JP4383131B2 - Method for industrial crystallization of transglutaminase and method for producing transglutaminase preparation - Google Patents

Description

  The present invention relates to a method for crystallizing transglutaminase that can be used mainly for food use on an industrial scale, and a method for producing a transglutaminase preparation using this method.

  Transglutaminase (hereinafter sometimes referred to as TGase) is an enzyme that catalyzes an acyl conversion reaction of a γ-carboxylamido group of a glutamine residue in a peptide chain, and serves as an acyl acceptor of a lysine residue in a protein. When the ε-amino group acts, an ε- (γ-Gln) -Lys crosslink is formed within or between protein molecules. Therefore, since the modification of protein or peptide can be carried out by utilizing the action of TGase, TGase (see Patent Document 1), which is a partially purified microbial enzyme derived from the genus Streptomyces, is used to bind meat, sausage, tofu, Used in the manufacture of bread and noodles.

By the way, crystallization of an enzyme is generally performed using an enzyme purified to a uniform level by a complicated purification operation in multiple stages, and conventionally, there are various methods. However, enzyme crystallization cannot be easily succeeded even by using an enzyme purified to homogeneity, and it is known that whether or not crystallization succeeds is highly empirical. Yes. Thus, there are many enzymes that have not been successfully crystallized so far. As for TGase, a crystallized one has already been reported (see Patent Document 2). However, the preparation method is based on an ultra-small laboratory-scale preparation method based on the hanging drop method. Substances unsuitable for food use are used. In the case of TGase, simple crystallization on an industrial scale is said to be difficult because of the complexity of the manufacturing process, low yield, and high cost, such as the need to purify the enzyme uniformly. The fact is that it has not been successful yet.
Therefore, TGase currently produced industrially is a crude enzyme solution obtained by removing cells from a fermentation mixture, desalted and concentrated with an ultrafiltration membrane, and then partially purified by alcohol fractionation or the like. However, due to the fact that it is a partially purified enzyme, there have been various problems as described below, and therefore industrial crystallization of TGase has been strongly desired.
(1) Contaminating enzymes such as protease and amylase and coloring substances are included, which may be undesirably affected by the purpose of use, and manufacturing management of products with strict quality control is necessary and complicated. In addition, when trying to improve the quality excluding these impurities, there is a problem that the yield is low and the cost is high.
(2) There is a limit to the kind and amount of stabilizer added due to a decrease in specific activity, and there has been a problem of a decrease in yield due to deactivation during drying.
(3) If it is a crystalline enzyme, it can be easily formulated into a liquid or high-concentration suspension. However, in partial purification, for example, if it is made liquid, there is a risk of inactivation due to contaminating enzymes such as proteases. When adding a precipitant or the like to form a suspension or paste, the specific activity is low, so the amount used is increased, and the effect of adding a stabilizer or precipitant to the final product is increased. In fact, it was difficult to formulate other than powder.
(4) For the reason of (3) above, TGase is currently supplied as a powder product, but the powder is likely to scatter during handling, such as dissolving the powder in water. There was a risk of causing inflammation.
Japanese Patent No. 2849773 JP 2002-253272 A

  The present invention has been made in view of the above circumstances, a method for easily crystallizing transglutaminase produced by a microorganism of the genus Streptomyces on an industrial scale, and a transcrystallized using this method. It is an object of the present invention to provide a method for easily producing a transglutaminase preparation by adding an additive to glutaminase.

As a result of repeated studies to solve the above problems, the present inventors have found that precipitation of contaminating proteins other than TGase is less if salting out is performed using sodium chloride or potassium chloride. It came to be completed.
That is, the present invention provides a crude enzyme comprising a culture filtrate by culturing a microorganism of Streptomyces mobaraensis or Streptomyces lavendulae, and the crude enzyme per absorbance of ultraviolet light at a wavelength of 280 nm. It is characterized by salting out by adding sodium chloride and / or potassium chloride by a batch method to a partially purified enzyme that is not purified until the specific activity measured by the enzyme activity is at least 2u (2u / Ab280nm) or more and uniform. The gist is an industrial crystallization method of transglutaminase. Here, the crude enzyme refers to a culture filtrate containing TGase produced by a microorganism belonging to the genus Streptomyces. Moreover, what refine | purified the crude enzyme partially means TGase which is not refine | purified until a simple purification operation is added to a crude enzyme and TGase becomes uniform.

