KR101651239B1 - Microorganism of Pseudomanas species and method for deamination of 6-aminocaproic acid using thereof - Google Patents

Abstract

The present invention relates to newly separated Pseudomonas stutzeri which can deaminate an amino compound, and to a method for deaminating an amino compound using the same. A compound without an amino group can be produced in an industrial manner by using the newly separated Pseudomonas stutzeri.

Description

[0001] The present invention relates to novel Pseudomonas sp. Strains, and to a method of using 6-aminocaproic acid for the deaminization of 6-aminocaproic acid using the same.

The present invention relates to novel Pseudomonas stutzeri which can deaminate an amino compound and a process for deaminating an amino compound using the same.

Adipic acid is a dicarboxylic acid compound having the molecular formula (CH ₂ ) ₄ (COOH) ₂ . Adipic acid is widely used as a raw material for nylon resin raw materials, plastic plasticizers, dyes, medicines and the like, and is an intermediate product particularly important for the production of polyamides such as nylon 6,6 and has a very high commercial value.

Adipic acid is produced primarily by chemical processes that begin with petroleum compounds as raw materials and undergo a two-step process. Specifically, cyclic compounds such as phenol, cyclohexane, cyclohexene, and benzene, which are starting materials, are converted to ketone-alcohol oil (KA oil) named cyclohexanone or cyclohexanol, To produce adipic acid. Although this chemical process has high efficiency and economical efficiency, there is a problem that benzene is used as a raw material and an enormous amount of nitrogen oxide is generated as a by-product. In addition to these problems, the recent environmental regulations have led to the need for environmentally friendly processes for the production of adipic acid, and efforts are underway to produce adipic acid through microorganisms. However, the precise biosynthesis or biodegradation pathway of 6-aminocaproic acid, which can be used as an intermediate in adipic acid production, is not yet known. When the 6-aminocaproic acid group of 6-aminocaproic acid is removed and the ketone group is introduced, it becomes an adipate semialdehyde, and adipic acid can be synthesized through the reaction of aldehyde dehydrogenase (Guerrillot L. et al., Eur J Biochem., 1977, 81 (1): 185-92; Vandecasteele, JP et al., Methods Enzymol., 1982, 89: 484-490). The conversion of an enzyme from 6-aminocaproic acid to adipic acid of the same number of carbons is highly evaluated as a novel technology as well as a commercial value, but enzymes or microorganisms that can participate in the actual reaction have not been known.

Thus, the present inventors have completed the present invention by separating a new microorganism having a deamines activity while studying a method of deaminizing 6-aminocaproic acid.

It is an object of the present invention to provide a novel microorganism which deamines an amino compound.

Another object of the present invention is to provide a deamination method of an amino compound using the microorganism.

In one aspect of the present invention, there is provided a novel isolated Pseudomonas stutzeri strain KCCM11587P which deamines the amino compound.

The term "amino compound" used in the present invention means an organic compound having an amino group (-NH ₂ ), specifically, a compound in which hydrogen of a hydrocarbon is substituted with an amino group, which is also referred to as a primary amine. The amino compound may be, for example, at least one selected from the group consisting of N-acetyl ornithine, gamma aminobutyric acid, 5-aminovaleric acid and 6-aminocaproic acid, but is not limited thereto. Specifically, the amino compound may be 5-aminovaleric acid, or 6-aminocaproic acid.

As used herein, the term "deamination" refers to the removal of an amino group from a compound having an amino group.

The terms used in the present invention, "Pseudomonas stu cherry (Pseudomonas Stutzeri is a gram-positive, rod-shaped, single polar flagellated, soil-borne bacterium. So far, Pseudomonas stuccoi strains have been found to contain amino compounds, especially 6-amino However, the present inventors have newly isolated and identified a Pseudomonas Stucheri strain capable of deaminating an amino compound containing 6-aminocaproic acid.

The Pseudomonas stuccoi KCCM11587P strain of the present invention can use an amino compound as a nitrogen source. In one embodiment, it was confirmed that Pseudomonas stucherii CJ-MKB (KCCM11587P) according to the present invention was immobilized in cells using an amine group of 6-aminocaproic acid as a single nitrogen. In the case of 6-aminocaproic acid, it has one amine group. To use it as a nitrogen source, a desalting process is necessary.

The Pseudomonas Stucheri strain of the present invention can deaminate an amino compound, thereby producing a compound having an amino group removed.

The ability of the Pseudomonas Stucheri strain to deaminate the amino compound may be due to the transaminase of the microorganism. The natural demineralization enzymes are divided into the ammonia lyase family (EC 4.3 .-.) And the transaminase family (EC 2.6 .- .-). In the case of ammonia lyase, the substrate containing an amine group has an electron withdrawing group capable of retaining electrons, but in the case of 6-aminocaproic acid, it has a functional group capable of retaining electrons around the amine group The ammonia lyase reaction will be difficult. On the other hand, in the case of the transaminase, it is possible to cause 6-aminocaproic acid to undergo a desamination reaction because a co-substrate can be reacted with an electron-withdrawing functional group. Therefore, Pseudomonas stuccoi KCCM11587P of the present invention can deaminate 5-aminovaleric acid or 6-aminocaproic acid which does not have an electron-withdrawing functional group around the amine group by the transaminase of the strain.

