JPS6341558B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel and useful leucine dehydrogenase and a method for producing the same, and more specifically, it is used in measuring leucine aminopeptidase activity in clinical tests in the medical field. This invention relates to leucine dehydrogenase and its production method. Recently, enzymes have attracted attention for their excellent reaction, substrate, and optical specificity, which are characteristics of enzymatic reactions, and are widely used as catalysts in medical analysis, food analysis, etc. Among them, leucine aminopeptidase activity in serum is known to increase due to liver disease and impaired bile excretion, and is an important test item in clinical tests. In recent years, methods using leucine dehydrogenase have been used to measure this leucine aminopeptidase activity, which is an excellent reaction substrate for the enzyme.
It is being used to take advantage of the unique characteristics of optics. While microanalytical methods that generally use enzymes have the above advantages, enzymes are generally very unstable and typically lose their catalytic activity within days to weeks at room temperature; This instability is a major obstacle in trace component analysis using enzymes. Previously known leucine dehydrogenases are similarly unstable;
Biological Chemistry (J.Biol.
Chem.), vol. 253, p. 5719 (1978).
Leucine dehydrogenase obtained from A. Sphaericus usually loses most of its activity within 1 to 3 weeks in an aqueous solution (room temperature), and has a major drawback in that it lacks thermostability and long-term stability. have. Therefore, in order to maximize the advantages of analytical methods using leucine dehydrogenase,
The emergence of a leucine dehydrogenase that is heat-stable and does not lose its activity for long periods at room temperature is eagerly awaited. Therefore, from this perspective, the present inventors conducted extensive research in search of a leucine dehydrogenase that is heat-stable and does not lose its activity over a long period of time. The present invention was completed based on the discovery that there is a leucine dehydrogenase having the following properties. That is, the present invention provides a solution for about 10
The activity after treatment is approximately 80% of the activity before treatment.
Leucine dehydrogenase and bacteria belonging to the genus Bacillus that have the above values and the following physical and chemical properties are cultured, and the culture is heated to approximately 70°C.
Collect a leucine dehydrogenase whose activity after treatment in a buffer solution for about 10 minutes retains approximately 80% or more of the activity before treatment, and which has the following physical and chemical properties. A method for producing leucine dehydrogenase, characterized by: (a) Action and substrate specificity L-leucine + H ₂ O + NAD ⁺ ↑↓ α-ketoisocaproic acid + NH ₄ ⁺ +NADH Catalyzes the above reaction. In addition to L-leucine, L-
It also acts on valine and L-isoleucine. (b) Molecular weight The value measured by gel chromatography is
Approximately 300,000 yen. (c) Optimum pH is approximately 11 at 55℃. (d) Stable pH range of 5.5 to 10.5. When the leucine dehydrogenase of the present invention is treated in a buffer solution at about 70°C for about 10 minutes, the residual activity of the leucine dehydrogenase is about 80% or more of the original activity.
It preferably maintains a value of about 90% or more, optimally about 100%, and in particular, the activity after processing in a buffer at about 70°C for about 30 minutes is about 80% of the activity before treatment. It has excellent properties, holding a value of % or more. The concentration and pH of the buffer are not particularly limited, but generally the concentration is 5mM to 500mM and the pH is 6 to 10. In particular, in the present invention, it contains 0.01% by volume of 2-mercaptoethanol.
