WO2021193893A1 - Culture medium and method for producing laccase - Google Patents

Abstract

A culture medium according to the present embodiment is for growing a fungus and for producing a laccase in the fungus by growing the fungus while including TEMPO-oxidized cellulose nanofibers. A method for producing a laccase according to the present embodiment includes a step for producing a laccase in a fungus by growing the fungus by using a culture medium that includes TEMPO-oxidized cellulose nanofibers.

Description

Method for producing medium and laccase

The present invention relates to a medium and a method for producing laccase.
The present application claims priority based on Japanese Patent Application No. 2020-058272 filed in Japan on March 27, 2020 and Japanese Patent Application No. 2021-034473 filed in Japan on March 4, 2021. The contents are used here.

Laccase is an enzyme that is widely distributed in nature, and the main laccase-producing fungi include wood-rotting fungi, especially basidiomycetes and ascomycetes. Laccase has actions such as decomposition of lignin, oxidation of phenols such as urushiol, and polymerization, and is highly useful. Therefore, in the industrial world, it is used for removing lignin during pulp production, treating wastewater containing pigments, producing lacquer, and the like. Furthermore, it is expected that laccase will be used for decomposing environmental pollutants such as polychlorinated biphenyls (PCBs).

On the other hand, the amount of laccase that can be extracted from nature is limited. The synthesis of laccase using fermentation technology is also being considered, but since it is necessary to use additives such as copper ions and aromatic compounds, there is a concern about environmental pollution, and detoxification of the fermentation broth is necessary. , It cannot be said that it is highly practical.
As described above, although laccase has high industrial utility value, it cannot be expected to increase in production volume and has not been used on a large scale.

As a method for producing laccase different from these, in the fermentation process for laccase production by a specific species of laccase-producing filamentous fungus, a specific species of carotenoid-producing microorganism is added to produce the laccase by the specific species of filamentous fungus. A method for promoting production and producing laccase is disclosed (see Patent Document 1). In this production method, as a combination of a laccase-producing filamentous fungus and a carotenoid-producing microorganism, a combination of Agitake (Pleurotus ferulae) and Spolidiobolus pararoseus; Pleurotus and Pleurotus osteos A combination with Rhodotorula mucilaginosa; a combination of Rhodotorula Lucidum and Rhodotorula mucilaginosa is adopted.

Japanese Patent No. 6560301

In the production method disclosed in Patent Document 1, not a carotenoid-producing microorganism but an extract thereof and a production method when β-carotene is added are also disclosed, but a carotenoid-producing microorganism was added. The lacquerze having higher enzyme activity is obtained. However, in that case, since it is necessary to use a carotenoid-producing microorganism in combination, there is a problem that the process is complicated due to the use and control of this microorganism. Therefore, it has been desired to develop a new method for producing laccase, which has never existed before.

An object of the present invention is to provide a new means for producing laccase.

The present invention is a medium for growing a fungus, wherein the medium contains TEMPO-oxidized cellulose nanofibers, and the medium is a medium for causing the fungus to produce laccase by growing the fungus. The medium is provided.
In the medium of the present invention, the fungus may be a mushroom.
In the medium of the present invention, the laccase may be bilirubin oxidase.
The present invention is a method for producing laccase, which comprises a step of causing the fungus to produce laccase by growing a fungus using a medium containing TEMPO-oxidized cellulose nanofibers. Provide a manufacturing method.

According to the present invention, a novel means for producing laccase is provided.

It is a graph which shows the measurement result of the enzyme activity of the laccase obtained in Examples 1 and 2 and Comparative Examples 1 and 2. It is a graph which shows the measurement result of the enzyme activity of the laccase obtained in Example 3 and Comparative Examples 3-6.

<< Medium >>
The medium according to one embodiment of the present invention is a medium for growing a fungus, and the medium is TEMPO-oxidized cellulose nanofibers (in this specification, it may be abbreviated as "TEMPO-oxidized CNF"). The medium is a medium for causing the fungus to produce laccase by growing the fungus.
Since the medium of the present embodiment contains TEMPO-oxidized CNF, it is possible to make the fungus produce laccase having sufficiently high enzyme activity (laccase activity) by growing the fungus, which is a novel method. Achieve the production of laccase in Japan.

In the present specification, the enzymatic activity of laccase means laccase activity unless otherwise specified.

<Fungus>
The fungus to which the medium of the present embodiment is used has a laccase-producing ability and is a laccase-producing bacterium.
The fungus grows utilizing TEMPO-oxidized CNF and produces laccase.

