Art
[1]
The present application relates to a process for the preparation of L- leucine using this microorganism and having a production capacity of L- leucine.[2]
BACKGROUND [3]
Branched-chain amino acids (branched-chain amino acid) is L- valine, L- leucine, refers to the three kinds of L- isoleucine, known to be primarily metabolized in the muscle used as an energy source during the activity. As the branched-chain amino acids are known to play an important role in muscle maintenance and increase activity increases during its usage. In particular, the L- leucine or the like are widely used pharmaceuticals, foods, feed additives and industrial chemicals as a sort of essential amino acids.
[4]
On the other hand, the production of branched-chain amino acid using a microorganism, primarily Escherichia spp or Corynebacterium is done through Solarium spp, Kane several steps from pyruvate toy small caproic acid (2-ketoisocaproate) the biosynthesis as a precursor it is known that [Republic of Korea Patent No. 10-0220018 call, the Republic of Korea Patent No. 10-0438146 call. However, the enzyme involved in the biosynthesis of leucine are to the feedback inhibition by the final product of L- leucine, or derivatives thereof, there has been caused a problem in mass producing the L- leucine industrially.
[5]
Detailed Description of the Invention
SUMMARY
[6]
The present inventors has tolerance to example efforts result, a norleucine the mutants obtained using a glutamic acid-producing microorganism leucine derivative (Norleucine, NL) to develop L- leucine-producing microorganism yield is better than the conventional strain L- a leucine or a feedback inhibition of the derivatives thereof is turned off, thereby completing the present application discovered that by producing L- leucine in high yield.
[7]
Problem solving means
[8]
One object of the present application is Corynebacterium compartment (having a novel L- leucine-producing ability Corynebacterium glutamicum to provide a) mutants.
[9]
It is another object of the present application is to provide a process for preparing L- leucine by using the mutant strain.
[10]
Effects of the Invention
[11]
Coryneform microorganisms of the present application, L- leucine and did not receive the feedback inhibition of L- leucine by having a resistance to a derivative thereof, is improved compared to the microbial production ability of the L- leucine parent strain. Accordingly, with the L- leucine production method of the present application of using the microorganism, a high efficiency and high L- leucine can be produced in a high yield.
[12]
Brief Description of the Drawings
[13]
1 is a diagram showing the biosynthesis of L- leucine The final product of the present application.
[14]
Best Mode for Carrying Out the Invention
[15]
This application is In some embodiments, Corynebacterium compartment (having a novel L- leucine-producing ability Corynebacterium glutamicum provides a) mutants. More specifically, it provides for the production of L- leucine Corynebacterium Com mutant KCCM11661P or KCCM11662P.
[16]
The term, "L- leucine" in the present application are structurally one of the essential amino acids, L- valine, L- isoleucine with the formula HO for the branched-chain amino acids 2 CCH (NH 2 ) CH 2 CH (CH 3 ) 2 wherein L - refers to the amino acid.
[17]
On the other hand, lactic acid acetonide (acetolactic acid), dihydroxy isovaleric acid (dihydroxy isovaleric acid), Kane small toy acid (ketoisovaleric acid) from pyruvic acid via the synthesis process shown in FIG. 1 biosynthesis of L- leucine in the microorganism, 2-isopropyl-known to be biosynthesized by way of the acid (isopropylmalic acid), 3- isopropyl-malate (isopropylmalic acid), Kane toy small caproic acid (isocaproic acid). In addition, this biosynthetic process are acetonitrile hydroxy acid synthase (acetohydroxy acid synthase), acetonitrile-hydroxy acid isopropyl merori reductase kinase (acetohydroxy acid isomeroreductase), dihydroxy acid-dihydroxy Alpharetta claim (dihydroxy acid dehydratase), isopropyl malate synthase the (isopropylmalic acid synthase), isopropyl malate dihydric Alpharetta claim (isopropylmalic acid dehydratase), isopropyl malate dehydrogenase now (isopropylmalic acid dehydrogenase), transaminase B is a catalyst by the enzyme, such as (transaminase B) biosynthesis do. However, these enzymes are the branched-chain amino acids, i.e., L- valine, L- isoleucine, L- leucine There is for producing industrially a branched chain of one amino acid by the fermentation, since the same procedure used for the synthesis difficult. In addition, the feedback inhibition by the final product of L- leucine, or derivatives thereof, there has been caused a problem in mass producing the L- leucine industrially. Thus, the mutant strain of the present application may have a resistance to L- leucine, or derivatives thereof.
[18]
Term in this application, the terms "derivative" may refer to the well-known that can inhibit the production capacity of L- leucine from a microorganism to cause a feedback inhibition relative to the biosynthesis of L- leucine The final product of the present application compounds. Examples isoleucine (isoleucine), Terre leucine (terleucine), norleucine (norleucine), but the like cycloalkyl leucine (cycloleucine), but is not limited to this. Specifically, the mutant strain may have a resistance to one or more materials selected from the group consisting of leucine, isoleucine, leucine Termini, norleucine and leucine cycloalkyl, may have resistance to norleucine more specifically.
