JP2639803B2 - Production of thermostable isocitrate dehydrogenase - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing thermostable isocitrate dehydrogenase, and more particularly to a method for producing conventionally unknown thermostable isocitrate dehydrogenase. Things.

(Prior Art) Isocitrate dehydrogenase (hereinafter sometimes referred to as ICDH) is widely present in animal and plant tissues, yeasts, and the like, and has conventionally been produced from fermented or animal tissues such as porcine myocardium ("""Chemical Encyclopedia 1" Kyoritsu Shuppan, 42-9-10, p5
98).

However, the previously known ICDH is unstable to heat and chemicals, and furthermore, in its reaction,
Those using only NAD (EC1.1.1.41) and those using only NADP (EC1.1.1.42) are known, but NAD, NADP
At present, none of the coenzymes can be used.

(Problems to be Solved by the Invention) As described above, the conventionally known ICDH lacks physicochemical stability and must be subjected to many restrictions for industrial use. In addition, the method of extracting from animal tissue, such as porcine myocardium, has disadvantages such as difficulty in obtaining raw materials, complicated operation, low yield, and high cost, and cannot be an industrial method.

(Means for Solving the Problems) The present invention has been made in order to solve the above problems all at once, and in order to simultaneously solve the disadvantages in both the production method and the product enzyme, From the viewpoint of industrial production, it is preferable to use a fermentation method using a microorganism instead of an extraction method from a tissue.

From this point of view, despite the screening of a huge number of microorganisms, the target IC
DH was not obtained. We changed our idea and examined archaebacteria, especially acidophilic thermophilic bacteria, and obtained new knowledge that Sulfolobus produces ICDH, and also obtained new knowledge that this ICDH is a novel enzyme that was previously unknown. Obtained. As a result of further screening, a very useful new finding that the thermophilic bacterium of the genus Termus similarly produces a novel ICDH enzyme was obtained.

The present invention has been finally completed as a result of further studies based on these new findings.

In the present invention, a thermostable ICDH-producing bacterium belonging to the genus Thermus or Sulfolobus is used. For example, Thermus thermophus isolated from hot springs (Thermus thermophus)
ilus), Sulfolob acidocardarium (Sulfolob)
It is preferred to use, for example, US acidocaldarius). These bacteria may be cultured in a normal bacterial medium,
Since all of them are thermophilic bacteria, they are cultured by a conventional method at a temperature of 65 to 85 ° C, preferably around 75 ° C. The culture pH may be about neutral, but since the latter is also an eosinophilic bacterium, it is preferable to bring the pH to the acidic side.

The target enzyme is secreted out of the cells, but a considerable portion remains in the cells. After the cultivation, the cells are collected by centrifugation, filtration, etc., and washed. The cells thus cleaned are mechanically, physically, chemically or biologically disrupted by ultrasonic treatment, mechanical stirring, bacteriolysis, etc., to release the contents of the cells to the outside. .

This is subjected to a separation operation such as centrifugation and filtration to collect the supernatant. The supernatant is fractionated with ammonium sulfate to obtain an active fraction. In addition, in order to remove nucleic acids in the cells, the released intracellular components are used.
After treating with RNase and DNase to decompose RNA and DNA, centrifuge and separate the precipitate by filtration, etc. to separate the precipitate, add buffer and KCl, homogenize, centrifuge and filter. Then, a supernatant, which is an enzyme-containing fraction, is obtained.

The ICDH-rich fraction thus separated (for example, the above-mentioned ammonium sulfate fraction or supernatant) is removed, if necessary, by removing contaminating proteins from the supernatant. The target ICDH is separated and purified with high efficiency and high purity without reducing the activity, by treating it in combination or repeatedly. For example,
Salting out, concentrated sulfuric acid, pH adjustment, dialysis, low temperature ethanol fractionation, chromatography using ammonium sulfate or sodium sulfate (DEAE-
Known purification methods such as ion exchange chromatography, partition chromatography, and adsorption chromatography using cellulose, CM-cellulose, etc.), high performance liquid chromatography, thin layer chromatography, gel filtration, electrophoresis, and sucrose density gradient ultracentrifugation. Separation and purification are carried out by using or appropriately combining or repeating.

