KR102072311B1 - Enterococcus faecium KA84 and method for production of ornithine using the same - Google Patents

Abstract

The present invention relates to an Enterococcus faecium KA84 strain, and a composition and a method for producing ornithine by using the same. Therefore, the Enterococcus faecium KA84 strain of the present invention has an activity of decomposing cellulose, protein, lipid, and phosphate, and particularly, can decompose insoluble phosphate. Accordingly, ornithine can be produced by bioconversion using arginine as a substrate, and thus the Enterococcus faecium KA84 strain can be effectively used in the composition and method for producing ornithine. In particular, the strain of the present invention shows a high yield of ornithine bioconversion rate in various media, thereby being highly industrially applicable.

Description

Enterococcus faecium KA84 and method for production of ornithine using the same}

The present invention relates to a composition for producing ornithine (ornithine) production method using the Enterococcus faecium KA84 strain and the same.

 Ornithine is a non-essential amino acid widely found in plants, animals, and microorganisms as a precursor for the biosynthesis of arginine, proline, and polyamines. Ortinine is not found in nature as a protein component and is reported to be present in antibiotic peptides such as tyrosidine or gramicidine.

Ornithine plays an important role in the pathway of the production and release of urea from amino acids or ammonia through the ornithine cycle in the in vivo metabolism of higher animals. In addition, L-ornithine is widely used in the United States as a food material to increase the synthesis of muscles by secreting growth hormone, and to promote basic metabolism and to prevent obesity. It was found to have functionality. In addition, it shows the effect of removing harmful ammonia from the liver and is used as an active ingredient of medicines for improving cirrhosis and liver dysfunction.

In addition, ornithine is used as a nutritional supplement because it has an effect on muscle production and body fat reduction, and ornithine-alphakedoglutarate salt containing ornithine and alpha ketoglutalylic acid in a 2: 1 ratio (OKG) is used as an immune enhancing substance. The use of ornithine as an active ingredient in medicines or dietary supplements is known worldwide. In Europe, pharmaceutical products for improving hepatic disorders in the form of L-ornithine-L-aspartinate are commercially available. In Japan, it can be used as a food material in the form of L-ornithine hydrochloride, and can be used as a dietary supplement in the United States.

As ornithine can be utilized in various fields as described above, various studies are being conducted to industrially mass-produce ornithine. Currently used mass production of ornithine is to separate the milk protein casein with digestive enzymes to obtain ornithine, or to use microorganisms that can convert the substrate to ornithine in the biosynthetic pathway. have. In the method using a biosynthetic pathway, arginine (L-arginine) amino acid is mainly used as a substrate for producing ornithine, and as the microorganism, Corynebacterium strain or transformed Escherichia coli is used. Mainly used.

The strain Corynebacterium is a representative industrial microorganism widely used in the production of amino acids, nucleic acids, enzymes or antibiotic analogs. In particular, arginine can be synthesized from glutamate by an enzyme expressed from an arginine operon structure gene in the form of argCJBDFRGH, and in the process, ornithine is known to be produced as an intermediate (FIG. 1).

Therefore, in order to prepare a strain with improved ornithine production, there are attempts to mutate the arginine operon of the arginine producing strain, or to change the medium or culture environment. Korean Patent Laid-Open Publication No. 10-2010-0060909 discloses a method for increasing the production of arginine through regulation of argR that regulates the expression of arginine operon gene and argB that is inhibited by arginine concentration.

In addition, a method of increasing the production of ornithine by the action of ornithine cyclodeaminase (ocd) by culturing the microorganism of Corynebacterium in a medium with proline, culture of the microorganism Methods of improving ornithine productivity by changing the impeller and feeding conditions are known. When using transformed Escherichia coli, glutamate is added to the medium when culturing argF- and argR-deficient strains, or the gamma glutamyl kinase (γ-glutamylkinase), which is involved in the first step of the proline-producing pathway, is produced. There is known a method of improving the productivity of ornithine by deleting the gene proB.

