JP2016127809A - Method for producing protein having tannase activity using thraustochytrids - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a protein having a tannase activity.SOLUTION: A protein having a tannase activity is produced by culturing at least one type of microorganisms classified as thraustochytrids.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a method for producing a protein having tannase activity derived from a microorganism.

Tannase is an enzyme that catalyzes a reaction that decomposes tannin into phenol carboxylic acid and sugar, and is widely used industrially. Specifically, it is often used for the purpose of flavor improvement such as prevention of white turbidity of tea drinks and removal of astringency. As a method for producing industrial tannase, a method of producing using an Aspergillus genus microorganism that is a mold is known (Non-Patent Documents 1 to 3).

  On the other hand, it is known that the jellyfish fungi, which are marine eukaryotic microorganisms, accumulate a significant amount of fats and oils in the cells. The fats and oils accumulated by the fungus are highly concentrated fatty acids such as docosahexaenoic acid (Patent Document 1). Hitherto, the tannase activity ability of the brown mold has not been studied.

JP 2005-287380 A

Advances in Biological Research, 2009, Vol. 3 (1-2), pp. 34-39 International Journal of Scientific Engineering and Technology, 2013, Vol. 2, Issue No. 8, pp. 752-755 Microbiology, 2003, Vol. 149, pp. 2941-2946

Conventionally, an enzyme derived from the genus Aspergillus , which is a mold, has been widely used as an industrial tannase production method. However, these cannot be said to have a sufficiently high specific activity (enzyme activity per unit weight of protein), and a protein having a novel tannase activity is still required.

The present inventors have found that microorganisms classified into Thraustochytrids such, i.e., Arutorunia (Althornia) genus, Apra eaves thorium (Aplanochytrium) genus, Au lunch Oki thorium (Aurantiochytrium) genus Botryococcus key thorium (Botryochytrium) genus, Japonoki thorium (Japonochytrium) genus of Longhi key thorium (Oblongichytrium) genus, Palier Chiki thorium (Parietichytrium) genus, Schizochytrium (Schizochytrium) genus, perilla Lee Doki thorium (Sicyoidochytrium) genus Thraustochytrium (Thraustochytrium) genus, and Ulkenia (Ulkenia It has been found that a protein having tannase activity can be produced by culturing at least one species selected from microorganisms belonging to the genus.

  According to the method of the present invention, it is possible to efficiently produce a protein having tannase activity.

FIG. 1 shows an example of a plate assay in a tannic acid-containing medium. FIG. 2 shows the time course of tannase specific activity in the culture supernatant in liquid culture. FIG. 3 shows the specific activity of tannase in the culture supernatant subjected to salting-out treatment (and dialysis treatment) and in the untreated culture supernatant. Fig. 4 shows changes over time in the number of cells and glucose concentration in the culture medium when A. limacinum mh0186 strain was cultured in a liquid medium containing 0%, 0.1%, 0.5%, 1.0% (w / v) tannic acid. Indicates. FIG. 5 shows the dry biomass weight when the A. limacinum mh0186 strain was cultured in a liquid medium containing 0%, 0.1%, 0.5%, 1.0% (w / v) tannic acid. FIG. 6 shows the total fatty acid content in the cells when A. limacinum mh0186 strain is cultured in a liquid medium containing 0%, 0.1%, 0.5%, 1.0% (w / v) tannic acid. FIG. 7 shows the amount of fatty acids in the cells present per medium volume when culturing A. limacinum mh0186 strain in a liquid medium containing 0%, 0.1%, 0.5%, 1.0% (w / v) tannic acid. Indicates. FIG. 8 shows the fatty acid composition of cells when A. limacinum mh0186 strain is cultured in a liquid medium containing 0%, 0.1%, 0.5%, 1.0% (w / v) tannic acid. FIG. 9 shows the results of measuring tannase activity of the culture supernatant of A. limacinum mh0186 strain by a plate assay. FIG. 10 shows the results of measuring the tannase activity of the culture supernatant of A. limacinum mh0186 strain by thin layer chromatography.

(Tannase activity)
In the present invention, tannase activity refers to the activity of hydrolyzing ester bonds and depside bonds present in hydrolyzable tannins such as tannic acid, and particularly means tannic acid decomposing activity. In the present specification, tannase activity is defined as 1 unit of enzyme activity in which 1 mg of protein degrades 1 mg of tannic acid in 1 minute at 30 ° C.

(Microorganism used in the present invention)
The method for producing a protein having tannase activity according to the present invention includes culturing at least one kind of microorganism classified as a jellyfish, and causing the microorganism to produce a protein having tannase activity.

