WO2012148249A2 - Glycoside hydrolase issue de bactéries lactiques et ses utilisations - Google Patents

Glycoside hydrolase issue de bactéries lactiques et ses utilisations Download PDF

Info

Publication number
WO2012148249A2
WO2012148249A2 PCT/KR2012/003387 KR2012003387W WO2012148249A2 WO 2012148249 A2 WO2012148249 A2 WO 2012148249A2 KR 2012003387 W KR2012003387 W KR 2012003387W WO 2012148249 A2 WO2012148249 A2 WO 2012148249A2
Authority
WO
WIPO (PCT)
Prior art keywords
ginsenoside
glycoside hydrolase
lane
compound
transformant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2012/003387
Other languages
English (en)
Korean (ko)
Other versions
WO2012148249A3 (fr
Inventor
김성건
이은지
이서연
안동선
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Korea Research Institute of Bioscience and Biotechnology KRIBB
Original Assignee
Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Research Institute of Bioscience and Biotechnology KRIBB filed Critical Korea Research Institute of Bioscience and Biotechnology KRIBB
Publication of WO2012148249A2 publication Critical patent/WO2012148249A2/fr
Publication of WO2012148249A3 publication Critical patent/WO2012148249A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

Definitions

  • the present invention relates to glycoside hydrolase derived from lactic acid bacteria, specifically, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Glycoside hydrolase from Leuconostoc citreum and its use.
  • Ginseng has been used as a medicine for promoting health and longevity in Asia, including Korea, China, and Japan. It is also used for therapeutic purposes at the plant level in the United States, and for herbal treatment in Europe, especially Germany.
  • Saponin means a substance consisting of several cyclic compounds in the glycosides widely present in the plant system, and triterpene saponin, a saponin component included as a main bioactive component in ginseng or red ginseng, is used in other plants. Since ginseng saponins are different from other saponins, they are called ginsenosides in the sense of Ginseng glycosides to distinguish them from other plant-based saponins.
  • ginsenosides have been purified, characterized and classified.
  • the basic structure of ginsenosides comprises one to three residues, ie, glycofyranosyl, arabinofyranosyl, arabinofuranosyl, xylopyranosyl, or rhamnopyranosyl groups, attached at specific positions to the main backbone. It consists of a tetracyclic triterpene dammarane or pentacyclic oleanane skeleton.
  • Damaren type ginsenosides are mainly classified according to their intrinsic aglyconic residues: protopanaxanadiol (PPD), protopanasatriol (PPT), and ocotillol.
  • Ginsenoside composition is diversified into various ginseng species.
  • Korean ginseng Panax ginseng
  • US ginseng Panax notoginseng
  • Ginsenosides In certain species of Panax , the composition of ginsenosides also varies in other parts of the ginseng body such as roots, leaves, and fruits.
  • ginsenosides In general, the effectiveness of ginsenosides increases with the degree of deglycosylation, which enhances their hydrophobicity and cell wall permeability.
  • deglycosylated ginsenosides are traces in ginseng extracts, whereas Rb1 ⁇ 3- O- [ ⁇ -D-glycopyranosyl- (1-2) - ⁇ -D-glycopyranosyl] -20- O -[ ⁇ -D-glycopyranosyl- (1-6) - ⁇ -D-glycopyranosyl] -20 ( S ) -protopanaxadianiole ⁇ , Rb2 ⁇ 3- O- [ ⁇ -D-glycopyra Nosyl- (1-2) - ⁇ -D-glycopyranosyl] -20- O- [ ⁇ -L-arabinopyranosyl- (1-6) - ⁇ -D-glycopyranosyl] -20 ( S )
  • a method for producing deglycosylated ginsenosides is required.
  • Physicochemical processes such as steaming (heating) and acid treatment have been developed to convert the major constituents of ginsenosides into deglycosylated ginsenosides.
  • the most preferred method is a bioconversion method in which the enzymes with known activities are sequentially reacted with a desired plan or a combination of two or more enzymes with known activities, according to a preliminary plan. This avoids such problems and produces known, defined products.
  • Rb1 or Rc is represented by Compound (C Thermostable ⁇ -glucosidase converting to) -K [20- O- ( ⁇ -D-glycopyranosyl) -20 ( S ) -protopanaxadiol based]; (2) ⁇ -glucosidase from soil metagenome that converts Rbl to Rd; And (3) Rb1 to CK, Rb2 to CK, and Rc to C-Mc ⁇ 20- O- [ ⁇ -L-arabinofuranosyl- (1-6) - ⁇ -D-glycopyranosyl] ⁇ -galactosidase from Sulfolobus acidocaldarius converting to -20 ( S ) -protopanaxadiol .
  • Rb1 is Gypenoside 17 ⁇ 3- O- ⁇ -D-glycopyranosyl-20- O- [ ⁇ -D-glycopyranosyl- (1-6) - ⁇ -D-glycopyranosyl] -20 ( S )- Protopanaxidaiol-based ⁇ , followed by Gypenoside LXXV ⁇ 20- O- [ ⁇ -D-glycopyranosyl- (1-6) - ⁇ -D-glycopyranosyl] -20 ( S ) -protopanaxadie Enzymes of GH family 3 that catalyze the continuous conversion to all systems and finally to CK have recently been reported by the inventors.
  • probiotics were defined by R. Fuller in 1989 as "probiotics that have beneficial effects on host animals by improving the intestinal microflora of host animals.”
  • Strains used as probiotics include bacteria such as Lactobacillus, Enterococcus, Bifidobacterium and Bacillus, yeasts including Saccharomyces, and fungi including Aspergillus.
  • Probiotics are classified as GRAS (Generally Recognized As Safe). They do not contain toxic genes for humans and animals and do not produce pathogenic substances. And it should be a microorganism confirmed the improvement effect on the livestock productivity.
  • the probiotics should be non-pathogenic, easy to in vitro proliferation, fast growth in vivo, acid and bile resistant.
  • the activity by the feed ingredients should not be inhibited, the effect should be maintained at room temperature, and convenient to mix with the feed.
  • probiotics in aquaculture are either added to feed or incidentally administered to water. Recently, active research has been conducted on disease improvement due to increased immunity to pathogenicity and improved water quality for feeding probiotics to fish and shrimp (Jiqiu Li et al. Aquaculture 291 (2009) 35-40).
  • the present inventors have identified ginsenosidase which bioconverts ginsenosides in lactic acid bacteria Luconosestock mesenteroids, Luconosestock citrium, bisella confusa, pediococcus pentosassius, isolated from kimchi. ) Enzyme activity, preferably glycoside hydrolase activity was confirmed. That is, Luconosstock mesenteroids, Luconosstock citrium, Bisella confusa, Pediococcus pentosassius bioconverts PPTs such as Rb1, Rc, Rd, Gyp 17 or F2, or PPT such as Re. And the present invention was completed.
  • 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 comprising the nucleic acid, the nucleic acid or a transformant transformed with the recombinant vector.
  • Still another object of the present invention is to provide a method for preparing glycoside hydrolase by culturing the transformant according to the present invention.
  • Still another object of the present invention is 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, bisella confusa ( Weissella confusa ), Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Leuconostoc citreum , cultures of the strains, or mixtures thereof It provides a method for producing a deglycosylated second ginsenoside from the first ginsenoside including the step of using.