The compound in which the amino group is removed may be, for example, at least one selected from the group consisting of N-acetylglutamate 5-semialdehyde, succinate semialdehyde, glutarate semialdehyde and adipate semialdehyde, but is not limited thereto . Specifically, the compound in which the amino group is removed may be glutarate or adipate semialdehyde.

The Pseudomonas stuccoi KCCM11587P strain of the present invention may have the sequence of the 16s rRNA of SEQ ID NO: 1. In addition, the Pseudomonas stuccoi strain may specifically have the property of forming resistance to ampicillin and / or biofilm in addition to the sequence of the sequence of 16s rRNA of SEQ ID NO: 1.

In addition, the Pseudomonas stuccoi strain specifically includes an N-acetylornithine transaminase gene comprising the nucleotide sequence of SEQ ID NO: 4 and / or a 4-aminobutyrate aminotransferase gene comprising the nucleotide sequence of SEQ ID NO: 6 .

As used herein, the term "4-aminobutyrate transaminase" refers to the reaction of 4-aminobutyrate + 2-oxoglutarate-succinate semialdehyde + L-glutamate Refers to an enzyme catalyzing, and is also referred to as '4-aminobutyrate aminotransferase' or 'GABA transaminase'.

The term, "N- acetyl ornithine transaminase (N-acetylornithine aminotransferase)" used in the present invention 'N ₂ - acetyl -L- ornithine 2-oxoglutarate + ↔ N- acetyl-5--L- glutamate Semialdehyde + L-glutamate ', which is also called' acetylornithin aminotransferase '.

In one embodiment, it was confirmed that the newly isolated and identified Pseudomonas stuchryi KCCM 11587P has the ability to deaminate 5-aminovaleric acid and 6-aminocaproic acid, respectively (see FIG. 2). In one embodiment, the Pseudomonas stacheri KCCM11587P was found to be a novel microorganism having the ability to deaminate the amino compound unlike the known strains (see Fig. 3).

Another aspect of the present invention provides a method for deaminating an amino compound comprising culturing the Pseudomonas Stucherii strain in a medium containing an amino compound.

The Pseudomonas stuccoi strain is as described above.

The cultivation may be carried out in accordance with a culture medium and an appropriate medium known in the art. In addition, a person skilled in the art can easily use the culture medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, for example, a batch culture, a continuous culture, a fed-batch culture, or a combination culture thereof.

The medium must meet the requirements of a particular strain in a suitable manner and may be suitably modified by a person skilled in the art. In addition, the medium may contain various nitrogen sources, carbon sources, trace element components, and the like.

The nitrogen source contained in the culture medium may include an amino compound to be subjected to decaramination. The nitrogen source may specifically be at least one amino compound selected from the group consisting of N-acetyl ornithine, gamma aminobutyric acid, 5-aminovaleric acid and 6-aminocaproic acid. But are not limited to, peptone, yeast extract, juice, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds other than the amino compound, such as ammonium sulfate, ammonium chloride, ammonium carbonate, Ammonium and the like may be further included.

Carbon sources that may be used include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture, but are not limited thereto.

Sources of phosphorus that may be used include, but are not limited to, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth, but is not limited thereto. In addition, essential growth materials such as amino acids and vitamins may be included.

Suitable precursors may also be used in the culture medium. Specifically, the medium may further include, but is not limited to, one or more and / or pyridoxal phosphate selected from the group consisting of pyruvate, oxaloacetate, and? -Ketoglutarate. The culture medium or the individual components may be added to the culture medium in the culture process in an appropriate manner, either batchwise or continuously, but the present invention is not limited thereto.

In addition, during culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microorganism culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture. In addition, oxygen or an oxygen-containing gas (e.g., air) may be injected into the culture medium to maintain the aerobic state of the culture medium. The temperature of the culture medium may be usually 20 to 45 캜, for example, 25 to 40 캜. The incubation period may be continued until the amount of the desired amino group-removed compound is obtained, and may be, for example, 10 to 160 hours.

The method for deaminating the amino compound may further include recovering a compound from which the amino group has been removed from the medium. The compound in which the amino group is removed may specifically be at least one selected from the group consisting of N-acetylglutamate 5-semialdehyde, succinate semialdehyde, glutarate semialdehyde and adipate semialdehyde, but is not limited thereto . The method of recovering the amino group-removed compound from the cell or culture can be carried out by collecting or recovering the amino group-free compound produced from the culture using a suitable method known in the art depending on the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC may be used, but the present invention is not limited to these examples.

Another aspect of the present invention provides a method of deaminating an amino compound comprising reacting an amino compound with a cell lysate of the Pseudomonas stachery cells. The Pseudomonas stuccoi strain and the reactable amino compound are as described above.

In the present invention, the term "cell lysate of Pseudomonas stacheri cells" means a cell fragment of Pseudomonas stacheri KCCM11587P produced by the disappearance or destruction of the membrane of Pseudomonas stacheri KCCM11587P cells, Or a nucleic acid or the like.

The solution for reacting the pulverized product of Pseudomonas stachery cells with the amino compound may contain one or more and / or pyridoxal phosphate selected from the group consisting of pyruvate, oxaloacetate, and? -Ketoglutarate . In a specific embodiment of the present invention, a cell lysate of Pseudomonas stucheri CJ-MKB was added to a solution containing 6-aminovaleric acid or 6-aminocaproic acid, alpha-keto glutarate and pyridoxal phosphate, . The temperature of the reaction may be from 25 캜 to 35 캜, specifically from 25 캜 to 30 캜. The reaction time may be 0.5 to 6 hours, 1 to 5 hours, 2 to 4.5 hours, or 3 to 4 hours. The decarboxylation method may further include recovering a compound from which the amino group has been removed from the reaction product.

