KR20150001330A - gene, enzyme and enzyme extraction method from Lactobacillus johnsonii PF01 for reducing hypercholesterolemia - Google Patents

Abstract

The present invention relates to genes and enzymes derived from Lactobacillus johnsonii PF01, and a method for extracting the enzyme for reducing hypercholesterolemia. More specifically, the present invention investigates characteristics of recombinant bshA, bshB, and bshC by amplifying the bshA, bshB, and bshC genes which code bile salt hydrolase derived from the Lactobacillus johnsonii PF01, cloning and overexpressing the same in Escherichia coli. The bile salt hydrolase bsh coded by the bshA, bshB, and bshC genes derived from the Lactobacillus johnsonii PF01 decomposes taurine binding and glycine binding of the bile salt, thereby confirming the fact that single strain of the Lactobacillus johnsonii PF01 has two kinds of bile salt hydrolases. The bile salt hydrolase bsh hereby: more efficiently lowers serum cholesterol levels in hypercholesterolemia patients using the efficacies of increasing intestinal viability and lowering cholesterol levels better than conventional bsh at the same time when adding the Lactobacillus johnsonii PF01 into foods and taking the same; and is useful for preventing people with normal cholesterol levels from having hypercholesterolemia.

Description

(Gene, enzyme and enzyme extraction method from Lactobacillus johnsonii PF01 for reducing hypercholesterolemia) gene derived from Lactobacillus Johnson PF01 strain for the reduction of hypercholesterolemia

The present invention relates to a method for separating genes, enzymes and enzymes derived from Lactobacillus johnsonii PF01 strain for the reduction of hypercholesterolemia, and more particularly to a method for separating genes, enzymes and enzymes from bile acids derived from Lactobacillus johnsonii PF01 The bsh A, bsh B, and bsh C genes encoding the salt hydrolase were amplified and cloned and overexpressed in E. coli to identify the characteristics of the recombinant bsh A, Bsh B, and Bsh C.

As the current economic development and income levels have improved, various adult diseases have increased rapidly, and cancer and arteriosclerosis have become the most important factors of modern death. Among the factors that cause adult disease, the most important is the high cholesterol in the blood due to excessive intake of animal fat. Lactic acid bacteria are beneficial and safe microorganisms for adult diseases such as inhibitory action of enteropathogenic bacteria in the intestines, function of reducing cholesterol in blood, function of anti-cancer, immunity enhancement, and lactose intolerance. Cholesterol is the precursor of the first bile acid in the body. In the case of humans, about 0.5g of free bile is excreted as feces. The same amount of bile acid is produced in the liver, and a constant bile acid is maintained in the body. In the liver, the primary bile acid is combined with taurine or glycine to form a complex bile acid. Binding of bile acid to amino acid is similar to peptide binding, but does not degrade any proteolytic enzymes. Even salt hydrolase ( bsh ) Is known to be degraded only by the enzyme. One strain was reported to have multiple bsh genes, but not all genes were active. It is also known that there are eight major complex bile acids in the body, which are bound to taurine or glycine, and that one bsh does not hydrolyze all of them.

Lactobacillus Johnson survives in the gastrointestinal tract, adheres to in vitro host epithelial cells, modulates host immune responses, inhibits pathogenic bacteria such as Helicobacter pyroli , and can provide liver protection in mice . Thus, although Lactobacillus Johnson plays a very important biological role in mammalian intestinal tract, the characteristics of bSh enzyme derived from Lactobacillus Johnson have not yet been elucidated. Therefore, in the present invention, the gene for bile acid hydrolysis and bsh was cloned from the Lactobacillus johnsonii PF01 strain, and the gene was analyzed. Also, the cloned bsh gene was expressed in E. coli, followed by isolation and purification, By investigating the characteristics, we want to make industrial applications possible.

Patent application No. 10-2004-0102428 Method for increasing bile acid-activating enzyme through co-culture of Bifidobacterium bifidum and method for enhancing bile acid-activating enzyme using the same

It is an object of the present invention to provide a pharmaceutical composition containing Lactobacillus Johnson PF01 johnsonii PF01) from the strain bsh A, bsh B, bsh C amplify the gene, followed by mass expressed after cloning E. coli completed the present invention by identifying Lactobacillus zone bile salt hydrolytic effect of Sony PF01 by irradiating the substrate of each bsh Respectively.

In order to accomplish the above object, the present invention provides a recombinant vector comprising Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01) derived bsh A (even salt hydrolase A) gene.

The present invention also provides a bsh B (bile salt hydrolase B) gene derived from Lactobacillus johnsonii PF01 comprising the nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a bsh C (bile salt hydrolase C) gene derived from Lactobacillus johnsonii PF01 comprising the nucleotide sequence of SEQ ID NO: 3.

The present invention also provides a vector comprising the gene.

The present invention also provides a host cell transformed with the recombinant vector.

In another aspect, the present invention Lactobacillus bacteria zone Sony PF01 (Lactobacillus having the nucleotide sequence of SEQ ID NO: 7 and 8 and a primer set for amplifying the bsh A (bile salt hydrolase A) gene derived from P. johnsonii PF01.

The present invention also relates to a nucleic acid encoding a lactobacillus Johnson PF01 ( Lactobacillus strains having the nucleotide sequences of SEQ ID NOS: 9 and 10, and a primer set for amplifying the bsh B (bile salt hydrolase A) gene derived from P. johnsonii PF01.

The present invention also relates to a nucleic acid encoding a Lactobacillus Johnson PF01 ( Lactobacillus < RTI ID = 0.0 > and a primer set for amplifying the bsh C (bile salt hydrolase A) gene derived from P. johnsonii PF01.