[19]
Typically, when more than a certain concentration L- leucine accumulation in cells known to be the L- leucine biosynthesis inhibition. Accordingly, the strain resistant to the derivative is the inhibition by L- leucine is turned off may be in high concentrations of L- leucine to generate L- leucine.
[20]
Microorganism having a production capacity of L- leucine of the present application is to obtain a desired mutant strain by mutation of the parent strain. At this time, the mutagenesis of microorganisms can be used for a method of more widespread, and can be carried out by various means as is known, physical or chemical mutagenesis in the art. For example, N- methyl chemical mutagens suitable for the present application -N'- nitro -N- nitrosoguanidine (N-Methyl-N'-nitro-N-nitrosoguanidine, NTG), diepoxy butane, ethyl methane sulfonate, but you can use the mustard compounds, hydrazine, and nitrous acid, but is not limited to these compounds. In addition, physical mutagens is not available, but the ultraviolet and gamma radiation, limited.
[21]
When the parent strain to mutagenesis is affected by the mutation inducer at a concentration of about to leave the surviving populations of a particular size. The size varies depending on the type of mutagenesis factor, it depends on the amount of mutations a mutation factor is induced in surviving population at a constant rate of killing. For example, the killing rate for NTG can place leave approximately a 10% to 50% of the starting population. Mutagenesis by nitrous acid is not, and can have approximately the left as a 0.01% to 0.1% of the starting population, mutations caused by UV light, but can leave approximately 1.0% of this limit.
[22]
The present application, and as another aspect provides a process for preparing L- leucine recovering the L- leucine from step and the mutant strain or its culture culturing said mutant strain.
[23]
The term "culture" in this application means that the growth in the environmental conditions which control the microorganisms with artificially appropriately. How to culture the microorganism of the present application are known Corynebacterium compartment (in the art Corynebacterium glutamicum can be performed using a culture method). Specifically, in the example of the culture method, but includes the batch culture (batch culture), continuous culturing (continuous culture), and fed-batch culture (fed-batch culture), but is not limited to this. These various methods, for example, are disclosed in "Biochemical Engineering" (James M. Lee , Prentice-Hall International Editions, pp138-176, 1991).
[24]
Terms of this application, "culture" refers to a material containing the growth or the growth medium being completed under the environmental conditions moderately artificially control microorganism. In a narrow sense, but not include the growth of the microorganism culture, the light means can be included. The "culture" can contain a leucine in a microorganism specifically target substance, and contains a variety of substances was discharged into the medium during the growth with the composition of medium components for culturing microorganisms.
[25]
The medium used to culture should meet the requirements of the particular strains in a suitable manner. There are four Corey culture medium for bacterial strains is known (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981). Which can be used members include glucose, saccharose, lactose, paroxetine lactose, maltose, starch, cellulose and and carbohydrates, soybean oil, sunflower oil as per, castor oil, oils and fats such as coconut oil, palmitic acid, stearic acid, , an alcohol such as a fatty acid, glycerol and ethanol, such as linoleic acid, may be included the organic acids such as acetic acid, but is not limited thereto. These materials may be used in separation or in combination. The nitrogen source which can be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, but may include the ammonium carbonate and ammonium nitrate, this is not limited. Nitrogen sources may also be used separately or as a mixture. Personnel to that may be used include phosphate, sodium or potassium phosphate or the corresponding potassium susoyi-containing salt may be included it is, not limited to this. Also, culture media may contain metal salts such as magnesium sulfate or iron sulfate needed for growth. In addition, in addition to the above material it may include essential growth substances such as amino acids and vitamins. Also may be used, suitable precursors to the culture medium. The above described material may be added in a batch or continuous system by a suitable method to the culture in culture.
[26]
On the other hand, using an acid compound such as phosphoric acid or sulfuric acid or a basic compound such as sodium hydroxide, potassium hydroxide, ammonia in a suitable manner it is possible to adjust the pH of water culture. It is also possible to use anti-foaming agents such as fatty acid polyglycol ester can suppress foam generation. Oxygen or oxygen into the culture in order to maintain aerobic condition-containing gas (e.g., air) injection. Incubation temperature of water may be usually 20 ℃ to 45 ℃. The culture is continued until the desired amount of Leucine L- obtained the maximum. For this purpose can be achieved in a normal 10 to 160 hours. L- leucine can be included in, or released into the culture medium, cells.
[27]
A method for recovering L- leucine that can be used in this application method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, etc. could be used, but is not limited to these examples.
[28]
Mode for the Invention
[29]
It will be described in detail below by the following examples of the instant application. However, it will be limited by the examples of this disclosure, it may make the present application to illustrate the present application.
[30]
[31]
Example 1: Artificial mutations law mutants by screening
[32]
Using the same method, and to get to the mutant microorganisms having the ability of producing L- leucine induced mutation of microorganisms.
[33]
Specifically, Corynebacterium Com ATCC 14067 and Corynebacterium Com ATCC for the strains activated by incubation for 16 hours in the activation medium 13869 sterilized at 121 ℃ 5 bungan one kinds of producing parent strain of glutamic acid It was inoculated into the medium for 14 hours to recover the culture then the culture medium 5 ㎖. The collected culture solution 100 mM citrate buffer solution (citric buffer) to a three and then, after the addition of NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) so to have a final concentration of 200 mg / L, processing for 20 minutes and washed with 100 mM phosphate buffer solution (phosphate buffer). To smear the strains treated with NTG in a minimal medium results mortality rate ever calculating the mortality rate was 85%.
