KR910007821B1 - Improvement in the method for producing l-glutamic acid - Google Patents

Abstract

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Description

Improved manufacturing method of L-glutamic acid

The present invention relates to a method for preparing L-glutamic acid.

In known fermentation methods for producing L-glutamic acid using microbial strains that require biotin, the amount of biotin added to the medium should be selected in the range below the amount necessary for maximum growth of the microbial cells. The addition of biotin above this range significantly reduces the yield of L-glutamic acid. [cf. Kinigoigokyo Kagaku (Modern Industrial Chemistry) vol, 23. Seibutsu Kogyo Kagaku (Biological Industrial Chemistry), p. 135, Asakura Shoten, 1969, Japan; Journal of the Agricultural Chemical Society of Japan, vol. 34, page 193, 1960] It is known that the reduction of L-glutamic acid yield due to excess biotin can be recovered to some extent by supplying oxygen in sufficient amounts during the cultivation (Agricultural and Biological Chemistry, vol. 32, page 855, 1968). However, as the amount of biotin added increases, the oxygen supply effect gradually decreases. Therefore, when an excessive amount of biotin-containing material such as molasses is used as a carbon source, an industrially satisfactory result cannot be expected even if a sufficient amount of oxygen is supplied.

Under these conditions, the present inventors have conducted extensive research to develop a new fermentation method for producing L-glutamic acid, and an excess of oleic acid is used to produce L-glutamic acid producing microorganism strains that require oleic acid for growth but do not require biotin. It has been found that the production of L-glutamic acid can be dramatically increased by culturing in a source containing medium through a medium having a higher oxygen concentration than air. The present invention is based on these findings and further studies.

Therefore, the present invention produces and accumulates L-glutamic acid in the culture medium by culturing L-glutamic acid producing microorganism strains that require oleic acid for growth but do not require biotin in the culture medium, and extract L-glutamic acid from the culture medium. The present invention relates to an improved method for producing L-glutamic acid to be recovered, wherein oxygen is higher in oxygen than normal air in the presence of a sufficient amount of oleic acid source to maximize the accumulation of L-glutamic acid when the culture is carried out with normal air. The culture is carried out while supplying the containing gas to the medium.

The oleic acid source used in the production method of the present invention may be oleic acid itself, a salt or ester thereof, or an oleic acid containing material. Examples of oleic acid salts are sodium salts, potassium salts and magnesium salts. Esters of oleic acid include sorbitan monooleate, polyoxyethylene sorbitan monooleate and polyoxyethylene glycol monooleate, as well as the corresponding sesquioleates and trioleates. Examples of oleic acid containing materials are laard oil, fats and other oils.

The concentration of oleic acid in which the accumulation of L-glutamic acid is maximized when cultured with aeration through normal air is the concentration of the oleic acid source in which the accumulation of L-glutamic acid under aeration is maximized under normal culture conditions. Depending on the microbial strain used, the carbon source used and other conditions, the oleic acid concentration in question is generally in the range of about 90-150 mg (oleic acid) per liter of medium.

In the practice of the present invention, about 180-600 mg (oleic acid) per 1 liter of medium is added to the medium, more preferably about 240-480 mg (oleic acid) per liter of medium.

In the practice of the present invention, a specified amount of oleic acid can be added to the medium to start culturing, or half of the amount can be added prior to culturing and the rest added during culturing.

Cultivation of the microorganism according to the present invention is carried out by a conventional method, for example, by aeration in agitation stirred water.

According to the present invention, a gas having a higher oxygen concentration than normal air is bubbled into the medium.

The gas used in the present invention may of course be pure oxygen gas itself or a mixture with air, and also a high concentration oxygen-containing gas released from an oxygen generator, for example, by molecular sieve method or membrane separation method. .

A gas having a higher oxygen concentration than air may be continuously supplied at a constant rate or automatically at regular intervals during the incubation period while measuring the amount of oxygen dissolved in the medium.

The supply of gas having a high oxygen concentration can also be carried out by supplying air and oxygen normally along each medium through each nozzle.

In carrying out the process of the invention, the amount of air and oxygen supplied to the medium during the fermentation period is calculated according to the following equation.

In the above formula, ^Q air and ^Q oxygen are the amounts of air and oxygen to be supplied to the medium (l / min), respectively, C is the oxygen concentration (%) in the mixed gas in which the two are mixed, and Q ₀ is performed in a conventional manner. Usually the amount of air supplied (l / min). The value of C can be easily known from the following relation based on the amount of oleic acid in the medium.

OA×10³+9C

OA × 10 ³ +9

OA in the formula is the concentration of oleic acid expressed in oleic acid concentration (weight / volume,%).

