JPS5847489A - Preparation of laccase - Google Patents

Abstract

PURPOSE:To prepare laccase in high yield, by cultivating a microorganism belonging to the genus Coriolus. CONSTITUTION:A microorganism such as Coriolus consors IFO8348, Coriolus versicolor IFO6481, etc. belonging to the genus Coriolus, capable of producing laccase is inoculated into a nutrient medium, cultivated at about 25 deg.C at the starting pH of 4-8 under aerobic conditions for about 3-4 days, and laccase is collected mainly from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing laccase, and more particularly, to a method for producing laccase produced by a fungus belonging to the genus Coriolus.

Conventionally, enzyme numbers [1, 10, 6, 2] have been cited as known laccases that oxidize p-diphenol and convert it into quinone (p-quinone), and this enzyme is shown by the following formula. catalyze a reaction.

Laccase exists equally in sumac juice and fungi (Polyporus versicolor), and has been isolated from each and its properties have been investigated (
Published by Academic Press 3rd edition The Enzymes No. 1
(See Vol. 2, p. 507).

In order to establish a method for industrially producing laccase from microorganisms, the present inventors searched for the production ability of the enzyme in various bacteria, yeast, and fungi, and found that various fungi had the production ability. Among them, we discovered that fungi of the genus Coriolus produce strong laccase activity, established a method for cultivating the strain, and a method for extracting and purifying the enzyme, thereby completing the present invention.

Bacterial strains that can be used in the present invention include, for example, Coriolus@
Ccriolus consora
IFO8648, Coriolus vashikara
(usversicolor) IFO6481 and the like; however, in addition to these bacteria, all bacteria of the genus Coriolus that produce laccase can be used in the method of the present invention.

To carry out the present invention, Utsucase-producing bacteria of the genus Coriolus are cultured by a conventional method. The form of culture may be liquid culture or solid culture, but industrially it is advantageous to inoculate a production medium with laccase-producing bacteria and perform deep culture. Nutrient sources for the culture medium include a wide variety of sources commonly used for culturing microorganisms. meat extract, yeast extract, glutamic acid, ammonium sulfate, ammonium chloride, etc. Furthermore, carbon sources such as glucose, glycerin, molasses, and starch hydrolyzate are added as necessary, and some inorganic salts,
It is used with appropriate amounts of vitamins added. Although the culture temperature can be changed as appropriate within the range that allows the bacteria to grow and produce laccase, a particularly preferred temperature is around 25C. The culture time varies depending on the conditions, but is usually about 3 to 4 days, and the culture may be terminated at an appropriate time, taking into consideration the time when laccase reaches its maximum titer. In addition, it is appropriate to adjust the initial pH of the medium to 4 to 8, and it is preferable to culture aerobically by submerged culture with aeration and shaking, or by shaking culture.

In the liquid culture of the laccase-producing bacteria thus obtained, the enzyme is mainly produced in the culture solution.

Various methods are used to collect and purify laccase, and an efficient method will be described below.

First, use cultured t-F paper, such as Toyoguchi Paper 42 (registered trademark,
A clear culture solution is obtained by rinsing the culture solution with a filter such as the one manufactured by Toyo Kuchushi Co., Ltd., and then salting out is carried out by adding ammonium sulfate to the culture solution until it becomes saturated with 601s.

The precipitate is separated by centrifugation and then dissolved in 10 mM phosphate buffer. Next, 67% acetone (to a volume of 1) cooled with dry ice is added. The precipitate is collected by centrifugation and dissolved in 1' OmM phosphate buffer to obtain a crude enzyme-containing solution. This crude laccase-containing solution is further purified using known means for isolating and purifying proteins, enzymes, etc. to obtain an enzyme solution.For example, this crude enzyme solution tDEAE cellulose, such as DK52 (registered trademark, manufactured by Whatman) Let it be adsorbed to
Elute by increasing the potassium chloride concentration. A concentrated solution is obtained by concentrating the eluate by salting out, dialysis against a polyethylene glycol solution, or by using an ultrafiltration device. Further, this concentrated solution is subjected to gel filtration using a Bio-Gel A-15m (registered trademark, manufactured by Bio-Ratsu F Co., Ltd.) column to obtain a purified enzyme solution. The purified laccase obtained by the present invention has the effect of oxidizing para-diphenol, which is the property of known laccases, and is a protein containing sugar and copper.
It exhibits the properties shown below.

t When applied to bilirubin solution, visible region 460 nm
Bilirubin with the maximum of icgL light is <58011m
Bili, which has an extinction maximum at , and a lower extinction coefficient than bilirubin, oxidizes to lugin, and piri oxidizes lugin to an unknown substance that has little absorption. At this time,
Dissolved oxygen is consumed, but unlike the enzyme described in JP-A-54-151193, hydrogen peroxide is not observed in the reaction product.1 In other words, the color or reducing power caused by bilirubin is eliminated. If 3-methyl-2-pencillinone hydrazone (MBTH) is present at this time, a dye having a maximum absorption of 520 nmK is quantitatively formed with bilirubin.

2. When reacted with ascorbic acid solution and uric acid solution, it oxidizes respectively.

(v) When a system that is frequently used to detect hydrogen peroxide in the clinical testing field, such as a 4-aminoantipyrine (4AA)-phenol system, is operated in the absence of hydrogen peroxide,
4AA and phenol are oxidized and condensed to quantitatively develop color. Utilizing this effect, the enzymatic activity unit of laccase can be measured. That is, 1M acetate buffer (pH 4,5) 100μm 125mM JAA 10
μm, 8.3mM N-ethyl-N-hyP-tetraoxyethyl-m-)luidine (ICHET) 20μ! , water 770μ! Reaction solution consisting of 900μjK enzyme solution 100μ
After reacting at 57C in addition to l 1 , the absorbance increase ΔA 7min per unit time at 550 nm is measured.