  Further, the present invention uses the above-described industrial crystallization method for transglutaminase, and the crystallized transglutaminase is converted from a protein partial degradation product, trehalose, cysteine, glutathione, sodium bisulfite, citrate and phosphate. The gist is a method for producing a transglutaminase preparation characterized by containing one or more selected one or more.

  The present invention has the following effects. Since the crude enzyme or TGase obtained by partially purifying the crude enzyme can be crystallized on an industrial scale and at a high recovery rate, cost reduction and production management can be simplified. In addition, the crystallized TGase can easily produce liquid products such as liquids, suspensions, and pastes, and avoid problems such as eye and nasal inflammation similar to proteases produced by TGase. Furthermore, since the crystallized TGase has a high specific activity, a sufficient amount of stabilizer can be added, and a TGase preparation excellent in storage stability can be provided. In addition, a high-quality transglutaminase preparation can be easily produced by using transglutaminase which is crystallized on an industrial scale.

  A crude enzyme solution of TGase comprising a culture filtrate obtained by removing cells from a fermentation mixture produced by fermentation of a strain having TGase production ability is prepared. As TGase, those produced by microorganisms of the genus Streptomyces can be used. Examples of the microorganism belonging to the genus Streptomyces include Streptomyces mobaraensis (formerly Streptomyces mobaraensis), Streptomyces lavendulae, and the like. Streptomyces mobaraensis is strain S-8112 (Agric. Biol. Chem., 53 (10), 2613-2617, 1989, FERM P-18980), Streptomyces lavendulae is No. 466 (see Patent Document 1, FERM P-11657). In addition, by using conventional methods such as ultraviolet irradiation and NTG (N-methyl-N'-nitrosoguanidine), productivity is increased, production of contaminating proteins such as protease and amylase is reduced, and physiologically active substances such as antibiotics are added. It is possible to use mutant strains of microorganisms belonging to the genus Streptomyces that are suppressed or lacked, and furthermore, it is also possible to use genetically modified bacteria. Streptomyces mobaraensis No.140226 and Streptomyces mobaraensis No.W42228 can be exemplified as mutants of the previously described Streptomyces mobaraensis S-8112 strain. These have been deposited by the applicant under the Budapest Treaty at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (IPOD, 1st, 1st, 1st Street, 1st Street, Tsukuba, Ibaraki 305-8586), The accession numbers are FERM P-19448 and FERM P-19449, respectively.

  Medium used for fermentation of microorganisms belonging to the genus Streptomyces includes carbon sources such as starch, sucrose, lactose, glycerol, glucose, peptone, meat extract, yeast extract, corn steep liquor, ammonium nitrate, ammonium chloride, ammonium sulfate, etc. Commonly used medium materials such as trace nitrogen salts such as monopotassium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, manganese sulfate, and calcium carbonate can be used. Moreover, in order to suppress foaming, addition of an antifoamer can also be performed as needed. The culture is generally in the range of 25 ° C. to 35 ° C., and is performed in various fermentation vessels, and aeration and agitation are usually performed for 3 to 6 days. This is not limited depending on the strain and fermentation medium culture conditions. For example, when the medium raw material is fed or a high concentration medium raw material is included, the culture time may generally be longer. The medium pH is also controlled as necessary.

