JPH08131165A - New glycerol kinase and its production - Google Patents

Abstract

PURPOSE: To obtain the subject new enzyme having specific thermal stability, useful as e.g. a stable liquefied clinical diagnostic agent for glyceride or lipase activity assay, by culturing glyserol kinase-producing bacteria belonging to Flavobacterium. CONSTITUTION: Glycerol kinase-producing bacterum belonging to Flavobacterium [e.g. Flavobacterium meningosepticum GK54 (FERM BP-4868) strain] is subjected to seed culture and then transferred to a defoaming agent-contg. medium in a jar fermenter, where the microbes are cultured at 28 deg.C for 22hr the resultant cultured product is centrifuged to recover the microbes, which are then suspended in a 20mM tris-hydrochloric acid buffer solution (pH8.6) to which lysozyme is added to effect bacteriolysis; the resultant system is centriguged and the supernatant formed is purified by e.g. ion exchange chromatography, hydrophobic chromatographpy, etc., thus obtaining the objective new glycerol kinase retaining >=80% of its activity even after treated at 70 deg.C for 10min, useful for e.g. assaying glyceride or lipase activity and enabling to be used as a highly thermally stable liquefied reagent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is characterized by retaining 80% or more of activity after heat treatment at 70 ° C. for 10 minutes.
And Flavobacterium
The present invention relates to a method for producing glycerol kinase, which comprises culturing a glycerol kinase-producing bacterium belonging to the genus um) in a medium and collecting glycerol kinase from the culture.

[0002]

BACKGROUND ART Glycerol kinase uses glycerol as a substrate from 1 mol of glycerol and 1 mol of ATP to 1 mol.
An enzyme that catalyzes a reaction that produces moles of ADP and 1 mole of glycerol-3-phosphate (Enzyme No. 2.7.1.
30) (Enzyme Handbook 341-
Page 342, Asakura Shoten Co., Ltd., published in 1982)
As a glycerol kinase-producing bacterium, conventionally, Streptomyces canus (JP-A-55-162987) and Cellulomonas sp. (JP-A-56-1214) have been used.
84), Bacillus stearothermophilus (JP-A-53-26389), Arthrobacter sp. (JP-A-59-2688), and the like, and are in the field of clinical diagnostic agents. Used in.

Further, the present inventors have screened excellent glycerol kinases in the field of clinical diagnostics, and found that Serratia macesense IFO-3736 strain, Pseudomonas fluorescens IFO-3925 strain,
When the Pseudomonas putida IFO-12653 strain was cultured in the medium of Reference Example described later,
It was found that glycerol kinase was produced at a culture titer of 5 U / ml, 0.8 U / ml, and 1.30 U / ml, but none of them was excellent in Km value and thermostability.

[0004]

Glycerol kinases have traditionally been used in the field of clinical diagnostics, for example, in combination with glycerol-3-phosphate oxidase or glycerol-3-phosphate dehydrogenase in the presence of lipase or glyceride to detect glyceride or lipase activity. It has been used as a measuring reagent, and has also been used as a measuring reagent for glycerol by the same enzymatic reaction.

However, in recent years, in the field of clinical diagnostic agents, it has been required to make a reagent in a liquid state, not a freeze-dried state, and a glycerol kinase or an industrial product having excellent storage stability that can be used as a liquefied reagent. It has been desired to develop a glycerol kinase having excellent thermostability that can be used for production.

[0006]

[Means for Solving the Problems] The inventors of the present invention have identified a glycerol kinase having excellent thermostability and, more preferably, stability that can be applied to a liquefying reagent, and a production method capable of industrial production thereof. As a result of repeated earnest research, microorganisms identified as the Flavobacterium meningosepticum GK-54 strain isolated from the field soil of Ohi-cho, Takata-gun, Shizuoka Prefecture have excellent stability and are capable of producing glycerol kinase. The present invention has been completed for the first time by having found that

The present invention has been completed based on the above findings, and a glycerol kinase having good thermostability, which retains 80% or more of activity by heat treatment at 70 ° C. for 10 minutes, and preferably a stabilizer. Without addition, glycerol kinase with good storage stability that retains 80% or more activity at 37 ° C for 2 weeks and glycerol kinase-producing bacteria belonging to the genus Flavobacterium are cultured in a medium, and glycerol kinase is collected from the culture. And a method for producing glycerol kinase.

