JPH0956378A - Laccase ii and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce laccase II, good in thermostability and having high properties of acting on an aromatic amine and provide a method for mass-producing the laccase II at a low cost. SOLUTION: This laccase II has the following characteristics: (1) about 62,000 molecular weight; (2) range of optimum action temperature: >=78 deg.C optimum temperature; (3) thermostability: stable to about 55 deg.C when kept at pH7.8 for 30min; (4) optimum pH: about 5.0; (5) stable pH range: pH5.0-10.0 when treated at 30 deg.C for 3hr and (6) isoelectric point: about 5.9. The laccase II is obtained from a cultured product of a new strain Trametes sp. Ha1 strain (FERM P-14472), etc., belonging to the genus Trametes in the presence of p-xylidine.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laccase II having excellent thermostability, which has excellent activity on aromatic amines and also phenol compounds, and a method for producing such laccase II from a basidiomycete culture. .

[0002]

Laccase, also called urushiol oxidase or lignin-degrading enzyme, exists in the presence of oxygen.
It is an enzyme that oxidizes phenolic compounds. Since laccase has a lignin decomposing action and an oxidative polymerization action of aromatic compounds such as urushiol and laccol, for example, treatment of waste liquid containing highly toxic aromatic amine, lignin removal in pulp production treatment, production of artificial lacquer , Synthesis of concrete admixture (Nikkei Biotech, October 25, 1993), browning treatment of cocoa, coffee and black tea (Motoda et al., Journal of Japan Food Industry Society, Vol. 29, No. 1, p11-15, ( 1982)), melanin production for cosmetics (JP-A-6-90776), clinical test reagent
Various applications such as JP-A-59-227300 are expected. In the production of artificial lacquer, in order to enhance the effect of lacquer dry to the touch, laccase with strong oxidizing power and good heat resistance,
It is especially necessary (Terada et al., Painting Engineering, Vol.26, No.6, (1
991)). In addition, even in the lignin removal and aromatic amine waste liquid treatment in the pulp manufacturing process, when it is carried out outdoors in the summer, the temperature becomes high, so that a laccase excellent in thermal stability is required. In particular, aromatic amines are difficult to remove by the activated sludge method using microorganisms, and therefore treatment with an enzyme having a high action on aromatic amines is desired.

On the other hand, laccases derived from various microorganisms have been conventionally known. For example, wood-destroying fungi, Schizophyllan commune, Coriolas versicolor (JP-A-62-220190), Lentites Bethulina (JP-B7-46989), Pycnoporas coccineus (Terada et al., Painting Engineering, Vol. .26, No. 6, (1991)), and a laccase derived from an imperfect fungus, Botulinum cinerea (Industrial Bioprocessing, June (1994)). In addition, the presence of laccase is known in animals and plants.

However, none of the laccases have been put into practical use for the above-mentioned uses, particularly for treating waste liquid containing aromatic amines, because of their low specificity to aromatic amines and low heat resistance. Is the current situation.

[0005]

SUMMARY OF THE INVENTION The present invention is to solve the above-mentioned conventional problems.
It is intended to provide a laccase II which has a high activity on an aromatic amine and also a phenol compound, and which has good heat resistance, a method for producing the same, and a bacterium producing the same. So far, a thermostable laccase highly effective for aromatic amines and a microorganism producing it have not been known.

[0006]

The present inventors have completed the present invention by inducing a novel laccase II from a newly isolated basidiomycete belonging to the genus Trametes .

The laccase II of the present invention has the following properties. (1) Molecular weight: about 62,000. (2) Optimum temperature range of action: The optimum temperature is 78 ° C or higher. (3) Thermal stability: Approximately 55 when kept at pH 7.8 for 30 minutes
Stable up to ° C. (4) Optimum pH: about 5.0. (5) Stable pH range: pH of 5.0 to 10.0 when treated at 30 ° C. for 3 hours. (6) Isoelectric point: about 5.9. This achieves the above object.

