JP4761430B2 - Production method of high molecular weight poly-γ-glutamic acid by Bacillus natto - Google Patents

Description

  The present invention relates to a method for producing poly-γ-glutamic acid (hereinafter referred to as γ-PGA), and more particularly to a method for producing high molecular weight poly-γ-glutamic acid by Bacillus natto.

  As described in Non-Patent Document 1, it is well known that Bacillus bacteria produce γ-PGA from glucose or citric acid.

  However, γ-PGA production by Bacillus subtilis var. Natto, which has a long dietary experience and is highly biosafe, is stable in solid culture but extremely unstable in liquid culture, which is essential for mass production. It is.

  This is due to the fact that the amount of γ-PGA accumulated in the medium is not constant in liquid culture, and γ-PGA may be hardly recovered even under the same culture conditions.

  This instability increases at an accelerated rate when the culture scale is increased, and the proportion of almost no viscosity observed at a culture scale of 50 liters or more may exceed 90%. It is extremely difficult to stably obtain. Thus, instability in liquid culture in Bacillus natto means that γ-PGA that is not uneasy in terms of safety to the human body cannot be mass-produced.

  Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis and Bacillus megaterium (Bacillus megaterium) other than natto bacteria can produce and accumulate γ-PGA relatively easily. Patent Document 1 proposes a method of additionally supplying saccharides using a glycine.

  However, this method aims to supply insufficient nutrients while preventing the formation of polysaccharides as impurities, and to improve the yield of γ-PGA. The concentration of saccharides is 2 to 3 g / l or less. The fed-batch culture is performed so as to maintain the temperature.

  However, maintaining the sugar concentration at a desirable level is effective for Bacillus bacteria that are in a state of accumulating γ-PGA, but is not effective for Bacillus natto that has fallen into the state of not accumulating γ-PGA in the medium. Does not show any effect.

  The average molecular weight of γ-PGA produced by Bacillus bacteria is several thousand to 2 million as described in Patent Documents 2 to 5, and the average molecular weight of γ-PGA produced by Bacillus natto in solid culture. This is less than 3 million.

  In addition, these Bacillus bacteria are not sufficiently examined for biological safety, and there is anxiety in terms of their effects on the human body.

  The gene coding for the production of γ-PGA is pgsBCA, which encodes γ-PGA degrading enzyme YwtD that cuts the bond between D-glutamic acid and L-glutamic acid and fragments it to a molecular weight of 100,000 to 500,000 immediately below it. It is reported in Non-Patent Document 2 and Patent Document 6 that a gene, ywtD exists.

  In addition to YwtD, Patent Document 7 describes the presence of an enzyme that cleaves into fragments with a molecular weight of 100,000, polyglutamate hydrase (hereinafter referred to as PghA), and Non-Patent Documents 3, 4 and Patent Documents. 8 and 9 are also reported to have an enzyme that sequentially cleaves glutamic acid from the end, γ-glutamyltranspeptidase (hereinafter referred to as GGT).

  In addition, γ-PGA is considered to be produced for the purpose of accumulating nutrient sources for future starvation and protecting the cells themselves from external enemies such as amoeba. In solid culture, it is recognized that some receptors present on the cell surface are exposed to the air, and the production of the above-described γ-PGA degrading enzymes such as YwtD, PghA and GGT is suppressed, A considerable amount of γ-PGA is accumulated.

  On the other hand, in the liquid culture, since the surface of the bacterial cell is not exposed to the air, the production of γ-PGA degrading enzymes is hardly suppressed, and as a result, the accumulation of γ-PGA is considered to be unstable. .

  In order to deal with this, Patent Documents 10 and 11 listed below propose to delete genes related to the γ-PGA degrading enzyme group.