  • Still another object of the present invention is 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, bisella confusa ( Weissella confusa ), Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Leuconostoc citreum , cultures of the strains, or mixtures thereof It is to provide a kit for the conversion from the protopanaxadiol (PPD) or PPT (Protopanaxatriol) type ginsenoside containing as an active ingredient to a deglycosylated rare ginsenoside.
  • PPD protopanaxadiol
  • PPT Protopanaxatriol
  • Still another object of the present invention is 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, bisella confusa ( Weissella confusa ), Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Leuconostoc citreum , cultures of the strains, or mixtures thereof It is to provide a food or food additive comprising a.
  • Lactobacillus according to the present invention specifically, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Luconostock citrium ( Glycoside hydrolase from Leuconostoc citreum ) exhibits excellent activity of converting ginsenosides into deglycosylated rare ginsenosides.
  • Figure 1 shows the chemical structure of ginseng saponin of the PPD and PPT type.
  • Figure 2 shows the SDS-PAGE analysis of the purification of the proteins GL24GH1 and GL24GH43 from the Weissella confusa strain GL24.
  • M protein marker
  • lane 1 flow through
  • lanes 2,3 and 4 maltose-eluted fractions of GL24GH1
  • lane 5 flow through
  • Lanes 6,7 and 8 maltose-eluted fractions of GL24GH43
  • lane 3 is a TLC result of analyzing whether ginsenoside conversion of glycoside hydrolase GL24GH1 derived from strain GL24 belonging to the lactic acid bacterium Bisella confusa ( Weissella confusa ).
  • Figure 4 is a TLC result of analyzing the ginsenoside conversion of the glycoside hydrolase GL24GH43 derived from strain GL24 belonging to the lactic acid bacterium Bisella confusa ( Weissella confusa ).
  • lane 5 is a TLC result of analyzing the PPD type ginsenoside conversion of glycoside hydrolase GL24GH3 derived from strain GL24 belonging to the lactic acid bacterium Bisella confusa ( Weissella confusa ).
  • Figure 7 is lactic acid bacteria Bisella Confusa ( Weissella confusa ) The result of TLC analysis of the bioconversion of ginsenoside of strain GL24 belonging to. (Lane 1: 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 bisella confusa ( Weissella confusa ) Reaction products between coenzymes of strain GL24 belonging to; Lane 9: reaction product between Gyp17 and the coenzyme; Lane 10: reaction product between Rd and the coenzyme)
  • Figure 11 is lactic acid bacteria Lukonostok ( Leuconostoc ) Belonging to the genus Luconostock Citrium ( Leuconostoc citreum ) TLC analysis of ginsenoside conversion of (lane 1: 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 protopanaxadiol aglycon (PPDA); lane 14: standard Re; lane 15: standard Rg1; lane 16: standard Rg2; lane 17: standard F1; lane 18: standard Rh1 (S); Lane 19: standard protopanaxatriol aglycon (PPTA); lane 8: Rb1 and luconostock citrium ( Leuconostoc citreum ) Reaction products between coenzymes of strain GL29 belonging to; Lane 9: reaction product between Gyp17 and the enzyme; Lane 10: reaction product between Rd and the 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)
  • Luconosestock mesenteroides Leuconostoc mesenteroides subsp. mesenteroides
  • TLC analysis of the ginsenoside conversion of the strain belonging to. (Lane 1: 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: standard protopanaxatriol aglycon (PPTA); lane 8: Rb1 and Lukonostoke Mesenteroides ( Leuconostoc mesenteroides subsp.
  • Figure 13 is a schematic diagram showing the reaction path of the PPD type ginsenosides produced in the present invention.
  • the present invention provides a glycoside hydrolase derived from lactic acid bacteria, specifically, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. mesenteroides ), or glycoside hydrolase from Leuconostoc citreum .
  • glycoside hydrolase means a glycoside hydrolase derived from lactic acid bacteria, preferably Weissella confusa , Pediococcus pentosaceus , and lucono .
  • Stock mesen steroid's (Leuconostoc mesenteroides subsp. mesenteroides), or base stock sheet Pocono Solarium (Leuconostoc citreum) can glycosidic hydroxyl of the roller the best results.
  • Glycoside hydrolase isolated from the Weissella confusa GL24 strain is named GL24GH1, GL24GH43 or GL24GH3, and glycoside hydrolase isolated from Pediococcus pentosaceus is PedGH1. It is named.
  • the amino acid sequence of GL24GH1 is shown by SEQ ID NO: 1
  • the amino acid sequence of GL24GH43 is shown by SEQ ID NO: 2
  • the amino acid sequence of GL24GH3 is shown by SEQ ID NO: 3
  • the amino acid sequence of PedGH1 is shown by SEQ ID NO: 4.
  • Glycoside hydrolase amino acid sequence isolated from the Weissella confusa GL24 strain is a sequence consisting of SEQ ID NO: 1, 2 or 3, glycoside hydride isolated from Pediococcus pentosaceus
  • the rollase amino acid sequence is a sequence consisting of SEQ ID NO: 4, preferably not only the sequence but also an amino acid sequence having at least 70% similarity with the sequence, preferably at least 80% similarity, more preferably at least 90% similarity, Even more preferably, an amino acid sequence exhibiting at least 95% similarity, most preferably at least 98% similarity, comprises a sequence having substantially the activity of glycoside hydrolase.
  • sequence having such similarity is an amino acid sequence having a biological activity substantially the same as or corresponding to glycoside hydrolase
  • a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention. Inclusion is self-evident.
  • lactic acid bacteria refers to Gram-positive, microaerobic or anaerobic bacteria which ferment sugars to produce acids, including lactic acid as the predominantly produced acid.
  • the present inventors have a ginsenosidase enzyme activity that bioconverts ginsenosides in lactic acid bacteria such as Lukonostok mesenteroids, Lukonostok citrium, Bicella confusa or Pediococcus pentosassius, Preferably glycosidic hydrolase activity was confirmed (see Example 4).
  • the ginsenosidase enzymatic activity is ⁇ -1,6 glucopyranosyl residues of ginsenosides, ⁇ -1,2 glucopyranosyl residues or glucose, ⁇ -arabinofuranosides, arabinopyranosides, or ⁇ It may be a glycoside hydrolase having a selective hydrolytic ability to oligosaccharide residues, including the rhamnopyranosyl residues.
  • PPD protopanaxadiol
  • the lactic acid bacteria-derived glycosidic hydroxyl hydrolase specifically a base Pocono stock mesen steroid's (Leuconostoc mesenteroides subsp. Mesenteroides), Lu Pocono stock sheet Solarium (Leuconostoc citreum), Bar Sela konpu four (Weissella confusa) Or a nucleic acid encoding a glycoside hydrolase derived from Pediococcus pentosaceus , a recombinant vector comprising the nucleic acid, a transformant transformed with the nucleic acid or the recombinant vector.