The method of deaminating the amino compound can be used for the production of adipic acid. In the production of adipic acid, 6-aminocaproic acid as an amino compound can be converted into adipate semialdehyde by adding a nitrogen source to the culture medium of the Pseudomonas stuccoi strain of the present invention or reacting with a cell crush of Pseudomonas stuccoi strain . The adipic acid can then be synthesized from the converted adipate semialdehyde using methods known to those of ordinary skill in the art. The synthesis of the adipic acid from the adipate semialdehyde may be specifically performed by an aldehyde dehydrogenase reaction.

The amino compound can be easily deaminated using the newly isolated Pseudomonas stucco strain KCCM11587P of the present invention and the compound in which the amino group is removed can be industrially produced. Further, the novel Pseudomonas stuccarelus strain of the present invention can be used in a process for producing adipic acid by deaminating 6-aminocaproic acid.

Figure 1 is a photograph showing that a selected strain using a minimal medium using 6-aminocaproic acid as a nitrogen source forms a biofilm. Specifically, a single colony is cultured and centrifuged, and the resultant product is separated into biofilm and strain using distilled water.
Figure 2 shows the results of TLC analysis of the production of glutamate and the degree of substrate degradation depending on various concentrations of 5-aminovaleric acid, 6-aminocaproic acid, alpha-keto glutaric acid, and pyridoxal phosphate and the presence or absence of each substrate .
[5AVA: 10mM 5-aminovaleric acid (standard); 1: 20 mM 6-aminocaproic acid (standard); 2: Pseudomonas stucheri CJ-MKB + 20 mM 6-aminocaproic acid; 3: Pseudomonas stutzeri CJ-MKB + 10 mM 6-aminocaproic acid and 20 mM 5-aminovaleric acid; 4: Pseudomonas stutzeri CJ-MKB + 20 mM 6-aminocaproic acid, 10 mM alpha-ketoglutarate and 0.1 mM pyridoxal-phosphate; 4-1: Pseudomonas stutzeri CJ-MKB + 20 mM 6-aminocaproic acid and 10 mM alpha-ketoglutarate; 5: Pseudomonas stutzeri CJ-MKB + 10 mM 6-aminocaproic acid, 20 mM 5-aminovaleric acid, 10 mM alpha-ketoglutarate and 0.1 mM pyridoxal-phosphate; 5-1: Pseudomonas stutzeri CJ-MKB + 10 mM 6-aminocaproic acid, 20 mM 5-aminovaleric acid and 10 mM alpha-ketoglutarate; 6: Pseudomonas stutzeri CJ-MKB + 20 mM 6-aminocaproic acid and 20 mM glutamate; E: 10 mM glutamate (standard)]
FIG. 3 shows TLC results of deaminization by reacting cell microparticles with 5-aminovaleric acid and 6-aminocaproic acid, respectively. Specifically, 10 mM 5-aminovaleric acid or 6-aminocaproic acid, 10 mM alpha-keto glutarate, and 0.1 mM pyridoxal phosphate were added to the cell lysate of each strain to carry out the reaction.
[1: 10 mM 5-aminovaleric acid, 10 mM 6-aminocaproic acid (standard); 2: Clostridium aminovalericum + 10 mM 5-aminovaleric acid; 3: Pseudomonas fluorescein + 10 mM 5-aminovaleric acid; 4: Pseudomonas memantine + 10 mM 5-amino valeric acid; 5: Pseudomonas stucheri CJ-MKB + 10 mM 5-aminovaleric acid; 6: Clostridium aminovalericum + 10 mM 6-aminocaproic acid; 7: Pseudomonas fluorescein + 10 mM 6-aminocaproic acid; 8: Pseudomonas memantine + 10 mM 6-aminocaproic acid; 9: Pseudomonas stutzeri CJ-MKB + 10 mM 6-aminocaproic acid; 10: 10 mM glutamate (standard)]

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1: Identification of microorganisms using 6-aminocaproic acid as a nitrogen source

1) Selection of microorganisms using 6-aminocaproic acid as a nitrogen source and 16S rRNA analysis

A novel strain, Pseudomonas stutzeri CJ-MKB, which can deaminate 6-aminocaproic acid through a subculture method was fixed with a nitrogen source fixed with 6-aminocaproic acid (6-ACA).

The minimum medium in which the microorganisms can be cultured for selection was prepared as shown in the following Table 1, and 6-aminocaproic acid was used as a nitrogen source for culturing the strains.

Medium composition Final concentration Na ₂ HPO ₄ .H ₂ O 15.1 mM KH ₂ PO ₄ 22 mM NaCl 8.6 mM 6-aminocaproic acid 20 mM MgSO ₄ 1 mM CaCl ₂ 100 mM _{(NH 4) 6Mo 7 O 24} · H 2 O 3 nM H ₃ BO ₃ 400 nM CoCl ₂ · H ₂ O 30 nM CuSo ₄ H ₂ O 10 nM MnCl ₂ · H ₂ O 80 nM ZnSO ₄ .H ₂ O 10 nM FeSO ₄ .H ₂ O 1 mM Glucose 11.1 mM

Specifically, a soil sample of a Kimpo plant of CJ CheilJedang in Gayang-dong, Seoul, was cultured at 37 ° C and 200 rpm using a medium having the composition shown in Table 1 above. The cultured candidate strains were cultured until the optical density reached 0.5 at an incubation optical density (OD600) of 0.05 and the same culture conditions as above. The microorganisms cultured in the medium having the composition shown in Table 1 could be selected through five subcultures. The selected microorganism was named as 'CJ-MKB' and the following experiment was conducted to confirm that it was a new microorganism.

