Method for producing algal-derived agarotriose and use thereof as probiotic
阅读说明：本技术 用于产生海藻衍生的琼胶三糖的方法和其作为益生菌的用途 (Method for producing algal-derived agarotriose and use thereof as probiotic ) 是由 金京宪 陈勇秀 尹银珠 金东玄 俞素萝 赵庆纹 朴娜庭 于 2018-11-15 设计创作，主要内容包括：本发明涉及一种用于产生海藻衍生的琼胶三糖的方法和其作为益生菌的用途。更具体来说,本发明研究作为益生菌的琼胶三糖的特性,其通过益生菌微生物选择性地代谢,从而使琼胶三糖能够在食品和药品领域中用作抗癌剂或抗炎剂,并且使琼胶三糖能够在红藻衍生的多糖在未经预处理的情况下酶水解之后以最小损失经由高效纯化以高产率获得。(The present invention relates to a method for producing algal derived agarotriose and its use as probiotic. More specifically, the present invention studies the properties of agarotriose as a probiotic, which is selectively metabolized by probiotic microorganisms, thereby enabling agarotriose to be used as an anticancer agent or an anti-inflammatory agent in the fields of foods and pharmaceuticals, and enabling agarotriose to be obtained in high yield via high-efficiency purification with minimal loss after enzymatic hydrolysis of red algae-derived polysaccharides without pretreatment.)
1. A pharmaceutical composition comprising:
one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose;
common Bacteroides (Bacteroides plexius) strains; and
bifidobacterium (Bifidobacterium) strains.
2. The pharmaceutical composition of claim 1, wherein the strain of Bacteroides vulgatus (Bacteroides plexiius) comprises the strain of Bacteroides vulgatus (Bacteroides plexiius) DSM 17135.
3. The pharmaceutical composition according to claim 1, wherein the Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum subsp matter of infant (Bifidobacterium longum subsp. infanis) ATCC15697, Bifidobacterium longum subsp. infanis (Bifidobacterium longum subsp. infanis) ATCC 17930, Bifidobacterium longum subsp. infanis (Bifidobacterium longum subsp. infanis) ATCC15702, Bifidobacterium bifidum (B.bifidum) DSM20082 and Bifidobacterium arborvitae (B.kashiwanensis) DSM 21854.
4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared by the method of SEQ ID NO: 1 (beta-agarase) and seq id NO: 2 (neo-agarobiose hydrolase) and the sequence of SEQ ID NO: beta-galactosidase (beta-galactosidase) of 3 or 4 degrades the substrate into 3, 6-anhydro-L-galactose.
5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is for preventing or treating cancer or an inflammatory disease.
6. A food composition comprising:
one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose;
common Bacteroides (Bacteroides plexius) strains; and
bifidobacterium (Bifidobacterium) strains.
7. The food composition according to claim 6, wherein the strain of Bacteroides vulgaris (Bacteroides plexius) comprises the strain of Bacteroides vulgaris (Bacteroides plexius) DSM 17135.
8. The food composition according to claim 6, wherein the Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum subsp matter of infant (Bifidobacterium longum subsp. infanis) ATCC15697, Bifidobacterium longum subsp. infanis ATCC 17930, Bifidobacterium longum subsp. infanis (Bifidobacterium longum subsp. infanis) ATCC15702, Bifidobacterium bifidum (B.bifidum) DSM20082 and Bifidobacterium arborvitae (B.kashiwanensis) DSM 21854.
9. The food composition of claim 6, wherein the food composition is prepared by the steps of isolating a polypeptide from the amino acid sequence of SEQ ID NO: 1 (beta-agarase) and seq id NO: 2 (neo-agarobiose hydrolase) and the sequence of SEQ ID NO: beta-galactosidase (beta-galactosidase) of 3 or 4 degrades the substrate into 3, 6-anhydro-L-galactose.
10. The food composition according to claim 1, wherein the food composition is for preventing or alleviating cancer or inflammatory diseases.