In this case, the enzyme activity unit is the molecular extinction coefficient 2 of the dye produced.
．． It can be expressed by the following equation using 6×104.

Enzyme activity (u/1Lt) = AXfX0.385 (where ΔA is the increase in absorbance per minute, f is the enzyme solution σ) and represents the dilution factor. ) As is clear from the above properties, the laccase of the present invention also acts on bilirubin, ascorbic acid, uric acid, etc. in serum, so it is useful for measuring various substances in serum, such as triglyceride V. By using the laccase of the present invention, it is possible to avoid the adverse effects of bilirubin, ascorbic acid, uric acid, etc. on the measurement system.

It can also be applied to the determination of bilirubin, etc. using a method of reducing the absorbance of bilirubin itself, and therefore the laccase obtained by the method of the present invention is extremely useful.

Next, the method of the present invention will be specifically explained with reference to Examples, but the method of the present invention is not limited thereto.

Example 1 Medium consisting of defatted soybean 2-1 lactic acid α116 (pH 4,5
)20111 di'r-77me7fi-C501 volume) K, autoclaved and sterilized under high pressure. After inoculating 1 volume of the pre-cultured Coriolus connulus IFO8348 inoculum and carrying out aeration-stirring deep culture (aeration 1vvm, stirring 250 rpm) for 25C for 3 days, bacterial cells were separated by suctioning through a mouthpaper. A clear culture solution was obtained.

Ammonium sulfate 5850JF (60% saturation) was added to this culture solution to dissolve it, and the resulting precipitate was collected by centrifugation and dissolved in 1()oM phosphate buffer (pH 7,0) K to bring the total volume to 9501. . To this solution, 1900 jlJ of acetone previously cooled with dry ice was added, and the resulting precipitate was collected by centrifugation and collected at 1.21 to 1M.
Total amount Y1B6 dissolved in phosphate buffer (pH 7,0)
I made it 1!7. Prepare 2mM phosphate buffer (pH 7) in advance.
DE52 column (φ5aI4x30
cs) ic This solution was allowed to flow down and the enzyme was adsorbed.

Concentration gradient elution with potassium chloride was performed, and the enzyme active portion was placed in a dialysis tube and dialyzed against 50 polyethylene glycol solution to concentrate to a total volume of 1:98. 1
Bio-gel A-t5m*lam (φ28mm x 1) equilibrated with 9mM phosphate buffer (pH 7,0)
00 side), the gel p
I went through the trouble. The active part was again dialyzed and concentrated using polyethylene glycol to obtain purified laccase 50III.

The purified laccase thus obtained exhibited a bright blue color.

The above purification process is summarized in Table 1K.

This n-made laccase exhibits the following properties.

(1) Optimal pH When the bilirubin decolorizing activity of this enzyme (measured as 460 nm absorbance decrease 1 in the reaction solution) and 4AA-EHET coloring activity were measured using phosphate buffer and acetate buffer, it was found that The optimum pH was around 5.5 and 4.5, respectively.

(21 pH stability: pH 2: Gumacine-HCl buffer; pH 5-5: Acetate buffer; pH 6-8: Phosphate buffer; pH 9: Tris-Hy-P hydroxyaminomethane buffer; pH 10-12: Glycine-NaOH buffer. , each with a final concentration of 0.5MK
Enzyme solution 1■/yil (60u/8
7) was left at each pH for 18 hours and the residual activity was measured, and it was found to be stable at pH 6 to 12.

(3) Thermostability After leaving 2/1 enzyme solution dissolved in 10 mM phosphate buffer (pH 1O) at each temperature for 20 minutes, the residual activity was measured and the temperature reached 60C. It gradually became inactive.

(4) Optimum temperature bilirubin decolorizing activity is pH 5.5, 4AA-EHET
The coloring activity was measured at pH 4 and 5, and both were 45C.
The reaction rate reached its maximum.

(5) Uniformity When protein staining was performed on the yeast μ obtained by the above method by electrophoresis on polyacrylate 9 amide and sodium lauryl sulfate (SDS) polyacrydamide, one stained band was observed. there were.

(6) The absorption quantity was determined by measuring the absorption quantity of this enzyme solution6.
The maximum is observed at 10 nm and 280 nm, and it is a colored blue protein. The extinction coefficient of each is
The molecular weight was estimated to be 58,000 as a result of measurement using a gel filtration method and an SDS electrophoresis method.

(8) Isoelectric point p) (3' As a result of electrophoresis at 400 V to 800 V for 40 hours using carrier ampholyte (registered trademark manufactured by LKB) with pH 10, the isoelectric point pH = 4.3.J was determined. .

(9) Quantification of copper in enzyme Quantification of copper in 1~/Ml of enzyme solution using CU-Test Wako (registered trademark manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) kit. and 0 μl copper/~protein. That is, it is a copper protein containing 0.3-copper.

Kan sugar content in enzyme Sugar content was measured by the orcinol sulfuric acid method.

The sugar content was estimated to be 3.4% in terms of glucose.

Example 2 The same medium 10 as in Example 1 was prepared in a QiuQ 500 ml shake flask, sterilized, and inoculated with each of the fungi shown in 2.
2511:' Cultured on a reciprocating shaker for 3 to 7 days. Table 2 shows the results of measuring the laccase activity of the culture solution.

Coriolus consolus IF085484! exhibited a productivity 200 times higher than that of the known laccase-producing bacterium Polyporus bashikara.

Table 2

Claims (1)

[Claims] A method for producing laccase, which comprises culturing laccase-producing bacteria belonging to the genus Coriolus in a normal culture medium and collecting laccase from the resulting culture.