  Removal of bacterial cells and the like from the fermentation mixture after completion of the culture can be performed by filtration using diatomaceous earth, filtration using a membrane or ceramic filter, or centrifugation. In the case of filtration, pressure filtration with diatomaceous earth added is preferable, and it is preferable to carry out at room temperature or lower. The obtained culture filtrate, ie, the crude enzyme solution of TGase, is cooled as necessary. In addition, as described in Eur, J, Biochem., 257, 570-576 (1998), TGase is known to be produced as a precursor, and the fermentation mixture is fixed for conversion to a mature form. The temperature may be kept as it is, or by adding an enzyme such as trypsin that can be used for other processing.

  Partial purification of the crude enzyme solution can be performed by concentration treatment. The concentration method is not particularly limited, but it is preferable to use an ultrafiltration membrane capable of simultaneously concentrating and purifying. Concentration can be performed up to about 10 to 100 times, but there is no particular problem as long as it reaches a concentration that can be used for the purification and precipitation of other purification operations and salting out crystallization in the next step. Higher is preferable in consideration of properties and recovery rate. In addition, the ultrafiltration membrane can be said to be preferable to use, for example, ACP manufactured by Asahi Kasei Co., Ltd. having an average pore diameter of 13,000 or less, considering the molecular weight of TGase of about 38,000, but is not necessarily limited thereto. Furthermore, even when a membrane having a pore size of 50,000 is used, almost no enzyme leaks, so that the degree of purification can be increased by selecting a membrane as necessary. Although precipitation may occur in desalting and concentration, it can be dissolved by adding an appropriate buffer or salt solution, and the recovery rate can be increased. Moreover, the temperature at the time of concentration is not particularly limited, but is preferably 10 ° C to 30 ° C. The higher the temperature, the more efficient the concentration, but deactivation must be taken into account. Concentration can be performed twice or more, and the concentrated solution may be decolorized with activated carbon in advance.

  Further, the partial purification of the crude enzyme solution can be carried out using an adsorbent, an ion exchange resin, or a precipitating agent such as ethanol or polyethylene glycol, in addition to simple desalting and concentration and decolorization with activated carbon as described above. In particular, when the enzyme concentration is low, it is also effective to use ammonium sulfate or the like having a high precipitation property in order to increase the recovery rate.

  TGase can be crystallized by salting out by adding sodium chloride or potassium chloride to TGase crude enzyme or partially purified crude enzyme. Enzyme crystallization is generally carried out using an enzyme solution that has been purified to a uniform concentration through a multi-step and complicated purification procedure, and according to the present invention, sodium chloride or potassium chloride is a TGase. Addition to crude enzyme or partially purified crude enzyme reduces the precipitation of other contaminating proteins, facilitates crystallization, and enables crystallization with high yield, so that TGase crystallized on an industrial scale can be produced. . If the enzyme unit (u) per absorbance at Ab 280 nm in the TGase crude enzyme or a partially purified crude enzyme is 2 units or more, crystallization can be ensured by addition of sodium chloride or potassium chloride. However, salting out on the crude enzyme is preferable because it contains more contaminating proteins, the enzyme concentration is low, the enzyme recovery rate is low, or the addition of a large amount of salts is required compared to the partially purified enzyme. Although it is not a method, it will not be limited if productivity becomes high by improvement of a production strain, production medium culture conditions, etc. Sodium chloride and potassium chloride may be used in combination.

  Crystallization of TGase may be carried out without adding a crystal seed in advance. Also, depending on the method of addition of sodium chloride or potassium chloride, an amorphous precipitate may be formed instead of crystals (it seems to be due to subtle effects of changes in enzyme purity and sodium chloride concentration or potassium chloride concentration). It is preferable to gradually add and saturate sodium chloride or potassium chloride instead of saturating at once. The amorphous precipitate can be easily crystallized by re-dissolving and salting out. Usually, enzyme crystallization is carried out by increasing the salt concentration over time from the supersaturated state that produces a slightly amorphous precipitate, or by applying some kind of stimulation to the enzyme solution. In the case of sodium chloride or potassium chloride, As described above, it is considered that crystallization can be easily performed because there is little precipitation of other contaminating proteins. In addition, the stability of the crystal suspension obtained using sodium chloride or potassium chloride is very excellent due to the crystalline state and the high salt concentration, and can be stored for a long time. Therefore, if necessary, the crystal suspension can be used as it is, or a preparation containing an appropriate stabilizer can be prepared.