The “microorganisms classified into the genus Amaranthus” are marine eukaryotic microorganisms that produce highly unsaturated fatty acids as explained in the background art. As microorganisms classified in Thraustochytrids class, Arutorunia (Althornia) genus, Apra eaves thorium (Aplanochytrium) species, Au lunch Oki thorium (Aurantiochytrium) genus, Botryococcus key thorium (Botryochytrium) genus, Japo eaves thorium (Japonochytrium) genus, of Longhi key thorium (Oblongichytrium) genus Palier Chiki thorium (Parietichytrium) genus Schizochytrium (Schizochytrium) genus perilla Lee Doki thorium (Sicyoidochytrium) genus Thraustochytrium (Thraustochytrium) genus, or include Ulkenia (Ulkenia) microorganism belonging to the genus It is done. In the present invention, a microorganism that is classified as a jellyfish and has the ability to produce a protein having tannase activity may be used.

As the Agrobacterium, particularly a microorganism having an ability to produce a protein having tannase activity belonging to the genus Aurantiochytrium is preferable.

Examples of the microorganism belonging to the genus Aplanochytrium and having the ability to produce a protein having tannase activity include at least one species belonging to Aplanochytrium kerguelensis . As a specific strain of a microorganism belonging to the genus Aplanochytrium, an Aplanochytrium kerguelensis SEK535 ( Aplanochytrium kerguelensis SEK535) strain is preferably used.

Examples of microorganisms belonging to the genus Aurantiochytrium and capable of producing a protein having tannase activity include at least one species belonging to Aurantiochytrium limacinum or Aurantiochytrium mangrovei . As a specific strain of microorganism belonging to the Aurantiochytrium genus, Aurantiochytrium limacinum SR21 ATCC MYA-1381 , Aurantiochytrium limacinum mh0186 ( accession number FERM P-19755), Aurantiochytrium mangrovei SEK218 ( accession number NBRC 103269), Aurantiochytrium mangrovei SEK243, Aurantiochytrium sp. ATCC20888 or Aurantiochytrium sp. ATCC26185 is preferably used.

Examples of the microorganism belonging to the genus Botryochytrium and having the ability to produce a protein having tannase activity include at least one species belonging to Botryochytrium radiatum . As a specific strain of microorganisms belonging to the genus Botryochytrium, Botryochytrium radiatum SEK353 (accession number NBRC 104107) is preferably used.

Specific strains of microorganisms having the ability to produce a protein having a tannase activity belonging to Oblongichytrium genus Oblongichytrium sp. SEK347 (accession number NBRC one hundred and two thousand six hundred eighteen) is preferably used.

Examples of the microorganism belonging to the genus Parietichytrium and having the ability to produce a protein having tannase activity include at least one species belonging to Parietichytrium sarkarianum . Specific strains of microorganisms belonging to the Parietichytrium genus, Parietichytrium sarkarianum SEK351 (Accession No. NBRC 104108), Parietichytrium sarkarianum SEK364 (Accession No. FERM BP-11298), or Parietichytrium sp. SEK358 (accession number FERM BP-11405) are preferred Used.

Examples of the microorganism belonging to the genus Schizochytrium and having the ability to produce a protein having tannase activity include at least one species belonging to Schizochytrium aggregatum . As specific strains of microorganisms belonging to the genus Schizochytrium , Schizochytrium aggregatum ATCC28209, Schizochytrium sp. SEK210 (accession number NBRC 102615), or Schizochytrium sp. SEK345 (accession number NBRC 102616) is preferably used.

The microorganism having the ability to produce a protein having a tannase activity belonging to Thraustochytrium genus Thraustochytrium, Aureumu (Thraustochytrium aureum), Thraustochytrium roseum (Thraustochytrium roseum), or Thraustochytrium-Sutoriatamu to (Thraustochytrium striatum) There is at least one species that belongs. As specific strains of microorganisms belonging to the genus Thraustochytrium , Thraustochytrium aureum ATCC34304, Thraustochytrium roseum ATCC28210, or Thraustochytrium striatum ATCC24473 is preferably used.

Examples of microorganisms belonging to the genus Ulkenia and capable of producing a protein having tannase activity include at least one species belonging to Ulkenia amoeboidea . As specific strains of microorganisms belonging to the genus Ulkenia , Ulkenia amoeboidea SEK214 (Accession No. NBRC 104106), Ulkenia sp. ATCC28207, or Ulkenia sp. 175-01m2 (Accession No. NBRC 110830) is preferably used.