The microbial strain used in the present invention requires oleic acid for growth, but does not require biotin and may be any strain capable of producing L-glutamic acid. (These strains are then produced as L-glutamic acid according to the present invention. Mainly abbreviated.) Examples of the microbial strains include strains of the genus Brevibacterium or Corynebacterium. Specific examples of Brevibacterium strains include Brevibacterium thiogenitalis D-248 and Brevibacterium flavum BN-11, Corynebacte Specific examples of the Leeum strain include Corynebacterium glutamicum NO. 534-MS-023 (Corynebacterium glutamicum No. 534-MS-023). These strains are representative L-glutamic acid producing strains of the present invention.

The above-mentioned strains have been deposited with Japan's Osaka Fermentation Research Institute (IFO) and Japan International Trade Industry Association's Ministry of Industry, Science and Technology (FRI). Strain deposits in the FRI were converted to deposits under the Budapest Convention, which are stored in the FRI. Accession numbers and deposit dates are listed in Table 1.

These strains have also been deposited with KAIST and the accession numbers and deposit dates are listed in Table 1.

TABLE 1

Among these strains, Brevibacterium thiogenitalis has bacterial properties as described in Japanese Patent Application Publication No. 942/1970, except that this strain requires oleic acid for growth but does not require biotin. . The bacterial properties of Brevibacterium plaboom are the same as Japanese Patent Application Publication No. 17348/1964, except that this strain requires oleic acid for growth but does not require biotin. The bacterial characteristics of Corynebacterium glutamicum are the same as those described in Japanese Journal of Agricultural Chemistry, vol. 38, page 328 (1965), except that the strain requires oleic acid for growth but does not require biotin.

In general, microorganisms of the Brevibacterium and Corynebacterium species can change in character and can be artificially altered either spontaneously or by irradiation with L-rays, ultraviolet rays or other rays, or by treatment with various artificial variation sources. have. The resulting variant can be used for the purposes of the present invention if it is capable of producing L-glutamic acid and requires oleic acid for growth but does not require biotin.

In the method of the present invention, the medium for culturing the L-glutamic acid producing strain according to the present invention is a medium generally used for producing fermentation of L-glutamic acid. For example, glucose, fructose, sucrose, brown sugar, molasses (e.g. beet molasses, sugar cane molasses), saccharified starch (e.g. tapioca, sago palm, sugar cane, potatoes and corn Starch), acetic acid and ethanol may be used alone or in combination.

Nitrogen sources added to the medium include organic nitrogen sources such as peptone, soy flour, corn steep liquor, yeast extract, meat extract and urea and inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate and other ammonium salts, ammonia gas and ammonia water There is this. In addition, various inorganic salts necessary for the growth of microorganisms such as calcium, potassium, sodium, magnesium, manganese, sulfates, hydrochlorides, carbonates, nitrates, phosphates and acetates of iron and zinc, as well as amino acids, vitamins and other necessary for the growth of microorganisms The media components are appropriately selected and added to the media alone or in combination. If necessary, antifoaming agents such as silicone oil can be added.

The pH of the medium is generally selected in the range of about 6.5 to 8.5. The culturing is preferably carried out at a pH range of about 7-8. The pH can be changed to the required range by observing the pH change of the medium during incubation and adding an appropriate amount of ammonia gas, ammonia water, urea or alkali hydroxide. Cultivation is carried out at a temperature suitable for the growth of microorganisms, and generally ranges from about 25 ° C to 37 ° C. Incubation is continued until the accumulation of L-glutamic acid is maximized. In general, incubation of about 20 to 48 hours can achieve the goal.

Recovery of L-glutamic acid formed in the fermentation broth can be easily carried out by a known method.

According to the method of the present invention, a large amount of L-glutamic acid accumulates in the culture. More specifically, the method can convert large amounts of carbon sources to L-glutamic acid because the method of the present invention increases the growth rate of microorganisms and increases the consumption rate of carbon sources by microorganisms. Therefore, when performed with a given amount of tool, the method of the present invention can increase the amount of L-glutamic acid product required. Since the obtained culture is rich in L-glutamic acid, the required product can be easily recovered. In view of the above, the method of the present invention is an industrially advantageous method.

The following reference examples and examples further illustrate the invention. Unless otherwise indicated, “%” means “weight / volume percentage”.

