KR0136642B1 - Novel enterobacter which produce l-leucine from methyl isobutyl ketone and l-leucine producing method - Google Patents

Abstract

The present invention provides a method for producing L-Leucine by oxidizing a methyl ketone material having a carbonyl group in an alpha position such as methyl isobutyl ketone using a microorganism having acetone magnetization and from methyl isobutyl ketone. It relates to a new microorganism that produces L-leucine. That is, acetone is used as a single carbon source to separate growing microorganisms and triton-X-100 is treated with microbial catalysts to enhance cell permeability. It relates to a method of biosynthesis. It is the first manufacturing method to produce el-leucine using a microbial catalyst from methyl isobutyl ketone.

Description

New microorganisms producing L-leucine from methyl isobutyl ketone and a method for producing L-leucine using the microorganisms

The present invention grows using acetone as a single carbon source and grows L-Leucine from methyl isobutyl ketone and amino acid from methyl isobutyl ketone using the microorganism. It relates to a method for producing el-leucine which is a kind of. L-leucine can be made in several ways. The method for synthesizing leucine through organic synthesis is well known. In this case, however, it is mainly made of a mixture of optical isomers of D, L-Leucine, which can then be separated by various methods to obtain only L-leucine. L-leucine is also produced through fermentation using Coryneform bactera. L-leucine is produced industrially by hydrolyzing proteins. In this process, various methods have been developed for separation from similar L-isosolecin (see US Pat. No. 4562153, 4731476, 4820869). Methyl isobutyl In the keto-isocarproic acid represented by the following molecular formula (I) in the oxidized form of the ketone, the carbonyl of the chito-isocaproxane is substituted with an amino group by transaminase of the microorganism. However, a method for producing el-leucine represented by the molecular formula (III) is known. (Biotech. Bioeng., 27 (II): 1616-1619, 1985) However, chito-isocaproxane is an easily obtainable substance at low cost. Because of this, there is a disadvantage that it is not suitable as a raw material. At the same time, there is a disadvantage in that coenzymes must be continuously regenerated in order to supply energy required for the transfer of amino groups. In view of the above problems, the present invention provides a microorganism which biosynthesizes L-leucine represented by the following molecular formula (III) using methyl isobutyl carton represented by the following molecular formula (II) as a starting material. Pure separation from natural soils. Methyl isobutyl ketone is a reduced form of the carbonic acid group of the chito-isocaproxane, and is used as a solvent. Biosynthesis of amino acids directly with butyl ketone. However, the method of producing el-leucine by the microbial catalyst from methyl isobutyl ketone claimed in the present invention is a novel one that has not been reported yet.

The present invention is described in detail as follows. Microorganisms that grow with acetone as the sole carbon source grow by using pyruvic acid produced by oxidizing the methyl group of acetone to turn into acetol, which in turn oxidizes to make acetal, and continue to oxidize it. That is, various components of the cell are synthesized using pyruvic acid synthesized by continuously oxidizing the terminal methyl group. The present invention utilizes the characteristics of the new microorganisms to magnetize acetone and el-leucine which directly synthesizes el-leucine by oxidizing methyl-ketone substances having an alpha-carbonyl group such as methyl isobutyl ketone in the same manner. It relates to a manufacturing method of. Next, the method for producing the present strain will be described. Soil samples from all over Korea were incubated for 2 to 10 days at 30 ° C in a minimum medium containing 0.5% acetone as the only carbon source, and the culture medium was the only carbon source containing 0.5% methyl ethyl ketone. Recultured in minimal medium and plated in agar nutrient medium. Thereafter, microbial colonies (Coloy) grown in nutrient medium were separated and cultured in a minimal medium containing 0.5% of acetone to check acetone magnetization ability. These strains were grown in a minimal medium, where acetone was the only carbon source, and then cultured at 30 ° C with 5% methyl isobutyl ketone. The supernatant was then analyzed by thin layer chromatography (Thin-Layer Chromatography; TSC) to isolate microorganisms producing substances with developmental values, such as leucine. The bacteriological properties of the disclosed strains isolated by the inventors are as follows.