Examples of the fungus include wood-rotting fungi, aspergillus, and the like, and may be fruiting bodies (mushrooms). That is, in the present embodiment, the growth of the fungus includes both the culture of the microorganism and the cultivation (growth) of the fruiting body (mushroom).

Wood-destroying fungi decompose the major components of any wood, such as cellulose, hemicellulose and lignin, to cause decay.
Examples of wood-destroying fungi include white-rot fungi such as shiitake mushrooms, oyster mushrooms, maitake mushrooms, and eryngii; brown-decay fungi such as shiitake mushrooms, sardine mushrooms, and Namidatake mushrooms; microorganisms belonging to the genus Trichoderma, and microorganisms belonging to the genus Caetomium. Examples include soft-rot fungi.

Aspergillus is a microorganism belonging to the genus Aspergillus, and specific examples thereof include Aspergillus, White Jiuqu, and Black Jiuqu.

The fungus to be grown using the medium may be only one kind, two or more kinds, and when there are two or more kinds, the combination and ratio thereof can be arbitrarily adjusted.

Among the fungi, mushrooms have high utility value by themselves and are particularly suitable as fungi that produce laccase.
That is, the fungus is preferably a mushroom.

<TEMPO Oxidized Cellulose Nanofiber>
TEMPO Oxidized Cellulose Nanofiber (TEMPO Oxidized CNF) is TEMPO, that is, by the catalytic action of 2,2,6,6-tetramethylpiperidine 1-oxyl (2,2,6,6-tetramethylpiperidine 1-oxyl). Cellulose nanofibers obtained by chemically modifying cellulose (in this specification, they may be abbreviated as "CNF").

Examples of cellulose nanofibers include cellulose or derivatives thereof, which are microfibrils or microfibril aggregates having a fiber width of 3 to 200 nm.

In the medium, the ratio of the content of TEMPO-oxidized CNF to the total mass of the medium may be appropriately adjusted so as to improve the growth of the fungus, and is not particularly limited.
Among them, the ratio is preferably 0.05 to 4% by mass, more preferably 0.1 to 2.5% by mass, for example, 0.2 to 1.5% by mass, and 0. It may be any of 3 to 1% by mass. When the ratio is equal to or higher than the lower limit, the enzymatic activity of the laccase produced by the fungus becomes higher. When the ratio is not more than the upper limit value, the excessive use of TEMPO-oxidized CNF is further suppressed.

The enzymatic activity of laccase can be measured by a known method using a liquid containing laccase as an enzyme (sometimes referred to as "enzyme solution" in the present specification).
For example, ABTS, that is, 2,2'-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt. , C ₁₈ H ₁₆ N ₄ S ₄ (NH ₄ ) ₂ ) is used as a substrate, an ABTS aqueous solution, a buffer solution, and an enzyme solution are prepared, and the reaction solution obtained by mixing these is used as a sample. .. In this sample, ABTS is oxidized by the enzymatic action of laccase. Since the oxide of ABTS has a large absorbance at a wavelength of 420 nm, the aqueous solution of ABTS is originally transparent, but when it is oxidized by laccase, its color changes to blue. After mixing (in other words, after the reaction starts), the absorbance A ₄₂₀ at a wavelength of 420 nm is continuously measured for this sample until a certain period of time (for example, several minutes) elapses, and within the range where _{A 420 increases linearly. The amount of increase in absorbance ΔA 420} at a wavelength of 420 nm and the corresponding reaction time (min) are determined. Then, the enzyme activity of laccase can be calculated by substituting each numerical value into the following formula (i). In formula (i), "36" is derived from ^{the molar extinction coefficient of ABTS (36000M -1} · cm ^{-1) at a wavelength of 420 nm.}
[Laccase enzyme activity (U / mL)] = {ΔA ₄₂₀ × [reaction solution volume (mL)]} / {[reaction time (min)] × 36 × [enzyme solution volume (mL)]} (i)
Here, the total enzyme activity 1U of laccase is defined as the amount of enzyme that oxidizes 1 μmol of ABTS per minute by the above reaction, and satisfies the relationship of the following formula (ii).
[Total enzyme activity of laccase (U)] = [Enzyme activity of laccase (U / mL)] x [Enzyme solution amount (mL)] (ii)

The medium may be either liquid or solid.
When the medium is liquid, the TEMPO-oxidized CNF may be unevenly distributed in the medium, but it is preferably uniformly dispersed.
When the medium is solid, the TEMPO-oxidized CNF may be unevenly distributed in the medium or may be uniformly dispersed, but when the medium is unevenly distributed, the site where the fungus grows in the medium. It is preferable that the mixture is unevenly distributed in the region in the vicinity of and the region.