In another aspect, the present invention Lactobacillus bacteria zone Sony PF01 (Lactobacillus amplifying bsh A, bsh B, and bsh C genes by PCR using amplification primers corresponding to the bsh A, bsh B, and bsh C genes from the johnsonii PF01 strain; A step of introducing the amplified bsh A, bsh B, and bsh C genes into vectors, respectively, and introducing them into an Escherichia coli strain; Digesting the recombinant plasmid produced in the cloning step with a restriction enzyme and recovering the insert DNA; Subcloning the recovered insert DNA by binding it to a vector, introducing the subcloned strain into an E. coli strain, and transforming the transformed DNA; Extracting a recombinant plasmid from the transformed strain; Introducing the extracted recombinant plasmid into E. coli which is a host cell for mass expression; Selecting a transforming enzyme showing a precipitation reaction by culturing the recombinant plasmid introduced into the E. coli; Lactobacillus Johnson PF01 for the reduction of hypercholesterolemia comprising Lactobacillus johnsonii PF01) strain-derived bile acid salt hydrolase.

In another aspect, the present invention Lactobacillus bacteria zone consisting of the amino acid sequence of SEQ ID NO: 4 Sony PF01 (Lactobacillus johnsonii PF01) derived bsh A (even salt hydrolase A).

The present invention also relates to a pharmaceutical composition comprising Lactobacillus Johnson PF01 ( Lactobacillus < RTI ID = 0.0 > johnsonii PF01) derived bsh B (bile salt hydrolas B).

The present invention also relates to the use of Lactobacillus Johnson PF01 ( Lactobacillus < RTI ID = 0.0 > bsh C (bile salt hydrolase C) derived from johnsonii PF01.

The invention also has a purified bsh A having specific properties.

The present invention also has a purified bsh B having specific properties.

The present invention also has a purified bsh C having specific properties.

The present invention also provides Lactobacillus johnsonii PF01, wherein the bsh A, Bsh B, and Bsh C genes comprising the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are expressed.

In another aspect, the present invention the Lactobacillus zone Sony PF01 (Lactobacillus johnsonii PF01). < / RTI >

As described above, in order to reduce hypercholesterolemia according to the present invention, Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01) isolation method of the gene of the strains derived from, enzyme and enzyme, Lactobacillus zone Sony bile salts singer encoded by the bsh A, bsh B, bsh C gene of PF01 strain derived lyase (bsh) a bile acid salt of taurine bond And glycine bond, it was confirmed that a single strain of Lactobacillus Johnson PF01 had both kinds of bile salt hydrolases. Thus, when Lactobacillus Johnson PF01 is added to food, it is possible to increase the viability of the intestines and to lower the serum cholesterol level more effectively in patients with hypercholesterolemia using the effect of lowering the cholesterol level more effectively than conventionally found bsh , People with cholesterol levels can be useful in preventing hypercholesterolemia.

Figure 1. Bsh gene cleaved by restriction enzyme.
A is Lactobacillus zone Sony number 1 lane with the bsh PF01 gene amplified from strain A is bsh, No. 2 Lane bsh B, lane 3 is bsh C.
B is amplified and purified bsh gene, lane 1 is bsh A, lane 2 is bsh B, lane 3 is bsh C.
C was digested with restriction enzymes, and the purified bsh gene was amplified by using Nde I , Hind III digested bsh A, lane 2 Nde I , Bsh B, lane 3 is cut by Xho Ⅰ Nde Ⅰ, cut by Xho Ⅰ bsh C.
Fig. 2. Results of restriction enzyme treatment of plasmid DNA transformed into Escherichia coli DH5α.
Lane M is DNA size marker, lane 1 is pET21b vector, pET21b vector and bsh A for lanes 2, 3, 4 and 5, pET21b vector and bsh B for lanes 6, 7, Lane 12 is the pET21b vector and bsh C.
Figure 3. Transformed bsh selection.
The plates (A) and (B) contain 0.3% of taurodeoxycholic acid (TDC) and the plate (C) contains glycodeoxycholic acid (GDC).
Figure 4. Expression of bsh
Lane M is the protein size marker, lane 1 is the over-expression of bsh A, lane 2 is the over-expression of bsh B, lane 3 is the over-expression of bsh C, lanes N1, N2 and N3 are control.
Figure 5. Purified recombinant bsh
Lane 1 represents total cell lysate, lane 2 is loaded on the column, and lane 3 represents after washing.
Figure 6. Results of eight major bile salts use.
TC: taurocholic acid, TDC: taurodeoxycholic acid, TCDC: taurochenodeoxycholic acid, THDC: taurohyodeoxycholic acid, TUDC: tauroursodeoxycholic acid, GC: glycocholic acid, GDC: glycodeoxycholic acid, GCDC: glycochenodeoxycholic acid
Figure 7. Effects of temperature of bsh in recombinant E. coli.
Figure 8. Effect of pH of bsh in recombinant E. coli according to pH.
Fig. 9. Base sequence and amino acid sequence of bsh A gene derived from Lactobacillus Johnson PF01.
RBS is the ribosome binding site, and the upward arrow indicates the transcriptional start point (TSP)
Figure 10. Base sequence and amino acid sequence of bsh B gene derived from Lactobacillus Johnson PF01.
RBS is the ribosome binding site, and the upward arrow indicates the transcriptional start point (TSP)
Figure 11. Base sequence and amino acid sequence of bsh C gene derived from Lactobacillus Johnson PF01.
RBS is the ribosome binding site, and the upward arrow indicates the transcriptional start point (TSP)
Figure 12. Lactobacillus Johnson Alignment of the bsh gene from PF01.
Arrow pointing down the active site, called C, D, N, N, four conserved sequence that represents the R has a bsh have a high probability in the area indicated by the four boxes (FGRNXD, AGLNF, VLTNXP, GXGXGXXGXPGD ) which also codes .

