WO2000056892A1 - Detection de bacterie productrice d'epsilon-polylysine, production d'epsilon-polylysine, et bacterie productrice d'epsilon polylysine - Google Patents

Detection de bacterie productrice d'epsilon-polylysine, production d'epsilon-polylysine, et bacterie productrice d'epsilon polylysine Download PDF

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Publication number
WO2000056892A1
WO2000056892A1 PCT/JP2000/001698 JP0001698W WO0056892A1 WO 2000056892 A1 WO2000056892 A1 WO 2000056892A1 JP 0001698 W JP0001698 W JP 0001698W WO 0056892 A1 WO0056892 A1 WO 0056892A1
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WIPO (PCT)
Prior art keywords
polylysine
strain
producing
plasmid
detecting
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Ceased
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PCT/JP2000/001698
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English (en)
Japanese (ja)
Inventor
Satoshi Inouye
Hiroshi Takagi
Shigeru Nakamori
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JNC Corp
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Chisso Corp
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Publication of WO2000056892A1 publication Critical patent/WO2000056892A1/fr
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • the present invention relates to a method for detecting ⁇ -polylysine-producing bacteria, a method for producing ⁇ -polylysine, and ⁇ -polylysine-producing bacteria. More specifically, a method for detecting an ⁇ -polylysine-producing bacterium for detecting a strain having a specific base sequence, a method for producing ⁇ -polylysine using the strain obtained by the detection method, and a specific base sequence The present invention relates to an ⁇ -polylysine-producing bacterium having
  • ⁇ -polylysine which is useful as an antibacterial substance, is available from Streptomycesa / Refras, Streptomycesa 1 bu 1 us) IF 0 1 4 1 4 7 strains, and Streptomyces It is produced by using a strain obtained by treating a mutated agent with a S. cerebrolus (Streptomycesalbulus) IFO-417 strain as a parent strain.
  • the above-mentioned ⁇ -polylysine-producing strain that is currently used is prepared by adding a dragendorf reagent to the supernatant of a culture solution cultured for 2 to 4 days in a liquid medium, and determining the presence or absence of the precipitate at that time. It was confirmed whether or not it had the ability to produce lysine, detected, and selected. Disclosure of the invention
  • an object of the present invention is to provide a novel method for detecting ⁇ -polylysine-producing bacteria, which can easily detect ⁇ -polylysine-producing bacteria.
  • Another object of the present invention is to provide a method for producing ⁇ -polylysine using a strain obtained by the detection method, and a novel ⁇ -polylysine-producing bacterium.
  • the present invention has the following configurations (1) to (6).
  • the nucleotide sequence derived from the plasmid ⁇ ⁇ 033 shown in FIG. 1 is the nucleotide sequence shown in SEQ ID NO: 1 according to any one of the above items 1 to 3. For detecting ⁇ -polylysine-producing bacteria.
  • FIG. 1 shows a restriction map of plasmid pN033.
  • the black part is the determined part of SEQ ID NO: 1.
  • the first invention is a method for detecting an ⁇ -polylysine-producing bacterium, which detects a strain having a base sequence derived from plasmid pN033 shown in FIG.
  • Plasmid ⁇ ⁇ 0 3 3 is derived from ⁇ -polylysine high-producing strain, Streptomyces aenobrass (Streptomycesa 1 bu 1 us) IF 0 1 4 1 4 7 It is a replicable cyclic plasmid that can be obtained, has a total length of about 37 kb, and has a restriction enzyme recognition site shown in Table 1, Table 2 and FIG. 1. It has the base sequence shown below.
  • the determined positions of the restriction enzymes start from the position of the restriction enzyme HindIII, and the clockwise distances are as shown in Table 2.
  • the method for detecting a strain having a nucleotide sequence derived from the plasmid pN033 is not particularly limited, but specifically, the whole nucleic acid fraction of the strain to be detected is sampled. And was specifically designed to hybridize to the nucleotide sequence derived from the plasmid pN033.
  • Gene amplification of the target base sequence is carried out using an oligonucleotide (primer), and the amplified target base sequence is subjected to gel electrophoresis, whereby the amplification is carried out.
  • PCR method a nucleotide sequence derived from NO33 how to determine the (Po re main chain reaction method, hereinafter referred to as "PCR method”.) and the this include high pli die Zeshi tio down method using a DNA probe radiolabeled with 3 2 P Can be done.
  • the strain to be detected is a strain having a nucleotide sequence derived from the plasmid pN ⁇ 33
  • the strain to be detected has the ability to produce ⁇ -polylysine.
  • the PCR method is a method developed by Saiki et al. And can be performed based on Science 230 volumes, ppl 350, and 1985.
  • This method is a method for amplifying a specific gene sequence.It is rapid, highly sensitive, highly specific, and easy to use. It is used for rapid determination of disease, genetic disease, and rapid test for harmful bacteria in the food field.
  • the target base sequence sandwiched between the two primers is amplified and can be easily detected by simple agarose electrophoresis. Become.
  • an amplification reaction is possible if at least one target sequence is present, and the sample can be easily provided as a sample simply by pretreating the target strain with a lytic enzyme or a surfactant. And can be Higher value than conventional detection methods.
  • the strain to be detected is not particularly limited, but may be a strain collected from nature or a strain already collected and stored.
  • the strain to be detected may be an isolated strain or a strain containing a plurality of strains before isolation.
  • the oligonucleotide used for the primer is a nucleotide sequence derived from the plasmid PN33 in consideration of selectivity, detection sensitivity and reproducibility, and is 10 bases or more, preferably 1 base or more. Chemical synthesis to specifically hybridize to oligonucleotide fragments having a length of 5 bases or more or to the base sequence derived from the plasmid pN033 You can use what is provided. - Also, ply M a in the row cormorants PCR method in the present invention, 3 2 P, etc. also have good and rather name is radiolabeled with particularly the detection.
  • the amplification region of the nucleotide sequence derived from pNO33 defined by the primer should be from 450 bases to 4600 bases, preferably from 100 bases to 100 bases. .
  • Reaction buffer, T ris - HC 1, M g C 1 2, KC ], T ween 2 0 soil force Ranaru also the not good if you use the.
  • the DNA polymerase used in the gene amplification reaction may be heat-resistant at a temperature of 90 ° C. or higher, and may be of any species.
