JP2006271284A - Method for cryo-preserving phospholipase d and freezing-resistant phospholipase d composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for preserving phospholipase D obtained by isolating and purifying from a microorganism, etc., over a long period without deteriorating the activity thereof. <P>SOLUTION: The phospholipase D is isolated and purified from a culture solution of a bacterium of the genus Streptomyces at pH3-8 by adding a bivalent metal (calcium or magnesium) salt and subjected to a freezing treatment in the presence of L-serine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for cryopreserving phospholipase D that catalyzes various reactions. More specifically, the present invention relates to a novel cryopreservation method and freeze-resistant phospholipase for suppressing reduction or inactivation of enzyme activity of phospholipase D due to freezing treatment. D relates to the composition.

  It is well known that many microorganisms existing in nature possess or produce various useful substances represented by enzymes in order to maintain life activity. Conventionally, the above-mentioned useful substances possessed or produced by these microorganisms have been used in the field of pharmaceuticals and foods. Among them, enzymes are used as raw materials for pharmaceutical materials and functional food materials or for their production. It is used as a catalyst.

  As a typical example of such an enzyme, for example, phospholipase D, which is an enzyme used for producing phospholipids having various physiological effects that have been attracting attention in recent years, can be mentioned.

  This phospholipase D has an enzymatic action to hydrolyze the ester bond between the phosphatidyl group of the phospholipid and the base to liberate phosphatidic acid and the base. Depending on the origin, a compound having a phosphatidyl group and the above alcoholic hydroxyl group at the same time as hydrolyzing an ester bond between the phosphatidyl group and the base of glycerophospholipid in the presence of a compound having an alcoholic hydroxyl group such as glycerol or ethanol. It is well known that it is used as an enzyme for producing phospholipids. As typical phospholipids obtained using this enzyme, phosphatidylglycerol (PG), phosphatidylserine (PS) and the like are well known.

  By the way, this phospholipase D is derived from microorganisms such as Streptomyces, Micromonospora, Norcadiopsis, Actinomadura, and Nocardia in addition to those originating from plants such as cabbage, carrot and peanut. It is known as one of the enzymes produced, and conventionally, it has been reported that it is isolated and purified from the plant or microorganism.

  Such useful substances such as enzymes produced by plants and microorganisms are isolated and purified from cells or cultures obtained by crushing the plants or by culturing the microorganisms. However, in any case, the process generally requires a process of efficiently removing substances (impurities) other than the useful substance such as the target enzyme. Usually, the means used to remove impurities is cell separation, ultraconcentration, microfiltration, salting out, organic solvent fractionation, dialysis, gel filtration, adsorption chromatography, ion exchange chromatography, etc. Are known.

  Among them, the microfiltration means is often used as a suitable means for purification because impurities can be easily and efficiently removed. The microfiltration means is roughly classified into two types depending on the type of filter used. Specifically, a type that captures foreign matter on the filter membrane surface, and another type that captures foreign matter inside the filter membrane, are used depending on the purpose.

  However, there are cases where impurities cannot be sufficiently removed even if different types of microfiltration means as described above are used. In particular, in order to isolate and purify a target substance from a culture obtained by culturing microorganisms, an operation of removing impurities by combining a plurality of means is required, resulting in a decrease in work efficiency. In addition to affecting the yield and yield of useful substances such as enzymes derived from microorganisms, there is a problem that the activity or functionality of useful substances such as enzymes is reduced. There was also.

  Therefore, in order to effectively use useful substances such as microorganism-derived enzymes for the production of pharmaceuticals and food materials, a method has been proposed in which the microbial cell itself is used as a catalyst and the impurity removal process is simplified. .