Hereinafter, the present invention will be described in detail. First, the glycerol kinase-producing bacterium of the present invention is not limited at all as long as it is a microorganism having the ability to produce glycerol kinase belonging to the genus Flavobacterium, and it may be a variant or mutant strain having the ability to produce glycerol kinase. The GK-54 strain belonging to the genus Flavobacterium is preferable. The mycological properties of this strain are as follows.

In the identification of this strain, the identification experiment is a guide for identifying medical bacteria (2nd edition), Microbio.
The method was carried out in accordance with logical Methods (Vol. 3), and the experimental results were used as a guide for identifying medical bacteria (2nd edition), Ber.
gay's Manual of System
c Bacterology Vol. 1, Vol.
2, Vol. 3, Int. J. Syst. Bacter
Identification was performed in comparison with iology.

Characteristics of growth Normal agar plate medium It grows well on a line. The surface is smooth. The edges are round. It exhibits a yellow to orange color. Opacity. There is gloss. Ordinary agar slope medium It grows well on the line. The surface is smooth. The edges are round. It exhibits a yellow to orange color. Opacity. There is gloss. Liquid medium Good growth and uniform growth. Litmus milk and BCP media Litmus was not reduced but liquefaction and coagulation were observed. GC mol% of DNA 35.7 ± 1.0% (HPLC) Characteristics of main isoprenoid quinone MK-6 morphology It is a long rod with a round end and a size of 0.4-
0.7 × 1.8 to 2.5 μm. There is no motility. No spore formation. Physiological and biochemical properties Gram stain-KOH reaction test + anti-acid stain-OF test (Hugh-Leifson) 0 OF test (NH ₄ H ₂ PO _{4 for} N source) 0 Aerobic growth + Anaerobic growth- Growth temperature 12.3 ° C-16.0 ° C + 40.4 ° C + 43.6 ° C- Optimum temperature is 25 to 39 ° C Salt tolerance 0% + 3.0% + 5.0%-Growth pH 4.40- 4.76 + 10.41 + 10.80- Optimal pH is 7.5-8.0 Gelatin decomposition + Starch decomposition-Casein decomposition + Esculin decomposition + Cellulose decomposition-Tyrosine decomposition-Arginine decomposition-Tween80 decomposition-Catalase production + Oxidase production + phosphatase production + urease production (SSR) -urease production (Chris.)-Indole production-hydrogen sulfide production ( TSI) -DNase production + acetoin production-MR test-LV reaction test-Nitrate reduction test-Nitrite reduction test + Availability in Simmons medium Citrate-malate-maleate-malonate-propionate -Gluconate-Succinate-Availability in Kristensen medium Citrate (+) Malate-Maleate-Malonate-Propionate + Gluconate-Succinate-Gas production from glucose -Production of acid from sugar Adonitol-L (+)-arabinose-cellobiose-dulcitol-meso-erythritol-fructose + D-fucose + D-galactose-D-glucose + glycerol + inositol-inulin-lactose + maltose + mannitol + mannitol. Sucrose + melezitose-melibiose + raffinose-L (+)-rhamnose-D-ribose-salicin-L-sorbose-sorbitol-starch + sucrose-trehalose + D-xylose-ethanol (3%) + pigment production fluorescein is produced. Accumulation of PHB-Use of inorganic nitrogen source Nitrate-Ammonium salt + As described above, the main characteristics of this strain are Gram-negative bacilli that are non-motile and positive for catalase, oxidase, and phosphatase, which oxidatively decompose glucose, Produces acid,
GC mol% is 35.7 ± 1.0%, quinone type is M
K-6, non-spore-forming, whose main characteristics are Berg
ey's Manual of Systematic
Bacteriology Vol. 1, Vol.
2, Vol. As a result of comparison with 3, the microorganism was found to belong to the genus Flavobacterium. Therefore, four strains similar to this strain were selected from the genus Flavobacterium and compared with this strain. A: Flavobacterium
erium breve). B: Flavobacterium gleum
terium gleum). C: Flavobacterium indologenes (Flavo
Bacterium indologenes). D: Flavobacterium meningosepticum (Fl
avobacterium meningosesepti
cum).