[0008] The laccase II of the present invention is a basidiomycete of the genus Astragalus, especially Tramet deposited on August 12, 1994 at the Institute of Biotechnology, Institute of Biotechnology (abbreviated as Life Research Institute).
es sp. Ha1 Strain (NTT Bacteria No. 14472; FERM P-14472)
Produced by. This achieves the above object.

The laccase II of the present invention produced by the above basidiomycete is a blue-green protein having a copper molecule,
It is further inhibited by the inhibitors mercaptoethanol, cystine, dithiothreitol, sodium azide, potassium cyanide, and α-naphthol.

No laccase derived from Bacillus subtilis having the above-mentioned properties has been known so far.

The method for producing laccase II of the present invention includes a step of culturing the above-mentioned producing bacterium in the presence of p-xylidine and separating and purifying the laccase II from the culture. This achieves the above object.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The laccase II of the present invention is a basidiomycete of the genus Arctophyta, particularly Trametes sp. Ha1 strain (Biol.
No. 2 produced by FERM P-14472). This strain is
As a result of extensive screening of the microorganisms that produce laccase from soil, sea water and fresh water, and decay products by the inventors, it is a new strain newly isolated from dead leaves of bamboo.

(1) Morphology and Culture The laccase II-producing bacterium of the present invention (hereinafter referred to as the bacterium) is
White rot fungus with lignin degrading ability, skeletal hypha and clamp connection to culture mycelium
However, due to the absence of heteromorphic cells such as cystidisia and bristly hyphae, and the fact that conidiospores are not formed in the culture hyphae, it has a mycological property such that it is a basidiomycete belonging to the genus Trametes. Identified,
It was named Trametes sp. Ha1.

The more detailed mycological properties of this bacterium are as follows.

(Potato-Glucose Agar Medium) On the 7th day after the start of the culture, both the front and back sides of the colony were white, and the colonies grew to have a diameter of 5 to 10 mm. Hyphae, with associated bound hyphae,
A small number of skeletal mycelia are found, with glare ligation. The surface of the hypha is flat and the width is 2 to 6 μm. No cystidisia or bristly hyphae are observed. Formation of conidia is not observed.

(Sabouraud agar medium) On the 7th day after the start of culture, the surface of the colony was white and the back surface was light brown.
It grows to a diameter of 3 to 7 mm and the aerial hyphae are short and sparse. Hyphae are accompanied by a characteristic combination of hyphae, a few skeletal hyphae are found, and they have glaze connections. The surface of the hypha is flat and has a width of 1 to 4 μm. Formation of conidia is not observed.

(Phenol oxidase reaction) 0.5%
The present strain is inoculated on Sabouraud agar medium containing gallic acid or 0.5% tannic acid, and a positive reaction is observed in which the periphery of the fungus is browned under culture conditions at 30 ° C. Therefore, this fungus is a kind of lignin-degrading wood decay fungus (white decay fungus).

The growth range of this bacterium is potato glucose agar medium (4 g of potato extract powder, 20 g of glucose and 2 agars).
0 g is dissolved in 1 liter of distilled water. ), The pH is about 2 to 12, the suitable growth temperature is 25 to 35 ° C., and the suitable growth pH is 4.0 to 8.0. Particularly preferred growth conditions are 30 ° C and pH of about 5.5.
It is.