Japanese Patent Publication No. 5-42917 Japanese Patent Publication No. 5-60357 JP 2002-218991 A

  Among various useful substances produced by microorganisms, the present inventors can easily isolate and purify phospholipase D from the microorganism, and efficiently use the obtained enzyme for various applications. The means were examined. In the examination, in isolating and purifying the phospholipase D derived from the microorganism, a gel-like impurity is generated in the culture obtained by culturing the microorganism, and the state in which the effectiveness of the isolated phospholipase D is maintained. And when it preserve | saved for a long period of time, it acquired the knowledge that there exists a tendency for the contamination by various bacteria to arise or an enzyme activity to fall.

  In general, two methods are known as methods employed when preserving enzymes isolated and purified from microorganisms. One is a storage method in a sterile liquid state that prevents contamination by various bacteria by increasing the osmotic pressure by using an excessive amount of sugars such as glycerol, sucrose, and glucose, and the other is freezing. It is a method of performing processing and storing in an individual state. Even in the case of the freezing process, the above-mentioned carbohydrate is often used for the purpose of preventing damage to the storage object during the freezing process.

  However, since enzymes isolated and purified from microorganisms may catalyze various reactions, it is not always preferable to store them using carbohydrates. In particular, phospholipase D has an alcoholic hydroxyl group. Since it is an enzyme used as a substrate for the reaction, it has not been a preferable method to use a compound such as a sugar as a protective agent during storage.

  Furthermore, normally, the protective agent added at the time of storage must be used in an excessive amount in order to sufficiently obtain its effect. Therefore, when applying the above storage method on an industrially large scale, the cost, etc. In some cases, there was a problem with the economy.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have used L-serine as a protective agent when freezing and storing phospholipase D from microorganisms, It has been found that contamination with various bacteria can be significantly prevented without impairing the activity of the enzyme.

  In particular, the present inventors can easily remove the gel-like impurities generated in the culture of the microorganism by adding a divalent metal salt at any stage of the process of isolating and purifying phospholipase D from the microorganism. The microorganism-derived phospholipase D obtained through these steps is likely to cause a decrease in enzyme activity due to storage, and is subjected to a freezing treatment using L-serine as a protective agent. As a result, the inventors have found that it is possible to effectively suppress a decrease in the enzyme activity and completed the present invention.

  That is, the present invention is a method for cryopreserving phospholipase D, which comprises freezing phospholipase D in the presence of L-serine.

  The present invention is also a method for cryopreserving the above phospholipase D, wherein the phospholipase D is obtained by a production method comprising a step of adding a divalent metal salt at an arbitrary stage.

  Furthermore, the present invention is a freeze-resistant phospholipase D composition comprising phospholipase D and L-serine.

  According to the present invention, phospholipase D obtained by isolation and purification from microorganisms can be stored for a long time without impairing its activity, and when various reactions on a large industrial scale are performed. Phospholipase D compositions that are effective as catalysts can be provided. In particular, the present invention is very effective as a method for preserving phospholipase D used in reactions in which L-serine serves as a reaction substrate.

  The present invention is a method for cryopreserving phospholipase D, comprising freezing phospholipase D in the presence of L-serine. More specifically, after adding L-serine to various phospholipases D isolated and purified from microorganisms, this can be frozen and preserved for a long period of time while maintaining the phospholipase D enzyme activity. It is what.

  In the present invention, L-serine added at the time of cryopreservation of phospholipase D is a compound well known as one of amino acids and is widely available on the market. In the present invention, any commercially available L-serine can be suitably used.

  The method for adding L-serine into phospholipase D is not particularly limited, and commercially available L-serine may be added as it is during the freezing treatment, but when L-serine is in a powder form or the like. Is preferably added after being dissolved in an appropriate aqueous solvent in order to disperse uniformly. The L-serine may be added before the freezing treatment, and may be added several hours or days before the freezing treatment or immediately before the freezing treatment. .

  The amount of L-serine added in the present invention is not particularly limited, but is about 0.01% by mass to 35% by mass with respect to the amount of phospholipase D to be subjected to freezing treatment, and more preferably 0.0%. What is necessary is just to add so that it may become about 1 mass%-about 20 mass%, More preferably about 0.1 mass-10 mass%.