However, culturing large amounts of genetically modified organisms is difficult to implement because they are restricted by the guidelines set by each ministry and agency, and there is also a problem in terms of the image given to consumers.
Korean Patent No. 363434 (PCT / KR00 / 00761) Japanese Patent Publication No.43-24472 Japanese Patent Laid-Open No. 1-174397 JP-A-3-47087 Japanese Patent No. 3081901 JP 2003-235666 A JP 2003-230384 A JP 2003-230384 A JP 2003-235666 A JP 2003-230384 A JP 2003-235666 A F. B. Oppermann-Sanio and A.M. Steinbuchel, Naturewissenschaften, Vol. 89, pp. 11-22 (2002) T.A. Suzuki and Y.J. Tahara, J .; Bacteriol. , Vol. 185, no. 7, pp. 2379-2382 (2003) Y. Ogawa, H .; Hosoyama, M .; Hamano and H.M. Motai, Agri. Biol. Chem. , 55, pp. 2971-2977 (1991) K. Xu and M.M. A. Strauch, J.M. Bacteriol. 178, pp. 4319-4322 (1996) T.A. Nagai, L .; S. Phan Tran, Y.M. Inatsu and Y. Itoh, J. et al. Bacteriology, 182, pp. 2387-2392 (2000) T.A. Nagai, L .; -S. Phan Tran, Y.M. Inatsu, and Y.J. Itoh, J. et al. Bacteriol. , 182, pp. 2387-2392 (2000) Natto Test Method Study Group, Natto Test Method, pp. 65-73, Korin (1990) A. Goto and M.M. Kunioka, Biosci. Biotech. Biochem. , 56, pp. 1031-1035 (1992) M.M. Kambourova, M .; Tangney and F.M. G. Priest, Appl. and Environ. Microbiol. , Vol. 67, no. 2, pp. 1004-1007 (2001)

  The problem to be solved by the present invention is to use Bacillus natto in which the accumulation of γ-PGA in a liquid medium is very unstable due to the fact that the amount of γ-PGA accumulated in the medium is not constant. The present invention proposes a method for stably producing high molecular weight γ-PGA.

  More specifically, a factor that makes it difficult to produce γ-PGA by Bacillus natto using a liquid medium is a group of γ-PGA degrading enzymes such as YwtD, PghA, and GGT. When the degrading enzyme group is generated, γ-PGA produced by Bacillus natto is immediately decomposed and is not accumulated in the medium. This invention provides a technique for suppressing the production of γ-PGA degrading enzymes produced by Bacillus natto, regardless of genetic manipulation.

The present invention focuses on the fact that the production stability of γ-PGA is impaired by the production of γ-PGA degrading enzyme group, and that stable production is possible if the production of γ-PGA degrading enzyme group can be suppressed. The present inventors have found that the generation of γ-PGA degrading enzyme group can be effectively suppressed by applying external stress to, and have been completed based on this finding.
That is, the invention of claim 1 is a method for stably producing high molecular weight poly-γ-glutamic acid by culturing Bacillus natto used for the production of natto.
Sodium glutamate monohydrate is 1.0 to 5.0 (mass / volume)% (hereinafter% is (mass / volume)%), glucose is 1.0 to 5.0%, potassium dihydrogen phosphate 0.25%, disodium hydrogenphosphate dodecahydrate 0.17%, sodium chloride 0.05%, magnesium sulfate heptahydrate 0.05%, biotin 0.1 ppm The Bacillus natto is cultured in a liquid medium, and when the dissolved oxygen concentration starts to decrease rapidly from the middle to the later stage of the logarithmic growth phase, the temperature of the culture solution is changed from the range of 40 ° C to 50 ° C to less than 40 ° C. It is characterized by that.
The invention of claim 2 is characterized in that, in the invention of claim 1 , after changing the culture solution temperature to less than 40 ° C., glucose is fed at the time when glucose depletion is observed due to pH increase.

  The Bacillus natto targeted by the present invention refers to those used for the production of commercial natto. In the production of commercially available natto, a strain that requires biotin for production of γ-PGA and is classified into Bacillus subtilis is used. Representative natto bacteria used in the production of natto are Miyagino, Takahashi, and Naruse, but it has been confirmed by molecular genetic techniques that they are derived from a common strain (Non-patent Document 5). ).

  Any of these strains may be used for the production of γ-PGA, and Bacillus subtilis var. Arbitrary strains such as IFO3009, IFO3013, IFO3335, IFO3336, IFO3936, IFO13169, ATCC7058, ATCC7059, and ATCC15245 registered as “natto” can also be used.

  However, as described in Non-Patent Document 6, natto bacteria are genetically unstable, and in order to stabilize production, preferably a highly viscous expression strain selected from commercial natto is used after being expanded and used. It is desirable. This highly viscous expression strain can be obtained by a known method described in Non-Patent Document 7.

  The external stress referred to in the present invention refers to various external factors that affect the production of γ-PGA degrading enzymes such as nutrient depletion in the medium, temperature fluctuation, presence / absence of oxygen supply, and pH fluctuation. The items are not limited to the listed items. These external stresses may be applied alone or in combination.