Next, the conditions for producing the laccase of the present invention from this bacterium will be explained. The bacterium can be cultivated in either liquid culture or solid culture. The medium for growing this bacterium is not particularly limited, and an ordinary liquid medium or solid medium is used. Any carbon source may be used as long as the bacterium can assimilate, and sugars such as glucose, sucrose and molasses, starch, wood flour, fusuma and the like can be used. As the nitrogen source, organic nitrogen sources such as peptone, yeast extract, malt extract, meat extract, soybean degradation product, urea and amino acid, as well as inorganic nitrogen sources such as ammonium nitrate and sodium nitrate may be used. Phosphate, magnesium sulfate, potassium, calcium, copper, sodium, if necessary,
Inorganic salts such as manganese and zinc, vitamins, nucleic acid-related compounds and the like may be added. These medium components may have a concentration that does not inhibit the growth of the bacterium, and the carbon source is 0.1 to 2
0% by weight, preferably 5 to 10% by weight, the nitrogen source is 0.0
It is 1 to 5% by weight, preferably 0.1 to 2% by weight.

The pH of the medium is adjusted to 2.0 to 12.0, preferably 4.0 to 8.0, and sterilized before use. The culture temperature may be any temperature at which basidiomycetes can grow, and in practice,
The temperature is 10 to 40 ° C, preferably 25 to 35 ° C. When the present bacterium is liquid-cultured, aeration culture or shaking culture is preferable. The culture time varies depending on various culture conditions, but in the case of aeration or shaking culture, it is usually preferably 2 to 10 days.

The laccase II of the present invention is secreted and produced extracellularly after induction by p-xylidine and accumulated in the culture medium. p-xylidine has a final concentration of 1 mM to 20 mM in the medium,
More preferably, it is added so as to be 1 mM to 5 mM.

(2) Separation and Purification of Laccase II from Culture Solution To separate and purify the laccase II of the present invention (hereinafter referred to as the present laccase II) from the culture solution of the present bacterium, a known purification method,
For example, dialysis, salting out, various column chromatography, isoelectric focusing, gel filtration, and electrophoresis can be used alone or in combination. After culturing, insoluble matters such as bacterial cells are removed by centrifugation, filtration with a filter paper or filter cloth, etc., and a culture solution (crude enzyme solution) containing the present laccase II is recovered. By commonly used enzyme purification methods such as concentration, ammonium sulfate fractionation (salting out), dialysis, various chromatographies, gel filtration, etc.
The present laccase II can be purified from the obtained crude enzyme solution.
For example, the concentrated crude enzyme solution obtained above is concentrated, and then subjected to dialysis, various chromatographies, ultrafiltration, and isoelectric focusing in order to give a purified highly active laccase II.
A fraction containing is obtained.

(3) Measurement of laccase activity In the present invention, the laccase II activity is the laccase II activity.
It is measured by a color reaction by oxidative condensation of 4-aminoantipyrine and a hydrogen donor catalyzed by. Examples of hydrogen donors include phenol and N, N-diethylaniline (DEA). The absorbance at 505 nm (phenol) or 555 nm (DEA) was measured in a color reaction system by oxidative condensation. In the present application, it contains 7.5 micromoles of phenol and 1.25 micromoles of 4-aminoantipyrine
520 μl of 190 mM sodium acetate buffer (pH 4.5) was preincubated at 30 ° C. for 1 minute, 20 μl of the enzyme solution was mixed, and the change in absorbance at 505 nm was measured. The enzyme activity that increases the absorbance by 0.01 for 1 minute is defined as 1 unit (U; unit).

The present invention will be described in more detail with reference to the following examples, which, however, do not limit the present invention in any way.

[0026]

【Example】

Example 1 (Cultivation of Trametes sp. Ha1) 500 m in the following seed culture medium
Incubate with shaking in 2 L Sakaguchi flask (2) containing L at 28 ° C. for 7 days (130 rpm, reciprocal shaking).

[0027]

[Table 1]

Glucose is added to each of the two so that the concentration becomes 2% on the 4th day of the culture, and p-xylidine is added to the other so that the concentration becomes 20 mM.

Next, two 500 mL of these culture solutions were inoculated into a 50 L culture tank containing 30 L of the following medium for main culture, and 28
The culture was carried out with aeration and agitation (number of agitation: 300 rpm) at 10 ° C for 10 days.