ABCD Main bacterium GC content (%) 32.4 37.6 33.8 37.0 35.7 ± 0.6 ± 1.0 ± 0.5 ± 1.0 Casein degradation + + + + + Esculin degradation − + + + + Indole production + + + d − Nitrate reduction − d − d − Urease production − d − d − 5 ° C. growth − − + − − Sugar assimilation glucose + + + + + Arabinose − + − d − cellobiose − − − − − ethanol − − − d + fructose − + − d + glycerol − d − d + lactose − − − d + maltose d + + + + + mannitol − − − d + raffinose −−−−− Salicin −−−−− Saccharose −−−−− Trehalose − + − d + xylose−d −−− (+: 90% or more positive, d: 1 1-89% is positive,-: 90% or more is negative) From the comparison of properties, it was determined that this strain belongs to Flavobacterium meningosepticum. In addition, DNA-
The DNA homology was measured by the DNA hybridization method. As a measuring method, quantitative measurement was performed spectroscopically by a fluorescence method using a microplate. The reaction temperature for hybridization was 40 ° C. by calculating the melting temperature of DNA from the GC content. The following strains were used for comparison. No. 1 strain: Flavobacterium bleb IFO-14
93 strains: type culture (Flavobacteri
um breve IFO-1493: type culture). No. 2 strains: Flavobacterium gureum IFO-1
5054 strain: type culture (Flavobacter
ium gleum IFO-15054: type culture). No. 3 strains: Flavobacterium indologenes IF
O-14944 strain: type culture (Flavova
cterium indologenes IFO-1
4944: Type culture). No. 4 strains: Flavobacterium meningosepticum IFO-12535 strains: type culture (Flav
Obacterium meningosepticu
mIFO-12535: Type Culture) From the above results, this strain has a DNA homology of Flavobacterium meningosepticum IFO-12535 strain:
Type culture (F. meningoseepticu
(m IFO-12535: type culture) and the determination criterion is 70% or more, so it was determined to be the same species together with the above findings, and this strain was identified and named Flavobacterium meningosepticum GK-54 strain. , Institute of Biotechnology, Institute of Industrial Science, contract number FERM BP-4868
Was deposited as.

Next, glycerol kinase was purified from a glycerol kinase-producing bacterium belonging to the genus Flavobacterium and various properties were examined. In carrying out the present invention, either liquid culture or solid culture can be used as the culture form, but industrially, aeration and agitation culture is advantageous. As a culture source to be used, in addition to carbon sources, nitrogen sources, inorganic salts and other trace nutrients generally used for culturing microorganisms, any nutrient source that can be utilized by a microorganism belonging to the genus Flavobacterium can be used.

As the carbon source, for example, glucose, fructose, sucrose, xylose, maltose, glycerol, dextrin, starch, amino acids, etc., as well as fatty acids, oils and fats, organic acids, etc. may be used alone or in combination. As the nitrogen source, for example, an inorganic nitrogen source,
Any organic nitrogen source can be used, and examples of the inorganic nutrient source include ammonium sulfate, ammonium nitrate, urea, sodium nitrate, ammonium chloride and the like. As the organic nitrogen source, for example, soybean, rice, corn, flour such as wheat, corn steep liquor, peptone, meat extract,
Examples include casein, amino acids, yeast extract, bacto soyton and the like.

Examples of inorganic salts and micronutrients include salts such as phosphoric acid, magnesium, potassium, iron, calcium and zinc, as well as growth of bacteria such as vitamins, nonionic surfactants and defoamers, and glycerol kinase. Any material that promotes production can be used as needed. The culture is carried out under aerobic conditions, and the culture temperature may be in the range where the bacteria grow and glycerol kinase is produced, and is usually 15 ° C to 37 ° C, preferably 25 ° C to 35 ° C. The culturing time varies depending on the conditions, but it may be cultivated until the time when glycerol kinase is most produced, and is usually about 24 to 100 hours.

Glycerol kinase is mainly contained and accumulated in the cells, and it may be extracted from the cells. As an example of the extraction method of glycerol kinase, the culture is first subjected to solid-liquid separation, the obtained wet cells are dispersed in a solution such as a phosphate buffer solution or a Tris-hydrochloric acid buffer solution, lysozyme treatment, ultrasonic treatment, and French treatment. By appropriately selecting and combining various bacterial cell treatment means such as press treatment and dynomill treatment,
A crude glycerol kinase containing solution is obtained.

