JP2984584B2 - Laccase II and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a laccase II having excellent heat resistance to aromatic amines and also to a phenol compound and having good heat resistance, and a method for producing such laccase II from a basidiomycete culture. .

[0002]

2. Description of the Related Art Laccase is also called urushiol oxidase or lignin-degrading enzyme, and in the presence of oxygen,
It is an enzyme that oxidizes phenolic compounds. Since laccase has a lignin decomposition activity and an oxidative polymerization activity of aromatic compounds such as urushiol and lacquer, for example, treatment of waste liquid containing highly toxic aromatic amines, removal of lignin in pulp production treatment, production of artificial lacquer , Synthesis of concrete admixture (Nikkei Biotech, October 25, 1993), Browning treatment of cocoa, coffee and tea (Motoda et al., Journal of the Japan Food Industry Association, Vol. 29, No. 1, p11-15, ( 1982)), production of melanin for cosmetics (JP-A-6-90776), clinical test reagents
(Japanese Patent Application Laid-Open No. 59-227300), and various uses are expected. In the production of artificial lacquer, laccase with strong oxidizing power and good heat resistance is used to enhance the touch-drying effect of lacquer
Especially necessary (Terada et al., Painting Engineering, Vol.26, No.6, (1
991)). In addition, the lignin removal and the aromatic amine waste liquid treatment in the pulp production treatment require a laccase excellent in heat stability because the temperature is high when the method is carried out outdoors in summer. In particular, since aromatic amines are difficult to remove by activated sludge method using microorganisms, treatment using an enzyme having a high effect on aromatic amines is desired.

On the other hand, laccases derived from various microorganisms have been known. For example, wood rot fungi such as Shizofiran Commune, Coriolus Versicolor (Japanese Patent Application Laid-Open No. 62-220190), Lentites Vetulina (Japanese Patent Application Publication No. 7-46989), Pycnopora coccineus (Terada et al., Coating Engineering, Vol. 26, No. 6, (1991)) and a laccase derived from the imperfect fungus Botulinum cinerea (Industrial Bioprocessing, June issue (1994)). In addition, the presence of laccase is known in animals and plants.

However, none of the laccases has been put to practical use for the above-mentioned applications, particularly for the treatment of waste liquids 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.
An object of the present invention is to provide a laccase II having high heat resistance to aromatic amines and also to phenol compounds and having good heat resistance, a method for producing the same, and a producing bacterium. So far, no heat-resistant laccase having high action on aromatic amines and microorganisms producing the same have been known.

[0006]

Means for Solving the Problems The present inventors have derived a novel laccase II from a newly isolated basidiomycete belonging to the genus Trametes , thereby completing the present invention.

The laccase II of the present invention has the following properties. (1) Molecular weight: about 62,000. (2) Range of optimal temperature for operation: The optimal temperature is 78 ° C or higher. (3) Thermal stability: about 55 minutes when kept at pH 7.8 for 30 minutes.
Stable up to ° C. (4) Optimum pH: about 5.0. (5) Stable pH range: pH 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 has been deposited Hourokutake genus Basidiomycetes, on August 12, in particular 1994 Agency of life Institute of Advanced Industrial Science and Technology (referred to as Life Institute) Tramet
es sp. Ha1 strain (Biological Research Institute No. 14472; FERM P-14472)
Produced by This achieves the above object.

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

A laccase derived from Basidiomycete basidiomycetes having the above characteristics has not been known so far.

[0011] The method for producing laccase II of the present invention comprises a step of culturing the above-produced bacteria in the presence of p-xylidine and separating and purifying the laccase II from the culture. This achieves the above object.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

The laccase II of the present invention is a basidiomycete belonging to the genus Pleurotus, especially Trametes sp.
No. 2; produced by FERM P-14472). This strain is
As a result of extensive screening of microorganisms that produce laccase from soil, seawater and freshwater, and decaying substances, the present inventors have newly isolated a new strain from bamboo leaves.

(1) Morphology and Culture The laccase II-producing bacterium of the present invention (hereinafter referred to as the present bacterium)
It is a white rot fungus with lignin decomposability, skeletal hypha and clasp connection to cultured mycelium
Basidiomycete belonging to the genus Trametes due to its mycological properties such as the absence of atypical cells such as cystidia and bristle hyphae, and the absence of conidia in cultured mycelia. Identified,
Trametes sp. Ha1 was named.