The ICDH enzyme separated and purified in this way is completely different from the conventionally known ICDH, and has a completely new and useful property in which both NAD and NADP can be used as coenzymes. Heretofore known ICDHs have been distinguished so far from those using only NAD as a coenzyme (EC1.1.1.41) and those using only NADP (EC1.1.1.42). The Michaelis constant is 20 μM for NADP and 2.6 mM for NAD for the enzyme obtained in Example 1 using the genus Termus, and NA for the enzyme obtained in Example 2 using the genus Sulfolobus.
It was 35 μM in DP and 1.6 mM in NAD. The reason that NAD was not contaminated with NADP was that only one band was obtained by thin-layer chromatography using an aqueous solution of lithium chloride as an eluent. Commercially available porcine myocardial isocitrate dehydrogenase (NADP-specific) It was confirmed that the reaction did not occur even with an excessive amount of NAD.

Regarding divalent metal cation requirement, Mn ²⁺ or Mg ²⁺ was essential for both NAD and NADP coenzymes, but Sr ²⁺ and Co ²⁺ were effective only for NAD-dependent reactions Met.

The ICDH enzyme according to the present invention is extremely stable to heat, and the residual activity after incubation at 90 ° C. for 15 minutes is 40% (Example 1) and 90% (Example 2) Fig. 1 and 2
(Fig.), And the activation energies of the reactions were 10.7 Kcal / mol (Example 1) and 11.8 Kcal / mol (Example 2) (see
(FIG. 4 and FIG. 4), it can be seen that this enzyme is extremely excellent in heat resistance. In the known ICDH, there is no enzyme exhibiting such excellent thermostability, and the use at a high temperature has become possible for the first time.

The ICDH enzyme according to the present invention shows excellent resistance to various denaturants and is extremely stable. For example, when ICDH derived from commercially available porcine myocardium is used as a control, in 2M urea,
Although commercially available ICDH was completely inactivated, the present enzyme showed 70% (Example 1) (FIG. 5) and 90% (Example 2) activities of the untreated enzyme, respectively. Further, the enzyme according to Example 2
It showed 80% activity even in 5M urea. Commercially available ICDH was completely inactivated in 0.1 M guanidine hydrochloride, but this enzyme was 55% of the untreated enzyme (Example 1) (FIG. 6) and 80%, respectively.
% (Example 2). Furthermore, in 0.001% sodium dodecyl sulfate, commercially available ICDH was almost completely inactivated (3%), but the present enzyme (Example 1) showed an activity of 85% (FIG. 7).

 Next, examples of the present invention will be described.

Example 1 The following operations were all performed at room temperature unless otherwise specified.
Advanced thermophile Thermus thermophilus HB8 (ATCC 2763)
4) in a nutrient medium (3 g of yeast extract, 5 g of polypeptone, 1 g of glucose, 2 g of NaCl dissolved in 1 water and adjusted to pH 7.2)
After culturing at 5 ° C. for 8 hours (wet weight: about 4 g /), the cells were collected by centrifugation. Then, after washing, the mixture was subjected to ultrasonic treatment and separated into a supernatant and a sediment by centrifugation. This crude extract of thermus is fractionated with ammonium sulfate, and the active fraction is separated into 20 fractions.
Dialysis was performed using mM Tris-HCl (pH 7.5) and 1 mM EDTA as an external solution. The dialyzed solution was subjected to DEAE Sephacel column chromatography (equilibrated with 20 mM Tris-HCl (pH 7.5), 1 mM EDTA buffer) to fractionate the solution. This was fractionated by hydrophobic chromatography with Toyopearl HW55-S (developed with an ammonium sulfate concentration gradient of 2M-0M) at an ammonium sulfate concentration of 2M. This was adjusted to an ammonium sulfate concentration of 2M, and then subjected to hydrophobic chromatography using butyl toyopearl 650S (ammonium sulfate concentration).
2M-ammonium sulfate concentration 0M, developed with a 10% glycerol gradient). After concentrating this, it is fractionated by Sephacryl S200 column chromatography (developed with 20 mM Tris-HCl, 1 mM EDTA), and finally the active fraction is subjected to high-performance liquid chromatography (gel filtration carrier TSK Gel G3000, 0.2 M phosphate buffer) (Developed at pH 6.9)), a sharp peak was obtained at the position of molecular weight 85,000. This fraction was obtained as one band by polyacrylamide gel electrophoresis, and was obtained as a single band at a molecular weight of 50,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and consisted of the same subunit. It was thought to be a dimer enzyme. It was purified to about 200 times in specific activity.