In Japanese Patent Application No. PCT / JP2005 / 024280, biotin deficiency conditions of Corynebacterium glutamicum or penicillin G or fatty acid ester surfactants result in damage to the cell wall, resulting in glutamate. It is disclosed that the discharge capacity is increased.

However, the above-mentioned methods are indirect methods such as inducing the accumulation of ornithine by blocking the arginine biosynthetic pathway by controlling the expression of an enzyme encoded by the arginine biosynthetic gene operon, or using a change in medium or culture environment. It is then disadvantageous that industrial processes requiring mass production can result in higher or lower production of ornithine yields. Therefore, in order to overcome these disadvantages, researches are being conducted to discover microorganisms capable of producing ornithine with high efficiency and to enable efficient ornithine production at a lower cost.

 Kimchi is Korea's representative fermented food with a long history. It obtained the Codex International Food Standard in July 2001 and is a natural fermented food that is different from the pickles of the world. In addition, kimchi is rich in vitamin A, vitamin B, vitamin C, etc. It is found that there are many healthy bacteria, lactic acid bacteria, which help digestion and prevent cancer cell growth.

There are major strains such as Lactobacillus genus, Leuconostoc genus, Pediococcus genus and Weissella genus, and these strains are known to play an important role in kimchi fermentation. have. Among them, lactic acid bacteria rich in kimchi produce various substances such as lactate, gamma aminobutyric acid GABA, and bacteriocin, and play various roles such as preservation of food, provision of acidity and flavor, and inhibition of food poisoning bacteria. It is known. In particular, strains such as Leuconostoc citreum and Weissella koreensis are known as dominant species that determine the taste of Korean kimchi. In addition, kimchi lactic acid bacterium is used as an intermediate in medicine and cosmetics because it has effects such as improving lipids in the body, boosting immunity, and anti-AI (avian influenza) efficacy.

Accordingly, the present inventors are working to develop a method for producing ornithine using microorganisms showing bioconversion activity of arginine to ornithine, while Enterococcus exhibiting insoluble phosphate soluble activity from kimchi. faecium ) KA84 strain was isolated. Since the strain can effectively convert arginine to ornithine in various medium environments of minimal medium, synthetic medium or natural medium, it was confirmed that it can be usefully used for mass production of ornithine and completed the present invention.

It is therefore an object of the present invention to provide a novel Enterococcus faecium KA84 strain deposited with accession number KCTC 13657BP.

Still another object of the present invention is to provide a composition for producing ornithine, comprising an Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof.

Another object of the present invention, i) preparing an Enterococcus faecium KA84 strain or culture thereof deposited with accession number KCTC 13657BP; And ii) reacting the strain or culture of step i) with a composition comprising arginine; To include, to provide ornithine production method.

In order to achieve the above object, the present invention can provide a novel Enterococcus faecium KA84 strain deposited with accession number KCTC 13657BP.

According to a preferred embodiment of the present invention, the strain may be derived from kimchi.

According to a preferred embodiment of the present invention, the strain may be composed of the nucleotide sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the strain may have an activity of decomposing any one or more organic substances selected from the group consisting of cellulose, proteins, lipids and phosphates.

According to a preferred embodiment of the present invention, the strain may have an activity of converting arginine to ornithine.

In addition, the present invention can provide a composition for producing ornithine, comprising an Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof.

In addition, the present invention comprises the steps of i) preparing an Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof; And

ii) reacting the strain or culture of step i) with a composition comprising arginine; It can be provided, ornithine production method.

According to a preferred embodiment of the present invention, the reaction of step ii) may be performed for 40 to 80 hours at a temperature of 20 to 40 ℃.

Therefore, the Enterococcus faecium KA84 strain of the present invention has an activity of degrading cellulose, proteins, lipids and phosphates, and among them, particularly insoluble phosphates. Therefore, ornithine can be produced through bioconversion using arginine as a substrate, and thus can be effectively used in compositions or production methods for ornithine production.

In particular, the strains of the present invention are highly industrially useful because they exhibit high yields of ornithine bioconversion rates in various media.