The CJ-MKB strains cultured in the medium were plated on M9 agar plates to obtain colonies. The obtained colonies had similar morphology and were resistant to ampicillin at a concentration of 25 mg / ml. It was confirmed that a light colored biofilm was formed around the colonies. Two colonies were selected and genomic DNAs were extracted using a commercially available genomic DNA prep kit. The sequence of 16S ribosomal RNA (16S rRNA) was analyzed for identification of the obtained genome. Using the primer 27F (AGA GTT TGA TCC TGG CTC AG; SEQ ID NO: 7) and primer 1492R (GGT TAC CTT GTT ACG ACT T; SEQ ID NO: 8) which are commonly used in the sequencing of microbial 16S rRNA, the CJ- Of the 16S rRNA has the nucleotide sequence of SEQ ID NO: 1.

Using the BLAST program provided by the National Center for Biotechnology Information (NCBI), strains having high nucleic acid homology with SEQ ID NO: 1 were searched. (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). As a result, Pseudomonas stutzeri strain NBRIS11, Gamma proteobacterium BP44-iso8, Uncultured bacterium clone 9, and Escherichia coli BM0446 Escherichia coli strain BM0446, and enterobacteriaceae bacterium BM005, respectively, as compared to the 16S rRNA of the enterobacteriaceae bacterium BM005.

Among the identified microorganisms, several Pseudomonas sp. Microorganisms are known to form exopolysaccharides to form bio-flims. Pseudomonas sp. Microorganisms are also known to be resistant to beta-lactam antibiotics such as penicillin It has resistance. Considering that the sequence information of 16S rRNA of CJ-MKB strain and the resistance to biofilm and amphicillin seen in culture are considered, it is expected that the selected strain is a microorganism of Pseudomonas line. Single colonies obtained from M9 medium agar plates containing 25 mg / ml of ampicillin were cultured in LB broth and centrifuged at 13,000 rpm for 1 minute to obtain cultured strains (FIG. 1). When distilled water is added to the obtained strain and lightly shaken, the biofilm and the strain are easily separated.

2) Sequence analysis of redox enzyme

To determine whether the selected strains were Pseudomonas sp., We analyzed whether the novel strain CJ-MKB has the nucleotide sequence of oxidoreductase present in Pseudomonas species. Sequencing was carried out using the newly designed primer NCPPB 5P (ATGAGCAAGACTAACGAATCCC; SEQ ID NO: 9) and the primer NCPPB 3P (TCCAGAATGGCCAGCCCGCG; SEQ ID NO: 10). As a result, it was confirmed to have the nucleotide sequence of SEQ ID NO: 2.

Analysis of the nucleotide sequence of SEQ ID NO: 2 with NCBI BLAST revealed that SEQ ID NO: 2 had the same sequence as the molybdopterin-binding sequence of the redox enzyme of the known Pseudomonas stucco strain A1501 , And the selected new strain CJ-MKB was a microorganism belonging to the genus Pseudomonas.

3) Sequence analysis of transaminase

Since the homologous sequence was 714 short sequences, it was not possible to correctly classify the microbial dependence. Additional protein nucleic acid sequences were analyzed to confirm the dependency. When the microorganism uses 6-ACA as a nitrogen source, it is expected that N-acetylornithine transaminase or 4-aminobutyrate transaminase in transaminase, which is expected to be involved in its enzyme conversion reaction, The base sequence of the enzyme was confirmed.

Specifically, argD_F2 (5 'primer: ATTTAAGGATCCGTCCGCCCCGCACACCCCGG; SEQ ID NO: 11) was used in order to comparatively analyze the nucleotide sequence of N-acetyl ornithine transaminase existing in the genome of Pseudomonas stuccoi A1501 and the selected strain CJ- And argD_R2 (3 'primer: ATTTAAGAGCTCTCAGGCCTGGGTCAGCGTC; SEQ ID NO: 12). As a result, it was confirmed that Pseudomonas stuccoi A1501 and the N-acetylornithine transaminase of the new microorganism had the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Also, gabT_F (5 'primer: ATTTAACATATGCAACGCCGTGTCGCCGCCGTTCC; SEQ ID NO: 13) and gabT_R (3' primer: ATTTAAGAATTCTCAGGTCAGCTCGTCGAAACACT; SEQ ID NO: 13) were used in order to comparatively analyze the nucleotide sequences of Pseudomonas stuccoi A1501 and selected strains of 4-aminobutyrate transaminase. No. 14). As a result, it was confirmed that they had the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Multiple sequence alignment was used to compare the N-acetyl ornithine transaminase sequence of Pseudomonas stuccoi A1501 and selected strains. As a result, 13 of the 1,221 nucleic acid sequences were found to be different (nucleic acid homology: 98.9353%). Also, by comparing the nucleic acid sequences of Pseudomonas stuccoi A1501 and the 4-aminobutyrate transaminase of the new strain in the same manner, 21 nucleic acids of 1,257 nucleic acid sequences were found to be different (nucleic acid homology: 98.3294%).

