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WO1998048033A1 - SOUCHE PRODUISANT UNE QUANTITE IMPORTANTE DE ε-POLY-L-LYSINE ET PROCEDE POUR PRODUIRE DE LA ε-POLY-L-LYSINE AU MOYEN DE CETTE SOUCHE - Google Patents

SOUCHE PRODUISANT UNE QUANTITE IMPORTANTE DE ε-POLY-L-LYSINE ET PROCEDE POUR PRODUIRE DE LA ε-POLY-L-LYSINE AU MOYEN DE CETTE SOUCHE Download PDF

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Publication number
WO1998048033A1
WO1998048033A1 PCT/JP1997/001403 JP9701403W WO9848033A1 WO 1998048033 A1 WO1998048033 A1 WO 1998048033A1 JP 9701403 W JP9701403 W JP 9701403W WO 9848033 A1 WO9848033 A1 WO 9848033A1
Authority
WO
WIPO (PCT)
Prior art keywords
strain
lysine
poly
producing
streptomyces
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1997/001403
Other languages
English (en)
Japanese (ja)
Inventor
Toshiharu Iwata
Yumiko Iwasawa
Shinji Shiraishi
Jun Hiraki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
JNC Corp
Original Assignee
Chisso Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to JP11324096A priority Critical patent/JP3525190B2/ja
Application filed by Chisso Corp filed Critical Chisso Corp
Priority to PCT/JP1997/001403 priority patent/WO1998048033A1/fr
Priority to CNB971822530A priority patent/CN1161455C/zh
Priority to KR10-1999-7009787A priority patent/KR100433741B1/ko
Publication of WO1998048033A1 publication Critical patent/WO1998048033A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the present invention relates to a strain that produces a large amount of ⁇ PL-L-lysine (hereinafter referred to as PPL) and a method for producing ⁇ PL by fermentation using the strain.
  • PPL ⁇ PL-L-lysine
  • £ PL has high safety because it is a polymer of L-lysine, an essential amino acid, and has unique physical properties because of its high cation content. Therefore, applications such as toiletries, cosmetics, feed additives, pharmaceuticals, agricultural chemicals, food additives, and electronic materials can be expected. In particular, in the field of food additives, it is attracting attention as a natural additive. Background technology
  • the resulting mutant strain Streptomyces albras, Lysinopolymeras 11011A-1 strain (Shenzhen No. 1109) is cultured in a medium, and the resulting culture is separated and purified.
  • a method for obtaining the £ PL is also known (Japanese Patent Publication No. 3-42070, Japanese Patent Publication No. 3-78998).
  • an object of the present invention is to provide a strain which has a higher ⁇ PL-producing ability and can improve ⁇ PL productivity as compared with a conventional £ PL-producing strain or an improved strain thereof. It is also an object of the present invention to provide a method for producing an inexpensive and significant amount of PL using the strain. Disclosure of the invention
  • the present invention is constituted by the following technical means.
  • the B21021 strain described in the above (2) is aerobically cultured in a medium, and £ -poly-L-lysine produced and accumulated in the culture solution is collected. Manufacturing method.
  • AEC used in the present invention is a structural analog of L-lysine (analog substance).
  • L-lysine is a compound with a structure that differs only in that the carbon atom at the 4-position is replaced with a sulfur atom. is there.
  • the AEC is added to the medium, the growth of the bacteria is inhibited, but the combination of L-threonine clearly shows the growth inhibition more strongly.
  • the 11011A-1 strain disclosed in Japanese Examined Patent Publication No. 3-42070 is a £ PL-producing strain resistant to AEC, an analog of L-lysine, but was screened in the presence of AEC at a concentration of 2 mg / ml.
  • an improved strain of AEC concentration c present invention show no growth at 10mg / ml or more, 10mg / ml or more concentrations of It is a microorganism belonging to the genus Streptomyces that has a degree of resistance that can grow even at an AEC concentration of 40 mg / ml, and has a mycological property that is one of that of the parent strain 11011A-1 that has been screened in the presence of AEC. Differences are noted in some parts.
  • any microorganism of the genus Streptomyces that can produce ⁇ PL can be used.
  • Streptomyces. Albras • lysinopolymeras 1101 1A-1 strain (Microe Kenjiro No. 1109) is preferred.
  • mutation treatment refers to a treatment in which a microorganism (strain) of the genus Streptomyces producing ⁇ PL is mutated into an improved strain resistant to AEC at a high concentration of lOmg / ml or more.
  • strain a microorganism
  • NTG N-methyl-N'-nitro-N-nitrosoguanidine
  • Chemical or physical mutation treatment methods can be mentioned.