Purified glycerol kinase is obtained from the crude glycerol kinase-containing liquid by a known means for isolating and purifying proteins and enzymes. For example, glycerol kinase is precipitated and recovered by applying a fractional precipitation method using an organic solvent such as acetone, methanol, and ethanol, a salting-out method using ammonium sulfate, sodium chloride, etc. to a crude glycerol kinase-containing liquid.

Further, after subjecting this precipitate to dialysis and isoelectric point precipitation as needed, a column using an ion exchanger, a gel filter, an adsorbent, etc. until a single band is shown by electrophoresis or the like. Purify by chromatography or the like. Also,
When the purification degree of glycerol kinase is improved by appropriately combining these methods, they can be appropriately combined.

The enzyme obtained by these methods can be used as a stabilizer in a liquid form or by a method of ultrafiltration concentration and lyophilization without or with addition of various salts, saccharides, proteins, lipids, surfactants and the like. Solid glycerol kinase can be obtained, and may be appropriately lyophilized. In this case, sucrose as a stabilizer,
0.5-10% mannitol, salt, albumin, etc.
You may add about.

Next, the physicochemical properties of glycerol kinase obtained by the present invention and the method for measuring enzyme activity will be described. Glycerol kinase activity assay 0.2 M Tris-HCl buffer (pH 8.0) 0.2
ml, 5% glycerol 0.1 ml, 0.1M A
TP 0.02 ml, 0.1M MgCl ₂ 0.04
ml, 45 U / ml peroxidase (manufactured by Sigma)
0.1 ml, 0.3% 4-aminoantipyrine 0.1 ml, 0.25% phenol 0.1 ml, 1
0.2 ml of 00 U / ml glycerophosphate oxidase (manufactured by Asahi Kasei) and 0.14 ml of distilled water
After preliminarily heating 1 ml of the resulting reaction solution at 37 ° C. for 1 minute, 0.02 ml of the enzyme solution is added and reacted for 10 minutes. After the reaction, the reaction was stopped by adding 2 ml of 0.5% SDS, and within 5 minutes, a cell having a layer length of 1 cm was used and a wavelength of 50 was used.
The absorbance at 0 nm is measured (As). Further, as a blind test, the absorbance is measured by performing the same operation using 0.02 ml of distilled water instead of the enzyme solution (Ab). The absorbance (As) using this enzyme solution and the blind absorbance (Ab) are used. The enzyme activity is determined from the difference in absorbance (As-Ab). Enzyme activity 1
The unit is the amount of enzyme that produces 1 μmol of glycerophosphate per minute at 37 ° C., and the calculation formula is as follows.

[0020]

[Equation 1]

Physicochemical properties (1) Enzyme action: The enzyme action using glycerol as a substrate is shown below.

[0022]

Embedded image

(2) Substrate specificity: shows substrate specificity for glycerol. The specificity for various substrates is shown in Table 1.

[0024]

[Table 1]

(3) Km value: 3.9x10 ^-2 ± 0.5x
10 ^-2 mM (relative to glycerol) 5.3 x 10 ^-2
± 0.5 × 10 ^-2 mM (relative to ATP) (4) Isoelectric point: 4.4 ± 0.5 (by electrophoresis using carrier-ampholine) (5) Molecular weight: 145000 ± 5000 (TSK G-
(By gel filtration method with 3000SW), 50,000 ± 5
000 (by SDS polyacrylamide gel electrophoresis) (6) Optimum pH The optimum pH was determined according to the enzyme activity measuring method described above, and the results are shown in FIG. The pH range of 4.5 to 6.5 is 100 mM citrate buffer, pH 5.5 to 7.5.
Range is 100 mM Bis-Tris buffer, pH
The range of 6.5 to 7.5 is 100 mM phosphate buffer, p
The range of H7.0 to 8.5 is 100 mM Tris-hydrochloric acid buffer solution, and the range of pH 8.5 to 11.0 is the activity value when 100 mM glycine-sodium hydroxide buffer solution is used. The suitable pH was 7.5-8. (7) pH stability 100 mM various buffers (glycerol kinase 1 U / m
l) was treated at 37 ° C. for 60 minutes, and its residual activity was measured according to the above-mentioned enzyme activity measuring method. The result is shown in FIG. pH 0.5-4.0 is 100 mM Clark Andrus buffer, pH 3.5-7.0 is 100 mM acetate buffer, pH 6.5-8.0 is 100 mM phosphate buffer, pH 8.0-9. 0.0 is 100 mM Tris-HCl buffer, pH 9.0-11.0 is 100 mM glycine-
Sodium hydroxide buffer was used. The best stability was shown in the pH range of 3-11. (8) Optimum temperature The optimum temperature was determined according to the method for measuring enzyme activity by changing the temperature in the range of 37 ° C to 95 ° C as shown in Fig. 3. Was 80 ° C. (8) Thermostability 100 mM Tris-hydrochloric acid buffer solution (pH 8.0) (glycerol kinase 1 U / ml) was heat-treated at each temperature for 10 minutes, and the residual activity was measured according to the above-mentioned enzyme activity measuring method. As a result, as shown in Fig. 4, it is 100% stable up to at least 60 ° C, and is 8 at 10 ° C for 10 minutes.
It retained a residual activity of 0% or more. (9) Effect of metal ion The results of examining the effect of various metal ions (1 mM) on the activity of this enzyme are shown in Table 2, and weak inhibition by calcium ion was observed.

