KR20140047886A - Over-expression system of glucosidase from bifidobacterium animalis subsp. lactis sh5 and bifidobacterium bifidum bgn4 transformed with the over-expression system - Google Patents

Abstract

The present invention relates to an over-expression system of glucosidase from bifidobacterium animalis subsp. lactis SH5 and bifidobacterium bifidum BGN4 transformed thereby, and when transforming bifidobacterium bifidum BGN4 by using an expression system of beta-glucosidas of the present invention, beta-glucosidase having significantly high activity can be produced. The beta-glucosidase manufactured like this can be used for hydrolyzing glycosides in a form of non-glycosides present in plants.

Description

Overexpression system of Bifidobacterium animalis subspiris lactis SH5-derived glucosidase and thereby transformed Bifidobacterium bidpi BGN4 {Over-expression system of glucosidase from Bifidobacterium animalis subsp. lactis SH5 and Bifidobacterium bifidum BGN4 transformed with the over-expression system}

The present invention provides an effective overexpression system of glucosidase derived from Bifidobacterium animalis subspiris lactis SH5 and thereby transformed Bifidobacterium bidpi BGN4.

Beta-glucosidase is a hydrolase that breaks down beta-glucosidic linkage and is present in various microorganisms, including lactic acid bacteria. Beta-glucosidase (β-glucosidase) hydrolyzes a glycoside-like compound present in a plant into a non-glycoside form of a compound, which also exhibits more effective function than a glycoside in vivo. Therefore, the development of beta-glucosidase with excellent activity or the development of overexpression system can be a very important technology in the industry.

Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum ) BGN4 is a lactic acid bacterium commercially sold by Bifido Co., Ltd. (688-1, Sangoan-ri, Hongcheon-eup, Hongcheon-gun, South Korea). Bifidobacterium Bifidum strain is one of the most important probiotic strains, and is well known for its stability and functionality, and is suitable for use in the food industry.

As described above, if the expression (production) of beta-glucosidase, which is an industrially important enzyme, is performed through the Bifidobacterium bipidum strain, it may be a very useful technique. However, genetic information, or transgenic techniques well known Escherichia coli (E. coli) and Saccharomyces access to my Celebi jiae (Saccharomyces Unlike cerevisiae ), the transformation techniques for Bifidobacterium bifidum strains are not well known.

Therefore, it can be said that the development of a technique capable of effectively expressing beta-glucosidase in the Bifidobacterium bifidum strain is urgently required.

Korean Patent Publication No. 10-2010-0090885 (published date: August 18, 2010), Bifidobacterium bifidum containing phytic acid (Bifidobacterium bifidum) BGN4 culture medium and Bifidobacterium BP using the same Bifidobacterium bifidum is described in the mass production method of BGN4 polysaccharide.

"Esen, A. 1993. [beta] -Glucosidase, In [beta] -Glucosidase: Biochemistry and Molecular Biology, pp. 1-13. American Chemical Society, Washinton DC" includes beta-glucose that breaks down the beta-glucosidic linkage. Is described for β-glucosidase.

In the present invention, it is intended to develop and provide an expression system capable of effectively overexpressing beta-glucosidase in a Bifidobacterium bifidum strain and a Bifidobacterium bifidum transformed by the expression system.

The invention Bifidobacterium no Marlies sub speaker system lactis (Bifidobacterium animalis subsp lactis.) Derived beta from SH5-glucosidase of the nucleic acid sequence, the beta-glucosidase nucleotide sequence of the kinase promoter (promoter), and the beta Provided is a Bifidobacterium bifidum transformation vector characterized in that the nucleic acid sequence of SEQ ID NO: 1 including the nucleic acid sequence of the glucosidase terminator is inserted.

In the present invention, an effort was made to separate high activity glucosidase from lactic acid bacteria, and as a result, Bifidobacterium animalis subspirosis lactis ( Bifidobacterium) animalis subsp. lactis ) It was possible to isolate beta-glucosidase with excellent activity from SH5. In the present invention, an effort was made to develop an expression system that can further enhance the activity of the isolated beta-glucosidase as described above. After constructing a recombinant expression system by combining various promoters and terminators, the enzyme activity was compared. . As a result of the present invention, it was confirmed that the beta-glucosidase cloned in the present invention has excellent activity compared to other cases when using its own original promoter and its own terminator.