Preferably, 10 mM phosphate buffer (PH7.2) is used. Next, the physicochemical properties of the leucine dehydrogenase of the present invention will be shown. 1 Action and substrate specificity As described above. Note that the Michaelis constants (Km values) for L-leucine and NAD are approximately 1.54 mM and approximately 0.39 mM, respectively. 2 Optimum PH As mentioned above. 3 Stable PH range As described above (almost no deactivation occurs after treatment at 55°C for 5 minutes). 4 Suitable temperature range for action: 25℃ to 70℃ at PH11. 5 Heat resistance Stable to heat treatment at 70℃ for 30 minutes. 6 Molecular weight As described above. Note that Toyo Pearl 55-F (manufactured by Toyo Soda Co., Ltd.) was used as gel chromatography. 7 Measurement method of titer PH11, in 0.1M glycine-KCl-KOH buffer
Prepare a mixed solution containing 1.25mMNAD and 10mML-leucine, add an appropriate amount of leucine dehydrogenase to the mixture, and measure the increase in reduced NAD per unit time at 55℃ as the increase in absorbance at 340nm. , 1 micromol per minute
The amount of enzyme that increases the absorbance of NADH at 340 nm was defined as one unit. 8. Unity The purified sample migrated to the anode side by 7.5% acrylamide gel disc electrophoresis at pH 9.4, giving a single band. 9 Elemental analysis Elemental analysis values have not yet been measured. 10 Crystal structure Unknown as it has not yet been obtained as a crystal. To produce the leucine dehydrogenase of the present invention,
The following methods can be adopted. That is, it can be obtained by culturing bacteria belonging to the genus Bacillus and collecting the leucine dehydrogenase of the present invention from the culture. The bacteria used in the present invention are bacteria of the genus Bacillus that can produce the leucine dehydrogenase of the present invention,
Any such bacteria can be used. Preferred bacteria include, for example, Bacillus stearothermophilus.
Stearothermophilus). As a specific example of stearothermophilus,
ATCC7953, 7954, 8005, 10194, 12980,
Examples include NCA1503. As a carbon source in the nutrient medium used for culturing the bacteria in the present invention, for example,
Glucose, sucrose, fructose, starch hydrolyzate, molasses, sugars from sulfite pulp waste liquid,
Organic acids such as acetic acid and lactic acid, as well as alcohols, oils and fats, fatty acids, glycerin, etc. that can be assimilated by the bacteria used can be used, and as nitrogen sources, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, amino acids, peptone, Inorganic or organic substances such as meat extract and yeast extract can be used. Furthermore, as inorganic salts, for example, potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, cobalt salts, trace metal salts, corn steep liquor, vitamins, nucleic acids, etc. etc., and common nutrient media for bacteria can be used. Using these media, bacteria belonging to the genus Bacillus may be cultured aerobically at 20°C to 80°C, preferably 40°C to 70°C, optimally 55°C, for about 2 to 16 hours. In addition, industrially, for example, the maximum specific growth rate of a strain is determined using the dilution rate (the value obtained by dividing the rate of supplying the culture medium into the fermenter and the rate at which it is simultaneously withdrawn from the fermenter by the amount of culture solution in the fermenter). A continuous culture method can also be used, in which the concentration is controlled continuously in the range of 0.3 to 1.0, preferably 0.5 to 1.0, optimally 0.7 to 1.0. Next, the leucine dehydrogenase of the present invention is collected from the obtained culture, but it can be collected at any stage, such as culture, isolated viable cells, processed products of isolated cells, crude enzyme, purified enzyme, etc. As a purification method, a normal enzyme purification method can be used. That is, after obtaining bacterial cells by centrifugation, the cells are crushed by Manton-Gaulin, Dyno Mill, French press, ultrasonic treatment, mortar grinding, etc., cell debris is removed by centrifugation, and the cell extract is obtained. This is then treated with streptomycin sulfate or protamine sulfate, and further subjected to polyethylene glycol precipitation, acetone precipitation, heat treatment, etc., and purified using ion exchange chromatography such as DEAE-cellulose column, hydroxyapatite column, etc. It can be carried out in combination with chromatography such as adsorption chromatography, gel filtration chromatography such as sepadex chromatography. In this way, the leucine dehydrogenase of the present invention can be isolated and purified. Since the leucine dehydrogenase of the present invention is very stable against heat, it can be stored for a long period of time after isolation, compared to conventional leucine dehydrogenase. For this reason, biochemical research, food,
