JPH044888A - Production of amino acid by fermentation - Google Patents

Abstract

PURPOSE:To obtain an amino acid in high fermentation efficiency by making trehalase exist in a culture solution of microorganism. CONSTITUTION:Trehalase having 0.5-100 units is made to exist in a culture solution of microorganism and the microorganism is aerobically cultured at pH 4-9 at 40-60 deg.C to give the objective amino acid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing amino acids by fermentation.

[Prior Art] It is known that in amino acid fermentation using glucose, sucrose, blackstrap molasses, etc., treno\loss is also produced in the culture solution (Biosci.

Rep,, vol, 5. No, 6. pp509
-515.1985). However, it is not known that this trehalose can be processed or utilized in any way.

[Problem to be solved by the invention]

If trehalose can be assimilated by fermenting bacteria, it is possible to increase the fermentation yield of the target amino acid.

[Means to solve the problem]

As a result of intensive studies to achieve the above object, the inventors of the present invention found that
We have now focused on trehalase, which decomposes trehalose into D-glucose. Furthermore, we succeeded in obtaining a microorganism that significantly produces trehalase, and by adding this trehalase to the culture solution for amino acid fermentation, we can effectively utilize the trehalose produced and accumulated in the culture solution to increase the fermentation yield of the target amino acid. This discovery led to the completion of the present invention.

That is, the present invention relates to a method for producing an amino acid using a microorganism, which is characterized in that trehalase is present in the culture solution of the microorganism.

Trehalase (E, C, 3, 2, 1, 28) is 2.2'
It is an enzyme that decomposes -1-levalose to produce D-glucose, and its existence is known in insects, animals, microorganisms, etc. An inexpensive trehalase is preferable, and from this point of view, the present inventors have developed 1.1 tubelase produced by a bacterium of the genus Corynebacterium (Japanese Unexamined Patent Publication No. 62-275682).
'3) is particularly preferred.

A known method may be used to obtain L.rehalase 4 by culturing Corynebacterium bacteria.

That is, Corynebacterium bacteria capable of producing trehalase are mixed with carbon sources such as glucose, sucrose, and soluble starch, inorganic substances such as ammonium sulfate, ammonium chloride, and ammonium phosphorus, or yeast extract, meat extract, soybean meal hydrolyzate, and peptone. The cells are cultured in a liquid medium containing an organic nitrogen source, trace organic nutrients such as vitamins, and inorganic salts such as potassium phosphate and magnesyl sulfate. The culture conditions may be the usual conditions for culturing bacteria of the genus FERM, but the present inventors have previously developed Corynebacterium sp.
In the case of P-8485), it is a thermophilic bacterium, so the
It is best to culture at around °C. Cultivation is carried out under aeration and agitation according to a conventional method, and is usually terminated when the accumulation of trehalase reaches its maximum or the most economically preferable accumulation amount. After culturing, the culture solution can be used as it is, or just concentrated and used as an enzyme source, or it can be used as an enzyme source by using a known enzyme method such as ultrafiltration, gel filtration, ion exchange chromatography, ammonium sulfate chromatography, etc. It can also be used after further purification by a purification method. It goes without saying that if L-levarase is an intracellular enzyme, the bacterial cells are used, or the bacterial cells are destroyed and the enzyme is extracted for use. The trehalase used in the method of the present invention is not limited to those derived from microorganisms, but may be derived from animals or plants. Alternatively, it may be prepared by genetic engineering techniques or the like.

The type of amino acid fermentation is not particularly limited, and examples include glutamic acid fermentation, lysine fermentation, glutamine fermentation, suI/onine fermentation, amino acid fermentation, phenylalanine fermentation, valine fermentation, isoleucine fermentation, etc. The type of fermenting bacteria is not particularly limited as long as it produces trehalose. So-called coryneform bacteria such as bacteria of the genus Corynebacterium or bacteria of the genus Brevibacterium, such as Corynebacterium lilyum,
The method of the present invention is particularly effective against Corynebacterium glutamate, Brevibacterium lactofermentum, and the like.