Therefore, in the present invention, Bifidobacterium animalis subspirosis lactis containing B-glucosidase of the present invention ( Bifidobacterium) animalis subsp. lactis ) Primer for cloning, including all open reading frame (ORF), promoter and terminator of beta-glucosease from cDNA of SH5 By designing and cloning, and inserted into the transformation vector of the Bifidobacterium bifidum, the Bifidobacterium bifidum transformation vector of the present invention was prepared. After transforming the Bifidobacterium bifidum with the thus prepared vector, and measuring the activity of beta-glucosidase, the wild type B. lactis which is the parent strain belonging to the beta-glucosidase used in the present invention was cloned. An excellent effect was exerted 31.32 times higher than the activity of beta-glucosidase of SH5 (FIG. 2).

Meanwhile, in the Bifidobacterium bifidum transformation vector of the present invention, the transformation vector is preferably a ssamy gene (SL Rhim, MS Park and GE Ji (2006. 2) Expression and secretion, which is a signal peptide of amylase). of Bifidobacterium adolescentis amylase by Bifidobacterium longum.Biotechnology Letters.28 (3), 163-168) It is preferable that the nucleic acid sequence of SEQ ID NO: 2, which is the sequence, is not inserted. In general, when a signal peptide is attached to an enzyme which is a target protein, the amount of the enzyme secreted out of the cell increases, thereby increasing the extracellular activity of the enzyme. As confirmed by the present invention, when Bifidobacterium vifidum is used as a host, Even with the addition of the signal peptide, the extracellular activity did not increase. Moreover, in the Bifidobacterium bifidum, the intracellular activity was rather increased without attaching the signal peptide.

On the other hand, the present invention provides a Bifidobacterium bifidum transformed with the transformation vector of the present invention. When using the transformed Bifidobacterium bifidum as described above, it is possible to mass-produce beta-glucosidase. The host Bifidobacterium bipidum is one of the most important probiotic strains, because it is widely known for its stability and functionality and is one of the most suitable strains for use in the food industry.

On the other hand, the Bifidobacterium bifidum ( Bifidobacterium bifidum ), preferably Bifidobacterium bipiduum ( Bifidobacterium bifidum ) is good for BGN4. Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum ) BGN4 is a lactic acid bacterium commercially sold by Bifido Co., Ltd. (688-1, Sangoan-ri, Hongcheon-eup, Hongcheon-gun, Korea) and is isolated from the colon of healthy adults.

When transforming Bifidobacterium bifidum BGN4 using the beta-glucosidase expression system of the present invention, it is possible to produce beta-glucosidase having a very high activity.

The beta-glucosidase thus produced can be used to hydrolyze glycosides present in plants in a nonglycoside form.

1 is B. lactis The results of sequence analysis of the beta-glucosidase of the invention derived from SH5 are shown.
2 shows recombinant B transformed with wild type and various vectors of the invention. For bifidum BGN4, enzyme enzymatic activity of beta-glucosidase is shown.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of the same or similar technical spirit.

On the other hand, in the present invention, in the gene and protein names, italic letters of the same spelling mean a gene name and gothic means a protein name.

Meanwhile, in the following examples, experiments were performed using the genes, vectors, and strains described in Tables 1-3. However, it should be noted that experimental materials and methods not specifically described in the Examples may be used using known genetic engineering knowledge and protocols.