Hereinafter, the present invention will be described in more detail by way of specific examples. However, it is to be understood that the present invention is not limited by the following examples, and that various changes and modifications may be made therein without departing from the spirit and scope of the present invention.

Example  1. Use strains and preservation

The strain used in the present invention was Lactobacillus johnsonii PF01 isolated from piglets stored in the laboratory of the Department of Animal Resources and Industrial Microbiology, College of Life Science and Natural Resources, Dankook University, and lactobacilli de Man-Rogosa-Sharpe (MRS, Difco LAB, USA), incubated at 37 ° C for 24 hours (in an incubator; jeio tech, Korea) and incubated in two subcultures to maintain the viability of the strain. Lactobacillus Johnson PF01 is a lactic acid bacterium derived from the feces of pigs, and is known to have excellent ability to degrade bile acids through previous studies.

Example  2. Use badge  And culture

The strains used were cultivated and stored in deMan-Rogosa-Sharpe (MRS) medium, a lactic acid culture medium. For short-term storage strains, they were cultured in MRS medium and stored under refrigeration conditions. To maintain vitality. For long-term storage, cells were collected by centrifugation at 3,000 × g for 20 min in a MRS medium. The collected cells were diluted with 15% glycerol and stored at -80 ° C in a cryogenic freezer.

Example  3. even salt hydrolase ( bsh ) Amplification of A, B and C genes

Polymerase chain reaction (PCR) was used to amplify the bsh (bile salt hydrolase) gene derived from Lactobacillus Johnson PF01. Chromosomal DNA was isolated from the strain of Lactobacillus Johnson PF01 cultured in MRS liquid medium and used as template DNA.

Based on the gene sequences of Lactobacillus Johnson PF01 bsh A, B, and C, specific primers were prepared as shown in Table 1 and PCR was performed.

For gene cloning and expression, Pst I, Nde I and Hind III are attached to both ends of the bsh A primer, Pst I, Nde I and Xho I restriction sites are attached to both ends of the bsh B and bsh C primers, Were added.

bsh A, bsh B, bsh C Primer sequences used for gene amplification Gene name Base sequence bsha Forward AAA CTG CAG CAT ATG TGT AACC TCA ATT GTT TA Reverse AGC CTG CAG AAG CTT ATT TTG ATA ATT AAT TG bsh B Forward AAA CTG CAG CAT ATG TGT ACT GGT TTA AGA T Reverse CTG CAG CTC GAG GTA AGT CAC AAG ACT bsh C Forward CTG CAG CAT ATG TGT ACA TCA ATT TTA T Reverse AAA CTG CAG CTC GAG ATT TTC AAA TTT AAT

PCR (Polymerase Chain Reaction) was performed using GeneAMP PCR system 9700 under the following conditions. 1 μl of each primer pair, 2 μl of 10 × Buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2, pH 8.3), 1 μl of dNTP, 1 μl of Taq Polymerase (10 units / μl) was added to the reaction solution, and the total amount of the PCR reaction solution was adjusted to 20 μl to carry out the PCR reaction. The reaction was repeated 35 times at 95 ° C for 5 minutes, at 95 ° C for 1 minute, at 55 ° C for 1 minute, at 72 ° C for 1 minute and 30 seconds, and finally at 72 ° C for 7 minutes to complete the reaction. After completion of the PCR, 2 μl of the PCR product was electrophoresed on 2% agarose gel to confirm whether the DNA was amplified (FIG. 1).

Example  4. bsh A , B and C genes ( cloning )

The combined DNA preparation and gene manipulation were performed according to the method of Sambrook et al. (2001). The pUC19 vector and the PCR product were treated with Pst I and ligated at 16 ° C before being introduced into Escherichia coli strain DH5α (Takara, Japan). The transformed Escherichia coli was plated on Luria-Bertani agar (Difco, USA) supplemented with ampicillin (50 g), isopropyl thiogalactopyranoside (IPTG 0.1 mM / ml) and X-gal After culturing at 37 ° C for 16-24 hours, bsh A, B, and C colonies were selected. Recombinant plasmids were extracted from the colonies using QIAPrep Miniprep kit (Qiagen, Netherlads). For the subcloning, the recombinant plasmid was digested with restriction enzymes ( bsh A: Nde I / Hind III, bsh B, C: Nde I / Xho I), and only the insert DNA (insert DNA) , Netherlands). The pET21b vector was digested with the same restriction enzyme, bound at 16 ° C, and then introduced into Escherichia coli DH5α. After the transformation, the recombinant plasmid was extracted and treated with a restriction enzyme, and the transformed enzyme was selected by confirming presence or absence of insert DNA through electrophoresis (FIG. 2). The recombinant plasmids extracted from the selected transformants were introduced into E. coli BLR (DE3), which was an expression host, in order to express a large amount of recombinant plasmids. Amphicrin (50 μg / ml), isopropylthiogalactopyranoside (0.1 mM / (LB agar, Taurodeoxycholic acid or Glycodeoxycholic acid, 0.3%) was added to the culture medium after selection of white colony ( bsh A, B and C colonies) after culturing in Luria-Bertani agar (Difco, USA) glucose 1%, CaCl ₂ 0.035%, ampicillin 50 μg / ml) and cultured at 37 ° C. for 24 hours. Transforming enzymes showing a precipitation reaction around the colonies were selected (FIG.

Example  5. bsh A , B, and C genes

A pET21b expression vector (Novagen, Germany) was used in which the His-tag was linked to the N-terminal portion of the cloned bsh A, B and C genes in E. coli.

