KR101514775B1 - Glycoside hydrolase From lactic acid bacteria And Use Thereof - Google Patents

Abstract

The present invention relates to a glycoside hydrolase derived from a lactic acid bacterium, more specifically a Weissella confusa , Pediococcus, pentosaceus , Leuconostoc citreum ) or Leuconostoc mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroides and their uses. < Desc / Clms Page number 2 >

Description

Glycoside Hydrolase Derived from Lactic Acid Bacteria and Use Thereof [

The present invention relates to a glycoside hydrolase derived from a lactic acid bacterium, more specifically a Weissella confusa , Pediococcus, pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum , and uses thereof.

Ginseng has been used as a medicinal product for promoting health and longevity in Korea, China and Japan including Asia, Japan, and also for therapeutic use at the plant level in the United States and in Oriental medicine treatment in Europe, especially Germany.

Saponin refers to a substance composed of a plurality of ring compounds in a part of glycosides which are widely present in the plant system, and triterpene saponin, which is a saponin component contained in ginseng or red ginseng as a major physiologically active ingredient, Since the chemical structure of saponin is different from that of saponin, it is called ginsenoside in order to distinguish saponin from other plant saponins.

To date, over 180 ginsenosides have been purified, characterized and classified. The basic structure of the ginsenoside is one to three residues attached to the main skeleton in a specific position, i.e., glycopyranosyl, arabinopyranosyl, arabinofuranosyl, xylopyranosyl, or rampopyranosyl groups A tetracyclic triterpene damermanane or a pentacyclic oleanane skeleton. Damaran-type ginsenosides are classified mainly by their own aglycone residues: protopanaxel (PPD), protopascan triol (PPT), and ocotillol.

The ginsenoside composition is diversified into various ginseng species. For example, Korean ginseng ( Panax ginseng contains 22 PPD-type ginsenosides and 14 PPT-type ginsenosides whereas the American ginseng ( Panax notoginseng ) contains 13 PPD type ginsenosides and 5 PPT type ginsenosides. In certain species of Panax , the composition of ginsenosides also varies in other parts of ginseng bodies such as roots, leaves, and fruit.

In general, the efficacy of ginsenosides increases with the degree of deglycosylation, which enhances its hydrophobicity and cell wall permeability. However, while deglycosylated ginsenosides are trace amounts in ginseng extract, Rb1 {3- O- [? - D-glycopyranosyl- (1-2) -? - D-glycopyranosyl] -20- O - [? - D-glycopyranosyl- (1-6) -? - D-glycopyranosyl] -20 ( S ) protopanaxanthooler system}, Rb2 {3- O- [ ( S ) -β-D-glycopyranosyl] -20- O- [α-L-arabinopyranosyl- (1-6) - Prototype wave incident yidayi olgye}, Rb3 {3- O - [ β-D- glycoside pyrazol nosil - (1-2) -β-D- glycoside pyrazol nosil] -20- O - [β-D- xylene to pyrazole O- [beta] -D-glycopyranosyl] - 20 ( S ) - protopanaxadiolide system), Rc {3- ? - D-glycopyranosyl] -20 ( S ) -protophenecarbocyanuric acid} -20- O- [? -L-arabinofuranosyl- (1-6) and Rd {3- O - [β- D- glycoside pyrazol nosil - (1-2) -β-D- glycoside pyrazol nosil] -20- O -β-D- glycoside pyrazol nosil -20 (S) - protocol It's like The larger ginsenoside of the PPD class constitutes more than two thirds of the total ginsenoside extract.

There is a need for a method of producing deglycosylated ginsenosides in order to facilitate the medical application of rare ginsenosides, which are known to be very low in physiological activity, but also to study the biological properties at the molecular level. Physicochemical processes such as steaming (heating) and acid treatment have been developed to convert the main constituents of ginsenosides to deglycosylated ginsenosides. However, the most preferable method is a bioconversion method in which an enzyme having known activity is sequentially reacted with a desired product for a desired product, or an enzyme having a known activity is used in combination of two or more, , And produce known, defined products. In recent years, some progress has been made in the identification of wild-type enzymes capable of converting large molecular weight ginsenosides from fungi and bacteria into low-molecular-weight ginsenosides and characterizing them for industrial applications there was. However, these studies confronted two limitations: 1) there was little progress in the discovery of the biochemical properties of the ginsenoside biotransformation enzyme due to the lack of sequence information for the enzyme; And 2) the method of culturing the wild type microorganism and purifying the wild type enzyme is considered to lack economic feasibility.

Recently, the sequence information of three enzymes exhibiting biosynthesis activity of ginsenoside from microorganisms has been identified and identified as an enzyme belonging to the family of Glycoside Hydrolase: (1) Rb1 or Rc as a compound (C ) -K [20- O - (? - D-glycopyranosyl) -20 ( S ) -protophenecarbocyanuric acid]; (2) β-glucosidase from soil metagenomes converting Rb1 to Rd; And (3) reacting Rb1 with CK, Rb2 with CK, and Rc with C-Mc {20- O - [? - L-arabinofuranosyl- (1-6) -? - D-glycopyranosyl] Beta] -galactosidase from Sulfolobus acidocaldarius , which converts to -20 ( S ) -proteinase . Gypenoside the Rb1 17 {3- O -β-D- glycoside pyrazol nosil -20- O - [β-D- glycoside pyrazol nosil - (1-6) -β-D- glycoside pyrazol nosil] -20 (S) - a protocol wave incident yidayi olgye}, then Gypenoside LXXV {20- O - [β -D- glycoside pyrazol nosil - (1-6) -β-D- glycoside pyrazol nosil] -20 (S) - Prototype wave incident yidayi Recently, the present inventors have reported an enzyme of GH family 3 that catalyzes the continuous conversion to CK and finally to CK.

Probiotics, on the other hand, was defined by R. Fuller in 1989 as "Probiotic for beneficial effects on host animals by improving intestinal microflora of host animals". Examples of the strain used as a probiotic agent include fungi such as Lactobacillus, Enterococcus, Bifidobacterium, and Bacillus, yeasts including Saccharomyces, and fungi including Aspergillus. Probiotics are classified as Generally Recognized As Safe (GRAS), do not have toxic genes for humans and animals, and do not produce pathogenic substances. And microorganisms whose improvement efficacy against animal productivity has been confirmed. Characteristics of probiotics are non-pathogenic, easy in vitro propagation, fast growing in vivo, acidic, and biliary. In addition, the activity by the feed ingredient should not be inhibited, the effect should be maintained at room temperature, and it should be convenient to be mixed with the feed.

The efficacy of these probiotics competitively drops out pathogens that cause intestinal diseases by sticking to the intestinal mucosa with a strong adherence, and it quickly restores destruction of intestinal microflora upon administration of antibiotics. In addition, it inhibits the infection and proliferation of pathogenic microorganisms, promotes the optimal environment and proliferation for the development of beneficial bacteria in the intestines, produces lactic acid and acetic acid, which are major constituents of intestinal organic acids, and reduces intestinal pH, . Thus, by the various complex actions, the productivity of the livestock can be improved while keeping the germ cell gun at the time of feeding. In addition, probiotics in aquaculture are added to feed or incidentally in water. Recently, there has been a lot of research on the improvement of the immunity against pathogenicity and the improvement of the disease due to water quality improvement in feeding probiotics to fish and shrimp (Jiqiu Li et al. Aquaculture 291 (2009) 35-40).

The present inventors have found that ginsenosides which biosynthesize ginsenosides in lactic acid bacteria, such as lactic acid bacteria, such as lactic acid bacteria, such as lactic acid bacteria, such as Luconostomyceteloids, Luconostocitrium, Baiceloconfusa, and Pediococcus pentosaceus, Enzyme activity, preferably glycoside hydrolase activity was confirmed. That is, it is possible to biotransform PPTs such as Rb1, Rc, Rd, Gyp17 or F2, or PPT such as Re, such as Luconostomenseolide, Luconostocitrium, Baicelacognus, Pediococcus pentosaceus And completed the present invention.

One object of the present invention is to provide a glycoside hydrolase derived from lactic acid bacteria, a nucleic acid encoding the same, a recombinant vector containing the nucleic acid, and a transformant transformed with the nucleic acid or the recombinant vector.

Yet another object of the present invention is to provide a method for producing glycoside hydrolase by culturing the transformant according to the present invention.

Still another object of the present invention is to provide a method for producing a glycoside hydrolase according to the present invention, a glycoside hydrolase prepared according to the present invention, a transformant according to the present invention, a culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum , a culture of the strain, or a mixture thereof to produce a deglycosylated second ginsenoside from the first ginsenoside.

Still another object of the present invention is to provide a method for producing a glycoside hydrolase according to the present invention, a glycoside hydrolase prepared according to the present invention, a transformant according to the present invention, a culture of the transformant, Weissella confusa , Pediococcus, pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides), or Lu Pocono stock sheet Solarium (Leuconostoc citreum), rare the culture, or a mixture of the strain of the active ingredient talgeul from PPD (protopanaxadiol) or PPT (Protopanaxatriol) type ginsenosides containing the lycopene misfire Gene And a kit for converting Seno side into Seno side.

Still another object of the present invention is to provide a method for producing a glycoside hydrolase according to the present invention, a glycoside hydrolase prepared according to the present invention, a transformant according to the present invention, a culture of the transformant, Weissella confusa , Pediococcus, pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , Leuconostoc citreum , a culture of the strain, or a mixture thereof.

As one embodiment for achieving the above object, the present invention relates to a glycoside hydrolase derived from a lactic acid bacterium, more specifically, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum- derived glycoside hydrolase.

In the present invention, the term "glycoside hydrolase" refers to glycoside hydrolase derived from lactic acid bacteria, and preferably is selected from the group consisting of Weissella confusa , Pediococcus pentosaceus , Stock mesen's steroid (Leuconostoc mesenteroides subsp. mesenteroides), or Lou Pocono sheet Stock Leeum (Leuconostoc citreum- derived glycoside hydrolase.

The < RTI ID = 0.0 > Weissella & confusa ) GL24 strain is named GL24GH1, GL24GH43 or GL24GH3, and Pediococcus pentosaceus ) is named PedGHl. The amino acid sequence of GL24GH1 is SEQ ID NO: 1, the amino acid sequence of GL24GH43 is SEQ ID NO: 2, the amino acid sequence of GL24GH3 is SEQ ID NO: 3, and the amino acid sequence of PedGH1 is SEQ ID NO: 4.

The < RTI ID = 0.0 > Weissella & confusa ) GL24 is a sequence consisting of SEQ ID NO: 1, 2 or 3, and the glycoside hydrolase amino acid sequence isolated from Pediococcus pentosaceus is SEQ ID NO: 4, preferably not only the above sequence but also an amino acid sequence having 70% or more similarity with the sequence, preferably 80% or more similarity, more preferably 90% or more similarity, still more preferably 95% Similarity, and most preferably greater than 98%, of the amino acid sequence shown in SEQ ID NO: 1, and substantially comprises the sequence having the activity of the glycoside hydrolase. Also, as the sequence having such similarity, if the amino acid sequence is substantially the same or has the same biological activity as the glycoside hydrolase, the protein having an amino acid sequence in which some of the sequences are deleted, modified, substituted or added is also within the scope of the present invention Included is self-explanatory.

As used herein, the term "lactic acid bacteria" refers to gram-positive, microaerophilic, or anaerobic bacteria that ferment sugars to produce acid, including lactic acid as the predominantly produced acid.

The present inventors have found that Rucono Stokes centenoids, Luconostoc citri, Baicelakonpusa or Pediococcus pentosaceus biologically convert PPDs such as Rb1, Rd or Gyp17 to produce Rh 2, F2 or Compound K and so on. In other words, the present inventors have found that a ginsenoside enzyme activity which biosynthesizes ginsenosides in lactic acid bacteria such as Luco stomaceenside, Luconostocitrium, Bissella conpusa or Pediococcus pentosaceus, Preferably, the glycoside hydrolase activity was confirmed (see Example 4).