[0030]

[Table 2]

However, inorganic salts and Pluronic were separately sterilized and then added to the sterilized medium. Although the pH of the culture solution decreased from the 5th day of culture, it was adjusted to 5.5 to 6.0 with 0.5M caustic soda. As a result of culturing in this way, the culture solution in the tank showed the highest activity of 94 (U / mL) on the 8th day and decreased to 71 (U / mL) on the 10th day.

Example 2 (Purification of the present laccase II) The culture solution obtained in the above Example 1 was centrifuged (8,000 rpm, 20 min), and the obtained supernatant was filtered with a filter paper, and the recovered filtrate was used as a crude enzyme. It was a liquid.

The crude enzyme solution was adjusted to pH 7 with 0.5 M caustic soda, concentrated by ultrafiltration, dialyzed against 20 mM Tris-hydrochloric acid buffer (pH 7.5) and frozen (-80 to -20 ° C). This was thawed and insoluble matter was removed by centrifugation to obtain a supernatant.

The above supernatant was treated with 10 mM Tris-HCl buffer (pH
7.5) DEAE-Sephacel column (5.5 x 18.7)
cm; manufactured by Pharmacia). After washing the column with the same buffer, 10 mM potassium phosphate buffer (pH 5.7) to elute the present laccase II adsorbed on the column,
Fractions were collected. This laccase II appears as a green-blue band during elution and migration on the column.
The procedure was started immediately before the laccase II was eluted from the column outlet.

A part of each of the obtained fractions was tested for the activity of the present laccase II, and the specific activity (activity value (U / mL) / OD ₂₈₀ ) was measured. Fractions having a specific activity of 45 or more were combined and concentrated by ultrafiltration. The concentrated solution was dialyzed against 0.5 mM sodium chloride-containing 20 mM Tris-hydrochloric acid buffer solution (pH 7.5; hereinafter abbreviated as STHB) to obtain a dialysate solution.

The above dialyzed solution was equilibrated with STHB to ConA-
It was tested on a Sepharose column (5.5 × 6 cm; manufactured by Pharmacia). After washing and elution of the non-adsorbed substance, the laccase II was eluted using the above buffer containing 1 M D-glucose.
Since tailing of activity was observed, elution was completed with 0.1 M boric acid-containing potassium phosphate buffer. The solution containing the laccase II was adjusted to pH 7 with 0.5 M caustic soda, concentrated by ultrafiltration, and dialyzed with 10 mM Tris-HCl buffer (pH 7.5). The insoluble matter generated in the dialysate was removed by centrifugation.
The resulting supernatant was filtered with a filter having a pore size of 0.2 μm. The filtrate was tested by a preparative isoelectric focusing device (rotophor; manufactured by Bio-Rad) and further fractionated and purified to obtain an enzyme purified solution.

The present laccase II in each of the above purification steps
Table 3 shows the degree of purification and the specific activity per protein solution.

[0038]

[Table 3]

Example 3 (Molecular weight of the present laccase II) SDS polyacrylamide gel electrophoresis (SDS-PAGE) method (Laemli, UK, Nature, 227,
680 (1970)) was used to measure the molecular weight of the laccase II.

Purified laccase II obtained in Example 2 above
And 6 kinds of proteins known as molecular weight markers (molecular weights of about 97,400, 66,300, 42,400, 30,000, 20,100 and 14,400) were co-migrated. After completion of the electrophoresis, the cells were stained with Coomassie Brilliant Blue, and the molecular weight of the present laccase II was measured from the relative migration distance between the present laccase II and each molecular weight marker.

By this SDS-PAGE, this laccase II showed a single band. The molecular weight of this laccase II by SDS-PAGE is about 62,000.

Example 4 (Adequate temperature range and thermostability of the present laccase II) (1) Optimum temperature range of action The activity of the purified laccase II obtained in Example 2 above was varied in the color reaction system. Temperature conditions (15, 2
(0, 30, 40, 50, 60, 70, 78 ° C). The results are shown in FIG. In the figure, the horizontal axis represents the reaction temperature (° C), and the vertical axis represents the relative activity (%) when the maximum activity value is 100. The optimum temperature of this laccase II is 78 ° C or higher.