Strains and Vectors (Plasmids) Used in Examples Strain or vector Characteristic or genotype Source or reference Strain SH5 B. lactis SH5, wild type; Glu + (original host of bbg572 ) RD68 B. lactis RD68, wild type; Glu + BGN4 B. bifidum BGN4, wild type; Glu-; Transformation host DH5α E. coli DH5α ,
F- (80 dlacZ M15 ) ( lacZYA - argF ) U169hsdR17 (r-m +) recA1 endA1 relA1 deoR ; Cloning host Hanahan, 1983 XL1 blue MR E. coli XL1 blue MR , Δ ( mcrA ) 183 Δ ( mcrCB - hsdSMR -mrr) 173 endA1 supE44 thi -1 recA1 gyrA96 relA1 lac ; Cloning host Stratagene Bpamyss572 B. bifidum BGN4 harboring pamyss572 Bp919ss572 B. bifidum BGN4 harboring p919ss572 Bp572ss572 B. bifidum BGN4 harboring p572ss572 Bp919bbg572 B. bifidum BGN4 harboring p919bbg572 Bp572bbg572 B. bifidum BGN4 harboring p572bbg572 Bp572bbg572t B. bifidum BGN4 harboring p572bbg572t Plasmid SuperCos1 Cosmid Δ ( mcrA 183 Δ ( mcrCB - hsdSMR - mrr 173 endA1 supE44  thi-1 recA1 gyrA96 relA1 lac Stratagene pUC18 Amp ^r pBES2 Amp ^r , Cm ^r ; E. coli - Bifidobacterium shuttle vector ¹⁹ Park et al ., 2003 pBESAF2 Amp ^r , Cm ^r ; E. coli - Bifidobacterium shuttle vector ¹⁸ Park et al ., 2005 pUC572PT Amp ^{r ;} : pUC18 with 2.276 kb insert of B. lactis DNA containing the bbg572pt

Primer used to amplify the beta-glucosidase gene and its potential promoter Name of the primer Sequence of primer Product Forward direction (5 → 3) Reverse (5 → 3) primerbbg572pt ggatccATGCTGCTCCTTATGTGTC gaattcGCTCCCCGCAGCTTCG bbg572pt primerbbg572p tctagaATGCTGCTCCTTATGTGTC gaattcCTACTTGGCGGAGTGCTC bbg572p
( p572 + bbg572 ) primerbbg572 ggatccATGACGATGACGTTCCC ^a gaattcCTACTTGGCGGAGTGCTC bbg572 agtactATGACGATGACGTTCCC ^b primerbbg572t ggatccATGACGATGACGTTCCC ^a gaattcGCTCCCCGCAGCTTCG bbg572t
( bbg572 + 572 t ) agtactATGACGATGACGTTCCC ^b primerPamy tctagaGAAATACCGCAATGCACG ggatccGGCTCCTTTATTCCTTTTC pamy primer919P tctagaTGAAGTGTGTCGTGTGG ggatccTGGTGTACCTTTTCTTG p919 primer572P tctagaATGCTGCTCCTTATGTGTC ggatccTGCTGATTCCTCC p572 primerSSamy ggatccATGAAACATCGGAAACC agtact GGCCTGTGCTGCGG ssamy Note) Restriction enzyme cleavage sites are shown in lowercase letters. Xba I tctaga, Bam H ggatcc, Eco RI gaattc, Sca I agtact. a) Primers were used to construct p919bbg572, p572bbg572 and p572bbg572t. b) Primers were used to construct pamyss572, p919ss572, p504ss572 and p572ss572.

Vector (plasmid) constructed by inserting the beta-glucosidase gene of the present invention Promoter Signal sequence ORF Terminator Plasmid pamy p919 p572 ssamy bbg572 572t o o   p919bbg572 o o   p572bbg572 o o o   p572bbg572t o o o   pamyss572 o o o   p919ss572 o o   p504ss572 o o o   p572ss572

[ Example  1: separated from the present invention Bifidobacterium lactis Derived beta- Glucosidase sequence  analysis]

B. lactis among 46 Lactic Acid Bacteria Beta-glucosidase isolated from two strains (SH5 and RD68) showed relatively high activity. After constructing a cosmid library in Escherichia coli using genomic DNA derived from these two strains, about 30 clones showing beta-glucosidase activity were isolated. B. lactis in clones derived from each of the screened colonies One clone from SH5 showed the highest activity. DNA fragments inserted into the cosmid were cut with BamH I and subcloned into the pUC18 vector.