  The mechanism for inhibiting the production of γ-PGA degrading enzymes by external stress is similar regardless of the type of external stress.

  For example, as one of the external stresses, the mechanism for suppressing the production of γ-PGA degrading enzymes for the depletion of nutrients in the medium is considered as follows.

  That is, as described in Non-Patent Document 8, γ-PGA produced by Bacillus natto is not derived from glutamic acid present in the medium, but a sugar component such as glucose or an organic acid such as citric acid as a carbon source. After being synthesized. In Natto, glutamic acid is known to be necessary for efficient expression of the γ-PGA synthesis pathway, but as described in Non-Patent Document 9, it is hardly consumed throughout the culture period in the presence of glucose. When the carbon source in the medium is depleted, glutamic acid is taken into the TCA cycle as a nutrient source, and a group of γ-PGA degrading enzymes is actively produced in order to use γ-PGA stored outside the cells. At this time, when sugar such as glucose is used as a carbon source, when consumption of glutamic acid is started, ammonia starts accumulating together with consumption of organic acid in the culture medium, so that the pH rapidly increases. . By using this rapid increase in pH as an index, it is possible to easily detect the depletion of the carbon source and automatically feed the carbon source.

  When the carbon source is fed again in a state where the carbon source in the medium is depleted, the incorporation of glutamic acid into the TCA cycle is quickly stopped. The produced γ-PGA degrading enzyme group is inhibited simultaneously with the stop of glutamate uptake, and γ-PGA is efficiently accumulated.

  Similarly, as another external stress, when the temperature is set in the range of 40 ° C. to 50 ° C., when the supply of oxygen is stopped, further, the pH is set within the range of 5 or more and less than 6. When it does, production | generation of (gamma) -PGA degrading enzyme group is accelerated | stimulated. The conditions under which the generation of the γ-PGA degrading enzyme group is suppressed are below 40 ° C. for temperature, and when oxygen is supplied for oxygen supply, the pH ranges from 6 to 8.

  Thus, for any external stress factor, external stress is applied to Bacillus natto by switching external conditions from an environment in which the generation of enzymes is promoted to an environment that is suppressed, and a γ-PGA degrading enzyme It suppresses the formation of groups and promotes the accumulation of γ-PGA.

  In order to obtain pure γ-PGA, the medium used for production is sodium glutamate monohydrate of 1.0 to 5.0 (mass / volume)% (hereinafter,% is (mass / volume)%. ), Glucose 1.0-5.0%, potassium dihydrogen phosphate 0.25%, disodium hydrogen phosphate dodecahydrate 0.17%, sodium chloride 0.05%, magnesium sulfate 7 What consists of 0.05% hydrate and 0.1 ppm biotin is desirable.

  In some cases, polysaccharides such as fructan, which is an impurity, may be mixed. In this case, sucrose may be used instead of glucose. For the same reason, when impurities may be mixed, 1.0% to 2.0% of soy protein degradation products such as polypeptone S and phyton may be added as additional nutrients of the above composition.

  Moreover, while a carbon source exists, pH falls under the influence of various organic acids discharged | emitted out of a microbial cell. At this time, if the pH is kept between 6.4 and 7 by adding an alkaline solution such as sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia, the activity of Bacillus natto can be maintained at a high level. Is possible.

  In the above description, the target is specifically described as Bacillus natto, but it is also effective against Bacillus bacteria other than Bacillus natto in a state where γ-PGA is not accumulated by the γ-PGA degrading enzyme group. Therefore, these Bacillus bacteria are also included in the Bacillus natto in this invention.

  In the present invention, the production target is specified as γ-PGA, but in a bacterium in which the production of biopolymers other than γ-PGA is inhibited by the production of the biopolymer degrading enzyme. Is also effective. In the present invention, any biopolymer produced by such bacteria is also included in γ-PGA.

  According to the present invention, high molecular weight γ-PGA having an average molecular weight of 3 million or more can be stably produced by Bacillus natto, whose biological safety is confirmed by eating experience.

  In addition, the γ-PGA obtained by the present invention has a higher molecular weight than those conventionally provided and has no anxiety in terms of biosafety, so that it can be applied particularly to applications related to cosmetics, pharmaceuticals and foods.

  It is extremely useful industrially in that γ-PGA that can be actively used for these applications can be stably supplied.