Very effective in the field of clinical testing. For example, the L-leucine content in a test solution can be measured with high accuracy using a reaction solution consisting of the leucine dehydrogenase of the present invention and NAD. This quantitative determination of L-leucine can be used to measure the activity of leucine aminopeptidase, which generates L-leucine from L-leucineamide.Measurement of leucine aminopeptidase activity in serum is useful for liver diseases, biliary excretion disorders, etc. It is used for diagnosis and is an important item in clinical tests. Next, the present invention will be specifically explained using examples. Example 1, Comparative Example 1 Polypeptone 10g/yeast extract 2.5g/,
Meat extract 2g/, glycerin 2g/, sodium chloride 5g/, dipotassium phosphate 2g/,
Monopotassium phosphate 2g/, magnesium sulfate
After heat sterilizing medium 3 adjusted to PH7.2 at 120°C for 10 minutes, Bacillus stearothermophilus ATCC12980 strain was inoculated and cultured aerobically at 55°C for 15 hours. . After culturing, the cells were collected using a centrifuge to obtain 11 g of wet cells. After grinding the obtained bacterial cells with about 30 g of aluminum oxide powder in a mortar for about 20 minutes, 50 ml of 0.6M phosphate buffer containing 0.01% by volume of 2-mercaptoethanol was added, and the aluminum oxide and cells were separated by centrifugation. The pieces were removed to obtain a crude extract containing leucine dehydrogenase. Polyethylene glycol (average molecular weight 7500) was added to this crude extract to give a final concentration of 9% (W/V) and stirred.The resulting precipitate was removed by centrifugation and the supernatant (crude enzyme solution ) was obtained. Contains 0.01% by volume of 2-mercaptoethanol in advance.
Equilibrated with 10mM phosphate buffer (PH7.2)
When the above crude enzyme solution was passed through a DEAE/cellulose column and eluted with a solution containing salt added to the above buffer, the target leucine dehydrogenase was eluted at a salt concentration of approximately 0.35M. The fractions were collected and solid ammonium sulfate was added slowly to 30% saturation (4°C). The generated precipitate was removed by centrifugation, and solid ammonium sulfate was gradually added to the resulting supernatant again to achieve 60% saturation (4°C). The generated precipitate was collected by centrifugation, and 2-
Mercaptoethanol 0.01% by volume and potassium chloride
Gel chromatography was performed using Sephadex G-150 using 10 mM phosphate buffer (PH7.2) containing 0.1 M as the eluent. Next, the eluted active fraction was dialyzed against a 2mM phosphate buffer containing 0.01% by volume of 2-mercaptoethanol, and passed through a hydroxyapatite column equilibrated with the same buffer in advance to continuously adjust the buffer concentration to 100mM. When the buffer concentration was increased to 20 to 35 mM, the target leucine dehydrogenase was eluted. Collect this classification and 2-
When passed through a DEAE-cellulose column equilibrated with 10mM phosphate buffer (PH7.2) containing 0.01% by volume of mercaptoethanol and eluted with a solution containing salt added to the above buffer, the salt concentration ranged from 0.2 to 0.25M. The target leucine dehydrogenase was eluted. This fraction was further collected, solid ammonium sulfate was added to achieve 70% saturation (at 4°C), the precipitate formed was collected by centrifugation, and 2-mercaptoethanol was extracted.
Gel chromatography was performed using Sephadex G-150 using 10 mM phosphate buffer (PH7.2) containing 0.01% by volume and 0.1M potassium chloride as the eluent. As a result, purified leucine dehydrogenase 1.2
mg was obtained. The leucine dehydrogenase obtained in this way has a pH of
In 7.5% acrylamide gel disc electrophoresis of 9.4, it migrated to the anode side and gave a single band, and in Toyopearl 55-F gel chromatography, it also gave a single peak at a molecular weight of about 300,000. In addition, the molecular weight was determined by 12% acrylamide gel slab electrophoresis containing sodium lauryl sulfate.
gave a single band at position 49000-50000. In addition, the yield of activity is approximately 9%, which is approximately 9% per mg of enzyme.