(2) Thermostability The purified laccase II obtained in Example 2 above was treated under various temperature conditions (0, 30, 40, 45, 50, 55, 60, 65, 70,
The activity was measured after incubation in phosphate buffer (pH 7.8) for 30 minutes at 95 ° C. The results are shown in FIG. The horizontal axis in the figure represents the treatment temperature (° C), and the vertical axis represents the maximum activity value.
The relative residual activity (%) when 100 is shown. Book laccase
II is stable because it retains 70% or more of the enzyme activity even when the treatment temperature is 60 ° C. This laccase II retains 100% of its enzymatic activity at the treatment temperature of 55 ° C.

Example 5 (Optimal pH and stable pH range of the present laccase II) (1) Optimum pH Depending on the pH range, 50 mM acetate buffer, 50 mM phosphate buffer or 50 mM Tris-HCl buffer was used. , PH 1.5 to 7.8 (1.
5, 3.0, 4.0, 4.5, 5.0, 5.5, 6.1, 7.1, 7.3, 7.5, 7.
After adjusting to 8), the activity of the purified present laccase II obtained in Example 2 above was measured. The results are shown in FIG. The horizontal axis in the figure represents the reaction pH, and the vertical axis represents the relative activity (%) when the maximum activity value is 100. Optimal pH of this laccase II
Is about 5.0.

(2) Stable pH range The pH of the laccase II solution obtained in Example 2 is 1.5-12.0.
(1.5, 3.0, 4.0, 4.6, 5.0, 5.5, 6.1, 7.1, 7.3, 7.
5, 7.8, 8.9, 9.0, 10.3, 11.3, 12.0), 30 ℃
And incubated for 3 hours. Then, the activity in each pH-treated solution was measured. FIG. 4 shows the results. The horizontal axis in the figure is processing
The pH is shown, and the vertical axis shows the relative residual activity (%) when the maximum activity value is 100. The stable pH range of this laccase II is 5.0.
~ 10.0.

Example 6 (Isoelectric point of the present laccase II) The isoelectric point of the present laccase II was measured by the isoelectric focusing method. As a result of the measurement, the isoelectric point of this laccase II is about 5.9.

Example 7 (Substrate specificity of the present laccase II) The substrate specificity of the present laccase II purified in the above Example 2 was examined. As a substrate, catechol, resorcinol, hydroquinone, caffeic acid, hydrocaffeic acid, guaiacol, pyrogallol, o-cresol, m-cresol, p-cresol,
p-hydroxybenzoic acid, phenol, 2,6-dimethoxyphenol, p-phenylenediamine, p-toluidine, dimethylaniline, diethylaniline, TOOS (N-ethyl-N-
(2-hydroxy-3-sulfopropyl) -3-methylaniline), ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), 3-indoleacetic acid, 3-indoleacetonitrile , L (+)-ascorbic acid, 3-methylcatechol, 4-methylcatechol or 4-chlorocatechol was used. The measurement was performed by the oxygen electrode method in which the oxygen consumption accompanying the oxidation of the substrate is converted into an electric signal by the oxygen electrode.
Specifically, 1980 μl of 200 mM sodium acetate buffer (pH 4.5) containing 10 μmol of the above substrate was equilibrated in air at 30 ° C. for 10 minutes, and then 20 μl of enzyme solution was added to the reaction. Starting, oxygen consumption was measured with a Clark oxygen electrode. In addition to the present laccase II, laccases derived from Pychnoporus coccineus and Coriolas versicolor were also examined for comparison. Table 4 shows the relative values (%), where the measured value when catechol was used as the substrate was 100.