The amino acid fermentation method may be carried out according to a known method except for adding trehalase, and the culture medium for culturing these bacteria may be an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and other organic micronutrients as necessary. is used.

Examples of carbon sources include carbohydrates such as glucose, sucrose, fructose, cane molasses, beet molasses, and starch hydrolysates. Suitable nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, and the like. The cultivation is carried out under aerobic conditions, with the temperature usually adjusted between 25°C and 39°C during cultivation control p)I between 4 and 9. However, in order to shorten culture time and prevent bacteria, it is necessary to
It is preferable to carry out fermentation at a certain high temperature, and in that case, a thermostable trehalase whose optimum temperature for production is 40 to 60°C, such as Corynebacterium sp. 1512 strain, is particularly preferable.

1-The amount of levalase added is determined taking into consideration the type and method of each fermentation, the type of trehalase, etc., but is usually 0.5 to 1.
It is about 00 units/-. It may be added to the medium in advance prior to culturing, or it may be added once during culturing.
It may be added at one time or in several parts. Trehalase can also be used in an immobilized state. This immobilized enzyme may be mixed with an immobilized amino acid-fermenting bacterium when used, or it may be used by adding it to a fermenter.

Thus, if the culture is continued for 1 to 7 days, a significant amount of amino acids will be produced in the medium. Amino acids are collected from the culture solution using conventional methods.

[Effect] Trehalose is a non-reducing dicarbon sugar in which 1.1 D-glucose is linked. This reduces the conversion rate of carbon sources such as glucose into amino acids. By treating trehalose produced in the culture solution of amino acid fermentation with trehalase, it is possible to efficiently convert it to glucose and increase the yield of amino acids.

〔Example〕

Example 1 Trehalase is derived from Corynebacterium sp.
Those obtained from 12 strains were used. Its preparation method is as follows.

First, -512 strain (FERM P-8485) was mixed with 1% soluble starch, 1% glucose, 1% polypeptone, and 0.5
% yeast extract, 0.1% to 211P04 and 0.02
The cells were inoculated into a liquid medium containing %Mg5O, 71120, and cultured aerobically at 55°C for 72 hours. The resulting culture solution was centrifuged at 11,000 Orp and 0°C for 10 minutes to remove bacterial cells and obtain a supernatant. Then, the supernatant was concentrated about 10 times using an ultrafiltration membrane with an average molecular weight of 10,000, and dialyzed against 10 mM phosphate buffer (pl+7) overnight. The precipitate generated after this dialysis was removed by centrifugation, and the resulting supernatant was equilibrated with 10 mM phosphate buffer (pH 7).
Adsorbed on EAE-Toyopearl 650M column, 0 to 0
．． The enzyme was eluted using a gradient method using the same buffer as above containing 7M NaCl. The eluted active fractions were collected, concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and further dialyzed overnight against 10 mM phosphate buffer (pH 7). The obtained active fraction was adsorbed onto a DEAE Toyo Pearlvac 6508 column equilibrated with 10 mM phosphate buffer (pH 7), and the enzyme was eluted using a concentration gradient method using the same buffer as above containing 0 to 0.5 M NaCl. I let it happen. The active fraction was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000, and then concentrated using an Asahi Back G5-520 P column equilibrated with the above buffer containing 0.0, IM NaCl. IM
Elution was performed with the above buffer containing NaCl. The obtained active fraction was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 10,000 to a concentration of 400 units/relative to give an enzyme solution.

Glucose 5g/side, Urea 0.4g/d1, KH2P0
．． 0.1g/df! ,,MgSO4・711200.0
4g/d1, FeSO4・711z01mg/company, Mn
SO4・nHzo 1mg/d1, thiamine・)IC
120μg/company, biotin 30μg/dll, soybean protein acid hydrolyzate 90mg#J! (as total nitrogen) was adjusted to pH 7.0, and the 50rd was adjusted to 500d.
It was placed in a shoulder flask and sterilized by heating. In addition to this, Corynebacterium glutamicum AJ 11441 (FERM
P-5138) and incubated at 31.5°C.
The culture was incubated with shaking for 5 hours.