<Other ingredients>
In addition to TEMPO-oxidized CNF, the medium may contain other components necessary for fungal growth.
The other components are not particularly limited and can be appropriately selected depending on the type of fungus and the like.
The other component may be, for example, either an organic component or an inorganic component.

The medium may be, for example, a known medium to which TEMPO-oxidized CNF is added.

The other components contained in the medium may be only one kind, two or more kinds, and when there are two or more kinds, the combination and ratio thereof can be arbitrarily adjusted.

Examples of the other components include base materials such as shavings and corn cobs; nutritional materials such as rice bran, bran, okara, corn bran, and bean skin; water; peptone; yeast extract; iron sulfate, magnesium sulfate, and dipotassium phosphate. Examples thereof include metal salts such as potassium, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, manganese sulfate (II), zinc sulfate, and copper sulfate (II).
Among the metal salts, those in which hydrate is present may be blended with the metal hydrate at the time of producing the medium.

When the medium contains the base material, the ratio of the content of the base material to the total mass of the medium in the medium may be appropriately adjusted so as to improve the growth of the fungus, and is not particularly limited.
Among them, the ratio is preferably 30 to 70% by mass, more preferably 35 to 65% by mass, and is, for example, 40 to 60% by mass or 45 to 55% by mass. May be good. When the ratio is equal to or higher than the lower limit, the fungus grows better and the amount of laccase produced increases. When the ratio is not more than the upper limit value, the excessive use of the base material is further suppressed.

When the medium contains the nutrient material, the ratio of the content of the nutrient material to the total mass of the medium in the medium may be appropriately adjusted so as to improve the growth of the fungus, and is not particularly limited.
Among them, the ratio is preferably 3 to 40% by mass, more preferably 5 to 35% by mass, and is, for example, any one of 10 to 30% by mass and 15 to 25% by mass. May be good. When the ratio is equal to or higher than the lower limit, the fungus grows better and the amount of laccase produced increases. When the ratio is not more than the upper limit value, the excessive use of the nutritional material is further suppressed.

When the medium contains water, the ratio of the water content to the total mass of the medium in the medium may be appropriately adjusted so as to improve the growth of the fungus, and is not particularly limited.
Among them, particularly in the case of a solid medium, the ratio is preferably 55 to 75% by mass, more preferably 60 to 70% by mass. When the ratio is equal to or higher than the lower limit, the effect of using water can be obtained more remarkably. When the ratio is equal to or less than the upper limit value, excessive use of water is further suppressed. In the examples described later, all of these conditions are satisfied.

When the medium is liquid, the ratio of the water content to the total mass of the medium in the medium is preferably 89.5 to 99.3% by mass.

Peptone, yeast extract and metal salts are preferred as components in the liquid medium.
When the medium is liquid, the ratio of the content of peptone to the total mass of the medium in the medium is preferably 0.5 to 5% by mass.
When the medium is liquid, the ratio of the content of yeast extract to the total mass of the medium in the medium is preferably 0.05 to 0.5% by mass.
When the medium is liquid, the ratio of the content of the metal salt to the total mass of the medium in the medium is preferably 0.1 to 1% by mass.

In the medium, the ratio of the total content of one or more of the above-mentioned components of the medium to the total mass of the medium does not exceed 100% by mass.

When the medium contains TEMPO-oxidized CNF, the base material, the nutrient material and water, the ratio of the total content of the TEMPO-oxidized CNF, the base material, the nutrient material and water to the total mass of the medium in the medium is , It may be appropriately adjusted so that the growth of the fungus becomes good, and is not particularly limited.
Among them, the ratio is preferably 80 to 100% by mass, more preferably 85 to 100% by mass, further preferably 90 to 100% by mass, for example, 95 to 100% by mass. There may be. When the ratio is equal to or higher than the lower limit, the fungus grows better.

When the medium contains TEMPO oxide CNF, peptone, yeast extract, metal salt and water, the total content of TEMPO oxide CNF, peptone, yeast extract, metal salt and water in the medium with respect to the total mass of the medium. The ratio may be appropriately adjusted so that the growth of the fungus is good, and is not particularly limited.
Among them, the ratio is preferably 90 to 100% by mass, more preferably 93 to 100% by mass, and may be, for example, 95 to 100% by mass. When the ratio is equal to or higher than the lower limit, the fungus grows better.

