JPH0779687B2 - Tannase manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing tannase.

Tannase is, for example, an inhibitor of tea cream down,
Alternatively, it is an extremely useful enzyme such as used for clarification during beer production.

[Conventional technology]

Conventionally, as a method for producing tannase, for example, extracted from a pod of a crushed cod tree with methanol, and a medium containing a product obtained by spray-drying the extract, a mutant strain of Aspergillus niger. A method for producing tannase by culturing is known [J. Furment. Techno. (J.Ferment.Technol.), Vol.50, No.
6, p.361-370 (1972)].

[Problems to be Solved by the Invention] When the conventional tannase production method such as the above method is used, the yield of tannase is not yet sufficiently satisfactory. The present invention solves this problem, and an object of the present invention is to provide a method for producing tannase in a significantly higher yield than conventional methods.

[Means for solving problems]

The present inventors have conducted various studies to solve the above problems, as a result, a strain belonging to the genus Aspergillus and having a tannase-producing ability, which contains a polar solvent extract of a tannin-containing plant, and copper ions in a medium and / or Alternatively, if iron ions are removed or reduced, or if the culture is carried out in a medium in which the copper ions and / or iron ions are complexed, the tannase in the culture is remarkably excellent in yield as compared with the case where an untreated medium is used. The present invention has been completed based on the knowledge that the yield of tannase is further improved by the addition of zinc ion to the above-mentioned medium.

That is, the present invention, a microorganism belonging to the genus Aspergillus and having a tannase producing ability, comprising a polar solvent extract of a tannin-containing plant, and removing or reducing copper ion and / or iron ion in the medium, or A method for producing tannase, which comprises culturing in a medium containing copper ions and / or iron ions as a complex and collecting tannase from the culture, and the present invention belongs to the genus Aspergillus and has tannase-producing ability. A medium containing a tannin-containing plant polar solvent extract and zinc ion, and removing or reducing the copper ion and / or iron ion in the medium, or a complex of the microorganism with the copper ion and / or iron ion. It is a method for producing tannase, which comprises culturing in tantalum and collecting tannase from the culture.

Hereinafter, the present invention will be specifically described.

First, the bacterium used in the present invention may be any bacterium that belongs to the genus Aspergillus and produces tannase, and may be a variant or mutant strain of these bacterium.

And as specific examples of this microorganism, for example, Aspergillus oryzae IFO 4206, Aspergillus flavus
Examples include IAM 2184 and Aspergillus niger IAM 2094.

Next, the medium used for tannase production includes a polar solvent extract of a tannin-containing plant, and removes or reduces copper ions and / or iron ions in the medium, or the copper ions and / or Any medium such as synthetic or natural medium can be used as long as it is a medium containing iron ions as a complex and further containing an appropriate amount of one selected from a nitrogen source, a carbon source, an inorganic substance, vitamins and the like, if necessary.

The tannin-containing plant may be any plant as long as it contains tannin, and examples thereof include pods of cod trees, bark and leaves of Nurede which is a plant of the family Rhusaceae. The polar solvent extract of these tannin-containing plants is, for example, water, methanol, ethanol, using a polar solvent such as ethyl acetate, from the crushed product obtained by treating the plant with a conventional crushing means, For example, temperature 60 ℃
Can be obtained by solid-liquid separation by a conventional method after heat treatment for about 1 hour.

The amount of the tannin-containing plant body polar solvent extract added to the medium is, for example, a tannin concentration of 0.5% (W / V) or more, preferably 2 to 5% (W / V).

Examples of the nitrogen source of the medium include casein, potassium nitrate, sodium nitrate, ammonium chloride, ammonium sulfate, 1 ammonium phosphate, 2 ammonium phosphate,
Urea, aspartic acid, asparagine, glutamic acid,
Methionine, tryptophan and the like can be mentioned.

The carbon source of the medium, for example, arabinose, glucose, mannose, rhamnose, sorbose, sucrose, xylose, melibiose, glycerose, lactose, maltose, mannitol, raffinose,
Starch, cellobiose, trehalose and the like can be mentioned, and as the inorganic substance, for example, magnesium sulfate, monopotassium phosphate and the like can be used.

Then, in order to remove or reduce copper ions and / or iron ions in the medium, for example, the prepared medium is treated with a cation exchange resin such as Diaion SK1B (manufactured by Mitsubishi Kasei Co., Ltd.), Dowex 50W (US). , Manufactured by Dow Chemical Company),
Duolite CG-120 (Diamond Alkali, USA), Amberlite IRC-50 (Rohm and Haas, USA), C-132 (Kurita Kogyo), etc. , For example, a temperature of 10 ° C or higher, preferably 20 to
Contact at 50 ° C. for 0.5 hours or more, preferably 2-3 hours with stirring, or in the case of continuous type,
For example, if a column packed with cation exchange resin is used,
By contacting under 20 SV or less, preferably 5 to 2 SV, copper ions and / or iron ions in the medium can be removed or reduced. And otherwise, unless copper ions and / or iron ions are added to the medium or mixed from tap water, copper ions and / or iron ions,
Since it is mainly derived from the polar solvent extract of the tannin-containing plant, the extract may be treated with the above-mentioned cation exchange resin in the same manner as described above.

