Method for producing d-serine

Abstract

To provide a method for producing D-serine with an improved conversion rate from L-serine to D-serine.SOLUTION: A method for producing D-serine involves converting at least a part of L-serine into D-serine through fermentation culture using Lactococcus lactis H74 strain (accession number: NITE P-03887) within a culture medium containing 0.5-1.0 mass% of L-serine, 0.1-5.0 mass% of D-arabinose, starch, melezitose, sorbitol, galactose, or sucrose, 1.0-2.0 mass% of yeast extract, 0.5-2.0 mass% of peptone, and 5.0-10 μg / L of pyridoxal hydrochloride or biotin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to methods for producing D-serine. [Background technology]

[0002] Amino acids include L-type amino acids that are widely present in nature, and D-type amino acids that are present in small amounts but are involved in the metabolism of living organisms. Amino acids obtained from normal foods are L-type amino acids because they are obtained by decomposing animal and plant proteins. On the other hand, D-type amino acids, which are one of the amino acids involved in metabolism in the body, are present in small amounts in nature and are difficult to obtain by decomposing animal and plant proteins. Thus, in addition to fermentation methods, chemical production methods using heavy metal catalysts have also been proposed (see, for example, Patent Document 1), but from the perspective of hygiene, it is difficult to apply these methods to food and other applications, and L-type amino acids often become contaminated in products.

[0003] On the other hand, it has become possible to detect D-type amino acids in various foods in recent years, and it has been found that higher concentrations of D-type amino acids are detected in fermented and aged foods than in fresh foods, and that D-type amino acids are involved in various metabolic functions in the body. Therefore, various D-type amino acids have been produced by fermentation (see, for example, Patent Documents 2 and 3), but the productivity is currently low, L-type amino acids are mixed in, and there have been few reports on the production of D-serine, one of the D-type amino acids. In recent years, it has been reported that D-serine is involved in diseases related to neurotransmission in the brain, such as schizophrenia, Alzheimer's disease, and stroke, and may improve the symptoms of these diseases (see, for example, Patent Document 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5478035 [Patent Document 2] Patent No. 3834960 [Patent Document 3] Patent No. 5965875 [Patent Document 4] Patent No. 6978874 Summary of the Invention [Problem to be solved by the invention]

[0005] When attempting to convert L-serine to D-serine by fermentation with conventional lactic acid bacteria, there was a problem that L-serine and D-serine were produced in equal amounts, i.e., only 50 weight percent of the added L-serine was converted to D-serine, with the remaining L-serine remaining as an impurity.

[0006] In view of the above circumstances, an object of at least one embodiment of the present disclosure is to provide a method for producing D-serine with an improved conversion rate from L-serine to D-serine. [Means for solving the problem]

[0007] In order to achieve the above object, the method for producing D-serine according to the present disclosure involves fermentation culture of Lactococcus lactis H74 strain (deposit number: NITE P-03887) in a medium containing 0.5 to 1.0 mass% L-serine, 0.1 to 5.0 mass% D-arabinose, starch, melezitose, sorbitol, galactose, or sucrose, 1.0 to 2.0 mass% yeast extract, 0.5 to 2.0 mass% peptone, and 5.0 to 10 μg / L pyridoxal hydrochloride or biotin, thereby converting at least a portion of the L-serine into D-serine. Effect of the Invention

[0008] According to the method for producing D-serine of the present disclosure, the conversion rate from L-serine to D-serine can be improved. [Brief description of the drawings]

[0009] [Figure 1]1 is a graph showing the verification results regarding the types and amounts of vitamins added to a culture medium. [Diagram 2] 1 is a graph showing the verification results regarding the types of sugars added to the medium. [Diagram 3] 1 is a graph showing the results of verification of the amount of D-arabinose added to a medium. [Figure 4] 1 is a graph showing the results of verification of the concentration of L-serine added to the medium. [Diagram 5] 1 is a graph showing verification results regarding fermentation temperature. [Figure 6] 1 is a graph showing verification results regarding fermentation culture time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] (Overview of the method for producing D-serine according to the present disclosure) The method for producing D-serine according to the present disclosure involves fermentation culture of Lactococcus lactis H74 strain (deposit number: NITE P-03887) to convert at least a portion of the L-serine into D-serine.