  Also, by increasing the temperature of the crystal mother liquor obtained after crystallization of TGase from the crude enzyme or partially purified crude enzyme, changing the pH, or adding a small amount of phosphate or sulfate salt TGase can be crystallized from the crystal mother liquor, and the TGase recovery rate can be increased.

  Crystals can be collected by a general method such as filtration or centrifugation. In any case, it is preferable to perform washing in order to sufficiently remove contaminating proteins contained in the crystal mother liquor. Further, after the obtained crystal (crude crystal) is dissolved using water or a salt solution or the like, the so-called recrystallization operation in which crystallization is performed again using the salt can further remove impurities. is there.

  The obtained crystals are dissolved using water or a salt solution, etc., and then desalted and concentrated using an ultrafiltration membrane or other methods to remove the salts used for salting out. Can be powdered. Other drying methods are possible, such as spray drying, vacuum drying, film drying, or precipitation with an organic solvent such as alcohol, followed by vacuum drying. In desalting and concentration, it is preferable to increase the concentration as much as possible in consideration of the drying efficiency, but TGase may precipitate as a precipitate by increasing the concentration. In this case, a salt solution with a low concentration is added and dissolved. Can increase the sex.

  For the purpose of increasing the recovery rate, it is preferable to add a substance having a high effect that contributes to stabilization of TGase. For example, a partial protein degradation product such as glutamine peptide and peptino, trehalose, cysteine, glutathione, sodium bisulfite In addition, one or more of citrate and phosphate can be added as an additive to prepare a formulation. In addition, excipients such as saccharides can be added as additives to prepare a formulation. These additives can be used in any partial purification step, but are preferably performed after crystallization.

  EXAMPLES Next, although an Example is given and this invention is demonstrated, this invention is not limited to a following example.

[Example 1] (Examination of salts used for crystallization)
The types of salts that crystallize TGase were examined.
Dissolved in 0.2M Tris-HCl buffer solution pH6.0 for the purpose of removing excipients from commercially available TGase (ActivaTG manufactured by Ajinomoto Co., Inc., a partially purified enzyme preparation of TGase produced by Streptomyces mobaraensis) Thereafter, ammonium sulfate salting out was performed, and the resulting precipitate was dissolved in the same buffer. This solution was desalted and concentrated using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Corporation). By this operation, a partially purified enzyme solution of TGase from which excipients and the like were removed was obtained. The enzyme solution had a TGase activity of 326 u / ml and a specific activity of 8.7 u / Ab 280 nm.

The TGase activity was measured by the method described in JP-A No. 64-27471. Namely, benzyloxycarbonyl-L-glutaminylglycine and hydroxylamine as substrates are reacted in the absence of Ca ²⁺ to form an iron complex in the presence of trichloroacetic acid, and the absorption at 525 nm is measured. The amount of hydroxamic acid is calculated from a calibration curve. Specific description will be given below.
Reagent A: 0.2 M Tris-HCl buffer (pH 6.0), 0.1 M hydroxylamine, 0.01 M reduced glutathione, 0.03 M benzyloxycarbonyl-L-glutaminylglycine
Reagent B: 3N hydrochloric acid, 12% trichloroacetic acid, 5% FeCl ₃ · 6H ₂ O (dissolved in O.1N-HCl)
A 1: 1 mixture of these solutions was designated as reagent B.
Add 0.5 ml of reagent A to 0.05 ml of the enzyme solution, mix and react at 37 ° C. for 10 minutes, add reagent B to stop the reaction and form an Fe complex, and then measure the absorbance at 525 nm. As a control, the absorbance of an enzyme solution that has been reacted in the same manner using a previously heat-inactivated enzyme solution is measured, and the difference in absorbance from the enzyme solution is determined. Separately, a calibration curve is prepared using L-glutamic acid-γ-monohydroxamic acid instead of the enzyme solution, the amount of hydroxamic acid generated from the difference in absorbance is obtained, and 1 μmol hydroxamic acid is produced per minute The activity was defined as 1 unit.
Further, the specific activity was measured by measuring the absorbance of ultraviolet rays at a wavelength of 280 nm and expressed as enzyme activity per absorbance. The measurement of the activity of TGase and the measurement of the specific activity were carried out in the same manner in the examples described later.

