JP2984552B2 - Laccase and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a laccase having high thermostability and a method for producing such a laccase from a basidiomycete culture.

[0002]

2. Description of the Related Art Laccase, also called phenol oxidase or lignin degrading enzyme, is an enzyme that oxidizes phenolic compounds in the presence of oxygen. Since laccase has a lignin decomposition action and an oxidative polymerization action of phenolic substances such as urushiol, for example, it is expected to be used for the synthesis of concrete admixtures, lignin removal in pulp production processing, and the production of artificial lacquer. . Especially,
For the production of artificial lacquer, a laccase that has a strong oxidizing power and does not deactivate at high temperatures is particularly necessary in order to enhance the touch-drying effect of lacquer (Terada et al., Painting Engineering, Vol. 26, No. 6,
(1991)). In addition, even when lignin is removed in the pulp manufacturing process, the temperature is high when the lignin is removed outdoors in summer, so that a laccase having excellent heat resistance is required.

Hitherto, laccases derived from various fungi have been known. For example, the basidiomycetes Coriolus hirstus and Coriolus versicolor (JP-A-62-2)
No. 20190), Pycnopora, a wood rot fungus
Coconesius (Terada et al., Painting Engineering, Vol.26, No.6,
(1991)), and a laccase derived from the zygote Mucor Mayhei (Japanese Patent Application Laid-Open No. 61-115488) is known. In addition, the presence of laccase is known in animals and plants.

However, none of the laccases is thermally unstable and it is difficult to purify the enzyme on a large scale. Therefore, at present, the laccase is not practically used for the above-mentioned applications.

[0005]

SUMMARY OF THE INVENTION The present invention is to solve the above-mentioned conventional problems.
It is an object of the present invention to provide a laccase excellent in thermostability, a method for producing the same, and a producing bacterium. High heat resistance,
Laccase and the fungi producing it are not known.

[0006]

Means for Solving the Problems The present inventors have found a novel laccase from newly isolated basidiomycetes belonging to the genus Trametes, and have completed the present invention.

[0007] The laccase of the present invention has the following properties. (1) Molecular weight: about 62,000. (2) Range of suitable temperature for operation: The optimum temperature is about 70 ° C. (3) Thermal stability: stable up to about 65 ° C. when kept at pH 7.0 for 30 minutes. (4) Optimum pH: about 5.0. (5) Stable pH range: pH 7.0 to 10.0 when treated at 30 ° C. for 3 hours. (6) Isoelectric point: about 3.0. This achieves the above object.

The laccase of the present invention is a basidiomycete belonging to the genus Psyllium, in particular, Tramet deposited on August 12, 1994, with the Institute of Biotechnology and Industrial Technology (AIST).
es sp. Ha1 strain (Biological Research Institute No. 14472; FERM P-1
4472). This achieves the above object.

Various properties of the laccase of the present invention produced from the above basidiomycetes are shown in Table 1.

[0010]

[Table 1]

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

The method for producing laccase of the present invention comprises a step of culturing the above-mentioned producing bacteria and separating and purifying the laccase from the culture. This achieves the above object. Hereinafter, the present invention will be described in detail.

The laccase of the present invention is a basidiomycete belonging to the genus Pleurotus, especially Trametes sp.
No. 72; FERM P-14472). This strain is a new strain newly isolated from dead bamboo leaves as a result of extensive screening of fungi that produce laccase from soil, seawater and freshwater, and decayed substances.

(1) Morphology and culture The laccase-producing bacterium of the present invention (hereinafter referred to as the present bacterium) is a white rot fungus having lignin decomposability, and has a skeletal hypha and a clasp connection in a cultured mycelium. Is identified as a basidiomycete belonging to the genus Trametes due to the absence of atypical cells such as cystidia and bristle hyphae, and the absence of conidia in cultured mycelia. , Tr
ametes sp. Ha1

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 present bacterium, and sugars such as glucose, sucrose, molasses, starch, and wood flour can be used. As the nitrogen source, an organic nitrogen source such as peptone, yeast extract, malt extract, meat extract, soybean decomposed product, urea and the like, and an inorganic nitrogen source such as ammonium nitrate and sodium nitrate can be used. If necessary, inorganic salts such as phosphate, magnesium sulfate, potassium, calcium, copper, sodium, manganese and zinc, vitamins and the like can be added. These medium components may be in any concentration that does not inhibit the growth of the present bacterium.
The nitrogen source is 0.01 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.