  The range of microorganisms used in the present invention is not limited to microorganisms that are currently classified into the above genera or species, but are currently classified into other genera or species, or the classification is not clear However, in terms of molecular evolution, microorganisms to be classified into any of the above genera or species are also included.

Among the above-mentioned specific strains, Aurantiochytrium limacinum mh0186 strain is disclosed in Japanese Patent Application Laid-Open No. 2005-287380, and is an independent administrative agency, National Institute of Advanced Industrial Science and Technology. This is the same as the Schizophytrium sp. M-8 strain deposited in Japan on March 29, 2004 under the accession number FERM P-19755 in the center No. 6). Genus Schizochytrium at the time of deposit of FERM P-19755 has been reorganized into Schizochytrium , Aurantiochytrium and Oblongichytrium (Rinka Yokoyama, Daiske Honda (2007) Mycoscience Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience, 48, 199-21). Aurantiochytrium limacinum mh0186 strain, JP is 2005-287380 discloses the disclosure of Schizochytrium sp. How to obtain the M-8 strain (thraustochytrid M-8 strain was obtained as follows. First, Ishigaki mangrove forests Place the seawater and deciduous leaves collected in step 3 into a 300 ml Erlenmeyer flask, and add about 0.05 g of pine pollen (here, collected from the coast around Miyazaki city). Seawater was collected so as to contain, and 0.1 ml was applied on a potato dextrose agar medium prepared in a petri dish, cultured at 28 ° C. for 5 days, picked up a creamy, non-glossy colony and applied on a new agar medium Three days later, the grown microorganisms were observed under a microscope and stored in a slant medium, judging from the size and form of the cells as being Labyrinthula.

Aurantiochytrium limacinum SR21 ATCC MYA-1381, Aurantiochytrium sp. ATCC20888, Aurantiochytrium sp. ATCC26185, Schizochytrium aggregatum ATCC28209, Thraustochytrium aureum ATCC34304, Thraustochytrium roseum ATCC28210, Thraustochytrium striatum ATCC24473, and Ulkenia sp. ATCC28207, from American Type Culture Collection (ATCC) It is available.

It should be noted, Aurantiochytrium limacinum SR21 ATCC MYA-1381 is a Schizochytrium limacinum Honda et Yokochi (ATCC MYA -1381), Aurantiochytrium sp. ATCC26185 is Thraustochytrium sp. And (ATCC 26185), Aurantiochytrium sp. ATCC20888 is Schizochytrium sp. And (ATCC 20888) , Schizochytrium aggregatum ATCC28209 the Schizochytrium aggregatum Goldstein et Belsky (ATCC 28209 ), Thraustochytrium aureum ATCC34304 and Thraustochytrium aureum Goldstein (ATCC 34304), Thraustochytrium roseum ATCC28210 and Thraustochytrium roseum Goldstein (ATCC 28210_TT), Thraustochytrium striatum ATCC24473 is Thraustochytrium striatum Schneider ( ATCC 24473) and Ulkenia sp. ATCC 28207 are the same as Japonochytrium sp. (ATCC 28207), respectively, and the genus or species classification has been reconstructed by the present inventors.

Aurantiochytrium mangrovei SEK218 (Accession number NBRC 103269), Botryochytrium radiatum SEK353 (Accession number NBRC 104107), Oblongichytrium sp. SEK347 (Accession number NBRC 102618), Parietichytrium sarkarianum SEK351 (Accession number NBRC 104108), Schizochytrium sp. ), Schizochytrium sp. SEK345 (Accession No. NBRC 102616), Ulkenia amoeboidea SEK214 (Accession No. NBRC 104106), and Ulkenia sp. 175-01m2 (Accession No. NBRC 110830) have been deposited with the National Institute for Product Evaluation and Technology. And is generally available.

Parietichytrium sp. SEK358 (Accession No. FERM BP-11405) and Parietichytrium sarkarianum SEK364 (Accession No. FERM BP-11298) have been deposited with the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary and are generally available. is there.

(Microbial culture)
The method of the present invention includes a culturing step of culturing the microorganism and causing the microorganism to produce a protein having tannase activity.

  In the culturing step, a fixed medium, a liquid medium, or the like suitable for cultivating the jarlet is used as the medium. The medium is not particularly limited, and examples of the medium that is usually used include GY medium (Journal of Fisheries Science, Vol. 68, No. 5, 674-678 (2002)), B1 agar plate medium (Appl. Microbiol. Biotechnol., 72 , 1161-1169 (2006)), and the like can be used as appropriate.