Figure 1 shows the process of biosynthesis from glutamate to arginine in the microorganism, it can be seen that ornithine is involved as an intermediate in the biosynthetic pathway.
Figure 2 shows the cellulose, protein, lipid and phosphate degradability of the Enterococcus faecium KA84 strain.
Figure 3 shows the results of bioconversion from arginine to ornithine when the Entrococcus pesium KA84 strain was cultured in TYE + NA complex medium. The strain specifically confirmed that 100% bioconversion of arginine to ornithine.
Figure 4 shows the results of bioconversion from arginine to ornithine when cultured in a natural medium of the entrococcus pesium KA84 strain. In addition to arginine, the strain was able to confirm that citrulline (citrulline; Cit) was also bioconverted to arginine.
5 shows the results of bioconversion from arginine to ornithine when the Entrococcus pesium KA84 strain was cultured in a complex medium of minimal medium. The strain was confirmed that the bioconversion of about 70% of arginine to ornithine.

Hereinafter, the present invention will be described in more detail.

As described above, ornithine can be utilized in various fields, particularly in the pharmaceutical and food industries, and efforts to mass produce ornithine continue. Currently, a representative method of industrially producing ornithine is to induce accumulation of ornithine by blocking the arginine biosynthetic pathway of recombinant microorganisms, but this is an indirect method that is disadvantageous in terms of production cost and production efficiency. Therefore, there is a need for a direct method showing a high yield while mass producing ornithine, or a study of microbial resources for use therein.

 Kimchi-derived entrococcus pessium KA84 strain according to the present invention exhibits a soluble activity that can be used to decompose insoluble phosphate, the strain effectively arginine to ornithine in a variety of medium environment of minimal medium, synthetic medium or natural medium Since the organism can be converted, the entrococcus pessium KA84 strain of the present invention can be usefully used in the mass production method of ornithine.

Thus, the present invention provides a novel Enterococcus faecium KA84 strain deposited with accession number KCTC 13657BP.

The present inventors intended to purely isolate microorganisms exhibiting insoluble phosphate degrading activity in order to provide a strain capable of converting arginine to ornithine. As a result of analyzing the 16S rRNA sequence by separating the strain having both phosphate degrading ability and ornithine bioconversion ability from kimchi, it was confirmed that the strain was a novel strain belonging to the entrococcus pessium and this was entrococcus pessium KA84. It was named strain (Figures 2 and 3).

The strain of the present invention is preferably composed of the nucleotide sequence of SEQ ID NO: 1.

The strain of the present invention preferably has the activity of decomposing any one or more organic substances selected from the group consisting of cellulose, proteins, lipids and phosphates, more preferably it has activity to decompose lipids and phosphates, Most preferably, it has the activity to decompose phosphate.

The strain of the present invention preferably has the activity of converting citrulline or arginine to ornithine, but more preferably, has the activity of converting arginine to ornithine.

The present invention also provides a composition for producing ornithine, comprising an Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof.

The strain contained in the composition is the same as the novel strain invention, so description is replaced by the above description.

Culture of the strain refers to the result of the strain is cultured so that the bioconversion of citrulline or arginine to produce ornithine, the culture may be used as a medium usually used for strain culture, but preferably TYE + NA complex medium, It is preferred that the MEM natural medium or M minimum medium, more preferably TYE + NA composite medium or MEM natural medium is most preferred, and most preferably TYE + NA composite medium (Table 1). And [FIG. 3]).

The strain of the present invention or a culture thereof is preferably included in the composition of the present invention 10 to 20 parts by weight, but is not limited thereto. The culture is preferably contained by diluting the culture supernatant and the culture supernatant from which the strain is removed, specifically, diluted 0.5 to 500 times. The composition of the culture preferably further includes all components known to act synergistically to the growth of the genus Enterococcus, as well as components necessary for the strains of the genus Enterococcus.

The invention also comprises the steps of i) preparing an Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof; And

ii) reacting the strain or culture of step i) with a composition comprising arginine; It includes, provides ornithine production method.