  The effect of suppressing the production of the γ-PGA degrading enzyme group can be known from the amount of γ-PGA accumulated in the culture solution. And the accumulation | storage amount of (gamma) -PGA in a culture solution is in a proportional relationship with the apparent viscosity of a culture solution.

  FIG. 1 shows the relationship between the apparent viscosity of the culture solution and the accumulated amount of γ-PGA. From this figure, it can be seen that the apparent viscosity of the culture solution and the accumulated amount of γ-PGA are in a proportional relationship.

  Embodiments of the present invention are shown in the following Examples, and the effect of suppressing the production of γ-PGA degrading enzymes was examined by the apparent viscosity mPa · s and γ-PGA concentration g / l of each culture solution.

Comparative example

  An example in which γ-PGA is cultured by a conventional method that does not give stress is shown as a standard for confirming the inhibitory effect on the production of γ-PGA degrading enzyme group.

  The sticky material on the surface of commercially available natto was scraped with platinum ears and suspended in 10 ml of sterilized water. Suspension 1 platinum ears were continuously streaked on a sticky product-producing agar medium and cultured at 37 ° C. for 24 hours. .

  The mucilage producing agar medium includes sodium glutamate monohydrate 2.0%, saccharose 3%, potassium dihydrogen phosphate 0.25%, disodium hydrogen phosphate dodecahydrate 0.17%, sodium chloride 0 A medium containing 0.05% magnesium sulfate heptahydrate 0.05%, biotin 0.1 ppm and agar 2% adjusted to pH 6.8 was used.

The tip of the platinum ear was applied to the colony after culture, and the colony that pulled the string most strongly was isolated. The isolated Bacillus natto was cultured on a standard agar medium at 37 ° C. for 3 days to obtain a spore-forming bacterial flora. This spore-forming bacterial flora was suspended at 1 platinum loop per 1 ml of sterilized water and stored in an environment at 20 ° C. or lower until it was used for production of γ-PGA. The obtained spore suspension contained 10 ⁸ to 10 ⁹ cfu / ml of Bacillus natto spores.

This Bacillus natto spore suspension was inoculated into 30 liters of γ-PGA production medium so as to be 10 ⁴ cfu / ml. For the cultivation, MSJ-U2 50L type fermenter manufactured by Maruhishi Bioengineer was used.

  The γ-PGA production medium consists of sodium glutamate monohydrate 2.0%, glucose 2%, potassium dihydrogen phosphate 0.25%, disodium hydrogen phosphate dodecahydrate 0.17%, sodium chloride 0. It consisted of 05%, magnesium sulfate heptahydrate 0.05%, biotin 0.1ppm, and was adjusted to pH 6.8 before inoculation. When culture was performed at 37 ° C. with an aeration rate of 1 VVM and a stirring speed of 120 rpm, an increase in pH was observed with the depletion of glucose, which is a carbon source, 32 hours after the start of the culture. At this time, the apparent viscosity of the culture solution was 100 mPa · s, and the γ-PGA concentration was 6.1 g / l. The apparent viscosity was measured with a B-type viscometer. Apparent viscosity was obtained under the condition of 2.

  γ-PGA was roughly purified by subjecting the γ-PGA-containing culture solution to an alcohol precipitation method which is a known separation method.

  This crude product was redissolved in water and dialyzed overnight and then freeze-dried to obtain purified γ-PGA. The above-mentioned γ-PGA concentration is the concentration of the purified γ-PGA thus obtained. Purified γ-PGA was a white solid, 90% or more was composed of glutamic acid, and no polysaccharide was contained. When the molecular weight of each γ-PGA powder was examined, the average molecular weight was 3 million or more.

The average molecular weight is based on a high performance liquid chromatograph using a gel filtration column, and a peak is observed at a retention time of about 10 minutes as shown in FIG. 2 (a). From the figure shown in FIG. 2 (b), the average molecular weight is obtained. Was confirmed to be 3 million or more. The measurement uses TSK-GEL GMPW _XL (inner diameter 7.8 mm, length 300 mm) for the column, 50 mM potassium phosphate buffer (pH 6.5) for the mobile phase, column temperature 40 ° C., flow rate 0.5 ml / min. It went on condition of. A UV detector was used to detect the peak, and a peak derived from γ-PGA was detected with absorption at 214 nm. The same applies to Examples 1 to 3 below.

  This example shows an example of re-administration after depleting nutrients as applied stress.

