JPH05244941A - Nadh-dependent p-hydroxybenzoic acid hydroxylase and its production - Google Patents

Abstract

PURPOSE:To obtain the subject hydroxylase having specific enzymological properties on an industrial scale at a low cost by culturing a microbial strain belonging to the genus Rhodococcus and capable of producing an NADH dependent p-hydroxybenzoic acid hydroxylase. CONSTITUTION:A microbial strain belonging to the genus Rhodococcus, e.g. Rhodococcus SP.L-1 strain (FERM P-10614) is cultured and the objective hydroxylase having the following enzymological properties is separated from the cultured product. Action, acting to p-hydroxybenzoic acid in the presence of NADH to form protocatechuic acid; substrate specificity, p-hydroxybenzoic acid is taken as the specific substrate; optimum pH and stable pH, optimum pH is 7.5-7.8 and stable pH is 7.4-7.8 when kept at 25 deg.C for 1hr; optimum temperature and stable temperature range, optimum temperature is 33 deg.C, the enzyme is stable up to 25 deg.C when heated at pH7.6 for 60min and is completely inactivated at >=60 deg.C ; molecular weight, 6.7X10<4> measured by high-speed gel-filtration column; effect of inhibitors, etc., inhibited by halide ion and thiocyanate ion and inert to cations such as sodium ion and potassium ion.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing NADH-dependent parahydroxybenzoate hydroxylase and a bacterium belonging to the genus Rhodococcus.

[0002]

2. Description of the Related Art Protocatechuic acid is widely used as a raw material for producing rust preventives, antioxidants for foods, vanillin used as flavors, and raw materials for various pharmaceuticals. .. As a method for producing this protocatechuic acid, one using an organic synthesis reaction,
It is known to be based on an enzymatic reaction using an enzyme derived from a microorganism having a high substrate specificity. As the latter method, by reacting para-hydroxybenzoic acid with para-hydroxybenzoic acid hydroxylase as an enzyme,
A method for producing protocatechuic acid is known.

Such conventionally reported parahydroxybenzoic acid hydroxylase is, for example, one produced by a microorganism of the genus Pseudomonas (J. Biol. Che.
m., 241, 2453 (1966), Biochem.Biophys.Res.Communs., 3
4, 1 (1969)) and those produced by microorganisms of the genus Micrococcus (Current Science, 54, 419 (1985)). All of these enzymes use NADPH (nicotinamide adenine dinucleotide phosphate) as a coenzyme, and para-hydroxybenzoate hydroxylase produced by a microorganism is a NADPH-dependent enzyme that reacts in the presence of NADPH. At present, only sex-parahydroxybenzoic acid hydroxylase is generally known.

However, since NADPH which is a coenzyme of NADPH-dependent parahydroxybenzoic acid hydroxylase is expensive, it is industrially disadvantageous to use the NADPH-dependent enzyme for the actual production of protocatechuic acid. Is. Furthermore, at pH 7 and above, NADPH is N
Since there is a problem that it is less stable than ADH,
In the art, it is a fact that development of parahydroxybenzoic acid hydroxylase suitable for industrial production is desired.

[0005]

[Means for Solving the Problems] In order to solve the above problems, the present inventors extensively searched for microorganisms having the ability to produce parahydroxybenzoate hydroxylase from the natural world.
As a result, the present inventors previously found that Rhodococcus sp. It was found that protocatechuic acid can be collected by culturing the L-1 strain (Microtechnology Research Institute, No. 10614) (Japanese Patent Application No. 1-573).
62) further proceeded with research and found that this microorganism unexpectedly produced NADH-dependent parahydroxybenzoic acid hydroxylase having NADH (nicotinamide adenine dinucleotide) as a coenzyme, and the present invention It came to completion.

That is, the gist of the present invention is (1) NADH
In the presence of (nicotinamide adenine dinucleotide), NADH-dependent parahydroxybenzoate hydroxylase having predetermined enzymatic properties such as acting on parahydroxybenzoic acid to produce protocatechuic acid, and (2) A NADH-dependent parahydroxyl which comprises culturing a microorganism belonging to the genus Rhodococcus and having the ability to produce NADH-dependent parahydroxybenzoic acid hydroxylase, and collecting NADH-dependent parahydroxybenzoic acid hydroxylase from the culture. It relates to a method for producing benzoate hydroxylase.