  The culture conditions in the culture step are not particularly limited, but typically, the microorganism is used in an appropriate medium for about 1 day to 2 weeks, preferably 2 to 12 days, more preferably 3 to 10 days, 25 to 35. Incubate at ℃. In the case of a liquid medium, culture is performed under shaking conditions as necessary. The light / dark conditions during the culture are not particularly limited, but are usually performed under dark conditions.

  The present inventors have found that by culturing the microorganism in the presence of tannic acid, the production amount of the protein having tannase activity by the microorganism is remarkably increased. Therefore, it is particularly preferable to use a medium containing tannic acid as the medium. Preferably, a medium added so that the tannic acid concentration is 0.01 to 10% (w / v), preferably 0.1 to 1.0% (w / v) is used.

  As explained in the background art, since microorganisms classified as a kind of mold are accumulating a significant amount of highly unsaturated fatty acid, by culturing the microorganisms, tannase produced in the culture solution is recovered. Recovery of fatty acids produced in biomass (bacteria) can also be performed simultaneously. In the examples described later, in a medium containing a low concentration of tannic acid, it was found that the DHA production amount in the cells per medium volume was the highest, so that the recovery of tannase produced in the culture solution was recovered. And a fatty acid produced in biomass (bacteria) simultaneously, a medium containing a low concentration of tannic acid, for example, a tannic acid concentration of 0.01 to 0.3% (w / v), preferably 0.05 It is preferable to use a medium added so as to be in a range of% to 0.2% (w / v).

(Protein recovery)
The protein having tannase activity produced by the cultured microorganism can be accumulated in the body of the microorganism, but is mainly secreted outside the body of the microorganism.

  When the protein having tannase activity accumulates in the microbial cell, it is contained in the microbial culture, microbial cell-containing fraction, microbial cell lysate, microbial cell extract (crude enzyme extract), etc. It is possible to directly use a protein having tannase activity. Further, when a protein having tannase activity is secreted outside the body of the microorganism, the protein having tannase activity in a state contained in the culture supernatant fraction can be used as it is.

  As a means for recovering a protein having a higher concentration of catalase activity from a culture of the microorganism, a microbial cell-containing fraction, a microbial cell lysate, a microbial cell extract (crude enzyme extract), a culture supernatant fraction, etc. Various gel chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc., salting out of ammonium sulfate fraction, sodium sulfate fraction, sodium chloride fraction, dialysis, or isoelectric focusing One or more general means such as these can be used in combination.

  In particular, the present inventors have found that the specific activity of a protein having tannase activity is significantly increased by salting out the protein having tannase activity contained in the cell culture supernatant and optionally performing dialysis. It was. Therefore, the method of the present invention preferably includes a step of salting out a protein having tannase activity. Salting out is performed, for example, by adding a salt such as ammonium sulfate to an excess medium. In addition, the method of the present invention preferably includes a step of dialysis of a culture supernatant or a solution containing salted out protein having tannase activity. Dialysis can be performed using, for example, a buffer solution known to those skilled in the art, and an example of the buffer solution is a citrate buffer solution. When dialysis is performed after salting out, the salted-out protein can be dissolved in a solution, for example, a buffer solution, and then dialyzed against the buffer solution.

  In the method of the present invention, how much the concentration of the protein having tannase activity is increased is not particularly limited, and can be appropriately determined according to the purpose of use. The tannase activity (specific activity) per 1 mg of protein is preferably 0.1 unit / mg or more, more preferably 1 unit / mg or more, and the upper limit of the specific activity is not particularly limited. The above-mentioned means can be combined appropriately.

  The protein having tannase activity obtained by the present invention can be used for industries that require tannase treatment, for example, flavor improvement such as prevention of white turbidity and improvement of astringency in tea beverages.

  Examples of the present invention will be described below, but the present invention is not limited thereto.

<Example 1: Screening of tannase producing strain>
The presence or absence of tannase production was determined using a plate assay method. That is, it grows on B1 agar medium (yeast extract 0.2g, polypeptone 0.2g, glucose 0.5g, vitamin mixed solution 0.1ml, agar 1.5g, 50% artificial seawater (ASW) 100ml [pH 6.8]) -7 days) Collected colonies of 21 jellyfish molds with platinum wire, agar plate medium containing 0.5% tannic acid (0.5g tannic acid, yeast extract 0.2g, polypeptone 0.2g, vitamin mixture 0.1ml , Agar 1.5g, 50% ASW 100ml [pH 6.8]) grown at 28 ° C for 5 days, and a tannase with clear zone (tannin degradation zone) or blackening (Berbendam reaction) around the grown colony It was determined as a positive strain. In addition, the size of a clear zona pellucida was measured.