  When the amount of L-serine added is less than 0.01% by mass with respect to the amount of phospholipase D to be frozen, the desired effect cannot be sufficiently obtained, and the enzyme can be used in various reactions. This is not preferable because it may become unsuitable for use as a catalyst. On the contrary, when the amount of L-serine added is more than 35% by mass with respect to the amount of phospholipase D to be frozen, it is not preferable because L-serine is difficult to uniformly disperse. Even if L-serine is added in an amount of 35% by mass or more, a remarkable effect is not recognized.

  The method for freezing phospholipase D and L-serine may be carried out in accordance with the conditions and methods used for ordinary cryopreservation, and is not particularly limited. For example, after adding a predetermined amount of L-serine to phospholipase D, it may be frozen using a refrigerator or a freeze freeze dryer set at -20 to -80 ° C.

  On the other hand, the phospholipase D preserved by the method of the present invention is possessed or produced by animals and plants that exist in nature, and in particular, is isolated and purified from microorganisms. There is no particular limitation as long as it can be used in production.

  Preferable phospholipase D used in the present invention is phospholipase D isolated and purified from a microorganism. This can be obtained by culturing a microorganism producing phospholipase D using a desired medium according to a conventional method, and isolating and purifying the desired phospholipase D from the obtained culture.

  Here, the medium for culturing the microorganism producing phospholipase D is not particularly limited, as long as it is configured by appropriately combining necessary nutrients depending on the type of microorganism to be cultured. . Specific nutrients constituting this medium include carbon sources such as glucose, fructose, sucrose, lactose, molasses, starch, dextrin, glycerin, fatty acids, fats and oils, lecithin, alcohol, etc., ammonium sulfate, ammonium nitrate, Nitrogen sources such as ammonium chloride, urea, sodium nitrate, peptone, meat extract, yeast extract, corn steep liquor, casamino acid, defatted soy flour, soy protein, destilers solubles and the like can be mentioned.

  In addition, the medium contains salt, potassium chloride, phosphate, magnesium salt, calcium salt, potassium salt, iron salt, manganese salt, various vitamins, and other microorganisms to promote the growth of microorganisms and the production of the desired phospholipase D. It is also possible to add an effective substance as needed. In addition, what is necessary is just to adjust and use the culture medium prepared in this way to pH suitable for the microorganisms to culture, if the pH is normally adjusted to about 5-8, Preferably it is about 6-7. Good.

  In addition, microorganisms may be cultured according to a conventional method, and specifically, a deep culture method or a solid culture method may be employed. The culture temperature at this time may be appropriately set according to the microorganism, and is usually 20 to 40 ° C, preferably about 25 to 35 ° C. In addition, the culture time may vary depending on the type of microorganism to be cultured and the composition of the medium to be used. Therefore, it is preferable to carry out by appropriately setting appropriate conditions. For example, the production amount of phospholipase D produced by the microorganism is The maximum stage, usually about 1 to 6 days, is sufficient.

  In order to isolate and purify the desired phospholipase D from the culture of microorganisms obtained as described above, after removing the microbial cells from the culture, ultrafiltration, concentration under reduced pressure, separation, organic What is necessary is just to perform using existing means, such as precipitation processing with a solvent, dialysis, gel filtration, adsorption chromatography, and ion exchange chromatography.

  The phospholipase D produced by the microorganism can be obtained by isolation and purification as described above. However, in the culture obtained by culturing the microorganism, substances other than the target phospholipase D are present as gel-like impurities. Since it may be contained, it can be efficiently removed in the isolation / purification process, thereby improving the efficiency of the process and dramatically improving the yield and yield of the target phospholipase D. it can.