The NADH-dependent parahydroxybenzoate hydroxylase of the present invention will be described in detail below. 1) Action of enzyme In the presence of NADH (nicotinamide adenine dinucleotide), it acts on para-hydroxybenzoic acid,
Protocatechuic acid is produced.

2) Substrate specificity As a result of examining the activity of 14 similar compounds of parahydroxybenzoic acid, when the activity against parahydroxybenzoic acid was taken as 100%, the compounds showing 10% or more activity were 2, 4-dihydroxybenzoic acid (16%) was the only compound, and 4 other compounds showed activity of 5% or less. Therefore, this enzyme is an enzyme that uses parahydroxybenzoic acid as a specific substrate.

3) Method for measuring titer Enzyme activity was measured by using 40 μM parahydroxybenzoic acid and a reduced pyridine nucleotide (NADH or NA).
50 mM Hepes buffer (pH) containing 40 μM (p
H7.6) in 0.6 ml for 1 minute at 33 ° C.
This was done by measuring the absorbance at 40 nm.
As a control, the absorbance was measured when the reaction was carried out without the addition of parahydroxybenzoic acid, and the value was subtracted as the control value. In addition, the enzyme unit has an enzyme activity of reducing 1 μM of reduced pyridine nucleotide in 1 minute.
The unit was used.

4) Optimum pH and stable pH range The optimum pH is pH 7.5 to 7.8 as shown in FIG. Stable pH range is 25 ° C for 1 hour,
As shown in FIG. 2, pH was 7.4 to 7.8.

5) Optimum temperature and stable temperature The optimum temperature for the reaction was 33 ° C (Fig. 3). The stable temperature range is pH 7.6 and 6 at each temperature in 50 mM Hepes buffer.
It was kept warm for 0 minutes and the residual activity was measured. As a result, the enzyme of the present invention was stable up to 25 ° C, showed a residual activity of about 50% at 45 ° C, and was completely inactivated at 60 ° C or higher (Fig. 4).

6) Km value of NADH The Km value of NADH for this enzyme is 5 as shown in FIG.
It was 5.3 μM.

7) Km value of NADPH The Km value of NADPH for this enzyme was 333 μM as shown in FIG.

8) Km value of parahydroxybenzoic acid The Km value of parahydroxybenzoic acid for this enzyme was 6.7 μM when the coenzyme NADH was used (FIG. 7),
It was 17.2 μM when the coenzyme NADPH was used (FIG. 8).

9) Extraction / Purification Method NADH-dependent parahydroxybenzoic acid hydroxylase is an inducing enzyme and is produced by culturing a microorganism belonging to the genus Rhodococcus in a medium containing isophthalic acid as a carbon source as described later. Can be made After completion of the culture, cells are separated from the culture by a method such as centrifugation.

Although the separated bacterial cells may be used as they are as an enzyme source, it is preferable to further extract and purify the enzyme. When the enzyme is separated from the bacterial cells, the collected bacterial cells are suspended, and then, for example, ultrasonic waves and pressure changes (French) are used.
Press, Huges press), stirring (homogenizer,
A cell-free extract can be obtained by destroying the cells by a physical method such as bead mill, blender), freeze-thawing, or a chemical method such as enzyme, surfactant, chaotropic ion, acid alkali, antibiotic, autolysis. You can This enzyme can be extracted and purified by general
It can be carried out by appropriately selecting the purification method and is not particularly limited. For example, salting out method, ultrafiltration method, column chromatography (ion exchange, affinity, gel filtration, etc.), electrophoresis method and the like can be mentioned.

10) Molecular Weight The molecular weight of this enzyme is Clearpack GFS-3 (8.0 m).
mID × 30 cm: manufactured by ATTO) and TSK gelG3
000SW (7.5 mm ID x 30 cm: manufactured by Tosoh Corporation)
Approximately 6.7 x 1 by the high-speed gel filtration column method used by connecting
It was estimated to be 0 ⁴ .

11) Effects of inhibitors, etc. When the effects of various added substances on this enzyme were examined, it was inhibited by halogen ions and thiocyanate. Further, it was not inhibited by cation ions such as sodium ion and potassium ion.