The sub-cloned recombinant plasmid was introduced into Escherichia coli BLR (DE3), and the transformed enzyme was selected. The transformed enzyme was cultured in an LB medium containing ampicillin (50 μg / ml) until the OD (600 nm) value became 0.5, and then isopropylthiogalactopyranoside (IPTG) was added so as to be 0.1 mM To induce protein expression. The cells were further cultured at 25 ° C for 5 hours to express the protein sufficiently, and the cells were harvested (3,000xg, 20 min). The cell pellet was suspended in lysis buffer (50 mM Na-Pi, 300 mM NaCl, 1 mM DTT) to obtain over-expressed recombinant proteins, and the cells were disrupted with an ultrasonic disintegrator (Sonicator; Sonics, USA) sec, off 10 sec, 1.5 min, 35% amplitude) and then centrifuged at 14,000 rpm for 30 minutes to separate the cell lysate. The cell lysate was subjected to 12% SDS-PAGE electrophoresis to confirm the mass expression of the enzyme (Fig. 4).

Example  6. Expressed recombination bsh A , B, C purification

To purify bsh A, bsh B, and bsh C expressed in cell lysate, Ni ^{2 + N} NTA affinity cloumn (Qiagen, netherlands) was used. The column was equilibrated by lysing buffer (50mM Na-Pi, 300mM NaCl, 1mM DTT) several times, and the cell lysate was loaded and loaded into a 50mM imidazole buffer Washed and eluted with 250 mM imidazole buffer. The presence of purified protein was confirmed by 12% SDS-PAGE (Sodium dodecyl sulfate polyacrylamide) electrophoresis and the protein band was stained with Coomassie Brilliant Blue R250 (Bio-Rad, USA).

As a result, it was confirmed that other protein bands other than bsh expressed from the cell lysate were removed, and the recombinant bsh A, B and C of about 35 kDa, 34 kDa and 37 kDa were respectively purified (FIG. 5).

Example  7. Investigation of substrate specificity

(TC), taurodeoxycholic acid (TDC), taurochenodeoxycholic acid (TCDC), taurohyodeoxycholic acid (THDC) and tauroursodeoxycholic acid (TUDC), glycocholic acid (GC), glycodeoxycholic acid (GDC) and glycochenodeoxycholic acid There are eight kinds of major bile acids such as taurine-based bile acid and glycine-based bile acid. Since no single bsh hydrolysis of both taurine and glycine-based bile acids has been reported so far It was examined that decomposition by the enzyme-linked-Lactobacillus bsh John Sony PF01 strain derived from that of any sequence bile acids and specific to the substrate, respectively.

The substrate specificity of the purified recombinant bsh A, B and C was investigated using 8 bile acids (Sigma, USA) shown in Table 2 and slightly modified based on Tanaka et al. (1999). Specifically, purified bsh was mixed with a total volume of 0.2 ml of 10 mM conjugated bile acid and 100 mM sodium phosphate (pH 6.0). The reaction was carried out at 37 ° C for 30 minutes, stopped with 15% trichloroacetic acid, and centrifuged at 1500 rpm for 5 minutes to remove the precipitate. 20 μl of supernatant was mixed with 80 μl of distilled water and 1.9 ml of ninhydrin reagent containing 0.5 ml of 1% ninhydrin, 1.2 ml of glycerol and 0.2 ml of 0.5 M citrate (pH 5.5) And then cooled for 3 minutes. The degree of color development was measured at OD 570 nm using a spectrophotometer.

As a result, bsh A and bsh B specifically hydrolyzed all of the taurine-based bile acids, but the degradation ability of glycine-based bile acids was very poor. On the other hand, bsh C was found to be superior to bile acid of taurine type in the degradation of glycine-based bile acid (Fig. 6). Lactobacillus Johnson PF01 strain is capable of degrading both taurine and glycine-based bile acids by three kinds of bile acid hydrolases.

The bile acid used in the present invention Glycocholic acid (GC) Glycodeoxycholic acid (GDC) Glycochenodeoxycholic acid (GCDC) Taurocholic acid (TC) Taurodeoxycholic acid (TDC) Taurochenodeoxycholic acid (TCDC) Taurohyodeoxycholic acid (THDC) Tauroursodeoxycholic acid (TUDC)

Example  8. Recombination Complex bile acid salt  Investigation of reaction conditions on the activity of hydrolytic enzymes

bsh To investigate the effect of temperature and pH on the enzyme activity and reaction of A, B and C, purified bsh was used in the reaction.

In order to examine the optimal temperature of the enzyme reaction, in 0.1M sodium phosphate (sodium phosphate) buffer (pH 7.0) bsh A, B is bsh 10mM TDC (10mM DTT), bsh C is as a substrate for (10mM DTT) 10mM GDC The enzyme activity was measured by the enzyme activity assay as in Example 7 after the reaction was performed at intervals of 10 ° C from 20 ° C to 90 ° C. In order to investigate the optimal pH of the enzyme, purified bsh was added to each of the following pH buffer solutions, and the activity of bsh A, bsh B at 60 ° C and bsh C at 70 ° C were compared. The buffer solution used was 0.1 M sodium acetate buffer for pH 3.0 to 6.0, 0.1 M sodium phosphate buffer for pH 6.0-80, 0.1 M tris buffer for pH 8.0-9.0 Respectively.

The optimal temperature for enzyme activity was 55 ℃ for bsh A and 60 ℃ for bsh B. The activity of both enzymes was 80% or more up to 70 ℃ and thus it was judged to have some degree of heat resistance. In case of bsh C, And exhibits activity and maintains an activity of 90% or more even at a high temperature of 80 캜, indicating that it has excellent heat resistance (Fig. 7).

On the other hand, when the effect of pH on the activity of enzyme was investigated, it was found that bsh A had the highest activity at pH 6.0, bsh B at pH 4.5, and bsh C at pH 5.0, indicating that all three enzymes were weakly acidic 8).