  In order to remove the gel-like impurities generated in the culture of the microorganism, it is possible to easily carry out by adding a divalent metal salt to the culture obtained by culturing the microorganism at any stage. . Thereby, since the gel-like substance in the culture is separated as a precipitate, impurities can be easily removed by simple means such as filtration and centrifugation, and phospholipase D can be obtained in a purified state. It is particularly advantageous to apply the method of the present invention to phospholipase D in such a state.

  The divalent metal salt added at any stage of the process of isolating and purifying the phospholipase D derived from microorganisms can be used without particular limitation. Specific examples include salts of calcium, magnesium, iron, zinc, manganese, copper and the like, for example, hydrochloride, nitrate, sulfate, acetate, chloride, sulfide, hydroxide, etc. These may be used alone or in combination of two or more. These may be used as they are, but in the case of powders or solids, it is preferable to use them dissolved in an appropriate aqueous medium so that they are uniformly dispersed in the culture.

  The amount of the divalent metal salt used is not particularly limited, but the concentration of the metal salt in the culture obtained by culturing the microorganism is 5 mM to 1 M, preferably 10 mM to 100 mM. What is necessary is just to add so that it may become. At this time, if the addition amount of the divalent metal salt is less than 5 mM, it is not preferable because the effect of sufficiently removing gel-like impurities generated in the culture obtained by culturing the microorganism cannot be obtained. If the addition amount exceeds 1M, the excessively added metal salt becomes an impurity, and it is necessary to take a separate means for removing this, which may lead to a decrease in work efficiency. It is not preferable. Furthermore, since excessive addition of a divalent metal salt may cause damage to the activity of the target phospholipase D, it is not preferable to use more than 1 M from this.

  Moreover, the addition time of the above-mentioned divalent metal salt may be any stage of the process of isolation / purification from the microbial culture. For example, the culture obtained by culturing or the microbial fungus from the culture What is necessary is just to add after removing a body.

  Furthermore, when adding a divalent metal salt to a culture, it is preferable to adjust the pH of the culture to a specific range. Thereby, the effect which removes the gel-like impurity which arises by culture | cultivation becomes remarkable. The specific pH range is about 3 to 8, more preferably about 4.5 to 6.5.

  Depending on the type of microorganisms to be cultivated, acidic substances and basic substances may be produced during cultivation, so the pH of the culture may be out of the above range. What is necessary is just to adjust the pH of a culture to a suitable range using the existing pH adjuster. Further, the lower the pH of the culture, the more effective the removal of impurities generated in the culture when the divalent metal salt is added. However, when the pH is too low, the activity of the target phospholipase D is simple. It is not preferable because it is lowered or deactivated in the separation / purification stage.

  EXAMPLES Hereinafter, although an Example, a test example, and a reference example are given and this invention is demonstrated further in detail, this invention is not restrict | limited at all.

Example 1
Acquisition of phospholipase D by microbial culture:
(A) Preparation of seed (1) Into a 500 ml Sakaguchi flask, 100 ml of the yeast extract medium shown in Table 1 was taken, and 2 ml of a cryopreserved bacterial solution of Streptomyces Prunicolor (FERM BP-1365) was inoculated into this. The culture was carried out at 28 ° C. with a shaking speed of 120 spm for 24 hours to obtain a culture solution.

なお、酵母エキス培地は、１２１℃で１５分間オートクレーブ滅菌して使用した。 Composition of yeast extract medium;

The yeast extract medium was used after autoclaving at 121 ° C. for 15 minutes.

  (2) Into a 500 ml Sakaguchi flask, take 100 ml of the soybean powder medium shown in Table 2, inoculate 0.3 ml of the culture solution obtained in (1), and incubate for 24 hours at 28 ° C. with a shaking speed of 120 spm. I got a seed.

なお、大豆粉培地は、水酸化ナトリウムでｐＨを６.８に調整し、１２１℃で１５分間オートクレーブ滅菌して使用した。 Composition of soy flour medium;

The soy flour medium was used after adjusting the pH to 6.8 with sodium hydroxide and autoclaving at 121 ° C. for 15 minutes.

