JPS5820186A - Cultivating method for microorganism capable of producing tryptophan synthetase - Google Patents

Abstract

PURPOSE:To improve the yield and activity of the resulting enzyme, by adding glucose to a culture medium of a microorganism capable of producing tryptophan synthetase to keep the concentration of the glucose at 1% or below. CONSTITUTION:Escherichia coli MT-10232 (FERM BP-19) of Escherichia coli MT-10242 (FERM BP-20) which is a microorganism capable of producing tryptophan synthetase is cultrivated in a culture medium containing a carbon source (glucose), a nitrogen source (an ammonium salt or nitrate), inorganic salt and others. In the process, the cultivation is carried out while adding glucose to the culture medium to keep the glucose concentration in the culture medium at 1% or below, preferably 0.3% or below. The glucose concentration is measured by using an analytical apparatus with an enzymic electrode.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses tryptophan synthetase belonging to the genus Escherichia to catalyze the reaction t-catalyzing $1) production of tryptophan (L-) from indole and L-serine.
The present invention relates to a culture method for culturing with high celluloid activity and high yield.

In recent years, L-)! Buto-7-an has attracted the world's attention for its effectiveness not only for medicinal purposes but also for medicinal purposes, and expectations for inexpensive production of this substance on an industrial scale are increasing. L
- One of the most promising methods for producing tryptophan is from indole and L-serine to L-serine.
) There is an elementary production method for producing lipdoan tubes. The element III that mediates the reaction tM is produced in large quantities by tryptophan synthetase69, mainly in the genetically modified organisms of microorganisms belonging to the genus Escherichia. This enzyme is used as it is, or in a cell-free extract, etc., but in order to use this enzyme commercially, it must be #inactive (unit time, unit recommended amount). A method for efficiently culturing the present enzyme producing l1It with a high yield (L-)ligtophan synthesis capacity of 9) will be developed.