(1) Appearance of colony: yellow, round

(2) Gram dye: Gram negative

(3) mobility: voice

(4) biochemical properties

Growth temperature below 37 ℃

Catalase production positive

Generation voice of indole

Gelatin hydrolytic negative

Positive acid production from cellobiose

Negative acid production from glycerol

Acid production negative from maltose

Acid production negative from mannitol

Positive acid production from Raffinose

Acid production positive from L-Rhamnose

Positive acid production from salicycin

Positive acid production from sucrose

Lysine decarboxylase negative

Arginine dehydroxylase negative

Ornithine decarboxylase negative

Nitrate reduction

Esculin hydrolysis positive

Urea Hydrolysis Negative

Based on the above bacteriological properties, this strain was recognized by Bergy's Systematics of Microbiology, a representative microbial identification method. MKO62) and was deposited on November 18, 1994, with the deposit number KCTC8634P at the Institute of Genetic Engineering, Korea Advanced Institute of Science and Technology.

On the other hand, according to the present invention the culture of the above strain is carried out at pH 4.5-0, preferably at pH 6.0-7.5. At this time, the culture is usually carried out at a temperature of 20-40 ℃, preferably at 25-35 ℃. As a carbon source for growth, acetone is usually used at an initial concentration of 1% (V / V). Hereinafter, the present invention will be described in detail with reference to the production examples of leucine using the present strain Enterobacter MKO62. However, the present invention is not limited to the examples. Analysis of the acetone and methyl isobutyl ketone used was gas chromatography (GS). The generated amino acid was analyzed by chromatography (Thin-Layer Chromatography or High Performance Liquid Chromatography).

Example 1

The cultured test strains were carried out in the following manner to obtain a large amount of cells.

(Badge composition)

Yeast extract 0.1%, potassium phosphate 0.5%, ammonium sulfate 0.5%, magnesium sulfate 0.001% (pressurized by sterilization at a temperature of pH 7.0, 121 ℃ for 15 minutes and added 1% of acetone volume)

(culture)

200 ml of the medium was added to a 2 l winged Erlenmeyer flask, inoculated with Entobacterium MKO62 isolated from the soil, and incubated at 20 ° C. at 200 rpm for 36 hours. The absorbance at 600 nm was recovered at about 2 and the culture was centrifuged at 500 rpm for 20 minutes to remove the supernatant and the cells were frozen at -70 ° C.

(Reaction conditions)

After dissolving the cells with buffer solution (0.1% potassium phosphate, ammonium sulfate 0.5%, sodium chloride 0.01%, pH 7.0), the cells were concentrated and absorbed at 600 nm until the absorbance was about 20. 2% was added and shake cultured at 30 degreeC. Samples are taken every 2 hours while incubating for 6 hours to analyze amino acids, and the productivity of L-leucine is shown in Table 1.

Table 1. Production of L-Leucine (Unit: L-leucine g / l)

Example 2

Under the same reaction conditions, the initial concentration of methyl isobutyl was added to 0%, 1%, 2%, 4%, 8%, 16%, 32% on the dispersed microbial catalyst and reacted at 30 ° C. for 6 hours.

Example 3

It is a type of nonionic surfactant known to increase the permeability of microbial cell membranes such as TRITON X-100, TWEEN80, calcium chloride and E.D.T. After pretreatment, 2% methyl isobutyl ketone was added in the same manner and reacted at 30 ° C. for 6 hours. An increase in production of L-leucine was observed compared to the control that was not pretreated.

Triton X-100 1% (v / v) production of L-leucine increased

Twin 80 1% (v / v) No effect

Calcium chloride (10mM) No effect

EDTA (10mM) No effect

Sodium Dodecyl Sulfate (0.1%) Increased L-Leucine Production

Example 4

0.1%, 0.5%, 1% of Triton X-100 was added to the reaction solution, and the reaction was carried out at 30 ° C. for 6 hours. The increase in production of L-leucine by the addition of Triton X-100 was confirmed as compared to the control.

Claims (5)

Enterocacter sp, MKO62, KCTC8634P, a strain of enterobacter sp. That grows acetone and methyl ethyl ketone as a single carbon source and produces el-leucine from methyl isobutyl ketone A method for producing L-leucine, which obtains L-leucine from methyl isobutyl ketone by culturing the strain Enterobacta genus MKO62 of claim 1. The method for producing L-leucine according to claim 2, wherein the initial concentration of methyl isobutyl ketone is at least 2%. The method for producing L-leucine according to claim 2, wherein any one of Triton X-100 and sodium dodecyl sulfate is used as a substance for increasing microbial cell membrane permeability. The method of claim 4, wherein up to 1% Triton X-100 is added from 0.1% (v / v) to 1% (v / v) to produce L-leucine.