Abstract:
A process for producing L-isoleucine, which comprises culturing a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium which has a methyllysine resistance or α-ketomalonic acid resistance and which is capable of producing L-isoleucine in a liquid medium, and obtaining the accumulated L-isoleucine from said medium.

Description:
BACKGROUND OF TH INVENTION 
     1. Field of the Invention 
     L-Isoleucine is an important component for parenteral and enteral nutrition and complex amino acid preparations. The present invention involves an improvement to a process for producing L-isoleucine by fermentation. 
     2. Description of the Prior Art 
     It is known that in order to impart L-isoleucine productivity to microorganisms belonging to the genus Brevibacterium or the genus Corynebacterium, it may be sufficient for the microorganisms to have reslstances to α-amino-β-hydroxyvaleric acid (hereafter simply referred to as AHV), etc. (Published Examined Japanese Patent Application No. 20316/73, U.S. Pat. No. 3,767,529). It is also known that the productivity of L-isoleucine may be improved by imparting to the microorganisms, in addition to the above-mentioned chemical resistances, O-methylthreonine resistance, β-hydroxyleucine resistance or trichloroalanine resistance, and by imparting thereto auxotrophy for purine type substances or lysine, etc. (Published Examined Japanese Patent Applications Nos. 21077/76, 4629/77, 29998/81 and 3035/81, and Published Unexamined Japanese Patent Application No. 2687/82). 
     From a viewpoint of industrial production, it is of importance to improve the fermentation yield or accumulation of L-isoleucine. 
     SUMMARY OF THE INVENTION 
     The present invention has been made to solve the foregoing problem. As a result of investigations on hitherto known microorganisms belonging to the genus Brevibacterium or the genus Corynebacterium and having L-isoleucine productivity with an attempt to improve these microorganisms and to find strains providing improved fermentation yields, it has been found that among those strains having imparted thereto resistance to methyllysine (hereafter simply referred to as ML) or a α-ketomalonic acid (hereafter simply referred to as α-KM), are found strains having L-isoleucine productivity in a higher yield than with known L-isoleucine-producing bacteria. 
     That is, the present invention relates to a process for producing L-isoleucine, which comprises culturing a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium, having ML resistance or α-KM resistance and capable of producing L-isoleucine in a liquid medium and obtaining the accumulated L-isoleucine from said medium. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Examples of ML&#39;s of the present invention include methyllysines such γ-ML, β-ML, δ-ML, ε-ML, etc. Bacteria having resistance thereto are effective for improving L-isoleucine productivity. 
     The microorganisms used in the present invention are mutants which belong to the genus Brevibacterium or the genus Corynebacterium, possess ML resistance or α-KM resistance and are capable of producing L-isoleucine. 
     To obtain the mutants of the present invention, wild strains described below may first have imparted thereto L-isoleucine productivity and then have imparted thereto ML resistance or α-KM resistance; alternatively, ML resistance or α-KM resistance may be first imparted to the wild strains followed by having L-isoleucine productivity imparted thereto. 
     Examples of the wild strains that are parent strains of the mutants are known as Coryneform L-glutamic acid-producing bacteria belonging to the genus Brevibacterium or the genus Corynebacterium, examples of which are shown below. 
     Brevibacterium lactofermentum ATCC 13869 
     Brevibacterium divaricatum ATCC 14020 
     Brevibacterium saccarolyticum ATCC 14066 
     Brevibacterium flavum ATCC 14067 
     Corynebacterium glutamicum ATCC 13032 
     Corynebacterium acetoamidophilum ATCC 13870. 
     To obtain the mutants of the present invention from these parent strains, a conventional process for mutation and induction, such as by treating the parent strains with N-methyl-N&#39;-nitro-N-nitrosoguanidine, etc. is applicable. Separation of the mutants of the present invention from the bacterial solution obtained by mutation treatment may be carried out by collecting strains that grow on a medium containing ML or α-KM. 
     The relationship between a specific process for mutation of the mutants of the present invention and the degree of growth of strains in the presence of γ-ML or α-KM is shown below. 
     Process for mutation 
     Brevlbacterium flavum AJ 3686 FERM-P 2433, FERM-BP 755 (AHV resistant strain induced from ATCC 14067) and Corytnebacterium glutamicum AJ 12150 FERM-P 7674, FERM-BP 756 (AHV resistant strain induced from ATCC 13032) were grown on bouillon agar slant at 30° C. for 24 hours. The thus grown cells were suspended in M/30 phosphate buffer to a cell concentration of 10 8  to 10 9  /ml. To the cell suspensions, 500 μg/ml of N-methyl-N&#39;-nitro-N-nitrosoguanidine was added and the mixture was maintained at 30° C. for 20 minutes. Then, the system was centrifuged to collect the bacteria. After the bacteria were thoroughly washed with M/30 phosphate buffer, the bacteria were inoculated on medium having the composition shown in Table 1, and were cultured at 31.5° C. for 2 to 10 days. 
     
