JP3653766B2 - Production method of ε-poly-L-lysine - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a method for producing ε-poly-L-lysine (hereinafter abbreviated as εPL) with improved yield to carbon source.
Since εPL is a polymer of L-lysine, which is an essential amino acid, it is highly safe and has a high cation content, so it has unique physical properties. Therefore, it is not only widely used as a food additive, but also can be expected to be used for toiletries, cosmetics, feed additives, medicines, agricultural chemicals, electronic materials and the like.
[0002]
[Prior art]
A method for producing εPL using Streptomyces albulus species microorganisms is disclosed in Japanese Patent Publication No. 59-20359. Streptomyces albulus subsp. Lysinopolymerus strains are resistant to L-lysine analogs. A method for producing εPL is characterized in that the mutant strain obtained by mutating the mutant strain is cultured in a medium, εPL is produced and accumulated in the culture solution, and this is collected. A medium in which L-lysine is added to a strain that produces εPL obtained by amplifying a plasmid containing a gene involved in εPL production using Chloramphenicol from a Streptomyces alblus subspecies lysinopolymelas strain A method for producing εPL, characterized in that εPL is produced and accumulated in the culture medium and collected. It is described in Rights 3-42075 JP. However, although the medium used for the above-described production method of εPL contains L-lysine, ammonium sulfate and phosphate, the concentration of phosphate is high and the concentration of L-lysine in the culture solution is controlled. There was no manufacturing method. Therefore, since a large amount of carbon source in the medium is consumed for the production of by-products other than εPL, the yield of carbon source in εPL production is poor, and when considering industrial production, the production cost is high. There was a point.
[0003]
[Problems to be solved by the invention]
As a result of intensive studies on a method for producing εPL with a good yield relative to the carbon source, the present inventors have cultivated Streptomyces albula spp. By limiting the concentration and using a medium containing a certain amount of L-lysine and ammonium sulfate in the medium, or controlling the concentration of L-lysine in the culture medium, the production of by-products can be suppressed. , ΕPL was found to improve the carbon source yield, and the present invention was made based on this finding.
An object of the present invention is to provide an inexpensive and advantageous production method of εPL by suppressing the amount of carbon source consumed by by-products other than εPL and improving the yield of εPL with respect to the carbon source. .
[0004]
[Means for Solving the Problems]
The present invention has the following configuration.
(1) In a method in which Streptomyces albulus species microorganisms are aerobically cultured in a medium and ε-poly-L-lysine is collected from the obtained culture solution, the concentration of phosphate in the medium Is 50 to 300 mg / l, the content of L-lysine is 2 to 20 g / l, and the content of ammonium sulfate is 10 to 20 g / l.
(2) A method of aerobically culturing Streptomyces albulus species microorganisms in the medium described in (1) above and collecting ε-poly-L-lysine from the obtained culture solution , Wherein L-lysine is added to the culture solution sequentially or continuously so that the L-lysine content in the culture solution is 1 to 10 g / l. .
[0005]
(3) Either of the above paragraph (1) or (2), wherein the strain producing ε-poly-L-lysine is Streptomyces albulus subsp. Lysinopolymerus (Streptomyces albulus subsp. Lysinopolymerus) A method for producing ε-poly-L-lysine according to item 1.
(4) A strain producing ε-poly-L-lysine added glycine to S-aminoethyl-L cysteine of Streptomyces albulus subsp. Lysinopolymerus No346-D strain The method for producing ε-poly-L-lysine according to any one of (1) or (2) above, which is a mutant strain 11011A-1 having a resistance to the above (Microtechnical Laboratories No. 1109) .
[0006]
Any microorganism can be used in the present invention as long as it is a Streptomyces albulus strain that accumulates εPL in the fermentation broth, and in particular, Streptomyces albulus subspices lysinopolymelas (Streptomyces albulus). subsp. lysinopolymerus) 11011A-1 strain (Mikken Kenjo No. 1109) and Streptomyces albulus subsp. lysinopolymerus No346-D strain are preferred.
[0007]
The content of L-lysine in the medium used in the present invention is 2 to 20 g / l, preferably 5 to 10 g / l. When the content of L-lysine is small, the production amount of εPL decreases. Moreover, when there is too much content of L-lysine, the proliferation of a microbe will be suppressed. Further, L-lysine used in the medium may be in the form of hydrochloride or other salt. The content of ammonium sulfate in the medium used in the present invention is 10 to 20 g / l . In a medium not containing ammonium sulfate, the production amount of εPL is significantly reduced. When the ammonium sulfate content in the culture medium is lowered during the culture, it is preferable to add so that the ammonium sulfate content is 2 to 20 g / l.
[0008]
The phosphoric acid component added to the medium may be in any form such as potassium phosphate, sodium phosphate, phosphate contained in yeast.