The reaction of step ii) of the present invention is preferably carried out for 40 to 80 hours at a temperature of 20 to 40 ℃, more preferably for 70 to 75 hours at a temperature of 25 to 30 ℃.

The reaction can be terminated if all citrulline or arginine in the medium is converted to ornithine.

The entrococcus pesium KA84 strain derived from kimchi of the present invention can effectively convert arginine to ornithine in various media environments, and thus, the entrococcus pessium KA84 strain of the present invention can be usefully used in the mass production method of ornithine. Can be.

In particular, ornithine may be produced using a low-cost minimal medium, thereby reducing the cost required for the medium, thereby lowering the production cost. In addition, it exhibits a high level of bioconversion in converting arginine to ornithine and citrulline in a minimal medium, and since production time can be very easily separated, harvested and purified as a target bioconversion product, expensive functional ornithine and citrulline. This can be shortened and is effective when applied to industrial mass production.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Isolation of Microorganisms Showing Insoluble Phosphate Degrading Activity from Kimchi

<1-1> Pure separation of Kimchi-derived microorganisms

To provide a strain capable of bioconversion of ornithine, microorganisms were first isolated from kimchi.

Specifically, samples of the kimchi broth were diluted in steps, and cultured in tryptone yeast (TYE) and nutrient medium (NA) containing the composition shown in the following [Table 1] to isolate and select the microorganisms first. Then, the microorganisms isolated and selected were cultured by inoculating the minimally flat medium each containing insoluble phosphate 3-magnesium phosphate, 3-calcium phosphate or calcite. . After cultivation, microorganisms showing transparent rings in a plate medium were obtained as insoluble phosphate solubilizing strains, which were purely cultured. Pure cultured strains were inoculated and cultured again in a minimal plate medium containing 3-magnesium phosphate, 3-calcium phosphate, or calcium phosphate, which are insoluble phosphates, respectively. The solubilization range was identified as the size of the clear ring. In Table 1 below, when TYE, NB, MEM or M medium is used as the solid medium, 20 g / l agar is added to each composition.

Badge Name Substance Concentration (g / ℓ)
TYE
Tryptone 15.0 Yeast extract  5.0 Sodium chloride  8.0 Nutrition medium
(NB) Peptone Yeast extract MEM
Natural medium Malt extract  5.0 Glucose 20.0 M medium
(Minimal
Medium) glucose 15.0 MgSO ₄   0.25 KH ₂ PO ₄   0.25 KCl   0.12 Ca (NO ₃ ) ₂  1.0 FeCl ₃ trace

As a result, as shown in FIG. 2 and Table 2, kimchi-derived strains showing insoluble phosphate solubilizing ability were isolated, and the strains contained organic substances of cellulose, starch, lipids and proteins contained in minimal plate medium. By showing the biodegradable ability to decompose it was confirmed that the organic material can be used as a growth source. In addition, the solubilization ability of 3-calcium phosphate or 3-magnesium phosphate was shown to assist in the decomposition and digestion of bone-derived foods, it was confirmed that 3-calcium phosphate or 3-magnesium phosphate can be used as a growth source.

CA AA PA LA Tri-Mg Tri-Ca Lig Resolution ○ ○ ○ ◎ ◎ ◎ × Transparent ring diameter (mm) - - - 35 20 7 - Transparent ring thickness (mm) - - - 15 5 2 -

In the table, CA is a cellulolytic enzyme (cellulase), AA is a starch decomposition activity (Amyloytic enzyme, amylase), PA is a proteolytic enzyme (protease), LA is a lipid degradation Tri-Mg is trimagnesium phosphate, Tri-Ca is tricalcium phosphate, and Lig is Ligninase. Indicates. The 'decomposition', ◎: strain growth, strong activity; ○: strain growth, weak activity; And x: No strain growth, no activity. The 'transparent ring thickness' represents the distance from the colony end of the strain to the edge of the active ring.

