JPH06327471A - Thermostable glutamate dehydrogenase and its production - Google Patents

Abstract

PURPOSE:To obtain a new glutamate dehydrogenase, useful for determining glutamic acid or clinical diagnosis, a sensor element, etc., and excellent in thermostability. CONSTITUTION:The thermostable glutamate dehydrogenase is obtained by culturing a hyperthermophilic archaebacterium Thermococcus litoralis DSM5473, etc., which is a strain belonging to the genus Thermococcus such as Thermococcus litoralis. This thermostable glutamate dehydrogenase has about 90 deg.C optimum action temperature and 7.8 optimum pH, manifests the maximum activity at 90-95 deg.C and is stable to 30min at 95 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel thermostable glutamate dehydrogenase and a method for producing the same.

[0002]

2. Description of the Related Art Glutamate dehydrogenase is widely present in animals, plants and microorganisms, and enzymes of various origins are known. Hitherto, a highly productive glutamate dehydrogenase derived from a Proteus bacterium (JP-A-54-126789) and a glutamate dehydrogenase derived from a Coprinus bacterium (JP-A-62-1)
07784) and a glutamic acid dehydrogenase derived from Bacillus bacterium (Japanese Patent Application No. 4-194245).

[0003]

The conventional glutamate dehydrogenase has low thermostability, and for example, the above-mentioned glutamate dehydrogenase derived from Proteus bacterium has a sharp decrease in activity above 40 ° C. Glutamate dehydrogenase derived from a genus bacterium is stable up to 45 ° C after 10 minutes of treatment, but its activity decreases to 70% at 60 ° C. In addition, the glutamate dehydrogenase derived from Bacillus is 2M N
It is stable up to 95 ° C in the presence of aCl, but loses its activity at 95 ° C in the absence of NaCl, and is only stable up to 70 ° C.

Therefore, the present invention aims to solve these problems and provide a glutamate dehydrogenase having excellent thermostability.

[0005]

As a result of intensive studies on the above problems, the present inventors have found a novel thermostable glutamate dehydrogenase and a method for producing the same, and completed the present invention. That is, the present invention is a thermostable glutamate dehydrogenase having a suitable action temperature of around 90 ° C.

Further, the present invention is characterized in that a microorganism belonging to the genus Thermococcus having the ability to produce the thermostable glutamate dehydrogenase is cultured in a medium, and the thermostable glutamate dehydrogenase is collected from the culture. Is a method for producing a thermostable glutamate dehydrogenase. The microorganisms belonging to the genus Thermococcus include Thermococcus
Litralis (Thermococcus litoralis) and the like.

The present invention will be described in detail below. First,
The physicochemical properties of the thermostable glutamate dehydrogenase of the present invention are described in detail below. (1) Action: Presence of nicotinamide adenine dinucleotide phosphate (hereinafter, oxidized nicotinamide adenine dinucleotide phosphate is abbreviated as “NADP” and reduced nicotinamide adenine dinucleotide phosphate is abbreviated as “NADPH”). The following formula (I) below:

[0008]

[Chemical 1]

It catalyzes the oxidative deamination reaction of L-glutamic acid shown in. (2) Substrate specificity: Shows substrate specificity for L-glutamic acid. That is, the deamination reaction shows almost no reactivity with amino acids other than L-glutamic acid. Further, it also shows high reactivity with α-ketoglutarate in the amination reaction. (3) High activity is observed at optimum pH: 7.8. (4) Stable pH range: The stable pH range is 7.0 to 11.0. (5) Optimum temperature range for action: 80 ° C to 100 ° C, with a suitable temperature around 90 ° C. (6) Thermal stability: Stable up to 95 ° C by heat treatment for 10 minutes, and stable up to 30 minutes at 95 ° C. (7) Inhibitor: The activity is inhibited by mercuric chloride (1 mM). (8) Molecular weight: The molecular weight is about 300,000 by gel filtration (using TSKgel G3000SWXL). (9) Purification method: Thermococcus litoralis DSM547 which is a bacterium producing the enzyme
Incubate 3 at 88 ° C. to disrupt cells in culture. Then, solid matter such as cell lysate is removed by a means such as centrifugation, and the present enzyme is separated and collected as a supernatant. The enzyme can be purified by combining the means such as affinity chromatography and ion exchange chromatography. (10) Method for measuring titer: the present enzyme having the composition shown in Table 1
A reaction solution containing NADP as a coenzyme and L-glutamic acid as a substrate was incubated at 37 ° C., and NADP accompanying the reaction was incubated.
The reaction rate is determined by quantifying the increase in H 2.
However, the reaction is started by addition of NADP. NAD
Since PH exhibits a spectrum having an absorption maximum at 340 nm, the change with time of the absorbance at 340 nm is measured with a spectrophotometer. At the time of measurement, the amount of enzyme is adjusted so that the absorption increases linearly.