[0026]

[Table 2]

(10) N-terminal amino acid sequence Met-Asp-Glu-Lys-Leu-Ile-L
eu-Ala-Leu-Asp-Glu-Gly-Th
r-Thr- (11) Storage stability For the purpose of dealing with a liquefaction reagent, 0.05% NaN ₃
20 mM Tris-HCl buffer (p) containing (preservative)
H7.5), the glycerol of the present invention was prepared to a concentration of 0.8 U / ml, and after 2 days at 37 ° C. and 1 week later,
After 2 weeks, 3 weeks, and 4 weeks, the residual activity was measured by the above-mentioned activity measurement method, and the results were 94%, 91%, and 81%, respectively.
%, 71% and 60% of the activity was retained. From this, this enzyme retained 80% or more activity at 37 ° C. for 2 weeks in the absence of a stabilizer (eg, BSA or saccharide).

From the above results, the properties of the glycerol kinase of the present invention and the conventionally known glycerol kinase are compared as follows. (Km value for ATP) This enzyme: 0.053 mM Streptomyces canas origin: 0.2 mM Cellulomonas sp. Origin: 0.43 mM Arthrobacter altranyaneus origin: 3.7 m
M (Thermal stability) The present enzyme: 80% or more of activity was retained by heat treatment at 70 ° C for 10 minutes.

Derived from Cellulomonas canus: pH 7.
Treatment at 5, 45 ° C. for 15 minutes retains 96% residual activity. It is completely deactivated by the treatment at 70 ° C. Derived from Arthrobacter Altorianius: pH 7.
Stable at a temperature of 45 ° C or lower after treatment for 0 to 15 minutes. From Bacillus stearothermophilus: 65 ° C, 10
After the heat treatment for 1 minute, the residual activity was 10% (FIG. 5). (Optimal temperature) This enzyme: 80 ° C. Cellulomonas sp. Origin: around 50 ° C. Arthrobacter sp. Origin: around 50 ° C. Bacillus stearothermophilus origin: 65 ° C. ± 5 ° C. The glycerol kinase of the present invention as described above Has a distinctly different property from the conventionally known enzymes, and has the most excellent thermal stability in particular.

[0030]

EXAMPLES Next, examples of the present invention will be described in detail.
The present invention is not limited to this.

[0031]

Example 1 Flavobacterium meningosepticum GK-54 strain was prepared by adding 1.0% glycerol, 3.0% meat extract, and 0.5% bactoisoton (manufactured by Difco).
After inoculating the medium (pH 7.0) consisting of 150 ml into a 500 ml Erlenmeyer flask, 28
Culturing was performed at 30 ° C. for 30 hours to obtain a seed culture solution.

Two seed cultures were added to each of four 20 l jar fermenters in a medium (pH 7.0) containing an antifoaming agent in the same composition medium as above, and cultured at 28 ° C. for 22 hours. After completion of the culture, the culture was centrifuged at 4000 rpm to collect 3.0 Kg of bacterial cells. To the cells, 9 l of 0.1% lysozyme solution containing 10 mM EDTA, 0.3% Triton X-100 and 20 mM Tris-hydrochloric acid buffer (pH 8.6) was added, and the mixture was reacted at 37 ° C for 90 minutes. And solubilized. Then, this is 2 at 4000 rpm.
Centrifuge for 0 minutes to remove insoluble matter and remove the supernatant 9 l (720
00U) was obtained.