As a medium containing TEMPO-oxidized CNF, a medium for cellulose-degrading bacteria has been disclosed in "Japanese Patent Laid-Open No. 2019-41731". In the above publication, the cellulolytic bacterium is a bacterium having cellulase which is a cellulase hydrolyzing enzyme, and by using cellulase, cellulose is decomposed into glucose or oligosaccharide which is the final product. , Usually, it is disclosed that this end product is used as a nutrient source. Further, the publication discloses that the cellulolytic bacteria decompose TEMPO-oxidized CNF as cellulose by growing the cellulolytic bacteria using such a medium.
However, the publication does not disclose at all about causing laccase to produce laccase by using the culture medium for cellulose-degrading bacteria. And, in general, it cannot be said that cellulose-degrading bacteria always produce laccase.
Further, the publication does not specifically disclose that fruiting bodies (mushrooms) are grown using the culture medium for cellulolytic bacteria.

On the other hand, by growing the fungus using the medium of the present embodiment, the fungus grows using a specific range of CNF called TEMPO-oxidized CNF, produces laccase, and its enzymatic activity (laccase activity). ) Is high.
In addition, by using the medium of the present embodiment, fruiting bodies (mushrooms) actually grow well and produce laccase with high enzymatic activity.
The medium of the present embodiment that brings out these laccase-producing characteristics of the fungus cannot be easily conceived from the contents of the above-mentioned gazette.

On the other hand, as a method for producing laccase, as described above, in "Patent No. 6560301 (Patent Document 1)", a specific species of laccase-producing filamentous fungus and a specific species of carotenoid-producing microorganism are used. , Is disclosed in combination. However, as described above, this production method requires the use and control of carotenoid-producing microorganisms, and the process is complicated. In the above publication, the enzymatic activity of laccase obtained by all the disclosed production methods including this production method is 80.3 to 10000 U / L.

On the other hand, by growing the fungus using the medium of the present embodiment, the enzyme activity is, for example, 12 U / mL (12000 U / L) or more, 30 U / / without requiring the combined use of other microorganisms. It is possible to obtain laccases of mL (30,000 U / L) or more, and in a simplified process, laccases with remarkably high enzyme activity can be obtained.
The enzymatic activity of laccase obtained by using the medium of the present embodiment will be described in detail later.

Since the medium of the present embodiment contains TEMPO-oxidized CNF, it is possible to produce laccase having sufficiently high enzyme activity (laccase activity). On the other hand, when a CNF other than TEMPO-oxidized CNF, for example, mechanically defibrated cellulose nanofibers (mechanically defibrated CNF) is used, such an effect cannot be obtained.

The mechanical defibration CNF is a cellulose nanofiber dispersion liquid (CNF) by subjecting a cellulose nanofiber precursor (CNF precursor) to a mechanical defibration treatment in a dispersion medium such as water without using TEMPO. CNF produced by a production method having a step of obtaining a dispersion liquid).

The medium can be produced by blending TEMPO-oxidized CNF and the other components.
The compounding of each component can be carried out under temperature conditions of, for example, 15 to 40 ° C., and may be carried out at room temperature.
In the present specification, "room temperature" means a temperature that is not particularly cooled or heated, that is, a normal temperature, and examples thereof include a temperature of 15 to 25 ° C.
Further, each component can be blended under normal pressure, for example.

The TEMPO-oxidized CNF used in the production of the medium may be blended in a pure product state by, for example, removal or purification, or may be in a state of a mixture with components other than the TEMPO-oxidized CNF by post-treatment. It may be blended as it is, or it may be blended as it is in the state of a mixture with components other than TEMPO oxide CNF obtained at the time of producing TEMPO oxide CNF.

<< Manufacturing method of laccase >>
The method for producing laccase according to an embodiment of the present invention is a step of causing the fungus to produce laccase by growing the fungus using a medium containing TEMPO-oxidized CNF (in the present specification, "laccase production". It may be abbreviated as "process").
The medium used in the production method of the present embodiment is the medium according to one embodiment of the present invention described above.
The fungus used in the production method of the present embodiment is the fungus described above.

In the laccase production step, the growth of the fungus can be carried out by the same method as a known method for growing the fungus, except that, for example, the medium and the fungus are used. That is, the method of using the medium may be the same as the method of using a known medium.