<1-2> Identification of biological characteristics of Kimchi-derived KA84 strain

Physiological and chemical characteristics of the strains isolated from kimchi exhibiting phosphate soluble activity were confirmed.

Specifically, the degradation activity of various strains of Enterococcus pessium KCCM12118 strain as a strain and a control group isolated in Example <1-1> was analyzed.

As a result, as shown in the following [Table 3], the KA84 strain was able to degrade a variety of substrates, it was confirmed that the L-arginine (L-arginine) is included.

temperament KA84 KCCM12118 sodium pyruvate + + hippuric acid + + esculin ferric citrate + + pyroglutamic acid-β-napthylamide + + 6-bromo-2-naphthyl-αD-galactopyranoside + + naphthol ASBI-glucuronic acid - - 2-naphthyl-βD-galactopyranoside + + 2-napthyl phosphate + + L-leucine-β-napthylamide - - L-arginine + + D-ribose + + L-arabinose + + D-mannitol - - D-sorbitol + + D-lactose + + D-trehalose + + inulin - - D-raffinose - - starch (2) - - glycogen - -

Identification of Microorganisms Showing Insoluble Phosphate Degradation Activity from Kimchi

It was identified to identify the strain isolated in Example <1-1>.

Specifically, the strain isolated in Example <1-1> was inoculated into 5 ml of NB liquid medium, followed by incubation at 37 ° C. for 12 to 16 hours, and then inoculated again with the strain cultured in 50 ml of NB liquid medium. Main culture was for 12-16 hours. After the end of the culture, the culture was obtained and centrifuged at 13,000 rpm for 10 minutes at 4 ° C to discard the supernatant to obtain cells. 567 µl of TE buffer solution, 30 µl of 10% SDS, and 3 µl of protease K (20 mg / ml) were added to the obtained cells, followed by vortexing, homogenization, and incubation at 37 ° C for 1 hour. 100 μl of 5M NaCl was added, homogenized, and 80 μl of CTAB / NaCl solution was added thereto, followed by vortexing and reaction at 65 ° C. for 10 minutes. Equivalent amount of chloroform: isoamyl-alcohol (24: 1) was added to the mixture, vortexed, and centrifuged for 5 minutes to transfer the supernatant to a new tube. An equal amount of phenol: chloroform: isoamyl-alcohol (25: 24: 1) was added to the supernatant, vortexed, and centrifuged for 10 minutes to transfer the supernatant to a new tube. 0.6 times volume of isopropanol was added to the obtained supernatant, and then slowly inverted to release genomic DNA. When the white genomic DNA strands were visible to the naked eye, they were removed with a glass rod, and 1 ml of 70% ethanol was added, followed by centrifugation at 4 ° C and 13,000 rpm for 10 minutes to discard the supernatant and to dry. After suspension with sterile distilled water added with RNA hydrolase, the reaction was carried out at 37 ° C. for 30 minutes to remove RNA and then stored at −20 ° C.

Genomic DNA extracted from the strain was used as template DNA, and PCR amplification was performed using Gene Cycle ^™ (BIO-RAD Co.). For PCR reaction solution, 3 μl of forward (SEQ ID NO: 2) and reverse (SEQ ID NO: 3) primers (10 pmol) prepared with the sequences shown in the following [Table 4] were added, and template DNA (10 ng genomic DNA) 5 39 μl of sterile distilled water was added to the PCR Mixer (Bioneer, Korea), mixed, and mineral oil was added. PCR reaction conditions, once denature (denaturation) for 4 minutes at 94 ℃; After 30 cycles of denaturation at 94 ° C. for 1 minute, binding at 60 ° C. for 1 minute, and elongation at 72 ° C. for 1 minute 30 seconds; Final amplification at 72 ° C. for 10 minutes. Amplified DNA was electrophoresed on 0.8% agarose gel to identify the amplified product of strain 16s rRNA.