Example  9. Recombination bsh  Analysis of genes

The nucleotide sequence of the bsh gene cloned in pET21b was analyzed. The amino acid sequence of the encoded protein was deduced based on the analyzed nucleotide sequence, and the homology of the nucleotide sequence and the amino acid sequence was examined using the NCBI Blast Searching program. Multiple sequence alignment of amino acids was analyzed using the Clustal W program.

As a result, it was confirmed that bsh A, B and C genes were all one ORF (open reading frame). The size of bsh A gene was 981 bp and consisted of 326 amino acids. The ribosome binding site site) and a nucleotide sequence presumed to be a promoter (Fig. 9, SEQ ID NO: 1, SEQ ID NO: 4). The size of the bsh B gene was 951 bp and consisted of 316 amino acids. Similarly, there existed a base sequence presumed to be a ribosome binding site and a base sequence presumed to be a promoter (Fig. 10, SEQ ID NO: 2, SEQ ID NO: 5).

In the case of the bsh C gene, the size of the gene was 978 bp, which was composed of 325 amino acids and had a nucleotide sequence and a promoter sequence presumed to be a ribosome binding site (FIG. 11, SEQ ID NO: 3, SEQ ID NO: 6).

Example  10. bsh  Gene Amino acid sequence  analysis

The homology of the cloned bsh A, bsh B, and bsh C genes was compared using the present invention. All of the bsh The gene conserves five amino acid sequences of C, D, N, N, and R, which are called active sites. Three bsh genes from Lactobacillus Johnson PF01 also have five active sites, bsh Four conserved sequences (FGRNXD, AGLNF, VLTNXP, GXGXGXXGXPGD) with high probability of the genes were also coded (FIG. 12).

Example  11. bsh Use of

Cholesterol is the precursor of the first bile acid in the body. In humans, about 0.5 g of free bile acid is released as feces and a large amount of bile acid is produced in the liver, and a certain amount of bile acid is maintained in the human body. In the liver, the first bile acid is combined with taurine or glycine to form a complex bile acid. The bile acid-amino acid bond is similar to the peptide bond, but is not degraded by any proteolytic enzymes but is degraded only by salt hydrolase ( bsh ) .

There are eight major types of complex bile acids in the body associated with taurine or glycine, and it is known that one bsh does not hydrolyze all.

Therefore, the above-mentioned example was used to clone a bile acid hydrolase gene from L. johnsonii PF01 strain having three kinds of bsh gene, analyze the gene characteristics, and cloned the bsh gene in Escherichia coli After separation and purification, the characteristics of enzyme were investigated and obtained as basic data for industrial application.

The present invention provides a food containing Lactobacillus Johnson PF01 in which the bsh A, bsh B, and bsh C genes as described above are expressed.

When Lactobacillus Johnson PF01 expressing the bsh A, bsh B, and bsh C genes of the present invention is used as a food additive, the strain may be added as it is or may be used together with other food or food ingredients, Can be used.

Examples of the foods to which the strains can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gourmet, ice cream, soups, drinks, tea, , Alcoholic beverages and vitamin complexes, and it is preferable that they are made of fermented milk, yogurt, cheese, milk beverage or milk powder, and more preferably fermented milk, yogurt, beverage, powdered milk, food additive or health supplement. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the Lactobacillus Johnson PF01 strain of the present invention is added to 1 x 10 ⁷ to 10 x 10 ⁸ bacteria (the number of bacteria) of the present invention per 1 g (or ml) of the specific food material at the time of producing food or beverage , Preferably 1 x 10 < ⁸ > to 5 x 10 < ⁸ > The effective dose of the strain of the present invention may be used in accordance with the effective dose of the composition, but may be less than the above range for health and hygiene purposes or long-term intake for the purpose of health control, It can be used in an amount exceeding the range described above.