11. A method for producing agarotriose, comprising:
reacting the reaction product with SEQ ID NO: 2 or 6, wherein the reaction product is prepared by reacting any one of agar, agarose or neoagarohexaose with the sequence of SEQ ID NO: 1 or 5 by beta-agarase reaction; and
the agarotriose was purified from the resulting product via a size exclusion column.
12. The method for producing agarotriose according to claim 11, wherein SEQ ID NO: 1 is an enzyme derived from the usual Bacteroides (Bacteroides plexius) DSM17135 strain, and agar, agarose or neoagarohexaose is used as the substrate to degrade to neoagarotetraose and neoagarobiose.
13. The method for producing agarotriose according to claim 11, wherein the reaction of the substrate with the β -agarase is carried out at 0rpm to 200rpm under the temperature condition of 30 ℃ to 60 ℃ for 5 minutes to 12 hours.
14. The method for producing agarotriose according to claim 11, wherein SEQ ID NO: 2 is an enzyme derived from the usual Bacteroides (Bacteroides plexius) DSM17135 strain, and degrades neoagarotetraose or neoagarobiose into agarotriose, galactose or 3, 6-anhydro-L-galactose.
15. The method for producing agarotriose according to claim 11, wherein the reaction of the substrate and the reaction product of the β -agarase with the neoagarase is carried out at 0 to 200rpm for 30 minutes to 12 hours under the temperature condition of 25 to 45 ℃.
16. A method for treating cancer or an inflammatory disease, comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition,
wherein the pharmaceutical composition comprises: one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose;
common Bacteroides (Bacteroides plexius) strains; and
bifidobacterium (Bifidobacterium) strains.
17. The method for treating cancer or inflammatory disease of claim 16, wherein the Bacteroides vulgatus (Bacteroides plexius) strain comprises Bacteroides vulgatus (Bacteroides plexius) DSM17135 strain.
18. The method for treating cancer or inflammatory disease according to claim 16, wherein the Bifidobacterium (Bifidobacterium) strain is selected from the group consisting of Bifidobacterium longum subsp.
19. The method for treating cancer or inflammatory disease according to claim 16, wherein the cancer is any one of colon cancer, cervical cancer, breast cancer, gastric cancer and liver cancer.
Technical Field
The present invention relates to a method for producing algal derived agarotriose and its use as probiotic.
Background
Probiotics (probiotics) refers to substances that are selectively fermented by beneficial gut bacteria to improve gut flora and benefit human health. Although studies on the correlation between human diseases and intestinal flora have been recently reported, the intestinal flora has been considered as a second human genome, so that studies in the field have been rapidly developed. In particular, studies on the intestinal flora have received much attention because there are studies reporting that obesity, diabetes and immune function are improved as the distribution of beneficial intestinal flora increases.
The probiotic effect of the hydrolysate of agarose, which is the main polysaccharide constituting red algae, has been predicted by animal experiments. As a result of orally administering the agar oligosaccharide mixture to rats suffering from obesity induced by high-fat diet, it was confirmed that the degree of distribution of bifidobacteria as intestinal beneficial bacteria was increased. In addition, the agar oligosaccharide mixture promotes the synthesis of low molecular weight fatty acids in the intestinal tract, and induces the expression of genes related to immune and anti-inflammatory functions. In addition, it was confirmed that in the case of producing a neoagaro-oligosaccharide mixture from agarose by two types of endo-beta-agarase enzymatic reactions, bifidobacterium and lactobacillus were grown under the carbon source condition of the neoagaro-oligosaccharide mixture. However, in this experiment, since the growth test of bifidobacterium and lactobacillus was not performed in the absence of the new agaro-oligosaccharide mixture as a control, there were the following problems: it is not known exactly whether growth is caused by other carbon sources in the medium or by the metabolism of the new agaro-oligosaccharides. In addition, it was confirmed that when the neoagaro-oligosaccharide mixture was administered to the rat model, the degree of distribution of bifidobacteria and lactobacilli increased.