When the medium is liquid, the medium may be allowed to stand or shake (that is, shake culture) when the fungus grows.
When the medium is solid, the medium may be allowed to stand during the growth of the fungus.

In the laccase production step, for example, after sterilizing the medium, the fungus is inoculated into the obtained sterilized medium, and the inoculated fungus is grown in the sterilized medium or on the sterilized medium. It can be carried out.

In the laccase production step, for example, it is preferable to carry out a so-called "fungal scraping treatment" in which the fungus after inoculation is allowed to grow to some extent and a part of the grown fungus is removed in or on the medium. The growth of fungi can be improved by performing the fungal scraping treatment.
When growing fruiting bodies (mushrooms) as fungi, for example, it is preferable to grow inoculated inoculum and perform fungal scraping treatment at a stage where growth in the form of fruiting bodies is not clearly observed. .. By doing so, the growth rate of fruiting bodies is remarkably improved.

In the laccase production step, the temperature at the time of fungal growth may be appropriately selected depending on the type of fungus, and is not particularly limited.
Usually, the temperature during fungal growth is preferably 13 to 50 ° C, more preferably 15 to 35 ° C. When the temperature is within such a range, the growth rate of the fungus is further improved.
When the fungal scraping treatment is performed, the temperature at the time of fungal growth may be the same or different before and after the fungal scraping treatment.

When the medium is in a solid state, the relative humidity during fungal growth in the laccase production step may be appropriately selected depending on the type of fungus, and is not particularly limited.
Usually, the relative humidity during fungal growth is preferably 50-99%, more preferably 60-85%. When the relative humidity is equal to or higher than the lower limit, the growth rate of the fungus is further improved. When the relative humidity is equal to or less than the upper limit value, it is possible to prevent the relative humidity from becoming excessive.
When the fungal scraping treatment is performed, the humidity at the time of fungal growth may be the same or different before and after the fungal scraping treatment.

In the laccase production step, the growth time of the fungus may be appropriately selected according to the type of fungus, the temperature at the time of fungus growth, the relative humidity at the time of fungus growth, the mode of the medium (solid or liquid), and the like. Not limited.
Usually, the growth time (number of days) of the fungus is preferably 10 to 180 days, more preferably 25 to 70 days. When the growth time is at least the lower limit value, the growth degree of the fungus is further improved. When the growth time is not more than the upper limit value, it is possible to prevent the time required for the laccase production step from becoming excessively long.

In the laccase production step, the fungus uses TEMPO-oxidized CNF in the medium to grow laccase well and produce laccase having sufficiently high enzyme activity.

The method for producing laccase of the present embodiment may include the laccase production step and other steps.
The other steps are not particularly limited and can be arbitrarily selected depending on the intended purpose.
Examples of the other step include a step of separating the produced laccase from the medium after the laccase production step (in the present specification, it may be abbreviated as "separation step").

The separation step can be performed, for example, by extracting or separating a liquid containing laccase (for example, an aqueous solution of laccase) from the medium after the laccase production step.
In the separation step, the laccase may be further precipitated in the liquid containing the laccase, and the precipitate of the laccase may be taken out from the liquid.

According to the production method of the present embodiment, by using the medium, the fungus can produce laccase having sufficiently high enzyme activity, and laccase having sufficiently high enzyme activity can be produced.
For example, the enzymatic activity of the laccase obtained by the production method of the present embodiment is preferably 12 U / mL or more, for example, 30 U / mL or more, 100 U / mL or more, 150 U / mL or more, 200 U / mL or more, 250 U / mL or more. It may be any of mL or more, 300 U / mL or more, and 340 U / mL or more.
On the other hand, the upper limit of the enzyme activity of the laccase obtained by the production method of the present embodiment is not particularly limited, but for example, laccase of 500 U / mL or less can be produced relatively easily.

After growing the fungus, for example, the medium after growing fruiting bodies (mushrooms) and harvesting is generally called a waste fungus bed or the like, and is conventionally discarded. However, the medium at this stage of this embodiment contains fungal-produced laccase. That is, the medium of the present embodiment is remarkably useful in that it not only grows fungi but also serves as a place for producing laccase, and is far superior to the conventional medium in terms of effective utilization.