                       TABLE 1______________________________________Composition of MediumComponent              Content______________________________________Glucose                1.0    g/dlUrea                   0.2    g/dlKH.sub.2 PO.sub.4      0.1    g/dlMgSO.sub.4.7H.sub.2 O  0.1    g/dlFeSO.sub.4.7H.sub.2 O  0.002  g/dlMnSO.sub.4.7H.sub.2 O  0.002  g/dlBiotin                 100    μg/lThiamine hydrochloride 100    μg/lγ-ML or α-KM                  0.2    g/dlAgar                   2.0    g/dl______________________________________ 
    
     From the bacteria that grew in agar medium containing γ-ML, Brevibacterium flavum AJ 12149 FERM-P 7677, FERM-BP 759 (AHV resistance, γ-methyllysine resistance) and Corynebacterium glutamicum AJ 121251 FERM-p 7675, FERM-BP 757 (AHV resistance, γ-methyllysine resistance) were selected as having high productivity of L-isoleucine. 
     Further, from the strains that grew in agar medium containing α-KM, Brevibacterium flavum AJ 12152, FERM-P 7678, FERM-BP 760 (AHV resistance, α-KM resistance) and Corynebacterium glutamicum AJ 12153, FERM-P 7676, FERM-BP 758 (AHV resistance, α-KM resistance) were obtained as strains having high productivity of L-isoleucine. 
     The strain FERM-P was originally deposited on Jan. 8, 1974 and the strains FERM-P 7674, FERM-P 7675, FERM-P 7676, FERM-P 7677 and FERM-P 7678 were deposited on June 22, 1984 at the Fermentation Research Institute, Agency of Industrial Sciences and Technology, Ministry of International Trade and Industry (FRI), 1-3, Migashi 1-chome, Yatabe-machi, Tsukuba-gun, Ibaragi-ken 305, Japan. These deposited strains were then converted into deposits under the Budapest Treaty on Apr. 10, 1985, and were accorded the corresponding FERM-BP numbers. 
     The degree of γ-ML resistance or the degree of α-KM resistance of the thus obtained variants was compared with that of the parent strains. 
     The bacteria obtained by culturing in natural medium (1 g/dl peptone, 1 g/dl yeast extract, 0.5 g/dl NaCl, pH 7.0) slant for 24 hours were suspended in sterilized water. The suspension was inoculated on medium containing 0.5 g/dl glucose, 0.2 g/dl urea, 0.15 g/dl ammonium sulfate, 0.3 g/dl KH 2  PO 4 , 0.1 g/dl K 2  HPO 4 , 0.01 g/dl MgSO 4  ·7H 2  O, 0.1 mg/dl CaCl 2  ·2H 2  O, 100 μg/l biotin, 100 μg/l thiamine hydrochloride, 0.002 g/dl FeSO 4  ·7H 2  O, and γ-ML or α-ML in the amount shown in Table 2, the pH of which was adjusted to 7.0. After culturing for 24 hours, the degree of growth was determined by turbidity. 
     