  • heat denaturation for converting double-stranded DNA into single strand is 90-98 ° C
  • annealing reaction for hybridizing primers is 3 ⁇ - At 8 ° C
  • DNA polymerase acts The chain length reaction to be performed is performed at a temperature of 50 to 75 ° C, and this cycle is performed for several tens of cycles, so that the target base sequence can be amplified. it can.
  • the reaction products are separated by electrophoresis, the nucleic acid is stained with bromide thidium bromide, etc., and the amplified base sequence has the length of the target base sequence expected from SEQ ID NO: 1. As long as the length is equal, the presence of high ⁇ -polylysine-producing bacteria in the cells to be detected can be determined.
  • a chromatographic method or a method for directly determining the nucleotide sequence is also effective.
  • a second invention is the first invention is a £ Ichipo using Li lysine detected strains in detection method of producing bacteria epsilon - is a manufacturing method of the port re lysine.
  • the production method of the present invention comprises the steps of: It is characterized by collecting resin.
  • the liquid medium may be any as long as it contains a carbon source, a nitrogen source, inorganic salts and other nutrients.
  • the carbon source includes glucose or glycerin as a carbon source
  • the nitrogen source includes ammonium sulfate or yeast excrete or polypeptone; Phosphorus ion as an inorganic salt.
  • the power stream a nutshell, a magnetion, etc.
  • the culture can be performed by culturing under aerobic conditions, and performing shaking culture, stirring culture, or the like.
  • the culture temperature is 20 to 40 ° C
  • the pH of the medium is 3 to 9
  • the culture period is 1 to 10 days.
  • ⁇ -polylysine is produced and accumulated in the culture solution.
  • the ⁇ -polylysine produced and accumulated in the culture solution can be collected by centrifuging or filtering the culture solution to remove the cells, and then using a known method from the obtained cell removal solution.
  • ⁇ -polylysine can be isolated.
  • the cell-removing solution is removed through a column of anion exchange resin to remove most of the impurities, and then purified and concentrated through a column of a cation exchange resin. This gives ⁇ -polylysine.
  • Isolate liquid medium of plasmid ⁇ ⁇ 0 3 3 (10 g of butatripton, 5 g of yeast strain, and 5 g of salt are dissolved in 1 L of tap water) After that, 50 ml of the medium (pH adjusted to 7.3) was placed in a Sakaguchi flask with a capacity of 50 ml, and Streptomycesa 1 bu 1 us) Gray spores of IFO1447 and IFO13410 were inoculated into each loop of platinum, and cultured at a shaking speed of 140 rpm at 30 ° C for 16 hours.
  • the cells are collected from the culture by centrifugation at 500,000 G, and TE buffer (25 mM Tris (hydroxy) aminomethan (hereinafter referred to as "Tris") is collected. After washing with 25 mM EDTA: pH 8.0), the cells were lysed (0.3 M sucrose, 25 mM tris, 25 mM MEDTA, 2 mg / mg). m 1 lysozyme) suspended in 20 ml, left at 4 ° C for 5 minutes, and a solution containing 2% sodium raurylsulfate and 0.3 N sodium hydroxide powder 1 0 ml was added to the mixture, mixed slowly, and allowed to stand at 4 ° C for 5 minutes to obtain a cell lysate.
  • Tris 25 mM Tris (hydroxy) aminomethan
  • the cell lysate was neutralized and mixed with 15 ml of 5 M ammonium acetate, allowed to stand at 4 ° C for 10 minutes, and then subjected to high-speed centrifugation at 1200 G for 10 minutes. The supernatant was collected. To this supernatant, add an equal volume of phenol saturated with TE buffer, mix vigorously for 5 minutes, centrifuge at 1200 G for 10 minutes, and extract the water-soluble fraction A. It was sorted. To this water-soluble fraction A, add an equal amount of black-mouthed form, mix vigorously for 5 minutes, and centrifuge at 1200 G for 10 minutes, then collect water-soluble fraction B. did.
  • liponucleolytic enzyme To remove ribonucleic acid, add liponucleolytic enzyme to the lysate to a final concentration of 50 g Zm1 and react at 37 ° C for 30 minutes. To this was added 0.6 volumes of 20% polyethylene glycol Z 5 M salt mixture, and the mixture was left at 4 ° C for 1 hour.
  • the generated white precipitate was collected by high-speed centrifugation and dissolved in ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ buffer. This was subjected to 0.7% agarose gel electrophoresis (100 volts, 20 minutes), and the gel was stained with a bromide reagent to confirm the presence of the high molecular nucleic acid.
  • the plasmid pN ⁇ 33 DNA was converted to a restriction enzyme, Eco. After digestion with RI and SmaI, the fragment can be replicated in E. coli by the known plasmid vector pGreen 19 lnouye et al., Genevol. 189, pp 159-16-12: 19. 97 7) A certain subcloning method was used to subclone the EcoRI and SmaI sites of pBluescript SK + (Stratagene) in a conventional manner. Confirmation of the inserted fragment was performed by plasmid separation based on the plasmid isolation method described in Example 1, digestion with EcoRI or SmaI, and then 0.7% agarose gel electrophoresis. I confirmed more.
  • the nucleotide sequence of the inserted fragment was determined using Applied Biosystems' fluorescence sequence sensor (ABI 373) using the dye terminator-cycle sequencing method. did. Based on SEQ ID NO: 1, translatable regions were searched by using DNASIS Ver. 3.6 Gene Analysis Software manufactured by Takara Shuzo, and SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO The translatable region shown in Fig. 5 was found. These translatable regions are presumed to be fully functional proteins.
  • Example 1 Detection of f-polylysine-producing bacteria
  • ⁇ -Polylysine high-producing bacterium IFO l4147 and ⁇ - Polylysine low-producing bacterium IFOl obtained in “(1) Detection of plasmid pN033” A part of the cultured cells of the strain 340 and the Streptomyces sp.
  • SP-72 strain (FERM strain-16810) disclosed in JP-A-2000-69988 were identified as (1) ) Detection of Plasmid ⁇ 3 0 3 3 ”
  • a chemically synthesized primer derived from the plasmid pN033 shown in Table 3 was used.
  • a gene amplification treatment (PCR method) was performed.
  • the processing conditions were denaturation at 96 ° C for 1 minute, elutriation at 59 ° C for 1 minute, and elongation at 72 ° C for 1 minute for 25 cycles.
  • ⁇ ⁇ ⁇ of the reaction solution is subjected to 1.2% agarose gel electrophoresis, stained with ethidium gel, and irradiated with ultraviolet light to detect the target base sequence. went.
  • a ⁇ -polylysine-producing bacterium of the present invention According to the method for detecting a ⁇ -polylysine-producing bacterium of the present invention, a ⁇ -polylysine-producing bacterium can be easily detected. In addition, a method for producing ⁇ -polylysine using a strain obtained by the detection method can produce ⁇ -polylysine with high efficiency.

