JPS5913194B2 - Method for producing L-serine by fermentation method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for producing L-serine.

More specifically, the present invention involves culturing a microorganism that belongs to the genus Nocardia and has the ability to directly produce L-serine from a carbon source in a nutrient medium without adding glycine, thereby producing and accumulating L-serine in the culture solution. The present invention relates to a method for producing L-serine, which comprises collecting perilla-serine from the culture solution.

The purpose is to provide an industrially advantageous method for producing L-serine used in pharmaceuticals, cosmetics, and other products.

Conventionally, regarding the production and accumulation of L-serine by all yeasts,
A method of adding and culturing various substances such as DL-glyceric acid, betaine, and glycine as precursors (Japanese Patent Publication No. 42-177
28, 45-11114, 46-32793, 4
7-38994, 51-6236, 51-9391
, JP-A-49-75783 and JP-A-49-134890) are known.

As for the method using strained direct fermentation, Arthrobacterium spp.
Method using mutant strains of microorganisms such as Brevibacterium, Corynebacterium, Micrococcus, Escherichia, and Bacillus (Japanese Patent Publication No. 46-29191, No. 48
-6558 (see JP-A-51-54984) is known.

The present inventors conducted various studies on industrially more advantageous L-serine production methods, and found that microorganisms belonging to the genus Nocardia directly accumulate L-serine in the culture medium in a nutrient medium without adding glycine. The present invention was completed based on the discovery of this fact.

Conventionally, a method for producing L-serine from glycine using microorganisms belonging to the genus Nocardia (Japanese Patent Publication No. 51-9391)
), but the method of the present invention does not require any precursors such as glycine as raw materials, and uses inexpensive carbohydrates, hydrocarbons, and other carbon sources to directly add L- to the culture medium.
It produces serine, and was published in 1986-939.
This is an excellent method that is clearly different from the method described in No. 1.

The present invention will be explained in detail below.

According to the present invention, if a microorganism belonging to the genus Nocardia and having the ability to directly produce L-serine from a carbon source is cultured in a nutrient medium without adding glycine, L-serine can be produced and accumulated in the culture solution. can.

As the microorganism used in the present invention, any strain of microorganism that belongs to the genus Nocardia and has the above-mentioned properties can be used.

Among these strains, strains having particularly enhanced glycine assimilation ability are suitable for producing L-serine.

Conventionally, when microorganisms belonging to the genus Nocardia are added to a culture medium at a concentration of about 10 mVTIL, their growth is usually severely inhibited, or even if they grow, they are unable to assimilate glycine, or are unable to assimilate it at all. The speed is also very slow.

In any case, a considerable amount of glycine remains even after culturing for about 3 days.

When such microorganisms are cultured in a medium to which glycine is not added, the amount of L-serine produced in the culture solution is minute.

Unlike these, the mutant strain with enhanced glycine assimilation ability used in the present invention has the ability to accumulate a significant amount of L-serine even when cultured in a medium without added glycine.

The L-serine producing strain that has the ability to directly produce L-serine from the carbon source used in the present invention is preferably obtained as a mutant strain that has an enhanced ability to assimilate glycine. It can be obtained from glycine analog-resistant strains and serine analog-resistant strains of microorganisms to which it belongs, and it can also be isolated from nature, and any of them can be used in the present invention.

In addition to the ability to directly produce L-serine from carbon sources, the present invention also has various amino acid analogs (
analogs such as glycine, serine, methionine, tryptophan, cysteine, histidine, tyrosine, phenylalanine, leucine, isoleucine, and valine)
resistance, resistance to various drugs (e.g. glycine, L-serine, sulfa drugs), various auxotrophies (e.g. histidine,
tryptophan, leucine, isoleucine, valine, glycine, choline, purine, etc.), or L-
It is also possible to use a strain that has one or more of the following properties: the property that it is difficult to degrade serine, the property that the inhibition of glycine decomposition by glucose or its metabolites or nitrogen is removed, and the property that it converts to glycine and L-serine.