(2) Separation and Purification of Laccase from Culture Solution To separate and purify the laccase of the present invention (hereinafter referred to as the present laccase) from the culture solution of the present bacterium, known purification methods such as dialysis, salting out, Column chromatography, isoelectric precipitation, and gel filtration can be used alone or in combination.

The present laccase is secreted and produced outside the cells,
Accumulates in culture. Therefore, after culturing, insoluble matter such as bacterial cells is removed by centrifugation, filtration with a filter paper or filter cloth, or the like, and a culture solution (crude enzyme solution) containing the present laccase is recovered. The obtained crude enzyme solution is concentrated, fractionated with ammonium sulfate (salting out),
The present laccase can be purified by commonly used enzyme purification methods such as dialysis, various types of chromatography, and gel filtration. For example, the crude enzyme solution obtained above is fractionated with ammonium sulfate, and then subjected to dialysis, various types of chromatography, and ultrafiltration in that order to obtain a purified fraction containing the present highly active laccase.

(3) Measurement of Laccase Activity In the present invention, the present laccase activity is measured by a color reaction catalyzed by the present laccase by oxidative condensation between 4-aminoantipyrine and a hydrogen donor. Examples of the hydrogen donor include phenol and N, N-diethylaniline (DEA). 505 nm in a color reaction system by oxidative condensation
(Phenol) or the absorbance at 555 nm (DEA) was measured. In the present application, a 190 mM sodium acetate buffer (pH 7.5) containing 7.5 μmol phenol and 1.25 μmol 4-aminoantipyrine was used.
4.5) After pre-incubating 520 μl at 30 ° C. for 1 minute, 20 μl of the enzyme solution was mixed, the change in absorbance at 505 nm was measured, and the enzyme activity for increasing absorbance 0.01 per minute was 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]

EXAMPLES Example 1 (Culture of Trametes sp. Ha1) 10 ml of medium (5% glucose, 0.5% peptone, 0.1% yeast extract, 0.2%)
_{% KH 2 PO 4, 0.1%} K 2 HPO 4, 0.02% MgSO 4 · 7H 2 O, pH
A 60 ml test tube containing 6.0) was inoculated with one platinum loop of Trametes sp. Ha1, and cultured with shaking at 28 ° C. for 4 days. Next, 20 ml of this culture was inoculated into a 5 liter mini-jar fermenter containing 2.5 liters of the above medium, and incubated at 28 ° C. for 7 minutes.
The culture was carried out with aeration and stirring for days.

Example 2 (Purification of the present laccase) The culture obtained in Example 1 was filtered through filter paper to remove bacteria, and the recovered filtrate was used as a crude enzyme solution.

The crude enzyme solution was concentrated by evaporation or ultrafiltration, salted out with 50% saturated ammonium sulfate, and the precipitated protein was removed by centrifugation. Next, a portion precipitated with 75% saturated ammonium sulfate was recovered by centrifugation. The recovered fraction was dialyzed against a 10 mM Tris-HCl buffer (pH 7.5) to obtain a dialysate.

The dialysis solution was subjected to a DEAE-Sephacel column (5.0 × 5.1 cm; manufactured by Pharmacia) equilibrated with the buffer. After the column was washed with the same buffer, the present laccase adsorbed on the column was eluted by eluting the buffer with a concentration gradient of sodium chloride from 0 M to 1 M, and fractionated and collected. This laccase is approx.
Eluted at 2M.

A portion of each of the obtained fractions was subjected to measurement of the present laccase activity, and the fractions having the activity were combined and dialyzed against 10 mM potassium phosphate buffer (pH 5.7). I got

The above dialysate was subjected to a hydroxyapatite packed column (2.6 × 18.8 cm; manufactured by Seikagaku Corporation),
After washing and eluting the non-adsorbed substance, the present laccase was eluted with the above buffer containing 1 M sodium chloride. The eluate containing this laccase was concentrated by ultrafiltration, and 10 mM Tris-HCl buffer containing 0.2 M sodium chloride (pH
A Sephacryl S-300 HR column (2.6 × 86 cm; manufactured by Pharmacia) equilibrated in 7.5) was tested to obtain a further purified and purified enzyme solution.

A part of the purified enzyme solution was subjected to polyacrylamide gel electrophoresis, proteins were detected by Coomassie brilliant blue staining, and laccase activity was stained in the above color reaction system using phenol and 4-aminoantipyrine. Was. The results are shown in FIG. Lane A shows the protein band detected by staining, and lane B shows the laccase active band detected by the above color reaction system. As shown by the arrows in the figure, the positions of the detected homogeneous protein band and the laccase activity band coincided, confirming that the present laccase can be purified by the above steps without losing the enzyme activity. Further, the gel after the electrophoresis was subjected to a Schiff staining method, and it was confirmed that the present laccase was a glycoprotein.