  • inoculation is performed on a minimal agar medium supplemented with AEC of 10 mg or more per 1 ml of medium and L-threonine at 1 mg per 1 ml of medium.
  • a method of collecting a growing strain can be mentioned.
  • the present invention will be described in detail.
  • One specific method for obtaining the improved strain of the present invention is to use the spores of Streptomyces albras subsp. Lysinopolymeras 11011A-1 strain, which is a PL-producing strain, by tris-maleic acid.
  • the suspension is suspended in a buffer (pH 6.0), NTG is added to the buffer at 1.5 mg / mL of the buffer, and the mixture is contacted at 37 ° C. for 60 minutes.
  • the cells are collected by centrifugation, washed with phosphate buffer (0.05 M, pH 7.0), and then added to a minimal agar medium containing 20 mg of AEC per 1 ml of medium and 1 mg of L-threonine per ml of medium (glucose 5 %, Ammonium sulfate 1%,
  • the productivity of ⁇ PL was evaluated for the thus obtained AEC high-concentration resistant strains, and the strain with the highest productivity was Strep-Small Myces, Albraz, Subspecies, Lysinopolymereras B21021 strain (Ministry of International Trade and Industry Technical Research Institute ( ⁇ 305, 1-3 1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan), International Accession Number: FERM BP-52926, 1997 (April 18) Transfer from the original deposit (FERM P-1 513, original deposit date: September 20, 1995) to an international deposit based on the Busu-Busto Treaty], followed by the B22107 strain, ⁇ 222 ( ⁇ shares.
  • the degree of AEC resistance of the resulting improved strain which is resistant to high concentrations of AEC, is measured as follows. That is, the resistant strain was applied to each of the minimal agar medium (described above) to which AEC and L-threonine at the respective concentrations shown in Table 1 described below were added at a rate of 1 ml per 1 mg of the medium. After culturing for 7 to 7 days, the degree of resistance is compared by observing the growth with the naked eye. Table 1 shows the results.
  • the parent strain, 11011A-1 grows at an AEC concentration of 5 mg / nd, but shows no growth at a concentration of 10 mg / ml, whereas the highly resistant strain, B2102 vermilion, £ (: Even if the concentration is 401 ⁇ / 1111 Growth is observed.
  • the improved strain of the present invention can be clearly distinguished from the parent strain in that it has resistance to high concentrations of AEC.
  • the spores are circular or elliptical, about 1.2.5 in size, and their surface structure is Spiny.
  • Optimal growth temperature around 30.
  • the bacteriological properties of one of the improved strains of the present invention are different in some respects from the bacteriological properties of the parent strain, 11011A-1 c .
  • 11011A-1 strain cannot use salicin, B2102 Red is available.
  • 11011A-1 strain grows a gray-brown aerial mycelium on sucrose / nitrate agar medium, while B21021 strain does not grow on c- nutrient agar medium. The mycelium grows abundantly, but the aerial hyphae grows only slightly in the B21021 strain.
  • a soluble pigment is also observed in the glucose-asparagine agar medium and the glycerol-asparagine agar medium for 11011A-red, but not for both mediums in the B21021 strain.
  • the B21021 strain can be clearly distinguished from the parent strain 11011A-1 also in bacteriological properties.
  • the improved strain is inoculated into a medium, cultured, and ⁇ PL produced and accumulated from the culture solution is separated and purified. Any medium can be used as long as it contains a carbon source, a nitrogen source, inorganic substances and other nutrients.
  • the carbon source is not limited as long as the improved strain such as glucose, fructose, glycerin, starch and the like can be assimilated, and its content is preferably 1 to 5% (g / dl%).
  • the nitrogen source peptone, casein hydrolyzate, amino acids, but may be any inorganic Anmoniu ⁇ like, the content of preferably c nitrogen source is sulfuric Anmoniu ⁇ is from 0.2 to 2% (% is g / dl %) Is preferred.
  • a carbon source and a nitrogen source may be added sequentially.
  • Inorganic substances include phosphate ion, potassium ion, sodium ion, magnesium ion, zinc ion, iron ion, manganese ion, nickel ion, sulfate ion and the like.
  • yeast extract is contained in an amount of 0.1 to 0.5% (% is g / dl%), the growth of the bacterium is improved, and a favorable result is also obtained in the production of ⁇ PL.
  • Culture is performed under aerobic conditions, such as shaking culture or stirring culture.
  • the culture temperature is preferably 25 to 35 ° C.
  • the pH of the medium is preferably around neutral (pH 6 to 8), but after the culture starts, the pH decreases as the bacteria grow. When the pH drops to 4, add alkali to maintain the pH at 4.
