JPS5813150B2 - Hatsukouhou Niyor El - Isoleusinno Seizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-inleucine from methanol by fermentation.

More specifically, it belongs to the genus Protaminobacter (according to the classification of Virgie's Manual of Bacteriology, 7th edition), has methanol assimilation ability, and has the ability to assimilate L-lysine and L-lysine.
At least one of homoserine, L-methionine, L-threonine, L-inleucine, L-leucine, L-valine, α-aminobutyric acid or analogs of these amino acids
A mutant strain having resistance to
Production of L-inleucine by a fermentation method characterized by inoculating and culturing a medium containing appropriate amounts of inorganic substances and other nutrients to produce and accumulate L-isoleucine in the culture solution, and then isolating and collecting it from the culture. It is about law.

The purpose is to industrially produce L-isoleucine, which is an essential amino acid and has a wide range of uses in human and animal nutrition, feed, and medicine, at low cost and in large quantities.

Conventionally, methods for producing L-inleucine by fermentation have relied primarily on carbohydrates (mainly blackstrap molasses) as the carbon source.

However, when carbohydrates are used, their production volume and price are unstable, and in addition, waste liquid treatment is often required, and these disadvantages affect the economic efficiency of L-isoleucine production.

From this point of view, it is considered advantageous to use carbon sources other than carbohydrates, such as methanol and acetic acid, and among them, methanol, which can be obtained in large quantities and at low cost through the organic synthetic chemical industry, is selected as the main carbon source. It is considered particularly advantageous.

Regarding the production method of L-inleucine using methanol as the main carbon source,
Method using bacteria of the genus Bacillus, Plevibacterium, Micrococcus, and Sarcina (Special Publication No. 45-252
73, JP-A-48-98092) is known, but L
- The production amount of iso-isin is at most 0.1 g/l, which cannot be said to be sufficient for industrial scale production.

The present inventors have invented a method for producing L-isoleucine and/or L-leucine from methanol using bacteria of the genus Protaminobacter and Methanonas, and are currently filing an application (Japanese Patent Application No. 85867/1983).

As a result of further improvement research on this inventive method, we found that Protaminobacter bacteria include L-lysine, L-homoserine, L-methionine, L-threonine, L-valine, L-inleucine, L-leucine, When a mutant strain with resistance to α-aminobutyric acid or one or more of these amino acid analogs is cultured in a medium with methanol as the main carbon source, the amount of isoleucine accumulates in the medium and the amount of isoleucine increases dramatically. The present invention has been completed based on the discovery that the

Until now, there has been no example of using a mutant strain endowed with the above-mentioned resistance properties in a method for producing L-isoleucine that uses methanol as the main carbon source, and there is no example of using a mutant strain that has been endowed with the above-mentioned resistance properties. I've never seen one.

The present invention is based on such novel findings.

The present invention will be explained in detail below.

The microorganisms used in the present invention belong to the genus Protaminobacter and have the ability to assimilate L-lysine, L-homoserine, L-methionine, L-threonine, L-inleucine, L-leucine, and L-valine. , α-aminobutyric acid, or one or more of these amino acid analogs.

Such mutant strains can be obtained by subjecting bacteria belonging to the genus Protaminobacter to mutation treatments such as ultraviolet irradiation, γ-ray irradiation, drug treatment, etc. using known methods, and by using an appropriate known selection method in combination. can.

As analogs of the above-mentioned amino acids, various analogs already known for microorganisms other than the genus Protaminohacter can be used.

Typical analogs among these include L-lysine analogs such as S-(β-aminoethyl)-L-cysteine, hydroxylysine, lysine hydroxamer, D-lysine, and 4-azaridine. L-homoserine analogs include hydroxylamine and D-homoserine; L-methionine analogs include ethionine, trifluoromethionine, selenomethionine, and α-methylmethionine; L-threonine analogs include α-amino-β-hydrokine valerate; L-inleucine analogs include threonine hydroxamate, β-hydroxynorleucine, β-hydroxynorvaline, and D-threonine; L-inleucine analogs include inleucine hydroxamate, O-methylthreonine, α-amino-β-methylmercaptoylbutyric acid, Examples of L-leucine analogs, such as D-aloin, include norleucine, norvaline, leucine hydroxamate, D-leucine, and trifluoroleucine; examples of L-valine analogs include valine hydroxamate, D-valine, and the like.

Suitable examples of microorganisms used in the present invention include the strains shown in Table 1.

In the table, the analog resistance abbreviations have the following meanings.

Hse, L-homoserine: H-Lys, hydroxylysine; Eth, ethionine: Thr, L-threonine: α
AB, a-aminobutyric acid; S-Ile, thiaisoleucine; AHV1 α-amino-β-hydroxyvaleric acid; OM
T10-methylthreonine: Nle1 norleucine; N
Va, norvaline;

The number in brackets indicates the concentration of analog at which the mutant strain was obtained, indicating that the mutant strain is resistant to at least this concentration of analog.

The parent strain (wild strain) of the above mutant strain is Protaminobacter ruber KY4064 (Feikoken Bacteriology No. 2903), and its mycological properties are described in Purge Aids Manual of Determinative Bacteriology, 7th edition 201-202.
There is a description on the page.