An example of a preferred medium of the present embodiment is a medium for growing a fungus.
The medium is a solid medium containing TEMPO-oxidized cellulose nanofibers, a base material, a nutrient material, and water.
In the medium, the ratio of the content of the TEMPO-oxidized cellulose nanofibers to the total mass of the medium is 0.05 to 4% by mass.
In the medium, the ratio of the content of the base material to the total mass of the medium is 30 to 70% by mass.
In the medium, the ratio of the content of the nutritional material to the total mass of the medium is 3 to 40% by mass.
In the medium, the ratio of the water content to the total mass of the medium is 55 to 75% by mass.
However, in the medium, the ratio of the total content of the TEMPO-oxidized cellulose nanofibers, the base material, the nutrient material and the water to the total mass of the medium does not exceed 100% by mass.
Examples of the medium include a medium that is a medium for causing the fungus to produce laccase by growing the fungus.

Another example of the preferred medium of the present embodiment is a medium for growing a fungus.
The medium is a liquid medium containing TEMPO-oxidized cellulose nanofibers, peptone, yeast extract, a metal salt, and water.
In the medium, the ratio of the content of the TEMPO-oxidized cellulose nanofibers to the total mass of the medium is 0.05 to 4% by mass.
In the medium, the ratio of the content of the peptone to the total mass of the medium is 0.5 to 5% by mass.
In the medium, the ratio of the content of the yeast extract to the total mass of the medium is 0.05 to 0.5% by mass.
In the medium, the ratio of the content of the metal salt to the total mass of the medium is 0.1 to 1% by mass.
In the medium, the ratio of the water content to the total mass of the medium is 89.5 to 99.3% by mass.
However, in the medium, the ratio of the total content of the TEMPO-oxidized cellulose nanofibers, peptone, yeast extract, metal salt and water to the total mass of the medium does not exceed 100% by mass.
Examples of the medium include a medium that is a medium for causing the fungus to produce laccase by growing the fungus.
In this medium, the laccase may be bilirubin oxidase.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the examples shown below.

[Example 1]
<< Production of medium >>
By mixing the retained sawdust (327 g), rice bran (130 g), water (193 g) and TEMPO oxidized CNF (4 g), a medium (654 g) containing TEMPO oxidized cellulose nanofibers was obtained.

<< Manufacture of laccase >>
<Sterilization of medium>
The entire amount of the medium obtained above was filled in a transparent mushroom cultivation bottle, a hole was made in the center of the medium, and the bottle was covered.
Next, the medium was placed in an autoclave device together with the bottle, and the medium was pre-sterilized under the conditions of 98 ° C. for 1 hour, and then the medium was sterilized under the conditions of 120 ° C. for 45 minutes.
Then, after lowering the temperature in the autoclave device to 80 ° C., the bottle was moved into the clean bench and allowed to stand until the temperature of the bottle (medium) reached 15 ° C.
From the above, a sterilized medium was obtained.

<Growth of fungi (manufacturing of laccase)>
In the clean bench, the lid of the bottle filled with the sterilized medium obtained above was opened, and the inoculum H67 (10 g) of Oyster Mushroom was placed on the sterilized medium.
Then, the bottle was immediately covered, and the inoculated sterilized medium was placed in the incubator together with the bottle, and the inoculum was cultured for 30 days under the conditions of a temperature of 20 ° C. and a relative humidity of 70%.

Next, the bottle was taken out of the incubator, the lid was removed, and the surface of the medium was scraped to perform a fungal scraping treatment. And I put a lid on the bottle.
Next, this medium was placed in an incubator together with the bottle, and the inoculum was grown on fruiting bodies (Oyster mushrooms) under the conditions of a temperature of 15 ° C. and a relative humidity of 90%, and laccase was produced on the mushrooms. The growing time during this period was 14 days.
Based on the above, the laccase production step was carried out.

The bottle was then removed from the incubator, the lid was removed and the oyster mushrooms were harvested.
From the above, a medium containing laccase was obtained.

<< Evaluation of medium >>
<Preparation of crude enzyme solution>
Samples were taken from the upper, middle and lower parts of the medium in the bottle after harvesting Oyster Mushroom, and this sample (15 g) was placed in sodium phosphate buffer (pH 7.0) (30 mL) having a concentration of 10 mM. The mixture was added and the resulting mixture was shaken overnight at 127 rpm and 4 ° C. using a shaker.
Then, the mixed solution after shaking was centrifuged at 10000 G for 30 minutes at 4 ° C. to remove solid content from the mixed solution.

Next, bentonite in an amount of 5% by mass with respect to the amount of the liquid after removing the solid content was added, and the obtained mixed solution was stirred at 4 ° C. for 30 minutes.
Then, the mixed solution after stirring was centrifuged at 10000 G for 30 minutes at 4 ° C. to remove fine insoluble matter from the mixed solution.