B. lactis of the present invention Beta-glucosidase gene clones derived from SH5 have been analyzed by sequence analysis, indicating that open reading frames (ORFs), potential promoter regions and potential terminator regions are derived from B. lactis RD68. Same as glucosidase (FIG. 1).

In Figure 1, the transcription start time is shown in dark capital letters, and the predicted promoter region is underlined. Translational termination sites are marked with an asterisk.

B. lactis of the present invention Beta-glucosidase derived from SH5 was named bbg572pt and used in the following experiment. bbg572pt consists of 2,276 bp and has one ORF. One inverted repeat sequence (indicated by an arrow in FIG. 1), presumed to be a transcriptional terminator, was located downstream of the ORF. A potential ribosome binding site (RBS), AGGAGGA (grey box in FIG. 1), was located upstream of the ORF, which is B. longum It was similar to the RBS of MG1.

The deduced amino acid sequence had a molecular weight of 50.71 kD and had 460 amino acid residues. This amino acid sequence of B. breve clb, B. breve CECT 7263, B. dentium ATCC 27679 and B. longum subsp. infantis There was 85%, 68%, 70% and 69% amino acid homology with each beta-glucosidase from ATCC 15697, respectively.

Based on the homology of such amino acid sequences, the beta-glucosidase coding gene of the present invention was named bbg572 and encoded by The beta-glucosidase of the present invention has been inferred into the glycosyl hydrolase family 1. In general, the total GC content of Bifidobacterium is 55.0-67.0%, which is higher than other bacteria. The GC content of the present invention bbg572 was 63%, which is B. breve It is slightly lower than the GC content of 65.1% of beta-glucosidase derived from clb.

[ Example  2: Of transcription start point  analysis]

As a result of the analysis of Example 1, it was confirmed that a potential promoter exists upstream of bbg572 (FIG. 1). Comparing the promoter regions, the sequences of the potential -10 and -35 regions of the present invention were identical to those of E. coli. Potential promoters of the present invention are E. coli Successful expression of bbg572 was induced in DH5α.

Meanwhile, 5'RACE analysis on bbg572 Two possible transcriptional start points were shown (G and A residues in FIG. 1). The -10 region (tgtatt) and -35 region (ttgcgc) upstream from the G residue showed 66.7% and 50% homology, respectively, when compared to that of E. coli. The -10 region (tatatt ) and -35 region (ttgcgg) upstream from the residue A showed 83.3% and 50% homology, respectively, when compared to that of E. coli. In the -35 region, the diversity of sequence homology is also confirmed from many previous studies, which suggests that transcription in E. coli can easily occur without the absolute similarity of the -35 region [Collado-Vides, J., B. Magasanik, and JD Gralla. 1991.Control site location and transcriptional regulation in Escherichia coli . Microbiol . Rev. 55: 371-394.

[ Example  3: B . bifidum BGN4 In beta- Glucosidase  Expression]

Strong promoters are required for enhanced expression of target genes. Through our previous research, we used B. bifidum P919 from BGN4 [Wang, Y., JY Kim, MS Park, and GE Ji. 2012. Novel Bifidobacterium promoters selected through microarray analysis lead to constitutive high level expression. J. Microbiol . 50: 638-643 and B. pseudocatenulatum From alpha-amylase pamy Park, MS, JM Seo, and JY Kim. Heterologous gene expression and secretion in Bifidobacterium longum . Containing Lait 85: 1-8] Various promoters were used to successfully express foreign genes in B. longum .

On the other hand, in the present embodiment, the alpha-amylase cloned in the present invention For the gene ( bbg572 ) To compare the strength of the promoters, various vectors with promoter and report gene ( bbg572 ) were constructed and transformed with B. bifidum BGN4, a beta-glucosidase negative strain. Promoter strength was analyzed by measuring the activity of beta-glucosidase using pNPG. The experimental results, in a system for the expression of bbg572, the bbg572 Unique promoter p572 was identified as a stronger promoter than the foreign promoter pamy or p919 (FIG. 2).