Wherein the ginsenoside enzyme activity is selected from the group consisting of a β-1,6 glucopyranosyl residue of ginsenoside, a β-1,2 glucopyranosyl residue or glucose, α-arabinofuranoside, arabinopyranoside, α - glycoside hydrolases with selective hydrolysis ability to oligosaccharide moieties containing a ramnopyranosyl moiety. Preferably, a β-1,2 glucopyranosyl residue and a β-1,6 glucopyranosyl residue located at the 3 nd and 20 th carbons of the protopanaxadiol type of PPD (protopanaxadiol), respectively, or α-ara Can exhibit selective hydrolysis ability to the? -Rhampopyranoside or arabinopyranoside, or an? -Rhampopyranosyl residue located at the 6th carbon of the protopanaxtriol (PPT) type ginsenoside.

In another embodiment, the glycoside hydrolase derived from lactic acid bacteria, specifically, Leuconostoc mesenteroides subsp. mesenteroides , Leuconostoc citreum , Weissella confusa ) or Pediococcus ( Pediococcus pentosaceus- derived glycoside hydrolase, a recombinant vector containing the nucleic acid, and a transformant transformed with the nucleic acid or the recombinant vector.

Nucleic acid sequences of Bysella congo GL24GH1, GL24GH43 and GL24GH3 are shown in SEQ ID NOS: 5, 6 and 7, respectively, and the nucleic acid sequence of PedGH1 of Pediococcus pentosius is shown in SEQ ID NO: The nucleic acid of the present invention is a nucleic acid encoding a glycoside hydrolase derived from a lactic acid bacterium, and preferably a nucleic acid encoding a glycoside hydrolase derived from Weissella confusa , Pediococcus pentosaceus , Leuconostoc, mesenteroides subsp. mesenteroides ) or a glucoside hydrolase derived from Leuconostoc citreum , and more preferably a nucleic acid encoding a glycoside hydrolase derived from Weissella confusa , Pediococcus, pentosaceus- derived glycoside hydrolase. The nucleic acid sequence encoding the glycoside hydrolase isolated from Weissella confusa is a nucleic acid sequence consisting of SEQ ID NO: 5, 6, or 7, and is a nucleic acid sequence consisting of the glycoconjugate isolated from Pediococcus pentosaceus The nucleic acid sequence encoding the seed hydrolase is a nucleic acid sequence consisting of SEQ ID NO: 8, preferably a sequence having not less than 70% homology with the above sequence, preferably 80% or more homology, more preferably 90% More preferably at least 95% homology, most preferably at least 98% homology with the amino acid sequence of SEQ ID NO: 1.

The term " similarity " in the present invention refers to a similar degree to the amino acid sequence of a wild-type protein. The term " similarity " Sequence. Comparison of these similarities can be performed using a visual program or a comparison program that is easy to purchase. Commercially available computer programs can calculate the similarity between two or more sequences as a percentage, and the similarity (%) can be calculated for adjacent sequences.

As used herein, the term "vector" refers to a nucleic acid construct comprising an essential control element operatively linked to the expression of a nucleic acid insert, as an expression vector capable of expressing a desired protein in a suitable host cell. The present invention can produce a recombinant vector comprising a nucleic acid encoding a glycoside hydrolase.

Meanwhile, according to the present invention, a nucleic acid encoding glycoside hydrolase or a recombinant vector containing the nucleic acid is transformed or transfected into a host cell, whereby the activity of the glycoside hydrolase of the present invention Lt; / RTI >

The recombinant vector of the present invention can be obtained by linking (inserting) the nucleic acid of the present invention into an appropriate vector. The vector into which the nucleic acid of the present invention is to be inserted is not particularly limited as long as it can be replicated in the host. For example, plasmid DNA, phage DNA, etc. may be used. Specific examples of plasmid DNA include commercial plasmids such as pCDNA3.1 + (Invitrogen). Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis subtilis) - a plasmid origin (YEp13, YEp24 and YCp50) - derived plasmid (pUB110 and pTP5), and yeast. Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used.

As a vector of the present invention, a fusion plasmid (for example, pJG4-5) to which a nucleic acid expression-activating protein (such as B42) is linked can be used. Such fusion plasmids include GST, GFP, His- tag, Myc-tag, etc. However, the fused plasmid of the present invention is not limited by the above examples.

In order to insert the nucleic acid of the present invention as a vector, a method of inserting the purified DNA into a restriction site or a cloning site of a suitable vector DNA by cleaving the DNA with an appropriate restriction enzyme can be used.

The nucleic acid of the present invention is preferably operably linked to a vector. The vector of the present invention may further comprise a promoter and a nucleic acid of the present invention in addition to a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker ), A ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include, but are not limited to, chloramphenicol resistant nucleic acids, ampicillin resistant nucleic acids, dihydrofolate reductase, neomycin resistant nucleic acids, etc., but limiting the additional components operatively linked by the above examples no.

In the present invention, the term 'transformation' refers to the introduction of DNA as a host and DNA can be cloned as a factor of chromosome or as a result of chromosome integration. Thus, by introducing external DNA into cells, This is the phenomenon that causes it.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. Generally, the transformation methods include the CaCl ₂ precipitation method, the Hanahan method which uses a reducing material called DMSO (dimethyl sulfoxide) for the CaCl ₂ method, the electroporation method, the calcium phosphate precipitation method, the protoplasm fusion method, Agrobacterium-mediated transformation, transformation with PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

The method for transforming the nucleic acid encoding the glycoside hydrolase of the present invention or a vector containing the same is not limited to the above examples and the transforming or transfection methods commonly used in the art can be used without limitation .

A transformant of the present invention can be obtained by introducing a nucleic acid encoding glycoside hydrolase, which is a target nucleic acid, or a recombinant vector containing it into a host.

The host is not particularly limited as long as it is capable of expressing the nucleic acid of the present invention. Specific examples of the host which can be used in the present invention include bacteria belonging to the genus Escherichia such as E. coli; Bacillus subtilis Bacillus genus such as subtilis ; Pseudomonas Pseudomonas such as putida ; Lactobacillus such as Lactobacillus and Enterococcus; Saccharomyces S. cerevisiae , yeast such as Schizosaccharomyces pombe ; Animal cells and insect cells.

Specific examples of the Escherichia coli strain which can be used in the present invention include CL41 (DE3), BL21 (DE3) and HB101, and specific examples of Bacillus subtilis strains include WB700 and LKS87.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention can autonomously replicate in a host, and is composed of a promoter, a ribosome binding sequence, a nucleic acid of the present invention, and a transcription termination sequence have.

The promoter of the present invention may be any promoter as long as the nucleic acid of the present invention is expressed in a host such as Escherichia coli. For example, Escherichia coli or phage-derived promoters such as trp promoter, lac promoter, PL promoter or PR promoter; Escherichia coli-infected phage-derived promoters such as the T7 promoter may be used. Artificially modified promoters such as the tac promoter may also be used.

Since the host of the present invention can also be used for food or feed, it is preferable to use lactic acid bacteria suitable for food and feed as a host.

Examples of the lactic acid bacteria include Lactococcus genus, Streptococcus genus, Lactobacillus genus, Leuconostoc genus, Pediococcus genus, Brevibacterium genus, and Propionibacterium genus. Bifidobacteria (ie, lactic acid-producing bacteria belonging to the genus Bifidobacterium spp.), Which are strictly anaerobic groups, which may be used alone or in combination with lactic acid bacteria, are also included in the lactobacillus family. As the lactic acid bacteria, Lactobacillus lactis is particularly preferable.

The nucleotide sequence encoding the glycoside hydrolase according to the present invention can be optionally linked to the appropriate regulatory nucleotide sequence (s) using methods known in the art to control the expression of the coding sequence (e. See Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY). It is also possible to analyze the codon usage patterns of a large number of sequenced genes from different Lactobacillus species to develop an approach to bypass codon bias as needed. See, e.g., Pouwels and Leunissen, 1994, Nucleic Acids Res. 22: 929-936.

The recombinant lactic acid bacteria may comprise one or more constitutive promoters, or one or more regulatable promoters operably linked to a coding nucleotide sequence. As used herein, the term "operably linked" indicates that the nucleotide sequence elements are linked in a functional relationship. For example, a "operably linked" promoter is one in which the regulatory element is in the proper position and orientation relative to the nucleotide coding sequence to control RNA polymerase initiation and expression of the nucleic acid (e.g., Effect. The promoter region may be a promoter present in any prokaryotic cell and a promoter substrate capable of functioning in a lactic acid bacterium (e.g., capable of promoting the expression of a operably linked nucleic acid sequence), but in some embodiments, Lt; / RTI > For example, in some embodiments, the promoter region is selected from the group consisting of Lactococcus lactis subsp. ≪ RTI ID = 0.0 > lactis subspecies l actis), for example, strains, referred to as MG1363 (In addition, in the literature, Lactococcus lactis subspecies Crescent Morris (cremoris), also referred to as search) (lit. [Nauta et al, 1997, Nat Biotechnol 15:.. 980 -983), and Lactococcus lactis subsp. ≪ RTI ID = 0.0 > Lactis < / RTI > May be derived from the promoter region of lactococcus lactis, including lactis biovar. Diacetylactis. Examples of other promoter regions suitable for use in constructing recombinant lactic acid bacteria are disclosed in WO 94/16086 and include regions comprising promoter P170 and derivatives thereof, examples of which are described in WO 98/10079 and US 2002 / 0137140, the disclosures of which are all incorporated herein by reference.

In some embodiments, the lactic acid bacteria used for expression of the recombinant glycoside hydrolase may be a mutant in which the extracellular housekeeping protease HtrA is inactivated. See, for example, Miyoshi, et al., 2002, Appl Environ Microbiol. 68: 3141-3146, the entire disclosures of which are incorporated herein by reference.

In some embodiments, the promoter used in the recombinant lactic acid bacteria may be an adjustable promoter or an inducible promoter. Factor (s) controlling or inducing a promoter includes any physical and chemical factors that can modulate the activity of the promoter sequence, including, for example, physical conditions such as temperature and light; Chemicals such as IPTG, tryptophan, lactate or nisin; And environmental or growth condition factors such as pH, incubation temperature and oxygen content. Other conditions that regulate promoter activity include temperature shifts leading to expression of heat shock genes; The composition of the culture medium, such as ionic strength / NaCl content; Accumulation of metabolites (including lactate / lactate) in cells or in media; Presence / absence of essential cell components or precursors thereto; And the growth or growth rate of the bacteria. See, for example, U.S. Application No. 2002/0137140, the entire disclosure of which is incorporated herein by reference.

Many inducible gene expression systems used in lactic acid bacteria have been developed (see, for example, Kok, 1996, Antonie Van Leeuwenhoek. 70: 129-145; Kuipers et al., 1997, Trends Biotechnol. (Kleerebezem, et al., 1997, Appl. Environ Microbiol. 63: 4581-4584); [Djordjevic and Klaenhammer, 1998, Mol Biotechnol. 9: 127-139]; A useful lactic acid bacterial expression system is the NICE system (de Ruyter et al., 1996, Appl Environ Microbiol. 62: 3662-3667) Is based on gene elements from a two-component system that controls the biosynthesis of antimicrobial peptide nisin in lactis. Another useful inducible expression system is El. Lactis bacteriophage f 31 (O'Sullivan et al., 1996, Biotechnology (NY), 14: 82-87 and Walker and Klaenhammer, 1998, J Bacteriol. 180: 921-931) and rlt (Natta et al., 1997, Nat Biotechnol. 15: 980-983), a promoter induced by environmental changes such as pH (Israelsen et al., 1995, Appl Environ Microbiol. 61: 2540 -2547]), Zn2 + (Llull and Poquet, 2004, Appl Environ Microbiol. 70: 5398-5406), salt concentration (Sanders et al., 1998, Mol Gen Genet, 257: 681-685) And the use of metabolites produced by host cells or naturally occurring conditions during host cell growth. Such promoters include pH inducible P170 promoters and derivatives thereof (WO 94/16086, WO 98/10079, U.S. Patent Application Publication No. 2002/0137140 and Madsen et al., 1999, Mol Microbiol. 107 : 75-87).

