JPS5816677A - Storage of serine-racemase - Google Patents

Abstract

PURPOSE:To store serine.racemase contained in microbial cells, for a long period, without lowering the enzymatic activity, by suspending the microbial cells in a specific aqueous solution and freezing the suspension. CONSTITUTION:Cells of bacteria belonging to Pseudomonas putida or Aeromonas punctate subsp. caviae and containing serine.racemase, e.g. Pseudomonas putida IFO 12996, are suspended in a >=0.0001%, preferably 0.001-0.1% aqueous solution of pyridoxal phosphate at a concentration of 5-200g/l preferably 30- 100g/l in terms of dried cell. After adjusting the pH of the suspension to preferably 7-9, the suspension is frozen at -5--50 deg.C, preferably -10--30 deg.C.

Description

[Detailed Description of the Invention] Pseudomonas putida 4 or Aeromonas punctata subsp.
ubsp. microorganisms belonging to O. caviae).
．． o o o This relates to a method for preserving serine racemase, which involves suspending it in an aqueous solution of pyridoxal phosphate of i% or more and then freezing, thereby preserving the serine racemase contained within the bacterial cells without reducing its activity. .

In recent years, L-serine has attracted public attention not only for medicinal purposes but also as a raw material for L-tryptophan synthesis, and expectations for inexpensive production of this substance on an industrial scale are increasing. A so-called fermentation method is known as a method for producing L-serine, but there are problems with accumulation amount, yield, purification, waste liquid treatment, etc., and it has not yet become an inexpensive industrial production method. An alternative method is to synthesize DL-serine organically and use it instead of L-serine as a raw material for the enzymatic production of L-tryptophan, but in this case, It is necessary to racemize the remaining D-serine and ultimately convert it all to L-serine.

Thus, the enzyme that racemizes D- or L-7 phosphorus is serine racemase, which is produced in large quantities within the cells of microorganisms belonging to the genus Sheetmonas or Aeromonas. Since this enzyme is produced within the orchid body, it is industrially advantageous to use the bacterial body itself as an enzyme source.
In order to use this enzyme on a commercial scale, it is necessary to preserve the bacterial cells containing this enzyme for a long time without reducing the enzyme activity (ability to racemize serine per unit time and unit amount of bacterial cells). A method must be established.

Conventionally, it has been generally known to cryopreserve bacterial cells containing enzymes, but in the case of bacterial cells containing serine and racemase, little is known about how to preserve them. According to their tests, it was difficult to say that simply freezing and preserving wet bacterial cells was a practical preservation method because the enzymatic activity of the bacterial cells decreased significantly after thawing.

The present inventors have developed bacterial cells containing serine racemase,
As a result of examining various methods for preserving the bacteria for a long time without reducing the enzyme activity, we found that the wet bacteria were frozen as they were by suspending the bacteria in an aqueous solution of pyridoxal phosphate with a concentration of 0.00014 or more and then freezing the cells. It was discovered that the decrease in enzyme activity was significantly less than in the case of
The invention has been completed.

Bacterial strains that produce large amounts of serine racemase in their cells, which can be used in the present invention, include Sheetmonas putida and Aeromonas punctata subsp. caviae. The concentration of pyridoxal phosphate is o, o
ooi 4 or more, preferably 0001 to 0.1 h, and the bacterial cell concentration during suspension is 5 to 200 f//l, preferably 30 to 10
Furthermore, after suspending the bacterial cells in an aqueous pyridoxal phosphate solution, the pH of this suspension was adjusted to 7.
It is desirable to adjust it to ~9. The temperature for freezing the bacterial cell suspension is -5 to -50°C, preferably -10 to -3°C.
The temperature range is 0°C, and the temperature when melting the frozen bacterial cell suspension is 5 to 50°C, preferably 15 to 40°C.
A range of °C is used.

According to the preservation method of the present invention, serine and
Since racemase can be stored for a long time with little loss of enzyme activity, the present invention is expected to greatly contribute to the industrial use of serine racemase.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 500ml of Pseudomonas putida IFO12996, a serine racemase producing bacterium! The cells were inoculated into 100 mJ of a medium having the composition shown in Table 1 in a Sakaro flask, and cultured at 30°C for 24 hours. 200ml of this culture solution (2 flasks
This product was inoculated into a medium ISZ with the composition shown in Table 2 in a 10 ton Jar Fermentor, and heated at 50°C, pH 6.8 (28
% ammonia water control). After the culture was completed, the cells were collected by centrifugation to obtain 150 S' wet cells (312 dry cells). Five volumes of the wet bacterial cells were suspended in an aqueous solution of pyridoxal phosphate at the concentration of each test group, adjusted to pH 8.5, and stored frozen at -15°C for the period of each test group. After thawing this frozen product at 20° C., serine racemase activity was measured using a reaction solution having the composition shown in Table 3. To measure serine racemase activity, react the reaction solution for activity measurement at 35°C for 2 hours, measure the amount of L-tryptophan produced by liquid chromatography, and measure the enzyme activity by unit time and unit cell mass. The winning L
-) Expressed as the amount of IJ butophane produced. The results obtained are shown in Table 4.

Table 1 Ehrlich Meat Extract 1of Polypeptone 101Nacz
Dilute to 1t with 5ij' distilled water and use (
pH68) Table 2 Glucose 10 g(NH,)2So, 1 fKH2P
0. 0.5 fK2HP0.
0.5 fMgSO, 7H201t Polypeptone 0.5 f Yeast extract 052 Table 3 Indole 2 Tyroserine
18 Tidriton X-1005 Tipyridoxal phosphate 0001
Chi (NH,) 2So,
2.5 pH8.5 Note) Enzyme activity is 4.1' (f ・Try/?・dry
eel I fist hr) Table 4 PLP: Biritoquinal phosphate Example 2 Aeromonas punctata subsp. caviae MT-10243, a serine racemase producing bacterium
The same operation as in Example 1 was performed using (FKRM BP-21). The results obtained are shown in Table 5.

Table 5 Patent applicant: Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] The feature is that microbial cells belonging to Sheetomonas putida or Aeromonas punctata subspecies φ caviae containing serine racemase are suspended in an aqueous solution of pyridoxal phosphate of 0.0001% or more, and then frozen. A method for preserving serine racemase inside bacteria.