JP2799690B2 - Fermentation method for improving phenylalanine production rate - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to Corynebacterium glutamic
micum) using phenylalanine (L-phen).
Fed batch (Fed) of the producing strain of Lalanine)
During the production process employing the (Batch) fermentation method, by increasing the supply rate of dissolved oxygen in an appropriate amount and at the appropriate time to reduce the feedback inhibitory effect of the final product, phenylalanine (L-phenalanine), on the growing strain, Dissolved oxygen injection method (DO-stat) by shortening fermentation time to increase production rate and by automatically adding substrate
The present invention relates to a method for controlling a low substrate concentration and a low oxygen state of a fermentation liquor, thereby making the production state of phenylalanine advantageous, and achieving a high phenylalanine production rate.

[0002]

2. Description of the Related Art Phenylalanine (L-phenolal)
Anine) is a kind of amino acid necessary for the human body, and is also a raw material for the synthesis of aspartase, an artificial sweetener. The main industrial production methods include an oxygenation method and a fermentation method using microorganisms. Regarding the oxygenation method, it is difficult to obtain the necessary raw materials and the price of the raw materials varies greatly.
In the world, there is a tendency to mainly adopt the microbial fermentation method. In the fermentation process, a fed batch (Fed-Bat)
ch) -type method is most often adopted, which is a batch (Ba) method.
The production rate is much higher than that of the tch) method, and the possibility of high contamination generation by the continuous fermentation method can be avoided. However, when phenylalanine is produced by the fed-batch fermentation method, the production rate is mostly limited by the feedback suppressing action of phenylalanine, which is the final product.

[0003] Past research literature includes anti-phenylalanine analogs such as fluorophenylalanine (p-fl).
uorophenylalanine or m-flu
orophenylalanine) to better isolate strains producing high concentrations of anti-phenylalanine, or anti-aromatic amine analogs such as aminotyrosine (3-ami
No-L-tyrosin) or methylethyltryptophan (5-methyltryptophan) is used to separate strains producing high-concentration strains. The more separated mutants have higher resistance to phenylalanine, The problem of the inhibitory effect on producing strains under high concentrations of phenylalanine remained unsolved.

[0004] In the past, studies have been made to recover and purify phenylalanine. For example, there is a method in which copper ions are used to bind to and precipitate phenylalanine in a fermentation solution, and phenylalanine is adsorbed by a resin. However, the conditions for separation and recovery in the fermentation stage were not appropriate. Therefore, the current technology has not been able to effectively separate phenylalanine produced at a high concentration during fermentation in an economically compatible production plan.

[0005]

Problems to be solved by the present invention based on the above-mentioned problems will be described below. Among the control methods of the substrate addition, the dissolved oxygen injection method (DO-stat
e) is the most economical and efficient automatic addition method.
As is evident from previous studies, this method not only effectively controls the timing of substrate addition, but also maintains the fermentation broth at a low substrate concentration, and thus the addition of a high substrate concentration inhibits the production of bacteria. Can be avoided. The method also has the property of maintaining the fermentation liquor in a low oxygen state. In a previous study, this dissolved oxygen injection method was used to produce a phenylalanine-producing fermentation process using Escherichia coli, but E. coli produced large amounts of acetic acid to produce phenylalanine under hypoxic conditions. Suppress. therefore,
There is no example of successful production of phenylalanine using a simple dissolved oxygen injection technique.

[0006] In the literature, the introduction of the process for producing phenylalanine is limited, and in particular, there is a common recognition regarding the production of phenylalanine by supplying oxygen. In other words, since hypoxia is advantageous for the production of phenylalanine, based on this insight, a fermentation process as described in the literature is devised, and the latter half of fermentation is maintained in a relatively low oxygen state or a fixed oxygen supply rate. However, the effect of high-concentration phenylalanine on the latter half of the fermentation was not considered.Therefore, it was not possible to increase the concentration due to the effect of phenylalanine concentration on feedback suppression. could not.