Kinds designation Information Plasmid pBluescript II pBluescriptⅡ SK (+/-) phagemid, Amp ^r , lacz gene, MCS Host strain E. coli DH5α F ^- Φ80d lac Z △ ( lac ZYA-argF) U160 end A1 rec A1 hsd R17 (r _k -m _k +) deo R thi -1 sup E44 λ- gyr A96 relA1 Primer sequence
16s 63F (Forward) 5'-CCCAAGCTTAACTGCAGCAGGCCTAACACATGCAAGTC-3 ' 16s 1406R (Reverse 5'-CGGAATTCGGGATCCACGGWGTRCAAG-3 '

0.6이 될 때까지(약 3 시간)더 배양하였다. 배양물을 4℃에서 원심분리(4,000 rpm, 10 분)한 후 10% 글리세롤을 첨가하여 3 회 세척하였다. 이렇게 제조된 competent cell을 각각 80 ㎕씩 분주하여 전기천공법(electroporation)을 위해 -70℃에 보관하여 사용하였다. EcoRⅠ으로 절단된 pBluescriptⅡ KS 벡터를 self-ligation한 것과 pBluescriptⅡ vector 1㎕을 각각 제조한 competent cell에 electroporator(BioRad)를 이용하여 전기충격으로 형질전환(transformation)을 수행하여 TYE-Amp 고체배지에서 형질전환율을 확인하였다. E. coli DH5α was plated on TYE plate medium and incubated at 37 ° C. for 12-16 hours to obtain a single colony. A single colony was inoculated in 1/100 volume in 5 ml SOB (2% tryptone, 0.5% yeast extract, 0.058% NaCl, 0.019% KCl, pH 7.0) with an A ₆₀₀ value of 0.4

The cells were further incubated until 0.6 (about 3 hours). The cultures were centrifuged at 4 ° C. (4,000 rpm, 10 min) and washed three times with the addition of 10% glycerol. 80 μl of competent cells thus prepared were dispensed and stored at −70 ° C. for electroporation. TYE-Amp solid medium was transformed by electroshock using electroporator (BioRad) to competent cells prepared by self-ligation of p BluescriptII KS vector digested with Eco RI and 1 μl of p BluescriptII vector. The transformation rate was confirmed in.

In DNA cloning, the amplified product of the KA84 strain 16s rRNA was double-cut into Bam HI and Hind III, and the p BluescriptII KS vector was also cut into Bam HI and Hind III. The cleaved PCR product and the vector were mixed and bound with T4 ligase (Elpis) at 16 ° C. for 2 hours. Then, the reaction solution was dialyzed for 20 to 30 minutes to remove salt, and then mixed with E. coli DH5α and the reaction solution in a cuvette and left on ice for 30 minutes, followed by electroporation (eletroporation). Samples of cuvettes were transferred to 1 ml SOC (980 μl SOB 980 μl 2 M Mg ²⁺ 10 μl, 40% glucose 10 μl) and incubated for 1 to 2 hours at 37 ° C. in a shaker. Recombinant cultured in SOC was plated in 80 μl of X-gal (20mg / DMF 1ml) smeared in TYE Amp solid medium, incubated for 16 hours at 37 ° C, and colonies selected through Blue-White screening were PCR. In order to confirm the insertion of the product into the vector, the plasmid was isolated. The isolated plasmid was digested by Bam HI and Sal I, Eco RI and Sal I at 37 ° C. for 2 hours, and the insert DNA was identified on 0.8% agarose gel.