[34]
Norleucine for the derivatives of the L- leucine in order to screen the mutants for resistance (Norleucine, NL), so the NTG is 20 mM, 30 mM a processing strain, each have a final concentration of NL, 40 mM and 50 mM is added the spread on a minimal medium, and cultured at 30 ℃ 5 ilgan acquires NL-resistant mutants.
[35]
[36]
Mutants obtained by the above method is Corynebacterium Com KCJ-24 ( Corynebacterium glutamicum KCJ-24) and Corynebacterium Com KCJ-28 ( Corynebacterium glutamicum KCJ-28) La was named, January 2015 22 date of accession to the international deposit agency, Korea Culture Center of microorganisms under the Budapest Treaty and were each given an accession number and KCCM11661P KCCM11662P.
[37]
[38]
Embodiment the composition of the medium used in Example 1 and 2 is as follows.
[39]
[40]
< Enabled badges >
[41]
Juicy 1%, polypeptone 1%, 0.5% sodium chloride, 1% yeast extract, 2% agar, pH 7.2
[42]
<종배지>
[43]
Glucose 5%, Bacto peptone 1%, sodium chloride 0.25%, 1% yeast extract, urea 0.4%, pH 7.2
[44]
< Minimal medium >
[45]
Glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium phosphate 0.1%, 1, 0.1% urea, 0.001% thiamine, biotin 200 ㎍ / L, 2% agar, pH 7.0
[46]
[47]
Example 2: L- leucine L- of the mutant for the production of leucine productivity irradiation
[48]
[49]
The embodiments were cultured in the same way as for Example 1 to a high concentration obtained in the four NL Corey resistant mutants tumefaciens glue Tommy KCJ Com-24, and Corynebacterium to determine the L- leucine productivity KCJ Com-28.
[50]
Species 250 ㎖ containing medium 25 ㎖ corner-Cory four parent strain in bapul flask tumefaciens glue Tommy Com ATCC 14067 ( Corynebacterium glutamicum ATCC 14067), Corynebacterium Com ATCC 13869 ( Corynebacterium glutamicum ATCC 13869), and the after inoculation the two kinds of mutant, respectively, at 30 ℃ for 20 hours by shaking culture at 200 rpm to give a seed culture. Then, to 250 ㎖ corner containing the production medium in 24 ㎖-inoculated with the seed culture in 1 ㎖ in bapul flask, and cultured at 30 ℃ at 200 rpm for 72 hours to prepare a L- leucine.
[51]
[52]
The composition of the production medium used in this Example 2 were as follows.
[53]
[54]
< Production medium >
[55]
5% glucose, 2% ammonium sulfate, the first 0.1% of potassium phosphate, magnesium sulfate heptahydrate 0.05%, 2.0% CSL (corn steep liquor), biotin 200 ㎍ / L, pH 7.2
[56]
[57]
After the completion of the incubation, the production of L- leucine was measured by using a high-speed liquid chromatography, to the experiments L- leucine concentration in the culture medium of each strain are shown in Table 1 below.
[58]
[59]
[Table 1] Corynebacterium KCJ Com-24, and Corynebacterium Com L- leucine Productivity of KCJ-28
Corynebacterium Com ATCC 14067 (parent strain) Corynebacterium Com KCJ-24 (mutant) Corynebacterium Com ATCC 13869 (parent strain) Corynebacterium Com KCJ-28 (mutant)
L- leucine concentration (g / L) 0.1 2.7 0.3 3.1

[60]
[61]
As a result, as, although the parent strain Cory four produce L- leucine in tumefaciens glue Tommy Com ATCC 14067 and Corynebacterium Com ATCC 13869 in a concentration of 0.1 and 0.3 g / L, respectively shown in Table 1 , by the present application mutant Corynebacterium KCJ Com-24 and Corynebacterium KCJ Com-28 according to the producing L- leucine at a concentration of 2.7, 3.1g / L, from about than the parental strain 10 times it was confirmed that the L- leucine productivity increased.
[62]
The results of the above means that the mutants having a resistance to leucine and L- norleucine consequently did not receive the feedback inhibition for leucine or derivatives thereof, capable of producing L- leucine in high efficiency and high yield.

Claims
[Claim 1]
Accession No. KCCM11661P mutant and accession No. KCCM11662P Corynebacterium compartment (with L- leucine-producing ability is selected from the group consisting of a mutant strain Corynebacterium glutamicum ) mutants.
[Claim 2]
The method of claim 1, wherein the mutant strain is a mutant strain that has a resistance to L- leucine and derivatives thereof.
[Claim 3]
A mutant strain according to claim 2, wherein the L- leucine derivative norleucine (Norleucine, NL).
[Claim 4]
The method comprising culturing the mutant strain of claim 1 and a method for producing L- leucine recovering the L- leucine from the mutant strain or its culture.