[0007] Therefore, in order to increase the production of phenylalanine, it is necessary to reduce or prevent the feedback suppression effect of phenylalanine on the producing bacteria, and to improve the fermentation production process currently used,
An object of the present invention is to reduce the fermentation time.

[0008]

In order to achieve the above object, the present invention provides a mutant strain of Corynebacterium, Corynebacterium glutamicum (Corynebacterium).
glutamicum) CCRC18335 to produce phenylalanine. In the previous study, when examining the effect of oxygen supply on phenylalanine production, the concentration of phenylalanine was not taken into account.However, as the phenylalanine concentration gradually increased during the fermentation process, the feedback suppression produced by the producing bacteria showed that Affects the continuous generation of Therefore, in the present invention, an experiment was previously conducted on the effects of various oxygen supply amounts under various phenylalanine concentrations using a batch fermentation method.

The experiments were carried out in a 5 liter stirred fermenter. Each experiment contained 3 liters of culture medium, the composition of which was as follows: Molasses 10% ammonium sulfate, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.003% and five different initial concentrations, each 0%,
0.5%, 1.5% and 2.0% phenylalanine were taken. Experiments were conducted with three different oxygen supply rates for each composition. The supply rates of the three different types of oxygen were set at a flow rate of 1.0 L / min. , Three different rotational speeds of 400 rpm, 600 rpm, and 900 rpm, respectively. All experiments are at 30 ° C and pH
The reaction was performed under 7.0, and the pH was adjusted using 11.1 N sodium hydroxide. Silicon KN-72 was used as the defoaming agent. The dissolved oxygen value was measured with an Ingold brand probe. The inoculation amount of the bacteria was 5%, and the activation time was 18 to 10 hours.

During the fermentation process, samples are taken on average every 4 hours, analyzed for phenylalanine via HPLC, and analyzed for dinitrosalicylic acid.
The residual sugar concentration was measured by the acid) method, the bacterial concentration was determined by the turbidity measured at a transmittance of 660 nm, and the growth rate () during the fermentation process every 4 hours was determined.

The relationship between the growth rate and the phenylalanine concentration is shown in FIG. 1 (see FIG. 1). FIG.
As shown in the above, when the fermentation broth contains a relatively large amount of initial phenylalanine, the growth rate of the bacteria is also relatively greatly affected. By comparing different rotation speeds (different oxygen supply rates), it was found that when relatively high oxygen was supplied under the same initial phenylalanine concentration, the growth of the producing bacteria was relatively fast.

If the results of the fermentation experiments contained a slightly higher initial phenylalanine concentration, ie, 1% or more, the relationship between the growth rate of the cells and the phenylalanine concentration was shown in FIG. 2 (see FIG. 2). ). From FIG. 2, it is possible to determine the inhibitory effect on phenylalanine-producing bacteria under different oxygen supply rates. If the slope (Slo
If pe) is defined as the extent of its effect due to feedback suppression, this gives a slope of -1.6 under an oxygen supply of 900 rpm, -2.4 at 600 rpm,
At 400 rpm, it becomes -2.83, which indicates that the feedback suppression phenomenon of the product is increasing as the oxygen supply amount is gradually reduced. That is, if the oxygen supply amount is increased, the phenylalanine of the product is increased. Of the present invention can reduce the effect of suppressing feedback on the producing bacteria.

The relationship between the yield of the product and the effect of the high concentration of phenylalanine under different oxygen supplies is shown in FIG.
It was shown to. As can be seen from FIG. 3, the high oxygen supply (9
The yields obtained at 00 rpm) are all lower than the relatively low oxygen supplies (400 rpm and 600 rpm), and under high phenylalanine concentrations, the yields at 600 rpm oxygen supply are best. Therefore, with a moderate increase in the oxygen supply, the phenylalanine feedback suppression effect of the product is reduced, and at the same time the product yield is increased. A more detailed description of the present invention will be described in the following examples.