Further, in order to form a complex with copper ions and / or iron ions in the medium, for example, a chelating agent such as o-phenanthroline, α, α-dipyridyl, or 2 is added to the polar solvent extract of the tannin-containing plant for the medium or the above reasons. 5,4,6-tripyridyl-S-triazine, etc., 5 mg / l or more, preferably 30 to 1
Add about 00mg / l, temperature 5 ℃ or more, preferably 20-40 ℃
Then, the metal ions in the medium or the polar solvent extract of the tannin-containing plant can be formed into a complex by reacting as it is or with stirring for 0.5 hour or more, preferably 1 to 2 hours.

The chelating agent may be used as it is,
It is preferably used by dissolving it in a polar solvent such as ethanol.

Also, in the present invention, a polar solvent extract of a tannin-containing plant and zinc ions (eg, zinc sulfate, zinc chloride, etc.) are contained, and copper ions and / or iron ions in the medium are removed or reduced, or By culturing the above-mentioned tannase-producing bacterium in a medium containing a complex of copper ions and / or iron ions, it is possible to produce tannase in an even higher yield.

The concentration of zinc ions contained in the medium is 0.02 mg / l or more, preferably 0.05 to 0.2 mg / l, and specific means for containing zinc ions include, for example, a method of adding zinc ions to the medium. Can be mentioned.

The removal or reduction of copper ions and / or iron ions in the medium is performed by using a chelating resin such as UNICEREX (manufactured by Unitika Ltd.) and CR-10 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). You can also

In the present invention, the tannase-producing bacterium may be cultured by a solid culture method, but it is usually advantageous to employ a liquid culture method. Specifically, shaking culture, stirring culture, aeration culture, etc. are aerobically cultivated. Do.

The culture temperature is, for example, usually 20 to 40 ° C, preferably 25 to 35 ° C.
The initial pH is 5.5 to 5.7, and the pH during culture is 5.5, for example.
-2.5, preferably pH 5.0-3.5.

Under such culture conditions, although the culture time varies depending on the culture form, tannase is produced and accumulated in the culture by culturing for 2 days or more, preferably about 3 to 5 days.

After completion of the culture, to collect tannase from the culture,
It can be obtained using a conventional enzyme collection means. For example, the culture is subjected to filtration, centrifugation, etc. to remove solids, and ammonium sulfate, alcohol, acetone, etc. are added for fractionation, and the precipitate is collected and dialyzed against water to give a crude precipitate. An enzyme solution is obtained, or this is dried in vacuum to obtain a crude enzyme powder.

To obtain a further purified enzyme preparation from the above-mentioned crude enzyme or crude enzyme powder, for example, a gel filtration method using Sephadex G-200, an adsorption elution method using an ion exchanger, hydroxyapatite, etc., a sucrose density gradient centrifugation method. Sedimentation method,
A purified tannase preparation can be obtained by appropriately selecting and combining an affinity chromatography method, a fractionation method using a molecular sieve membrane or a hollow fiber membrane, and the like.

The physicochemical properties of the purified tannase obtained by the above-mentioned purification means are as follows. Biol. Chem. (Agr.Biol.Che
m.), Vol. 32, No. 7, p. 803 to 809 (1968).

〔Example〕

 Hereinafter, the present invention will be specifically described with reference to examples.

Example Cod powder (manufactured by Elsor, Peru) 4 kg of warm water 2
Add to 4 liters, extract tannin while stirring at a temperature of 60 ° C for 1 hour, squeeze this using cotton cloth by a conventional method to obtain a filtrate, and further centrifuge it at 9000 rpm for 15 minutes by a conventional method. Separation gave 20 l of tannin solution containing 7.2% (W / V) tannin.

10 l of the above tannin solution was passed through a column (4 × 60 cm) packed with Diaion SK1B strongly acidic cation exchange resin (manufactured by Mitsubishi Kasei Co., Ltd.) regenerated by a conventional method at a flow rate of 3 SV to remove metal ions or A tannin solution containing reduced 7.0% (W / V) tannin was obtained.

Then, using the tannin solution prepared in this way, glucose 1% (W / V), monoammonium phosphate 1.4
% (W / V), potassium phosphate 1% 0.2% (W / V), magnesium sulfate heptahydrate 0.1% (W / V) and tannin 3% (W / V)
V) (pH5.70) was used to prepare a medium of which 5
0 ml was dispensed into a 500 ml Sakaguchi flask, and 50 ml of medium containing zinc sulfate heptahydrate added to the above composition at 0.2 mg / l was dispensed into the flask.
Saturated steam having a pressure of 1 kg / cm ² (gauge pressure) was allowed to act at 120 ° C. for 5 minutes to obtain two sterilized medium sections (invention section).

The water used for the preparation of the above medium was distilled water, and the distilled water was also used for the preparation of the following medium.

On the other hand, a medium having the above composition was prepared using a tannin solution which had not been subjected to the strong acid cation exchange resin treatment, and o-phenanthroline was added thereto as a chelating agent so that the concentration was 30 mg / l, and the same procedure as described above was performed. Sterilization treatment was performed to obtain a sterilized medium (invention category).

