KR101127164B1 - Lactobacillus genus microorganism having the glutaminase gene of Lactobacillus reuteri - Google Patents

Abstract

The present invention relates to a strain of the genus Lactobacillus having a good glutaminase activity by introducing a glutaminase gene of Lactobacillus luterie.

According to the present invention, glutamate can be produced from glutamine by using a strain of the genus Lactobacillus having high glutaminase activity as a natural food additive, which can avoid side effects or pathological symptoms caused by existing artificial food additives.

Glutaminase, Lactobacillus, Transformation

Description

Lactobacillus genus microorganism having the glutaminase gene of Lactobacillus reuteri with the glutaminase gene of Lactobacillus luteri introduced

The present invention relates to a strain of the genus Lactobacillus having a good glutaminase activity by introducing a glutaminase gene of Lactobacillus luterie.

Currently, MSG (monosodium glutamate), a chemical seasoning, is widely used as a food additive, and its consumption is steadily increasing. However, chemically synthesized MSG is sometimes toxic when used as a food additive (Tiziana P et al., Food Chemistry 104 (4): 1712-1717, 2007). And by overingesting MSG, 'Chinese Restaurant Syndrome', 'Relevance to cancer' (which can turn into carcinogens at high temperatures), and 'Association with asthma' in people sensitive to MSG It has been reported to appear.

Therefore, recently, interest in natural food additives that benefit human health as a food additive or ingredient has emerged. With the westernization of diet, it is revealed that the main cause of various adult diseases is in the diet, and research is being conducted to find functional substances that improve the physiological functions of the human body from living organisms (animals, plants, microorganisms) and enzymes. Among them, glutaminase enzyme, which is known as a commercially important enzyme that can enhance the taste of food, is a representative example.

Glutaminase acts as a catalyst for the deamination of L-glutamine into L-glutamate and has a taste enhancing effect when added to foods (Hodson & Linden, Physiology & Behavior 89 (5): 711-717 , 2006), are generally known as important enzymes that provide taste to fermented foods such as soy. The sodium salt component of glutamate serves to taste when added to food, and is widely used in microorganisms including bacteria as well as yeast or mold, and is widely used as an additive in fermented foods (Naohisa M et al., J. Bioscience & Bioengineering, 2005). Glutaminase, which plays the largest role in the production of glutamate, is generally stable to temperature and heat, and has high salt resistance, which can withstand environmental changes in food processing (Lapufade P et al., Appl Environ Microbiol 64: 2485-2489, 1998). It has also been shown to be effective in treating leukemia in combination with asparaginase (Masuo N et al., Bioscience & Bioengineering, 2005).

Lactobacillus spp., Which lives in the normal flora of the human oral, digestive and urogenital systems, produces lactic acid to acidify the pH to neutralize pathogenicity, and bacteriocin, an antibiotic produced by Lactobacillus lactic acid bacteria. (bacterocin) inhibits the growth of several pathogenic bacteria (Goossens D et al., Dig Dis Sci 37: 44-50, 2005; Hiller SL et al., Clin Infect Dis 16: S273-281, 1991; Karine V et al., J Bacteriol 70: 2057-2064, 2002; Ouwehand AC et al., Antonie van Leeuvenhoek 82: 279-289, 2002). Lactic acid bacteria of the genus Lactobacillus having such characteristics have been studied a lot of probiotics (probiotics) used as dietary treatments and formal dressings of humans and animals (Talarico TL et al., Antimicrob Agents Chemother 32: 1854-1858, 1988; Wolf BW et al., Food Chem Toxicol 36: 1085-1094, 1998).

Representative examples of probiotic strains include Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus bulgaricus and Lactobacillus bulgaricus. Some species, such as Lactobacillus rhamnosus and Lactobacillus delbrueckii, have been studied to be resistant to salts and to heat stability (Erwin G et al., J Bacteriol 180: 4718-4723, 1998; Jaya) P et al., Appl Envir Microbiol 69: 917-925,2003).

In addition, some of the probiotic strains are known to exhibit glutaminase activity, and glutaminase gene sequences such as Lactobacillus luteri F275, Lactobacillus johnsonii NCC533, and Lactobacillus gasseri LGAS0507 Is open to the public.

However, studies on increasing glutaminase activity by introducing the glutaminase gene into a strain are unknown.

Therefore, the present inventors have studied and tried to improve the glutaminase activity, and found that by introducing the glutaminase gene of Lactobacillus luterie into a specific strain of Lactobacillus, excellent glutaminase activity can be obtained. The present invention has been completed.