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

(Potato / Glucose Agar Medium) On the seventh day after the start of the culture, both the front and back surfaces of the colonies are white, grow to a diameter of 5 to 10 mm, and the aerial hyphae are short, dense and fluffy. Hyphae are accompanied by characteristic binding hyphae,
A small number of skeletal hyphae are observed and have faint connections. The surface of the mycelium is flat and has a width of 2-6 μm. No cystidia and bristle hyphae are found. No conidiospore formation is observed.

(Sabouraud Agar Medium) On the seventh day after the start of the culture, the surface of the colony was white and the back was light brown,
It grows to a diameter of 3-7 mm, and the aerial hyphae are short and sparse. The hyphae are characteristically associated with the hyphae, with a small number of skeletal hyphae and a faint connection. The surface of the hypha is flat and 1-4 μm wide. No conidiospore formation is observed.

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

The growth range of this bacterium was determined on a potato glucose agar medium (potato extract powder 4 g, glucose 20 g and agar 2).
Dissolve 0 g in 1 liter of distilled water. )), About 4 to 40 ° C., pH is about 2 to 12, preferred temperature for growth is 25 to 35 ° C., and preferred pH for growth is 4.0 to 8.0. Particularly preferred growth conditions are 30 ° C. and a pH of about 5.5.
It is.

Next, conditions for producing the laccase of the present invention from the present bacterium will be described. The bacterium can be cultured in either liquid culture or solid culture. The medium for growing the present bacterium is not particularly limited, and a usual liquid medium or solid medium is used. Any carbon source can be used as long as it can be assimilated by the bacterium, and sugars such as glucose, sucrose, molasses, starch, wood flour, and fu (brass) can be used. As the nitrogen source, an organic nitrogen source such as peptone, yeast extract, malt extract, meat extract, soybean decomposed product, urea, amino acid and the like, and an inorganic nitrogen source such as ammonium nitrate and sodium nitrate can be used. Phosphate, magnesium sulfate, potassium, calcium, copper, sodium,
Inorganic salts such as manganese and zinc, vitamins, nucleic acid-related compounds and the like can be added. These medium components need only be at a concentration that does not inhibit the growth of the present bacterium, and the carbon source is 0.1 to 2
0% by weight, preferably 5 to 10% by weight, 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 used after sterilization. The culture temperature may be any temperature at which basidiomycetes can grow.
The temperature is 10 to 40C, preferably 25 to 35C. When the present bacterium is subjected to liquid culture, aeration culture or shaking culture is preferred. The culturing time varies depending on various culturing conditions, but in the case of aeration or shaking culturing, usually 2 to 10 days is preferable.

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

(2) Separation and Purification of Laccase II from the 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 is used.
For example, dialysis, salting out, various column chromatography, isoelectric point precipitation, gel filtration, and electrophoresis can be used alone or in combination. After the culture, insoluble substances such as bacterial cells are removed by centrifugation, filtration with a filter paper or a filter cloth, or the like, and a culture solution (crude enzyme solution) containing the present laccase II is recovered. Concentration, ammonium sulfate fractionation (salting out), dialysis, various chromatography, gel filtration and other commonly used enzyme purification methods,
The present laccase II can be purified from the obtained crude enzyme solution.
For example, the crude enzyme solution obtained above is concentrated, and then subjected to dialysis, various types of chromatography, ultrafiltration, and isoelectric focusing in order, thereby purifying the highly active present laccase II.
Is obtained.

(3) Measurement of Laccase Activity In the present invention, the laccase II activity is defined as the laccase II activity.
Is measured by a color reaction catalyzed by oxidative condensation of 4-aminoantipyrine with a hydrogen donor. Examples of the hydrogen donor include phenol and N, N-diethylaniline (DEA). In a color reaction system by oxidative condensation, the absorbance at 505 nm (phenol) or 555 nm (DEA) was measured. In the present application, containing 7.5 micromolar phenol and 1.25 micromolar 4-aminoantipyrine
After pre-incubating 520 µl of 190 mM sodium acetate buffer (pH 4.5) 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 per minute is defined as 1 unit (U; unit).

The present invention is described in more detail in the following examples, which do not limit the present invention in any way.