Table 2 shows the degree of purification of the present laccase and the specific activity per protein in each of the above purification steps.

[0034]

[Table 2]

Example 3 (Molecular weight of the present laccase) SDS polyacrylamide gel electrophoresis (SDS-PAGE) method (Laemmli, UK, Nature, 22)
7, 680 (1970)), the molecular weight of this laccase was measured.

The purified laccase obtained in Example 2 and six proteins known as molecular weight markers (molecular weights of about 97,400, 66,300, 42,400, 30,000, 20,100 and
14,400). After the electrophoresis, the sample was stained with Coomassie brilliant blue, and the molecular weight of the present laccase was measured from the relative migration distance between the present laccase and each molecular weight marker. The results are shown in FIG. The lane on the right shows the laccase, and the lane on the left shows the molecular weight markers that were simultaneously electrophoresed. The numbers on the left outside indicate the molecular weight size (kDa). According to the present SDS-PAGE, the present laccase showed a single band. The molecular weight of this laccase in SDS-PAGE is about 62,000. As a result of subjecting the purified enzyme solution to high performance liquid chromatography (HPLC) and measuring the molecular weight, it was about 62,000 as described above.

Example 4 (Appropriate Temperature Range and Thermal Stability of the Present Laccase) (1) Range of Appropriate Temperature The activity of the purified laccase obtained in Example 2 was measured at various temperatures in the color reaction system. Conditions (16, 30,
40, 50, 60, 70, 75, 80 ° C). Fig. 3 shows the results.
Shown in 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 the laccase is about 70 ° C.

(2) Thermostability The purified laccase obtained in Example 2 was used at various temperature conditions (0, 30, 40, 45, 50, 55, 60, 65, 70, 80).
After incubation for 30 minutes in a phosphate buffer (pH 7.0) under the same temperature, the activity was measured. FIG. 4 shows the results. 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. The present laccase is stable, maintaining 70% or more of the enzyme activity even when the treatment temperature condition is 65 ° C. The present laccase retains 100% of the enzymatic activity at a treatment temperature of 60 ° C.

Example 5 (Optimal pH and stable pH range of the present laccase) (1) Optimal pH Depending on the pH range, 50 mM acetate buffer, 50 mM phosphate buffer, 50 mM Tris-HCl buffer or 50 mM Tris-nitrate. The pH is adjusted to 2.5 to 9.0 (2.5, 3.5, 4.
4, 5.0, 5.7, 6.3, 7.3, 8.0, 9.0), and the activity of the purified present laccase obtained in Example 2 above was measured. FIG. 5 shows the results. 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. The optimum pH of the laccase is about 5.0.

(2) Stable pH Range The pH of the laccase solution obtained in Example 2 was adjusted to 1.0 to 1
2.0 (1.0, 1.9, 4.1, 5.0, 6.1, 7.0, 7.5, 8.0, 8.
2, 9.2, 10.0, 10.3, 10.9, 12.0) and incubated at 30 ° C. for 3 hours. Next, the activity in each pH-treated solution was measured. FIG. 6 shows the results. The horizontal axis in the figure is the treatment pH
And the vertical axis represents the relative residual activity (%) when the maximum activity value is set to 100. The stable pH range of this laccase is
7.0 to 10.0.

Example 6 (Isoelectric point of the present laccase) The isoelectric point of the present laccase was measured by a conventional isoelectric focusing method. As a result of the measurement, the isoelectric point of the present laccase is about 3.0.

Example 7 (Substrate specificity of the present laccase) The substrate specificity of the present laccase purified in Example 2 was determined using catechol, resorcinol, hydroquinone, caffeic acid, hydrocaffeic acid, pyrogallol, o-cresol. , M-cresol, p-cresol, p-hydroxybenzoic acid, phenol, p-phenylenediamine, p-toluidine, N, N-
Dimethylaniline, N, N-diethylaniline, or N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline were used as substrates, and the amount of oxygen consumed by the oxidation of these substrates was measured as oxygen. The study was performed using an oxygen electrode method in which an electrode converts the electric signal. In detail, a 200 mM sodium acetate buffer solution (pH
4.5) After equilibrating 1980 μl with air at 30 ° C. for 10 minutes, the reaction was started by adding 20 μl of an enzyme solution, and the oxygen consumption was measured with a Clark-type oxygen electrode. For comparison, laccases from Pycnopora coccineus and Coriolus versicolor were similarly examined. Table 3 shows relative values (%), where the measured value when catechol was used as the substrate was 100.