Table-1 Composition of reaction solution for measurement -200 μmol phosphate buffer (pH 7.8) -Enzyme -10 μmol L-glutamic acid -1.25 μmol NADP final reaction solution volume 1.0 ml The unit of enzyme activity is shown in Table-1. Using reaction composition, 37 ℃
The amount of enzyme that produces 1 μmol / min of NADPH is defined as 1 unit (U; unit). Specific activity is expressed as the number of units per mg of enzyme protein.

The above-mentioned novel enzyme of the present invention has extremely excellent thermostability. In addition, since the enzyme has a high substrate specificity, it can be expected to be applied to quantification of glutamic acid, clinical diagnosis, sensor devices and the like. Secondly, the method for producing the novel enzyme will be described in detail below.

First, the microorganism used in the present invention is
Is a species belonging to the thermo-genus, as this species, ultra-high fever archaea Thermococcus litoralis (Thermo
coccus litoralis ) DSM5473. A representative strain of this microorganism is the depository institution DSM (Deutsche Sammlung von Mikro) approved by the Budapest Treaty.
organismen, Mascheroder Weg 1b, D-3300, Braunschwe
ig, FDR), and its number is DSM5473. And this strain is available to many.

Next, the method for culturing the microorganism in the method of the present invention can be carried out according to a general culturing method. For example, a liquid culture medium (adjusted to pH 6.0) containing yeast extract, casein degradation product, maltose, cysteine, inorganic salts, and seawater is inoculated with the above microorganisms and anaerobically stirred at about 88 ° C while blowing nitrogen. Incubate for 24 hours. Thereafter, the cells are collected by centrifugation, suspended in a phosphate buffer and washed.

The present enzyme can be collected from the cultured cells described above as follows. That is, the microbial cells are disrupted using an ultrasonic disruptor or the like, and then centrifuged to obtain an enzyme-containing supernatant. The resulting crude enzyme solution can be purified by dye-ligand affinity chromatography such as red sepharose or blue sepharose. Furthermore, D
Purity can be increased by EAE Toyopearl.

The protein can be quantified using any of the following methods. (A): Method by Kalb and Bernlohr (Anal.Biochem., 82,362-371,1977) The absorbance at 230 nm and 260 nm was measured, and the following formula was used: Protein concentration (mg / ml) = {(A ₂₃₀ × 183)-(A
₂₆₀ x 75.8)} x (1/1000) x dilution rate to calculate the protein concentration.

Here, A ₂₃₀ and A ₂₆₀ are each 230n
Absorbance at m and 260 nm. (B): Folin-Lowry method (J. Bi
ol.Chem.193,265-371,1951) Since the absorption is affected by the suspension of fine gel after electrophoresis, the quantitative measurement is performed by the Foreign-Lowry method. In this method, the absorbance at 650 nm is measured, and the protein concentration is calculated by the following formula: protein concentration (mg / ml) = A ₆₅₀ × 0.255 × dilution ratio.

Here, A ₆₅₀ is the absorbance at 650 nm, and the coefficient 0.255 is the value obtained from the calibration curve.

[0018]

According to the present invention, a novel glutamate dehydrogenase having excellent thermostability and a method for producing the same can be provided. INDUSTRIAL APPLICABILITY The novel glutamate dehydrogenase of the present invention can be applied not only to quantification of glutamate but also to clinical diagnosis and sensor elements, and is industrially very useful.

[0019]

[Example 1]

(1) Cultivation of bacterial cells, cell collection and washing Bacteria preparation and sterilization treatment A medium having the composition shown in Table 2 below was used. The pH of the medium was adjusted with 1N-HCl.

[0020]

First, 4.9 L of the medium A having the above composition was placed in a 5 L flask and autoclaved (2 atm, 121 ° C.,
Sterilized for 20 minutes. To this, 50 ml of autoclaved medium B and 50 ml of filter-sterilized medium C were added under the same conditions to make a total volume of 5 L. Bacteria collection and washing The bacteria cultured in the main culture were collected by a centrifuge (10,000 xg, 10 minutes, 4 ° C). After 3% NaCl was added to the suspension to suspend it, centrifugation was performed again. The cells washed in this manner were treated with 0.2 M NaCl, 1 mM EDTA and
10 mM potassium phosphate buffer containing 0.1 mM dithiothreitol (hereinafter referred to as DTT) (p
H7.2).