On the other hand, the present invention alpha-to induce amylase extracellular expression of the gene (bbg572), saw by inserting the amylase gene signal sequence (ssamy) between the promoter and the reporter gene, switching all that as expected include ssamy transfected body B bifidum showed low beta-glucosidase activity in BGN4 (FIG. 2). In addition, ssamy is B. bifidum The extracellular transfer of bbg572 protein expressed in BGN4 has also been shown to fail. That is, intracellular beta-glucosidase activity of Bp572ss572 with ssamy was 1,186 times higher than that of extracellular activity. This is the host, promoter and ssamy used It was thought to have been caused by discord between them.

The inventors thought that the host change to B. bifidum BGN4 in this experiment had an effect on the activity of promoter and ssamy , which in turn affected low protein expression and secretion. Many previous studies have reported that -10 and -35 sequences influence promoter strength [McCracken, A., MS Turner, P. Giffard, LM Hafner, and P. Timms. 2000. Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species. Arch . Microbiol . 173: 383-389. In addition, TG motifs, spacers, UP elements, and three-dimensional structures of promoters have been reported to affect the efficiency of transcription.

Thus, in this example, after removing ssamy , we compared the activity of beta-glucosidase, but the expression of beta-glucosidase was significantly increased by the removal of ssamy . Among the ssamy removal strains, Bp572bbg572 showed about 24.35-fold increase in activity (1.5 mU / μg protein), and Bp919bbg572 strain showed about 137-fold increase (0.78 mU / μg protein) (Figure 2).

On the other hand, proteins known as transcription terminators or terminators are generally known to increase the expression of genes. In the present invention, a potential transcription terminator consisting of an inverted repeat sequence was found downstream of the beta-glucosidase bbg572 isolated from the present invention, which was named 572t . This was added downstream of bbg572 of p572bbg572 and this vector was named 72bbg572t.

Experimental results show that the addition of transcription terminator is B. bifidum This resulted in a 25.4% increase in beta-glucosidase specific activity (inactivity) in BGN4 (FIG. 2). B containing p572bbg572t. The beta-glucosidase activity of bifidum BGN4 was 1.89 mU / μg, which is wild type B. lactis It is about 31.32 times higher than beta-glucosidase of SH5 and 15.88 times higher than wild type B. lactis RD68 (FIG. 2).

[ Example  4: B . bifidum BGN4 Derived from Recombinant beta- Glucosidase  Substrate resolution]

In this example, to determine whether the extract of the Bp572bbg572t strain of the present invention decomposes beta-glucoside bonds, using various disaccharides, isoflavones, quercetin and ginsenosides as substrates. (Tables 4 and 5 below). As a control, a cell extract of pBESAF2 containing B. bifidum BGN4, which had no resolution for glucoside binding, was used [Park, MS, JM Seo, and JY Kim. Heterologous gene expression and secretion in Bifidobacterium longum . Lait 85: 1-8].

As a result, the extracts of Bp572bbg572t strains are β-1,2 (sophorose), β-1,3 (laminaribiose), β-1,4 (cellobiose), and β-1,6 (gentiobiose). It was confirmed that the bond could be hydrolyzed.

Substrate Specificity of Bp572bbg572t temperament Combination Enzyme activity
(O: active, X: no activity) Bbg572 Disaccharides   Cellobiose O -β-D-glucosyl- (1> 4) -D-glucose O   Sophorose O -β-D-glucosyl- (1> 2) -D-glucose O   Laminoribiose O -β-D-glucosyl- (1> 3) -D-glucose O Gentiobiose O -β-D-glucosyl- (1> 6) -D-glucose O Isoflavone Daidzin Daidzein 7- O -β-D-glucoside O Genistin Genistein 7- O -β-D-glucoside O Glycitin Glycitein 7- O -β-D-glucoside O Quercetine Isoquercetrin Quercetin-3- O -β-D-glucoside O Spiraeoside Quercetin-4- O -β-D-glucoside O Quercetin-3,4-di- O- β-D-glucoside O Quercetin-7- O -β-D-glucoside O