[0026]

【Example】

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

[0027]

[Table 1]

On both of the two plants, glucose was added to 2% on the fourth day of culture, and p-xylidine was added to one of them to 20 mM.

Next, two 500 mL of these culture solutions were inoculated into a 50 L culture tank containing 30 L of the following main culture medium.
C. for 10 days under aeration and stirring (300 rpm for stirring).

[0030]

[Table 2]

However, the inorganic salts and Pluronic were sterilized separately and then added to a sterilized medium. From the fifth day of the culture, the pH of the culture solution dropped, but the pH was adjusted to 5.5 to 6.0 with 0.5 M sodium hydroxide. As a result of cultivation in this manner, the culture solution in the tank showed the highest activity at 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 supernatant obtained by centrifuging the culture obtained in Example 1 (8,000 rpm, 20 min) was filtered through a filter paper, and the collected filtrate was subjected to crude enzyme treatment. Liquid.

The crude enzyme solution was adjusted to pH 7 with 0.5 M sodium hydroxide, concentrated by ultrafiltration, dialyzed against 20 mM Tris-HCl buffer (pH 7.5), and frozen (−80 to −20 ° C.). This was melted and insolubles were removed by centrifugation to obtain a supernatant.

The above supernatant was added to a 10 mM Tris-HCl buffer (pH
7.5) Equilibrated DEAE-Sephacel column (5.5 x 18.7)
cm; manufactured by Pharmacia). After the column was washed with the same buffer, the present laccase II adsorbed on the column was eluted with a 10 mM potassium phosphate buffer (pH 5.7),
Fractions were collected. Since this laccase II appears as a green-blue band during the elution transfer on the column, fraction collection is performed.
The procedure was started immediately before the present laccase II was eluted from the column eluate.

A part of each of the obtained fractions was used for measurement of the present laccase II activity, 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 concentrate was dialyzed against a 20 mM Tris-HCl buffer containing 0.5 M sodium chloride (pH 7.5; hereinafter abbreviated as STHB) to obtain an inner dialysate.

[0036] The above dialysate was used for ConA-
The test was performed on a Sepharose column (5.5 × 6 cm; manufactured by Pharmacia). After washing and eluting the non-adsorbed substance, the present laccase II was eluted using the above buffer containing 1M D-glucose,
Since the tailing of activity was observed, elution was completed with potassium phosphate buffer containing 0.1 M boric acid. The solution containing the laccase II was adjusted to pH 7 with 0.5 M sodium hydroxide, concentrated by ultrafiltration, and dialyzed against 10 mM Tris-HCl buffer (pH 7.5). Insoluble matter generated in the dialysis solution was removed by centrifugation.
The obtained supernatant was filtered with a filter having a pore size of 0.2 μm. The filtrate was subjected to a preparative isoelectric focusing apparatus (rotophore; manufactured by Bio-Rad) and further fractionated and purified to obtain a purified enzyme 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)), and the molecular weight of the present laccase II was measured.

Purified laccase II obtained in Example 2 above
And six proteins known as molecular weight markers (molecular weight of about 97,400, 66,300, 42,400, 30,000, 20,100 and 14,400) were simultaneously electrophoresed. After completion of the electrophoresis, staining was performed 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.

According to the present SDS-PAGE, the present laccase II showed a single band. The molecular weight of this laccase II on SDS-PAGE is about 62,000.

Example 4 (Appropriate temperature range of action and thermal stability of the present laccase II) (1) Range of actionable temperature The activity of the purified laccase II obtained in the above Example 2 was measured in various ways in the above color reaction system. Temperature conditions (15, 2
0, 30, 40, 50, 60, 70, 78 ° C). The results are shown in FIG. The horizontal axis in the figure 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 was used at various temperature conditions (0, 30, 40, 45, 50, 55, 60, 65, 70,
After incubation in a phosphate buffer (pH 7.8) at 95 ° C) for 30 minutes, the activity was measured. The results are shown in FIG. The horizontal axis in the figure represents the processing temperature (° C), and the vertical axis represents the maximum activity value.
The relative residual activity (%) when 100 is indicated. Book laccase
II is stable, maintaining 70% or more of the enzyme activity even at a treatment temperature of 60 ° C. The present laccase II retains 100% of its enzymatic activity at a treatment temperature of 55 ° C.