Selected strains were inoculated in 5 ml of TYE liquid medium to which antibiotics (Ampicillin, Amp) were added and incubated at 37 ° C. for 12 to 16 hours, followed by centrifugation at 4 ° C. and 12,000 rpm for 5 minutes to obtain cells. 100 μl of Solution I (0.9% glucose, 10 mM EDTA, 25 mM Tris-HCl pH 8.0, 4 mg / ml) was added to the obtained cells, mixed, homogenized, and then 200 μl of Solution II (0.2N NaOH, 1% SDS). Was dissolved and reacted for 5 minutes on ice. 150 μl of Solution III (60 ml of 5M phosphate acetate, 11.5 ml of acetic acid, 28.5 ml of dH ₂ O) was added, taking care not to let 10 minutes pass, followed by reaction on ice for 10 minutes and centrifugation at 4 ° C. for 10 minutes The resulting supernatant was transferred to a new tube, and then turbidized with the same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuged at room temperature for 5 minutes. Transfer the obtained supernatant to a new tube, add 1 ml of 100% ethanol (-20 ° C.) to remove salt, and centrifuge at 4 ° C. for 5 minutes to discard the supernatant. Then, precipitate by adding 1 ml of 70% ethanol (-20 ° C), centrifuged at 4 ° C for 5 minutes, discard the supernatant, dry, dissolve in sterile distilled water, decompose RNA with RNase (10mg / ml), and -20 ° C. Stored in. Plasmid DNA separation for sequencing was performed according to the manufacturer's protocol using a plasmid identity kit (Promega, USA). The sequencing of the isolated plasmid DNA was commissioned by Cosmojintech (Korea), and aligned using the blast N program of the National Center for Biotechnology Information (NCBI). ) Was identified as a strain that shows more than 98% homology.

As a result, the strain having insoluble phosphate soluble activity isolated from kimchi has a nucleotide sequence represented by SEQ ID NO: 1 of a total size of 1,350 bp, it was confirmed that this is a novel strain belonging to Enterococcus faecium ( Enterococcus faecium ). Thus, the novel strain was named as Enterococcus faecium KA84 strain.

Entrococcus Pesium  Ornithine from Arginine of KA84 Strain Conversion  Check whether

In order to check whether the entrococcus pesium KA84 strain (hereinafter referred to as the KA84 strain) exhibits the ability to convert arginine to ornithine, thin layer chromatography (TLC) is performed to determine whether to convert arginine to ornithine. I wanted to confirm.

Specifically, inoculated with KA84 strain in TYE and NA liquid medium and pre-incubated for 2 days, and then in each experimental condition medium prepared by adding 0.174% arginine to TYE + NA complex medium, M minimal medium or MEM natural medium, 100 μl of the precultured strain was inoculated and main culture was carried out at 28 ° C. for 72 hours. After the completion of the main culture, 500 µl of the culture was obtained, 5 ml of free amino acid extraction solvent (methanol: chloroform: distilled water = 12: 5: 3) was added, vortexed and mixed, followed by centrifugation at 3,000 rpm for 5 minutes. Supernatant was taken to extract the amino acids in the reaction mixture as extracts. The resulting extract was spotted in 100 μl on silica gel TLC plates for amino acid analysis. The drip plate was developed by placing it in a chamber containing a development buffer in which 1-butanol, acetic acid and water were mixed at 80:20:20 (v / v / v). Then, ethanol containing 1% ninhydrin was sprayed onto the developed plate with a color developing reagent, and heated at 110 ° C. for 5 to 10 minutes to form spots of the developed arginine and ornithine. By comparing the positions, the level of conversion of the ornithine produced was confirmed. Untreated control (KA84 strain cultured in a complex medium containing no arginine), Bacillus licheniformis (KAcillus Bacillus licheniformis ) KA6, KA89 or KA107 strain in the experimental condition medium containing arginine as main culture, said The production level of ornithine was confirmed in the same manner as.

As a result, as shown in [Fig. 3] to [5], the KA84 strain produced ornithine in both complex medium, minimal medium and natural medium. Specifically, after 72 hours of culture in TYE + NA complex medium and MEM natural medium, arginine was 100% converted to ornithine, and furthermore, it was confirmed that not only ornithine but also citrulline (citrulline; Cit) could be converted ([FIG. 3] and [FIG. 4]), it was confirmed that about 70% of arginine was converted to ornithine even in the M minimal medium (FIG. 5).