[0011] Fermentation culture using Lactococcus lactis H74 strain is carried out in a medium. The medium used for fermentation culture contains 0.5 to 1.0 mass% L-serine, 0.1 to 5.0 mass% D-arabinose, starch, melezitose, or sorbitol, 1.0 to 2.0 mass% yeast extract, 0.5 to 2.0 mass% peptone, and 5.0 to 10 μg / L pyridoxal hydrochloride or biotin. Fermentation culture is preferably carried out at a temperature of 15 to 30° C. for 2 to 8 days. EXAMPLES

[0012] (Summary of the Example) In the examples described below, the following items were verified. 1. Verification of the types and amounts of vitamins added to the medium 2. Verification of the type and amount of sugar added to the medium 3. Verification of the amount of L-serine added to the medium 4. Verification of fermentation temperature 5. Verification of fermentation incubation time

[0013] <1. Types and amounts of vitamins added to the medium> A basic medium containing 1.0% by mass of glucose, 1.0% by mass of powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of L-serine, 0.1% by mass of Tween 80, 0.2% by mass of sodium acetate, and 0.5% by volume of mineral solution (containing 40 mg of magnesium sulfate, 2 mg of manganese sulfate, 2 mg of iron sulfate, and 2 mg of sodium chloride) was supplemented with biotin or pyridoxal hydrochloride as vitamins, giving six medium samples with respective concentrations of 1.0 μg / L, 5.0 μg / L, and 10.0 μg / L, as well as a control medium sample without vitamins, for a total of seven medium samples. Lactococcus lactis H74 strain was pre-cultured in 30 mL of MRS medium obtained from OXOID. The culture was incubated at a final cell count of 1.0 × 10 per ml in each of these media samples. 7 The cells were added to the medium, and fermentation culture was carried out at 30°C for 48 hours. After the fermentation culture was completed, the concentration of D-serine in the medium was measured, and the conversion rate from L-serine to D-serine (relative conversion rate) was calculated. The results are shown in Figure 1. The relative conversion rate plotted on the vertical axis of Figure 1 is the percentage of the conversion rate obtained under each condition relative to the conversion rate obtained under the condition with the highest conversion rate (condition in which 5.0 μg / L of pyridoxal hydrochloride was added).

[0014] It was found that a higher conversion rate was obtained under the condition of adding biotin or pyridoxal hydrochloride than under the condition of not adding vitamins. In comparison between biotin and pyridoxal hydrochloride, a higher conversion ability was obtained under the condition of adding pyridoxal hydrochloride than under the condition of adding biotin. Since pyridoxal acts as a coenzyme in the enzyme reaction, it is considered that the activity was enhanced compared to the condition of adding biotin. Regarding the amount of addition, a higher conversion ability was obtained under the condition of 5.0 to 10.0 μg / L than under the condition of 1.0 μg / L for both biotin and pyridoxal hydrochloride. Therefore, the preferred amount of addition of biotin and pyridoxal hydrochloride in the method for producing D-serine according to the present disclosure is 5.0 to 10.0 μg / L.

[0015] <2. Types and amounts of sugars added to the medium> A total of 18 medium samples were prepared, including 17 medium samples in which various sugars were added to a basal medium containing 1.0 mass% powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5 mass% pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1 mass% L-serine, 0.1 mass% Tween 80, 0.2 mass% sodium acetate, 5.0 μg / L pyridoxal hydrochloride, and 0.5 vol% mineral solution (containing 40 mg magnesium sulfate, 2 mg manganese sulfate, 2 mg ferrous sulfate, and 2 mg sodium chloride), to give a concentration of 1.0 mass%. Also, one medium sample was prepared without added sugar. The sugars used in this verification experiment were D-arabinose, starch, methyletose, sorbitol, galactose, sucrose, raffinose, glucose, mannitol, lactose, maltose, fructose, salicin, mannose, L-arabinose, xylose, and trehalose. Lactococcus lactis H74 strain was pre-cultured in advance in 30 mL of MRS medium obtained from OXOID. The cultured cells were added to each of these medium samples so that the final cell count was 1.0 × 10 per ml. 7The cells were added to the medium and fermented at 30°C for 48 hours. After the fermentation, the concentration of D-serine in the medium was measured and the conversion rate (relative conversion rate) of L-serine to D-serine was calculated. The results are shown in Figure 2.