  • Ammonia water is preferably used as the additive, and sodium hydroxide, potassium hydroxide or the like may be used.
  • ⁇ PL is accumulated in the culture solution in 1 to 7 days.
  • the cell-removed solution After removing the cells from the culture by centrifugation or a filter, the cell-removed solution is purified, decolorized, and concentrated. Crystallization from the concentrated solution with an organic solvent such as acetone or ethanol gives PL.
  • the amount of PL produced in the culture solution was measured by the method described in Itzhaki et al., Analytical Biochemistry, 50, 569, (1972). That is, after removing the cells by centrifugation of the culture solution, 2 ml of the supernatant ( ⁇ L: 0 to 200 ⁇ g) and 2 mM of 1 mM aqueous solution of methyl orange are mixed and left at room temperature for 30 minutes. The resulting ⁇ PL-methyl orange complex is removed by centrifugation, and the supernatant is measured for absorbance at 465 nm to determine the ePL amount in the culture solution.
  • C % in Examples and Comparative Examples Is weight (g) / volume (dl)% unless otherwise specified. (Example 1)
  • Table 3 shows the amount of PL production and the yield to sugar after 72 hours of culture.
  • the culture was carried out according to Example 1 except that Streptomyces albras subsp. Lysinopolymeras B210107 strain was used instead of Streptomyces albras subsp. Lysinopolymeras B21021 strain.
  • the amount of PL was measured. Table 3 shows the amount of PL production and the yield to sugar after 72 hours of culture.
  • Streptomyces ⁇ Alblas ⁇ Sub-species ⁇ Lysinopolymeras B21021 instead of the strain, the cells were cultured according to Example 1 except that Streptomyces'Albras'Subspecies' Lysinopolymeras 222 (a strain was used), and the amount of PL in the culture solution was measured. Table 3 shows the amount of ⁇ PL produced and the yield relative to sugar.
  • Example 6 Culture was carried out in accordance with Example 4, except that Streptomyces 'Albras' Subsp. Was measured. Table 4 shows the sPL production amount and the sugar yield after the culture for 168 hours. (Example 6)
  • Streptomyces albus subsp. Lysinopolymeras The procedure was performed in accordance with Example 4, except that the parent strain Streptomyces albus brass subspecies lysinopolymeras 11011A-1 was used instead of the B21021 strain. Table 4 shows the amount of PL production and the yield to sugar after culturing for 168 hours.
  • Example 7 With respect to the culture solution cultured for 16 hours in Example 4, the culture solution was centrifuged to remove cells, adjusted to pH 7.5, and then treated with IRC-50 (cation exchange resin) and IR A- Separation and purification were performed using ion exchange resins of 402 (anion exchange resin) and XT-106 (cation exchange resin), and ⁇ PL was obtained by concentration with a reverse osmosis membrane (RO). The yield is shown in Table 5.
  • the improved strain having high resistance to AEC of the present invention has the ability to produce ⁇ PL in high production and high yield.
  • PL can be produced with high productivity and high yield. It is possible to manufacture.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Souche présentant une capacité de production de l ε-poly-L-lysine accrue par rapport aux souches productrices de ε-poly-L-lysine classiques ou aux variétés améliorées de ces dernières, et capable d'augmenter la productivité de ε-poly-L-lysine. L'invention porte en outre sur un procédé pour produire une quantité importante de ε-poly-L-lysine au moyen de cette souche . Ce procédé consiste à provoquer la mutagenèse d'un micro-organisme et de l'espèce Streptomyces albulus, capable de produire de la ε-poly-L-lysine, pour obtenir une souche tolérante à la S-(2-aminoéthyl)-L-cystéine en concentration élevée, égale ou supérieure à 10 mg/ml et capable de produire de la ε-poly-L-lysine, puis à incuber cette souche par voie aérobie et enfin à récolter la ε-poly-L-lysine dans la culture.
PCT/JP1997/001403 1995-10-24 1997-04-23 SOUCHE PRODUISANT UNE QUANTITE IMPORTANTE DE ε-POLY-L-LYSINE ET PROCEDE POUR PRODUIRE DE LA ε-POLY-L-LYSINE AU MOYEN DE CETTE SOUCHE Ceased WO1998048033A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP11324096A JP3525190B2 (ja) 1995-10-24 1996-04-09 ε−ポリ−Ｌ−リジンを著量に生産する菌株及びそれを用いたε−ポリ−Ｌ−リジンの製造法
PCT/JP1997/001403 WO1998048033A1 (fr) 1995-10-24 1997-04-23 SOUCHE PRODUISANT UNE QUANTITE IMPORTANTE DE ε-POLY-L-LYSINE ET PROCEDE POUR PRODUIRE DE LA ε-POLY-L-LYSINE AU MOYEN DE CETTE SOUCHE
CNB971822530A CN1161455C (zh) 1997-04-23 1997-04-23 大量产生ε-聚-L-赖氨酸的菌株和生产方法
KR10-1999-7009787A KR100433741B1 (ko) 1997-04-23 1997-04-23 ε-폴리-Ｌ-라이신을 현저한 양으로 생산하는 균주 및 이를 사용한 ε-폴리-Ｌ-라이신의 제조방법