In some embodiments, the promoter and nucleotide sequence encoding the glycoside hydrolase may be introduced into the lactic acid bacteria on a plasmid, a transfer factor, a bacteriophage or a cosmid and a spontaneous replication replicon. In some embodiments, the promoter and recombinant protein nucleotide coding sequences can be introduced under conditions that allow the recombinant protein nucleotide coding sequence to integrate into the lactobacillus cell chromosome and thereby ensure that the recombinant protein nucleotide coding sequence in the bacteria remains stable. Integration can be performed by an integrated system based on homologous recombination, transposon, junction migration, and phage integration (see, e.g., Frazier et al., 2003, Appl Environ Microbiol. 69 (2): 1121-8 [Romero et al., 1992, Appl Environ Microbiol. 58 (2): 699-702.); [Romero et al., 1994, J Bacteriol. 176 (4): 1069-76] Leenhouts et al., 1990, Appl Environ Microbiol. 57 (9): 2562-7.); [Leenhouts et al., 1991, J Bacteriol. 173 (23): 7599-606. Appl Environ Microbiol. 55 (7): 1769-74] and Scheirlinck et al., 1989, Appl Environ Microbiol. 55 (7) 9): 2130-7).

In another embodiment, the glycosidic hydrolase nucleotide coding sequence can be introduced into the lactic acid bacterial cell chromosome at a position operably linked to a naturally-occurring promoter in the chromosome of the selected host organism (see, for example, Rauch 1993, Appl Environ Microbiol. 59 (1): 21-26.) and [Maguin et al., 1996, J. Bacteriol. 174 (4): 1280-7] J Bacteriol. 178 (3): 931-5).

In some embodiments, the glycoside hydrolase nucleotide coding sequence may be operably linked to a nucleotide sequence encoding a signal peptide (SP) such that the gene product is secreted outside the bacteria and introduced into the culture medium. Suitable signal peptides for use in lactic acid bacteria, including Usp45, are disclosed in U.S. Publication No. 2002/0137140, the entire disclosures of which are incorporated herein by reference.

In some embodiments, additional nucleotide sequences, such as those used to improve the production and secretion of heterologous proteins in lactic acid bacteria, can be used in the methods and compositions described herein. For example, in some embodiments, the nucleotide sequence encoding Staphylococcus nuclease (Nuc) and the synthetic propeptide LEISSTCDA may be linked to a nucleotide sequence encoding a glycoside hydrolase. See, for example, Nouaille et al., 2005, Braz J Med Biol Res. 38: 353-359, the entire disclosures of which are incorporated herein by reference.

In some embodiments, pLF22 (see, eg, Trakanov, et al., 2004, Microbiology 73: 170-175) and pTREX (see, eg, Reuter, et al., 2003, "Vaccine Protocols , "In Methods in Molecular Medicine 87: 101-114 "), can be used to express a recombinant glycoside hydrolase in lactic acid bacteria.

In various embodiments, the lactic acid bacteria comprising the nucleotide sequence encoding the glycoside hydrolase are cultured, for example, as disclosed in U.S. Publication No. 2002/0137140 to produce an endotoxin-free glycoside hydrolase can do. The method can include culturing the transformed lactic acid bacteria under conditions suitable for expression of the apolipoprotein and recovering the glycoside hydrolase from the transformed lactic acid bacteria. Recombinant cells and / or glycoside hydrolases may be harvested using conventional techniques known to those skilled in the art. See, for example, U.S. Patent Application Publication No. 2002/0137140, the entire disclosure of which is incorporated herein by reference.

In some embodiments, short-chain acyl phospholipids are added to a medium (i. E., Fermentation medium) used for the cultivation of lactic acid bacteria comprising a recombinant glycoside hydrolase (as described above). Lactic acid bacteria may use short-chain acyl phospholipids as a source of nutrients. In addition, short-chain acyl phospholipids can be used as an adjuvant to dissolve the expressed apolipoprotein. The short-chain acyl phospholipid can be easily removed by adding a phospholipase which degrades the acyl chain and liberating the short-chain fatty acid and short-chain lysoPL.

In order to facilitate the purification of the target protein recovered in the present invention, the plasmid vector may further include other sequences as necessary. The sequence that may be further included may be a tag sequence for protein purification and may be a tag sequence such as glutathione S-transferase (Pharmacia, USA), MBP (Maltose binding protein, USA), FLAG (IBI, USA) and hexa histidine hexahistidine; Quiagen, USA), and most preferably MBP. However, the types of sequences necessary for purifying the target protein are not limited by the above examples.

Further, in the case of a fusion protein expressed by a vector containing the fusion sequence, it can be purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When MBP is used, desired protein can be easily recovered using an amylose column.

The host cell transformed by the above method for expressing the glycoside hydrolase of the present invention can be cultured by a conventional method used in the art. For example, the transformant expressing the glycoside hydrolase can be cultured in a variety of media, and can be fed-batch cultured and continuous cultured. However, according to the present invention, The method of culturing the transformant of the present invention is not limited. The carbon source that may be contained in the medium for the growth of the host cell may be appropriately selected according to the judgment of a person skilled in the art depending on the type of the transformant prepared and a suitable culture condition is adopted to control the culture time and amount .

When appropriate host cells are selected and the medium conditions are established, the transformant in which the target protein has been successfully transformed will produce glycoside hydrolase, and the glycoside hydrolase produced according to the constitution of the vector and the characteristics of the host cell Can be secreted into the cytoplasm, periplasmic space, or extracellular space of host cells. The target protein may also be expressed in soluble or insoluble form.

Proteins expressed in or outside the host cell can be purified in a conventional manner. Examples of purification methods include salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (eg, protein fraction precipitation using acetone or ethanol), dialysis, gel filtration, ion exchange, reverse phase column chromatography Chromatography, ultrafiltration and the like can be used alone or in combination to purify the protein of the present invention.

In vitro (in vitro) to the ginsenosides present binary using the glycoside hydrolase hydroxide separated by the above method or in vivo (in RTI ID = 0.0 > gancenoside, < / RTI > which is readily absorbable in vivo systems.

In at least one stage of the production of the deglycosylated second ginsenoside from the first ginsenoside, the present invention relates to the glycoside hydrolase of the present invention, the transformant of the present invention or a culture of the transformant, Weissella confusa , Pediococcus, pentosaceus , < / RTI > Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum , cultures of the strains, or mixtures thereof.

The first ginsenoside used as a starting material in the present invention may be a ginsenoside of PPD or PPT type and may be separated and purified form of ginsenoside or may be contained in powder or extract of ginseng or red ginseng You can also use the Gin Seno side. That is, the method of the present invention may be carried out by directly using ginseng or red ginseng powder or extract containing ginsenoside as a starting material. The ginsenosides of PPD or PPT type are shown in Fig.

As the ginseng to be used in the present invention, various known ginsengs can be used, and ginseng such as Panax ginseng , P. quiquefolius , P. notoginseng , P. japonicus , But are not limited to, P. trifolium , P. pseudoginseng , and P. vietnamensis .

The first ginsenoside, which is a substrate of the glycoside hydrolase, is selected from the group consisting of Rg1, Rg2, Rb1, Rc, Rb2, Rd, F2, Rg3, Gyp17, the compound O, the compound Y, the compound Mc1, And preferably Rb1, Gyp17, Rd or Re.

The glycoside hydrolase of the present invention can be selectively hydrolyzed if it is a non-reduced β-1,6 glucopyranosyl residue, a β-1,2 glucopyranosyl residue, or a ginsenoside having glucose, Beta-1,6 glucopyranosyl residue, beta-1,2 glucopyranosyl residue, alpha -laminopyranosyl residue, or glucose of any PPD or PPT type of ginsenoside known in the art, For example, since Rb1 among the ginsenosides of the PPD type has a non-reduced β-1,6 glucopyranosyl residue at C20 (meaning 20th carbon), the non-reduced β of the Rb1 -1,6 glucopyranosyl residue can be selectively hydrolyzed by the glycoside hydrolase of the present invention. Residues such as arabinofuranoside of C3 of ginsenoside Rc and arabinofiranoside of Rb2 can also be selectively hydrolyzed by the glycoside hydrolase of the present invention. Furthermore, since the? -Rhamnopyranosyl moiety at C6 of ginsenoside Re has the? -Rhamnopyranosyl moiety, it can be selectively hydrolyzed by the glycoside hydrolase of the present invention.

A glycoside hydrolase of the present invention, a transformant of the present invention or a culture of the transformant, a Weissella confusa , Pediococcus, pentosaceus , < / RTI > Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum , cultures of the strains, or mixtures thereof convert PPD (protopanaxadiol) or PPT type of ginsenosides to relatively deglycosylated rare ginsenosides Which may be a non-reduced β-1,6 glucopyranosyl residue, β-1,2 glucopyranosyl residue, α-rhamnopyranosyl residue, glucose, arabinofuranosyl residue of PPD type or PPT type of ginsenoside Hydroxysuccinimide, and the like, and more preferably, a non-reduced β-1,6 glucopyranosyl residue located at the 20th carbon of the PPD type ginsenoside, arabinofuranos Or an arabinopyranoside moiety, or a non-reduced? -1,2 glucopyranosyl moiety or glucose located at the third carbon, or an? -Carbonyl moiety located at the 6th carbon of the PPT type ginsenoside, No pyrazole can be carried out by optionally hydrolyzing nosil residue.

This conversion is accomplished by bioconversion and includes continuous or discontinuous non-reduced β-1,6 glucopyranosyl residues, β-1,2 glucopyranosyl residues or glucose of the PPD type of ginsenosides, Arabinofuranoside, arabinofuranoside, or an a-rhamnopyranosyl residue located at the 6th carbon of the PPT type ginsenoside.

Preferably, examples of biotransformation by the protein of the present invention include conversion of ginsenoside Rb1 to Rd, conversion of ginsenoside Rc to Rd, conversion of ginsenoside Rb2 to Rd, conversion of ginsenoside Rd to F2, Conversion of ginsenoside Rd to compound K, conversion of ginsenoside Rb1 to F2, conversion of ginsenoside Gyp17 to F2, conversion of ginsenoside Rb1 to compound K, conversion of ginsenoside F2 to compound K, The side Rd to Rg3, the ginsenoside Rb1 to Rh2, the ginsenoside Rg3 to Rh2, the ginsenoside Rb1 to Gyp17, the ginsenoside Gyp17 to GypLXXV, the ginsenoside gyp17 to the compound K , Conversion of ginsenoside Rb2 to compound O, conversion of ginsenoside compound O to compound Y, conversion of ginsenoside Rc to compound Mc, conversion of ginsenoside Re to Rg1, conversion of ginsenoside compound Mc to compound Y, How to switch to Mc1 More preferred methods include converting Rb1 to Rh2, converting Rb1 to compound K, Gyp17 to F2, Rd to F2, Re to Rg1, and Rb1 to F2 , But is not limited thereto. The reaction path of the PPD type ginsenosides which can be produced according to the invention is shown in Fig.