On the other hand, sucrose 20g/a (as total sugar content) KI
I2PO, 0.1g/d1, Mg5040.04g/d
1.FeSO4・711201mg/dl, MnSO
4.4Hz01■/d1, Thiamin・LC120μ
g/a, biotin 30μg/a, surfactant 0.2g/a
and antifoaming agent 0.005d/d1. Soybean protein acid hydrolysis solution 90cm/plane (as total nitrogen), 285m of culture medium adjusted to pt+ 7.0! 1! The mixture was placed in a fermenter and sterilized. After cooling, add 0 or 6.7 units of trehalase.
It was added so that the amount was 100 g/ml. In this case, one unit is 1 μm from trehalose per minute at a reaction temperature of 55°C.
This is the enzyme activity that produces ol glucose.

This was inoculated with the above seed culture solution for 15 days. Culture is 31.
The reaction was carried out at 5°C while maintaining the pl+7.5 with ammonia gas and adding sugar under aeration and stirring. After 48 hours of culture, the amounts of L-glutamic acid and trehalose in each culture solution were as shown in Table 1.

Table 1 Example 2 Trehalase is Corynebacterium sp. L512
strain (FERM P-8485) in the following GS medium.
The cells were cultured at 5°C for 72 hours and purified using ion exchange resin and gel filtration column chromatography.

GS medium 1% glucose 1% soluble starch 1% polypeptone 0.5% yeast extract 0.1% KzllPo.

0.02% Mg5O,・711□0 Using the strain, medium composition, and culture method of Example 1, the above-mentioned toto halase was added to 1 ml of the fermentation solution without addition of trehalase at a final concentration of O and 6.7 units/female. Add it so that it becomes 31
, 5"C for 7 hours. After the reaction, 1.
- The amounts of glutamic acid and trehalose are shown in Table 2.

Table 2 Example 3 Trehalase is Corynebacterium sp.) [-5
A culture solution obtained by culturing 12 strains (FERM p, -8485) (Japanese Unexamined Patent Publication No. 62-275682) was sterile-filtered and used.

Peptone Ig/d1, Yeast Aegis Ig#f, NaCl
O, 5g/mi, glucose 0.5 (H/mi, agar 2g
7 dl, pH 7.0 solid plate of 1-
Novibacterium rough 1-fermentum J 34
24 (FERM P-1711), temperature 31
．． The cells were cultured at 5°C for 24 hours to obtain seed germ cells.

On the other hand, glucose 10g/di, ammonium sulfate 5.5g/d1,
ToeSo, -711z0 1mg/d, Mn5Os ・
4H201mg/di! , 1(Il, PO40, 1g/
d, MgSO40, 1g/mi, thiamine lIc12
0μg/d1, biotin 50μg/d1, nicotinamide 0.5mg/Li1, soy protein acid hydrolysis 105■
20 d of culture medium adjusted to pl+ 8.0 by adding /mi (as total nitrogen) was placed in a 500 mp shoulder flask and heat sterilized 1-. Separately sterilized CaC0:+Ig was added to this to prepare a medium for lysine production. After inoculating 1 platinum bud of seed germ cells into this lysine production medium, the aforementioned trehalase, which had been sterilized with a Millipore filter, was added to a final concentration of O and 6.
It was added at the start of the culture and at 24 hours so that the amount was 20%)/d, and cultured with shaking at 31.5°C for 48 hours. 1 in each culture solution. - The amounts of lysine and trehalose are shown in Table 3.

Table 3 [Effects of the Invention] According to the present invention, the fermentation yield of amino acids can be increased.

Claims (1)

[Claims] A method for producing an amino acid using a microorganism, the method comprising making trehalase exist in the culture solution of the microorganism.