As previously described, since probiotic functional studies of red algae derived oligosaccharides have so far used mixtures rather than purified standards, it is not known at all which effective indicator ingredients will produce changes in the intestinal flora while actually having probiotic activity. Furthermore, it is not known how agarose derived oligosaccharides are metabolized by intestinal potent probiotic microorganisms.
Meanwhile, the main polysaccharide constituting red algae is agarose, and agarose is a polymer in which 3, 6-anhydro-L-galactose (hereinafter referred to as '3, 6-AHG') and D-galactose (hereinafter referred to as 'D-Gal') are alternately linked together via α -1, 3-bonds and β -1, 4-bonds. Previous studies established a process for producing AHG with anti-caries, anti-inflammatory, whitening and moisturizing functions. Agaro-oligosaccharides (agaroagosaccharides) were obtained by pretreating a substrate with a weak acid, acetic acid, or a low-concentration neutral buffer, Tris-HCl buffer (ph7.4), and neoagarobiose (neoagarobiose) was produced from the agaro-oligosaccharides via an excision-type β -agarase II enzyme (exo-type β -agarase II) promotion reaction. In this case, there are disadvantages as follows: agarotriose (agarotriose) is also produced as a by-product, and in order to degrade agarotriose into the monosaccharides AHG and galactose, an additional enzyme called agarolytic β -galactosidase (ABG) needs to be introduced. However, agarotriose in the form of Gal-AHG-Gal, which is an oligosaccharide considered as a byproduct for AHG production in previous studies, is currently known through various literatures to promote beneficial bacteria in intestinal functions of the body and to have a probiotic effect by itself, but detailed process techniques for obtaining purified and pure agarotriose are not known at home.
Disclosure of Invention
Technical problem
The invention aims to provide the application of agarotriose as a medicine or food substance by researching the metabolism of agarotriose by intestinal effective probiotic microorganisms.
It is another object of the present invention to provide a process for preparing agarotriose by enzymatic hydrolysis and purification.
Technical solution
To achieve the object, the present invention provides a pharmaceutical composition comprising: one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose; common Bacteroides (Bacteroides plexius) strains; and a Bifidobacterium (Bifidobacterium) strain.
The present invention also provides a method for treating cancer or an inflammatory disease, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition.
The present invention also provides a food composition comprising: one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose; common Bacteroides (Bacteroides plexius) strains; and a Bifidobacterium (Bifidobacterium) strain.
The present invention also provides a method for preparing agarotriose, which comprises: reacting the reaction product with seq id NO: 2 or 6, wherein the reaction product is prepared by reacting any one of agar, agarose or neoagarohexaose with the sequence of SEQ ID NO: 1 or 5 by beta-agarase reaction; and purifying the agarotriose from the resulting product via a size exclusion column.
Advantageous effects
The invention has the following effects: the present invention can be used as an anticancer or anti-inflammatory substance in the fields of medicines and foods by studying the characteristics of agarotriose as a probiotic bacterium selectively metabolized by probiotic microorganisms such as bacteroides and bifidobacteria.
In addition, the present invention has the effect of enabling agarotriose to be obtained in high yield via efficient purification with minimal loss after enzymatic hydrolysis of red algae-derived polysaccharides without pretreatment.
Drawings
Fig. 1 shows a schematic view for producing oligosaccharides with different degrees of polymerization, neoagarobiose and AHG from agarose via an enzymatic reaction.
Fig. 2 shows the results of purifying recombinant proteins of polysaccharide degrading bacteria (s.degradans) 2-40T-derived endo-beta-agarase Aga16B, exo-beta-agarase Aga50D and alpha-neoagarobiose hydrolase SdNABH (a), producing oligosaccharides with different degrees of polymerization, neoagarobiose and 3, 6-AHG from agarose via enzymatic reaction (B), and purifying saccharides via size exclusion chromatography of enzyme reaction products of agarose and usual Bacteroides sp (DSM 17135) derived endo-beta-agaropectinase BpGH16A (BACPLE _01670) and neoagarobiose hydrolase BpGH117(BACPLE _01671) (C).