(2) Purification of Enzyme, Measurement of Activity and Assay of Purity Purification of Enzyme and Measurement of Its Activity Step 1: Preparation of Crude Enzyme Solution Bacteria were adjusted to about 10 times the wet weight (volume / weight). Equivalent to,
0.2 M NaCl, 1 mM EDTA and 0.1 mM D
The cells were suspended in 10 mM potassium phosphate buffer (pH 7.2) containing TT, and disrupted intermittently for about 15 minutes using an ultrasonic disruptor (70 to 100 W). Centrifuge this (10,000 × g, 20
Minutes) to obtain a supernatant, and this supernatant was used as a crude enzyme solution. Volume of this crude enzyme solution, total protein amount (the above-mentioned quantitative method (a)
Quantified), total activity and specific activity were measured. The results are shown in Table-3.

Step 2: Red Sepharose CL-4
B affinity chromatography 0.2 M NaCl, 1 mM EDTA and 0.1 mM D
Red Sepharose 4B column (internal diameter 1.5 cm x) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing TT
(12 cm in length), and then the same buffer (pH 7.2)
Pour 200 ml through the column, then 0.5 M NaCl, 1
A 20 mM potassium phosphate buffer (pH 8.0) containing mM EDTA and 0.1 mM DTT was run.

The eluate from the column was fractionated by a fraction collector in an amount of about 5 ml, and the protein concentration and activity were measured. Glutamate dehydrogenase is 0.5M NaC
20m containing 1 mM EDTA and 0.1 mM DTT
It could be eluted with M potassium phosphate buffer (pH 8.0). Among the obtained fractions, fractions having high glutamate dehydrogenase activity were collected. The protein concentration at this time is the ultraviolet absorption (absorption maximum 2
80 nm) was directly measured. After measuring the volume and total activity of the collected enzyme solution, an ultrafilter (Toyo Roshi U
K-50) and concentrated. The volume of the enzyme solution after concentration, the total protein amount (quantified by the above-mentioned quantitative method (a)), the total activity and the specific activity were measured. The results are shown in Table-3.

Step 3: DEAE Toyopearl Chromatography 0.2 M NaCl, 1 mM EDTA and 0.1 mM D
The enzyme solution obtained in step 2 was dialyzed for about 20 hours in 10 mM potassium phosphate buffer (pH 7.2) containing TT. The enzyme solution after dialysis was equilibrated with the same buffer solution (pH 7.2).
After adsorbing to EAE Toyopearl (inner diameter 2.0 cm x length 30 cm), about 150 ml of the same buffer solution (pH 7.2) was applied to the column, and then 0.3 M NaCl, 1 mM EDTA and
A 20 mM potassium phosphate buffer (pH 7.2) containing 0.1 mM DTT was run.

The eluate from the column was fractionated by a fraction collector in an amount of about 5 ml, and the protein concentration and activity were measured. Glutamate dehydrogenase is 0.3M NaC
l 10m containing 1mM EDTA and 0.1mM DTT
Elution with M potassium phosphate buffer (pH 8.0) was performed, and among the obtained fractions, fractions having high glutamate dehydrogenase activity were collected. The protein concentration at this time is determined by the ultraviolet absorption (absorption maximum 280
nm) was directly measured. After measuring the volume and total activity of the collected enzyme solution, an ultrafilter (Toyo Filter Paper UK
-50) and concentrated. The volume of the enzyme solution after concentration, the total protein amount (performed by the above-mentioned quantitative method (a)), the total activity and the specific activity were measured. The results are shown in Table-3.

[0027]

Purity Assay of Purified Enzyme by Disc Gel Electrophoresis The purified enzyme obtained in step 3 of disc gel electrophoresis was subjected to a purity assay by disc gel electrophoresis by the Davis and Ornstein method. 7.5% (weight / volume) in a glass tube (inner diameter 0.5 cm x length 7.5 cm)
A polyacrylamide gel (pH 9.4) was prepared, and a current of 3 mA was applied to each gel, and the enzyme solution was electrophoresed for about 1 hour. The gel obtained was subjected to protein staining and activity staining. Protein staining was carried out using Coomassie brilliant blue R-250, and activity staining was carried out at 50 ° C. by the PMS-INT method using an active staining reaction solution having the composition shown in Table-4.

Table-4 Composition of active staining reaction solution 0.1 M phosphate buffer (pH 7.5) 10 mM L-glutamic acid 0.5 mM INT ¹⁾ 0.4 mM PMS ²⁾ 1.0 mM NADP INT ¹⁾ : iodine nitrotetrazolium chloride PMS ²⁾ : Phenazine methosulfate Since one band was observed in both protein staining and activity staining, it was confirmed that this enzyme could be purified to homogeneity by the above method.

As described above, the hyperthermophilic archaebacterium Thermococcus litoralis DSM
Glutamate dehydrogenase was collected from 5473 and could be purified rapidly and efficiently. (3) Examination of chemical properties of purified enzyme Substrate specificity (1-1) Deamination reaction Using the reaction solution having the composition shown in Table-5, the reactivity in deamination reaction of 12 kinds of substrates was examined. Examined. All substrates were adjusted to a final concentration of 10 mM, 50 reactions
Performed at ° C. The results are shown in Table-6. This enzyme showed very high substrate specificity for L-glutamic acid and did not act on other amino acids.