As described above, the present enzyme acts on parahydroxybenzoic acid to produce protocatechuic acid, and thus is a parahydroxybenzoic acid hydroxylase.
Furthermore, as described above, the Km value of this enzyme for NADH was 55.3 μM, and the Km value for NADPH was 333 μM.
Since it is M, the Km value for NADH is NADP
It shows a Km value that is 1/6 times lower than that for H, which indicates that the enzyme is a NADH-dependent parahydroxybenzoate hydroxylase.

Next, the microorganism capable of producing the NADH-dependent parahydroxybenzoate hydroxylase of the present invention will be described. Microorganisms used in the present invention,
Any substance belonging to the genus Rhodococcus and capable of producing NADH-dependent parahydroxybenzoate hydroxylase having the above-mentioned properties may be used. Among them, preferred strains are Rhodococcus sp. Isolated from the soil in Tokyo by the present inventors. L-1 strain (Rhodoc
occus sp. L-1 strain, Micro Engineering Research Institute, No. 10614).

The present bacterium has the following mycological properties. (1) Morphological properties Bacteria form: Rod size: 0.8 × 1.5-2.0 μm Polymorphism: 1-2 at a temperature of 30 ° C.
It forms hyphae in the day and then undergoes fragmentation to become bacilli. It does not form aerial mycelia. Acid resistance: None Gram stainability: Positive flagella: None spores: None

(2) Culture properties Meat agar plate culture: Good growth. The colony shape is circular,
The surroundings are smooth and the color is milky white. Broth agar slope culture: filamentous. The color of the colony is milky white. Broth liquid culture: Uniform turbidity. Isophthalic acid agar plate culture: Good growth. The colony shape is circular, the periphery is smooth, and the color is white.

(3) Physiological properties (1) Optimum growth temperature: 25 to 33 ° C (2) Growth temperature: 20 to 35 ° C (3) Optimum pH: 6.0 to 8.0 (4) Growth Growth pH: 5.0 to 10.
0 (5) Acid requirement: Aerobic (6) Chromine production: None (7) Gelatin liquefaction: Cationic (8) Catalase activity: Cationic (9) Oxidase activity: Anionic ( 10) Urease activity: positive (11) Production of indole: negative (12) Hydrolysis activity of starch: negative (13) Reduction of nitrate: negative (14) Growth in citrate medium: grow (15) Methyl red test ： Negative (16) VP reaction ： Negative

(17) OF test (Hugh Leifson method, 30 ° C., 10 days, open system, closed system): [sugar] [open system] [closed system] Glucose No change No change Sucrose No change No change D -Fructose No change No change D-Mannit No change No change L-Sorbose No change No change D-Raffinose No change No change D-Rhamnose No change No change D-Galactose No change No change meso-Eristol No change No change D-lactose No change No change myo-inosit No change No change D-xylose No change No change Inositol No change No change Trehalose No change No change D-Arabinose No change No change L-Arabinose No change No change Cellobiose No change No change

(18) Decomposition of sugar (Barsiekow medium, 30 ° C., 10 days): [Sugar] [Acid production] [Gas production] No glucose production No production Saccharose production No production D-lactose production No production Glycerol production Not produced Not produced D-arabinose Not produced Not produced

(19) Diaminopimelic acid isomer of cell wall: meso-DAP. (20) Cell wall acyl type (glycolyl test): There is a glycolyl group. (21) Presence of mycolic acid: present. (22) Separation source: Soil in Tokyo

Next, in order to produce NADH-dependent parahydroxybenzoic acid hydroxylase using such a microorganism belonging to the genus Rhodococcus, culture is carried out using the following medium. That is, a natural medium or a synthetic medium can be used as long as the medium appropriately contains isophthalic acid as a main carbon source, a nitrogen source, inorganic salts, vitamins and other nutritional factors. As a nitrogen source, ammonium nitrate,
Inorganic fluorine sources such as sodium nitrate, ammonium sulfate, ammonium chloride and ammonium phosphate, peptone, meat extract, constip liquor, yeast extract, casamino acid, soybean flour and the like can be used alone or in combination. As inorganic salts, potassium phosphate, sodium phosphate, magnesium sulfate, manganese sulfate, iron sulfate, zinc sulfate, copper sulfate, sodium molybdate, calcium chloride, potassium chloride and the like may be added. In addition to these, vitamins,
Examples include amino acids, nucleic acids and salts thereof. Also, peptone, meat extract, constip liquor, yeast extract,
Natural organic nutrients containing the above nutritional factors such as casamino acid can also be added as appropriate.