[0016] The sugar with the highest conversion rate was D-arabinose. Although the conversion rates of starch, methyletose, sorbitol, galactose, and sucrose were inferior to those of D-arabinose, the results were relatively sufficient for practical use. However, for raffinose and the like, whose relative conversion rate to the conversion rate obtained when D-arabinose is used is less than 10%, the conversion rate is insufficient for practical use. Therefore, the most preferable sugar that can be used in the method for producing D-serine according to the present disclosure is D-arabinose, and although it is less effective than D-arabinose, it can also be used for starch, methyletose, sorbitol, galactose, and sucrose.

[0017] Next, the appropriate range of the amount of D-arabinose added to the medium, which had the highest conversion rate in the above verification experiment, was verified. D-arabinose was added to a basic medium containing 1.0% by mass of powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1% by mass of L-serine, 0.1% by mass of Tween 80, 0.2% by mass of sodium acetate, 5.0 μg / L of pyridoxal hydrochloride, and 0.5% by volume of a mineral solution (containing 40 mg of magnesium sulfate, 2 mg of manganese sulfate, 2 mg of iron sulfate, and 2 mg of sodium chloride), to prepare four medium samples with D-arabinose concentrations of 0.1%, 1.0%, 5.0%, and 10% by mass. Lactococcus lactis H74 strain was pre-cultured in 30 mL of MRS medium obtained from OXOID Co., Ltd. The cultured cells were added to each of these medium samples at a final cell count of 1.0 × 10 per ml. 7The cells were added to the medium and fermented at 30°C for 48 hours. After the fermentation, the concentration of D-serine in the medium was measured and the conversion rate (relative conversion rate) from L-serine to D-serine was calculated. The results are shown in Figure 3.

[0018] The conversion rate was highest when the concentration of D-arabinose was 1.0% by mass, and when the concentration of D-arabinose was 0.1-5.0% by mass, the relative conversion rate was 80-90% compared to the conversion rate when the concentration of D-arabinose was 1.0% by mass. On the other hand, when the concentration of D-arabinose was 10% by mass, the relative conversion rate was about 20%, and a rapid drop was observed. From this result, it can be said that the appropriate concentration (addition amount) of D-arabinose is 0.1-5.0% by mass. It can be considered that the same can be said about the addition amounts of starch, methyletose, and sorbitol.

[0019] <3. Amount of L-serine added to the medium> L-serine was added to a basic medium containing 1.0% by mass of D-arabinose, 1.0% by mass of powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1% by mass of Tween 80, 0.2% by mass of sodium acetate, 5.0 μg / L of pyridoxal hydrochloride, and 0.5% by volume of a mineral solution (containing 40 mg of magnesium sulfate, 2 mg of manganese sulfate, 2 mg of iron sulfate, and 2 mg of sodium chloride), to prepare three medium samples with L-serine concentrations of 0.1%, 1.0%, 5.0%, and 1% by mass. Lactococcus lactis H74 strain was precultured in 30 mL of MRS medium obtained from OXOID. The cultured cells were added to each of these medium samples so that the final cell count was 1.0 × 10 per ml. 7 The cells were added and fermentation culture was carried out at 30° C. for 48 hours. After the fermentation culture was completed, the concentration of D-serine in the medium was measured. The results are shown in FIG.

[0020] The concentration of L-serine after the start of culture in which the L-serine concentration was 0.5-1% by mass was approximately six times higher than the concentration of D-serine after the start of culture in which the L-serine concentration was 0.1% by mass. From this result, it can be said that the appropriate concentration (addition amount) of L-serine is 0.5-1% by mass.

[0021] <4. Verification of fermentation temperature> Lactococcus lactis H74 strain, which had been precultured in 30 mL of MRS medium obtained from OXOID, was added to a basic medium containing 1.0% by mass of L-serine, 1.0% by mass of D-arabinose, 1.0% by mass of powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1% by mass of Tween 80, 0.2% by mass of sodium acetate, 5.0 μg / L of pyridoxal hydrochloride, and 0.5% by volume of a mineral solution (containing 40 mg of magnesium sulfate, 2 mg of manganese sulfate, 2 mg of ferrous sulfate, and 2 mg of sodium chloride) until the final cell count was 1.0 × 10 per ml. 7 The cells were added to the medium and fermented at 15°C, 30°C, and 37°C for 48 hours. After the culture was completed, the concentration of D-serine was measured, and the conversion rate from L-serine to D-serine (relative conversion rate to the conversion rate after the completion of fermentation culture) was calculated. The results are shown in Figure 6.