  • nucleic acid sequences of Bisella Confusa GL24GH1, GL24GH43 and GL24GH3 are shown in SEQ ID NOs: 5, 6 and 7, respectively, and the nucleic acid sequences of PedGH1 of Pediococcus pantosassius are shown in SEQ ID NO: 8.
  • the nucleic acid of the present invention is a nucleic acid encoding a glycoside hydrolase derived from lactic acid bacteria, preferably Weissella confusa , Pediococcus pentosaceus , Leuconostoc ( Leuconostoc) mesenteroides subsp.mesenteroides ), or a nucleic acid encoding a glycoside hydrolase from Leuconostoc citreum , more preferably Weissella confusa , Pediococcus pantosassius , Pediococcus pentosaceus ) is a nucleic acid encoding a glycoside hydrolase.
  • the nucleic acid sequence encoding the glycoside hydrolase isolated from the Weissella confusa is a nucleic acid sequence consisting of SEQ ID NOs: 5, 6 or 7, glyco 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 at least 70% similarity with the sequence as well as the sequence, preferably at least 80% similarity, more preferably 90%
  • a sequence exhibiting the above similarity, even more preferably at least 95% similarity, most preferably at least 98% similarity comprises a sequence having substantially the activity of ginsenoside glycosidase.
  • the term 'similarity' refers to a degree similar to the amino acid sequence of a wild type protein, and has a sequence encoding the glycoside hydrolase of the present invention and the same sequence as the percentage or more. Sequence. This similarity can be compared with the naked eye or using 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.
  • the term 'vector' refers to a nucleic acid construct that is an expression vector capable of expressing a protein of interest in a suitable host cell and that includes essential regulatory elements operably linked to express the nucleic acid insert.
  • the present invention can produce a recombinant vector comprising a nucleic acid encoding a glycoside hydrolase.
  • a nucleic acid encoding a glycoside hydrolase or a recombinant vector comprising the nucleic acid is transformed or transfected into a host cell to thereby activate the glycoside hydrolase of the present invention.
  • 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.
  • plasmid DNA, phage DNA and the like can 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 -derived plasmids pUB110 and pTP5
  • yeast-derived plasmids YEp13, YEp24 and YCp50.
  • phage DNA include ⁇ -phage (Charon4A, Charon21A, EMBL3, EMBL4, ⁇ gt10, ⁇ gt11 and ⁇ ZAP).
  • animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses can also be used.
  • a fusion plasmid (eg, pJG4-5) to which a nucleic acid expression activating protein (eg, B42) is linked may be used, and the fusion plasmid may be GST, GFP, His- tag, Myc-tag and the like, but the fusion plasmid of the present invention is not limited by the above examples.
  • a method of cutting the purified DNA with a suitable restriction enzyme and inserting it into a restriction site or a cloning site of the appropriate vector DNA can be used.
  • the nucleic acid of the present invention is preferably operably linked to a vector.
  • the vector of the present invention is a cis element such as an enhancer, a splicing signal, a poly A addition signal, and a selection marker. ), A ribosome binding sequence (ribosome binding sequence, SD sequence) and the like can be further included.
  • a selection marker chloramphenicol resistance nucleic acid, ampicillin resistance nucleic acid, dihydrofolate reductase, neomycin resistance nucleic acid, and the like may be used, but the additional components that are operably linked by the above examples are limited. no.
  • the term 'transformation' refers to the introduction of DNA into a host so that the DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration, thereby introducing an artificial genetic change by introducing an external DNA into a cell. It means the phenomenon that causes.
  • any transformation method may be used, and may be easily performed according to conventional methods in the art.
  • the Hanahan method, the electroporation method, the calcium phosphate precipitation method, the plasma fusion method, the silicon carbide which have improved efficiency by using a CaCl 2 precipitation method and a reducing material called DMSO (dimethyl sulfoxide) in the CaCl 2 method Agitation with fibers, agrobacterial mediated transformation, transformation with PEG, dextran sulfate, lipofectamine and dry / inhibition mediated transformation.
  • DMSO dimethyl sulfoxide
  • the method for transforming a nucleic acid encoding a glycoside hydrolase of the present invention or a vector comprising the same is not limited to the above examples, and transformation or transfection methods commonly used in the art may be used without limitation. .
  • the 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 comprising the same into a host.
  • the host is not particularly limited as long as the host is allowed to express the nucleic acid of the present invention.
  • Specific examples of hosts that can be used in the present invention include Escherichia bacteria such as E. coli; Bacillus Bacillus (Bacillus) in bacteria such subtilis (Bacillus subtilis); Bacteria of the genus Pseudomonas, such as Pseudomonas putida ; Lactic acid bacteria such as Lactobacillus and Enterococcus; Yeasts such as Saccharomyces cerevisiae , Schizosaccharomyces pombe ; Animal cells and insect cells.
  • E. coli strains that can be used in the present invention are CL41 (DE3), BL21 (DE3) and HB101, specific examples of Bacillus subtilis strains are WB700 and LKS87.
  • the recombinant vector of the present invention is capable of autonomous replication in the host and consists of a promoter, a ribosomal binding sequence, a nucleic acid of the present invention, and a transcription termination sequence. have.
  • the promoter of the present invention can be used as long as it allows expression of the nucleic acid of the present invention in a host such as E. coli.
  • E. coli or phage-derived promoters such as, for example, trp promoter, lac promoter, PL promoter or PR promoter; E. coli infected phage-derived promoters such as the T7 promoter can be used.
  • Artificially modified promoters can also be used, such as the tac promoter.
  • the host of the present invention can also be used in food or feed, it is preferable to use lactic acid bacteria suitable for food and feed as a host.
  • lactic acid bacteria examples include the genus Lactococcus, Streptococcus, Lactobacillus, Leukonostock, Pediococcus, Brevibacterium and Propionibacterium. Also included in the lactic acid bacteria class are strictly anaerobic military bifidus bacteria (i.e., lactic acid producing bacteria belonging to the genus Bifidobacterium spp.), which may be used alone or in combination with lactic acid bacteria as a food fermentation culture. Lactobacillus lactis is particularly preferable as the lactic acid bacteria.
  • Nucleotide sequences encoding glycoside hydrolase according to the present invention can be optionally linked to appropriate regulatory nucleotide sequence (s) using methods known in the art to control the expression of such coding sequences (e.g., See Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY.
  • codon usage patterns of multiple sequenced genes from different Lactobacillus species can be analyzed to develop an approach to bypass codon bias as needed. See, eg, Pouwels and Leunissen, 1994, Nucleic Acids Res. 22: 929-936.
  • Recombinant lactic acid bacteria may comprise one or more constitutive promoters, or one or more regulated promoters operably linked to a coding nucleotide sequence.
  • operably linked refers to the linking of nucleotide sequence elements in a functional relationship.
  • an “operably linked” promoter may have regulatory elements present at appropriate positions and orientations relative to the nucleotide coding sequence to control RNA polymerase initiation and expression of the nucleic acid (eg, it may affect transcription of the coding sequence). Effect).
  • the promoter region may be promoter based (eg, capable of promoting expression of operably linked nucleic acid sequences) in promoters and lactic acid bacteria present in any prokaryotic cell, but in some embodiments from lactic acid species Is derived.
  • the promoter region is Lactococcus lactis subspecies l actis , for example a strain referred to as MG1363 (also in the literature Lactococcus lactis subspecies Cremoris ( (also referred to as cremoris) (Nauta et al., 1997, Nat Biotechnol. 15: 980-983), and Lactococcus lactis subspecies Lactis biovar.
  • promoter region of Lactococcus lactis including lactis biovar.diacetylactis.
  • 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 include WO 98/10079 and US Application Publication No. 2002. / 0137140, all of which are incorporated herein by reference.
  • the lactic acid bacteria used for recombinant glycoside hydrolase expression may be variants in which extracellular housekeeping protease HtrA is inactivated. See, for example, Miyoshi, et al., 2002, Appl Environ Microbiol. 68: 3141-3146, all of which are incorporated herein by reference.
  • the promoter used for the recombinant lactic acid bacterium may be an adjustable promoter or an inducible promoter.
  • Factor (s) that modulate or induce the promoter include any physical and chemical factor capable of modulating 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; Composition of the culture medium, such as ionic strength / NaCl content; Accumulation of metabolites (including lactic acid / lactate) intracellularly or in the medium; Presence / absence of essential cell components or precursors to them; And growth stages or growth rates of bacteria. See, for example, US application 2002/0137140, the disclosure of which is incorporated herein by reference in its entirety.
  • lactic acid bacteria A number of inducible gene expression systems have been developed for use in lactic acid bacteria (see, eg, Kok, 1996, Antonie Van Leeuwenhoek. 70: 129-145; Kuipers et al., 1997, Trends Biotechnol. 15: 135 -40; Djordjevic and Klaenhammer, 1998, Mol Biotechnol. 9: 127-139; see Kleerebezem, et al., 1997, Appl Environ Microbiol. 63: 4581-4584.
  • Useful lactic acid bacterium expression systems include the NICE system (de Ruyter et al., 1996, Appl Environ Microbiol. 62: 3662-3667).
  • Such promoters include pH-inducible P170 promoters and derivatives thereof (WO 94/16086, WO 98/10079, US Appl. Pub. No. 2002/0137140 and Madsen et al., 1999, Mol Microbiol. 107). : 75-87).
  • promoter and nucleotide sequences encoding glycoside hydrolase may be introduced into the lactic acid bacteria on plasmids, transfer factors, bacteriophages or cosmids and spontaneously replicating replicons.
  • the promoter and recombinant protein nucleotide coding sequence can be introduced under conditions such that the recombinant protein nucleotide coding sequence is integrated into the lactic acid cell chromosome to maintain the recombinant protein nucleotide coding sequence stable in bacteria. Integration can be performed by integration systems based on homologous recombination, transposons, conjugation shifts and phage integrase, etc.
  • glycoside hydrolase nucleotide coding sequences may be introduced into the lactic acid cell chromosome at positions operably linked to promoters that occur naturally in the chromosome of the selected host organism (eg, Rauch et al., 1992, J Bacteriol. 174 (4): 1280-7; Israelsen et al., 1993, Appl Environ Microbiol. 59 (1): 21-26. and Maguin et al., 1996, J Bacteriol. 178 (3): 931-5).
  • the glycoside hydrolase nucleotide coding sequence can be operably linked to the nucleotide sequence encoding a signal peptide (SP) such that the gene product is secreted out of the bacteria and introduced into the culture medium.
  • SP signal peptide
  • Signal peptides suitable for use in lactic acid bacteria, including Usp45, are published in US Publication No. 2002/0137140, all of which is incorporated herein by reference.
  • 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.
  • nucleotide sequence encoding Staphylococcus nuclease (Nuc) and the synthetic propeptide LEISSTCDA can be linked to the nucleotide sequence encoding glycoside hydrolase. See, for example, Nouaille et al., 2005, Braz J Med Biol Res. 38: 353-359, all of which are incorporated herein by reference in their entirety.
  • pLF22 see, eg, Trakanov, et al., 2004, Microbiology 73: 170-175
  • pTREX eg, Reuter, et al., 2003, "Vaccine Protocols Recombinant glycoside hydrolase can be expressed in lactic acid bacteria using expression vectors developed for use in lactic acid bacteria, including "In Methods in Molecular Medicine 87: 101-114".
  • lactic acid bacteria comprising nucleotide sequences encoding glycoside hydrolase are cultured, for example, as published in US Publication No. 2002/0137140 to produce endotoxin-free glycoside hydrolase. can do.
  • the method may comprise culturing the transformed lactic acid bacteria under conditions suitable for expression of the apolipoprotein and recovering glycoside hydrolase from the transformed lactic acid bacteria.
  • Recombinant cells and / or glycoside hydrolases can be harvested using conventional techniques known to those skilled in the art. See, eg, US Application Publication No. 2002/0137140, all of which is incorporated herein by reference in its entirety.
  • short chain acyl phospholipids are added to the medium (ie, fermentation medium) used for the cultivation of lactic acid bacteria comprising recombinant glycoside hydrolase (as described above).
  • Lactic acid bacteria can use short-chain acyl phospholipids as a nutrient source.
  • Short-chain acyl phospholipids can also be used as adjuvants to dissolve expressed apolipoproteins.
  • Short-chain acyl phospholipids can be easily removed by the addition of phospholipase, which breaks down the acyl chain, and the release of short-chain fatty acids and short-chain lysoPL.
  • the plasmid vector may further comprise other sequences as necessary.
  • the sequence which may be further included may be a tag sequence for protein purification, such as glutathione S-transferase (Pharmacia, USA), MBP (maltose binding protein, USA), FLAG (IBI, USA) and hexahistidine ( hexahistidine; Quiagen, USA) and the like, and most preferably MBP, but the examples do not limit the type of sequence required for purification of the target protein.
  • a fusion protein expressed by a vector containing the fusion sequence it can be purified by affinity chromatography.
  • glutathione-S-transferase which is a substrate of the enzyme
  • MBP amylose column
  • Host cells transformed by the above method to express the glycoside hydrolase of the present invention can be cultured by conventional methods used in the art.
  • the transformant expressing the glycoside hydrolase may be cultured in various media, and may be fed-batch culture and continuous culture, but the present invention by the above examples
  • the method of culturing the transformant of is not limited.
  • the carbon source that may be included in the medium for the growth of the host cell may be appropriately selected according to the judgment of those skilled in the art according to the type of transformant produced, and appropriate culture conditions may be adopted to control the timing and amount of culture. Can be.
  • the transformant which has been successfully transformed with the target protein produces glycoside hydrolase, and the glycoside hydrolase produced according to the composition of the vector and the characteristics of the host cell.
  • the agent may be secreted into the cytoplasm of the host cell, into the periplasmic space or extracellularly.
  • the protein of interest may also be expressed in soluble or insoluble form.
  • Proteins expressed in or outside the host cell can be purified in a conventional manner.
  • purification methods include salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (eg, protein fraction precipitation with acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, reverse phase column chromatography. Techniques such as chromatography and ultrafiltration may be applied alone or in combination to purify the proteins of the invention.
  • Glycoside hydrolase isolated in this manner can be used to produce deglycosylated ginsenosides that can be readily absorbed in an in vitro or in vivo system in which ginsenosides are present. have.
  • the invention is directed to a glycosidic hydrolase of the invention, a transformant of the invention or a culture of said transformant, Weissella confusa , Pediococcus pentosaceus , Leuconostoc mesenteroides subsp. Mesenteroides , or Leuconostoc citreum , cultures of the strain Or mixtures thereof.
  • the first ginsenoid to be used as a starting material in the present invention may be a ginsenoside of PPD or PPT type, may use a ginsenoside in a separated and purified form, or may be included in a powder or extract of ginseng or red ginseng. Ginsenosides can also be used. That is, the method of the present invention may be carried out using a powder or extract of ginseng or red ginseng including ginsenosides as a starting material. Ginsenosides of the PPD or PPT type are shown in FIG. 1.
  • ginseng used in the present invention various known ginsengs can be used, and Korean ginseng ( Panax ginseng ), Gyegi ( P. quiquefolius ), Jeonchisam ( P. notoginseng ), bamboo ginseng ( P. japonicus ), and three leaf ginseng ( P. trifolium ), Himalayan ginseng ( P. pseudoginseng ) and Vietnamese ginseng ( P. vietnamensis) , but are not limited thereto.
  • Korean ginseng Panax ginseng
  • Gyegi P. quiquefolius
  • Jeonchisam P. notoginseng
  • bamboo ginseng P. japonicus
  • three leaf ginseng P. trifolium
  • Himalayan ginseng P. pseudoginseng
  • Vietnamese ginseng P. vietnamensis
  • the first ginsenoside which is a substrate of glycoside hydrolase
  • the glycoside hydrolase of the present invention can be selectively hydrolyzed if all non-reduced ⁇ -1,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues, or ginsenosides with glucose are all preferred. Selectively hydrolyzing non-reduced ⁇ -1,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues, ⁇ -ramnopyranosyl residues or glucose of all PPD or PPT types of ginsenosides known in the art.
  • Non-reduced ⁇ of Rb1 for example, because Rb1 in the ginsenosides of the PPD type has ⁇ -1,6 glucopyranosyl residues unreduced at C20 (meaning the 20th carbon)
  • the -1,6 glucopyranosyl moiety can be selectively hydrolyzed by the glycoside hydrolase of the present invention.
  • residues such as arabinofuranoside of C3 of ginsenoside Rc and arabinopyranoside of Rb2 can also be selectively hydrolyzed by the glycoside hydrolase of the present invention.
  • the ⁇ -lamnopyranosyl residue at C6 of ginsenoside Re the ⁇ -lamnopyranosyl residue may be selectively hydrolyzed by the glycoside hydrolase of the present invention.
  • Glycoside hydrolase of the present invention transformant of the present invention or culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Luconostock mesenteroids ( Leuconostoc mesenteroides subsp. Mesenteroides ), or Leuconostoc citreum , cultures of the strains, or mixtures thereof, are relatively deglycosylated rare deglycosylated ginsenosides of the PPD (protopanaxadiol) or PPT type.
  • cenosides which include non-reduced ⁇ -1,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues, ⁇ -rhamnopyranosyl residues, glucose of PPD type or PPT type ginsenosides.
  • residues such as arabinofuranoside residues, arabinopyranoside, and more preferably located at the 20th carbon of the PPD type ginsenoside.
  • This conversion is via bioconversion, either continuously or discontinuously, non-reduced ⁇ -1,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues or glucose of PPD type ginsenosides, It can be achieved by selectively hydrolyzing an a-ramnopyranosyl residue located at the sixth carbon of the arabinofuranoside, arabinopyranoside, or PPT type ginsenoside.
  • examples of bioconversion by the protein of the present invention include ginsenoside Rb1 to Rd, ginsenoside Rc to Rd, ginsenoside Rb2 to Rd, ginsenoside Rd to F2, Ginsenoside Rd to Compound K, Ginsenoside Rb1 to F2, Ginsenoside Gyp17 to F2, Ginsenoside Rb1 to Compound K, Ginsenoside F2 to Compound K, Ginsenoside Convert side Rd to Rg3, convert ginsenoside Rb1 to Rh2, convert ginsenoside Rg3 to Rh2, convert ginsenoside Rb1 to Gyp17, convert ginsenoside Gyp17 to GypLXXV, convert ginsenoside gyp17 to compound K Conversion, ginsenoside Rb2 to compound O, ginsenoside compound O to compound Y, ginsenoside Rc to compound Mc, ginsenoside Re to Rg1, ginsen
  • Deglycosylation of such ginsenosides can be achieved by non-reducing ⁇ -ramnopyranosyl residues, ⁇ -1,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues or glucose of the glycoside hydrolase of the present invention.
  • Deglycosylated rare ginsenosides produced through the hydrolysis as described above may be easily absorbed by the body.
  • this bioconversion facilitates the conversion of PPD type ginsenosides, such as ginsenoside Rb1, to compound K, its most deglycosylated form.
  • Glycoside hydrolase of the present invention ⁇ -1 of the protopanaxadiol type PPX (protopanaxadiol) type ginsenoside of the PPT type ginsenoside, at various temperature and pH conditions as long as activity and stability can be maintained
  • Selective hydrolysis of 6,6 glucopyranosyl residues, ⁇ -1,2 glucopyranosyl residues or glucose and rare ginsenosides in the form of easy absorption into the body of protopanaxadiol (PPD) or protopanaxatriol (PPT) type ginsenosides It can be used for the conversion of.
  • glycoside hydrolase of the present invention may be used with, but not limited to, one or more metals and chemical agents selected from the group consisting of MgCl 2 , EDTA, NaCl, KCl, DTT, and beta-mercaptoethanol.
  • the glycoside hydrolase of the present invention when preparing a deglycosylated second ginsenoside from the first ginsenoside, the glycoside hydrolase of the present invention, the transformant of the present invention or the culture of the transformant, bisella confusa ( Weissella confusa ), Pediocaucus Pantosassius ( Pediococcus pentosaceus ), Lukonostoke mesenteroids ( Leuconostoc mesenteroides subsp. mesenteroides ), Or leuconot citrium ( Leuconostoc citreum ), Cultures of the strains, or mixtures thereof, may be used with other enzyme (s) simultaneously or in any order.
  • bisella confusa Weissella confusa
  • Pediocaucus Pantosassius Pediococcus pentosaceus
  • Lukonostoke mesenteroids Leuconostoc mesenteroides subsp
  • Non-limiting examples of other enzymes include ⁇ - N Arabinofuranosidase, ⁇ -galactosidase, glycosidase, ⁇ -L-arabinopyranosidase, ⁇ -L-arabinofuranosidase, ⁇ -xylosidase, and ⁇ -L-lamnosidase and the like.
  • Second ginsenosides prepared by glycoside hydrolase alone or in combination with other enzymes may be different, and such second ginsenosides may be one or two or more second ginsenosides.
  • Glycoside hydrolase of the present invention transformant of the present invention or culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Luconostock mesenteroids ( Leuconostoc mesenteroides subsp. Mesenteroides ), or Leuconostoc citreum , cultures of the strains, or mixtures thereof may be provided in the same or different containers as the other enzyme (s).
  • the present invention provides a glycoside hydrolase, a transformant of the present invention, or a culture of the transformant, Weissella confusa , Pediococcus pentosaceus , Kono base stock mesen steroid's (Leuconostoc mesenteroides subsp. mesenteroides), or base stock sheet Pocono Solarium (Leuconostoc citreum), provides a food or a food additive comprising a culture, or a mixture of the strains.
  • Feed or feed additives are included in the food or food additives according to the invention.
  • the food or food additive may further further comprise ginsenosides, or hemps, of the PPD or PPT type.
  • the food includes not only the health functional food, but also general foods that humans consume in general and daily.
  • it When used as a nutraceutical, it may be formulated into a formulation of a conventional nutraceutical known in the art together with a pharmaceutically acceptable carrier or additive.
  • the health functional food may be prepared, for example, as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, extracts, teas, jelly, or beverages.
  • the food acceptable carrier or additive may be any carrier or additive known to be available in the art for the preparation of the formulation to be prepared.
  • the food may be fermented food
  • fermented foods include, but are not limited to, kimchi, vegetable fermented product, soybean paste, soy sauce, cheonggukjang, salted fermented dairy products (fermented milk, yogurt, cheese) and the like.
  • ginsenosides or intragranular ginsenosides may be hydrolyzed by glycoside hydrolase and may not be hydrolyzed.
  • the glycoside hydrolase of the present invention, the transformant of the present invention or the culture of the transformant Weissella confusa , Pediococcus pentosaceus , Lu Pocono stock mesen steroid's (Leuconostoc mesenteroides subsp. mesenteroides), or Lu Pocono stock sheet Solarium (Leuconostoc citreum), a mixture of the culture, or a said strain is from 20 to 90wt% high concentrated liquid or a powder or made from a granular form Can be.
  • the feed additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherols and rosemary Extracts, vitamin C, green tea extracts, licorice extracts, chitosan, tannic acid, phytic acid, such as any one or more may be further included.
  • the composition may be formulated in a conventional feed form and may include common feed ingredients.
  • the feed additives and feeds are cereals, for example milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as feed consisting mainly of rapeseed, soybean, and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, may further include a dry ingredient consisting of various powdered milk and whey powder, and may further include a nutritional supplement, digestion and absorption enhancer, growth promoter and the like.
  • the feed additive may be administered alone or in combination with other feed additives in an edible carrier.
  • the feed additive may be easily administered to the animal as a top dressing or directly mixed into the animal feed or in an oral formulation separate from the feed.
  • the feed additive can be prepared in immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier, as is well known in the art.
  • a pharmaceutically acceptable edible carrier may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol.
  • the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in microdisperse form.
  • the feed additive may be a gelatin soft capsule or a formulation of syrup or suspension, emulsion, or solution.
  • the feed may comprise any protein-containing organic flour commonly used to meet the dietary needs of the animal.
  • protein-containing flours typically consist primarily of corn, soy flour, or corn / bean flour mixtures.
  • the feed additives and feed may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifiers, solution promoters and the like.
  • the feed additive may be used by adding to animal feed by infiltration, spraying or mixing.
  • the feed or feed additive of the present invention is applicable to many animal diets, including mammals, poultry and fish.
  • the mammals can be used not only for pigs, cows, sheep, goats, experimental rodents, and experimental rodents, but also for pets (eg, dogs, cats), and the like as poultry such as chickens, turkeys, ducks, geese, pheasants, And can also be used for quail, etc., may be used in trout, etc. as the fish, but is not limited thereto.
  • the GL24 strain was cultured in MRS plate medium, and the cells were collected. Then, the genomic DNA extraction kit (MoBio) was used to generate genomic DNA ( genomic DNA) was isolated.
  • the pET21a vector used for cloning was KCTC11478 (pMBP-BglQM6775) with Maltose binding protein epitope added to the N-terminal side.
  • template DNA template DNA
  • gDNA genomic DNA
  • the sequence of the amplified fragment was analyzed by BLSTP and identified as an enzyme belonging to glycoside hydrolase family I, which was named GL24GH1.
  • the amplified fragment was cloned into pET21a expression vector using EcoRI and HindIII restriction enzyme delivery sites.
  • the cloned GL24GH1-pET21a was transformed into E. coli strain C41.
  • IPTG was added to a final concentration of 0.2 mM and incubated 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 mill. Supernatants centrifuged at 12,500 Xg for 20 minutes were used for the purification of enzyme proteins.
  • the strains were cultured in MRS plate medium, and the cells were collected and genomic DNA was isolated using a genomic DNA extraction kit (MoBio).
  • the vector used for cloning was KCTC11478 (pMBP-BglQM6775) with Maltose binding protein epitope added to the N-terminal side.
  • template DNA used 100ng of GL24 gDNA.
  • the amino acid sequence is shown in SEQ ID NO: 2, and BLSTP analysis showed that the enzyme belongs to the glycoside hydrolase family 43. Named GL24GH43.
  • the amplified fragment was cloned into pET21a expression vector using EcoRI and HindIII restriction enzyme delivery sites.
  • the cloned GL24GH43-pET21a was transformed into E. coli strain C41.
  • the strains were cultured in an MRS plate medium, and the cells were collected to separate genomic DNA using a genomic DNA extraction kit (MoBio).
  • the pET21a vector used for cloning was derived from KCTC11478 (pMBP-BglQM6775) in which the Maltose binding protein epitope was added to the N-terminal side. Used.
  • the template DNA used 100ng of the gDNA of W. confusa GL24.
  • the amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO: 3, BLSTP analysis showed that the enzyme belongs to the Glycoside hydrolase family3. Named GL24GH3.
  • the amplified fragment was cloned into pET21a expression vector using EcoRI and HindIII restriction enzyme delivery sites.
  • the cloned GL24GH3-pET21a was transformed into E. coli strain C41.
  • IPTG phosphate buffer
  • the cells were collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5) and then disrupted by an ultrasonic mill. Supernatants centrifuged at 12,500 Xg for 20 minutes were used for the purification of enzyme proteins.
  • Escherichia coli E. coli C41, DE3 containing an overexpressed plasmid of GL24GH1-pET21a or GL24GH43-pET21a bound with MBP tag was subjected to 37 ° C until the OD value was 600 to 0.6 in LB-Ampicillin medium. Incubated at the temperature of and treated with 0.2 mM isopropyl-D-thiogalactopyranoside for 6 hours to induce protein expression. The E. coli cells were centrifuged at 5,000 ⁇ g for 20 min at 4 ° C. and then obtained.
  • Cell pellets were resuspended using suspension buffer (50 mM sodium phosphate, 5 mM EDTA, pH 7.5), centrifuged at 12,500 ⁇ g after sonication (ultrasonification). The supernatant was loaded on an MBPTrap (GE) column using an FPLC system, washed with the phosphate buffer solution, eluted with maltose 200 mM, and the proteins separated and purified. Then, SDS-PAGE analysis was performed on GL24GH1 and GL24GH43.
  • suspension buffer 50 mM sodium phosphate, 5 mM EDTA, pH 7.5
  • the supernatant was loaded on an MBPTrap (GE) column using an FPLC system, washed with the phosphate buffer solution, eluted with maltose 200 mM, and the proteins separated and purified. Then, SDS-PAGE analysis was performed on GL24GH1 and GL24GH43.
  • GL24GH1 protein was expressed in lanes 2, 3 and 4 (95 kDa), and GL24GH43 protein (70 kDa) was expressed in lanes 6, 7 and 8 (FIG. 2).
  • the glycoside hydrolase gene was cloned from 100118TI-4 strain identified as Pediococcus pentosaceus .
  • the strains were cultured on an MRS plate medium, and then the cells were collected using a genomic DNA extraction kit (MoBio). Genomic DNA was isolated.
  • the pET21a vector used KCTC11478 (pMBP-BglQM6775).
  • template DNA used 100ng of gDNA of Pediococcus pentosaceus 100118TI-4.
  • the amino acid sequence of the cloned glycoside hydrolase is shown in SEQ ID NO: 4, and BLSTP analysis revealed that the enzyme belongs to the glycoside hydrolase family 1, which was named PedGH1.
  • Example 3 lactic acid bacteria culture and enzyme expression induction
  • lactic acid bacteria were inoculated into 100 ml MRS liquid medium and cultured in a 37 ° C shake incubator.
  • 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.
  • the absorbance (OD 600nm) of the culture medium was 0.5-0.6, and the glycosid hydrolase enzyme exhibiting ginsenosidase activity as a PPD or PPT mixture when the cells entered logarithmic growth phase. Expression was induced. After 1 or 2 days of culture, 50 ml of cell culture was recovered, respectively.
  • Example 1 The enzyme solution over-expressed and purified in Example 1, Rb1, Gyp17, Rd, F2, Re and Rg1 in which the PedGH1 enzyme solution of Example 2 and the crude enzyme solution of the wild type strain were dissolved in sodium phosphate buffer solution (50 mM, pH7.5). (1mg / ml) and the volume ratio of 1: 1 (200ul: 200ul) was reacted. After the reaction, the samples were sampled over time and extracted with the same volume of butanol for TLC analysis. Identification of the reaction product was determined by the development on TLC using a standard of purity such as enzyme reaction substrate.
  • Example 3 Cells of 100 ml of the culture solution 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) analysis of the results of confirming the conversion activity of ginsenosides for the recombinant GL24GH1 and GL24GH43 isolated and purified in Example 1.
  • 3 shows that GL24GH1 has a conversion activity of Rb1-> Rh2 and C-K, Gyp17-> F2, Rd-> F2, Re-> Rg1.
  • 4 shows that GL24GH43 has a conversion activity of Rb1-> F2, Gyp17-> F2, Rd-> F2, Re-> Rg1, which are PPD type ginsenosides.
  • 5 shows that GL24GH3 has a conversion activity of Rb1-> F2, Gyp17-> F2, Rd-> F2.
  • Figure 7 shows that the GL24 strain belonging to the lactic acid bacteria Weissella confusa has the activity of bioconversion of PPD ginsenosides Rb1, gyp17, Rd to F2.
  • strain 100118TI-4 belonging to the lactic acid bacterium Pediococcus pentosaceus has a bioconversion activity of Rb1-> F2, Gyp17-> F2, Rd-> F2 among ginsenosides of the PPD type. .
  • Figure 10 shows that the lactic acid bacteria Pediococcus pantosassius strain 100118TI-4 ( Pediococcus pentosaceus 100118TI-4) has the enzymatic activity of bioconversion of ginsenosides of the PPT type from Re-> Rg1.
  • Figure 11 is lactic acid bacteria Lukonostok strium ( Leuconostoc citreum ) GL29 has the activity of bioconversion from PPD type ginsenoside Rb1-> F2-> CK, gyp17-> CK, Rd-> CK, F2-> CK and PPT type ginsenoside Re-> Rg1 TLC analysis shows that it has bioconversion activity.
  • Figure 12 is a lactic acid base Pocono stock mesen steroid's (Leuconostoc mesenteroides subsp mesenteroides.) Ginsenoside Rb1 of strain 25, the PPD type - to> CK, F2 - -> F2 has the activity to convert organisms in> CK and the PPT type It shows the result confirmed by TLC analysis that it has the activity of bioconversion from ginsenoside Re-> Rg1.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une glycoside hydrolase issue de bactéries lactiques, en particulier une glycoside hydrolase issue de Weissela confuse, Pediococcus pentosaceus, Leuconostoc citreum ou Leuconostoc mesenteroides sous espèce mesenteroides, et ses utilisations.
PCT/KR2012/003387 2011-04-29 2012-04-30 Glycoside hydrolase issue de bactéries lactiques et ses utilisations Ceased WO2012148249A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20110041178 2011-04-29
KR10-2011-0041178 2011-04-29

Publications (2)

Publication Number Publication Date
WO2012148249A2 true WO2012148249A2 (fr) 2012-11-01
WO2012148249A3 WO2012148249A3 (fr) 2013-01-03

Family

ID=47072968

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2012/003387 Ceased WO2012148249A2 (fr) 2011-04-29 2012-04-30 Glycoside hydrolase issue de bactéries lactiques et ses utilisations

Country Status (2)

Country Link
KR (1) KR101488481B1 (fr)
WO (1) WO2012148249A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101557930B1 (ko) 2012-11-27 2015-10-12 한국식품연구원 신규한 진세노사이드 화합물
CN108066170A (zh) * 2013-04-24 2018-05-25 株式会社爱茉莉太平洋 含有人参皂苷Mc的皮肤外用剂组合物
CN108902600A (zh) * 2018-07-02 2018-11-30 东莞市农业科学研究中心 荔枝内生乳酸菌在制备低糖健康型发酵果汁中的应用
CN109010112A (zh) * 2013-05-03 2018-12-18 株式会社爱茉莉太平洋 含有作为主要有效成分的人参皂苷y的皮肤外用剂组合物的应用
CN109182441A (zh) * 2018-08-31 2019-01-11 吉林农业大学 人参皂苷Rb1发酵液的制备方法及其应用
CN109627303A (zh) * 2018-12-11 2019-04-16 昆明理工大学 三七病程相关蛋白PnPR3的基因及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2015324813B2 (en) 2014-09-30 2019-04-18 Rna Inc. Method for preparing microbial preparation and microbial preparation produced by the same
KR20170047007A (ko) * 2015-10-22 2017-05-04 (주)아모레퍼시픽 효소적 방법에 의하여 인삼의 사포닌으로부터 진세노사이드 F2, 컴파운드 Mc 및 컴파운드 O를 선택적으로 제조하는 방법

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101091138B1 (ko) * 2009-03-26 2011-12-09 한국생명공학연구원 류코노스톡 락티스로부터 유래된 글루칸수크라제 및 그 제조방법

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101557930B1 (ko) 2012-11-27 2015-10-12 한국식품연구원 신규한 진세노사이드 화합물
CN108066170A (zh) * 2013-04-24 2018-05-25 株式会社爱茉莉太平洋 含有人参皂苷Mc的皮肤外用剂组合物
CN109010112A (zh) * 2013-05-03 2018-12-18 株式会社爱茉莉太平洋 含有作为主要有效成分的人参皂苷y的皮肤外用剂组合物的应用
CN109010112B (zh) * 2013-05-03 2021-06-04 株式会社爱茉莉太平洋 含有作为主要有效成分的人参皂苷y的皮肤外用剂组合物的应用
CN108902600A (zh) * 2018-07-02 2018-11-30 东莞市农业科学研究中心 荔枝内生乳酸菌在制备低糖健康型发酵果汁中的应用
CN108902600B (zh) * 2018-07-02 2021-06-04 东莞市农业科学研究中心 荔枝内生乳酸菌在制备低糖健康型发酵果汁中的应用
CN109182441A (zh) * 2018-08-31 2019-01-11 吉林农业大学 人参皂苷Rb1发酵液的制备方法及其应用
CN109627303A (zh) * 2018-12-11 2019-04-16 昆明理工大学 三七病程相关蛋白PnPR3的基因及其应用
CN109627303B (zh) * 2018-12-11 2021-11-12 昆明理工大学 三七病程相关蛋白PnPR3的基因及其应用

Also Published As

Publication number Publication date
KR20140043421A (ko) 2014-04-09
KR101488481B1 (ko) 2015-01-30
WO2012148249A3 (fr) 2013-01-03

Similar Documents

Publication Publication Date Title
WO2012148249A2 (fr) Glycoside hydrolase issue de bactéries lactiques et ses utilisations
Avila et al. Physiological and biochemical characterization of the two α-L-rhamnosidases of Lactobacillus plantarum NCC245
KR101108428B1 (ko) 인간의 모유에서 분리한 프로바이오틱 활성 및 체중 증가억제 효과를 갖는 유산균
Xu et al. Extracellular secretion of feruloyl esterase derived from Lactobacillus crispatus in Escherichia coli and its application for ferulic acid production
JP5643756B2 (ja) バチルス・サーキュランス由来のβ−ガラクトシダーゼ
Youn et al. Identification of the beta-glucosidase gene from Bifidobacterium animalis subsp. lactis and its expression in B. bifidum BGN4.
CN107058366B (zh) 乳酸乳球菌食品级双筛选标记分泌表达载体、含有所述载体的工程菌株、其构建方法及应用
RO116211B1 (ro) Polipeptida cu activitate de degradare a arabinoxilanului, adn recombinant, ce o codifica, procedeu de obtinere si compozitie alimentara cu aceasta
KR101514775B1 (ko) 유산균 유래의 글리코시드 히드롤라제 및 이의 용도
KR102152138B1 (ko) 활성이 개선된 자일라나제 변이체 및 이의 생산방법
JP6364506B2 (ja) 新規β−ガラクトシダーゼ
US20140227789A1 (en) Stress tolerant bifidobacteria
JPH06500022A (ja) 真菌類由来のアラビナン分解酵素をコードするｄｎａ分子のクローニングおよび発現
CN106434598B (zh) 一种酯酶及其应用
KR101248191B1 (ko) 알긴산 분해 미생물 및 그의 용도
KR20160141123A (ko) 고당전이 활성의 베타-갈락토시다제 변이체 및 그 용도
WO2012070724A1 (fr) Alpha-n-arabinofuranosidase issue de rhodanobacter ginsenosidimutans et ses utilisations
Kim et al. Cloning and expression of β-glucosidases from Bifidobacterium lactis AD011
KR20090088630A (ko) 프로테우스 미라빌리스 유래 아미노산 탈아미노화 효소유전자, 단백질, 재조합 벡터 및 형질전환체, 및 이를사용하여 기능성 유기산을 제조하는 방법
KR101212106B1 (ko) 신규한 베타-아가라제 및 그 용도
KR102513450B1 (ko) 신규한 엑토인 합성효소 및 그 용도
JP2003174892A (ja) 新規のガラクタナーゼ
CN102181437A (zh) 猪脂联素球状结构域蛋白gAd基因及其编码的蛋白和应用
KR101825456B1 (ko) 고당전이 활성의 베타-갈락토시다제 변이체 및 그 용도
KR20050102955A (ko) 내산성과 내담즙성이 좋고 효소의 생산이 우수한 신규바실러스 서브틸리스 유비티-엠02 균주 , 이 신규 균주를이용 함유하는 사료첨가제 및 이 사료첨가제를 함유하는가축 사료

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12777453

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12777453

Country of ref document: EP

Kind code of ref document: A2