[0014]

[Effect] Phenylalanine (L-phenalanani)
During the fermentation process using the fed-batch fermentation method for the production strain of ne), the feed-supply rate of dissolved oxygen at an appropriate amount and at the appropriate time can be increased to reduce the feedback suppression effect of the final product phenylalanine on the growing cells. Therefore, the fermentation time can be shortened and the production rate can be increased.In addition, by adopting the dissolved oxygen injection method by automatic addition of the substrate, the low substrate concentration and the low oxygen state of the fermentation liquor are controlled, and the phenylalanine Make the production situation advantageous.

[0015]

【Example】

Example 1: Activation and storage of bacterial species Corynebacterium glutamic (Corynebact)
erium glutamicum) CCRC1833
5 in complete medium
And culture. The composition of one liter of culture medium is as follows: 10 grams of glucose (Glucose), 2.5 grams of sodium chloride (NaCl), yeast extract (Ye).
ast extract, 10 grams, peptone (Pe)
ptone) containing 10 grams, the culture medium can be stored in an ice box at 4 ° C. for 1 to 2 weeks, or a culture medium containing 10% glycerol (Glycerol) is
Store in a freezer at 80 ° C. A large number of bottles of the above culture solution are prepared, one bottle is taken out every month, and activated on a plate of a complete culture medium.

Example 2: Fermentation culture in a 5 liter stirred fermenter Corynebacterium glutamic (Corynebact)
erium glutamicum) CCRC1833
5 was cultivated in a liquid complete culture medium, and vibrated at 30 ° C. for 24 hours. The seed culture was seeded at 2.5% in seed medium.
Inoculate a grooved triangular jar containing
Incubate for 18-20 hours in a shaker at 0 rpm. The composition of this seed culture is as follows: molasses 3.5% (W
/ V), ammonium sulfate 0.3%, soy proteins (hydrolyis)
From chloricacid) 5%, dipotassium hydrogen phosphate 0.1%, potassium dihydrogen phosphate 0.01%, magnesium sulfate 0.03%, calcium carbonate 2%, and the like. p
Adjust H to 7. The seed culture after inoculation is inoculated in a ratio of 6.7% into a 5 liter fermenter containing 3 liters of fermentation medium, at 30-50 ° C. and 11.1 N concentrated aqueous ammonia (NH 4 OH). The pH to 6.8-7.5 using
The stirring speed ratios prepared during
At 900 rpm, the ventilation flow rate was 1.0 L / min. , A batch fermentation was carried out.

Fed-batch production method The seed culture is inoculated at a rate of 14% into a 5-liter fermenter containing a 1.4-liter fermentation culture medium, and a stirring rate of 6%.
00 rpm, aeration flow rate is 1.0 L / min. , Temperature and p
H added a culture medium on the second day after the inoculation as in the batch fermentation described above. The composition of the seed culture medium is 3.5% molasses, 0.3% ammonium sulfate, 5% soy protein, 0.03% magnesium sulfate, 2% calcium carbonate, 0.1% potassium dihydrogen phosphate, pH 7.0. 0. -The composition of the fermentation broth is molasses 7%, potassium dihydrogen phosphate 0.1%, ammonium sulfate 0.3%, pH 7.0.
The composition of the additional culture medium prepared as described above is 50% molasses.

Example 3 Phenylalanine is produced by fermentation by a dissolved oxygen injection method. When utilizing the dissolved oxygen injection method, several basic devices are basically used. Dissolved oxygen electrode and indicator. Computer data acquisition and digital controller. Electric signal operated pump.

When the fermentation is performed in a fed-batch mode, in the batch mode, the fermentation is performed under a fixed oxygen supply (1 L /
min. )), The dissolved oxygen value of the fermented liquor that has been fermented for 20-26 hours drops to near zero, and the dissolved oxygen value is maintained near zero and the fermentable liquor in the fermentation liquor is used up.
The dissolved oxygen injection value rises rapidly and the signal of this change is captured by the computer, and the signal from the computer is pumped to add the raw material. The average amount is 6
0-80 ml / min. Once the raw material enters the fermentation liquor, the dissolved oxygen value returns to near the original zero value again, and this operation is repeated until the fermentation ends.

