 37 ° C. overnight.

  Subsequently, the obtained transformant was inoculated into an LB liquid medium (5 ml / 30 ml test tube preparation) containing 100 μg / ml ampicillin and cultured with shaking at 37 ° C. overnight. After completion of the culture, the cells were collected by centrifugation, and pCGK14 was purified using a MagExtractor-Plasmid-kit (manufactured by Toyobo). The base sequence corresponding to the open reading frame encoding the glycerol kinase gene of the recombinant plasmid pCGK14 is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of glycerol kinase deduced from the base sequence is shown in SEQ ID NO: 2 in the sequence listing. .

Example 2: Construction of a recombinant expression plasmid encoding a modified glycerol kinase (1)
(A) QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using the recombinant plasmid pCGK14 containing the glycerol kinase gene, a synthetic oligonucleotide described in SEQ ID NO: 3 in the sequence listing and a synthetic oligonucleotide complementary thereto. The recombinant plasmid (pCGK14M1) encoding a modified glycerol kinase in which the 50th glutamine of the amino acid sequence shown in SEQ ID NO: 2 is substituted with glutamic acid is subjected to a mutation treatment operation according to the protocol. ).
(B) In addition, using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pCGK14, a synthetic oligonucleotide described in SEQ ID NO: 4 in the sequence listing and a synthetic oligonucleotide complementary thereto, the same as above By the operation, a recombinant plasmid (pCGK14M2) encoding a modified glycerol kinase in which the 137th glycine and the 138th proline of the amino acid sequence shown in SEQ ID NO: 2 were respectively substituted with alanine and serine was obtained.
(C) In addition, using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pCGK14, a synthetic oligonucleotide described in SEQ ID NO: 5 in the sequence listing and a synthetic oligonucleotide complementary thereto, the same as above By the operation, a recombinant plasmid (pCGK14M3) encoding a modified glycerol kinase in which the 163rd asparagine and 164th threonine of the amino acid sequence shown in SEQ ID NO: 2 were respectively replaced with threonine and isoleucine was obtained.
(D) In addition, using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pCGK14, a synthetic oligonucleotide described in SEQ ID NO: 6 in the sequence listing and a synthetic oligonucleotide complementary thereto, the same as above By the operation, a recombinant plasmid (pCGK14M4) encoding a modified glycerol kinase in which the 274th leucine in the amino acid sequence shown in SEQ ID NO: 2 was replaced with methionine was obtained.
(E) In addition, using QuickChangeTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) using pCGK14, the synthetic oligonucleotide described in SEQ ID NO: 7 in the sequence listing and a synthetic oligonucleotide complementary thereto, the same as above By the operation, a recombinant plasmid (pCGK14M5) encoding a modified glycerol kinase in which 386 threonine and 388 th phenylalanine of the amino acid sequence shown in SEQ ID NO: 2 were substituted with isoleucine and tyrosine, respectively, was obtained.

Example 3: Production of modified glycerol kinase (1)
Recombinant plasmids pCGK14M1, pCGK14M2, pCGK14M3, pCGK14M4, and pCGK14M5, and a glycerol kinase-deficient Escherichia coli KM1 strain (see Japanese Patent Application Laid-Open No. 11-9279) was obtained by electroporation using a gene pulser. Each transformant was obtained by transformation.

  Dispense 5 ml of Terrific medium into a 20 ml Sakaguchi flask, autoclave at 121 ° C. for 20 minutes, allow to cool, and then separate ampicillin and isopropyl-β-D-thiogalactoside that were separately sterile filtered to a final concentration of 100 μl / ml. It added so that it might become 1 mM. 50 μl of the culture solution of each transformant previously cultured at 37 ° C. for 12 hours in LB medium containing 100 μl / ml ampicillin was inoculated into this medium, and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, the cells are collected by centrifugation, suspended in 50 mM potassium phosphate buffer (pH 7.5), crushed with ultrasonic waves, further centrifuged, and modified glycerol kinase CGKM1, A supernatant containing CGKM2, CGKM3, CGKM4, and CGKM5 was obtained as a crude enzyme solution.

Comparative Example 1: Production of wild type glycerol kinase As a comparative example, Escherichia coli KM1 strain was transformed with pCGK14, the transformant was cultured in the same manner as in Example 3, and glycerol kinase was extracted from the culture solution. Wild type glycerol kinase was obtained.

Example 4: Evaluation of modified glycerol kinase (1)
After heat-treating the modified glycerol kinase obtained in Example 3 (CGKM1, CGKM2, CGKM3, CGKM4, CGKM5) and the wild-type glycerol kinase obtained in Comparative Example 1 for 15 minutes each in the range of 40 ° C to 70 ° C. The residual enzyme activity rate (%) was measured, and the temperature at which the residual activity rate reached 50% (Tm (° C.)) was calculated from the plot of the residual activity rate by each temperature treatment. The results are shown in Table 1 below. As can be seen from Table 1, it was confirmed that the modified glycerol kinase obtained in Example 3 had improved thermal stability compared to the wild-type glycerol kinase before modification.