The culture is carried out under aerobic conditions such as a shaking culture method,
The aeration and agitation culture method is preferable, but the liquid static culture method can be appropriately combined. The culture temperature is 20 to 35 ° C,
Particularly, 25 to 33 ° C. is preferable, and the pH during culture is 7 to 9. The concentration of isophthalic acid in the medium is usually 0.1-10.
%, Preferably 0.5 to 3.0% is suitable. Isophthalic acid as main carbon source, medium pH around 7.0, 30 ℃
Shake culture or culture with aeration and stirring. The culture time is usually about 65 to 75 hours. After the completion of the culture, the NADH-dependent parahydroxybenzoate hydroxylase of the present invention is collected from the culture medium and purified by the method described above.

[0029]

The present invention will be described in more detail with reference to examples and reference examples, but the present invention is not limited thereto. Example 1 Isophthalic acid 2%, ammonium sulfate 0.2%, monopotassium phosphate 0.15%, dipotassium phosphate 0.15%, magnesium sulfate 0.05%, sodium chloride 0.01%, calcium chloride 0. A medium consisting of 002%, 0.001% iron sulfide, 0.0002% manganese sulfide, 0.0002% zinc sulfide, 0.0002% sodium molybdate, 0.0002% pH 7.2 was added to a 100 ml Erlenmeyer flask with 1 platinum loop Rhodococcus. L1 (Microtechnology Research Institute, No. 10614) was inoculated and shake-cultured at 30 ° C. for 70 hours, and 100 μl of what was shake-cultured was extracted and then inoculated into 40 ml of a new medium. Shake culture was performed.

After the completion of the culture, 2 liters of the culture solution was centrifuged to obtain 25.7 g of bacterial cells (wet cell weight). The cells thus obtained were suspended in 50 mM Hepes buffer (pH 7.6) containing 5 mM Mg ²⁺ so as to have a concentration of 25.7 g / 100 ml, and Sony Cater 250 (BRANSO
After crushing the bacterial cells by N company), centrifugation (100,000 xg)
Then, a cell-free extract was obtained. The specific activity of this extract is 1
The enzyme preparation was 1.6 × 10 ^-2 U / mg.

Next, this extract was added to 5 mM Mg ²⁺ containing 5
0 mM Hepes buffer (pH 7.6) flow rate 1 ml / min
HiTrap Blue column (2 cmI)
D × 4 cm: manufactured by Pharmacia), washed with the same equilibration buffer, and then subjected to stepwise elution with 50 mM Hepes buffer (pH 7.6) containing 50 mM and 150 mM KCl to obtain an active image of the target enzyme. Minutes (fraction No.
46-49) was obtained (FIG. 9). This active fraction was desalted using a centrifugal type ultrafiltration device Ultracent-30 (manufactured by Tosoh Corporation) and equilibrated with 50 mM Hepes buffer clear pack IEP / DEAE (60 mm ID × 1).
0 cm: manufactured by Ato Co., Ltd.) and adsorbed by 0.0 to 0.5 M
By the linear gradient elution method using the ionic strength of KCl,
An enzyme active fraction was obtained at a flow rate of 8 ml / min. This enzyme active fraction was desalted to 500 μl using Ultracent-30 and concentrated to give Clearpack GFS-3 (8.0 mm).
ID × 30 cm: manufactured by ATTO) and TSK gelG30
00SW (7.5 mm ID x 30 cm: manufactured by Tosoh Corporation) was connected to a high-speed gel filtration column, and separated and eluted with 50 mM Hepes buffer solution (pH 7.6) containing 0.5 M KCl to give a single electrophoresis gel. An active fraction was obtained (Fig. 10).
Electrophoresis was carried out on a 10% polyacrylamide gel by the slab gel electrophoresis method according to the Davis method, and then stained by the silver staining method. The molecular weight of this active fraction was estimated to be about 6.7 × 10 ⁴ . The specific activity of the purified product obtained here was 18.8 × 10 ^-2 U / mg.