Next, 90 ml of 5.0% protamine sulfate was added to the supernatant to remove the precipitate by centrifugation, and the supernatant was heated at 50 ° C. for 30 minutes to remove the precipitate by centrifugation.
The centrifugation supernatant was treated with 20 mM Tris-HCl buffer (pH
7.5) overnight dialysis, Q-Sepharose (2.7 x
30 cm) (Pharmacia) ion exchange chromatography was performed. Elution is 0 to 0.5 of sodium chloride
0.2 to 0.3M with a linear gradient of M
The sodium chloride elution fraction (29000 U) was collected.

Sodium chloride was dissolved in this enzyme solution to a concentration of 2.5 M, and octyl sepharose (2.6 × 20 cm) (manufactured by Pharmacia) was subjected to hydrophobic chromatography. Elution was performed with a linear gradient of 2.5 M to 0 M sodium chloride, and an elution fraction of 0 M sodium chloride (25000 U) was collected.
Then, this enzyme solution was added to 20 mM Tris-HCl buffer (p
It was dialyzed against H7.5) overnight and subjected to hydroxyapatite (1.6 × 26.5 cm) (Pentax) chromatography. Elution is performed with a linear gradient of 5-0.2 M phosphate buffer,
Elution fraction of 0.12 M phosphate buffer (16300 U)
Was recovered.

Then, the enzyme solution was dialyzed against 20 mM Tris-hydrochloric acid buffer solution (pH 7.5) overnight, and DEAE-Sepharose (1.6 × 20 cm) (Pharmacia).
Ion exchange chromatography was performed. Elution is 0
Performed by a linear gradient with 0.5 M sodium chloride to collect an elution fraction of 0.2 to 0.3 M sodium chloride (12000 U), dialyzed against 20 mM Tris-hydrochloric acid buffer (pH 7.5) overnight, BSA was added to 1% and freeze-dried to obtain purified glycerol kinase (12000U, 9.9U / mg).

The physicochemical properties of the glycerol kinase thus obtained are as described above.

[0037]

[Reference Example 1] Serratia macesense IFO-3736
The strain was inoculated into a 500 ml Erlenmeyer flask in which 150 ml of a medium (pH 7.0) consisting of 2.0% glycerol and 2.0% yeast extract was inoculated, and at 28 ° C.
Culture was performed for 30 hours. After completing the culture,
Centrifugation was carried out at rpm to collect 30 mg of bacterial cells. The obtained cells were treated with 10 mM Tris-hydrochloric acid buffer solution (pH 7.5) 30
It was suspended in ml and solubilized by ultrasonication.

Then, the solubilized solution was centrifuged at 16000 rpm to obtain 29 ml (70 U) of a supernatant in which insoluble matter was removed. The centrifuged supernatant was then dialyzed against 8 l of 10 mM Tris-HCl buffer overnight, followed by Q-Sepharose (2x8).
cm) (Pharmacia) ion exchange chromatography was performed. Elution was performed with a 0 to 0.5 M linear gradient of sodium chloride, and a 0.2 to 0.3 M sodium chloride elution fraction (67 U) was collected to obtain crude glycerol kinase (67 U, 25 U / mg). Physicochemical properties (1) Enzyme action and substrate specificity: The enzyme action and substrate specificity of this enzyme for glycerol are flavobacterium meningosepticum GK-54 (FERM B
P-4868) strain. (2) Km value: 2.4x10 ^-2 ± 0.5x10 ^-2 mM
(Relative to glycerol), 5.0x10 ^-1 ± 0.5x
10 ^-1 mM (relative to ATP) (3) Optimum pH: 7.5 (50 mM PIPES buffer)

[0039]

[Reference example 2] Pseudomonas fluorescens IF
The O-3925 strain was inoculated into a 500 ml Erlenmeyer flask in which 150 ml of a medium (pH 7.0) consisting of 2.0% glycerol and 2.0% yeast extract was inoculated, and then at 28 ° C. for 30 hours. Culture was performed.