[0043]

[Table 3]

The present laccase has a particularly high specificity for caffeic acid, and also has a different specificity for hydrocaffeic acid from other laccases.

Example 8 (Study of culture conditions) A 60 ml test tube with a cotton plug and 50
Using a 0 ml flask, the amount of the medium was changed little by little, and the change in laccase productivity due to the change in the oxygen supply was examined. The volume of the culture medium was 5, 1 in a 60 ml test tube.
For 0 and 15 ml, 500 ml flasks, 25,
50, 75 and 100 ml. Laccase activity was measured for each medium volume, and the result was taken as the amount of laccase produced. The respective results are shown in FIG. 7 and FIG.

In all cases, when the amount of the medium was increased to decrease the oxygen supply, the laccase production decreased. Similar results were obtained in experiments using 2 liter flasks.

From the results of these cultures, it was suggested that the production of the enzyme of the present bacterium varies depending on the amount of oxygen supplied to the cells.

Therefore, the cotton plug was further changed to a silicon plug to reduce the amount of oxygen supplied into a 500-ml flask in which the amount of oxygen was reduced.
0 ml of the medium solution was added and cultured at 28 ° C., and the time-dependent change in laccase activity was examined. In FIG. 9, changes in dry cell weight, pH, and laccase activity are shown by − □ − and − ●, respectively.
Indicated by-and -O-.

The laccase activity reached its maximum on the sixth day, but was about one tenth of the activity when cultured with a cotton plug. This revealed that the present bacterium increased laccase productivity by increasing the oxygen supply.

[0050]

According to the present invention, a novel laccase having high heat resistance and a method for producing the same are provided. Since the present laccase has higher heat resistance than conventional laccase, it is useful for the production of artificial lacquer, the pulp manufacturing process, and the like. Since the present laccase is produced by cultivation of basidiomycetes, the present laccase 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 is a gel electrophoresis photograph showing a migration pattern of the present laccase by polyacrylamide gel electrophoresis.

FIG. 2 is a gel electrophoresis photograph showing an electrophoresis pattern of the present laccase by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

FIG. 3 shows the relative activity when the present laccase activity was measured in the range of 16 to 80 ° C.

FIG. 4 shows the relative residual activity of this laccase measured after incubation at 0 to 80 ° C. for 30 minutes.

FIG. 5 shows the relative activity when the present laccase activity was measured in the pH range of 2.5 to 9.0.

FIG. 6 shows the relative residual activity of this laccase measured after incubation at 30 ° C. for 3 hours under conditions of pH 1.0 to 12.0.

FIG. 7 shows the oxygen supply effect on the production amount of the present laccase during the culture of the present bacteria in a 60-ml test tube.

FIG. 8 shows the oxygen supply effect on the production amount of the present laccase during the culture of the present bacteria in a 500 ml flask.

FIG. 9 shows changes with time in the amount of bacterial growth, the activity of the present laccase, and the pH of the culture solution in the culture of the present bacteria in a 500-ml flask.

──────────────────────────────────────────────────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) range of suitable working temperature: optimum temperature is about 70 ° C .; (3) thermal stability: 30 at pH 7.0. when held minutes is stable up to about 65 ° C., (4) optimum pH: about 5.0; Contact and (5) substrate specificity: acting on caffeic acid; a has laccase. 2. The laccase according to claim 1, wherein:
The following characteristics: (6) Stable pH range: when treated at 30 ° C for 3 hours
PH 7.0 to 10.0; and (7) Lacquer further having an isoelectric point: about 3.0;
Ze. 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 the laccase according to 2 . 4. The following properties: (1) molecular weight: about 62,000; (2) range of suitable working temperature: optimum temperature is about 70 ° C .; (3) thermal stability: 30 at pH 7.0. when held minutes is stable up to about 65 ° C., (4) optimum pH: about 5.0; Contact and (5) substrate specificity: acting on caffeic acid; in a method of producing laccase having There, a step of cultivating a basidiomycete belonging to the genus Pleurotus producing the laccase,
And a step of separating and purifying the laccase from the culture. 5. The laccase having the following properties: (6) Stable pH range: when treated at 30 ° C for 3 hours
PH 7.0 to 10.0; and (7) The method according to claim 4, further comprising an isoelectric point of about 3.0;
The described method.