Reference Example 1 10 μl of parahydroxybenzoic acid (final concentration 40 nmol)
And 8 μl of NADH (final concentration 40 nmol) was dissolved in 432 μl of 50 mM Hepes buffer (pH 7.6), and then the enzyme extract obtained in Example 1 (100,000 × g of supernatant, 687.
9 μg protein / ml) 250 μl was added to give 0,1
The reaction was carried out for 0, 20 minutes at a reaction temperature of 33 ° C., and the reaction was stopped by adding 600 μl of 1M perchloric acid + 0.2% EDTA-Na solution. Then, the reaction solution is 1500 rp
Centrifugation was performed for 10 minutes to remove proteins, and the resulting supernatant was analyzed by high performance liquid chromatography. As a result, the peak of para-hydroxybenzoic acid observed at 0 hour of the reaction was greatly reduced after 10 minutes of the reaction and completely disappeared after 20 minutes of the reaction.

Reference Example 2 Para-hydroxybenzoic acid (final concentration 40 nmol) 10
50 [mu] l and NADH (final concentration 40 nmol) 10 [mu] l and 3,4-dihydroxyphenylacetic acid (final concentration 400 nmol) 10 [mu] l which is an internal standard substance for quantification.
After dissolving in 470 μl of M Hepes buffer (pH 7.6), the crude enzyme (HiTrap Blu) obtained in Example 1 was used.
e-column (2 cm ID x 4 cm) eluate, 105 μg
/ Ml) 10 μl was added to 0, 3, 10, 20, 40,
Perform the reaction for 60, 100 minutes at a reaction temperature of 33 ° C., and
M perchloric acid + 0.2% EDTA-Na solution 500μ each
1 was added to stop the reaction. Then the reaction solution is 1500r
pm was centrifuged for 10 minutes to remove protein, and the resulting supernatant was analyzed by high performance liquid chromatography. As a result, the peak of protocatechuic acid increased with time. The amount of protocatechuic acid at each elapsed time was quantified with an internal standard substance, and an increase curve of the amount of protocatechuic acid produced as shown in FIG. 11 was obtained.

[0034]

INDUSTRIAL APPLICABILITY The NADH-dependent parahydroxybenzoic acid hydroxylase of the present invention has the property of acting on parahydroxybenzoic acid in the presence of NADH to produce protocatechuic acid. It is extremely useful for commercializing industrial production.

[Brief description of drawings]

FIG. 1 is a diagram showing the optimum pH of the present enzyme.

FIG. 2 is a diagram showing the pH stability of the present enzyme.

FIG. 3 is a diagram showing the optimum temperature of the present enzyme.

FIG. 4 is a diagram showing the temperature stability of the present enzyme.

FIG. 5 is a diagram showing Km values of the present enzyme with respect to NADH.

FIG. 6 is a diagram showing Km values of the present enzyme with respect to HADPH.

FIG. 7 is a graph showing the Km value of this enzyme for parahydroxybenzoic acid when NADH is used.

FIG. 8 is a view showing a Km value of this enzyme for parahydroxybenzoic acid when NADPH was used.

FIG. 9 is a HiTrap Blue in Example.
It is a figure which shows the elution pattern of this enzyme using a column.

FIG. 10 shows an electrophoretogram of the present enzyme active fraction using 10% polyacrylamide gel in Examples.

FIG. 11 shows an increase curve of the amount of protocatechuic acid produced, obtained in Reference Example 2.

Claims (2)

[Claims] 1. A NADH-dependent parahydroxybenzoate hydroxylase having the following enzymatic properties. Action In the presence of NADH (nicotinamide adenine dinucleotide), acting on parahydroxybenzoic acid,
Protocatechuic acid is produced. Substrate specificity Parahydroxybenzoic acid is used as a specific substrate. Optimum pH and stable pH range The optimum pH is pH 7.5 to 7.8, and the stable pH range is pH 7.4 to 7.8 under a holding condition of 25 ° C. for 1 hour.
Is. Optimum temperature and stable temperature range The optimum temperature of the reaction is 33 ° C., it is stable up to 25 ° C. when it is kept at pH 7.6 for 60 minutes, and is completely deactivated at 60 ° C. or higher. Molecular weight The molecular weight measured by the high speed gel filtration column method is about 6.7.
It is × 10 ⁴ . Effect of inhibitors, etc. It is inhibited by halogen ions and thiocyan ions, but not by cation ions such as sodium ions and potassium ions. 2. A microorganism belonging to the genus Rhodococcus, which has the ability to produce NADH-dependent parahydroxybenzoate hydroxylase, is cultivated, and NADH-dependent parahydroxybenzoate hydroxylase is collected from the culture. A method for producing NADH-dependent parahydroxybenzoic acid hydroxylase.