(B) Isolation / Purification of Phospholipase D (1) Into a 3 L jar, 1.5 L of yeast extract medium shown in Table 1 was added, and 30 ml of the seed prepared in (A) (2) was inoculated at 28 ° C The culture was performed at a stirring speed of 500 rpm and an aeration amount of 0.5 vvm until the production amount of phospholipase D was maximized. After removing the cells from the obtained culture by centrifugation and concentrating with an ultra-concentrator, impurities are removed by adding a calcium chloride solution to a calcium concentration of 40 mM to remove the phospholipase D solution. Got.

  After adding L-serine, glucose, sucrose, and trehalose as protective agents to these solutions to 0.5%, 1%, 1.5%, 2%, and 2.5%, respectively. These were frozen at −20 ° C., and after 1 week, they were thawed, and the enzyme activity of phospholipase D was measured. The result is shown in FIG. The enzyme activity of phospholipase D was measured by the following method.

<Measurement of production amount and enzyme activity of phospholipase D>
To a test tube containing 16.7 μL of a measurement sample diluted to an appropriate concentration with an enzyme solution ^{* 1} , 33.3 μL of a substrate solution ^{* 2} containing phosphatidylcholine was added and reacted at 37 ° C. for exactly 10 minutes. After the reaction, the enzyme is inactivated by heating with a block heater preheated to 130 ° C. for 10 minutes, and then 1.5 ml of coloring solution ^{* 3} is added and reacted at 37 ° C. for 20 minutes. The absorbance was measured. At the same time, a coloring solution was added to and reacted with a known amount of choline chloride, the absorbance at 500 nm of the obtained solution was measured to prepare a calibration curve, and the hydrolysis activity of the measurement sample was measured therefrom. In addition, it subtracted from the value of the light absorbency of a measurement sample by making the light absorbency of what measured the sample before adding a substrate solution beforehand at 130 degreeC and heat-inactivated for 10 minutes as a blank. The hydrolysis activity was determined by defining 1 unit as the activity that liberates 1 μmol of choline per minute.

* 1 Enzyme solution: 100 mM sodium acetate buffer containing 0.5% Triton-X100
(PH 6.0)
* 2 Substrate solution: 1.5% phosphatidylcholine, 13.5% 2-propanol,
Enzyme solution containing 0.6% Triton-X100 * 3 Coloring solution; 0.74 mg of calcium chloride (anhydrous) per 1.5 ml, 4-amino
Antipyrine 0.3mg, phenol 0.47mg, cholineoxy
1.5 units of oxidase, 30 units of peroxidase and Triton-X100
50 mM Tris-HCl buffer (pH 8.0) containing 1.5 mg of

  As shown in FIG. 1, the effect of suppressing the decrease in enzyme activity after freezing and thawing was observed by freezing phospholipase D isolated from a microorganism in the presence of L-serine. In addition, general cryoprotectants (glucose, sucrose, and trehalose) have a remarkable effect when added in large amounts, whereas L-serine has an excellent effect at a lower concentration. It was.

Example 2
Relationship between added concentration of L-serine and cryopreservation stability of phospholipase D;
In the same manner as in Example 1, microorganisms ( Streptomyces prunicolor ) were cultured, phospholipase D was isolated and purified, 0 to 10% by mass of L-serine was added, and the mixture was frozen. At this time, phospholipase D was isolated and purified by adding calcium chloride to the concentrated solution to 20 mM. After melting these, the enzyme activity of the enzyme was examined. The result is shown in FIG.

  The addition of L-serine during the freezing treatment was about 0.1% by mass, and it was confirmed that there was an effect of significantly suppressing the decrease in the enzyme activity of phospholipase D after sufficiently freezing and thawing. Moreover, the tendency for the effect of suppressing the decrease in enzyme activity to decrease due to an increase in the concentration of L-serine added is that the effect of diluting the enzyme solution due to the increase in the amount added cannot be ignored. it is conceivable that.

