JPS58129975A - Method for suppressing activity of serine decomposition enzyme in bacterial cell - Google Patents

Abstract

PURPOSE:To suppress the activity of serine-decomposition enzyme, and improve the productivity of L-tryptophan, by heat-treating bacterial cells belonging to a specific genus and containing serine racemase. CONSTITUTION:Bacterial cells belonging to Pseudomonas Putida or Aeromonas punctata subspecies caviae and containing serine racemase which is an enzyme for racemizing serine, e.g. cultured liquid of Pseudomonas Putida IFO 12996, Aeromonas punctata subspecies caviae MT-10243 (FERM BP-21) or wet bacterial cells collected from the cultured liquid, are heat-treated at 40-60 deg.C to suppress the activity of the serine decomposition enzyme without lowering the activity of serine racemase. Accordingly, the yield of L-tryptophan based on serine is increased without adding chemical substances to the reaction liquid, and the industrial productivity of L-tryptophan can be improved.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of Pseudomonas putida (Pbtu) containing serine racemase, an enzyme that racemizes serine.
d-nv courtesy αIμ, ii) Mata is treated with Aeromonas punctata subspecies cam to suppress only the serine-degrading enzyme activity, which is a side reaction, without reducing the activity of serine racemase contained within the bacterial body. In recent years, L-serine has attracted attention from the world not only for medicinal purposes, but also as a raw material for L-)ributophane synthesis, and has become a #f song due to industrial compatibility. tc student] The next four samurai are growing up and making holes. A so-called fermentation method is known as a method for producing L-serine, but there are problems with accumulation of bones, yield, production, waste liquid production, etc., and it has not yet become an inexpensive industrial method. . I bow down to this and DL it as an organic combination.
- Synthesize serine and use DL- instead of indole and L-serine as a raw material for enzymatic production of L-tryptophan.
There is a method using serine and tryptophan synthetase and serine racemase as the r-polymer.
In this case, it is necessary to sequentially racemize the remaining D-serine and eventually convert it all to I4-serine. The enzyme that racemizes D- or L-serine in this way is serine racemase, and Pseudomonas kamaroku is a microorganism that lives in Aeromonas t.
I can do it. It is known from the T-layer egg base that the fungal body itself is used as an enzyme γI with θ) produced within the fungal body, but the fungal body is t[Since it contains an enzyme that decomposes any serine, serine, which is a raw material, is simultaneously decomposed during the tryptophan synthesis W reaction, resulting in a major problem in that the reaction yield for serine decreases.

Conventionally, a method of adding ammonium ions to the nuclear reaction solution has been known as a method of suppressing the decomposition of serine during the tryptophan synthesis reaction, but this method has the disadvantage of placing a burden on the purification process after the reaction is completed. Therefore, a more practically advantageous method was desired.

The present inventors have demonstrated that by physically treating the serine racemase-containing bacterial cells without adding any chemical substances to the reaction solution, only the serine degrading enzyme activity is reduced without reducing the enzyme activity. As a result of examining various methods, we found that the bacterial culture solution or the moist bacterial cells obtained by collecting the bacteria were heated at 40° to 60°C.
The present invention was completed based on the results of the present invention, which showed that when heat-treated with 1 egg, the serine degrading enzyme activity can be significantly reduced while retaining the serine racemase activity.

The microorganisms used in the present invention that produce serine racemase in multiple forms within their cells include Pseudomonas putida and Aeromonas O. punctata subs. caviae. The cells to be subjected to the heat treatment may be a cell culture solution, a suspension of wet cells obtained by collection in a buffer solution, or the wet cells themselves.

The pH during the heat treatment is not particularly limited, but is preferably in the range of 4 to 10. The heating time is usually 5 at 40 to 60℃.
~30 minutes, preferably 10-30 minutes, more preferably about 5-15 minutes at 50-60°C. Regarding the relationship between heat treatment temperature and time, if the treatment temperature is high, it is preferable to perform the treatment for a short time, and conversely, if the treatment temperature is low, it is preferable to conduct the treatment for a long time.

This is because the higher the treatment temperature and the longer the treatment time, the lower the serine degrading enzyme activity.1. ), but serine racemase also shows a similar tendency.

According to the method of the present invention, the seri contained in the bacterial cells
without reducing the activity of 1-in racemase.
Since only the serine degrading enzyme activity can be reduced, the bacterial cells according to the method of the present invention can be used to synthesize tryptophan by 1 old size + 1
n,! By adding chemical substances during the reaction, the reaction yield for serine can be increased to 11τ1-hi.
Therefore, the present invention can greatly contribute to the enzymatic industrial production of L-IJbutophane.

The present invention will be more clearly explained by carrying out the experiment below.

Example 1 Pseudomonas ll putida IFO12996, which is a serine racemase-producing bacterium, was inoculated into a concave medium 100 shown in Table 1 in a 5 n Q ml Sakalo flask.
The cells were incubated at ℃ for 24 hours and cultured under FR. After completing the culture, collect the bacteria by centrifugation and dry the cells.
/e diluted with culture P solution.

This bacterial cell suspension was subjected to the treatment and treatment time shown in Table 4, and then centrifuged and scorched to determine the serine decomposition ability and serine racemase activity of the nuclear heat-treated bacterial cells. Serine content N
The activity of serine racemase was determined by shaking the reaction composition shown in Table 2 at 30°C for 24 hours, and removing the remaining serine. The medium reaction solution was incubated at 35°C for 2 hours, and the generated L
-Tryptophan levels were measured by liquid chromatography, and enzyme activity was expressed as L-) tryptophan production per unit time and monoacetic acid cell leaf. The obtained margins are shown in Table A4.

Table 1 Ehrlich 1 Momme Extract 10 9 Polypeptone
10 9 NaC159 Dilute to 11 with distilled water and... (PH6, 8) Table 2 DL-Serine 60 Cupyridoxalline 0.019 Amount of bacterial cells (converted to dry bacterial cells)
8゜5 Steamed water and used for locking (P1
4s, 5)[)-Serine l・8
% Triton X-1005% Pyridoxal phosphate 0. On1% Cy\-Tomonas putida I FO1299fi bacterial cell collection (v;,
, l; i/X'rJ i body structure) O, n2%
Etschierhia coli MT 10242 (r=”[
(M BP-70) Kantaisaki (dry bacterial cell deterioration) Note) 0゜2%PH8,5 n) '1 happiness activity kt, 5,0 (q''r4.p
, 10-c<11-phase Table 4 [Agitation] The blank mark in the middle of the column indicates comparison l111.

Example 2 Aeromo+s punctata subsp. caviae MT-10243 around the time of serine racemase production
(FERM RP-21), real frfa'
! ; Perform the same operation as above. The results obtained are shown in Table 5.

Table 5

Claims (1)

[Claims] 1. A method for inhibiting intracellular serine degrading enzyme activity, which comprises heat-treating a microbial cell belonging to Pseudomonas putida or Aeromonas fistula subspecies capie containing serine racemase at 40 to 60°C.