[Trusted agency]

Depositary Name: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13657BP

Deposit Date: 20181005

<110> PAICHAI UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Enterococcus faecium KA84 and a Method for bioconverting arginine          to ornithine using the strain <130> 1064550 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> Enterococcus faecium KA84 <400> 1 ccggaaaaag aggagtggcg aacgggtgag taacacgtgg gtaacctgcc catcagaagg 60 ggataacact tggaaacagg tgctaatacc gtataacaat cgaaaccgca tggttttgat 120 ttgaaaggcg ctttcgggtg tcgctgatgg atggacccgc ggtgcattag ctagttggtg 180 aggtaacggc tcaccaaggc cacgatgcat agccgacctg agagggtgat cggccacatt 240 gggactgaga cacggcccaa actcctacgg gaggcagcag tagggaatct tcggcaatgg 300 acgaaagtct gaccgagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaaactct 360 gttgttagag aagaacaagg atgagagtaa ctgttcatcc cttgacggta tctaaccaga 420 aagccacggc taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg 480 gatttattgg gcgtaaagcg agcgcaggcg gtttcttaag tctgatgtga aagcccccgg 540 ctcaaccggg gagggtcatt ggaaactggg agacttgagt gcagaagagg agagtggaat 600 tccatgtgta gcggtgaaat gcgtagatat atggaggaac accagtggcg aaggcggctc 660 tctggtctgt aactgacgct gaggctcgaa agcgtgggga gcaaacagga ttagataccc 720 tggtagtcca cgccgtaaac gatgagtgct aagtgttgga gggtttccgc ccttcagtgc 780 tgcagctaac gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag 840 gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcaaga 900 accttaccag gtcttgacat cctttgacca ctctagagat agagcttccc cttcgggggc 960 aaagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1020 cgcaacgagc gcaaccctta ttgttagttg ccatcattca gttgggcact ctagcaagac 1080 tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1140 tgggctacac acgtgctaca atgggaagta caacgagttg cgaagtcgcg aggctaagct 1200 aatctcttaa agcttctctc agttcggatt gcaggctgca actcgcctgc atgaagccgg 1260 aatcgctagt aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca 1320 ccgcccgtca caccacgaga gtgtatcaca 1350 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer_F <400> 2 cccaagctta actgcagcag gcctaacaca tgcaagtc 38 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer_R <400> 3 cggaattcgg gatccacggw gtrcaag 27

Claims (8)

A novel Enterococcus faecium KA84 strain deposited with accession number KCTC 13657BP, characterized in that the strain is derived from kimchi and consists of the nucleotide sequence of SEQ ID NO: 1,
The strain has the activity of degrading any one or more organics selected from the group consisting of cellulose, proteins, lipids and phosphates; And the activity of converting citrulline or arginine to ornithine; Characterized in having a
The strain is characterized in that the strain is cultured in TYE + NA complex medium or MEM natural medium for 70 to 75 hours at a temperature of 25 to 30 ℃.
delete delete delete delete Ornithine production composition comprising Enterococcus faecium KA84 strain deposited with Accession No. KCTC 13657BP or a culture thereof, wherein the strain is derived from Kimchi and consists of the nucleotide sequence of SEQ ID NO: 1 As a composition for producing ornithine,
The strain has the activity of degrading any one or more organics selected from the group consisting of cellulose, proteins, lipids and phosphates; And the activity of converting citrulline or arginine to ornithine; Characterized in having a
The strain is characterized in that the composition is cultured in TYE + NA complex medium or MEM natural medium for 70 to 75 hours at a temperature of 25 to 30 ℃.
i) preparing an Enterococcus faecium KA84 strain deposited with accession number KCTC 13657BP or a culture thereof; And
ii) reacting the composition comprising arginine with the strain or culture of step i) in a TYE + NA complex medium or MEM natural medium at a temperature of 25-30 ° C. for 70-75 hours;
As comprising, ornithine production method, wherein the strain of step i) is derived from kimchi, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1,
The strain has the activity of degrading any one or more organics selected from the group consisting of cellulose, proteins, lipids and phosphates; And the activity of converting citrulline or arginine to ornithine; Ornithine production method characterized in that having.

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