[0048]

[Table 4]

The present laccase II contains hydrocaffeic acid and
It showed high specificity to some phenolic compounds such as 2,6-dihydroxyphenol and aromatic amines such as p-toluidine, dimethylaniline, diethylaniline and TOOS. Specificity for L (+)-ascorbic acid also differs from other laccases.

Example 8 (Inhibitor of the present laccase II) The inhibitor of the present laccase II purified in the above Example 2 was examined. As an inhibitor
Using α, α-dipyridyl, acetylacetone, cystine, dithiothreitol, potassium cyanide, neocuproine, mercaptoethanol, α-naphthol, barium thiocyanate, phenylhydrazine or sodium azide to measure enzyme activity inhibition by these inhibitors The inhibitory activity was investigated by Specifically, it was carried out in a microcell in a spectrophotometer under constant temperature conditions of 30 ° C., 550 μl of 218 mM sodium acetate buffer (pH 5.0) containing 0.6 μmol of the above inhibitor was added, and 20 μl of enzyme solution was added.
After reacting for 3 minutes, 30 μl of 0.1 M p-phenylenediamine solution was added, and the change in absorbance at a wavelength of 460 nm was examined. The inhibitory activity was expressed by setting the amount of increase in absorbance per minute when no inhibitor was added as 100. The results are shown in Table 5.

[0051]

[Table 5]

The present laccase II was strongly inhibited by disulfide bond reducing compounds (cystine, dithiothreitol, mercaptoethanol, etc.) and electron transfer system inhibitors (potassium cyanide, α-naphthol, sodium azide, etc.). This was the same as a conventionally known inhibitor of laccase.

[0053]

According to the present invention, a novel laccase having good heat resistance and a method for producing the same are provided. The present laccase II has a higher action on aromatic amines than a conventional laccase, acts on a phenol compound, and has good heat resistance. Therefore, the present laccase II is useful for treatment of waste liquid containing aromatic amine or phenol, production of artificial lacquer, pulp manufacturing treatment process, synthesis of useful polyphenols, and the like. Since the present laccase II is produced by culturing basidiomycete, it can provide the laccase II in large quantities at low cost and can be widely used for reagents or industrial applications.

[Brief description of drawings]

FIG. 1 shows the relative activity when the present laccase II activity is measured in the range of 15 to 78 ° C.

FIG. 2 shows the relative residual activity of the present laccase II measured after incubation at 0 to 95 ° C. for 30 minutes.

FIG. 3 shows the relative activity when the present laccase II activity is measured in the pH range of 1.5 to 7.8.

[Fig. 4] The present laccase II was treated at pH 1.5 to 12.0 at 30 ° C.
The relative residual activity measured after 3 hours of incubation at.

Claims (3)

[Claims] 1. The following characteristics (1) molecular weight: about 62,000; (2) optimum temperature range for action: optimum temperature is 78 ° C or higher; (3) thermal stability: when kept at pH 7.8 for 30 minutes. About 55
Stable up to ℃; (4) Optimum pH: about 5.0; (5) Stable pH range: pH 5.0 to 10.0 when treated at 30 ℃ for 3 hours; and (6) Isoelectric point: about 5.9; Laccase II with. 2. The laccase II according to claim 1, which is produced by a basidiomycete belonging to the genus Trametes , Trametes sp. Ha1 strain (Life Research Institute No. 14472). 3. The following characteristics: (1) molecular weight: about 62,000; (2) optimum working temperature range: optimum temperature is 78 ° C. or higher; (3) thermal stability: kept at pH 7.8 for 30 minutes. In case of about 55
Stable up to ℃; (4) Optimum pH: about 5.0; (5) Stable pH range: pH 5.0 to 10.0 when treated at 30 ℃ for 3 hours; and (6) Isoelectric point: about 5.9; A method for producing laccase II having laccase II, comprising the steps of:
A method comprising culturing in the presence of xylidine, and separating and purifying the laccase II from the culture.