Test example 1
According to the same method as in Example 1, microorganisms ( Streptomyces prunicolor ) were cultured and phospholipase D was isolated and purified. At this time, calcium chloride was added to the concentrate so as to be in the range of 0 to 40 mM, and phospholipase D was isolated and purified. The obtained phospholipase D solution was frozen, thawed after one week, and the enzyme activity of phospholipase D was measured. The result is shown in FIG. In addition, the enzyme activity was similarly examined when the freezing treatment was not performed.

  As is apparent from the results, the enzyme activity of phospholipase D after freezing and thawing tended to decrease depending on the calcium concentration added in the isolation / purification step. Moreover, even when a calcium salt was added, it was shown that the enzyme activity was not impaired when the freezing treatment was not performed.

Reference example 1
(A) culture of Streptomyces bacteria and concentration of the culture solution;
(1) Into a 500 ml Sakaguchi flask, 100 ml of the yeast extract medium shown in Table 3 was taken, and this was inoculated with 2 ml of a cryopreserved bacterial solution of Streptomyces prunicolor (FERM BP-1365) at 28 ° C., 120 spm. Culturing was carried out at a shaking speed of 24 hours to obtain a culture solution.

Composition of yeast extract medium;

  (2) Take 15 L of soybean flour medium 15 L shown in Table 4 in a 30 L jar, inoculate about 50 ml of the culture solution obtained in (1) above, with a stirring speed of 28 ° C., 150 rpm, and an aeration rate of 1.0 vvm, Culture was performed for 24 hours.

なお、大豆粉培地は、水酸化ナトリウムでｐＨを６.８に調整したのちに滅菌処理を行った。 Composition of soy flour medium;

The soybean flour medium was sterilized after adjusting the pH to 6.8 with sodium hydroxide.

  (3) Into a 200 L jar, 180 L of the yeast extract medium shown in Table 3 was placed, and 30 ml of the seed prepared in (2) above was inoculated, and the mixture was stirred at 28 ° C., 200 rpm stirring speed, 0.5 vvm aeration rate, 24 Time culture was performed. The cells were removed from the obtained culture solution by centrifugation, and the concentrated solution after cell separation was concentrated using an ultraconcentrator. Furthermore, 5 times the volume of RO water with respect to the concentrate was added to the concentrate, and the concentration operation was performed again to remove low-molecular substances. After the batch water addition, the concentration operation was repeated three times to obtain a sample.

(B) Effect of removal of gel-like substances in Streptomyces bacteria culture by calcium chloride:
To the concentrated sample obtained in (A) (3) above, 1M calcium chloride solution is added to a final concentration of 10, 50, 100, 250, and 500 mM, immediately after addition, 1.5 hours, 3 hours, Centrifugation was performed after standing overnight (about 18 hours), and the turbidity (0. 660 nm) and PLD activity of the supernatant were measured. Each sample was prepared to finally have a liquid volume of 5 ml, and the PLD activity yield was determined using as a control the same amount of water added as the 1 M calcium chloride solution. Centrifugation was performed at 15,000 × g for 8 minutes. The results are shown in FIG.

  The OD 660 nm of the concentrated sample before the centrifugation operation was 1.28, whereas the one with calcium chloride added was significantly more than the one with the same amount of water added. It can be seen that the turbidity of the centrifugal supernatant is reduced. That is, this indicates that the gel-like substance can be removed by adding calcium chloride.

  In addition, in the case of adding high concentration calcium chloride, the turbidity of the centrifugal supernatant is not different from that in which water was added in the centrifugation operation immediately after the addition, but after 1.5 hours the water was added. In comparison, the centrifugal supernatant turbidity decreased. The precipitate remaining at the bottom of the centrifuge tube was in a gel state and was in a clay-like state. In addition, when the activity of phospholipase D in the concentrate sample was evaluated, it showed an activity of 95% or more under any condition with respect to the activity of the concentrate sample before the centrifugation operation. It was confirmed that the activity of phospholipase D therein was not impaired.