Further, as a control section, a medium having the above composition was prepared using a tannin solution that had not been subjected to the above treatment, and sterilized in the same manner as described above to prepare a sterilized medium.

The thus obtained culture mediums of the 3rd section of the present invention and the control section were each inoculated with 1 platinum loop of Aspergillus oryzae IFO 4206 obtained by preculturing using a koji juice agar slant medium,
Culture was carried out at a temperature of 30 ° C. for 96 hours with shaking (140 rpm.min / min) to obtain a culture solution.

The culture solution was centrifuged at 12000 rpm for 15 minutes by a conventional method to obtain a supernatant, and the tannase activity in the solution was measured by
S. Ibuchi. Wai. Minoda and Kay. Yamada (S.Ii
buchi, Y. Minoda and K. Yamada): Agric. Biol. Chem. Vol.
The results obtained by measuring in the same manner as the method described in 31, No. 5, p. 513 to 518 (1967) are shown in Table 1 below.

9 l of culture solution obtained by repeating the above culture operation
Each of them was subjected to suction filtration by a conventional method to separate the bacterial cells to obtain a filtrate, to which 500 g of Celite (manufactured by Wako Pure Chemical Industries, Ltd.) was added, mixed well, and then suction filtered again by a conventional method to clarify. Ammonium sulphate was added to each of the above so as to reach 0.8 saturation to form a precipitate, and 9000 r.
A precipitate was obtained by centrifugation at pm for 15 minutes.

The precipitate was dissolved in 500 ml of distilled water according to a conventional method 1
The supernatant obtained by removing the insoluble matter by centrifugation at 2000 rpm for 15 minutes was dialyzed at 5 ° C. for 24 hours by a conventional method to obtain a dialysate. This dialysate was again mixed with 0.01M citrate buffer (pH
The sample obtained by dialysis in 5.0) was passed through a column (4 × 10 cm) packed with DEAE Sephadex A-50 (Pharmacia Fine Chemical Co., Ltd.) that had been equilibrated with the same buffer solution, and 200 ml of the same sample was added. After washing the column with a buffer solution, the tannase adsorbed on the resin was eluted by a linear concentration gradient elution method from 0 to 1 M sodium chloride in 0.01 M citrate buffer (pH 5.0) to obtain tannase activity. The fractions containing the same were collected, and were collected in 0.01M citrate buffer (pH 3.5)
It dialyzed in and the dialyzate was obtained.

Furthermore, this dialyzate was treated with SP that had been equilibrated with the same citrate buffer.
It is passed through a column (2.6 × 20 cm) packed with Sephadex C-50 (Pharmacia Fine Chemicals Co., Ltd.) and washed with 100 ml of the same citrate buffer solution to remove tannase adsorbed on the resin with 0.01 M citrate buffer solution. The operation of elution by a linear concentration gradient elution method of 0 to 0.25 M sodium chloride in a liquid (pH 3.5) and collecting the tannase activity section was repeated twice to obtain a tannase activity section.

Then, the tannase activity fraction obtained was concentrated to 2 ml using an ultrafiltration membrane (Amicon Far East Limited, cutoff molecular weight: 50,000) and a collodion bag (Zertreuse). Sephadex G-200 equilibrated with M citrate buffer (pH 5.0)
Gel filtration using a packed column (2.6 x 100 cm) (Pharmacia Fine Chemical Co.) was performed to collect tannase activity fractions, and the weight and specific activity of the purified tannase obtained by gel filtration under the same conditions were determined. The results are shown in Table 1 below.

It can be seen from Table 1 that in the present invention category, the yield of tannase is remarkably increased as compared with the control category, and in the case of using the cation exchange resin treatment medium containing zinc ion, the cation containing no zinc ion. It can be seen that the yield of tannase is further increased as compared with the case where the exchange resin-treated medium is used.

〔The invention's effect〕

As described above, according to the present invention, tannase can be obtained in a remarkably high yield in a short time. Therefore, the present invention is industrially very useful.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kosuke Takei 1249-6 Yamazaki, Noda City, Chiba Prefecture (72) Inventor Yasuhiko Imai 101 Miyazaki, Noda City, Chiba Prefecture (72) Inventor Katsumi Yuasa 6-Miyamoto Town, Funabashi City, Chiba Prefecture 2829-5

Claims (2)

[Claims] 1. A microorganism belonging to the genus Aspergillus and having a tannase-producing ability, which contains a polar solvent extract of a tannin-containing plant and removes or reduces copper ion and / or iron ion in the medium, or A method for producing tannase, which comprises culturing in a medium containing copper ions and / or iron ions as a complex and collecting tannase from the culture. 2. A microorganism belonging to the genus Aspergillus and capable of producing tannase containing a polar solvent extract of a tannin-containing plant and zinc ions, and removing or reducing copper ions and / or iron ions in the medium. , Or culturing in a medium containing the copper ion and / or iron ion as a complex,
A method for producing tannase, which comprises collecting tannase from a culture.