Conventionally, it is known that glucose is used as a carbon source for culturing tryptophan synthetase production 1, but in both cases, the amount of glucose used in the culture is small, and the body yield obtained is also low. , No attempt has been made to obtain high-yield enzymes with high bulk activity.Furthermore, little is known about the relationship between culture conditions and enzyme activity.
Conventionally, glucose was only added at a relatively low concentration (mainly 2 or less) at the start of the culture and cultured. The present inventors have developed a method for producing tryptophan synthetase, an enzyme with high activity. , and as a result of examining various culture conditions for culturing with high yield, we found that the glucose concentration in the medium was reduced to 1.
- If the culture is carried out while adding glucose gold continuously or intermittently, the yield will be significantly higher and the fermentation rate will be higher than when glucose is added all at once at the start of the culture.
A tryptophan cunsetase producing plant with high activity can be obtained.All headings The present invention has been completed.
0P-19) or Escherichia coli M'r-
10242 (FIRM Bl'-20) Tryptophan synthetase activity was increased by culturing with continuous and intermittent addition of glucose so that the glucose concentration in the medium was kept below 1. According to the results of various studies conducted to complete the present invention, the glucose concentration in one can of medium at the start of culture is relatively low. If the cocoons are grown at a low concentration, usually less than 1 hour, the culture time required to obtain tryptophan synthetase-producing cocoons at a constant concentration is relatively short, but the disadvantage of not being able to produce cocoons at a high concentration is To'p. , on the other hand, the glucose concentration of the medium at the start of culture # is set to a high concentration, usually 2.
441: It takes a very long time to obtain a certain number of images t - 441: time t - required and obtained one #
According to the present invention, tryptophan synthetase production with high self-talk property a
Since tS can be obtained in a high yield and in a short culture time of 1 hour, it is considered to have extremely great significance in engineering.As a nitrogen source for the culture medium used in the present invention, ammonium sulfate 1. 'JL(t) Ammonium salts such as ammonium, ammonium phosphate, nitrates such as sodium nitrate, potassium nitrate, ammonium nitrate, ammonia, etc. are suitable, and as inorganic substances, potassium phosphate, magnesium sulfate, iron, manganese, zinc, etc. Growth promoting substances include vitamins such as suiamin and biotin, methionine,
Amino acids such as cysteine, or substances that contain these substances in whole or in part *i Minikiscone steep liquor, molasses, meat syrup, polypeptone, casamino acids, etc. are used.In addition, genetically improved Trit-7an synthetase production requires tryptophan or indole for growth, so when culturing, one of these substances must be added to the entire medium. Furthermore, the above nutrients 2#I other than Tatsuju Ji (glucose) must be added in sufficient amounts to the medium at the end of culturing the main production fji so that they do not become limiting substrates. °C, preferably 50-40 °C
&)l of the medium is 5 to 9, preferably 6.
- 8, and it is desirable to keep the sodium hydroxide and potassium chloride constant during the culture using ammonia, etc. The culture should be carried out aerobically, so that the dissolved oxygen in the medium during the culture period does not support growth. It is preferable to carry out aeration and stirring so as not to become a rate-limiting factor.As a method of controlling the glucose concentration in the culture medium of the present invention to a total of 1 or less, the glucose concentration in the culture medium can be controlled by glucose analysis using an immobilized enzyme. Glucose concentration is added so that the glucose concentration does not exceed 1%, preferably does not exceed 0.6%, and more preferably does not exceed αSSt when determined continuously or intermittently using an analytical instrument such as Alternatively, if the relationship between glucose concentration and dissolved oxygen or - is determined in advance, for example, if a correlation is obtained that when glucose concentration is zero, dissolved oxygen or - increases, then dissolved oxygen or - It is also possible to adopt a method in which glucose is added so that its concentration does not exceed 11 Gt when the The present invention is believed to greatly contribute to the industrial production of tryptophan synthetase because enzyme-producing enzymes can be obtained. Escherichia coli MT-10252 and FFRMBF-19, which are tryptophan synthetase producing bacteria, were
0 Qrnl's Sakarov 2 Sco was inoculated into 100 m of a medium with the composition shown in Table 1, and cultured at 65°C for 24 hours.
This culture solution 200rnj (7; y 2 bottles) t-50/
Medium 1 with the composition shown in Table 2 in a jar 7 armer of
51 and cultured at 55°C in PI (ta (adjusted with 28% ammonia water)) ◎The aeration rate and stirring rotation speed were adjusted to wj4 so that the dissolved ill elements in the medium did not fall below 5% of the gun value. Dear friend, I confirmed in advance that dissolved oxygen or - in the culture solution increases when the glucose concentration becomes zero, and at the 4 points when these values start to increase, glucose is added so that it reaches 1%. Repeat this operation until the final amount of glucose added corresponds to the 5% concentration.As a comparative example, at the start of the culture, the glucose concentration was 5- (other medium compositions are the same as in Table 2). The culture medium was used in the summer without adding glucose. Table 1 Ehrlich meat extract 10 g Polypeptone 109 NaCl 51 l/l with distilled water
(pH 6,8) fM 2 Human Glucose 10 9KHIP04
2 11, I(PO42II (NHa)t80a t51
Mg80+ 07 HtO2Ji' Polybegtone 2g Yeast extract 21 Cu(J'2 @2 Hto 1
qMn804” 5 u, o 1
0 qlZn804”7H202lll NalMo04” 2 uto 2
QHJOs
O,5qCaClt” 2 n,o
40 “lCoC1z” 6 u, o
4 ``91'e80a''
7 HtO40”1kecls” 6 Hzo
1 0 119L-) Lipdoan 500 η Adekanol LG805
Use by diluting 1jK with 5 ml distilled water (i"6.
8) After culturing for a certain period of time, the bacterial cells were collected by centrifugation, and the dry image weight and tryptophan synthetase activity were measured. It's 1 o'clock at 55℃! II
J reaction, unit dry g/ka body mass and L- per nest time
) If the result is 90 or more expressed as the amount of liptophan produced, the fourth
0 shown in the table 3. Table Indole 2 - L-serine ta S triton X-
1005-Biridoquinal 5'-phosphate α019g (NHa)
*80* 2.5% bacterial cells (dry matter equivalent) o, o 6--a, 5 Table 4 Example 2 The glucose linearity in the culture solution was continuously quantified, and fkH-
Glucose was continuously supplied so that the concentration of glucose was Q, 5 ill, and the culture was performed until the amount of glucose added ma- tially corresponded to the degree of 5-chain.
The same operations as in tA Example 1 were performed and the results are shown in Table 5.

Table 5 Example 3

Claims (1)

[Claims] Tryptophan synthetase production characterized by continuously or intermittently adding glucose so that the glucose concentration in the medium is maintained at 1% or less - Escherichia coli (H. cbertchiacoli) MT
-10252, or Escherichia coli MT-10
242 culture method)