The deglycosylation of such ginsenosides may be carried out in the presence of a non-reduced α-rampopyranosyl residue, β-1,6 glucopyranosyl residue, β-1,2 glucopyranosyl residue or glucose of the glycoside hydrolase of the present invention , And the deglycosylated rare ginsenoside produced by the hydrolysis as described above is easily absorbed into the body. In addition, this bioconversion facilitates the conversion of PPD type ginsenosides, such as ginsenoside Rb1, to its most deglycosylated form, compound K.

The glycosidic hydrolase of the present invention can be administered at various temperature and pH conditions as long as its activity and stability can be maintained by administering to the patient an effective amount of an α-ramanopyranosyl residue of PPT type ginsenoside, β-1 of protopanaxadiol type ginsenoside , 6-glucopyranosyl residue,? -1,2-glucopyranosyl residue, or glucose, and a PPD (protopanaxadiol) type or PPT (protopanaxatriol) type ginsenoside as a rare ginsenoside Lt; / RTI >

In addition, the glycoside hydrolase of the present invention may be used in combination with at least one metal selected from the group consisting of MgCl ₂ , EDTA, NaCl, KCl, DTT, and beta-mercaptoethanol, and a chemical agent.

On the other hand, when producing the deglycosylated second ginsenoside from the first ginsenoside, the glycoside hydrolase of the present invention, the transformant of the present invention or a culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum ), The culture of the strain, or a mixture thereof, can be used with other enzyme (s) simultaneously or in a certain order. Non-limiting examples of other enzymes include α- N -arabinofuranosidase, β-galactosidase, glycosidase, α-L-arabinopyranosidase, α-L-arabinofuranoside Beta -xylosidase, and alpha -L-rhamocidase. The second ginsenosides produced by the glycoside hydrolase alone or in combination with other enzymes may be different, and the second ginsenoside may be one or more second ginsenosides.

The glycoside hydrolase of the present invention, the transformant of the present invention or a culture of the transformant, Weissella confusa , Pediococcus pentosaceus , ( Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum , a culture of the strain, or a mixture thereof may be provided in the same container as the other enzyme (s) or in a different container.

In another embodiment, the present invention relates to a method for producing a transformant of the present invention, which comprises administering to the transformant or a transformant of the present invention a glycoside hydrolase, a culture of the transformant of the present invention, Weissella confusa , Pediococcus pentosaceus , Lou's Pocono Stock mesen steroid (Leuconostoc mesenteroides subsp. mesenteroides), or Lou Pocono sheet Stock Leeum (Leuconostoc citreum ), a culture of the strain, or a mixture thereof.

The feed or feed additive is included in the food or food additive according to the present invention. The food or food additive may further preferably further comprise ginsenosides of PPD or PPT type, or hemp. The foods include not only health functional foods, but also general foods that are widely consumed by humans on a daily basis. When used as a health functional food, may be formulated into a conventional health functional food formulation known in the art together with a pharmaceutically acceptable carrier or additive. The health functional food may be prepared, for example, as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a liquid, an extract, tea, jelly, The pharmaceutically acceptable carrier or additive may be any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared.

The food may be a fermented food, and examples of the fermented food include, but are not limited to, kimchi, fermented vegetables, soybean paste, soy sauce, chonggukjang, fermented dairy products (fermented milk, yogurt, cheese) and the like.

In food, food additives, feeds or feed additives, the ginsenoside or trinucleoside may be hydrolyzed by glycoside hydrolase and not hydrolyzed.

When used as a feed additive, the glycoside hydrolase of the present invention, the transformant of the present invention or a culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Lou's Pocono Stock mesen steroid (Leuconostoc mesenteroides subsp. mesenteroides), or Lou Pocono sheet Stock Leeum (Leuconostoc citreum ), the culture of the strain, or a mixture thereof may be 20 to 90 wt% highly concentrated, or may be prepared in powder or granular form. The feed additive may be selected from the group consisting of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, Extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. When used as a feed, the composition may be formulated in conventional feed form and may contain conventional feed ingredients.

The feed additives and feeds may be selected from the group consisting of cereals, such as ground or crushed wheat, oats, barley, corn and rice; Feeds based on vegetable protein such as rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A sugar or a milk product, for example, a dry component comprising various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered to animals singly or in combination with other feed additives in edible carriers. The feed additives can also be administered to the animal either as a top dressing or they can be mixed directly with the animal feed or in a separate oral form separate from the feed. When the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive can be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution formulation.

The feed may comprise any protein-containing organic fructose commonly used to meet an animal's dietary needs. These protein-containing flours usually consist mainly of corn, soy flour, or corn / soy flour mix.

The feed additives and feeds may also contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. The feed additive may be used by being added to animal feed by infiltration, spraying or mixing.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry, and fish. The above-mentioned mammals can be used for pets (for example, dogs and cats) as well as pigs, cows, sheep, goats, laboratory animals and laboratory animals as well as poultry such as chicken, turkey, duck, goose, And quail, and the fish can be used as trout, but the present invention is not limited thereto.

The lactic acid bacteria according to the present invention, specifically, Weissella confusa , Pediococcus, pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides , or Leuconostoc citreum- derived glycoside hydrolase exhibits excellent activity of converting ginsenosides into deglycosylated rare ginsenosides.

Figure 1 shows the chemical structure of PPD and PPT type ginseng saponin.
Figure 2 is a four-by-Cellar konpu (Weissella confusa ) strain GL24-derived proteins GL24GH1 and GL24GH43. (M: protein marker; lane 1: flow through; lane 2, 3 and 4: maltose-eluted fractions of GL24GH1); lane 5: flow- ; Lanes 6, 7 and 8: maltose-eluted fractions of GL24GH43 from GL24GH43)
FIG. 3 is a TLC result of a glycoside hydrolase GL24GH1 derived from a strain GL24 belonging to the lactic acid bacteria Weissella confusa , which is analyzed for the conversion of ginsenosides. (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s), lane 6: standard CK, lane 7: standard PPDA protopanaxadiol aglycon) lane 14: standard Re lane 15: standard Rg1 lane 16: standard Rg2 lane 17: standard F1 lane 18: standard Rh1 (S) lane 19: protopanaxatriol aglycon lane 8: Reaction product of Re and Enzyme after Reaction with Rb1 and Enzyme Reaction product of Reaction Reaction with Enzyme Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Reaction Lane 13: reaction product of Rg1 and enzyme)
FIG. 4 is a TLC result of a glycoside hydrolase GL24GH43 derived from a strain GL24 belonging to the lactic acid bacterium, Weissella confusa , analyzed for the conversion of ginsenosides. (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s), lane 6: standard CK, lane 7: standard PPDA protopanaxadiol aglycon) lane 14: standard Re: lane 15: standard Rg2 lane 16: standard Rg1 lane 17: standard F1 lane 18: standard Rh1 lane 19: protopanaxatriol aglycon lane 8: Lane 9: reaction product of Gyp17 and the enzyme, lane 10: reaction product of Rd and the enzyme, lane 11: reaction product of F2 and the enzyme, lane 12: reaction product of Re and the enzyme, lane 13: reaction product of Rg1 and enzyme Lt; / RTI >
FIG. 5 is a TLC result of analyzing the conversion of glycoside hydrolase GL24GH3 derived from strain GL24 belonging to the lactic acid bacteria Weissella confusa to PPD type ginsenosides. (Lane 1: standard Rb1) lane 2: standard Gyp17 lane 3: standard Rd lane 4: standard F2 lane 5: standard CK lane 6: enzyme reaction product of GL24GH3 and Rb1 Lane 7: Reaction product between Gyp17 and enzyme; lane 8: reaction product between Rd and enzyme)
FIG. 6 is a TLC result of analysis of the conversion of glycoside hydrolase PedGH1 derived from Pediococcus pentosaceus to PPD type ginsenosides of lactic acid bacterium Pediococcus pentosaceus . (Lane 1: standard Rb1) lane 2: standard Gyp17 lane 3: standard Rd lane 4: standard F2 lane 5: standard CK lane 6: reaction product between PedGH1 and Rb1 enzyme lane 7 : Reaction product between Gyp17 and enzyme; lane 8: reaction product between Rd and enzyme)
FIG. 7 is a graph showing the results of measurement of the concentration of lactic acid bacteria ( Weissella confusa ) Of the strain GL24 belonging to the genus Senoside. (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s), lane 6: standard CK, lane 7: standard PPDA protopanaxadiol aglycon); 8: Rb1 and Weissella confusa The reaction product between the coenzyme of strain GL24 belonging to the genus GL24; Lane 9: reaction product between Gyp17 and the coenzyme; Lane 10: reaction product between Rd and the coenzyme)
Fig. 8 is a graph showing the results of the measurement of the activity of the lactic acid bacteria Weissella confusa Of the strain GL24 belonging to the genus Senoside. Lane 2: Standard Rg1; Lane 3: Standard Rg2; Lane 4: Standard Rh1 (S) Lane 5: Standard F1 Lane 6: Standard PPTA Lane 7: Weissella confusa The reaction product between the coenzyme of strain GL24 belonging to the genus GL24; Lane 8: reaction product between Rg1 and the coenzyme; Lane 9: reaction product between Re and coenzyme; Lane 10: reaction product between Rg1 and the coenzyme; Lane 11: reaction product between Re and coenzyme; Lane 12: reaction product between Rg1 and the coenzyme)
FIG. 9 is a TLC result of the conversion of the strain 100118TI-4 belonging to the lactic acid bacterium Pediococcus pentosaceus to PPD type ginsenoside biotransformation. (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s), lane 6: standard CK, lane 7: standard PPDA lane 9: reaction product between Gyp17 and the coenzyme lane 10: reaction product between Rd and the coenzyme): lane 8: reaction product between Rb1 and the crude enzyme of Pediococcus pangotusii strain 100118TI-4;
FIG. 10 is a TLC result of analysis of PPI type ginsenoside biotransformation of strain 100118TI-4 belonging to the lactic acid bacterium Pediococcus pentosaceus . Lane 1: standard Re; lane 2: standard Rg1; lane 3: standard Rg2; lane 4: standard F1; lane 5: standard Rh1 (S); lane 6: standard CK; lane 7: standard PPTA; Lane 8: Reaction product between Re and crude enzyme of Pediococcus pan Tosausi strain 100118TI-4; lane 9: reaction product between Rg1 and coenzyme)
Fig. 11 is a graph showing the effect of lactic acid bacteria ( Leuconostoc ) Belonging to Leuconostoc citreum (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s) Lane 6: standard CK lane 7: protopanaxadiol aglycon lane 14: standard Re: lane 15: standard Rg1 lane 16: standard Rg2 lane 17: standard F1 lane 18: standard Rh1 (S); Lane 19: Standard PPTA (protopanaxatriol aglycon) Lane 8: Rb1 and Leuconostoc citreum The reaction product of the coenzyme of strain GL29 belonging to the genus; Lane 9: reaction product between Gyp17 and the enzyme; Lane 10: reaction product between Rd and enzyme; Lane 11: reaction product between F2 and the enzyme; Lane 12: reaction product between Re and the enzyme; Lane 13: reaction product between Rg1 and the enzyme)
Fig. 12 is a graph showing the activity of the lactic acid bacterium Leuconostoc mesenteroides subsp. mesenteroides ) Of the strain belonging to the genus Senoside. (Standard Rb1), lane 2: standard Gyp17, lane 3: standard Rd, lane 4: standard F2, lane 5: standard Rh2 (s), lane 6: standard CK, lane 7: standard PPDA protopanaxadiol aglycon) lane 14: standard Re lane 15: standard Rg1 lane 16: standard Rg2 lane 17: standard F1 lane 18: standard Rh1 (S) lane 19: protopanaxatriol aglycon lane 8: Rb1 and Luconostoc mesenteroides ( Leuconostoc mesenteroides subsp. the reaction product between the coenzyme of strain 25 belonging to the mesenteroides ; Lane 9: reaction product between Gyp17 and the enzyme; Lane 10: reaction product between Rd and enzyme; Lane 11: reaction product between F2 and the enzyme; Lane 12: reaction product between Re and the enzyme; Lane 13: reaction product between Rg1 and the enzyme)
13 is a schematic view showing the reaction path of the PPD type ginsenoside produced in the present invention.