Reference example 2
Detailed examination of calcium chloride addition concentration;
1M calcium chloride solution was added to the concentrated sample obtained in the same manner as in Reference Example 1 except for the concentration ratio, so that the final concentration was 0, 5, 10, 20, 40, 100, and 200 mM. Centrifugation was performed on the supernatant, and the turbidity (OD 660 nm) of the supernatant was measured. Centrifugation was performed under conditions of 3000 × g and 2.5 minutes. The results are shown in FIG.

  The OD 660 nm of the concentrated solution sample before the centrifugation operation was 4.6, whereas calcium chloride was added so that the concentration was 10 to 100 mM. It can be seen that the turbidity of the centrifugal supernatant is significantly reduced compared to 0 mM which was not present. Since the turbidity of the centrifugal supernatant was higher than that before centrifugation at an addition concentration of 200 mM, it was confirmed that there was an optimum range for the addition concentration of calcium chloride when centrifugation was performed immediately after the addition.

Reference example 3
Comparison with other metal salts;
In order to evaluate the influence of metal salts other than calcium chloride, magnesium chloride, sodium chloride and potassium chloride were used in addition to calcium chloride, and the influence on the clarification of the centrifugal supernatant was evaluated.

  Calcium chloride and magnesium chloride were added to a concentrated sample obtained by the same method as in Test Example 2 so as to have final concentrations of 20 mM and 40 mM. In addition, 40 mM sodium chloride and potassium chloride were added so that the number of moles was the same as the amount of 40 mM calcium chloride, 80 mM sodium chloride and the same amount of chlorine contained in the concentration of 40 mM calcium chloride, and What added potassium chloride was prepared. All of these were centrifuged immediately after the addition of the metal salt, and the turbidity (OD 660 nm) of the supernatant was measured. Centrifugation was performed under conditions of 3000 × g and 2.5 minutes. The results are shown in FIG.

  As shown in the above results, it was found that the same effect as calcium chloride can be obtained when magnesium chloride, which is a divalent metal salt, is added similarly to calcium chloride. Moreover, since the effect was not seen by the addition of 80 mM sodium chloride and potassium chloride with the same amount of chlorine, this effect was not due to the action of chlorine, and at the same time, the effect was seen with a monovalent metal salt. Not shown.

Reference example 4
Influence of the pH of the culture medium at the time of addition on the effect;
The pH of the concentrated sample obtained by the same method as in Test Example 2 was adjusted to an arbitrary pH of 4.5 to 6.5, and calcium chloride was added to a final concentration of 20 mM. Moreover, only pH was adjusted and the thing which does not add calcium chloride was also prepared. All of these were centrifuged immediately after the addition of the metal salt, and the turbidity (OD 660 nm) of the supernatant was measured. Centrifugation was performed under conditions of 3000 × g and 2.5 minutes. The results are shown in FIG.

  From this result, it was confirmed that the effect of calcium chloride on clarification of the centrifugal supernatant was stronger as the pH was lower. In addition, it was found that clarification can be achieved by adjusting the pH to 5 or less even when calcium chloride is not added. However, at pH 5 or less, a decrease in phospholipase D activity in the centrifugal supernatant was observed and it was inactivated. It was suggested. From this, it was found that when obtaining a useful substance whose activity such as an enzyme is affected by pH, an effect equal to or higher than that when the pH is extremely lowered by adding calcium chloride was obtained.

Example 3
Relationship between addition concentration of metal salt and cryoprotective action of L-serine;
In the same manner as in Example 1, microorganisms ( Streptomyces prunicolor ) were cultured, phospholipase D was isolated and purified, and added or 1% by mass of L-serine was added and frozen. At this time, phospholipase D was isolated and purified by adding calcium chloride to the concentrated solution so as to be 10 to 100 mM. After melting these, the enzyme activity of the enzyme was examined. The result is shown in FIG.