Korea Microorganism Conservation Center (Domestic) KFCC11549P 20130228

<110> DanKook university Industry-Academic Cooperation Foundation <120> gene, enzyme and enzyme extraction method from Lactobacillus          johnsonii PF01 for reducing hypercholesterolemia <130> P13-0026 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 981 <212> DNA <213> Lactobacillus johnsonii PF01 bshA DNA sequence <400> 1 atgtgtacct caattgttta tagttcaaat aatcatcatt attttggccg aaatctagac 60 ttggaaattt catttggtga acatcctgta attacaccaa gaaattatga gtttcaatat 120 cgtaaattac caaataaaaa ggcaaaatat gccatggttg ggatggcgat tgtagaagat 180 aattatccat tatattttga tgcatcaaat gaagaagggc taggaattgc tggtcttaat 240 tttgatggac catgtcatta tttcccagaa gtgtctggaa aaaataatgt tacaccattt 300 gaattaattc cttatttgtt aagtcaatat actacagtgg ctgaagtaaa agaagcattg 360 aaaagtgtta acttagtaaa gataaacttt tcagaaaaac tccaactttc tccactgcat 420 tggttaatgg ctgataagac tggagagtca attgttgtag aatctacttt aagtggatta 480 cacgtttatg ataatccagt tcatgtttta accaataatc ctgaatttcc aggccagtta 540 agtaatttag ctaattatag caatatagca ccttcacagc ctaaaaatac tcttgttcca 600 ggtgttgacc ttaatttata tagtcgcggg ttagggactc attttttgcc aggaggaatg 660 gattcagcca gtcgttttgt gaaagtagct tttgttcgtg cacattctcc tcaagggaat 720 aatgaactaa gtagtgtaac aaattacttc catattctac attcagtcga acagccaaaa 780 ggaacagatg aagtaggacc aaattcttat gagtacacaa tttactctga tggaactaat 840 ttagagacag gaacatttta ttatacaaat tatgaaaata atcaaattaa cgccattgaa 900 ttaaataaag aaaacttaaa tggtgatgag ttaatagatt acaagttgat tgaaaagcaa 960 acaattaatt atcaaaatta g 981 <210> 2 <211> 951 <212> DNA <213> Lactobacillus johnsonii PF01 bshB DNA sequence <400> 2 atgtgtactg gtttaagatt cacagatgat caaggaaatt tatactttgg ccgtaatcta 60 gatgttggac aggattatgg cgaaggcgtt attattacgc cgcgtaatta tcctcttcca 120 tataagttct tagataacac cactactaaa aaggctgtta ttggaatggg aattgtggtt 180 gatggctatc catcatactt tgactgctat aacgaagatg gattaggcat tgcaggttta 240 aacttcccac attttgctaa atttagtgat ggtcctattg acggtaaaat caacttagct 300 tcttacgaaa ttatgctctg ggttactcaa aactttactc atgttagtga agtaaaggaa 360 gcgttaaaga atgttaactt agtgaatgaa gctattaaca catcatttgc ggttgcccct 420 cttcactgga tcattagtga tagtgacgaa gccattattg ttgaagtttc aaaacaatat 480 ggaatgaaag tctttgatga taaagttggc gttttaacta atagccctga ctttaactgg 540 caccttacta accttggtaa ctatactggt ttaaatccac atgacgctac agcccaaagc 600 tggaacggtc aaaaagttgc tccttggggt gtaggaactg gtagtttagg tctgcctggt 660 gacagcattc cagccgaccg ttttgttaaa gctgcttact taaacgtaaa ctatccaact 720 gctaaaggtg aaaaagcaaa cgtcgctaaa ttctttaaca tcttaaagtc tgttgccatg 780 atcaaaggca gtgtagtcaa cgatcaaggc aaggacgaat atactgttta tactgcatgc 840 tactcttctg gaagcaagac ttactactgt aattttgaag atgattttga attaaagact 900 tataaactag atgatcacac gatgaattca accagtcttg tgacttacta g 951 <210> 3 <211> 978 <212> DNA <213> Lactobacillus johnsonii PF01 bshC DNA sequence <400> 3 atgtgtacat caattttata tagtccaaaa gataattatt ttggtagaaa tttagattat 60 gaaattgcct atggtcagaa agtggtaatt actcctagaa attatcaact taattatcgt 120 catttaccaa cacaagatac tcattatgca atgatcggtg tttcagtagt tgccaatgac 180 tatccattgt attgtgatgc tatcaatgaa aaaggattag gaatagccgg attaaatttc 240 actggtcctg gtaaatattt tgctgtagat gaaagcaaaa agaatgttac ttcttttgaa 300 ctgatcccat atttactaag cagttgtgaa actattgaag atgtaaagaa attattgtct 360 gaaactaata ttactgatga aagtttctct aaagatttac cagttactac tcttcattgg 420 ttaatgggtg ataaaagtgg taagagtata gttattgaat caacagaaac tggtttacac 480 gtttatgaca acccagttaa tactttaaca aataatcctg tctttccagc tcaagttgaa 540 accttggcta actttgcttc ggtttctcca gctcaaccta aaaatacact tgtacctaat 600 gcagatatta atctgtatag tcgtggatta gggacccatc atttaccagg cggaacagat 660 tcaaattctc gctttattaa ggcatctttt gtattagctc attcaccaaa aggtaatgat 720 gaagttgaaa atgtaactaa tttctttcat atcttacatt cagttgaaca agcaaagggt 780 accgatgaag ttgaagataa tgtatttgaa tttaccatgt attcagactg tatgaatttg 840 gataagggaa ttttatattt tactacttac gataataacc aaattaatgc tgtggatatg 900 aataatgaaa atttggatac ttctgacttg attacctatg aattatttaa ggatcaagcc 960 attaaatttg aaaattaa 978 <210> 4 <211> 326 <212> PRT <213> Lactobacillus johnsonii PF01 bshA amino acid sequence <400> 4 Met Cys Thr Ser Ile Val Tyr Ser Ser Asn Asn His His Tyr Phe Gly   1 5 10 15 Arg Asn Leu Asp Leu Glu Ile Ser Phe Gly Glu His Pro Val Ile Thr              20 25 30 Pro Arg Asn Tyr Glu Phe Gln Tyr Arg Lys Leu Pro Asn Lys Lys Ala          35 40 45 Lys Tyr Ala Met Val Gly Met Ala Ile Val Glu Asp Asn Tyr Pro Leu      50 55 60 Tyr Phe Asp Ala Ser Asn Glu Glu Gly Leu Gly Ile Ala Gly Leu Asn  65 70 75 80 Phe Asp Gly Pro Cys His Tyr Phe Pro Glu Val Ser Gly Lys Asn Asn                  85 90 95 Val Thr Pro Phe Glu Leu Ile Pro Tyr Leu Leu Ser Gln Tyr Thr Thr             100 105 110 Val Ala Glu Val Lys Glu Ala Leu Lys Ser Val Asn Leu Val Lys Ile         115 120 125 Asn Phe Ser Glu Lys Leu Gln Leu Ser Pro Leu His Trp Leu Met Ala     130 135 140 Asp Lys Thr Gly Glu Ser Ile Val Glu Ser Thr Leu Ser Gly Leu 145 150 155 160 His Val Tyr Asp Asn Pro Val His Val Leu Thr Asn Asn Pro Glu Phe                 165 170 