Next, a saturated aqueous solution of ammonium sulfate having a concentration of 70% by mass was added to the liquid after removing the insoluble matter to precipitate the protein, and the liquid containing this precipitate was prepared under the conditions of 10000 G for 30 minutes and 4 ° C. The precipitate was taken out by centrifugation in.
The steps up to this point correspond to the separation step.

The removed precipitate was added to sodium phosphate buffer (pH 7.0) (5 mL) having a concentration of 50 mM, and the mixture was stirred and suspended to obtain a crude enzyme solution.
Based on the above, three kinds of crude enzyme solutions corresponding to the three kinds of samples taken out from the upper part, the middle part and the lower part of the medium were prepared.

<Measurement of enzyme activity of laccase>
An ABTS solution having an ABTS concentration of 10 mM was prepared by adding ABTS as a substrate to a sodium citrate buffer solution (pH 3.0) having a concentration of 50 mM and dissolving it.
Obtained by mixing the ABTS solution (250 μL), sodium citrate buffer (pH 3.0) (730 μL) having a concentration of 50 mM, purified water (19 μL), and the crude enzyme solution (1 μL). The reaction solution (1 mL) was used as a sample. In the obtained sample, the concentration of ABTS was 2.5 mM and the concentration of sodium citrate was 36.5 mM.

The absorbances of the three types of samples obtained above were measured at a wavelength of 420 nm for 3 minutes. Then, the amount of increase in absorbance ΔA ₄₂₀ at a wavelength of 420 nm and the reaction time (min) were determined, and the enzymatic activity (U / mL) of laccase was determined by the above formula (i). The results are shown in FIG. In FIG. 1, "upper", "middle", and "lower" mean the extraction sites of the culture medium samples, respectively.

<< Production of medium, production of laccase, and evaluation of medium >>
[Example 2]
As the inoculum of Oyster Mushroom, KH-83 was used instead of H67, and the inoculum was cultured for 15 days instead of 30 days. And laccase was prepared and the medium was evaluated. The results are shown in FIG.

[Comparative Example 1]
The medium and laccase were produced by the same method as in Example 1 except that TEMPO-oxidized CNF was not used when the medium was produced and the number of days for culturing the inoculum was 35 days instead of 30 days. , Medium was evaluated. The results are shown in FIG.

[Comparative Example 2]
The medium and laccase were produced by the same method as in Example 2 except that TEMPO-oxidized CNF was not used when the medium was produced and the number of days for culturing the inoculum was changed from 15 days to 20 days. , Medium was evaluated. The results are shown in FIG.

As is clear from FIG. 1, in Examples 1 and 2, the enzymatic activity of the laccase produced by Oyster Mushroom was 45 U / mL or more (45 to 346 U / mL) in all the samples, which was extremely high. Focusing on the sample having the highest enzyme activity, in Examples 1 and 2, the enzyme activity of laccase was more than 220 U / mL, which was extremely high. Among them, in Example 1, the enzyme activity of laccase was 346 U / mL, which was particularly high.
In Examples 1 and 2, since the type of medium is the same, the oyster mushroom used in Example 1 is superior to the oyster mushroom used in Example 2 in terms of producing laccase having high enzymatic activity. rice field.

On the other hand, in Comparative Examples 1 and 2, the enzymatic activity of the laccase produced by Oyster Mushroom was extremely low. From the comparison of Example 1 and Comparative Example 1 and the comparison of Example 2 and Comparative Example 2, it can be confirmed that the presence or absence of TEMPO-oxidized CNF in the medium has a great influence on the degree of enzyme activity of laccase. rice field.

[Example 3]
<< Production of medium >>
Peptone with a concentration of 10 g / L, yeast extract with a concentration of 1.0 g / L, potassium dihydrogen phosphate with a concentration of 1.0 g / L, disodium hydrogen phosphate twelve hydrate with a concentration of 0.25 g / L , Calcium chloride dihydrate with a concentration of 13.2 mg / L, Manganese (II) sulfate pentahydrate with a concentration of 1.08 mg / L, Zinc sulfate heptahydrate with a concentration of 1.0 mg / L, Copper (II) sulfate pentahydrate with a concentration of 2.0 mg / L, iron (II) heptahydrate with a concentration of 0.1 g / L, magnesium sulfate heptahydrate with a concentration of 0.5 g / L , And TEMPO oxide CNF having a concentration of 5.0 g / L were mixed, and distilled water was further added to adjust the total liquid volume to 50 mL.
Then, the pH of the obtained aqueous solution was adjusted to 6.00 to obtain a medium containing TEMPO-oxidized CNF.