The content of the phosphoric acid contained in the medium is 50 to 300 mg / l, preferably 50 to 150 mg / l, and is limited to about 1/5 to 1/30 compared with a normal medium. If the phosphoric acid content is high, the production of by-products increases, and if the content is low, the growth of microorganisms is affected.
[0009]
In addition to the above-mentioned L-lysine, ammonium sulfate, and phosphate, a carbon source and a mineral are used for the medium used in the present invention. The carbon source is preferably glucose, but is not limited as long as it can assimilate εPL-producing bacteria such as fructose, glycerin, and starch, and the carbon source is contained in the medium. The amount is preferably 10 to 50 g / l. When the carbon source content of the culture solution decreases during the culture, the carbon source content is added so as to be 10 to 50 g / l. Examples of the mineral include potassium ion, magnesium ion, zinc ion, iron ion and the like, and these are preferably contained in the medium in the range of 0.01 to 1 g / l. Further, when yeast extract is contained in the medium in an amount of 1 to 5 g / l, the growth of the bacteria is improved, and preferable results are obtained in the production of εPL.
[0010]
When L-lysine is added sequentially, it is added so that the content of L-lysine in the culture solution becomes 1 to 10 g / l when it becomes 1 g / l or less, for example. Moreover, when adding continuously, it adds so that L-lysine in a culture solution may always become 1-10 g / l .
[0011]
The culture is performed under aerobic conditions such as shaking culture and stirring culture. The culture temperature is preferably 25 to 30 ° C. The pH of the medium is preferably near neutral (pH 6-8), but the pH decreases with the growth of bacteria after the start of culture. Since the pH of the medium is preferably around 4 for the production of εPL, alkali is added to maintain the pH at 4 when the pH of the culture solution falls to 4 or less. The alkali to be added is preferably ammonia water, but it may be an aqueous solution of sodium hydroxide or potassium hydroxide. Usually, εPL accumulates in the culture solution in 1 to 5 days.
[0012]
After removing the cells from the culture solution by centrifugation or filter, the cell-removed solution is purified, decolorized, and concentrated. ΕPL is obtained by crystallization from an organic solvent such as acetone or ethanol from the concentrated solution.
[0013]
Examples The present invention will be described in more detail than examples.
εPL concentration, residual glucose concentration, and residual L-lysine concentration were measured by the following methods.
(1) The εPL concentration was measured by the method of Itzhaki (Analytical Biochemistry, 50, 569). That is, 2 ml of a solution containing 0 to 200 μg of εPL and 2 ml of 1 mM methyl orange aqueous solution were mixed and allowed to stand at room temperature for 30 minutes. The resulting εPL-methyl orange complex was removed by centrifugation, and the absorbance of the supernatant water at 465 nm was determined. Measurement was made to determine the amount of εPL.
[0014]
(2) Residual glucose concentration was determined by mutarotase / glucose oxidase method, that is, 20 μl of sample (containing 0-100 μg of glucose) and a coloring reagent (mutarose 0.13 unit / ml, glucose oxidase 9.0 unit / ml, Peroxidase 0.65 unit / ml, 4-aminoantipyrine 0.50 mM, 3 ml of 60 mm phosphate buffer (pH 7.1) containing 5.3 mM phenol to which ascorbate oxidase 2.7 unit / ml was added, and mixed at 37 ° C. for 5 minutes After heating, the absorbance at 505 nm was measured and determined.
(3) The residual L-lysine concentration was measured by the L-lysine-α-oxidase method. That is, 100 μl of 70 mM phenol and 100 μl of 30 mM 4-aminoantipyrine are added to 200 μl of 1M phosphate buffer (pH 7.4), and 450 μl of a solution containing 0 to 300 μg of L-lysine is added and mixed. After adding 50 μl of oxidase and heating at 30 ° C. for 20 minutes, the absorbance at 500 nm was measured and determined.
[0015]
Example 1
Glucose 50 g / l, yeast extract 5 g / l (containing about 100 mg / l phosphoric acid concentration), ammonium sulfate 10 g / l, L-lysine 10 g / l, MgSO4 · 7H2O 0.5 g / l, ZnSO4 · 7H2O 0.04 g / l, 2L of FeSO4 · 7H2O 0.03g / l pH6.8 medium was prepared and placed in a 3L jar. Streptomyces albulus subsp. lysinopolymerus 11011A-1 strain (fine Kokenjoyo 1109) was inoculated and aerobic culture was performed at 30 ° C. and 700 rpm for 96 hours.
After sterilizing the fermentation broth by centrifugation, the εPL concentration, residual glucose concentration, and residual L-lysine concentration of the supernatant water were measured.
As a result, the yield of carbon source for εPL was 22%. Moreover, the εPL production activity per 1 g of the bacterial cells was 0.51 g / g · dry cell / day.
[0016]
Example 2
Culturing was performed according to Example 1 except that the L-lysine content was 5 g / l. As a result, the yield of carbon source for εPL was 22%. Moreover, the εPL production activity per 1 g of bacterial cells was 0.50 g / g · dry cell / day.