175 Pro Gly Gln Leu Ser Asn Leu Ala Asn Tyr Ser Asn Ile Ala Pro Ser             180 185 190 Gln Pro Lys Asn Thr Leu Val Pro Gly Val Asp Leu Asn Leu Tyr Ser         195 200 205 Arg Gly Leu Gly Thr His Phe Leu Pro Gly Gly Met Asp Ser Ala Ser     210 215 220 Arg Phe Val Lys Val Ala Phe Val Arg Ala His Ser Pro Gln Gly Asn 225 230 235 240 Asn Glu Leu Ser Ser Val Thr Asn Tyr Phe His Ile Leu His Ser Val                 245 250 255 Glu Gln Pro Lys Gly Thr Asp Glu Val Gly Pro Asn Ser Tyr Glu Tyr             260 265 270 Thr Ile Tyr Ser Asp Gly Thr Asn Leu Glu Thr Gly Thr Phe Tyr Tyr         275 280 285 Thr Asn Tyr Glu Asn Asn Gln Ile Asn Ala Ile Glu Leu Asn Lys Glu     290 295 300 Asn Leu Asn Gly Asp Glu Leu Ile Asp Tyr Lys Leu Ile Glu Lys Gln 305 310 315 320 Thr Ile Asn Tyr Gln Asn                 325 <210> 5 <211> 316 <212> PRT <213> Lactobacillus johnsonii PF01 bSB amino acid sequence <400> 5 Met Cys Thr Gly Leu Arg Phe Thr Asp Asp Gln Gly Asn Leu Tyr Phe   1 5 10 15 Gly Arg Asn Leu Asp Val Gly Gln Asp Tyr Gly Glu Gly Val Ile Ile              20 25 30 Thr Pro Arg Asn Tyr Pro Leu Pro Tyr Lys Phe Leu Asp Asn Thr Thr          35 40 45 Thr Lys Lys Ala Val Ile Gly Met Gly Ile Val Val Asp Gly Tyr Pro      50 55 60 Ser Tyr Phe Asp Cys Tyr Asn Glu Asp Gly Leu Gly Ile Ala Gly Leu  65 70 75 80 Asn Phe Pro His Phe Ala Lys Phe Ser Asp Gly Pro Ile Asp Gly Lys                  85 90 95 Ile Asn Leu Ala Ser Tyr Glu Ile Met Leu Trp Val Thr Gln Asn Phe             100 105 110 Thr His Val Ser Glu Val Lys Glu Ala Leu Lys Asn Val Asn Leu Val         115 120 125 Asn Glu Ala Ile Asn Thr Ser Phe Ala Val Ala Pro Leu His Trp Ile     130 135 140 Ile Ser Asp Ser Asp Glu Ala Ile Ile Val Glu Val Ser Lys Gln Tyr 145 150 155 160 Gly Met Lys Val Phe Asp Lys Val Gly Val Leu Thr Asn Ser Pro                 165 170 175 Asp Phe Asn Trp His Leu Thr Asn Leu Gly Asn Tyr Thr Gly Leu Asn             180 185 190 Pro His Asp Ala Thr Ala Gln Ser Trp Asn Gly Gln Lys Val Ala Pro         195 200 205 Trp Gly Val Gly Thr Gly Ser Leu Gly Leu Pro Gly Asp Ser Ile Pro     210 215 220 Ala Asp Arg Phe Val Lys Ala Ala Tyr Leu Asn Val Asn Tyr Pro Thr 225 230 235 240 Ala Lys Gly Glu Lys Ala Asn Val Ala Lys Phe Phe Asn Ile Leu Lys                 245 250 255 Ser Val Ala Met Ile Lys Gly Ser Val Val Asn Asp Gln Gly Lys Asp             260 265 270 Glu Tyr Thr Val Tyr Thr Ala Cys Tyr Ser Ser Gly Ser Lys Thr Tyr         275 280 285 Tyr Cys Asn Phe Glu Asp Asp Phe Glu Leu Lys Thr Tyr Lys Leu Asp     290 295 300 Asp His Thr Met Asn Ser Thr Ser Leu Val Thr Tyr 305 310 315 <210> 6 <211> 325 <212> PRT <213> Lactobacillus johnsonii PF01 bSHC amino acid sequence <400> 6 Met Cys Thr Ser Ile Leu Tyr Ser Pro Lys Asp Asn Tyr Phe Gly Arg   1 5 10 15 Asn Leu Asp Tyr Glu Ile Ala Tyr Gly Gln Lys Val Ile Thr Pro              20 25 30 Arg Asn Tyr Gln Leu Asn Tyr Arg His Leu Pro Thr Gln Asp Thr His          35 40 45 Tyr Ala Met Ile Gly Val Ser Val Val Ala Asn Asp Tyr Pro Leu Tyr      50 55 60 Cys Asp Ala Ile Asn Glu Lys Gly Leu Gly Ile Ala Gly Leu Asn Phe  65 70 75 80 Thr Gly Pro Gly Lys Tyr Phe Ala Val Asp Glu Ser Lys Lys Asn Val                  85 90 95 Thr Ser Phe Glu Leu Ile Pro Tyr Leu Leu Ser Ser Cys Glu Thr Ile             100 105 110 Glu Asp Val Lys Lys Leu Leu Ser Glu Thr Asn Ile Thr Asp Glu Ser         115 120 125 Phe Ser Lys Asp Leu Pro Val Thr Thr Leu His Trp Leu Met Gly Asp     130 135 140 Lys Ser Gly Lys Ser Ile Val Ile Glu Ser Thr Glu Thr Gly Leu His 145 150 155 160 Val Tyr Asp Asn Pro Val Asn Thr Leu Thr Asn Asn Pro Val Phe Pro                 165 170 175 Ala Gln Val Glu Thr Leu Ala Asn Phe Ala Ser Val Ser Ala Gln             180 185 190 Pro Lys Asn Thr Leu Val Pro Asn Ala Asp Ile Asn Leu Tyr Ser Arg         195 200 205 Gly Leu Gly Thr His His Leu Pro Gly Gly Thr Asp Ser Asn Ser Arg     210 215 220 Phe Ile Lys Ala Ser Phe Val Leu Ala His Ser Pro Lys Gly Asn Asp 225 230 235 240 Glu Val Glu Asn Val Thr Asn Phe Phe His Ile Leu His Ser Val Glu                 245 250 255 Gln Ala Lys Gly Thr Asp Glu Val Glu Asp Asn Val Phe Glu Phe Thr             260 265 270 Met Tyr Ser Asp Cys Met Asn Leu Asp Lys Gly Ile Leu Tyr Phe Thr         275 280 285 Thr Tyr Asp Asn Asn Gln Ile Asn Ala Val Asp Met Asn Asn Glu Asn     290 295 300 Leu Asp Thr Ser Asp Leu Ile Thr Tyr Glu Leu Phe Lys Asp Gln Ala 305 310 315 320 Ile Lys Phe Glu Asn                 325 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> bshA_F <400> 7 aaactgcagc atatgtgtaa cctcaattgt tta 33 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> bshA_R <400> 8 agcctgcaga agcttatttt gataattaat tg 32 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> bshB_F <400> 9 aaactgcagc atatgtgtac tggtttaaga t 31 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> bshB_R <400> 10 ctgcagctcg aggtaagtca caagact 27 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> bshC_F <400> 11 ctgcagcata tgtgtacatc aattttat 28 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> bshC_R <400> 12 aaactgcagc tcgagatttt caaatttaat 30