Such a no-serine producing mutant strain was exposed to UV irradiation, r
- Selection can be made using known selection methods from among mutant strains obtained by performing known mutation treatments such as radiation irradiation and drug treatment.

Preferred specific examples of microorganisms used in the present invention include Ci/Cardia butanica KY7992 and Microorganisms 3953.
(NRRL 11096).

This strain is a mutant strain obtained by the following method.

Namely. Nocardia butanica ATCC21197
(Parent strain) (The mycological properties of ATCC21197 are known as described in Japanese Patent Publication No. 47-48673. As per the law, N-methyl-mernitro-N-nitrosophanicin (0.5w
IILl) (pH 6,0) for 730 minutes at room temperature,
After dilution (104~'g), solid nutrient medium (yeast bouillon medium, yeast extract 0.5 g 41, meat extract 1
? /d/3, peptone 1'di, NaC10,5f
t/dl agar 2? /dl, pH 7, 2), 3
Grown at 0°C.

After 4 days, the resulting bacteria were harvested using a conventional method, and cultured in a medium containing glycine [glycine, glucose 51? /d1.
(NH4)2SO40, 5Vdl, KH2PO40
, 15S'/K2 loquat.

0.05? /di, MS'SO4・7 H2O0,05
flAl, NaC/10°011/d1%FeSO4
-7H200,001? A11゜MnSO4・nH2O
0-001ft/dl, CaCl20.00197
di, hyotine iooμg/l, thiamine hydrochloride 1■/
l.

The cells were grown for 3 days and 2 days in a large test tube (190 mm x 20 mm) into which 157 nl of CaCO33g1dA pH 7.2 was dispensed.

The remaining amount of glycine in the culture solution is that of the parent strain ATCC211.
97 (residual amount of glycine 16°5 m'17'yn/)
Among the fewer strains, we selected Kaikoken Bacteria No. 3953 [residual amount of glycine is trace amount (less than %)].

As the medium composition used in the present invention, either a synthetic medium or a natural medium can be used as long as it is a medium normally used for culturing microorganisms.

i.e. carbon sources (e.g. glucose, starch hydrolyzate,
Sucrose, fructose, molasses, acetic acid, fumaric acid, and depending on the assimilation ability of the bacteria used, hydrocarbons, methanol,
ethanol, ethylene glycol), nitrogen sources (e.g. ammonium sulfate, ammonium chloride, ammonia, urea, etc.), nitrogen-containing natural products (yeast extract, peptone, NZ-amine, meat extract, casamino acids, corn steep liquor, etc.), Inorganic substances (potassium phosphate, magnesium sulfate, magnesium phosphate,
Calcium carbonate, calcium salts, iron salts, trace metal salts such as manganese salts, etc.) can be used as long as they contain a suitable amount.

According to the method of the present invention, since L-serine can be produced by direct fermentation from a carbon source other than glycine, L-serine can be produced by culturing microorganisms in a nutrient medium to which no glycine is added. .

However, when using natural nutritional sources such as yeast extract, peptone, corn steep liquor, etc., contamination of the medium with glycine cannot be avoided, but this case is not excluded from the present invention.

That is, in the present invention, much more L-serine is produced from the carbon source in one medium than the amount of L-serine that may be derived from glycine in these natural nutritional sources.

In fact, in Example 2, a much more significant amount of L-serine was produced than could have been produced from the added peptone, and this L-serine served as the carbon source (
In this case, it is clearly derived from molasses).

The culture is carried out under aerobic conditions such as shaking culture or aerated agitation culture, at a culture temperature of 20°C to 40°C, and a pH of the culture solution of 3 to 9 (preferably maintained near neutrality using an acidic or alkaline substance). Do it with

The culture period is usually 1 to 10 days, and L-serine is produced and accumulated in the culture solution.