Reference Example 1

2% glucose, 0.4% urea, 0.1% KH ₂ PO ₄ , 0.04% MgSO ₄ . 7H ₂ O, 0.1% CaCO ₃ , 0.015% FeSO ₄ . 7H ₂ O, 0.001% CuSO ₄ . 10 parts (20 each in a 200 ml Erlenmeyer flask) of Brevibacterium thiaget containing 20 ml of sterile culture medium containing 5H ₂ O, 2% corn steep liquor and 0.03% sodium oleate (60% oleic acid content) Inoculate with gradient culture of Nitalis D-248 (FERM BP-433, IFO 12331, KAIST 840124-12411). After 18 hours of incubation at 28 ° C., the entire cultures were prepared with 2% glucose, 0.2% urea, 0.15% KH ₂ PO ₄ , 0.06% MgSO ₄ . 7H ₂ O, 0.06% of CaCl ₂ . 2H ₂ O, 5 mg / l MnSO ₄ . 10 l of 4 sterile fermentation medium containing 4-6H ₂ O, 150 μg / l thiamine hydrochloride, 300 μg / l biotin and 0.2% corn steep liquor were transferred to a bottle fermenter and sodium oleate (oleic acid) Content 60%) to the temperature given in Table 2, and then start fermentation at 32 ° C at 600 rpm (rpm). Do this twice at each sodium oleate concentration. In the first run, only air (0.3VVM) is passed through the medium during the preculture period. In the second run, air (0.2 VVM) and pure oxygen (0.1 VVM) are passed through the medium 6 hours after the start of the culture. Sterilized 70% glucose solution is added separately from 8 hours after the start of the culture to maintain the glucose concentration in the fermentation broth below 0.5% or less. In this case fermentation is carried out for 36 hours. During fermentation, 25% ammonia water is automatically added to maintain the pH of the fermentation medium in the range of 7.4 to 7.6. The L-glutamic acid concentration in the fermentation broth thus obtained is determined by the Waberg Manometer method. The results obtained are shown in Table 2.

TABLE 2

Reference Example 2

For comparison with Reference Example 1, biotin is required for growth and the following is carried out using a microbial strain capable of producing L-glutamic acid. Of 2% glucose, 0.4% urea, 0.1% of KH ₂ PO _4, of 0.04% MgSO ₄ .7H ₂ O, of 0.015% FeSO _4. 7H ₂ O, 0.001% CuSO ₄ . Ten portions (each placed in a 200 ml Erlenmeyer flask) of 20 ml of sterile species culture medium containing 5H ₂ O and 1% corn steep liquor were each brebbacterium thiogenitalis No. Inoculate with a sloped culture of 594 (FERM-P No. 6096, IFO 12115, ATCC 19240) and incubate for 18 hours at 28 ° C. The entire seed culture was prepared with 2% glucose, 0.2% urea, 0.2% KH ₂ PO ₄ , 0.08% MgSO ₄ . 7H ₂ O, 5 mg / l MnSO ₄ . 10 L containing 4 L of sterile fermentation medium containing 4-6H ₂ O, 300 μg / L thiamine hydrochloride and 0.2% corn steep liquor are transferred to the bottle fermentor with the biotin concentrations listed in Table 3. Fermentation is started at 600 rpm at a temperature of 32 ° C. Do this twice for each biotin concentration. That is, in the first run, only air (0.3VVM) is passed through the medium during the entire culture. In the second run, air (0.2 VVM) and pure oxygen (0.1 VVM) were simultaneously supplied 6 hours after the start of the culture. Then fermentation is continued in the same manner as in Reference Example 1 until 36 hours.

Comparing Reference Example 1 and Reference Example 2, if the L-glutamic acid producing microorganism strain which requires biotin for growth is cultured in the presence of excess biotin, aeration of gas with high oxygen concentration has little effect, but the present invention When using the L-glutamic acid producing strain according to the above, venting with a gas with a high oxygen concentration in the presence of excess oleic acid significantly increases the accumulation of L-glutamic acid.

TABLE 3

Example 1

2% glucose, 0.4% urea, 0.1% KH ₂ PO ₄ , 0.04% MgSO ₄ . 7H ₂ O, 0.1% CaSO ₃ , 0.015% FeSO ₄ . 7H ₂ O, 0.001% of CuSO ₄ .5H ₂ O, _2% of corn steep liquor and 0.03% of sodium oleate (oleic acid content of 60%) sterilized seed culture medium each at 10 20ml portions of each 200ml Erlenmeyer flask containing Inoculated with a slope culture of Brevibacterium thiogenitalis D-248 (FERM BP-433, IFO 12331, KAIST 840124-12411), followed by incubation for 18 hours at 28 ° C. A total of 5% of the obtained species culture was sugarcane molasses (total sugar determined by the Antron-Sulfate method), 0.2% urea, 0.15% KH ₂ PO ₄ , 0.04% MgSO ₄ . 10 liters containing 4 liters of sterile fermentation fermentation medium containing 7H ₂ O and 100 μg / L thiamine hydrochloride were transferred to a bottle fermentor, and the sodium oleate concentrations (60% oleic acid content) of Table 5 were added to the medium. Start the fermentation at 600 ° C at 32 ° C. Twice for each sodium oleate concentration. That is, in the first run, only the air (0.3VVM) is aerated during the entire fermentation period, and in the second run, the air (0.2VVM) and pure oxygen (0.1VVM) are simultaneously aerated at 5 hours after the start of the culture. From 6 to 8 hours, the sterile 60% (total sugar) sugar cane molasses solution is continuously added separately to keep the total sugar concentration in the fermentation broth below 0.5%. Continue fermentation until 28 hours. 25% aqueous ammonia is automatically added to maintain the pH of the fermentation broth in the range of 7.4 to 7.6. L-glutamic acid in the fermentation broth thus obtained is analyzed by the Waberg Manometer method. The results obtained are shown in Table 5.

