JPH0453510B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-serine using a fermentation method that produces L-serine from glycine using the action of microorganisms. Conventionally, fermentation methods have been used to produce L-serine from glycine using microorganisms of the genus Brevibacterium (Japanese Patent Publication No. 45-11114), Arthrobacter genus, Pichia sp.
32793), and even the genus Corynebacterium (Special Publication No. 51
-6232) or Nocardia genus (Special Publication No. 51-9391)
A method is known that utilizes microorganisms that belong to the genus and have the ability to produce L-serine from glycine. In order to industrially advantageously produce L-serine from glycine by fermentation, it is necessary to increase the concentration of glycine in the substrate to improve the accumulation amount and increase the fermentation yield. If the temperature is too high, the growth of microorganisms will be significantly suppressed, which will retard fermentation and make it impossible to efficiently produce L-serine. On the other hand, if the concentration of the carbon source is increased in order to increase the fermentation yield, the initial growth of microorganisms will be inhibited, and the yield will decrease, so it has been difficult to increase the fermentation yield. Under these circumstances, the present inventors have conducted various studies with the aim of increasing the accumulated amount of L-serine and further improving the fermentation yield to develop a method for industrially advantageous production of L-serine. As a result, the culture was started at a low initial concentration of sugar, which is a carbon source, and the microorganisms were grown.
After the production of L-serine has started, a carbon source is supplied continuously or intermittently to reduce the carbon source concentration in the culture solution to 1.
4.0 by introducing the so-called sugar feed method in which fermentation is carried out while controlling the level to a low level of ~6g/dl.
It has been discovered that L-serine can be produced in high yield from a substrate with a high concentration of g/dl or higher. The present invention was completed based on this discovery. The method of the present invention will be explained below. The microorganism used in the present invention is a microorganism that has the ability to produce L-serine from glycine.
-32793 L-serine producing bacteria of the genus Arthrobacter, genus Candeida and genus Pichia, as well as
L-serine producing bacteria of the genus Corynebacterium No. 51-6232 are used. More specifically, for example, Corynebacterium glycinophilum
AJ3411 FERM−P1685 Corynebacterium glycinophyllum
AJ3412 FERM-P1686 (L-leucine and L-methionine auxotrophy) Corynebacterium glycinophyllum
AJ3413 FERM-P1687 (L-leucine and L-isoleucine requirement) etc. are used. The culture medium used in the present invention includes a carbon source, a nitrogen source,
It is a normal nutrient medium containing inorganic salts and, if necessary, organic micronutrients such as vitamins and amino acids. As the carbon source, saccharides such as glucose, fructose, sucrose, and maltose, starch saccharification solutions containing these saccharides, fruit juice, molasses, organic acids such as acetic acid and ethanol, and alcohols are used. The concentration of the carbon source in the medium is preferably about 2 to 4 g/dl, and if it is 10 g/dl or more, the growth of microorganisms will be suppressed. As nitrogen sources, ammonium salts such as ammonium sulfate and ammonium phosphate, nitrates, urea, ammonia, etc. are used, and amino acids, peptone, etc. are also used. As the inorganic salts, potassium salts, magnesium salts, phosphates, sulfates, manganese salts, etc. are used, and as the organic micronutrients, various vitamins, amino acids, nucleic acids, biotin, etc. are used as appropriate. The substrate glycine is added to the above medium at 4.0 g/dl or more. As for the addition method, the entire amount may be added to the culture medium in advance, or it may be added to the culture solution in divided portions. Cultivation of microorganisms is best carried out aerobically, with a culture temperature of 20 to 40℃, and a PH during culture of ammonia, urea,
It is adjusted to a range of 5.0 to 9.0 using mineral acids, etc. When cultured in this manner, the microorganisms grow well despite the high glycine concentration and start producing L-serine. After the production of L-serine has started, the above carbon source is supplied continuously or intermittently, and the culture is carried out while maintaining the concentration of the carbon source in the culture solution at 2 to 4 g/dl or more. The carbon source supply is stopped when the rate of consumption has substantially decreased, and approximately
Fermentation is completed in 70 to 90 hours. By culturing in this way, the amount of L-serine accumulated increases.
It can reach up to 2.4g/dl, and the fermentation yield can also be improved up to 52%. Examples will be described below. Example 1 A series medium with the composition shown in Table 1 containing 6.0 g/dl glucose and a B series medium containing 12.0 g/dl glucose were adjusted, and after adjusting the pH to 7.0, the pH was adjusted to 1.0.
Dispense 300ml into a small jar fermentor.
It was sterilized by heating at 120°C for 10 minutes. Table 1 Content per 1.0 of medium composition Glucose 60 or 120g KH ₂ PO ₄ 1.0〃 MgSO ₄・7H ₂ O 0.5〃 L-isoleucine 1.0〃 L-leucine 0.3〃 L-methionine 0.6〃 Biotin 200μg Thiamine hydrochloride 1mg Folic acid 1 Nicotinic acid amide 2.5 Soybean protein decomposition solution 0.4 g (TN conversion) A glycine aqueous solution that had been separately heat-sterilized was added to each medium, and the glycine concentration in each medium was adjusted to the second level.
The concentrations were adjusted as shown in the table. For each medium, pre-30
Inoculate 15 ml of seed culture of Corynebacterium glycinophilum AJ3412 that had been shaken in a flask for 40 hours at 34°C, and culture with aeration at 34°C (1/2 V.VM,
1200rpm). When using A series medium, after 20 hours, glucose is continuously supplied to reduce the glucose concentration in the culture solution to 2.0 g/dl.
Culture is carried out while controlling the pH level to 6.5 to 8.0 using ammonia gas.65
Glucose supply was stopped after 72 hours, and the culture was terminated after 72 hours. On the other hand, for medium B, only PH control was performed without supplying glucose, and the culture was completed in 72 hours. The amount of L-serine accumulated in each culture solution thus obtained was measured, and the yield per glycine was determined. The results are shown in Table 2.

[Table] 〓

(batch method)

Claims (1)

[Claims] 1. Cultivate a microorganism that has the ability to produce L-serine from glycine in a nutrient medium containing 4.0 g/dl or more of glycine, and after the production of L-serine has started, add a carbon source continuously or intermittently. Supply L
- A method for producing L-serine by a fermentation method, which comprises producing serine and collecting the L-serine.