  As shown in this result, the activity after thawing of phospholipase D, which was frozen without adding L-serine, decreased depending on the calcium chloride concentration, but 1% by mass of L-serine was added. In the case of freezing treatment, a decrease in enzyme activity was suppressed. Particularly for calcium chloride concentrations up to 40 mM, the decrease in enzyme activity was completely suppressed.

  According to the method of the present invention, phospholipase D, specifically, phospholipase D isolated and purified from a microorganism can be stored for a long period of time without damaging the enzyme activity by cryopreservation. It is possible to provide a phospholipase D composition that is effective as a catalyst for carrying out various reactions with a large scale.

  In particular, the method of the present invention is suitable for a method for preserving phospholipase D obtained by a production method comprising a step involving a divalent metal salt in order to remove impurities in the step of isolation and purification from microorganisms. Furthermore, the method of the present invention is very effective as a method for preserving phospholipase D used in a reaction using L-serine as a substrate, and can be advantageously used in the production of pharmaceuticals and food materials.

It is drawing which shows the cryopreservation stability of phospholipase D by various cryoprotectants. It is drawing which shows the relationship between the addition density | concentration of L-serine and the cryopreservation stability of phospholipase D. It is drawing which shows the influence which the addition amount of calcium salt and the presence or absence of a freezing process have on phospholipase D activity. It is drawing which shows the removal effect of the gel-like substance in the Streptomyces cell culture solution by calcium chloride. It is drawing which shows the relationship between the turbidity of a centrifugation supernatant, and a calcium chloride density | concentration. It is drawing which shows the influence which it has on clarification of various metal salts. It is drawing which shows the relationship between the culture pH at the time of calcium salt addition, and centrifugal supernatant turbidity. It is drawing which shows the relationship between the calcium salt addition density | concentration and the cryoprotective effect | action of L-serine. more than

Claims (14)

  A method for cryopreserving phospholipase D, comprising freezing phospholipase D in the presence of L-serine.   2. The method for cryopreserving phospholipase D according to claim 1, wherein L-serine is added in an amount of 0.01 to 35% by mass based on the amount of phospholipase D.   The method for cryopreserving phospholipase D according to claim 1 or 2, wherein the phospholipase D is obtained by a production method comprising a step of adding a divalent metal salt at an arbitrary stage.   The method for cryopreserving phospholipase D according to claim 3, wherein the divalent metal salt added at any stage during the production of phospholipase D is a calcium salt or a magnesium salt.   The method for cryopreserving phospholipase D according to claim 3 or 4, wherein the divalent metal salt is added within a pH range of 3 to 8.   The method for cryopreserving phospholipase D according to any one of claims 1 to 5, wherein the phospholipase D is obtained from a microorganism.   The method for cryopreserving phospholipase D according to claim 6, wherein the microorganism is a Streptomyces bacterium.   A freeze-resistant phospholipase D composition comprising phospholipase D and L-serine.   The freezing-resistant phospholipase D composition according to claim 8, wherein L-serine is contained in an amount of 0.01 to 35 mass% with respect to phospholipase D.   The freeze-resistant phospholipase D composition according to claim 8 or 9, wherein the phospholipase D is obtained by a production method comprising a step of adding a divalent metal salt at an arbitrary stage.   The freeze-resistant phospholipase D composition according to claim 10, wherein the divalent metal salt added at an arbitrary stage during the production of phospholipase D is a calcium salt or a magnesium salt.   The freeze-resistant phospholipase D composition according to claim 10 or 11, wherein the divalent metal salt is added within a pH range of 3 to 8.   The freeze-resistant phospholipase D composition according to any one of claims 8 to 12, wherein the phospholipase D is obtained from a microorganism. The freeze-resistant phospholipase D composition according to claim 13, wherein the microorganism is a Streptomyces bacterium.

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