[0022] Among the three experiments, the highest conversion rate was obtained at 15°C, while the conversion rate at 30°C was about 28% of the conversion rate at 15°C, and the conversion rate at 37°C was about 16% of the conversion rate at 15°C. Although not shown in Figure 5, in an experiment conducted under the same conditions except for the temperature of 40°C, the conversion rate was less than 10%. Furthermore, although not shown in Figure 5, in an experiment conducted under the same conditions except for the temperature of 10°C or less, the conversion rate was low at about several percent. Originally, D-amino acids used for the growth of bacteria are considered to be used as nutrients for decomposition and uptake in bacteria at 30°C to 37°C, where growth is vigorous. On the other hand, at low temperatures (below 15°C), bacteria cannot grow, and therefore conversion from L-serine to D-serine is considered to be impossible. Therefore, in the present disclosure, the temperature range in which conversion of L-serine to D-serine is possible is 15°C to 37°C, with a temperature range of 15°C to 30°C being more preferred, and fermentation at 15°C being most preferred.

[0023] <5. Verification of fermentation culture time> Lactococcus lactis H74 strain, which had been precultured in 30 mL of MRS medium obtained from OXOID, was added to a basic medium containing 1.0% by mass of L-serine, 1.0% by mass of D-arabinose, 1.0% by mass of powdered yeast extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% by mass of pancreatin-digested casein peptone (Kyokuto Pharmaceutical Co., Ltd.), 0.1% by mass of Tween 80, 0.2% by mass of sodium acetate, 5.0 μg / L of pyridoxal hydrochloride, and 0.5% by volume of a mineral solution (containing 40 mg of magnesium sulfate, 2 mg of manganese sulfate, 2 mg of ferrous sulfate, and 2 mg of sodium chloride) until the final cell count was 1.0 × 10 per ml. 7 The cells were added to the medium so that the cells would become cells, and fermentation culture was carried out at a temperature of 30°C for 8 days. The concentration of D-serine in the medium was measured three times during the fermentation culture and one time after the fermentation culture was completed, for a total of four times, and the conversion rate from L-serine to D-serine (the relative conversion rate to the conversion rate after the fermentation culture was completed) was calculated. The results are shown in Figure 6.

[0024] The conversion rate did not improve much for the first day after the start of fermentation culture, but after two days, it increased rapidly, and showed a tendency to increase slightly up to eight days. From these results, it was found that a minimum of two days was required for the fermentation culture period, and no adverse effects on the conversion rate were observed up to eight days after the start of fermentation culture. Therefore, it can be said that the appropriate period for fermentation culture is 2 to 8 days.

[0025] As described above, according to the method for producing D-serine of the present disclosure, the conversion rate from L-serine to D-serine can be improved.

[0026] <Considerations regarding other conditions> Regarding the amounts of yeast extract and peptone added, the only examples given were 1.0% by mass and 0.5% by mass, respectively. Although a larger amount of these added is preferable, there is an upper limit to the amount that can be dissolved in the medium, and adding too much increases the cost of fermentation culture. Therefore, the appropriate range for the amount of yeast extract added is preferably 1.0 to 2.0% by mass, and the appropriate range for the amount of peptone added is preferably 0.5 to 2.0% by mass.

Claims

1. 0.5 to 1.0% by mass of L-serine; 0.1 to 5.0% by mass of D-arabinose, starch, melezitose, sorbitol, galactose, or sucrose; 1.0 to 2.0% by mass of yeast extract; 0.5 to 2.0% by mass of peptone; 5.0 to 10 μg / L of pyridoxal hydrochloride or biotin The method for producing D-serine comprises fermenting and culturing Lactococcus lactis H74 strain (deposit number: NITE P-03887) in a medium containing the above-mentioned compound, thereby converting at least a part of the L-serine into D-serine.

2. The method for producing D-serine according to claim 1, wherein the fermentation culture is carried out at a temperature of 15 to 37° C. for 2 to 8 days.

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