Example 2 The following operations were all performed at room temperature unless otherwise specified.
Culture medium (yeast extract 1 g, casamino acid 1 g, glucose 1 g, Na acidophilic thermophilic archaeon Sulfolobus acidocardarius)
Cl 0.2 g, potassium phosphate 0.3 g, ammonium sulfate 1.3 g,
Magnesium sulfate heptahydrate 0.25g, calcium chloride dihydrate
Dissolve 0.05 g in 1 water and adjust the pH to 2.5 with sulfuric acid).
After shaking culture (wet weight about 1 g /) at 4 ° C. for 4 days, sodium bicarbonate was added to adjust the pH to 5, and the cells were collected by centrifugation. After washing, pH is adjusted with Tris-HCl (pH 8).
Was adjusted to 7.5, lysed, DNase and RNase were added, and the mixture was left at 37 ° C. for 1 hour to decompose DNA and RNA. This was centrifuged and separated into a supernatant and a sediment. The precipitate was suspended in 50 mM Tris-HCl, potassium chloride was added to a final concentration of 0.5 M, homogenized, and then centrifuged. The supernatant contains 0.2M citric acid-0.1M phosphoric acid 2
A buffer solution of sodium pH 2.6 was added to adjust the pH to 4, and proteins precipitated by centrifugation were removed. The supernatant was then saturated with 40% ammonium sulfate and adsorbed on a Toyopearl HW55-S column,
An active fraction was obtained by lowering the ammonium sulfate concentration to saturation, and an active fraction was obtained. This was subjected to butyl toyopearl 650S column chromatography (developed with a ammonium sulfate concentration of 40% -0% gradient) to collect an active fraction. . This was dialyzed against a 20 mM sodium phosphate buffer, adsorbed on a DEAE Sephacel column, and the active fraction eluted by linearly increasing the salt concentration from 0 to 0.5 M was collected. This was dialyzed again, adsorbed on a Brute Yopearl column, and eluted with a buffer containing 0.5 M KCl. This fraction was concentrated and subjected to high performance liquid chromatography (TSK Gel Type G3000 0.2 M sodium phosphate, 1 mM E
(Development with DTA)), and a sharp peak was obtained at the position of 85,000 in molecular weight. This fraction was obtained as a single band by polyacrylamide gel electrophoresis, and was obtained as a single band at a molecular weight of 42,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Was thought to be. About specific activity
It was purified up to 200 times.

(Effect of the Invention) According to the present invention, thermostable isocitrate dehydrogenase can be industrially mass-produced by a fermentation method using a microorganism.

Moreover, the heat-resistant ICDH obtained by the present invention has extremely high heat resistance and extremely high resistance to denaturants,
Although both NAD and NADP are used as coenzymes,
None of the ICDHs of bacterial origin having such excellent properties is known at all, and therefore the enzyme according to the present invention is a novel enzyme which is hitherto unknown.

As described above, this enzyme has excellent properties such as heat resistance and high stability, so that it can be used freely in reactions at high temperatures and in the presence of denaturing agents. It can be widely used in industrial applications, analytical chemistry applications, and other applications.

[Brief description of the drawings]

1, 2, 3, and 4 show Examples 1 and 2.
2 shows the residual activity and activation energy of the enzyme obtained after maintaining the enzyme at 90 ° C. for 15 minutes, respectively. 5 to 7 illustrate changes in the activity of the enzyme obtained according to Example 1 in urea, guanidine hydrochloride and sodium dodecyl sulfate at various concentrations, respectively.

Claims (2)

(57) [Claims] 1. A novel thermostable isocitrate dehydrogenase having the following physicochemical properties: (B) Thermostability The enzyme has a residual activity of 40% or more after holding at 90 ° C. for 15 minutes. (B) Utilization of coenzyme This enzyme utilizes both NAD and NADP coenzymes. (C) Resistance to denaturing agents This enzyme shows an activity of 70% or more in 2M urea. (D) Molecular weight As a result of subjecting this enzyme to high performance liquid chromatography,
A sharp peak is obtained at a molecular weight of 85,000. 2. A novel thermostable isocitrate dehydrogenase having the following physicochemical properties, comprising culturing a thermostable isocitrate dehydrogenase producing bacterium belonging to the genus Thermus and collecting the enzyme from the culture. Recipe. (B) Thermostability The enzyme has a residual activity of 40% or more after holding at 90 ° C. for 15 minutes. (B) Utilization of coenzyme This enzyme utilizes both NAD and NADP coenzymes. (C) Resistance to denaturing agents This enzyme shows an activity of 70% or more in 2M urea. (D) Molecular weight As a result of subjecting this enzyme to high performance liquid chromatography,
A sharp peak is obtained at a molecular weight of 85,000.