Accordingly, an object of the present invention is to provide a strain of the genus Lactobacillus into which the glutaminase gene of Lactobacillus luterie is introduced.

The invention is characterized by Lactobacillus plantarum KLB 213-13 (KCTC 18164P) into which the gene of SEQ ID NO: 1 is introduced.

In addition, the present invention is characterized by Lactobacillus paracasei KLB 58-7 (Accession No. KCTC 18163P) into which the gene of SEQ ID NO: 1 is introduced.

In another aspect, the present invention is characterized by another vector pGlu-16 for transformation of the genus Lactobacillus strain comprising the gene of SEQ ID NO: 1.

According to the present invention, glutamate can be produced from glutamine by using a strain of the genus Lactobacillus having high glutaminase activity as a natural food additive, which can avoid side effects or pathological symptoms caused by existing artificial food additives.

The invention is characterized by Lactobacillus plantarum KLB 213-13 (KCTC 18164P) into which the gene of SEQ ID NO: 1 is introduced.

In another aspect, the present invention is characterized by Lactobacillus paracasei KLB 58-7 (KCTC 18163P) in which the gene of SEQ ID NO: 1 is introduced.

The length of the gene of SEQ ID NO: 1 corresponds to 1293 bp, and the glutaminase gene present in the gene is present at positions 228 to 1167 in the gene sequence of Lactobacillus luteri F275, and its length corresponds to 939 bp. do.

In addition, the gene of SEQ ID NO: 1 includes a base sequence that exhibits glutaminase activity as a modified sequence in which not only SEQ ID NO: 1 but also some bases of SEQ ID NO: 1 are substituted, deleted or added.

The Lactobacillus plantarum KLB 213-13 transformed by introducing the gene of SEQ ID NO: 1 was deposited on June 19, 2009 with the accession number KCTC 18164P to KCTC, and the gene of SEQ ID NO: 1 was introduced and transformed. Lactobacillus paracasei KLB 58-7 was deposited with KCTC on accession number KCTC 18163P on June 19, 2009.

The present invention also features a vector pGlu-16 for transformation of a strain of the genus Lactobacillus comprising the gene of SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail by the following examples. The following examples are given for the purpose of illustrating the invention and do not limit the scope of the invention.

[ Example ]

1. Cloning of the Glutaminase Gene

Forward primer 5 for amplification of the glutaminase gene, based on the gene sequence of Lactobacillus luteri F275 (NC009513) through nucleotide search by the National Institute of Biological Information (NCBI) (www.ncbi.nlm.nih.gov) ′-AGT CAC GAG TGA CTT CTG-3 ′ (SEQ ID NO: 2) and reverse primer 5′- AGC GGT GGC AAA GTA GTC-3 ′ (SEQ ID NO: 3) were designed. And genomic DNA of SEQ ID NO: 1 was extracted from Lactobacillus reuteri KCTC 3594 (FIG. 1), PCR was performed using the primers as a template, and the gene of SEQ ID NO: 1 was used. Was amplified.

2. Preparation of vector for introduction of glutaminase gene

The amplified gene of SEQ ID NO: 1 was inserted into a pGEM-T easy vector (Promega), which was named pL3594. After pL3594 and pTRKL2, which is an E. coli - Lactobacillus shuttle vector, was ligated to prepare plasmid pGlu-16 of 7.5 kb.

3. Transformation of Lactobacillus plantarum

Lactobacillus plantarum KLB 213 strain was inoculated in 5 mL MRS medium containing 1% by weight glycine in 15 mL flasks (Difco, West Molesey), incubated for 3 hours at 37 ° C and stationary conditions, followed by centrifugation at 8,000 rpm. After suspension in 1 mL SM buffer and 100 ㎕ were dispensed to obtain a competent cell (competant cell).

1 μg of the glutaminase gene introduction vector pGlu-16 of Example 2 was added to the reactive cells, and placed in a 2 mm electroporation cuvette stored in ice, and then subjected to 1.2 kV, 2 ms. After puncture, 200 μl of MRS was mixed and incubated at 37 ° C. for 3 hours, and then plated on a MRS plate containing 5 μg / ml of erythromycin. The resulting colonies were once again confirmed on MRS plates containing 5 μg / ml of erythromycin.