Example 4 Production of Phenylalanine by Fed-Batch Method by Increasing the Oxygen Supply Rate By increasing the oxygen supply rate, the effect of suppressing the feedback of phenylalanine against the producing bacteria can be reduced, and the fermentation time can be shortened. This example was performed in a similar operating situation for comparison with Example 3. But fermentation is 2
1. Change the stirring speed from 600 rpm to 900 rpm in 1 hour
And after 44 hours of fermentation, increase the aeration flow rate to 1.0 L / mi.
n. To 2.0 L / min. FIG. 5 shows the production rates of phenylalanine described above.

The time required for fermentation to reach a phenylalanine concentration of 20 grams / liter can only be reached after 77 hours with a fixed oxygen supply, but if the oxygen supply is increased, Below, a fermentation time of only 54 hours could be reached and the fermentation time could be reduced by nearly 23 hours. If we calculate the yield of the product, the yield will reach 9.1%, an increase of about 7%.

[0022]

By increasing the oxygen supply rate, the effect of suppressing the feedback of phenylalanine to the producing bacteria can be reduced, and the fermentation time can be shortened. The fermentation time required to reach a phenylalanine concentration of 20 gram / L requires 77 hours with a fixed oxygen supply, but only with 54 hours of fermentation if the oxygen supply is increased. And the fermentation time could be reduced by nearly 23 hours. If we calculate the yield of the product, the yield will reach 9.1%, an increase of about 7%.

[Brief description of the drawings]

FIG. 1 shows the effect of different initial phenylalanine concentrations on the growth rate of Corynebacterium glutamic under different oxygen supply rates.

FIG. 2 shows the effect of different oxygen supply rates on different degrees of product feedback suppression produced with the growth of Corynebacterium glutamic under high phenylalanine concentrations.

FIG. 3 shows the effect of different initial phenylalanine concentrations on product yield under different oxygen supplies.

FIG. 4 shows a curve of phenylalanine production time in a typical fed-batch fermentation process under a fixed oxygen supply using dissolved oxygen injection.

FIG. 5 is a diagram showing a curve of phenylalanine production time in a fed-batch fermentation method in a state where the oxygen supply amount is increased using a dissolved oxygen injection method.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-62-289192 (JP, A) JP-A-61-216697 (JP, A) JP-A-62-289192 (JP, A) JOURNAL OF FERMENT TATION AND BIOENGI NEERING, VOL. 71, NO. 5 (1991), p. 350-355 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 1/00-41/00 CA (STN)

Claims (7)

(57) [Claims] When the concentration of phenylalanine is increased during the production of phenylalanine by fermentation, the rate of oxygen supply is increased to reduce the phenomenon of feedback suppression of the final product phenylalanine-producing strain. A fermentation method for improving the phenylalanine production rate, characterized by shortening the production rate and increasing the production rate. 2. The fermentation method for improving phenylalanine production rate according to claim 1, wherein in the phenylalanine production process by fermentation, a raw material is injected into a fermentation solution using a fed-batch method. 3. The process of producing phenylalanine by fermentation according to claim 2, wherein a timing of adding a raw material to the culture solution is controlled by using a dissolved oxygen injection method.
Fermentation method for improving the phenylalanine production rate according to the above. 4. The fermentation method for improving phenylalanine production rate according to claim 2, wherein the raw material to be injected into the fermentation broth contains molasses. 5. The fermentation method for improving phenylalanine production rate according to claim 1, wherein the strain used for the fermentation includes a bacterium of the genus Coryne. 6. The fermentation method for improving phenylalanine production rate according to claim 5, wherein the pH of the fermentation broth is in the range of 6.0 to 7.5. 7. The fermentation method for improving phenylalanine production rate according to claim 5, wherein the temperature of the fermentation broth is 25 ° C. to 35 ° C.