Substrate Specificity of Bp572bbg572t for Various Ginsenosides Gin Senocide R1 R2 R3 Enzyme activity
(O: active, X: no activity) Bbg572

20 (S) -Protopanaxadiol type Compound k OH H O-Glc ^a X F2 O-Glc H O-Glc X Rb1 O-Glc ² - ¹ Glc ^a H O-Glc ⁶ - ¹ Glc O Rb2 O-Glc ² - ¹ Glc ^a H O-Glc ⁶ - ¹ Arap ^a O Rc O-Glc ² - ¹ Glc ^a H O-Glc ⁶ - ¹ Araf ^a X Rd O-Glc ² - ¹ Glc ^a H O-Glc ^a X 20 (S) -PROTOPANAXATRIOL TYPE F1 OH OH O-Glc ^a X Re OH O-Glc ² - ¹ Rha ^a O-Glc ^a X Rg1 OH O-Glc ^a O-Glc ^a X Rg2 (S) OH O-Glc ² - ¹ Rha ^a OH X Rg3 (S) O-Glc-Glc ^a H H X Rg3 (R) O-Glc-Glc ^a H H X Rh1 OH O-Glc ^a OH X Rh2 (S) O-Glc ^a H H X a) Arap, α-L-arabinopyranosyl; Araf, α-L-arabinofuranosyl; Glc, β-D-glucopyranosyl; Rha, α-L-Rhamnopyranosyl

The above results, in the strain extract of the present invention, ginsenoside Rb1, Rb2 and seven flavonoids (daidzin, genistin, glycitin, isoquercetrin, spiraeoside, quercetin-3,4-di-O-β-D-glucoside and beta-glucosidase activity against quercetin-7-O-β-D-glucoside).