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP29893495 1995-10-24
JP11324096A JP3525190B2 (ja) 1995-10-24 1996-04-09 ε−ポリ−Ｌ−リジンを著量に生産する菌株及びそれを用いたε−ポリ−Ｌ−リジンの製造法
PCT/JP1997/001403 WO1998048033A1 (fr) 1995-10-24 1997-04-23 SOUCHE PRODUISANT UNE QUANTITE IMPORTANTE DE ε-POLY-L-LYSINE ET PROCEDE POUR PRODUIRE DE LA ε-POLY-L-LYSINE AU MOYEN DE CETTE SOUCHE

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Publication Number Publication Date
WO1998048033A1 true WO1998048033A1 (fr) 1998-10-29

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WO (1) WO1998048033A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102094066A (zh) * 2010-12-13 2011-06-15 华南农业大学 一种筛选ε-聚赖氨酸产生菌的方法
CN107164417A (zh) * 2017-05-17 2017-09-15 郑州拜纳佛生物工程股份有限公司 一种ε‑聚赖氨酸的生产方法
CN112359002A (zh) * 2019-12-04 2021-02-12 江南大学 一株小白链霉菌及其在生产ε-聚赖氨酸中的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002330797A (ja) * 2001-05-08 2002-11-19 Chisso Corp ε−ポリ−Ｌ−リジンの製造法
EP2100628B1 (fr) 2006-11-30 2015-04-15 BMG Incorporated Adhésif autodégradable à usage médical d'un système réactif à deux composants comprenant le mélange poudre-poudre
EP3674311A4 (fr) 2017-08-23 2021-05-12 Fukui Prefectural University Groupe fonctionnel clic ayant un dérivé de e-poly-l-lysine, procédé de production associé, et utilisation associée

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5372896A (en) * 1976-12-10 1978-06-28 Heiichi Sakai Production of polylisine
JPS6349075A (ja) * 1986-08-19 1988-03-01 Chisso Corp イプシロン―ポリ―ｌ―リシンを生産する菌株
JPH01187090A (ja) * 1988-01-19 1989-07-26 Chisso Corp ε−ポリリシンの製造方法およびε−ポリリシン生産菌
JPH01222790A (ja) * 1988-03-03 1989-09-06 Chisso Corp 精製ε―ポリリシンの製造法
JPH08163992A (ja) * 1994-12-15 1996-06-25 Chisso Corp εーポリーＬーリジンの製造法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5372896A (en) * 1976-12-10 1978-06-28 Heiichi Sakai Production of polylisine
JPS6349075A (ja) * 1986-08-19 1988-03-01 Chisso Corp イプシロン―ポリ―ｌ―リシンを生産する菌株
JPH01187090A (ja) * 1988-01-19 1989-07-26 Chisso Corp ε−ポリリシンの製造方法およびε−ポリリシン生産菌
JPH01222790A (ja) * 1988-03-03 1989-09-06 Chisso Corp 精製ε―ポリリシンの製造法
JPH08163992A (ja) * 1994-12-15 1996-06-25 Chisso Corp εーポリーＬーリジンの製造法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHOJI SHIMA et al., "Microbial Production of epsilon-Polylysine", ABSTR. ANNU. MEET., (1979), No. 79, p. 210. *
SHOJI SHIMA et al., "Polylysine Produced by Streptomyces", AGRIC. BIOL. CHEM., (1977), Vol. 41, No. 9, p. 1807-1809. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102094066A (zh) * 2010-12-13 2011-06-15 华南农业大学 一种筛选ε-聚赖氨酸产生菌的方法
CN102094066B (zh) * 2010-12-13 2013-07-31 华南农业大学 一种筛选ε-聚赖氨酸产生菌的方法
CN107164417A (zh) * 2017-05-17 2017-09-15 郑州拜纳佛生物工程股份有限公司 一种ε‑聚赖氨酸的生产方法
CN112359002A (zh) * 2019-12-04 2021-02-12 江南大学 一株小白链霉菌及其在生产ε-聚赖氨酸中的应用
CN112359002B (zh) * 2019-12-04 2022-08-02 江南大学 一株小白链霉菌及其在生产ε-聚赖氨酸中的应用

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JPH09173057A (ja) 1997-07-08
JP3525190B2 (ja) 2004-05-10

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