In addition, as a method of mutation treatment, 108ce11s/m
Add NTG (N-methyl-N'-12tro-N-nitrosoguanidine) dissolved in 0.01M phosphate buffer to a suspension of the parent strain at a final concentration of 0.5 mg/ml and incubate in a room. The treatment was carried out for 30 minutes.

As shown in the examples, the medium composition used in the present invention may be a synthetic medium or a natural medium as long as it contains methanol as the main carbon source, an assimilable nitrogen source, inorganic substances, and other nutrients in a suitable amount. Either can be used.

Methanol, which is used as the main carbon source, may inhibit the growth of microorganisms if used at high concentrations from the early stage of culture. Sequential additions of 0.1 to 3%) often give good results.

Nitrogen sources for the culture medium include ammonium salts of various inorganic and organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium nitrate, and ammonium acetate.
or ammonia, urea, amines, and other nitrogen-containing compounds, as well as peftone, NZ-amine, meat extract, corn steep liquor, yeast extract, casein hydrolyzate, cox hydrolyzate, fish meal or its digest, defatted soybean. Alternatively, various nitrogenous organic substances such as digested products thereof can be used.

Furthermore, as inorganic substances, potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate,
Manganese sulfate, calcium carbonate, etc. are used.

Of course, if the microorganisms used in the present invention require specific nutrients for growth, appropriate amounts of those nutrients must be present in the medium.

However, this type of nutrient may be added as being included in the nitrogenous organic substance exemplified as the nitrogen source, and in such cases there is no particular need to add it.

Culture is performed under aerobic conditions such as shaking or deep aeration.

Culture humidity is usually in the range of 20 to 40°C, and the pH of the medium is 3.
Although it is desirable to maintain the temperature in the range of ~9, preferably around neutrality, it is possible to carry out the experiment under other humidity conditions or pH conditions as long as the bacteria used grow.

The purpose of adjusting the pH of the culture medium is achieved by adding calcium carbonate, a pH buffer, or an acid or alkaline solution, but depending on the strain used, pH adjustment may not be necessary.

The culture period is usually 2 to 7 days, and L-isoleucine is produced and accumulated in the culture solution.

After the culture is completed, the bacterial cells and precipitates such as calcium carbonate are removed, and L-isoleucine is recovered from the culture by treatment with an ion exchange resin as shown in Examples.

It can also be recovered by using other known ion exchange resin treatment methods, concentration methods, adsorption methods, and the like.

Hereinafter, the present invention will be specifically illustrated by giving examples.

Example 1 Protaminobacter ruber ST-14K as inoculum
Y7859 (Feikoken Bibori No. 2906) was used.

This strain is a mutant strain obtained as a strain 7 that can grow in a medium containing 1 mg/ml of L-threonine.

(Growth of the parent strain was completely inhibited in the presence of 50 μg/ml of L-threonine.

) Methanol 20ml, (NH4)2HPO47.5g
, K2HP041g, NaCI0.1g, MgSO, -
7H20, 0.5g, FeSO4・7H2010■, M
nS04/nH20107%, CaCl210m9, phenolette (pH indicator) 10mg and CaCO
s 309 dissolved in 1 liter of distilled water (pH 7.1)
) This inoculum was inoculated into a 50 ml thick test tube containing 5 ml, and cultured with shaking at 30° C. for 90 hours.

At this time, 2 ml/dl of methanol (total 6 ml/dl) and 0.1 g/dl of urea were added at 40, 48, and 64 hours after the start of culture.

At this time, L-inleucine was produced and accumulated in the culture solution at a concentration of 0.9 g/l.

After culturing, remove bacterial cells, carunium carbonate, and other precipitates from 500 ml of the culture solution, and pass the F solution through a column of strongly acidic cation exchange resin Diaion SKI (H type 10) (manufactured by Mitsubishi Kasei Corporation) to L- Isoleucine is adsorbed and after washing with water, 0.
The L-isoleucine fraction was collected by elution with 5N aqueous ammonia, concentrated, and crystallized at an isoelectric point of pH 6.02 to obtain 310 mg of L-isoleucine with a purity level of 99.

Example 2 Protaminobacter ruber KY4064 as inoculum
Various analog-resistant mutant strains derived from the following were used.

Corn steep liquor was added to the medium used in Example 1.
When shaking culture was carried out in the same manner as in Example 1 except that a medium (pH 7.1) supplemented with 0.5% was used, L-isoloin was produced and accumulated in the culture solution as shown in Table 2. Ta.

For comparison, the results of inleucine production of the parent strain are also shown.

Claims (1)

[Claims] 1 Belongs to the genus Protaminobacter and has methanol assimilation ability, L-homoserine, hydroxylysine, ethionine, L-threonine, α-aminobutyric acid, thiaisoleucine, α-amino-β-hydroxyvaleric acid, 0-methyl A mutant strain having resistance to at least one of sluonine, norleucine, or norvaline and having the ability to produce L-inleucine is cultured in a medium containing methanol as the main carbon source to produce and accumulate L-isoleucine. A method for producing L-inleucine by a fermentation method, which comprises isolating and collecting L-inleucine from a culture.