An example of the plate assay is shown in FIG. 1, and the results of the plate assay in all 21 test strains are shown in Table 1. In Table 1, ND indicates not detected, and B indicates that blackening was observed. As a result of the plate assay, clear zona pellucida was confirmed for A. limacinum mh0186 strain and A. limacinum SR21 strain among the 21 strains tested . In addition, the Barbendam reaction was confirmed for the other three Aurantiochytrium strains.

<Example 2: Measurement of tannase activity in culture supernatant>
A. limacinum mh0186 strain, which showed remarkable tannase activity in the plate assay, was added to a TY liquid medium containing tannic acid (0.5 g tannic acid, 0.2 g yeast extract, 0.2 g polypeptone, 50 ml concentration ASW 100 ml [pH 7.0 ]) And shaking culture (28 ° C., 110 rpm, 120 hrs), and the tannic acid concentration in the culture and the tannase activity in the culture supernatant were measured according to the following method.

  The tannase activity in the culture supernatant was measured by the change in absorbance at 310 nm according to the method described in Iibuchi, S. et al., Agric. Biol. Chem., Vol. 31, pp. 513-518, 1967. That is, add 600 μl of citrate buffer (50 mM, pH 5.5) containing 0.35% tannic acid to 150 μl of culture supernatant and stir well, leave it at 30 ° C. for 15 minutes, and then add 3 ml of 90% ethanol solution. Stir well, transfer 150 μl of the solution to another disposable test tube, add 3 ml of 90% ethanol solution, stir well, measure absorbance at a wavelength of 310 nm using a spectrophotometer, and create a calibration curve prepared in advance. The tannase activity was calculated from The enzyme activity was defined as 1 unit of enzyme activity in which 1 mg of protein decomposes 1 mg of tannic acid in 1 minute at 30 ° C.

  As a result, the tannase activity of the culture supernatant increased after 48 hours of culture (FIG. 2). The tannase activity showed a maximum value after 96 hours of culture and decreased after 120 hours of culture.

<Example 3: Concentration of enzyme protein having tannase activity>
After shaking culture of A. limacinum mh0186 strain in TY liquid medium (28 ° C., 110 rpm, 24 hrs), the culture solution was centrifuged (3000 rpm, 30 min, 4 ° C.), and the culture supernatant was collected. Ammonium sulfate was added to the culture supernatant to a saturation concentration of 90%, and salting out was performed. Protein precipitated by salting out was dissolved in 5.0 mL of 50 mM citrate buffer (pH 5.5), and dialyzed against 50 mM citrate buffer (pH 5.5) using a cellulose dialysis membrane. After dialyzing overnight under ice-cooling, the protein exhibiting tannase activity remaining in the cellulose membrane was dissolved in 5.0 mL of the same buffer. The tannase activity was measured in the same manner as in Example 2 for the untreated culture supernatant and the culture supernatant subjected to salting-out treatment (and dialysis treatment).

  As a result, the tannase specific activity per protein in the sample was significantly increased, about 50 times higher than the untreated culture supernatant by salting out, and compared with the untreated culture supernatant by further dialysis. It increased about 100 times (Fig. 3).

<Example 4: Examination of influence of tannic acid content in culture medium on microorganisms>
Culturing mh0186 strain in GY liquid medium (glucose 0.5g, yeast extract 0.2g, polypeptone 0.2g, 100ml 50% concentration ASW) containing tannic acid 0%, 0.1%, 0.5%, 1.0% (w / v) Then, cell growth, glucose content in the medium, dry biomass amount, fatty acid content / fatty acid composition in the cells, and tannase activity in the culture supernatant were analyzed.

(Cell growth and glucose content of medium)
The cell number was directly counted using a Thomas blood cell plate under a light microscope. The glucose concentration was measured using a glucose analysis kit (Test Wako C-II, Wako Pure Chemical Industries).

In all the test groups, the number of cells rapidly increased after 24 hours of culture, and after about 24 hours of culture, the number of cells was about 10 ⁸ cells / ml. As the cells grew, the glucose concentration in the medium decreased sharply, almost depleted at 72 hours of culture, and was below 0.1 g / L (Fig. 4).