  Dispense 5 ml each of the partially purified enzyme solution obtained above into a test tube, add and dissolve various salts as solids to saturate, store at 4 ° C for 1 day, centrifuge, and deposit 0.2M Dissolved in Tris-HCl buffer pH 6.0. After making this solution 7 ml, TGase activity and absorbance at Ab 280 nm were measured to determine the recovery rate and specific activity, and the available salts were examined. The results are shown in Table 1.

  From Table 1, it was confirmed that sodium chloride and potassium chloride had a higher specific activity of the precipitate than the control, and this precipitate was already a crystalline precipitate, so that TGase can be easily and highly crystallized. Became clear. In the case of potassium chloride, the recovery rate from precipitation is as low as 48%, but TGase can be crystallized from the crystal mother liquor with a high recovery rate, so that crystallization can be performed with a high recovery rate as a whole. On the other hand, with ammonium sulfate, sodium / potassium tartrate, and a mixture of monopotassium phosphate and dipotassium phosphate, the specific activity is not increased compared to the control, but the precipitate recovery is high. It seems that it can be used for salting out. Moreover, these salts can also be supplementarily added in salting out using sodium chloride or potassium chloride. For sodium chloride and potassium chloride, crystallization was possible from a partially purified enzyme solution such as this enzyme solution, but for other salts, it was necessary to use the enzyme after complicated operations such as fractional precipitation. It was. Although the results are not shown, crystallization with sodium chloride or potassium chloride can be employed not only in the batch method but also in the hanging drop method useful for the preparation of trace crystals.

Example 2 (TGase crystallization 1)
Using the TGase producing strains Streptomyces mobaraensis S-8112 and Streptomyces lavendulae No.466, soluble starch 2%, sucrose 5%, polypeptone 2%, yeast extract 0.2 %, Magnesium sulfate 0.1%, dipotassium phosphate 0.2%, adecanol 0.05%, put into a 500 ml Sakaguchi flask and shake cultured at 30 ° C. for 4 days to obtain 450 ml and 930 ml of a crude enzyme solution, respectively. . Note that “%” is “% by weight” and is the same in the following examples.

  The above crude enzyme solution was desalted and concentrated using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.), and finally 194 ml and 255 ml concentrated solutions were obtained. The concentrated solution was dialyzed in advance with 50 mM phosphate buffer pH 7.0, passed through an approximately 40 ml Blue Sepharose CL-6B column (manufactured by Amersham Biosciences) equilibrated with the same buffer, and the enzyme was adsorbed. Elution was performed in only one step with the same buffer containing 0.5 M sodium chloride. Enzyme protein was collected as a precipitate as ammonium sulfate saturation and dissolved in about 10 ml of 0.2 M Tris-HCl buffer pH 6.0 to obtain a partially purified enzyme solution. Thereafter, sodium chloride was gradually added to the partially purified enzyme solution to crystallize TGase (crude crystal). The recovery rate was about 10% due to the low enzyme concentration. The specific activity of the partially purified enzyme solution (see each column eluate in Table 2) was 6.2u / Ab280nm and 1.74u / Ab280nm, respectively, but the specific activity of the crude crystals was 13.08u / Ab280nm and 6.86u, respectively. / Ab 280 nm. The results of each step in crystallization are shown in Table 2. The crystal was a needle-like crystal having a length of several tens of μm, as shown in the optical micrograph image of FIG.