<< Manufacture of laccase >>
<Sterilization of medium>
The medium obtained above was autoclaved at 121 ° C. for 20 minutes to obtain a sterilized medium.

<Growth of fungi (manufacturing of laccase)>
The sterilized medium obtained above was inoculated with the Hiratake NBRC104981 strain, and the inoculated sterilized medium was shaken at 26 ° C. and 100 rpm using a shaker to carry out shaking culture. The laccase production step was performed. The culture (shaking culture) was carried out for 30 days.
From the above, a medium containing laccase was obtained.
The Hiratake NBRC104981 strain used here can be sold from the Biotechnology Center of the Product Evaluation Technology Infrastructure Organization.

<< Evaluation of medium >>
<Preparation of crude enzyme solution>
From the start of culturing until 30 days have passed, a sample is appropriately taken out from the medium, and this sample (1 mL) is centrifuged at 12000 rpm (10020 G) at 4 ° C. for 30 minutes to obtain a supernatant. Got Then, this supernatant was used as a crude enzyme solution.
This step corresponds to the separation step.

<Measurement of enzyme activity of laccase>
Using the crude enzyme solution obtained above, the enzyme activity (U / mL) of laccase was determined by the same method as in Example 1. The results are shown in FIG.

<< Production of medium, production of laccase, and evaluation of medium >>
[Comparative Example 3]
The medium and laccase were produced and evaluated by the same method as in Example 3 except that the same amount of glucose was used instead of TEMPO-oxidized CNF at the time of producing the medium. The results are shown in FIG.

[Comparative Example 4]
The medium and laccase were produced and evaluated in the same manner as in Example 3 except that the same amount of mechanically defibrated CNF was used instead of TEMPO-oxidized CNF when producing the medium. The results are shown in FIG.

[Comparative Example 5]
The medium and laccase were produced and evaluated by the same method as in Example 3 except that the same amount of powdered cellulose was used instead of TEMPO-oxidized CNF at the time of producing the medium. The results are shown in FIG.

[Comparative Example 6]
The medium and laccase were produced and evaluated by the same method as in Example 3 except that the same amount of cellouronic acid was used instead of TEMPO-oxidized CNF at the time of producing the medium. The results are shown in FIG.

As is clear from FIG. 2, in Example 3, the enzymatic activity of the laccase produced by Oyster Mushroom was maximized on the 15th day after the start of culturing. At this time, the total activity in 50 mL of the medium was about 650 U, and the enzyme activity of laccase was 13.3 U / mL. Moreover, when the highly active laccase at this time was identified, it was bilirubin oxidase (EC 1.3.3.5). At this time, the crude enzyme solution obtained above is purified, and the target product separated by SDS polyacrylamide gel electrophoresis (SDS PAGE) is analyzed by liquid chromatography-mass spectrometry (LC / MS / MS). Identified that the laccase was bilirubin oxidase (EC 1.3.3.5).

On the other hand, in Comparative Examples 3 to 6, the enzymatic activity of the laccase produced by Oyster Mushroom was clearly lower than that in Example 3.
In Comparative Example 3, the enzyme activity of laccase was maximized on the 15th day after the start of culturing, and the enzyme activity at this time was 6.8 U / mL.
In Comparative Example 4, the enzyme activity of laccase was maximized on the 15th day after the start of culturing, and the enzyme activity at this time was 7.8 U / mL.
In Comparative Example 5, the enzyme activity of laccase was maximized on the 7th day after the start of culturing, and the enzyme activity at this time was 1.9 U / mL.
In Comparative Example 6, the enzyme activity of laccase was maximized on the 15th day after the start of culturing, and the enzyme activity at this time was 6.7 U / mL.

The present invention can be used for the production of laccase.

Claims (4)

1. A medium for growing fungi
   The medium contains TEMPO-oxidized cellulose nanofibers and contains.
   The medium is a medium for causing the fungus to produce laccase by growing the fungus.
2. The medium according to claim 1, wherein the fungus is a mushroom.
3. The medium according to claim 1 or 2, wherein the laccase is bilirubin oxidase.
4. It ’s a method of manufacturing laccase.
   The production method is a method for producing laccase, which comprises a step of causing the fungus to produce laccase by growing the fungus using a medium containing TEMPO-oxidized cellulose nanofibers.

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