It shows the titer in units, and the degree of purification was about 70 when the crude extract is 1. Next, we investigated the stability of the leucine dehydrogenase obtained in this way and the stability of the leucine dehydrogenase obtained in this way and the
The stability of the leucine dehydrogenase obtained from A. sphaericus (Comparative Example 1) was compared. As a result, at 70℃ in 10mM phosphate buffer with pH 7.2 containing 0.01% by volume of 2-mercaptoethanol.
When the residual activity was measured after heat treatment for 10 minutes, the leucine dehydrogenase of Bacillus sphaericus was found to be 100% inactivated. However, the leucine dehydrogenase of the present invention not only does not lose its activity at all when heated at 70°C for 10 minutes, but also loses its activity at 70°C.
℃, heat treatment for 30 minutes also has approximately 100% of the activity before treatment.
held the value of. Next, add 0.01% by volume of 2-mercaptoethanol.
4-8°C in 10mM phosphate buffer with pH 7.2
The storage stability was investigated. The results are shown in FIG. In FIG. 1, curve A is Example 1, and curve B is Comparative Example 1. As is clear from Figure 1, the activity of the leucine dehydrogenase obtained from Bacillus sphaericus decreases to about 50% in 4 days and almost loses its activity in 2 weeks, whereas the leucine dehydrogenase of the present invention The activity remained approximately 100% even after 2 weeks. Furthermore, when we investigated the storage stability at room temperature, we found that the leucine dehydrogenase obtained from Bacillus sphaericus was almost inactivated after 5 days, whereas the leucine dehydrogenase of the present invention remained at approximately 90% activity even after 10 days. % or more of the activity. Reference Example 1 Using the leucine dehydrogenase obtained in Example 1,
A calibration curve necessary for measuring L-leucine was prepared. The method of measurement is 70 micromoles of glycine.
Kcl−KOH buffer (PH11.0), 10 μM
Use 0.8 ml of NAD and 0 to 0.2 micromoles of L-leucine as a reaction solution, measure the absorbance at 340 nm, add about 0.3 units of leucine dehydrogenase, incubate at 55°C for 30 minutes, and measure the absorbance at 340 nm. The absorbance was measured again. As a result, a good proportional relationship was observed between the increase in absorbance at 340 nm and the amount of L-leucine added to the reaction solution, as shown in Figure 2. It was found that L-leucine could be measured with high accuracy using the method.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the residual activity of the leucine dehydrogenase of the present invention (curve A) and the leucine dehydrogenase obtained from Bacillus sphaericus (curve B) after being left at 4 to 8°C; The figure shows a calibration curve for L-leucine prepared using the leucine dehydrogenase of the present invention.

Claims (1)

[Claims] 1. Has the property that the activity after being treated for about 10 minutes in a buffer solution at about 70°C maintains a value of about 80% or more of the activity before the treatment, and the following physical and chemical Leucine dehydrogenase with properties. (a) Action and substrate specificity L-leucine + H ₂ O + NAD ⁺ ↑↓ α-ketoisocaproic acid + NH ₄ ⁺ +NADH Catalyzes the above reaction. In addition to L-leucine, L
-Also acts on valine and L-isoleucine. (b) Molecular weight The value measured by gel chromatography is approximately 300,000. (c) Optimum pH is approximately 11 at 55℃. (d) Stable pH range of 5.5 to 10.5. 2. Bacteria belonging to the genus Bacillus are cultivated and the culture is treated in a buffer solution at approximately 70°C for approximately 10 minutes, and the activity thereof retains approximately 80% or more of the activity before treatment. A method for producing leucine dehydrogenase, which comprises collecting leucine dehydrogenase having the following physical and chemical properties. (a) Action and substrate specificity L-leucine + H ₂ O + NAD ⁺ ↑↓ α-ketoisocaproic acid + NH ₄ ⁺ +NADH Catalyzes the above reaction. In addition to L-leucine, L
-Also acts on valine and L-isoleucine. (b) Molecular weight The value measured by gel chromatography is approximately 300,000. (c) Optimum pH is approximately 11 at 55℃. (d) Stable pH range of 5.5 to 10.5.