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 to these examples.

Example  1: Recombination GL24GH1, GL24GH43  And GL24GH3 Molecule Cloning , Expression and purification

(1-1) Strain GL24 Characterization of

(SEQ ID NO: 9) and a reverse primer (16S-1492R; 5'-GGTTACCTTGTTACGACTT-3 '(SEQ ID NO: 10)) to analyze the characteristics of the GL24 strain PCR amplification was performed, and the nucleotide sequence was determined by Solgent. The determined 16S rRNA gene sequence was collected and blasted in the NCBI database. As a result of 16S rRNA sequencing analysis of GL24 strain, it was identified as Weissella confusa . The glycoside hydrolase gene was cloned in the GL24 strain identified as Weissella confusa .

(1-2) Recombination GL24GH1 Molecule Cloning  And expression

In order to amplify the glycoside hydrolase gene by PCR, the GL24 strain was cultured on MRS plate culture medium, and then the cells were collected and genomic DNA extraction kit (MoBio) genomic DNA). The vector pET21a used for cloning was KCTC11478 (pMBP-BglQM6775) added to the N-terminal side of the maltose binding protein epitope.

Bioneer's PCR pre-mix was used to perform PCR, and the following primers were custom-made and used. At this time, 100 ng of gDNA (genomic DNA) of GL24 was used as a template DNA.

Forward primer:

5'-GAA GAA TTC ATG TCA TTA CGG GGC AAA GAA-3 '(SEQ ID NO: 11)

Reverse primer:

5'-CC A AGC TT C TAC TTC CCT CGA TAC TCT AA-3 '(SEQ ID NO: 12)

(The underlined portion of the primer sequence indicates restriction sites of EcoRI and HindIII, respectively)

The sequence of the amplified fragments was analyzed using BLSTP, and it was identified as an enzyme belonging to the Glycoside hydrolase family I, which was named GL24GH1.

The amplified fragments were cloned into the pET21a expression vector using EcoRI and HindIII restriction enzyme transfer sites. The cloned GL24GH1-pET21a was transformed into E. coli strain C41. The transformed Escherichia coli was inoculated into LB liquid medium and when OD ₆₀₀ = 0.4 to 0.6, IPTG was added to a final concentration of 0.2 mM and cultured at 37 for 6 hours. The cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5), and then disrupted by an ultrasonic disintegrator. The supernatant centrifuged at 12,500 xg for 20 min was used for the separation and purification of the enzyme protein.

(1-3) Recombination GL24GH43 Molecule Cloning , Expression

In order to amplify the gene by PCR, the strain was cultured on MRS plate culture medium, and then the cells were collected and genomic DNA was isolated using genomic DNA extraction kit (MoBio). The vector used for cloning was KCTC11478 (pMBP-BglQM6775) to which the maltose binding protein epitope was added at the N-terminal side.

BIONEER PCR premix was used for PCR, and the following primers were customized and used. At this time, 100 ng of GL24 gDNA was used as a template DNA.

Forward primer:

5'-CC G AAT TCA TGA CAG TTC GCT ACG AAA AC-3 '(SEQ ID NO: 13)

Reverse primer:

5'-CCCA AA GCT T TT AGT TAA AAG TAA ATG GTT GTG-3 '(SEQ ID NO: 14)

(The underlined portion of the primer sequence indicates restriction sites of EcoRI and HindIII, respectively)

The amino acid sequence is shown in SEQ ID NO: 2 and BLSTP analysis showed that the enzyme belonged to Glycoside hydrolase family 43. GL24GH43.

The amplified fragments were cloned into the pET21a expression vector using EcoRI and HindIII restriction enzyme transfer sites. The cloned GL24GH43-pET21a was transformed into E. coli strain C41.

When the transformed E. coli was inoculated into LB liquid medium and OD ₆₀₀ = 0.4 to 0.6, IPTG was added to a final concentration of 0.2 mM and cultured at 37 ° C for 6 hours. The cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5), and then disrupted by an ultrasonic disintegrator. The supernatant centrifuged at 12,500 xg for 20 min was used for the separation and purification of the enzyme protein.

(1-4) Recombination GL24GH3 Molecule Cloning , Expression

In order to amplify the gene by PCR, the strain was cultured on MRS plate culture medium, and the cells were collected and genomic DNA was isolated using a genomic DNA extraction kit (MoBio). The pET21a vector used for cloning was KCTC11478 (pMBP-BglQM6775) added to the N-terminal side of the maltose binding protein epitope Respectively.

BIONEER PCR premix was used for PCR, and the following primers were customized and used. At this time, 100 ng of the gDNA of W. confusa GL24 was used as a template DNA (template DNA).

Forward primer:

5'-CC G AAT TC A TGA CCC AGC AAC TCA CTC AC-3 '( SEQ ID NO: 15)

Reverse primer:

5'-GCC AAG CTT TCA ATG TGT TGT CTG ACT ACC-3 '(SEQ ID NO: 16)

(The underlined portion of the primer sequence indicates restriction sites of EcoRI and HindIII, respectively)

The amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO: 3, and BLSTP analysis revealed that the enzyme belonged to Glycoside hydrolase family 3. GL24GH3. ≪ / RTI >

The amplified fragments were cloned into the pET21a expression vector using EcoRI and HindIII restriction enzyme transfer sites. The cloned GL24GH3-pET21a was transformed into E. coli strain C41.

The transformed Escherichia coli was inoculated into LB liquid medium and when OD ₆₀₀ = 0.4 to 0.6, IPTG was added to a final concentration of 0.2 mM and cultured at 37 for 6 hours. The cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5), and then disrupted by an ultrasonic disintegrator. The supernatant centrifuged at 12,500 xg for 20 min was used for the separation and purification of the enzyme protein.

(1-5) Recombination GL24GH1  And GL24GH43 Purification of

E. coli containing the overexpressed plasmid of GL24GH1-pET21a or GL24GH43-pET21a with the MBP tag attached thereto C41, DE3) were cultured in LB-amphi sylline medium at a temperature of 37 DEG C until the OD value reached 0.6 at 600 nm, and isopropyl-D-thiogalactopyranoside was incubated for 6 hours. 0.2 mM to induce protein expression. The E. coli cells were centrifuged at 5,000 x g for 20 minutes at 4 DEG C and then obtained. The cell pellet was resuspended in suspension buffer (50 mM sodium phosphate, 5 mM EDTA, pH 7.5), sonicated, and centrifuged at 12,500 xg. The supernatant was loaded on an MBPTrap (GE) column using an FPLC system, washed with the phosphate buffer solution, eluted with 200 mM maltose, and the protein was then purified. SDS-PAGE analysis was then performed on GL24GH1 and GL24GH43.

As a result, it was confirmed that GL24GH1 protein was expressed in lane 2, 3 and 4 (95 kDa), and GL24GH43 protein (70 kDa) was expressed in lanes 6, 7 and 8 (FIG. 2).

Example  2. Recombination PedGH1 ( Pediococcus pentosaceus glycoside hydrolase  1) molecule Cloning  And expression

(SEQ ID NO: 10) and reverse primer (16S-1492R; 5'-GGTTACCTTGTTACGACTT-3 '(SEQ ID NO: 10)) in order to analyze the characteristics of the strain 100118TI- ) Was used for PCR amplification, and the nucleotide sequence was determined by Solgent. The determined 16S rRNA gene sequence was collected and blasted in the NCBI database. The 16S rRNA sequencing of the GL24 strain identified it as Pediococcus pentosaceus .

The glycoside hydrolase gene was cloned in 100118 TI-4 strain identified as Pediococcus pentosaceus . In order to obtain genomic DNA necessary for PCR amplification of the gene, the strain was cultured on an MRS plate culture medium, and then the cells were collected using a genomic DNA extraction kit (MoBio) The genomic DNA was isolated. Vector used for cloning was KCTC11478 (pMBP-BglQM6775) as the pET21a vector.

BIONEER PCR premix was used for PCR, and the following primers were customized and used. At this time, the template DNA is Pediococcus pentosaceus ) 100118TI-4 gDNA.

Forward primer:

5'-CC G AAT TC A TGA CTG AAA AGC ATT ATA AG-3 '( SEQ ID NO: 17)

Reverse primer:

5'-GCC AAG CTT CTA TAA ATC TTC ACC ATT AG-3 '(SEQ ID NO: 18)

(The underlined portion of the primer sequence indicates restriction sites of EcoRI and HindIII, respectively)

The amino acid sequence of the cloned glycoside hydrolase is shown in SEQ ID NO: 4. As a result of BLSTP analysis, an enzyme belonging to Glycoside hydrolase family 1 was designated as PedGH1.

Example  3: Culture of lactic acid bacteria and induction of enzyme expression

Lactic acid bacteria were inoculated in 100 ml of MRS liquid medium using the lactic acid bacteria and the culture medium described in the following [Table 1] and cultured in a 37 ° C shaking incubator.

Lactic acid bacteria and culture medium number Strain name Culture medium One Weissella confusa GL24 MRS 2 Pediococcus pentosaceus 100118TI-4 MRS 3 Leuconostoc mesenteroides subsp. mesenteroides strain 25 MRS 4 Leuconostoc citreum GL29 MRS

The base stock Pocono strain (strain) of the lactic acid bacteria, Lactococcus Phedi O fan soil siwooseu (Pediococcus pentosaceus) belonging to lactic acid bacteria and by Cellar konpu four (Weissella confusa) belonging to the (Leuconostoc) are brought up well on MRS medium.

After 8 hours, the absorbance (OD 600nm) of the culture solution was 0.5-0.6. When the cells entered the logarithmic growth period, the glycoside hydrolase enzyme showing ginsenosidase activity with the PPD mixture or the PPT mixture Expression was induced. One or two days after the culture, 50 ml of the cell culture medium was recovered, respectively.

Example 4: Bioconversion experiment of saponin (ginsenoside) of ginseng

Rg1, Gyp17, Rd, F2, Re, and Rg1 in which the crude enzyme solution of the PedGH1 enzyme solution and the wild type strain dissolved in the sodium phosphate buffer solution (50 mM, pH 7.5) (1 mg / ml) and a volume ratio of 1: 1 (200 ul: 200 ul). After the reaction, the sample was sampled by time, extracted with butanol of the same volume, and subjected to TLC analysis. Confirmation of the reaction product was judged by the developed view on TLC using the standard substance of the same purity as the enzyme reaction substrate.

Cells of 100 ml of the culture obtained in Example 3 were suspended in 2 ml of phosphate buffer and sonicated for 1 minute. The cell extract was centrifuged at 15,000 rpm for 20 minutes to obtain a crude enzyme solution.

3 to 5 show the results of TLC (thin layer chromatography) for the conversion of the result of confirming the conversion activity of ginsenoside into recombinant GL24GH1 and GL24GH43 isolated and purified in Example 1. FIG. 3 shows that GL24GH1 has the activity of converting Rb1 -> Rh2 and CK, Gyp17 -> F2, Rd -> F2, and Re -> Rg1. FIG. 4 also shows that GL24GH43 has conversion activity to PPD type ginsenosides Rb1 -> F2, Gyp17 -> F2, Rd -> F2 and Re -> Rg1. FIG. 5 shows that GL24GH3 has an activity of converting Rb1 -> F2, Gyp17 -> F2, and Rd -> F2.

Fig. 6 shows that PedGH1 has an activity of converting Rb1 -> F2 and Rd - > F2.

In addition, the crude enzyme solution was reacted with ginseng saponin to determine whether bioconversion was carried out using TLC. The results are shown in FIGS. 7 to 12.

FIG. 7 is a graph showing the effect of the lactic acid bacteria Weissella confusa ) has an activity of biotransformation of PPD type ginsenosides Rb1, gyp17, and Rd into F2.

FIG. 8 is a graph showing the activity of the lactic acid bacteria Weissella confusa ) Indicates that the strain GL24 belonging to the genus Escherichia has an activity of biotransformation of PPT type ginsenoside Re to Rg1.

FIG. 9 is a graph showing the activity of the lactic acid bacteria Pediococcus pentosaceus belonging to the strain 100118TI-4 has biotransformation activity of Rb1 -> F2, Gyp17 -> F2 and Rd -> F2 among PPD type ginsenosides.

10 shows that the lactic acid bacterium Pediococcus panthosiaus strain 100118 TTI-4 ( Pediococcus pentosaceus 100118 TTI-4) has an enzyme activity for biotransformation of PPT type ginsenoside into Re -> Rg1.

Fig. 11 is a graph showing the distribution of the lactic acid bacteria, Leuconostoc citreum , GL29 has the activity of biotransforming PPD type ginsenoside Rb1 -> F2 -> CK, gyp17 -> CK, Rd-> CK, F2 -> CK and PPT type ginsenoside Re -> Rg1 TLC analysis showed that the cells had a bioconversion activity.

12 shows that the strain 25 of the lactic acid bacterium Leuconostoc mesenteroides subsp. Mesenteroides has a biotransformation activity of PPD type ginsenoside Rb1 -> F2 -> CK and F2 -> CK, and PPT type And the activity of biosynthesizing Re -> Rg1 by ginsenoside TLC analysis.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Glycoside hydrolase From lactic acid bacteria And Use Thereof <130> PA120296KR <150> 10-2011-0041178 <151> 2011-04-29 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 415 <212> PRT <213> Weissella confusa <220> <221> PEPTIDE &Lt; 222 > (1) .. (415) <223> amino acid sequence of GL24GH1 <400> 1 Met Ala Leu Arg Gly Lys Glu Phe Leu Trp Gly Ala Ser Ile Ala Asn   1 5 10 15 Tyr Gln Thr Asp Gly Ala Glu Asn Thr Gln Trp Tyr Glu Trp Glu Leu              20 25 30 Ala Asn Ala Asp Arg Leu Ala Ser Glu Tyr Asp Thr Ala Phe Lys Asn          35 40 45 Val Pro Thr Val Asp Asp Phe Arg Val Ala Gly Asn Asp Pro Gln Asn      50 55 60 Tyr Ile Ser Gly Met Gly Ile Arg His Arg Glu Arg Tyr Asp Ala Asp  65 70 75 80 Phe Asp Lys Leu Gln Ala Leu His Phe Asn Ala Phe Arg Phe Ser Val                  85 90 95 Glu Trp Ala Arg Val Glu Pro Glu Pro Gly Val Tyr Asn Glu Gln Glu             100 105 110 Ile Ala Phe Leu Lys Asp Tyr Ile Ala Ala Ile Lys Ala His Gly Met         115 120 125 Thr Pro Val Leu Thr Leu Trp His Trp Thr Met Pro Val Trp Phe Thr     130 135 140 Glu Lys Gly Gly Phe Glu Lys Arg Glu Asn Glu Arg Tyr Phe Glu Glu 145 150 155 160 Phe Ala Ala Tyr Val Leu Gln Lys Leu Gln Asp Asp Ile Asp Ile Val                 165 170 175 Leu Thr Phe Asn Glu Trp Asn Val Tyr Thr Phe Ala Gly Tyr Ile Ala             180 185 190 Gly Glu Trp Ala Pro Met Gln Thr Ser Phe Leu Thr Ala Phe Lys Val         195 200 205 Ala Leu His Leu Thr Glu Ala His Asn Arg Val Tyr Asp Ile Ala Lys     210 215 220 Met Ile Lys Pro Ala Phe Lys Val Ser Val Ala His Asn Thr Ala Asp 225 230 235 240 Phe Ile Ala Leu Asn Arg Lys Val Thr Thr Lys Leu Gly Leu Ala Trp                 245 250 255 Asn Arg Trp Gln Arg Asp Asn Phe Leu Asp Arg Thr Tyr Gln Lys             260 265 270 Met Asp Phe Leu Gly Leu Asn Trp Tyr Asn Ala Asp Ser Tyr Asp Gly         275 280 285 Phe Thr Val Lys Asn Pro Asn Glu Lys Val Asn Asp Met Gly Trp Asp     290 295 300 Met Arg Pro Ile Arg Ile Glu Lys Thr Leu Val Arg Leu Tyr Asn Arg 305 310 315 320 Tyr Gln Leu Pro Ile Leu Ile Thr Glu Asn Gly Leu Ala Asp Gly Asp                 325 330 335 Asp Ser Asp Arg Glu Trp Trp Leu Ser Glu Thr Leu Gln Ala Leu Glu             340 345 350 Asn Ala Glu Ser Ala Gly Val Asp Leu Met Gly Tyr Leu His Trp Ser         355 360 365 Ala Phe Asp Asn Phe Glu Trp Asp Lys Gly Tyr Trp Pro Arg Phe Gly     370 375 380 Leu Ile Ala Val Asp Tyr Glu Asn Asp Tyr Ala Arg Asp Ile Arg Pro 385 390 395 400 Ser Ala Gln Tyr Tyr Ala Thr Arg Ile Leu Glu Tyr Arg Gly Lys                 405 410 415 <210> 2 <211> 324 <212> PRT <213> Weissella confusa <220> <221> PEPTIDE &Lt; 222 > (1) <223> amino acid sequence of GL24GH43 <400> 2 Met Thr Val Arg Tyr Glu Asn Pro Val Ile Ile Gln Arg Ala Asp Pro   1 5 10 15 Tyr Ile Tyr Lys His Thr Asp Gly Tyr Tyr Tyr Phe Val Ala Ser Val              20 25 30 Pro Ala Tyr Asn Leu Ile Glu Leu Arg Arg Ala Lys Thr Ile Asp Gly          35 40 45 Leu Ala His Ala Met Pro Arg Thr Ile Trp Arg Lys His Asp Ser Gly      50 55 60 Thr Gly Ala Gln Ser Glu Leu Ile Trp Ala Pro Glu Leu His Tyr Thr  65 70 75 80 Asp Gly Lys Trp Tyr Val Tyr Tyr Ala Ala Ser His Thr Thr Ala Phe                  85 90 95 Asp Glu Asn Gly Met Phe Gln His Arg Met Phe Ala Ile Glu Cys Asp             100 105 110 Ala Glu Asp Pro Met Glu Thr Glu Glu Asn Trp Val Glu Lys Gly Gln         115 120 125 Ile Glu Thr His Leu Asp Ser Phe Ala Leu Asp Ala Thr Ser Phe Glu     130 135 140 Leu Asn Asp Lys Leu Tyr Tyr Val Trp Ala Gln Lys Asp Pro Glu Ile 145 150 155 160 Lys Gly Asn Ser Asn Leu Tyr Ile Ala Glu Met Glu Asn Pro Trp Thr                 165 170 175 Leu Lys Thr Ala Pro Val Met Leu Ser Lys Pro Glu Phe Asp Trp Glu             180 185 190 Thr Lys Ile Phe Trp Val Asn Glu Gly Pro Ala Ile Leu Lys Arg Asn         195 200 205 Gly Lys Val Phe Leu Thr Phe Ser Gly Ser Ala Thr Asp Glu Asn Tyr     210 215 220 Ala Met Gly Met Leu Trp Ile Glu Asp Asp Lys Asp Val Leu Asp Ala 225 230 235 240 Ala Asn Trp His Lys Leu Asp His Pro Val Phe Gln Ser Asp Met Glu                 245 250 255 Asn Gly Leu Tyr Gly Pro Gly His Asn Ser Phe Thr Val Ala Glu Asp             260 265 270 Gly Glu Thr Asp Leu Leu Val Tyr His Val Arg Asn Tyr Leu Asp Ile         275 280 285 Lys Gly Asp Pro Leu Tyr Asp Pro Asn Arg His Thr Met Val Gln Pro     290 295 300 Phe Glu Trp Asp Asp Glu Gly Phe Pro Val Phe Gly Lys Pro Gln Pro 305 310 315 320 Phe Thr Phe Asn                 <210> 3 <211> 713 <212> PRT <213> Weissella confusa <220> <221> PEPTIDE &Lt; 222 > (1) <223> amino acid sequence of GL24GH3 <400> 3 Met Thr Gln Gln Leu Thr His Glu Glu Ala Arg Arg Gln Ala Lys Val   1 5 10 15 Ile Val Asp Gln Met Thr Ile Asp Glu Lys Ile Gly Gln Ile Lys Tyr              20 25 30 Glu Ala Pro Ala Ile Glu Arg Leu Asn Ile Pro Glu Tyr Asn Tyr Trp          35 40 45 Asn Glu Ala Leu His Gly Val Ala Arg Ala Gly Val Ala Thr Val Phe      50 55 60 Pro Gln Ala Ile Gly Leu Ala Ala Thr Phe Asp Asp Gln Leu Ile Asn  65 70 75 80 Asp Ile Ala Asp Val Ile Gly Thr Glu Gly Arg Ala Lys Tyr Asn Glu                  85 90 95 Phe Thr Lys His Glu Asp Arg Asp Ile Tyr Lys Gly Leu Thr Phe Trp             100 105 110 Ser Pro Asn Val Asn Ile Phe Arg Asp Pro Arg Trp Gly Arg Gly His         115 120 125 Glu Thr Tyr Gly Glu Asp Pro Phe Leu Thr Ser Lys Phe Gly Met Ala     130 135 140 Phe Ile Lys Gly Leu Gln Gly Gln Ala Lys Tyr Leu Lys Leu Ala Ala 145 150 155 160 Thr Ala Lys His Phe Ala Val His Ser Gly Pro Glu Gly Leu Arg His                 165 170 175 Gly Phe Asp Ala Val Val Ser Asp Lys Asp Leu Tyr Glu Thr Tyr Leu             180 185 190 Pro Ala Phe Lys Ala Ala Val Glu Glu Ala Asp Val Glu Ser Ile Met         195 200 205 Thr Ala Tyr Asn Ala Val Asp Gly Val Ala Ser Val Ser Glu Met     210 215 220 Leu Leu Arg Asp Ile Leu His Asp Lys Trp Ser Phe Glu Gly His Val 225 230 235 240 Val Ser Asp Tyr Met Ala Pro Glu Asp Val His Glu Asn His Lys Tyr                 245 250 255 Thr Gln Asp Ala Ala Glu Thr Met Gly Leu Ala Ile Lys Ala Gly Leu             260 265 270 Asn Leu Val Ala Gly His Ile Glu Gln Ser Leu His Glu Ala Leu Asp         275 280 285 Arg Gly Leu Val Thr Glu Glu Glu Ile Thr Asn Ala Val Ile Ser Leu     290 295 300 Tyr Ala Thr Arg Val Arg Leu Gly Met Phe Ala Thr Asp Asn Glu Tyr 305 310 315 320 Asp Ala Ile Pro Tyr Glu Ala Asn Asp Thr Lys Ala His Asn Asn Leu                 325 330 335 Ser Glu Ile Ala Ala Glu Lys Ser Phe Val Leu Leu Lys Asn Asp Gly             340 345 350 Val Leu Pro Leu Arg Lys Glu Thr Met Glu Ala Ile Ala Val Val Gly         355 360 365 Pro Asn Ala His Ser Glu Ile Ala Leu Leu Gly Asn Tyr Phe Gly Thr     370 375 380 Pro Ser Arg Ser Tyr Thr Ile Leu Glu Gly Ile Gln Glu Arg Leu Gly 385 390 395 400 Asp Asp Val Arg His Val Tyr Ser Ile Gly Ser Gly Val Phe Gln Asp                 405 410 415 His Ala Ala Glu Pro Leu Ala Lys Ala Asp Glu Arg Glu Ser Glu Ala             420 425 430 Ile Ile Ala Ala Glu His Ser Asp Val Val Val Ala Val Leu Gly Leu         435 440 445 Asp Ser Thr Ile Glu Gly Glu Glu Gly Asp Ala Gly Asn Ser Gln Gly     450 455 460 Ala Gly Asp Lys Pro Asn Leu Ser Leu Pro Gly His Gln Arg Gln Leu 465 470 475 480 Leu Glu Arg Leu Leu Ala Val Gly Lys Pro Val Val Val Leu Leu Ala                 485 490 495 Ser Gly Ser Ser Leu Gln Leu Asp Gly Leu Glu Asn His Pro Asn Leu             500 505 510 Arg Ala Ile Met Gln Ile Trp Tyr Pro Gly Ala Arg Gly Asn Leu Ala         515 520 525 Val Ala Asp Val Leu Phe Gly Thr Val Ser Ser Gly Lys Leu Pro     530 535 540 Val Thr Phe Tyr Lys Asn Thr Asp Asn Leu Pro Ala Phe Glu Asp Tyr 545 550 555 560 Asn Met Ala Gly Arg Thr Tyr Arg Tyr Met Thr Glu Glu Ala Leu Tyr                 565 570 575 Pro Phe Gly Tyr Gly Leu Thr Tyr Ser Ser Val Glu Leu Ser Asp Leu             580 585 590 Gln Val Lys Ser Tyr Glu Glu Ala Ala Thr Ala Thr Val Thr Ile Gln         595 600 605 Asn Thr Gly Asn Phe Asp Thr Asp Glu Val Val Arg Val Tyr Val Lys     610 615 620 Asp Leu Glu Ser Glu Phe Ala Val Pro Asn Ala Gln Leu Lys Gly Phe 625 630 635 640 Lys Arg Val Phe Leu Gly Lys Gly Ser Lys Gln Thr Ile Thr Phe Asp                 645 650 655 Leu Arg Pro Gln Asp Phe Glu Val Phe Asp Glu Gln Gly His Asn Phe             660 665 670 Ile Asp Ser Asn Arg Phe Glu Ile Ser Val Gly Val Ser Gln Pro Asp         675 680 685 Ala Arg Ser Ile Ala Leu Thr Gly Val Gln Pro Leu Gln Thr Glu Leu     690 695 700 Asn Leu Ala Gly Ser Gln Thr Thr His 705 710 <210> 4 <211> 481 <212> PRT <213> Pediococcus pentosaceus <220> <221> PEPTIDE &Lt; 222 > (1) .. (481) <223> amino acid sequence of PedGH1 <400> 4 Met Thr Glu Lys His Tyr Lys Met Pro Glu Asn Phe Leu Trp Gly Gly   1 5 10 15 Ala Val Ala Ala His Gln Phe Gly Gly Gly Trp Gln Glu Gly Gly Lys              20 25 30 Gly Val Ser Ile Asp Val Met Thr Ala Gly Asn Lys Asp Thr Ala          35 40 45 Arg Lys Val Thr Asp Gly Ile Lys Ala Gly Glu Val Tyr Pro Asn His      50 55 60 Trp Gly Ile Asp Phe Tyr His Arg Tyr Pro Glu Asp Ile Lys Leu Phe  65 70 75 80 Asn Glu Met Gly Phe Lys Cys Phe Arg Thr Ser Ile Ala Trp Thr Arg                  85 90 95 Ile Phe Pro Asn Gly Asp Glu Thr Glu Pro Asn Glu Ala Gly Leu Lys             100 105 110 Phe Tyr Asp Asp Met Phe Asp Glu Leu Leu Lys Tyr Asn Ile Gln Pro         115 120 125 Ile Ile Thr Leu Ser His Phe Glu Met Pro Tyr His Leu Val Lys Glu     130 135 140 Tyr Gly Gly Trp Thr Asn Arg Lys Leu Ile Asp Phe Phe Val Arg Phe 145 150 155 160 Ala Glu Thr Val Met Lys Arg Tyr Lys Gly Lys Val Lys Tyr Cys Met                 165 170 175 Thr Phe Asn Glu Ile Asn Asn Gln Thr Asp Trp Ser Asn Pro His His             180 185 190 Leu Leu Gln Asp Ser Ala Ile Lys Val Lys Ala Gly Thr Pro Gly Met         195 200 205 Glu Asp Met Met Tyr Gln Ala Ala His Asn *** Met Val Ala Ser Ala     210 215 220 Lys Val Val Glu Leu Gly His Gln Ile Asp Ser Glu Tyr Gln Ile Gly 225 230 235 240 Ala Met Ile Ala Met Cys Pro Ile Tyr Pro Leu Thr Ala Lys Pro Glu                 245 250 255 Asp Ile Met Met Ala Glu Arg Ala Met Gln Thr Arg Tyr Tyr Phe Gly             260 265 270 Asp Val Gln Ala Asn Gly Glu Tyr Pro Asn Trp Leu Pro Lys Tyr Trp         275 280 285 Glu Arg Gln Asn Ile His Val Glu Ile Ser Asp Glu Asp Arg Glu Ile     290 295 300 Leu Lys Lys Gly Ala Val Asp Tyr Ile Gly Phe Ser Tyr Tyr Met Ser 305 310 315 320 Phe Thr Thr Lys Ser Thr Asp Glu Asn Thr Asp Tyr Ala Tyr Lys Glu                 325 330 335 Ala Arg Asp Leu Val Asp Asn Pro Tyr Val Gln Lys Ser Asp Trp Gly             340 345 350 Trp Gln Ile Asp Pro Val Gly Leu Arg Tyr Ser Met Asn Trp Met Gln         355 360 365 Asp Arg Trp His Lys Pro Gln Phe Ile Val Glu Asn Gly Phe Gly Ala     370 375 380 Tyr Asp Gln Lys Glu Ala Asp Gly Ala Val His Asp Asp Tyr Arg Ile 385 390 395 400 Lys Tyr Phe His Asp His Ile Leu Glu Met Glu Lys Ala Val Ala Leu                 405 410 415 Asp Gly Val Asn Leu Ile Gly Tyr Thr Pro Trp Gly Cys Ile Asp Leu             420 425 430 Ile Ser Ala Ser Thr Gly Glu Met Ala Lys Arg Tyr Gly Phe Ile Tyr         435 440 445 Val Asp Gln Asp Asp Gln Gly Asn Gly Ser Leu Glu Arg Ser Ser Lys Lys     450 455 460 Asp Ser Phe Asp Trp Tyr Lys Gln Val Ile Ala Ser Asn Gly Glu Asp 465 470 475 480 Leu     <210> 5 <211> 1248 <212> DNA <213> Weissella confusa <220> <221> gene &Lt; 222 > (1) .. (1248) <223> DNA sequence of GL24GH1 <400> 5 atggcattac ggggcaaaga atttttatgg ggtgcatcaa ttgcaaatta tcaaacggat 60 ggcgcagaaa atacgcagtg gtatgagtgg gaacttgcga atgctgaccg cttagcaagt 120 gaatatgata cggcgttcaa gaatgtgcca acagtcgatg atttccgtgt agctgggaac 180 gatccacaaa attacattag cggcatgggt attcgccacc gtgaacgcta cgatgccgat 240 tttgataaac tgcaagcgtt acactttaac gcttttcgat tcagtgtgga atgggcccgg 300 gttgaaccag aaccaggtgt ttataatgaa caagaaattg cgtttctgaa agattatatt 360 gccgcgatta aagcgcatgg tatgacgcca gtcttaaccc tatggcattg gacaatgcca 420 gtatggttta ccgaaaaggg tggctttgaa aaacgtgaaa atgagcggta ctttgaagag 480 ttcgcggcat acgtcttgca gaaattgcaa gatgacattg atattgtctt aacttttaac 540 gagtggaatg tctatacatt tgctggctat atagcaggtg aatgggcccc gatgcagacg 600 tcatttttga cagcgtttaa ggtggctttg cacttaacgg aagcacacaa ccgcgtctac 660 gatattgcca aaatgattaa gccagcattc aaagtgtcgg tggctcacaa tacggctgat 720 tttatcgcgt taaaccggaa ggtaacaacg aaactcggtt tggcttggaa ccgttggcaa 780 cgtgacaact ttttcttaga tcggacatat caaaaaatgg atttcttagg gctgaattgg 840 tataacgctg atagttatga tggctttaca gttaaaaatc ctaacgaaaa ggtcaacgac 900 atgggatggg atatgcgacc aattcgcatt gaaaagactt tggttcgctt gtacaatcgt 960 tatcagttgc caattttaat tacagaaaat ggtttagctg atggtgacga ttcagatcgt 1020 gaatggtggc tttcggagac cctgcaagcg ctagaaaatg ctgaaagtgc cggtgttgat 1080 ttgatggggt atctgcattg gtcggcgttt gataactttg aatgggataa ggggtattgg 1140 ccgcgatttg gcctaattgc agttgattac gaaaacgatt atgcgcgtga cattcgacca 1200 tcagcacagt attatgcaac gcgaatttta gagtatcgag ggaagtag 1248 <210> 6 <211> 975 <212> DNA <213> Weissella confusa <220> <221> gene <222> (1). (975) <223> DNA sequence of GL24GH43 <400> 6 atgacagttc gctacgaaaa cccagtgatc atccaacgtg ctgatccata tatctacaag 60 cacacggatg gttattacta ctttgttgct tcggtaccgg cttacaactt aattgagttg 120 cgtcgtgcga agacgattga tggtttggct cacgcaatgc cacgcacgat ttggcgtaag 180 cacgattcag gtaccggcgc acaaagcgag ttgatttggg caccagagtt acactacacg 240 gatggtaagt ggtatgtgta ctatgctgct tcacatacaa cagcatttga tgaaaacggc 300 atgttccaac accgtatgtt tgccatcgag tgcgacgcag aagacccaat ggaaacagaa 360 gaaaactggg ttgaaaaggg acaaattgaa acgcaccttg attcatttgc cctggatgca 420 acgtcatttg aattaaacga caagctgtac tacgtttggg cgcaaaagga cccggagatt 480 aagggtaact caaacttgta catcgctgaa atggaaaacc cgtggacgtt gaagacggcg 540 ccagttatgt tgtcaaagcc agaatttgac tgggaaacta agattttctg ggtgaacgaa 600 ggaccggcta tcttgaagcg taacggcaag gtgttcttga cgttctctgg ctcagccacc 660 gatgaaaact acgcgatggg gatgttgtgg atcgaggatg acaaggatgt cttggacgct 720 gcaaactggc ataagttgga ccaccctgtt ttccaatcag atatggaaaa tggtttgtat 780 ggtccgggtc acaactcatt tacggttgct gaagatggtg aaactgattt gctggtttac 840 cacgtgcgta attacttgga tatcaagggt gatcctttgt atgatccaaa ccgccacacg 900 atggtgcagc catttgagtg ggacgatgag ggcttcccgg tgtttggtaa gccacaacca 960 tttactttta actaa 975 <210> 7 <211> 2142 <212> DNA <213> Weissella confusa <220> <221> gene &Lt; 222 > (1) .. (2142) <223> DNA sequence of GL24GH3 <400> 7 atgacccagc aactcactca cgaagaagct cgccgccaag ctaaggtgat tgtcgaccaa 60 atgacgattg atgagaaaat tggccaaatt aagtatgaag caccagcaat tgaacgattg 120 aacattccag aatacaacta ctggaatgaa gcattgcacg gtgtggcccg cgccggcgtt 180 gcgacggttt ttccacaagc aattggactt gcggcaactt ttgatgacca gttaattaat 240 gatattgccg atgtgattgg gacggaagga cgtgcaaagt ataacgaatt tacgaagcac 300 gaggatcgtg acatctacaa gggactgact ttttggtcgc ctaacgtcaa tattttccgt 360 gatccacgtt ggggacgtgg acatgaaaca tatggtgaag atccgttctt aacatcgaag 420 tttggtatgg cttttatcaa agggctgcaa gggcaagcta agtacttgaa gctggctgcg 480 acggcaaagc attttgcggt ccactcaggt ccggaaggtt tgcgtcacgg ttttgatgca 540 gttgtgtcag acaaggactt gtatgagacg tacttgccag ctttcaaggc agcggttgaa 600 gaggccgatg tcgaaagcat tatgacggca tataacgcag ttgatggcgt gccggcttcg 660 gttagtgaaa tgttgttgcg ggacattttg catgataagt ggtcatttga aggtcacgtt 720 gtctcagatt acatggcccc agaagatgtg catgagaacc acaagtacac acaggatgcc 780 gctgaaacga tgggattagc catcaaagca ggtttgaact tggttgccgg ccatattgaa 840 caatctttgc atgaggcgtt ggatcgtggt ttggtgactg aagaagaaat taccaatgcg 900 gttatttcac tttatgcaac ccgtgtccgt ctaggcatgt ttgcaactga taatgagtac 960 gatgccattc cgtatgaggc gaacgacacg aaggcgcaca ataatctgtc agaaattgca 1020 gctgagaagt catttgtgtt attgaagaat gatggtgttt tgccactacg caaggaaaca 1080 atggaagcaa ttgcagttgt tgggccaaat gcgcattcag aaatcgcatt gctaggtaac 1140 tactttggaa caccatcacg ttcatatacc atcttggaag gtattcagga acgacttggt 1200 gatgatgttc gggtccacta cagtattgga tcaggggtgt tccaagacca cgcagctgag 1260 ccgttggcta aggcagatga acgtgaatca gaagcgatta ttgctgctga acactcagac 1320 gtcgttgttg cagttttggg gcttgattca acgattgaag gcgaagaagg ggatgctggt 1380 aactcacaag gggctgggga taagccgaac ttgtcattgc cagggcatca acggcaactg 1440 ttggaacgtt tgttagctgt tggtaagcct gtagtagttc tgttagcttc gggttcatca 1500 ttgcaattgg atggcttgga gaaccacccc aacttacgag caattatgca aatttggtac 1560 ccaggtgctc gaggtaactt ggcggttgca gatgttttgt ttggtacagt aagtccatca 1620 ggaaagttgc cggtaacgtt ctacaagaac acagataatt tgccagcctt tgaagattac 1680 aatatggcag gacgcacata ccgttatatg acagaggagg ccttgtatcc atttggttac 1740 ggtttgacgt actcgtcagt tgaattgtca gatttgcagg tgaagtctta tgaagaggcg 1800 gcaacggcaa cggtgacgat tcaaaatacg ggtaattttg atacggatga agttgttcga 1860 gtctatgtga aggacttaga atcagaattt gctgtgccaa atgcccagtt gaagggattc 1920 aaacgcgtct ttttgggtaa gggatctaag caaacgatta cgtttgattt acgtccacag 1980 gacttcgaag tgtttgatga gcaagggcat aactttattg atagtaatcg ttttgagatt 2040 tcagttggtg ttagccaacc agatgcgcga tcgattgcgc tgacaggtgt gcaacctttg 2100 caaactgagt tgaatctggc aggtagtcag acaacacatt ga 2142 <210> 8 <211> 1446 <212> DNA <213> Pediococcus pentosaceus <220> <221> gene &Lt; 222 > (1) .. (1446) <223> DNA sequence of PedGH1 <400> 8 atgactgaaa agcattataa gatgccggaa aatttcttgt ggggcggtgc agtagccgct 60 catcaatttg aaggcggatg gcaagaaggc ggtaagggag ttagcattgc agacgtgatg 120 accgctggaa acaaagatac tgctcgaaaa gtaacggacg gtattaaagc tggagaagtt 180 tacccaaatc actgggggat tgatttttat catcgttatc ctgaagatat taaacttttt 240 aacgaaatgg gtttcaaatg cttccgtacc tcgattgcat ggactagaat ttttcctaat 300 ggagatgaaa ccgaacctaa cgaagctggt ttaaagttct atgatgatat gtttgatgaa 360 ttgttaaaat acaatattca acccatcatt actttgtcac acttcgaaat gccatatcat 420 ttagtaaaag aatatggtgg ttggactaat cgtaaattga ttgacttttt cgtacgtttt 480 gccgaaacag tgatgaaacg ctataaggga aaagttaagt actgcatgac cttcaacgag 540 attaataatc aaactgattg gagtaatcct catcatttac ttcaagattc agccattaag 600 gttaaagctg gaacaccagg aatggaagac atgatgtatc aagccgctca taattaaatg 660 gttgcaagtg ctaaagtagt tgaactaggc catcaaattg attcggaata tcaaattggt 720 gcaatgattg caatgtgtcc aatttatcca ctaactgcta agccagaaga catcatgatg 780 gctgaacgtg ctatgcaaac gcgttactac tttggagacg ttcaagctaa tggtgaatac 840 cctaattggc ttcctaaata ttgggaacgc caaaacatcc atgttgaaat tagtgatgaa 900 gccgtgaaa ttttgaaaaa gggtgcagtt gattatattg gatttagtta ctatatgtca 960 tttacaacta aatctacgga tgaaaatacc gactatgcct ataaagaagc acgtgattta 1020 gtcgacaatc cttatgttca aaaatcagat tggggctggc agattgatcc tgttggtcta 1080 cgttattcga tgaattggat gcaagatcgt tggcacaaac ctcaatttat tgtggaaaat 1140 ggttttggag catacgacca aaaagaagcc gatggcgctg tccatgatga ttaccgcatc 1200 aagtatttcc acgatcatat tttagagatg gaaaaagcag ttgccttaga tggggtcaac 1260 ttaattggtt atactccatg gggatgcatc gatttaattt cagcaagtac tggagaaatg 1320 gctaaacgtt atggatttat ctatgttgat caagatgacc aaggtaatgg tagtctagaa 1380 cgttcgaaga aggattcatt tgattggtac aaacaagtaa ttgcatctaa tggtgaagat 1440 ttatag 1446 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S-27F <400> 9 agagtttgat cctggctcag 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 16S-1492R <400> 10 ggttaccttg ttacgactt 19 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GL24GH1 <400> 11 gaagaattca tgtcattacg gggcaaagaa 30 <210> 12 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GL24GH1 <400> 12 ccaagcttct acttccctcg atactctaa 29 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GL24GH43 <400> 13 ccgaattcat gacagttcgc tacgaaaac 29 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GL24GH43 <400> 14 cccaaagctt ttagttaaaa gtaaatggtt gtg 33 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GL24GH3 <400> 15 ccgaattcat gacccagcaa ctcactcac 29 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GL24GH3 <400> 16 gccaagcttt caatgtgttg tctgactacc 30 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PedGH1 <400> 17 ccgaattcat gactgaaaag cattataag 29 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PedGH1 <400> 18 gccaagcttc tataaatctt caccattag 29

Claims (18)

A glycoside hydrolase derived from Weissella confusa , defined by the amino acid sequence of SEQ ID NO: 1, 2 or 3.
[Claim 2] The glycoside hydrolase according to claim 1, wherein the glycoside hydrolase derived from Baicela Conpusa, which is defined by the amino acid sequence of SEQ ID NO: 1, 2 or 3, is located at the 3rd or 20th carbon of the protopanaxadiol type ginsenoside glycopyranosyl residue or? -1,6 glucopyranosyl residue, and having a selective hydrolysis ability to? -1,2 glucopyranosyl residue or? -1,6 glucopyranosyl residue.
The glycoside hydrolase according to claim 1, wherein the glycoside hydrolase derived from Baicela Conpusa, which is defined by the amino acid sequence of SEQ ID NO: 1 or 2, is an α-ramanopyranosyl residue located at the 6th carbon of PPT (protopanaxtriol) &Lt; / RTI &gt; wherein said hydrolyzate has a selective hydrolyzability to said glycoside hydrolase.
A composition for the conversion of a protopanaxadiol (PPD) or protopanaxatriol (PPT) type ginsenoside to a deglycosylated rare ginsenoside comprising the glycoside hydrolase of claim 1.
A nucleic acid encoding the glycoside hydrolase according to claim 1.
A recombinant vector comprising the nucleic acid of claim 5. A transformant transformed with the nucleic acid of claim 5 or a recombinant vector comprising said nucleic acid. 8. The transformant according to claim 7, wherein the transformant is E. coli or lactic acid bacteria. (a) culturing the transformant of claim 7;
(b) producing glycoside hydrolase from the cultured transformant; And
and (c) recovering the produced glycoside hydrolase. A pharmaceutical composition comprising the glycoside hydrolase of any one of claims 1 to 3, the glycoside hydrolase of claim 9, the transformant of claim 7, the culture of the transformant, A method of producing a deglycosylated second ginsenoside from a first ginsenoside, comprising using a strain of Weissella confusa , a culture of the B. subtilis strain, or a mixture thereof.
11. The method of claim 10, wherein the first ginsenoside as a substrate of glycoside hydrolase is selected from the group consisting of Re, Rg2, Rb1, Rc, Rb2, Rd, and Gyp17.
11. The method of claim 10, wherein the method of making a deglycosylated second ginsenoside from the first ginsenoside comprises converting ginsenoside Rb1 to F2, converting ginsenoside Gyp17 to F2, converting ginsenoside Rd F2 and conversion of ginsenoside Re to Rg1. &Lt; Desc / Clms Page number 13 &gt;
A pharmaceutical composition comprising the glycoside hydrolase of any one of claims 1 to 3, the glycoside hydrolase of claim 9, the transformant of claim 7, the culture of the transformant, (Protopanaxadiol) or PPT (protopanaxatriol) type ginsenoside, which contains Aspergillus oryzae, Weissella confusa , a culture of the above Bacela communa strain, or a mixture thereof as an active ingredient. Switching kit.
14. The pharmaceutical composition according to claim 13, which comprises, in the same container or in a different container, a pharmaceutical composition comprising an α- N -arabinofuranosidase, a β-galactosidase, a glycosidase, α-L-arabinopyranosidase, Wherein the kit further comprises an enzyme selected from the group consisting of L-arabinofuranosidase,? -Xylosidase, and? -L-rhamnosidase, and mixtures thereof. The glycoside hydrolase of any one of claims 1 to 3, the glycoside hydrolase of claim 9, the transformant of claim 7, the culture of the transformant or a mixture thereof &Lt; / RTI &gt;
16. The food according to claim 15, further comprising a PPD (protopanaxadiol) type or PPT (protopanaxatriol) type ginsenoside, or hemp.
The glycoside hydrolase produced according to claim 9, the transformant according to claim 7, the culture of the transformant, the Weissella confusa , the culture of the above-mentioned Bicelapneumoniae strain, Compositions for the conversion of protopanaxadiol (PPD) or protopanaxatriol (PPT) type ginsenosides containing the mixture as an active ingredient to the deglycosylated rare ginsenosides. The glycoside hydrolase of any one of claims 1 to 3, the glycoside hydrolase of claim 9, the transformant of claim 7, the culture of the transformant, Food additive comprising a mixture.

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