(Dry biomass amount and fatty acid content and fatty acid composition in cells)
In the measurement of the dry cell weight, a 1.5 ml microtube that had been dried in a dryer overnight at 105 ° C. overnight and constant in weight was used. 1.0 ml of the culture solution was collected in the above microtube, centrifuged at 150 rpm for 5 minutes at room temperature, the supernatant was removed, and the precipitate was washed twice with distilled water. After washing, it was dried overnight at 105 ° C. with a dryer, and the weight was measured using a precision electronic balance. The value obtained by subtracting the weight of the microtube that had been constant in advance from the weight of the microtube containing the precipitate was taken as the dry cell weight.

  Fatty acids in the microbial cells were converted to methyl esters using 10% methanolic hydrochloric acid, and the composition and content were analyzed using a gas chromatograph. That is, the temperature of the gas chromatograph (GC-2014, SHIMADZU) inlet and detector (FID) is set to 250 ° C, and the column temperature is set to increase from 150 ° C to 220 ° C by 2 ° C per minute. Then, the fatty acid sample methylated was injected, and the fatty acid content was calculated using C19: 0 as an internal standard. Each fatty acid was identified by comparison with the retention time of the standard fatty acid.

  The amount of dry biomass after 72 hours showed a low value in the 0% test group, and increased as the tannic acid concentration in the medium increased (FIG. 5). On the other hand, the total fatty acid content in the cultured cells decreased as the tannic acid concentration increased (Fig. 6). The total fatty acid production in the microbial cells per medium volume was significantly higher in the 0.1% group than in the other test groups (FIG. 7).

  Fatty acids in the cells mainly contain saturated fatty acids, mainly palmitic acid, and highly unsaturated fatty acids, docosahexaenoic acid (DHA). The DHA content in the cells was highest in the 0% test group, and decreased as the tannic acid content in the medium increased. The highest DHA production in the cells per medium volume was obtained at 0.1% (FIG. 8).

(Tannase activity in culture supernatant)
The tannase activity of the culture supernatant was measured by a plate assay method using a tannic acid-containing plate. In other words, prepare an agar plate containing 0.5% tannic acid (0.5g tannic acid, 100ml citrate buffer (50mM, pH5.5), agar powder 1.5g) and place a well (hole, 5mm diameter) on the agar medium with a cork borer. The well was added with 40 μl of culture supernatant and incubated at 28 ° C. for 4 hours, and the presence or absence of activity was judged by the zona pellucida around the well.

  As a result, the activity of tannase in the culture supernatant was confirmed only in a medium containing 0.5% tannic acid (FIG. 9).

  The tannase activity in the culture supernatant was also confirmed by thin layer chromatography. That is, using butanol: acetic acid: water = 4: 1: 1 as a developing solvent, the culture supernatant was subjected to paper chromatography, and after development, sprayed with 30% ethanol containing 0.15% ferric chloride by spraying at 105 ° C. The tannic acid spots were colored by heating for 10 minutes.

  When tannic acid in 0.5% culture supernatant was subjected to thin layer chromatography, only tannic acid spots were observed at the start of culture (Figure 10 left), but after 48 hours of culture, in addition to tannic acid, A spot of gallic acid, a degradation product, was confirmed (Fig. 10, right).

Claims (6)

  A method for producing a protein having tannase activity, comprising a culture step of culturing at least one microorganism classified as a genus mold and causing the microorganism to produce a protein having tannase activity. Wherein the microorganism is at least one selected from microorganisms belonging to Au lunch Oki thorium-Rimashinamu (Aurantiochytrium limacinum), and Au lunch Oki thorium-Mangurobei (Aurantiochytrium mangrovei), The method of claim 1. The microorganisms are Aurantiochytrium limacinum SR21 ATCC MYA-1381 ( Aurantiochytrium limacinum SR21 ATCC MYA-1381), Aurantiochytrium limacinum mh0186 ( Aurantiochytrium limacinum mh0186) strain (FERM P-19755), Aurantiochytrium The method according to claim 1, wherein the method is at least one selected from the strains of Mangrobay SEK218 ( Aurantiochytrium mangrovei SEK218) and Aurantiochytrium sp. ATCC26185 ( Aurantiochytrium sp. ATCC26185).   The method according to any one of claims 1 to 3, wherein the culturing step is a step of culturing the microorganism using a medium containing tannic acid.   The method according to any one of claims 1 to 4, further comprising a step of salting out a protein having tannase activity.   6. The method of claim 5, further comprising dialyzing a solution containing the salted out tannase activity against a buffer.

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