Example 3 (TGase crystallization 2)
Using the TGase producing strain Streptomyces mobaraensis S-8112 described in Example 2, soluble starch 4%, Far East peptone 2%, yeast extract 0.2%, meat extract 0.2%, phosphoric acid 1 Cultivation was performed with a 3L mini jar fermenter using a medium containing 0.5% potassium, 0.5% dipotassium phosphate, 0.05% magnesium sulfate and 0.04% adecanol, and 2800 ml of crude enzyme solution was obtained by filtration using diatomaceous earth. .

  Perform primary desalination and concentration with an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) to make the volume 270 ml, then decolorize by adding 2% activated carbon, and remove the activated carbon by filtration using diatomaceous earth to clear enzyme 310 ml of liquid was obtained. At the same time as the concentration, an enzyme solution containing a phosphate buffer solution was subjected to secondary desalting and concentration using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) to obtain a partially purified enzyme solution having a volume of 54 ml. To this partially purified enzyme solution, sodium chloride was gradually added until saturation was reached, and at the same time, a small amount of seed crystal was added for salting out crystallization. Example 2 is the same as that of Example 2, except that the crude enzyme solution produced by Streptomyces mobaraensis S-8112 was partially purified by blue sepharose chromatography and crystallized. The difference is that the crude enzyme produced by Streptomyces mobaraensis strain S-8112 was partially purified by concentration and decolorization treatment with activated carbon, followed by crystallization. The recovery was about 34% for the crystallization step alone and about 18% for the total process yield, including recovery from the crystal mother liquor. The specific activity of the partially purified enzyme solution (see the secondary ultrafiltration membrane treatment solution in Table 3) was 2.03 u / Ab 280 nm, while the crude crystal enzyme had a specific activity of 4.37 u / Ab 280 nm (first time). And calculated from the results of the second crystal solution. The results of each step in crystallization are shown in Table 3.

[Example 4] (crystallization of TGase 3)
Using No. 140226 strain obtained by mutation of Streptomyces mobaraensis S-8112 strain described in Example 2, soluble starch 4%, Far East peptone 2%, yeast extract 0.2%, meat extract 0.2 %, Monopotassium phosphate 0.5%, dipotassium phosphate 0.5%, magnesium sulfate 0.05%, adecanol 0.04%, culture using a 3L mini jar fermenter, and 3800ml of crude enzyme solution by filtration using diatomaceous earth Obtained.

  The above crude enzyme solution is first desalted and concentrated using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) to make the liquid volume 750 ml, then decolorized by adding 2% activated carbon, and the activated carbon is filtered by using diatomaceous earth. Removal of 950 ml of a clear enzyme solution was obtained. In order to obtain an enzyme solution containing a phosphate buffer at the same time as the concentration, secondary desalting and concentration using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) was performed to obtain a partially purified enzyme solution having a liquid volume of 180 ml. Sodium chloride was added to this partially purified enzyme solution until saturation was reached, and a small amount of seed crystal was added, and the mixture was allowed to stand at 4 ° C. for 3 days for salting out crystallization. The second crystallization was performed by adding monopotassium phosphate to the crystal mother liquor and leaving it at room temperature for 7 hours. The recovery rate, including recovery from the crystal mother liquor, was about 68% for the crystallization step alone, and the recovery rate for all steps was about 28%. The specific activity of the partially purified enzyme solution (see the secondary ultrafiltration membrane treatment solution in Table 4) was 2.67 u / Ab 280 nm, while the specific activity of the crude crystal enzyme was 9.03 u / Ab 280 nm (first and second). It was calculated from the result of the first crystal solution. The results of each step in crystallization are shown in Table 4.

Example 5 (TGase crystallization 4)
Using No.W42228 strain obtained by mutation of Streptomyces mobaraensis S-8112 strain described in Example 2, soluble starch 4%, Far East peptone 2%, yeast extract 0.2%, meat extract 0.2 %, 1% potassium phosphate 0.5%, 2 potassium phosphate 0.5%, magnesium sulfate 0.05%, adecanol 0.04% culture medium with a 3L mini jar fermenter, crude enzyme by filtration using diatomaceous earth 3600 ml of liquid was obtained.