Example 5: Construction of recombinant plasmid encoding modified glycerol kinase (2)
(A) Using the recombinant plasmid pCGK14M1 encoding the modified glycerol kinase obtained in Example 2, the synthetic oligonucleotide described in SEQ ID NO: 6 in the sequence listing and a synthetic oligonucleotide complementary thereto, Example 2 By the same operation, a recombinant plasmid (pCGK14M6) encoding a modified glycerol kinase in which the 50th glutamine and 274th leucine of the amino acid sequence shown in SEQ ID NO: 2 were respectively replaced with glutamic acid and methionine was obtained.
(B) In addition, by using pCGK14M1, a synthetic oligonucleotide described in SEQ ID NO: 7 in the sequence listing and a synthetic oligonucleotide complementary thereto, by the same operation as in Example 2, the amino acid sequence 50 described in SEQ ID NO: 2 A recombinant plasmid (pCGK14M7) encoding a modified glycerol kinase in which the first glutamine, the 386th threonine and the 388th phenylalanine were replaced by glutamic acid, isoleucine and tyrosine, respectively, was obtained.
(C) In addition, using the pCGK14M7 obtained in (b) above, the synthetic oligonucleotide described in SEQ ID NO: 6 in the sequence listing and the synthetic oligonucleotide complementary thereto, 2. A recombinant plasmid (pCGK14M8) encoding a modified glycerol kinase in which the 50th glutamine, 274th leucine, 386th threonine and 388th phenylalanine of the amino acid sequence described in 2 are substituted with glutamic acid, methionine, isoleucine and tyrosine, respectively. ).

Example 6: Production of modified glycerol kinase (2)
A transformant was obtained by transforming Escherichia coli KM1 strain (see JP-A-11-9279) with the recombinant plasmids pCGK14M6, pCGK14M7, and pCGK14M8 in the same manner as in Example 3. By culturing and extracting in the same procedure as in 3, crude enzyme preparations of each modified glycerol kinase CGKM6, CGKM7, and CGKM8 were obtained.

Example 7: Evaluation of modified glycerol kinase (2)
Each of the modified glycerol kinases (CGKM6, CGKM7, and CGKM8) obtained in Example 6 was measured for the remaining enzyme activity (%) after heat treatment for 15 minutes in the range of 40 ° C to 70 ° C. The temperature (Tm (° C.)) at which the residual activity rate becomes 50% was calculated from the plot of the residual activity rate. The results are shown in Table 2 together with Tm data of modified glycerol kinases CGKM1, CGKM4, and CGKM5 obtained in Example 3 and Tm of wild type glycerol kinase obtained in Comparative Example 1. As can be seen from Table 2, the modified glycerol kinases CGKM6, CGKM7, and CGKM8 obtained in Example 6 are compared with the wild-type glycerol kinase before modification and the modified glycerol kinases CGKM1, CGKM4, and CGKM5 obtained in Example 3. It was confirmed that the thermal stability was improved.

  Since the glycerol kinase of the present invention has high heat stability in addition to high preservative resistance, clinical tests such as a neutral fat assay kit and a neutral fat sensor that require long-term stable storage of glycerol kinase in a liquid state It is a great place to contribute in the field of medicine.

SEQ ID NO: 1 is the sequence of the glycerol kinase gene contained in the plasmid pCGK14 prepared in Example 1.
SEQ ID NO: 2 is the amino acid sequence of glycerol kinase encoded by the gene of SEQ ID NO: 1.
SEQ ID NOs: 3 to 7 are the synthetic oligonucleotide sequences used for the mutation treatment in Example 2 or 5.

Claims (9)

  A modified glycerol kinase having improved thermal stability compared to wild-type glycerol kinase derived from a bacterium of the genus Cellulomonas, wherein position 50 and / or position 137 and position 138 of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and A modified glycerol kinase characterized by having an amino acid sequence introduced with amino acid substitutions at positions 163 and 164, and / or 274, and / or 386 and 388.   Amino acid substitutions are Q50E, (G137A + P138S), (N163T + T164I), L274M, (T386I + F388Y), (Q50E + L274M), (Q50E + T386I + F388Y), and (Q50E + L274M + T386I + F388) Modified glycerol kinase.   A gene encoding the modified glycerol kinase according to claim 1 or 2.   A recombinant vector comprising the gene according to claim 3.   A transformant transformed with the recombinant vector according to claim 4.   A method for producing a modified glycerol kinase, comprising culturing the transformant according to claim 5.   A neutral fat assay kit using glycerol kinase, wherein the modified glycerol kinase according to claim 1 or 2 is used as glycerol kinase.   A neutral fat sensor using glycerol kinase, wherein the modified glycerol kinase according to claim 1 or 2 is used as glycerol kinase.   A method for measuring neutral fat using glycerol kinase, wherein the modified glycerol kinase according to claim 1 or 2 is used as glycerol kinase.