                                           TABLE 2__________________________________________________________________________        Concentration of                    Concentration of        γ-ML (g/dl)                    α-KM (g/dl)        0  0.05              0.1                0.2                  0.3                    0  0.05                          0.1                            0.2                              0.3Strain       Degree of Growth (%)__________________________________________________________________________Brevibacterium flavum        100           90 58                20                   0                    100                        90                          45                             0                               0AJ 3686FERM-P 2433FERM-BP 755Brevibacterium flavum        100           90 90                85                  80                    -- -- --                            --                              --AJ 12149FERM-P 7677FERM-BP -Brevibacterium flavum        -- -- --                --                  --                    100                       100                          95                            95                              80AJ 12152FERM-P 7678FERM-BP 760Corynebacterium        100           88 75                30                   0                    100                        95                          50                             0                               0glutamicum AJ 12150FERM-P 7674FERM-BP 756Corynebacterium        100           88 85                85                  82                    -- -- --                            --                              --glutamicum AJ 121251FERM-P 7675FERM-BP 757Corynebacterium        -- -- --                --                  --                    100                       100                          90                            90                              85glutamicum AJ 12153FERM-P 7676FERM-BP 758__________________________________________________________________________ 
    
     In most cases, the yield can be further improved by additionally imparting to the aforesaid mutants a property that is already known to improve the productivity of L-isoleucine, such as O-methylthreonine resistance, β-hydroxyisoleucine resistance or trichloroalanine resistance. 
     The medium used for culturing such mutants is a conventional one containing carbon sources, nitrogen sources, inorganic ions, substances for satisfying the above-mentioned auxotrophy, and if necessary, vitamins and other organic trace nutrients. 
     As carbon sources, carbohydrates such as glucose, sucrose, etc., organic acids such as acetic acid, etc. are preferred and as nitrogen sources, ammonia water, ammonia gas, ammonium salts, etc. are preferred. If necessary, potassium ions, sodium ions, magnesium ions, phosphate ions and others are appropriately added to the medium as inorganic ions. 
     It is preferred that the cultivation be conducted under aerobic conditions. When the cultivation is carried out while adjusting the pH of the medium to 4 to 8 and the temperature to 25° to 37° C. during the cultivation, more preferred results are obtained. Cultivation for 1 to 7 days under the conditions produces and accumulates marked amounts of L-isoleucine. L-isoleucine may be harvested from the culture solution in a conventional manner such as using ion exchange resins, etc. 
     The invention now being generally described, the same will be better understood by reference to the following examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof, unless specified. 
     EXAMPLES 
     A medium containing 10 g/dl glucose, 7 g/dl (NH 4 ) 2  SO 4 , 0.1 g/dl KH 2  PO 4 , 0.04 g/dl MgSO 4  ·7H 2  O, 1 mg/dl FeSO 4  ·7H 2  O, 1 mg/dl MnSO 4  ·4H 2  O, CaCl 2  ·2H 2  O, 100 μg/l thiamine hydrochloride, 100 μg/l biotin, 60 mg/dl (as the total nitrogen) soybean protein acid hydrolysate solution and 5 g/dl calcium carbonate (separately sterilized) was adjusted to a pH of 7.0, 20 ml of which was charged in a 500 ml volume-flask followed by thermal sterilization. A platinum loop of the strain shown in Table 1 was inoculated on the medium. While being maintained at 31° C., the system was shaken for 4 days. In the culture solution of each strain, L-isoleucine was accumulated in the amount shown in Table 3. AJ 12149 was cultured in a similar manner to obtain 1 liter of the culture solution. From the culture solution, the bacteria were removed by centrifugation and the supernatant was passed through a strongly acidic ion exchange resin &#34;Diaion&#34; SK-1B (NH +  type). After rinsing the resin with water, elution was conducted with ammonia water. Then the eluate was concentrated and from the concentrate, 17.0 g of crude L-isoleucine crystals were obtained. 
     
                       TABLE 3______________________________________                    Amount of L-isoleucineStrain       Property    accumulated (g/dl)______________________________________Brevibacterium        AHV.sup.r   0.6flavum AJ 3686FERM-P 2433FERM-BP 755Brevibacterium        AHV.sup.r, γ-ML.sup.r                    1.9flavum AJ 12149FERM-P 7677FERM-BP 759Brevibacterium        AHV.sup.r, α-KM.sup.r                    1.8flavum AJ 12152FERM-P 7678FERM-BP 760Corynebacterium        AHV.sup.r   0.5glutamicum AJ 12150FERM-P 7674FERM-BP 756Corynebacterium        AHV.sup.r, γ-ML.sup.r                    1.7glutamicum AJ 12151FERM-P 7675FERM-BP 757Corynebactium        AHV.sup.r, α-KM.sup.r                    1.6glutamicum AJ 12153FERM-P 7676FERM-BP 758______________________________________