Table-5 Composition of reaction solution for measuring substrate specificity -200 μmol phosphate buffer (pH 7.8) -enzyme-10 μmol substrate- 1.25 μmol NADP final reaction volume 1.0 ml

[0032]

(1-2) Amination reaction Using the reaction solutions having the compositions shown in Table 7, the reactivity of the 6 types of substrates in the amination reaction was examined. All substrates were adjusted to a final concentration of 10 mM and the reaction was performed at 50 ° C.
I went there. The results are shown in Table-8. This enzyme showed high substrate specificity for α-ketoglutarate, but showed low reactivity for α-keto acids such as α-ketocaproic acid.

Table 7 Composition of reaction solution for measuring substrate specificity -200 μmol phosphate buffer (pH 7.8) -Enzyme-10 μmol substrate-200 μmol NH ₄ Cl -0.2 μmol NADPH final reaction volume 1.0 ml

[0035]

Optimum pH Using a reaction solution having the composition shown in Table 1, the pH of the phosphate buffer was changed and the enzyme activity of this enzyme on L-glutamic acid was measured. As a result, the optimum pH was pH 7.8. Met. Stable at 80 ° C for 20 minutes, it is stable in the range of pH 7.0 to 11.0, and no deactivation is observed.

Optimal temperature in the range of action As for the influence of temperature on the enzyme activity, the activity increases with increasing temperature and shows the maximum activity at 90 ° C to 95 ° C. At higher temperatures, the activity decreases. Thermal stability 10 minutes heat treatment did not undergo thermal denaturation up to 95 ° C.
The results are shown in Fig. 1. It was stable at 95 ℃ for up to 30 minutes. The results are shown in Figure 2.

Inhibitor The effect on the enzyme reaction was examined by adding an inhibitor to the reaction solution having the composition shown in Table 1 and measuring the activity (Table 9). The relative activity was determined by setting the activity in the absence of an inhibitor to 100% under the activity measurement conditions for the deamination reaction of L-glutamic acid. This enzyme was heat-treated at 37 ° C. for 60 minutes, and the residual activity was measured. This enzymatic reaction was completely inhibited by mercuric chloride (1 mM). Also, other metals, metal chelating agents, and nucleotides had almost no effect.

[0039]

Example 2 Determination of Glutamic Acid The purified enzyme obtained in Example 1 was used to determine the amount of glutamic acid. (Principle) Formula (II):

[0041]

[Chemical 2]

As shown in, glutamic acid produces NADPH by the glutamate dehydrogenase (GLDH) reaction. This NADPH is Meldola blue
INT (iodine nitrotetrazolium chloride) is reduced in the coexistence of e) to form formazan showing absorption around 492 nm. The amount of glutamic acid can be calculated using the molecular extinction coefficient of this formazan.

After stirring 3.15 ml of the solution containing the above components, the absorbance (E1) at 492 nm was measured. In this solution
0.05 ml of GLDH containing 0.3 to 1.0 U was added and reacted for 10 to 20 minutes, and then the absorbance (E2) was measured. Further, blanks E1 and E2 were obtained by using a blank to which distilled water was added instead of the glutamic acid standard solution. The following formula: ΔE = (E2-E1) standard solution- (E2-E1) blank The value ΔE is the increase in absorbance corresponding to the amount of glutamic acid, and the molecular extinction coefficient (ε) of formazan under this condition. = 19.0 to 20.0 cm ² / μmol) was used to calculate the amount of glutamic acid according to the following formula.

[0044]

[Equation 1]

As a result, as shown in FIG. 3, the calibration curve using the standard solution of glutamic acid gives a straight line passing through the origin, and it is clear that glutamic acid can be quantified accurately.

[Brief description of drawings]

Figure 1: Thermostability of purified enzyme (relationship between temperature and relative activity)
FIG.

FIG. 2 is a diagram showing the thermostability of purified enzyme (relationship between reaction time and relative activity).

FIG. 3 is a diagram showing a calibration curve using a glutamic acid standard solution.

Claims (3)

[Claims] 1. A thermostable glutamate dehydrogenase having a suitable temperature of action of about 90 ° C. 2. A microorganism belonging to the genus Thermococcus having the ability to produce a thermostable glutamate dehydrogenase according to claim 1, is cultured in a medium, and the thermostable glutamate dehydrogenase is collected from the culture. And a method for producing a thermostable glutamate dehydrogenase. 3. The method for producing a thermostable glutamate dehydrogenase according to claim 2, wherein the microorganism belonging to the genus Thermococcus is Thermococcus litoralis.