  The above crude enzyme solution is first desalted and concentrated with an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) to make the volume 320 ml, then decolorized by adding 2% activated carbon, and the activated carbon is removed by filtration using diatomaceous earth. As a result, 390 ml of a clear enzyme solution was obtained. In order to obtain an enzyme solution containing a phosphate buffer at the same time as the concentration, secondary desalting and concentration using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.) was performed to obtain a partially purified enzyme solution having a liquid volume of 58 ml. Sodium chloride was added to this partially purified enzyme solution until saturation was reached, and a small amount of seed crystal was added, and the mixture was allowed to stand at 4 ° C. for 3 days for salting out crystallization. The second crystallization was performed by leaving the crystal mother liquid obtained in the first crystallization for 7 hours at room temperature. Including the recovery from the crystal mother liquor, the recovery rate was about 48% in the crystallization step alone, and the recovery rate in all steps was about 19%. The specific activity of the partially purified enzyme solution (see the secondary ultrafiltration membrane treatment solution in Table 5) was 2.10 u / Ab 280 nm, while the specific activity of the crude crystal enzyme was 4.75 u / Ab 280 nm (first and second). It was calculated from the result of the first crystal solution. The results of each step in crystallization are shown in Table 5.

Example 6 (TGase crystallization 5)
After 200 g of commercially available TGase (ActivaTG manufactured by Ajinomoto Co., Ltd., partially purified enzyme preparation of TGase produced by Streptomyces mobaraensis) is dissolved in 1000 ml of 0.2M Tris-HCl buffer solution, excipient, etc. Insoluble matters were removed by filtration using diatomaceous earth (filtrate: 1000 ml), and 450 g of ammonium sulfate was added for salting out. After standing at 4 ° C. for 3 hours, the precipitate was collected by centrifugation, dissolved in about 1000 ml of the above buffer, and desalted and concentrated to 500 ml using an ultrafiltration membrane (ACP1010 manufactured by Asahi Kasei Co., Ltd.). Sodium chloride was gradually added to the partially purified enzyme solution for salting out crystallization. The specific activity of the obtained crude crystals was 10.9 u / Ab 280 nm, compared to 8.7 u / Ab 280 nm before crystallization. The specific activity of the product-dissolved filtrate filtered through diatomaceous earth was 7.1 u / Ab 280 nm. The recovery rate was 66% from the partially purified enzyme solution (see the ultrafiltration membrane treatment solution in Table 6) and 60% from the product dissolution filtrate (see the diatomaceous earth filtration treatment solution in Table 6). The results of each step in crystallization are shown in Table 6.

    In Examples 2 to 6 described above, the specific activity of the crystallized TGase is significantly higher than that before crystallization (see Tables 2 to 6). It is clear that is a partially purified enzyme that is not purified to homogeneity.

It is an optical microscope photograph image of the crystal | crystallization of transglutaminase by this invention.

Claims (2)

A crude enzyme comprising a culture filtrate was obtained by culturing a microorganism of Streptomyces mobaraensis or Streptomyces lavendulae, and the ratio of the crude enzyme measured by the enzyme activity per absorbance of ultraviolet rays at a wavelength of 280 nm Industrial production of transglutaminase, characterized in that sodium chloride and / or potassium chloride is added to a partially purified enzyme that is not purified until the activity becomes uniform at least 2u (2u / Ab280nm) and uniform, and salted out by batch method. Crystallization method.   The transglutaminase industrial crystallization method according to claim 1, wherein the crystallized transglutaminase is selected from a protein partial degradation product, trehalose, cysteine, glutathione, sodium bisulfite, citrate and phosphate. Or a method for producing a transglutaminase preparation, comprising one or more of them.

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