After the completion of the culture, the culture was centrifuged at 8000 rpm to recover 30 mg of bacterial cells. 10m of the obtained fungus body
It was suspended in 30 ml of M Tris-hydrochloric acid buffer (pH 7.5) and solubilized by ultrasonication. Then, the solubilized solution was centrifuged at 16000 rpm to obtain 29 ml (130 U) of a supernatant in which insoluble matter was removed. Then, centrifuge the supernatant 8 times.
After dialysis against 1 l of 10 mM Tris-HCl buffer overnight, Q-sepharose (2 × 8 cm) (Pharmacia) ion exchange chromatography was performed. Elution was performed with a 0 to 0.5 M linear gradient of sodium chloride, and a 0.2 to 0.3 M sodium chloride elution fraction (114 U) was collected, and the crude glycerol kinase (1
14 U, 30 U / mg) was obtained. Physicochemical properties (1) Enzyme action and substrate specificity: The enzyme action and substrate specificity of this enzyme for glycerol are flavobacterium meningosepticum GK-54 (FERM B
P-4868) strain. (2) Km value: 2.0x10 ^-2 ± 0.5x10 ^-2 mM
2.4 ± 0.5 mM (for glycerol) (AT
(For P) (3) Optimum pH: 7.5 (50 mM PIPES buffer)

[0041]

[Reference Example 3] Pseudomonas Puchida IFO-126
The 53 strain was inoculated into a 500 ml Erlenmeyer flask in which 150 ml of a medium (pH 7.0) consisting of 2.0% glycerol and 2.0% yeast extract was inoculated.
Culturing was performed at 30 ° C. for 30 hours. After completion of the culture,
The cells were centrifuged at 00 rpm to collect 30 mg of the bacterial cells. The obtained cells were treated with 10 mM Tris-HCl buffer (pH 7.5).
It was suspended in 30 ml and solubilized by ultrasonication. Then, the solubilized solution was centrifuged at 16000 rpm to obtain 29 ml (190 U) of a supernatant in which insoluble matter was removed.

Then, the centrifuged supernatant was dialyzed against 8 l of 10 mM Tris-hydrochloric acid buffer overnight, and then subjected to Q-sepharose (2 × 8 cm) (Pharmacia) ion exchange chromatography. Elution is 0 to 0 for sodium chloride
Performed with a 0.5 M linear gradient, 0.2-
The 0.3 M sodium chloride elution fraction (110 U) was collected and crude glycerol kinase (110 U, 30 U / m
g) was obtained. Physicochemical properties (1) Enzyme action and substrate specificity: The enzyme action and substrate specificity of this enzyme for glycerol are flavobacterium meningosepticum GK-54 (FERM B
P-4868) strain. (2) Km value: 2.0x10 ^-2 ± 0.5x10 ^-2 mM
(For glycerol), 2.0 ± 0.5 mM (AT
(For P) (3) Optimum pH: 7.5 (50 mM PIPES buffer)

[0043]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a glycerol kinase having a good thermostability and a method for producing a microbe thereof, which is useful for the measurement of glyceride and lipase activity using the present enzyme and has a high thermostability. It was possible to provide a measuring enzyme that enables a good liquefaction reagent.

[Brief description of drawings]

FIG. 1 shows the optimum p of the glycerol kinase of the present invention.
The H curve is shown.

FIG. 2 shows a pH stability curve of the glycerol kinase of the present invention.

FIG. 3 shows an optimum temperature curve of glycerol kinase of the present invention.

FIG. 4 shows a thermostable curve of the glycerol kinase of the present invention.

FIG. 5 shows thermostability curves of glycerol kinase of the present invention and glycerol kinase from Bacillus stearothermophilus.

Claims (4)

[Claims] 1. A glycerol kinase which retains an activity of 80% or more after heat treatment at 70 ° C. for 10 minutes. 2. The glycerol kinase according to claim 1, wherein glycerokinase retains an activity of 80% or more at 37 ° C. for 2 weeks in the absence of a stabilizer. 3. A method for producing glycerol kinase, which comprises culturing a glycerol kinase-producing bacterium belonging to the genus Flavobacterium in a medium and collecting glycerol kinase from the culture. 4. The method for producing glycerol kinase according to claim 3, wherein the glycerol kinase-producing bacterium belonging to the genus Flavobacterium is Flavobacterium meningosepticum GK-54 (FERM BP-4868) strain.