4. Transformation of Lactobacillus paracasei

Lactobacillus paracasei KLB 58 strains were inoculated in 5 mL MRS medium containing 1% by weight glycine in 15 mL flasks (Difco, West Molesey), incubated at 37 ° C. for 3 hours, and then centrifuged at 8,000 rpm. Thereafter, the cells were suspended in 1 mL SM buffer and dispensed into 100 μl to obtain competent cells.

After adding 1 ㎍ of the glutaminase gene introduction vector pGlu-16 of Example 2 to the reactive cell prepared as described above, put it in a 2 mm electroporation cuvette stored in ice and electroporated at 1.2 kV, 2 ms. 200 μl of MRS was mixed and incubated at 37 ° C. for 3 hours, and then plated on an MRS plate containing 5 μg / ml of erythromycin. The resulting colonies were once again confirmed on MRS plates containing 5 μg / ml of erythromycin.

5. Check whether the gene is introduced into the strain

Genomic DNA was extracted from the transformed Lactobacillus strains obtained in Examples 3 and 4. Primer pairs of SEQ ID NOs: 2 and 3 for amplification of the gene of SEQ ID NO: 1 using each genomic DNA as a template, and forward primers for amplification of the erythromycin resistance gene 5′- GAA ATT GGA ACA GGT AAA GG Each PCR was performed using 3 ′ (SEQ ID NO: 4) and reverse primer 5′- GAT ATT CTC TGA CCC AT-3 ′ (SEQ ID NO: 5) to finally confirm whether the gene was inserted.

6. Measurement of Glutaminase Activity

Lactobacillus luteri KCTC 3594, Lactobacillus plantarum KLB 213, Lactobacillus paracasei KLB 58, and the transformed Lactobacillus plantarum KLB 213-13, Lactobacillus paracaze of Example 4 KLB 58-7 was incubated in MRS plate medium containing 5 μg / ml of 1.5% agar smeared erythromycin at 37 ° C. anaerobic conditions. The MRS is based on 20 g of glucose, 10 g of peptone NO.3, 10 g of gravy, 5 g of yeast extract, 5 g of sodium acetate, 2 g of potassium dihydrogen phosphate (K ₂ HPO ₄ ), and twin 80 1 g. , trace salts solution _{(CaCl 2 5 g, FeSO 4} 125 mg, CaSO 4 75 mg, MnSO 4 4.3 mg, ZnSO 4 108 mg, Na 2 MoO 4? 2H 2 0 125 mg, nitrile acetate 7 g) containing 2 mL of It was made.

Colonies on MRS agar plate medium were transferred to 5 mL of MRS (broth) containing 5 μg / ml of erythromycin and incubated at 37 ° C. for 24 to 48 hours. The culture was centrifuged at 8000 x g for 5 minutes to obtain cultured cells.

To remove residual components, washed twice with TE buffer solution (50 mM Tris-HCl and 5 mM EDTA, pH 7.5) and then resuspended in 1 ml of TE buffer solution and homogenized by sonication for 1 minute.

The total protein concentration of each cell was then measured by the Bradford method using bovine serum albumin.

In addition, after equilibrating the mixed solution of 2 wt% L-glutamine solution and 100 mM Tris-HCl buffer solution (pH 7.5) for 5 minutes at 37 ℃, 0.1 mL of each of the homogenized cells was added and 10 minutes at 37 ℃ Reacted. The reaction was stopped by heating the reaction for 3 minutes, then centrifuged at 8000 xg for 5 minutes and 50 μl of supernatant was added to 1 mL of hydroxylamine buffer solution (0.25 M hydroxyl amine and 20 mM EDTA, pH 8.0), 10 mM NAD ⁺ 0.5 mL of solution, 1 mL distilled water and 20 U / ml glutamate dehydrogenase (GLDH) mixture were added. After reacting at 37 ° C. for 30 minutes, absorbance was measured at 340 nm. 1 U (unit) in glutaminase activity is defined as the amount of glutaminase required to produce 1 μmol NADH for 1 minute at 37 ° C.

The total protein and glutaminase activity of each cell measured are shown in Table 1 below, and a graph comparing unit activity is shown in FIG. 2.

cell Total protein (mg) Total active (U) Unit activity (U / mg) Lactobacillus luster KCTC 3594 9.03 4.660 0.516 Lactobacillus plantarum KLB 213 5.72 1.206 0.210 Lactobacillus paracazei KLB 58 5.82 - - Transformed Lactobacillus plantarum KLB 213-13 (Example 3) 5.72 4.486 0.784 Transformed Lactobacillus paracasei KLB 58-7 (Example 4) 1.77 1.206 0.249

As shown in Table 1, Lactobacillus plantarum KLB 213 showed about 1/2 glutaminase activity as compared to Lactobacillus luteri KCTC 3594, but the transformed Lactobacillus plantarum KLB 213 of Example 3 -13 showed more than 1.5-fold activity of the Lactobacillus luteri KCTC 3594.