In conclusion, the selected strain Pseudomonas stutzeri CJ-MKB (Pseudomonas stutzeri CJ-MKB) has the highest homology with Pseudomonas stuccoi A1501 among the known microbial genomic sequences and is a new strain. Accordingly, the selected Pseudomonas stucheri CJ-MKB strain was deposited on October 22, 2014 with the Korea Microorganism Conservation Center and received the accession number KCCM11587P.

Example  2: Deamines  Check reactivity

1) Pseudomonas Stucherry  CJ- MKB of Deamines  Check reactivity

An evaluation of 5-aminovaleric acid and 6-aminocaproic acid tamarinization reactivity of Pseudomonas stucheri CJ-MKB was performed. Since the reaction of the transaminase is a substitution reaction of the amine group and the ketone group, the reaction product can be easily identified through the amine coloring reaction of ninhydrin after the material separation using thin layer chromatography (TLC) have. The whole-cell reaction was carried out to confirm the transaminase activity of Pseudomonas stucheri CJ-MKB. When Pseudomonas stucheri CJ-MKB was cultured and the optical density reached 0.7, the cells were subcultured on a new M9 medium, and 6-aminocaproic acid and 5-aminovaleric acid were fixed with a nitrogen source and cultured. Alpha-ketoglutarate and pyridoxal-phosphate were added to the medium, and TLC was performed to confirm the degree of desamination of 5-aminovaleric acid and 6-aminocaproic acid. The reaction volume was 100 μl, and the concentration of 5-aminovaleric acid, 6-aminocaproic acid, alpha-ketoglutarate, and pyridoxal-phosphate and the production of glutamate according to the presence or absence of each substrate, Were compared on TLC. After the development of the TLC was completed, a substance having an amine group was developed with a 3% ninhydrin solution. As a result of TLC, it was confirmed that 5-aminovaleric acid, 6-aminocaproic acid disappeared and glutamate was formed (see FIG. 2).

As shown in 97 h reaction, lane 4 in FIG. 2, the strain which added 20 mM 6-aminocaproic acid, 10 mM alpha-keto glutarate, and 0.1 mM pyridoxal phosphate to the medium, Aminocaproic acid were all reacted. It was confirmed on TLC that the 6-aminocaproic acid decreases even in the absence of pyridoxalphosphate, though the reaction rate is slow compared to when the transaminase reaction is met (97 h reaction lane 4-1 of FIG. 2). When 5-aminovaleric acid and 6-aminocaproic acid were present at the same time, no decrease in 6-aminocaproic acid was observed (97 h reaction in FIG. 2, lane 5, and 5-1). Thus, when Pseudomonas stucheri CJ-MKB was cultured under minimal medium conditions, 6-aminocaproic acid and 5-aminovaleric acid were evaluated to compete with nitrogen source.

From the results of this experiment, Pseudomonas stucheri CJ-MKB can be used as a single nitrogen source of 6-aminocaproic acid, and 5-aminovaleric acid and 6-aminocaproic acid thalamination reaction in the presence of alpha-ketoglutarate and pyridoxalphosphate (Fig. 2). On the other hand, a small amount of glutamate was confirmed in the 24h reaction, lane 5 and 72h reaction, and lane 4 in Fig. 2, but it was not continuously accumulated. It is estimated that glutamate is produced in addition to the reaction of 6-aminocaproic acid, but that most of the glutamate is used as an amine source and therefore is not rapidly accumulated in the cell but is rapidly converted.

2) Other strains and Pseudomonas Stucherry  CJ- MKB Comparison of Reamerization Reactivity

In order to further verify the deamination reactivity of Pseudomonas stucheri CJ-MKB, the concentration of the cell lysate was increased to carry out the deamination reaction using 5-aminobalate and 6-aminocaproic acid as amine substrates, respectively. 10 mM 5-aminovaleric acid, 6-aminocaproic acid, 10 mM alpha-keto glutarate, and 0.1 mM pyridoxal phosphate were added to the CJ-MKB cell mixture of Pseudomonas stucco and reacted at 28 ° C for 4 hours 3).

As a comparative example, Clostridium aminovalericum, Pseudomonas fluorescens, and Pseudomonas mendocina were respectively cultured, and 5-aminovaleric acid and 5-aminobutyric acid were used as cell mills of each microorganism. The reaction results for 6-aminocaproic acid were compared by TLC. As shown in lanes 5 and 9 in FIG. 3, the production of glutamate could be confirmed in the Pseudomonas stucheri CJ-MKB cell crush. On the other hand, the production of glutamate was not confirmed in other strains.

Thus, unlike the known strains, the novel strain Pseudomonas stucheri CJ-MKB has the ability to deaminate an amino compound.

Korea Microorganism Conservation Center (overseas) KCCM11587P 20141022

<110> CJ CheilJedang Corporation <120> Microorganism of Pseudomonas species and method for deamination of          6-aminocaproic acid using thereof <130> PN107904 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 1384 <212> DNA <213> Pseudomonas stutzeri <220> <221> rRNA &Lt; 222 > (1) .. 1384 <223> a partial DNA sequence of CJ-MKB 16s rRNA <400> 1 tgcagtcgag cggatgaatg gagcttgctc catgattcag cggcggacgg gtgagtaatg 60 cctaggaatc tgcctggtag tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac 120 gtcctacggg agaaagtggg ggatcttcgg acctcacgct atcagatgag cctaggtcgg 180 attagctagt tggtgaggta aaggctcacc aaggcgacga tccgtaactg gtctgagagg 240 atgatcagtc acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 300 aatattggac aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc 360 ggattgtaaa gcactttaag ttgggaggaa gggcagtaag ttaatacctt gctgttttga 420 cgttaccaac agaataagca ccggctaact tcgtgccagc agccgcggta atacgaaggg 480 tgcaagcgtt aatcggaatt actgggcgta aagcgcgcgt aggtggttcg ttaagttgga 540 tgtgaaagcc ccgggctcaa cctgggaact gcatccaaaa ctggcgagct agagtatggc 600 agagggtggt ggaatttcct gtgtagcggt gaaatgcgta gatataggaa ggaacaccag 660 tggcgaaggc gaccacctgg gctaatactg acactgaggt gcgaaagcgt ggggagcaaa 720 caggattaga taccctggta gtccacgccg taaacgatgt cgactagccg ttgggatcct 780 tgagatctta gtggcgcagc taacgcatta agtcgaccgc ctggggagta cggccgcaag 840 gttaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 900 gaagcaacgc gaagaacctt accaggcctt gacatgcaga gaactttcca gagatggatt 960 ggtgccttcg ggaactctga cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020 tgttgggtta agtcccgtaa cgagcgcaac ccttgtcctt agttaccagc acgttaaggt 1080 gggcactcta aggagactgc cggtgacaaa ccggaggaag gtggggatga cgtcaagtca 1140 tcatggccct tacggcctgg gctacacacg tgctacaatg gtcggtacaa agggttgcca 1200 agccgcgagg tggagctaat cccataaaac cgatcgtagt ccggatcgca gtctgcaact 1260 cgactgcgtg aagtcggaat cgctagtaat cgtgaatcag aatgtcacgg tgaatacgtt 1320 cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgctcca gaagtagcta 1380 gtct 1384 <210> 2 <211> 714 <212> DNA <213> Pseudomonas stutzeri <220> <221> rRNA <222> (1). (714) <223> partial DNA of CJ-MKB oxidoreductase molybdopterin binding <400> 2 agcccttcct cgaacagggt gttgagcagg ccgaacaaca gcgcggcgtc ctgtccgggg 60 cgcacgaaca gatgttgatc ggcgatggcg gcggtctcgc tgcgccgcgg gtcgaccacc 120 accagtttgc cgccgcgggc gcggatcgcc ttcagccgct tctccacatc gggcacggtc 180 atgatgctac cgttggaggc caaagggttg ccgccgagaa tcagcatgaa atcggtgtga 240 tcgatgtccg gcaccggcag cagcagacca tggccgtaca tcaggtggct ggtcaggtgg 300 tgcggcagct ggtcgaccga cgtggccgag aagcggttgc gcgtcttcag cagaccgagg 360 aagtaattgc tgtgggtcgt cagcccgtag ttatgcacac tcgggttgcc ttggtagatc 420 gccacggcat tactaccgtg gcgctcgcgg atctcagcca ggcgctcggc gaccagttcg 480 taggcctcct cccagctgat ggcctgccac tcatcgccgc agcggcgcat cgggtggcgc 540 aggcgatcgg ggtcgttctg gatgtcctgc agcgccactg ccttcggaca gatatggccg 600 cggctgaagc tgtcgagggg atcgcccttg atcgaaagga tgcgctcgcc ttcggtctgg 660 atggtcaggc cgcagatggc ttcgcacagg tgacaggcac ggtgatggac gctg 714 <210> 3 <211> 1221 <212> DNA <213> Pseudomonas stutzeri <220> <221> gene &Lt; 222 > (1) .. (1221) <223> N-acetylornithine transaminase gene of P. stutzeri <400> 3 atgtccgccc cgcacacccc ggtagaacgc gccgatttcg accaggtcat tgttcccact 60 ttcgcccccg ccgccttcgt gccggtgcgt ggtctgggct cgcgagtatg ggatcagagc 120 ggtcgagaac tggtggactt cgctggcggc atcgcggtta acgccctggg ccatgcccat 180 ccggccatga tcgcggcact gacggagcag gctggcaagc tctggcacat ctccaacatc 240 tataccaacg agccggcgct gcgcctggcc aaaaagctgg tggcggcgac cttcgccgac 300 cgcgccttct tctgcaattc cggcgcagag gccaacgagt cggcgttcaa actggcccgt 360 cgctacgccc atgatgtgta tggcccgcag aaattcgaga tcatctcggc gctcaacagc 420 ttccacggcc gcaccctgtt caccgtcacg gtgggcgggc agtcgaagta ctccgatggc 480 ttcgggccga agatcgaagg catcactcac gtgccttaca acgacctcga agcgctgaag 540 gcggcgatct ccgacaagac ctgtgccgtg gtgctcgaac cgatccaggg cgagagcggc 600 atcctgccgg gcgagcaggc gtacctggaa ggtgcacgcc agctgtgcaa cgagcacaac 660 gcgctgctga tcttcgatga ggtgcagacc ggtctaggcc gtaccggtga gctgttcgcc 720 tacatgcact atggcgtcac gccggacatt ctgaccaacg ccaagagcct gggtggcggc 780 ttcccgatcg gtgcgatgct gaccaccaac gagatcgccg cccatttcag cgtcggtacc 840 cacggcacca cgtacggcgg caatccgctg gcctgcgcgg tggccgagac ggtgctggat 900 atcgtcaaca ccccggaggt gctggagggc gtcaaggccc gccacgagcg cttcaaggcc 960 cggctgttgc agatcggtga gcgctacggc gtgttcagcc aggtccgcgg ccgtggcctg 1020 ttgatcggct gcgtgctctc ggatgcctgg aaaggcaagg ccggtgcatt ctgggccgcg 1080 gcggagaagg aggcgctgat ggtgctgcag gcagggccgg acgtggttcg cctggcgccg 1140 agtctgatca tcgacgaagc tgacatagac gaggggctgg atcgtttcga gcgcgccatc 1200 gcgacgctga cccaggcctg a 1221 <210> 4 <211> 1221 <212> DNA <213> Pseudomonas stutzeri <220> <221> gene &Lt; 222 > (1) .. (1221) <223> N-acetylornithine transaminase gene of P. stutzeri CJ-MKB <400> 4 gtgtccgccc cgcacacccc ggtagaacgc gccgatttcg accaggtcat tgttcccact 60 ttcgcccccg ccgccttcgt gccggtgcgt ggtctgggct cgcgagtatg ggatcagagc 120 ggtcgagaac tggtggactt cgctggcggc atcgcggtca acgccctggg ccatgcccat 180 ccggccatga tcgcggcact gacggagcag gctggcaagc tctggcacat ctccaacatc 240 tataccaacg agccggcgct gcgcctggcc aagaagctgg tggcggcgac cttcgccgac 300 cgcgccttct tctgcaattc cggtgcagag gccaacgagg cggcgttcaa actggcccgt 360 cgctacgccc atgatgtgta cggcccgcag aagttcgaga tcatctcggc gctcaacagc 420 ttccacggcc gcaccctgtt caccgtcacg gtgggcgggc agtcgaagta ctccgatggc 480 ttcgggccga agatcgaagg cattactcac gtgccttaca acgacctcga agcgctgaag 540 gcggcgatct ccgacaagac ctgtgccgtg gtgctcgaac cgatccaggg cgagagcggc 600 atcctgccgg gcgaacaggc gtacctggaa ggcgcacgcc agctgtgcaa cgagcacaac 660 gcgctgctga tcttcgatga ggtgcagacc ggtctgggcc gtaccggtga gctgttcgcc 720 tacatgcact atggcgtcac gccggacatt ctgaccaacg ccaagagcct tggtggcggc 780 ttcccgatcg gtgcgatgct gaccaccaac gagatcgccg cccatttcag cgtcggtacc 840 cacggcacca cgtacggcgg caatccgctg gcctgcgcgg tggccgagac ggtgctggat 900 atcgtcaaca ccccggaggt gctggagggc gtcaaggccc gccacgagcg cttcaaggcc 960 aggctgttgc agatcggtga gcgctacggc gtgttcagcc aggtccgcgg ccgcggcctg 1020 ttgatcggct gcgtgctctc ggatgcctgg aaaggcaagg ccggtgcatt ctgggccgcg 1080 gcggagaagg aggcgctgat ggtgctgcag gcagggccgg acgtggttcg cctggcgccg 1140 agtctgatca tcgacgaagc tgacatagac gaggggctgg atcgtttcga gcgcgccatc 1200 gcgacgctga cccaggcctg a 1221 <210> 5 <211> 1257 <212> DNA <213> Pseudomonas stutzeri <220> <221> gene &Lt; 222 > (1) .. (1257) <223> 4-aminobutyrate aminotransferase gene of P. stutzeri <400> 5 atgcaacgcc gtgtcgccgc cgttccccgt ggcgtcggcc agatccaccc gatctttgcc 60 gaccacgcga agaacagcag cgtggtggat gtggagggcc gcgagttcat cgatttcgcc 120 ggcggcatcg ccgtgctgaa taccggccac ctgcacccga agatcgtcaa ggcggtggaa 180 gaccagctgc acaagctcac ccacacctgc ttccaggtgc tggcctacga gccgtacgtc 240 gagctgtgcg agaagatcaa cgcgcgggtg ccgggtgatt tcgccaagaa gactctgctg 300 gtcaccaccg gctccgaggc cgtggagaac gcggtgaaga tcgcccgcgc cgccaccggc 360 cgtgccggcg tgattgcctt caccggcgcc tatcacggcc gcaccatgat gaccctcggc 420 ctgaccggca aggtcgcccc gtactccgcc ggcatgggcc tgatgccggg cgggatcttc 480 cgtgcgcagt atccctgcgc gatccatggt gtcagcgtcg atgactcgat cgcgagcatc 540 gagcgcatct tcaagaacga tgccgagccg cgcgacatcg ccgcgatcat cgtcgaaccg 600 gtgcagggcg agggcggctt caatgtcgcg ccgaaggatt tcatggcccg cctgcgcgcg 660 ctctgcgacg agcacggcat cctgctgatc gccgacgagg tgcagaccgg cgccgggcgc 720 accggcacct tcttcgccat ggagcagatg ggcgtggtcg ccgacctgac caccttcgcc 780 aagtcggtcg gcggcggctt cccgatcgcc ggcgtgtgcg gcaaggccga gatcatggat 840 gccatcgcac ccggcgggct gggcggcacc tatgccggca acccgttgtc ctgcgcggcg 900 gcgctggcgg tcatggaaat cttcgaggag gagcatctgc tcgaccgctg caaggccgtg 960 gcggagaagc tcaccaccgg cctcaaggcg atccaggcca agcacaagga gatcggcgag 1020 gtgcgcggcc tcggcgcgat gatcgccatc gagctgttcg aggatggcga cctggcgcga 1080 ccggccgcgg cgctcacgtc gcagatcgtc gcccgggcgc gggagaaggg attgatcctg 1140 ctgtcctgcg gtacctacta caacgtgctg cgcgtgctgg tgccgctgac cgtcgaggac 1200 gagctgctcg agcgcggcct ggccatcctc ggcgagtgtt tcgacgagct gacctga 1257 <210> 6 <211> 1257 <212> DNA <213> Pseudomonas stutzeri <220> <221> gene &Lt; 222 > (1) .. (1257) <223> 4-aminobutyrate aminotransferase gene of P. stutzeri CJ-MKB <400> 6 atgcaacgcc gtgtctccgc cgttccccgt ggcgtcggcc agatccaccc gatctttgcc 60 gaccacgcga agaacagcag cgtggtggat gtggaggggc gccagttcat cgatttcgcc 120 ggcggcaacg ccgtgctgaa taccggccac ctgcacccga agatcgtcaa ggcggtggaa 180 gaccagctgc acaagctcac ccacacctgc ttccaggtgc tggcctacga gccgtacgtc 240 gagctgtgcg agaagatcaa cgcgcgggtg ccgggtgatt tcgccaagaa gactctgctg 300 gtcaccaccg gctccgaggc cgtggagaac gcggtgaaga tcgcccgcgc cgccaccggc 360 cgtgccggcg tcattgcctt caccggcgcc tatcacggcc gcaccatgat gaccctcggc 420 ctgaccggca aggtcgcccc gtactccgcc ggcatgggcc tgatgccggg cgggatcttc 480 cgtgcgcagt acccctgcgc gatccatggt gtcagcgtcg atgactcgat cgcgagcatc 540 gagcgcatct tcaagaacga cgccgagccg cgcgacatcg ccgcgatcat cgtcgaaccg 600 gtgcagggcg agggcggctt caatgtcgcg ccgaaggatt tcatggcccg cctgcgcgcg 660 ctctgcgacg agcacggcat cctgctgatc gccgacgagg tgcagaccgg cgccggacgc 720 accggcacct tcttcgccat ggagcagatg ggcgtggtcg ccgatctgac caccttcgcc 780 aagtcggtcg gcggcggctt cccgatcgcc ggcgtgtgcg gcaaggccga gatcatggat 840 gccatcgcgc ccggcgggct gggcggcacc tatgccggca acccgctgtc ttgcgcggcg 900 gcgctggcgg tcatggaaat cttcgaggag gagcatctgc tcgaccgctg caaggcggtg 960 gcggagaagc tcaccaccgg cctcaaggcg atccaggcca agcataagga gatcggcgag 1020 gtgcgcggcc tcggcgcgat gatcgccatc gagctgttcg aggatggcga cctggcgcga 1080 ccggccgcgg cgctgacgtc gcagatcgtc gcccgggcgc gggacaaggg gctgatcctg 1140 ttgtcctgcg gtacctacta caacgtgctg cgcgtgctgg tgccgctgac cgtcgaggac 1200 gagctgtacg agcgcggcct ggccatcctc ggcgagtgtt tcgacgagct gacctga 1257 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 27F <400> 7 agagtttgat cctggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 1492R <400> 8 ggttaccttg ttacgactt 19 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer NCPPB 5P <400> 9 atgagcaaga ctaacgaatc cc 22 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer NCPPB 3P <400> 10 tccagaatgg ccagcccgcg 20 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer argD F2 <400> 11 atttaaggat ccgtccgccc cgcacacccc gg 32 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer argD R2 <400> 12 atttaagagc tctcaggcct gggtcagcgt c 31 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer gabT F <400> 13 atttaacata tgcaacgccg tgtcgccgcc gttcc 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer gabT R <400> 14 atttaagaat tctcaggtca gctcgtcgaa acact 35

Claims (8)

A novel isolated Pseudomonas stutzeri KCCM11587P deaminating an amino compound, wherein the amino compound is at least one selected from the group consisting of 5-aminovaleric acid and 6-aminocaproic acid, Pseudomonas stutzeri KCCM 11587P. delete A method for deaminating an amino compound, comprising culturing Pseudomonas stacheri KCCM 11587P of claim 1 in a medium containing an amino compound, wherein the amino compound is selected from the group consisting of 5-aminovaleric acid and 6-aminocaproic acid Being one or more. delete 4. The method according to claim 3, wherein the medium further comprises at least one member selected from the group consisting of pyruvate, oxaloacetate and? -Ketoglutarate, and pyridoxal phosphate. A process for deaminating an amino compound comprising the step of reacting a cell lysate of Pseudomonas stucco KCCM 11587P according to claim 1 with an amino compound, wherein the amino compound is selected from the group consisting of 5-aminovaleric acid and 6-aminocaproic acid Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; delete The method according to claim 6, wherein the step of reacting the pulverized product of Pseudomonas stucco KCCM 11587P with an amino compound comprises reacting at least one compound selected from the group consisting of pyruvate, oxaloacetate, and? -Ketoglutarate and pyridoxal phosphate Lt; RTI ID = 0.0 &gt; (I) &lt; / RTI &gt;

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