<110> YEON SUNG INDUSTRY FOUNDATION <120> Over-expression system of glucosidase from Bifidobacterium          animalis subsp. lactis SH5 and Bifidobacterium bifidum BGN4          transformed with the over-expression system <130> AP-2012-0240 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 2270 <212> DNA <213> Bifidobacterium animalis subsp. lactis SH5 <400> 1 atgctgctcc ttatgtgtct ctgctgtgcc gattatatgc ctattctcgc ggtgtcgcgg 60 ccctcacttt gctgcgaaac tctgttgcga actctgttac gggccaagtt tgtgcgtacg 120 ggctaggatt ttcggttacg ggctagggca tctctgcctt ttcgtgaaaa acgggtagtc 180 ggaaaccgcc atgttccaat gatcccgttt gccggggccc cgactttctt tagcctgtaa 240 cgcaaaatcg tagcccgtac gcacaaactt ggcccgtaag caagaagtta gcccgtaaga 300 gatggggaat ctggcccgta acggatggaa gagagcgggg gggatgtgga agattgttgg 360 cgatgcgcgt gttgcgggcg gtgtggaggg gaggagcatg ctatatttgg catatgctga 420 ctcgagagta agtaaatgaa tgccggagca gatccgggat ttgcgcgggt ctgtgctcgg 480 atgcgacgtg tattgtgtcg gatgtatcga caggaggaat cagcaatgac gatgacgttc 540 ccgaagggct tccagttcgg caccgcgact gccgcctacc agatcgaagg cgcggtggac 600 gaagacggcc gcacgccgtc gatctgggat gtgttctcgc acgccccggg ccgcgtgctg 660 aatggcgaca ccggagacaa ggccgacgat ttctaccacc gctggcagga cgatctcaag 720 ctcgtgcgcg atctcggcgt gaacgcatac cggttctcga ttggcgtgcc gcgcgtcatt 780 cccaccccgg acggcaagcc gaacgagaag ggcctcgatt tctacgagcg cattgtcgac 840 cagctgctcg aatacggcat cgacccgatt gtgacgctct accattggga tctgccgcag 900 tatctgaacg aagatccgta ccgggatggc tggctgaacc gtgagaccgc gttccgcatg 960 gcggagtatg ccggcattgt ggccaagcgc ctcggcgacc gtgtgcacac ctacaccacg 1020 ctcaacgaac cgtggtgctc ggcgcacctg agctacggcg gcaccgagca tgcccccggc 1080 ctgggcgccg gcccgctcgc gttccgcgcc gcccatcacc tgaatctggc acatggtctg 1140 atgtgcgagg cagtgcgtgc cgaggccgga gcgaagccgg atctctcggt gacgctgaat 1200 ctgcaggtga accgcggtga tgcggatgcc gtgcaccgcg tggatctcat tgccaaccgc 1260 gtgttcctcg atccgatgct gcgcggctac tacccggacg agctgttcgc aatcaccaag 1320 ggaatctgcg attgggactt cgtgcatgac ggcgatctca agctcatcaa ccagccgatt 1380 gacgtcctgg ggcttaatta ttactcgacg aatctgctcg ccatgagcga ccgcccgcag 1440 ttcccgcaga gcaccgaggc ctccaccgcg ccgggcgcca gcgacatcga ctggctgcct 1500 accgacggcc cgcacacgca gatggggtgg aacatcgacc cggatgcgct ttataacacg 1560 ctggttcgcc tgaacgacga ctacgaccac attccgctcg tcgtcactga aaacggcatg 1620 gcgtgccccg acgaggtgga agtcggcccg gatggtgtga agatggtgca cgacgacgac 1680 cgcatcgact acctgcgtcg ccatctcgag gccgtccacc gcgcgatcga ggagggggcg 1740 aatgtcatcg gatacttcgt gtggtcgctg atggataatt tcgagtgggc gttcggctac 1800 gaccgccgct tcggcctgac ctacgtggac tacgacaccg aggagcgcat acggaaggac 1860 agctacaact ggtaccgtaa cttcatcgcc gagcactccg ccaagtagcg ggttcgggcg 1920 cgccgggcgc gggtcatgca atgtctgtgg atatgcacat gtatgagcat gcacagacga 1980 gttgcatatt cgcgcccggc tcgttttgat ttcaggatgt ggacggcgcg tcgcctgaag 2040 cgattaacta tttgggatta cttgcagaga atctcaccca gttttgtgtg acggtctaat 2100 aaatttgaaa tccgcggaaa ttcgggaaag cgcggcggag aggtaatttg cggcggtgtg 2160 aaatctgagg caaattggct cggtaaaaga acttaagcgc ttgataaact cgggtgaggg 2220 gaattattct ttcaatcatc gattcgcaag cccgcgaagc tgcggggagc 2270 <210> 2 <211> 132 <212> DNA <213> Bifidobacterium adolescentis INT57 <400> 2 atgaaacatc ggaaacccgc accggcctgg cataggctgg ggctgaagat tagcaagaaa 60 gtggtggtcg gcatcaccgc cgcggcgacc gccttcggcg gactggcaat cgccagcacc 120 gcagcacagg cc 132

Claims (4)

Bifidobacterium animalis subspisis lactis animalis subsp. lactis ) SEQ ID NO: 1 comprising a nucleic acid sequence of beta-glucosidase derived from SH5, a nucleic acid sequence of the beta-glucosidase promoter and a nucleic acid sequence of the beta-glucosidase terminator Bifidobacterium Bifidobacterium ( Bifidobacterium) characterized in that the nucleic acid sequence is inserted bifidum ) vector for transformation.
The method of claim 1,
The transformation vector is
Nucleic acid of ssamy gene, signal peptide of amylase SEQ ID NO: 2 of the sequence Bifidobacterium, characterized in that the nucleic acid sequence is not inserted Bifidobacterium bifidum ) vector for transformation.
Bifidobacterium ( Bifidobacterium) transformed with the transformation vector described in claim 1 bifidum ).
The method of claim 3,
The Bifidobacterium bifidum ( Bifidobacterium bifidum ),
Bifidobacterium Bifidobacterium Doom (Bifidobacterium bifidum ) Bifidobacterium Bifidobacterium, characterized in that the Bifidobacterium bifidum ).

Images