And Lactobacillus paracasei KLB 58 did not exhibit glutaminase activity, but transformed Lactobacillus paracasei KLB 58-7 of Example 4 showed 0.2 U / mg or more activity.

Therefore, it was confirmed that Lactobacillus plantarum KLB 213-13 into which the gene of SEQ ID NO: 1 was introduced showed significantly better glutaminase activity than wild type Lactobacillus plantarum KLB 213 which was not transformed. there was.

Figure 1 shows the gene sequence of SEQ ID NO: 1. ATG represents the start codon and TAA represents the stop codon.

FIG. 2 shows wild-types of Lactobacillus luteri KCTC 3594, Lactobacillus plantarum KLB 213 and Lactobacillus paracaze KLB 58 and Lactobacillus plantarum KLB 213 and Lactobacillus paracaze KLB 58 transformed according to the present invention. It is a graph comparing glutaminase activity.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Lactobacillus genus microorganism having the glutaminase gene of          Lactobacillus reuteri <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 1293 <212> DNA <213> Lactobacillus reuteri <400> 1 agtcacgagt gacttctgtc tcgctcccat ttcgttatct ttttttataa aaatatttat 60 tctaattatt tgatttattt ataatatatt tgttgcgact attacgatga ttagtatctg 120 agaagatgag ctcatgtaga tattaggcta ttatttcatc atgcaaatta agtgtaaagg 180 ggggctaatt tttcttgttt ggtacttgag gagctgcagg gttatttatg ccttgtgtga 240 gagaggtttt gacgatgcaa aaattagagc aattgattga taaaaacttt gctgaaacag 300 ctcatggtaa ggttgcaaac tatattccga tattaggaat tgttgatccg caacaactgg 360 gcattgccat ttatgacgtt gacgaggacg aaattgggac cgctggaatg gcaggaacgc 420 gatttgcgat cgagagtatt gcgaaggtca taactttaat attaacgatc aagagattag 480 gacataagcg cgtcttagca gaactagaaa atggttcggc agattatagc ctaagttcgg 540 tactattgga tgacgagtta accgagcagg tccaccgggt aaattacctt aataattcct 600 ccgctttgtt aacgacagcc ttaattgacc agttgatggg gcagaatagt tttaatgccc 660 ttctcggttt ctgccgtgag atctgcaatg acccatgcat tagcttgaat gagcgtttat 720 ttcggtcagc aattatgaat gataaagata ttcatgcact ggcttactat atgaaagata 780 aagatatttt agagactgtt gatcaaacat tgataactta ttttatgcaa agctcaatga 840 tggtgacatc acagagtctt gctaacctag gagcagtctt ggcaaatgat ggaattaaac 900 cttgggataa tgagcgtctt attagccatg aagataacga gttggtaaag aaattgctaa 960 caacagttgg ttcgtttgaa gaatcaaaag aatacacaat taaaattgaa ctccctatta 1020 aaagtggtac tggcggcggc ttattggctt gtgccccgca aaaatgtggt attggtattt 1080 ttagtccagc tcttgatcaa catggcaata gtttggcagg aatgagttta ttacaagatg 1140 ttgttgatca attagaatta gtagtttaat cataaccacc cgtcaaacgg gtggtttgca 1200 catcggctat aagccgataa taaaagccgg cgtctcaagg cgctggcttt cgctttgctc 1260 aagccaaaat gctctgacta ctttgccacc gct 1293 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer (Glutaminase) <400> 2 agtcacgagt gacttctg 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> Reverse primer (glutaminase) <400> 3 agcggtggca aagtagtc 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer (Erythromycin) <400> 4 gaaattggaa caggtaaagg 20 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> Reverse primer (Erythromycin) <400> 5 gatattctct gacccat 17  

Claims (3)

Lactobacillus plantarum KLB 213-13 (accession number KCTC 18164P) into which the gene of SEQ ID NO: 1 was introduced. Lactobacillus paracasei KLB 58-7 (accession number KCTC 18163P) into which the gene of SEQ ID NO: 1 was introduced. delete

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