Example 5 (Optimal pH and stable pH range of the present laccase II) (1) Optimal pH Depending on the pH range, a 50 mM acetate buffer, 50 mM phosphate buffer or 50 mM Tris-HCl buffer was used. , PH 1.5-7.8 (1.
5, 3.0, 4.0, 4.5, 5.0, 5.5, 6.1, 7.1, 7.3, 7.5, 7.
8), and the activity of the purified present laccase II obtained in Example 2 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. Optimum pH of this laccase II
Is about 5.0.

(2) Stable pH Range The pH of the laccase II solution obtained in Example 2 was adjusted to 1.5 to 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) at 30 ℃
For 3 hours. Next, the activity in each pH-treated solution was measured. FIG. 4 shows the results. The horizontal axis in the figure is processing
The vertical axis represents 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 isoelectric focusing. 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 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. The measurement was based on the oxygen electrode method in which the oxygen consumption accompanying the oxidation of the substrate was 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 the reaction was performed by adding 20 μl of the enzyme solution. Starting, oxygen consumption was measured with a Clark-type oxygen electrode. In addition to the present laccase II, for comparison, laccases derived from Pycnopora coccineus and Coriolus versicolor were similarly examined. Table 4 shows the relative values (%) when the measured value when catechol was used as the substrate was set to 100.

[0048]

[Table 4]

The laccase II is hydrocaffeic acid or
It showed particularly high specificity for some phenolic compounds such as 2,6-dihydroxyphenol and aromatic amines such as p-toluidine, dimethylaniline, diethylaniline and TOOS. The 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 Example 2 was examined. As inhibitors
Using α, α-dipyridyl, acetylacetone, cysteine, dithiothreitol, potassium cyanide, neocuproine, mercaptoethanol, α-naphthol, barium thiocyanate, phenylhydrazine or sodium azide to measure the degree of enzyme activity inhibition by these inhibitors To determine the inhibitory activity. Specifically, the measurement was carried out in a microcell at 30 ° C. in a spectrophotometer under constant temperature conditions, 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 the enzyme solution was added.
After reacting for 3 minutes, 30 μl of a 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. Table 5 shows the results.

[0051]

[Table 5]

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

[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 and also works on a phenol compound, and has good heat resistance. Therefore, the present laccase II is useful for treatment of waste liquid containing aromatic amines and phenol, production of artificial lacquer, pulp production treatment process, synthesis of useful polyphenol, and the like. Since the present laccase II is produced by cultivation of basidiomycetes, the present laccase II can be provided in large quantities at low cost and can be widely used for reagents or industrial uses.

[Brief description of the drawings]

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

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

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

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

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁶ , DB name) BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. The following properties: (1) molecular weight: about 62,000; (2) optimum temperature range of operation: optimum temperature is 78 ° C. or higher; (3) thermal stability: kept at pH 7.8 for 30 minutes. when, is stable up to about 55 ° C., (4) optimum pH: about 5.0; Contact and (5) substrate specificity: phenolic compounds and aromatic amino
Laccase II having an action of 2. The laccase II according to claim 1, wherein
(6) Stable pH range: treated at 30 ° C for 3 hours
PH 5.0 to 10.0; (7) Isoelectric point: about 5.9; Laccase II, further comprising: 3. A basidiomycete Trametes sp. Belonging to the genus Trametes. The strain of claim 1 or 2 wherein the strain is produced by the Hal strain (Deposited with No. 14472, LIKEN).
Is laccase II according to 2 . 4. The following properties: (1) molecular weight: about 62,000; (2) optimum temperature range of operation: optimum temperature is 78 ° C. or higher; (3) thermal stability: pH 7.8 for 30 minutes. If held, is stable up to about 55 ° C., (4) optimum pH: about 5.0; Contact and (5) substrate specificity: phenolic compounds and aromatic amino
Down to act; a method of producing laccase II having the step of culturing a basidiomycete belonging to Hourokutake genus of producing the laccase II in the presence of p- xylidine, and the laccase II from the culture A method comprising a step of separating and purifying. 5. The laccase II has the following properties: (6) Stable pH range: when treated at 30 ° C for 3 hours
PH 5.0 to 10 . 0; and (7) Isoelectric point: about 5.9; 5. The method of claim 4, further comprising: