JPS6262151B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glycerol kinase, which comprises culturing a glycerol kinase-producing strain belonging to the genus Arthrobacter and collecting glycerol kinase from the culture.

Glycerol kinase (Glycerol kinase.E.
C.2.7.1.30, hereinafter abbreviated as GK) is glycerol +
It is an enzyme that catalyzes the reaction of ATP → L → α-glycerophosphate + ADP and is used for quantifying glycerol, triglyceride, and phospholipids in serum.

Microorganisms that conventionally produce GK include Candida, Aerobacter, Propionibacterium, Acetobacter, Streptomyces,
Known species include Bacillus, Cellulomonas, and Corynebacterium.

As a result of searching for microorganisms that produce GK industrially at low cost, the present inventors found that strains belonging to the genus Arthrobacter, which is a different genus from the various microorganisms mentioned above, produce a significant amount of GK. I found it.

The present invention was completed based on the above knowledge, and involves culturing a bacterial strain belonging to the genus Arthrobacter and having the ability to produce GK in a medium, and
This invention relates to a method for producing GK, which is characterized by collecting GK.

The strain belonging to the genus Arthrobacter used in the production of GK in the present invention is, for example, Arthrobacter Iuteus.
Examples include ATCC21606 and Arthrobacter artrocyaneus IAM12339, but in addition to these bacteria, any strain belonging to the genus Arthrobacter and having GK-producing ability can be used in the method of the present invention.

As a method for culturing the bacterial strain in the method of the present invention, a conventional culture method for bacteria can be applied. As a nutrient source, a wide variety of commonly used nutrients can be used. For example, as a carbon source, sugars such as glucose, sucrose, lactose, xylose, and maltose, sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, acetic acid, and citric acid, or alcohols such as ethanol and glycerol are used. Ru. The nitrogen source may be any available nitrogen compound, such as yeast extract,
Peptone, polypeptone, meat extract, soybean flour, etc. are used. Other salts such as phosphates, sulfates, sodium, potassium, magnesium, manganese, and iron are used as necessary.

The culture temperature may be any temperature within the range that allows the bacteria to grow and produce GK, but preferably 25~25°C.
30℃ is good. In addition, as for the culture time, it is sufficient to finish the culture at the time when the maximum yield of GK is reached, which is usually 16 to 28 hours.
It takes about an hour.

The strain used in the present invention can be made to produce a larger amount of GK by adding glycerol or glycerols to the culture medium.

GK can be collected from the culture thus obtained by appropriately combining known methods. For example, a culture solution is centrifuged to collect a bacterial strain, then the bacterial cells are disrupted by various mechanical methods, and further centrifuged to obtain a clear enzyme solution. After removing impurities from this enzyme solution by salting out with ammonium sulfate or the like, it is purified by conventional methods such as adsorption chromatography, ion exchange chromatography, and gel filtration.

Next, the method for measuring GK activity in the method of the present invention,
and Arthrobacter altocyaneus
We will describe the physical and chemical properties of GK produced by IAM12339.

Activity measurement method 3ml of 0.2M glycine-hydrazine buffer (2μM, containing _MgCl2 , PH9.8), 14mMNAD
(nicotinamide adenine dinucleotide) 0.1
ml, 0.1M ATP (adenosine triphosphate) 0.05
ml, glycerophosphate dehydrogenase (Boehringer: 1mg/ml, 170u/ml) 10μ,
1M glycerol 10μ and GK solution of the invention
30μ was mixed and reacted at 25°C, and the change in NAD was measured by the increase in absorbance at 340nm. Activity is 1 micromole of NAD per minute
The amount of enzyme required to reduce was defined as 1 unit.

Physical and chemical properties (1) Action This enzyme catalyzes at least the reaction shown in the following formula.

Glycerol + ATP → L-α-glycerophosphate + ADP (2) Substrate specificity 0.2M phosphate buffer (PH7.5),
10mM ATP 0.1ml, 3mM phosphoenolpyruvate 0.1ml, 2mM NADH 0.1ml, 0.1M
Mix 20μ of MgCl ₂ , 10μ of pyruvate kinase-lactate dehydrogenase mixture (manufactured by Boehringer), and 0.1ml of 0.2M glycerol, 0.2M dihydroxyacetone or DL-glyceraldehyde as a substrate, and add this enzyme solution to the final solution. After the volume was reduced to 1 ml, the reaction was carried out at 25°C and the rate of decrease in NADH was measured. The relative activity of each substrate is as follows.

Substrate Relative activity Glycerol 100% Dihydroxyacetone 97% DL-glyceraldehyde 55% (3) Optimal PH The optimal PH of this enzyme is around PH9.5-10 as shown in Figure 1.

(4) PH stability The PH stability of this enzyme is PH5.5 to 9.5 when treated at 37°C for 1 hour, as shown in Figure 2.

(5) Thermostability This enzyme has a pH of
7.0, stable at temperatures below 45°C in a 15 minute treatment.

(6) Optimal temperature The optimal temperature for this enzyme is around 50°C as shown in Figure 4 under the condition of pH 7.0.

(7) Molecular weight Approximately 82,000 (by Cephadex G-100 gel filtration) (8) Isoelectric point around PH4.3 (by focused electrophoresis using ampholine) (9) Substrate affinity Michaelis constant (Km) for glycerol of this enzyme value) is 3.5×10 ^-5 M,
The Km value for ATP was 3.7×10 ^-5 M.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Culture medium (PH7.0) with the composition of 0.5% glycerol, 0.5% polypeptone, 0.3% yeast extract, and 0.02% antifoaming agent Adekanol LG-126 (manufactured by Asahi Denka) 2 to 3
In a jar of water, heat at 120℃, 20℃.
After autoclaving for 30 minutes, inoculation with Arthrobacter althrocyaneus IAM12339 was carried out for 30 minutes.
Cultured at ℃ for 20 hours.

The resulting culture solution was centrifuged to collect bacterial cells, which were suspended in 20mM phosphate buffer (PH7.0), crushed using Cellmil (manufactured by MS Kiki Co., Ltd.), and crude enzymes were extracted by centrifugation. 150 ml of liquid was obtained. The activity of this enzyme solution was 8.8 U/ml. After that, ammonium sulfate
The precipitated fractions at 20 to 60% saturation were collected, dissolved in the same phosphate buffer as above, and then dialyzed against the same buffer overnight.

The enzyme solution was transferred to an aminohexyl sepharose column (130ml) equilibrated with 20mM phosphate buffer in advance.
I passed it through (Yong). This enzyme adsorbed on the column
Elution was performed with 20mM phosphate buffer (PH7.0) containing 0.4M NaCl. Transfer 130ml of eluate to Hollow Eye Bar HI6000
It was desalted and concentrated using an ultrafiltration membrane (manufactured by Asahi Kasei), and then equilibrated with 20mM phosphate buffer (PH7.0).
It was passed through a DEAE-Sepharose column (150 ml volume).

Elution was performed using a linear concentration gradient method using 0 to 0.5M NaCl, and active fractions were collected. This was desalted and concentrated using the hollow eye bar, and then freeze-dried. In this way, 15 mg of GK with a specific activity of 30 U/mg was obtained.

Example 2 Arthrobacter luteus ATCC21606 was cultured in the same manner as in Example 1, and 170 ml of crude enzyme extract was obtained by disrupting the cells. The activity of this enzyme solution was 3.7 U/ml.

[Brief explanation of the drawing]

The first is a diagram showing the optimal PH of glycerol kinase produced by Arthrobacter althrocyaneus IAM12339 in the method of the present invention; similarly, Figure 2 shows the PH stability, and Figure 3 shows the thermostability.
FIG. 4 is a diagram showing the optimum temperature.

Claims (1)

[Claims] 1. A method for producing glycerol kinase, which comprises culturing a strain belonging to the genus Arthrobacter and having the ability to produce glycerol kinase, and collecting glycerol kinase from the culture.