Recovery of L-glutamic acid from the fermentation broth of Example No. 4 by a known method yields 631 g of crude crystals.

TABLE 5

Example 2

About 200 ml of a species culture of Brevibacterium thiogenitalis D-248 (FERM BP-433, IFO 12331, KAIST 840124-12411) obtained using the same medium and method as in Example 1 was diluted to 1% (acetic acid). Ammonium acetate, 0.15% KH ₂ PO ₄ , 0.06% MgSO ₄ . Transfer 10 liter bottle fermentor containing 4 liters of sterile fermentation medium containing 7H ₂ O, 100 μg / l thiamine hydrochloride, 20 μg / l biotin and 0.2% corn steep liquor, and transfer to sodium oleate (oleic acid content 60) %) Is added to the medium at the concentrations listed in Table 4. Fermentation starts at 32 ° C. at 600 rpm. Do this twice for each sodium oleate concentration. That is, in the first embodiment, only air (0.25VVM) is ventilated during the entire culture period, and in the second embodiment, air (0.2VVM) and pure oxygen (0.05VVM) are simultaneously ventilated after 6 hours. After 8 hours, sterilized 70% acetic acid adjusted to pH 4.0 with 25% aqueous ammonia before the sterilization was intermittently added to keep the acetic acid concentration in the fermentation broth below 0.5%. In this way fermentation is continued until 46 hours. During fermentation, 30% sodium hydroxide is automatically added to maintain the pH of the fermentation broth at 7.4 to 7.6. L-glutamic acid in the final fermentation broth is determined by the Waberg Manometer method. The results obtained are shown in Table 6.

Recovery of L-glutamic acid from the fermentation broth of Example No. 4 by a known method yields 595 g of crude crystals.

TABLE 6

Example 3

Using a sloped culture of Brevibacterium flavum BN-11 (FERM BP-534, IFO 12525, KAIST 840124-12311), the sodium oleate concentration was 0.06%, air (0.2VVM) and pure after 5 hours of incubation. Simultaneously venting oxygen (0.1 VVM) and performing the method of Example 1 as it is except that the sugar cane molasses supply volume is 2.33 L. The final fermentation broth amount is 7.27 L, and the accumulation of L-glutamic acid is 96.1 g / L.

Corynebacterium glutamicum no. Using a slope culture of 435-MS-023 (FERM BP-434, IFO 12523, KAIST 840124-12211), the concentration of sodium oleate was 0.06%, and air (0.2 VVM) and pure oxygen (0.1 VVM) after 5 hours of incubation. ), And the method of Example 1 is carried out as is, except that the sugar cane molasses supply volume is 2.33 L. The final fermentation broth volume is 7.30 L and the accumulation of L-glutamic acid is 96.5 g / L.

Claims (3)

Strains of Brevibacterium or Corynebacterium that require oleic acid for growth but do not require biotin and are capable of producing L-glutamic acid are cultured in the culture medium to produce and accumulate L-glutamic acid in the culture medium. In the method for producing L-glutamic acid, which recovers L-glutamic acid from the culture medium, in the presence of an oleic acid source having a sufficient amount of oleic acid so that the accumulation of L-glutamic acid can be maximized when the culture is carried out while being aerated with normal air. Improved method characterized in that the culture is carried out while supplying oxygen-containing gas having a higher oxygen concentration to the medium. The method of claim 1 wherein the amount of oleic acid added to the culture medium is about 180 to 600 mg (oleic acid) per liter of medium. The method of claim 1 wherein the amount of air and oxygen to be supplied to the medium during fermentation is calculated by the following equation.

[Wherein, ^Q air and ^Q oxygen are the amount of air and oxygen to be supplied to the medium (l / min), C is the oxygen concentration (%) in the mixed gas mixed with air and oxygen, and Q ₀ is known The amount of normal air (l / min) supplied to carry out the method, and the value of C based on the amount of oleic acid in the medium is represented by the following relationship. C

OA × 10 ³ +9 (OA is the concentration of oleic acid expressed in oleic acid concentration (weight / part,%))