  The culture conditions were the same as in the above comparative example. After 32 hours had passed, 2% glucose was fed when glucose depletion was observed due to an increase in pH. The viscosity of the culture solution immediately before feeding was 100 mPa · s as in the comparative example (No. 2 rotor, 30 rpm). Then, when culture | cultivation was performed until 48 hours passed, the apparent viscosity of the culture solution reached 2,000 mPa · s and its γ-PGA concentration reached 15.4 g / l (No. 3 rotor, 30 rpm). And the average molecular weight was 3 million or more. Thus, in the case of this example, the suppression effect reaches about 2.5 times as compared with the comparative example in which no stress is applied.

  This embodiment shows an example in which a temperature change is given as the applied stress.

  The culture was performed under the same conditions as in the comparative example except that the culture temperature was set to 45 ° C. When 7.5 hours passed and the middle of the logarithmic growth phase was reached, the temperature of the culture solution was changed to 37 ° C. when the dissolved oxygen concentration began to decrease rapidly. When 30 hours passed from the start of the culture, an increase in pH accompanying glucose depletion was observed. At this time, the viscosity of the culture solution reached 1,600 mPa · s, and the γ-PGA concentration was 12.2 g / l (No. 3 rotor, 30 rpm). The average molecular weight was 3 million or more. Thus, in the case of this Example, the suppression effect has almost doubled compared with the comparative example which does not give stress.

  This example shows an example in which nutrient depletion, re-administration, and temperature change are combined as applied stress.

  Cultivation was performed under the same conditions as in Example 2, and 2% glucose was fed when 30 hours passed and when an increase in pH was observed accompanying glucose depletion. The viscosity of the culture solution immediately before feeding was 1,600 mPa · s as in Example 2. Then, when it culture | cultivated until 48 hours passed, the culture solution viscosity reached 3,500 mPa * s and the (gamma) -PGA density | concentration reached 22.8 g / l (No. 3 rotor, 30 rpm). The average molecular weight was 3 million or more. Thus, in the case of this example, as compared with the comparative example in which no stress was applied, the suppression effect reached almost 3.7 times.

Table 1 summarizes the inhibitory effects of the production of γ-PGA degrading enzyme groups in the above Examples and Comparative Examples.

  As is clear from the table, when stress was added during culture according to the present invention, the amount of γ-PGA accumulated markedly increased compared to the case where no stress was applied, and different stresses were applied. It turns out that the effect becomes more remarkable when it adds in combination.

  In addition, a good correlation was observed between the viscosity of each culture solution of Examples 1 to 4 and the amount of γ-PGA accumulated in the medium as shown in FIG. 2, and the γ-PGA degradation pathway was suppressed. As a result, it was confirmed that γ-PGA was effectively accumulated and that the inhibitory effect on the degradation pathway could be enhanced by combining different stresses.

  The γ-PGA obtained by this invention can be used for a wide range of applications such as water purification agents, concrete cracking prevention agents, water retention materials for desert greening, and dew condensation prevention agents in addition to cosmetics, pharmaceuticals, and foods.

It is a graph which shows the relationship between (gamma) -PGA accumulation amount and a culture solution apparent viscosity. (A) is a chart which shows the molecular weight distribution measurement result by a high performance liquid chromatograph, (b) is a graph which shows the relationship between retention time and molecular weight.

Claims (2)

In a stable production method of high molecular weight poly-γ-glutamic acid by culturing natto bacteria used in the production of natto,
Sodium glutamate monohydrate is 1.0 to 5.0 (mass / volume)% (hereinafter% is (mass / volume)%), glucose is 1.0 to 5.0%, potassium dihydrogen phosphate 0.25%, disodium hydrogenphosphate dodecahydrate 0.17%, sodium chloride 0.05%, magnesium sulfate heptahydrate 0.05%, biotin 0.1 ppm Culturing the Bacillus natto in a liquid medium,
A high molecular weight poly-γ- characterized by changing the temperature of the culture solution from the range of 40 ° C. to 50 ° C. to less than 40 ° C. when the dissolved oxygen concentration starts to decrease rapidly from the middle to the late phase of the logarithmic growth phase. A stable production method of glutamic acid. The method for stable production of high molecular weight poly-γ-glutamic acid according to claim 1 , characterized in that glucose is fed at the time when the depletion of glucose is observed due to an increase in pH after changing the culture solution temperature to less than 40 ° C. .

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