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Abstract

La présente invention concerne un procédé de détection d'une bactérie productrice d'ε-polylysine comprenant la détection d'une souche bactérienne dont une séquence de bases provient du plasmide pN033. L'invention concerne également un procédé de production d'ε-polylysine caractérisé par l'utilisation de la souche détectée par le procédé de détection ci-dessus. En utilisant le procédé de détection tel que décrit ci-dessus, on peut facilement détecter une bactérie productrice d'ε-polylysine. En utilisant le procédé de production d'ε-polylysine par utilisation de la souche obtenue par le procédé de détection ci-dessus, on peut produire avec un bon rendement de l'ε-polylysine.
PCT/JP2000/001698 1999-03-23 2000-03-21 Detection de bacterie productrice d'epsilon-polylysine, production d'epsilon-polylysine, et bacterie productrice d'epsilon polylysine Ceased WO2000056892A1 (fr)

Applications Claiming Priority (2)

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JP11/77445 1999-03-23
JP7744599 1999-03-23

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WO2000056892A1 true WO2000056892A1 (fr) 2000-09-28

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10210995A (ja) * 1997-01-31 1998-08-11 Chisso Corp ε−ポリ−Ｌ−リジンの製造法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10210995A (ja) * 1997-01-31 1998-08-11 Chisso Corp ε−ポリ−Ｌ−リジンの製造法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKAGI H. ET AL.: "Isolation and sequence analysis of plasmid pN033 in the epsilon-poly-lisine-producing actinomycete streptomyces albulus IF014147", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 89, no. 1, January 2000 (2000-01-01), pages 94 - 96, XP000861925 *

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