Claims (18)

A gene for bsh A (bile salt hydrolase A) derived from Lactobacillus johnsonii PF01 comprising the nucleotide sequence of SEQ ID NO: 1. A bsh B (bile salt hydrolase B) gene derived from Lactobacillus johnsonii PF01, consisting of the nucleotide sequence of SEQ ID NO: 2. A bsh C (bile salt hydrolase C) gene derived from Lactobacillus johnsonii PF01 comprising the nucleotide sequence of SEQ ID NO: 3. A recombinant vector comprising the gene according to any one of claims 1 to 3. A host cell transformed with the recombinant vector of claim 4. 6. The method of claim 5,
Wherein the host cell is Escherichia coli. Lactobacillus johnsonii ( Lactobacillus johnsonii) having the nucleotide sequences of SEQ ID NOS: 7 and 8 A primer set to amplify the bsh A (even salt hydrolase A) gene derived from PF01. Lactobacillus johnsonii ( Lactobacillus johnsonii) having the nucleotide sequences of SEQ ID NOS: 9 and 10 A primer set to amplify the bsh B (bile salt hydrolase B) gene derived from PF01. Lactobacillus johnsonii ( Lactobacillus johnsonii) having the nucleotide sequences of SEQ ID NOS: 11 and 12 A primer set for amplifying the bsh C (bile salt hydrolase C) gene derived from PF01. Lactobacillus Johnson PF01 ( Lactobacillus &lt; RTI ID = 0.0 &gt; amplifying bsh A, bsh B, and bsh C genes by PCR using amplification primers corresponding to the bsh A, bsh B, and bsh C genes from the johnsonii PF01 strain; A step of introducing the amplified bsh A, bsh B, and bsh C genes into vectors, respectively, and introducing them into an Escherichia coli strain; Digesting the recombinant plasmid produced in the cloning step with a restriction enzyme and recovering the insert DNA; Subcloning the recovered insert DNA by binding it to a vector, introducing the subcloned strain into an E. coli strain, and transforming the transformed DNA; Extracting a recombinant plasmid from the transformed strain; Introducing the extracted recombinant plasmid into E. coli which is a host cell for mass expression; Selecting a transforming enzyme showing a precipitation reaction by culturing the recombinant plasmid introduced into the E. coli; ( Lactobacillus johnsonii PF01) for the reduction of hypercholesterolemia, comprising the steps of: (a) isolating a cholanic acid hydrolase from a strain of Lactobacillus johnsonii PF01. Lactobacillus johnsonii &lt; / RTI &gt; consisting of the amino acid sequence of SEQ ID NO: 4 PF01) derived from bsh A (bile salt hydrolase A). Lactobacillus johnsonii &lt; / RTI &gt; consisting of the amino acid sequence of SEQ ID NO: 5 PF01) derived from bsh B (bile salt hydrolase B). A Lactobacillus johnsonii ( Lactobacillus johnsonii &lt; RTI ID = 0.0 &gt; PF01) derived bsh C (bile salt hydrolase C). 12. The method of claim 11,
The Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01) derived bsh A (bile salt hydrolase A) enzyme is a purified bsh A:
(a) optimum temperature 50-70 DEG C;
(b) Optimum pH 6.0;
(c) SDS-PAGE measurement: molecular weight 35 kDa;
(d) has a hydrolytic activity specifically for taurine-based bile salts. 13. The method of claim 12,
The Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01) derived bsh B (bile salt hydrolase B) enzyme is a purified bsh B:
(a) optimum temperature 55-70 DEG C;
(b) Optimum pH 4.5;
(c) SDS-PAGE measurement: molecular weight 34 kDa;
(d) has a hydrolytic activity specifically for taurine-based bile salts. 14. The method of claim 13,
The Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01) derived bsh C (bile salt hydrolase C) enzyme is a purified bsh C enzyme having the following characteristics:
(a) optimum temperature 65-80 DEG C;
(b) Optimum pH 5.0;
(c) SDS-PAGE measurement Molecular weight 37 kDa;
(d) Hydrolytic activity specifically to glycine-based bile salts. Lactobacillus Johnson PF01 expressing the bsh A, bsh B and bsh C genes consisting of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 ( Lactobacillus johnsonii PF01). The method of claim 17, wherein the Lactobacillus Johnson PF01 ( Lactobacillus johnsonii PF01).

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