[0017]
Example 3
Culturing was performed according to Example 1 except that the L-lysine content was 2 g / l. As a result, the carbon source yield of εPL was 18%. Moreover, the εPL production activity per 1 g of the bacterial cells was 0.44 g / g · dry cell / day.
[0018]
Example 4
Culture was performed according to Example 1 except that 0.2 g / l of KH2PO4 was added (containing 240 mg / l as the total phosphate concentration). As a result, the carbon source yield of εPL was 14%. Moreover, the εPL production activity per 1 g of cells was 0.40 g / g · dry cell / day.
[0019]
Example 5
Streptomyces albulus subsp. Lysinopolymerus (Streptomyces albulus subsp. Lysinopolymerus) No346-D strain is used instead of Streptomyces albulus subsp. Lysinopolymerus 11011A-1 The culture was performed according to Example 1 except that. As a result, the carbon source yield of εPL was 15%. Moreover, the εPL production activity per gram of the bacterial cells was 0.35 g / g · dry cell / day.
[0020]
Example 6
The culture time is 288 hours, the glucose content in the culture solution is 10 g / l or less, the ammonium sulfate content is 5 g / l or less, and the L-lysine content is 1 g / l or less. Sequentially added. Other conditions were cultured according to Example 1. As a result, the carbon source yield of εPL was 26%.
[0021]
Comparative Example 1
Glucose 50g / l, yeast extract 5g / l (containing about 100mg / l phosphate concentration), ammonium sulfate 10g / l, K2HPO4 0.8g / l, KH2PO4 1.36g / l, MgSO4 ・ 7H2O 0.5g / l, ZnSO4 -2 L of a medium of 7H2O 0.04 g / l and FeSO4.7H2O 0.03 g / l pH 6.8 was prepared and cultured according to Example 1. As a result, the carbon source yield of εPL was 8%. Moreover, the εPL production activity per 1 g of the bacterial cells was 0.28 g / g · dry cell / day.
[0022]
Comparative Example 2
Culture was performed according to Comparative Example 1 except that 10 g / l of L-lysine was added. As a result, the yield of carbon source for εPL was 9%. In addition, the εPL production activity per 1 g of bacterial cells was 0.31 g / g · dry cell / day.
[0023]
Comparative Example 3
Culture was performed according to Comparative Example 1 except that K2HPO4 and KH2PO4 were not added. As a result, the carbon source yield of εPL was 10%. Moreover, the εPL production activity per 1 g of the bacterial cells was 0.32 g / g · dry cell / day.
[0024]
Comparative Example 4
Culturing was performed according to Example 1 except that 10 g / l of L-lysine was added and ammonium sulfate was not added. As a result, the carbon source yield of εPL was 3%. In addition, the εPL production activity per 1 g of bacterial cells was 0.04 g / g · dry cell / day.
[0025]
Comparative Example 5
Streptomyces albulus subsp. Lysinopolymerus (Streptomyces albulus subsp. Lysinopolymerus) No346-D for Streptomyces albulus subsp. Lysinopolymerus No. 11011A-1 The culture was carried out according to Comparative Example 1 except that. As a result, the carbon source yield of εPL was 5%. Moreover, the εPL production activity per gram of the bacterial cells was 0.22 g / g · dry cell / day.
[0026]
Comparative Example 6
As a result of culturing according to Example 6 except that L-lysine was not added, the yield of carbon source of εPL was 19%.
[0027]
【The invention's effect】
By the production method of the present invention, production of by-products can be suppressed, and the yield of εPL with respect to the carbon source can be increased to 2 to 3 times that of the conventional method, so that εPL can be produced with high yield.

Claims (4)

In a method in which Streptomyces albulus species microorganisms are aerobically cultured in a medium, and ε-poly-L-lysine is collected from the obtained culture solution, the phosphate concentration is 50 to 300 mg / l. A method for producing ε-poly-L-lysine, comprising using a medium having an L-lysine content of 2 to 20 g / l and an ammonium sulfate content of 10 to 20 g / l. In a method of aerobically culturing Streptomyces albulus species microorganisms in the medium according to claim 1 and collecting ε-poly-L-lysine from the obtained culture solution, A method for producing ε-poly-L-lysine, comprising adding L-lysine sequentially or continuously to a culture solution so that the content of L-lysine is 1 to 10 g / l. The ε-poly- of any one of claims 1 or 2, wherein the strain producing ε-poly-L-lysine is Streptomyces albulus subsp. lysinopolymerus. A method for producing L-lysine. A strain producing ε-poly-L-lysine is resistant to Streptomyces albulus subsp. lysinopolymerus No346-D strain added with glycine to S-aminoethyl-L cysteine The method for producing ε-poly-L-lysine according to any one of claims 1 and 2, which is a mutant strain 11011A-1 strain having a phenotype (Microtechnical Research Institute, No. 1109).