After the culture is completed, L- is removed from the culture by a combination of known ion exchange treatment methods, adsorption methods, precipitation methods, extraction methods, etc.
Serine can be recovered.

The present invention will be specifically illustrated with reference to Examples below.

Example 1 Nocardia butanica as seed fungus
No. 3 was used.

The inoculum was 297 dl of glucose. KH2PO40,15?
Al, 2HPO40,05H11MgSO4・7H2
00.05g%11 peptone%11 yeast extract 0.5
T? ' / dl, seed medium (pH 7.5 before sterilization) 7
It was inoculated into a thick test tube (190 mm x 20 mm) containing m7 and cultured at 30°C for 24 hours.

Add 0.25ml of this seed culture solution to Course 5 I? Al
l 1NH4・C1O,5? /dl, K2HPO40
,05g/dl.

KH2PO40, 15%7. Mg SO4・7H200
,05shimeng, NaC10,01fh”di, FeSO4
・7H200,001Vdl, MnSO4・nH2O
0,001riA11Cacts 3? /a'A' fermentation medium (pH 7.5 before sterilization) was inoculated into a large test tube containing 5 m/', and cultured with shaking at 28°C for 3 days.

At this time, L-serine in the culture solution was 0,5"?, fi,
i generated and accumulated.

The parent strain ATCC211 was carried out simultaneously using the same method.
The L-serine production amount of No. 97 was 0.05 to below.

After the completion of the culture, the culture solution 11 is collected, centrifuged to remove the bacterial cells and other precipitates, and the supernatant is purified with 400 TrLl of Diamond Ion 5K-IA (styrene-based strong acid ion manufactured by Mitsubishi Chemical Corporation). L-serine was adsorbed by passing it through a column of exchange resin (trade name, H type).
- Fractions containing serine were collected and concentrated to 30 m/ml.

After adjusting the pH of the concentrate to 3.41 with citric acid, add 0.1M citric acid buffer (pH 3.41) (21.01 ft of citric acid to 200 Tr of 1N caustic soda).
A column of Diaion S K-I A (Na 10 form) which was equilibrated with 110 m/N hydrochloric acid and 5 M thioglycol and made up to 11 with water) was used.
3,2CrrLX 85crIL) to adsorb L-serine, and a citric acid buffer solution (pH
3,41).

At this time, the column jacket was kept at 37.5°C. Collect both parts containing L-serine and add them again to Diaion 5K-IA (H
+ type) column (3851rLl), washed with water,
After elution with 0.5N aqueous ammonia, fractions containing L-serine were collected and concentrated to 100TrLl.

The concentrated solution was decolorized by activated carbon treatment, concentrated to 25 d, and then heated to 4°C.
While cooling to 4° C., 80% (V/V) ethanol cooled to 4° C. was gradually added to crystallize L-serine.

The crude crystals were recrystallized from an alcohol solution to obtain 275 cm of L-serine crystals.

Example 2 In Example 1, as a fermentation medium, molasses
/ -x- le conversion), ammonium sulfate 1? 7di, KH2PO
40,15Vdl, K2HPO40,05f/dl,
MgSO4・7H200,05e/d11NaCJ!
0.01? /di.

FeSO4・7H200,001Orchid11Mn5o4・n
I(2゜O,001?/d/, , peptone 1?/d1
%CaCO33? /dl (pH 7 before sterilization,
2), 20 ru of this medium was dispensed into 300Tr.
The cells were grown in the same manner as in Example 1, except that they were cultured with shaking in a L1 Erlenmeyer flask.

When applied, L-serine was present in the culture solution at 1.2Q/, 1
Generated and accumulated.

Claims (1)

[Claims] 1. A microorganism that belongs to the genus Nocardia and has the ability to directly produce L-serine from a carbon source is cultured in a nutrient medium without adding glycine, and L-serine is produced and accumulated in the culture solution, and L-serine is produced and accumulated in the culture solution. A method for producing L-serine, which comprises collecting L-serine.