FIG. 3 shows the results (A) of each recombinant enzyme of purified intestinal microorganisms, Bacteroides vulgares (Bacteroides sporolebeius), DSM 17135-derived endo-beta-agarases BpGH16A (BACPLE-01670) and BpGH50 (BACPLE-01683) and Neoagarobiose hydrolase BpGH117 (BACPLE-01671) after expression in E.coli and the results (B) of conducting the enzymatic reaction experiment.
FIG. 4 shows the results of the production of agarotriose and AHG via the enzymatic reaction of the usual Bacteroides (Bacteroides plexius) DSM 17135-derived endo-type β -agarase BpGH16A (BACPLE-01670) with neoagarobiose hydrolase BpGH117 (BACPLE-01671) from an agarose substrate.
FIG. 5 shows the results of culturing Bifidobacterium longum subsp.infantis ATCC15697 strain as a probiotic microorganism having a correspondingly purified sugar as a carbon source using biogrid C (A: AHG, B: NeoDP2, C: AgaDP3, D: NeoDP4, E: AgaDP5, F: NeoDP6, G: glucose, H: galactose, I: 2 FL).
FIG. 6 shows the results of analyzing the agarotriose fermentation curve of Bifidobacterium longum infantis ATCC15697 strain (A: results using AgaDP3, NeoDP2 and galactose as substrates; B: cell density and acetic acid and lactic acid as substrates).
Fig. 7 shows the results of degradation experiments of agarotriose (a) and neoagarobiose (B) using crude enzyme solutions of Bifidobacterium longum infantis ATCC15697 strain.
FIG. 8 shows the results of enzymatic reactions performed after cloning of four beta-galactosidase coding genes (Blon _2016, Blon _2123, Blon _2334 and Blon _2416) of Bifidobacterium longum subsp.
FIG. 9 shows the results of experiments on the fermentation ability of Bifidobacterium longum infantis (Bifidobacterium longum subsp. infantis) ATCC 17930 and Bifidobacterium longum infantis (Bifidobacterium longum subsp. infantis) ATCC15702 strains as other strains belonging to Bifidobacterium longum infantis (A: Bifidobacterium longum infantis. ATCC 17930) to agarotriose.
FIG. 10 shows the results of experiments on the fermentation capacity of Bifidobacterium bifidum (B.bifidum) DSM20082 and Bifidobacterium bifidum (B.kashiwanohense) DSM21854 strain for agarotriose (A: Bifidobacterium bifidum (B.bifidum) DSM 20082; B: Bifidobacterium bifidum (B.kashiwanohense) DSM 21854).
Fig. 11 shows the results of stability test of agarotriose to artificial gastric fluid (a: TLC results are shown as a graph; B: HPLC results).
FIG. 12 shows the metabolic pathway of the intestinal microorganism, Bacteroides thetaiotaomicron (Bacteroides plexius) DSM17135 and Bifidobacterium longum subsp.
FIG. 13 shows a schematic view of a process for producing and separating agarotriose from agarose (A: a process for producing agarotriose via acid treatment and enzymatic saccharification; B: a process for producing and purifying AHG, D-Gal and agarotriose via enzymatic saccharification).
FIG. 14 shows the results of producing agarotriose from agarose via a two-step enzymatic reaction and purifying the agarotriose using a size exclusion column (A: TLC results; B: HPLC results).
FIG. 15 is a result of HPLC quantitative analysis showing the ratio of neoagarotetraose to neoagarobiose produced from agarose via the enzymatic reaction of usual Bacteroides (Bacteroides plebeius) DSM 17135-derived endo-type β -agarase BpGH16A (BACPLE-01670).
FIG. 16 shows the TLC results using a size exclusion column to analyze the difference in the degree of separation of each fraction.
Fig. 17 shows the results of measuring the yield and purity of agarotriose using an HPLC KS-802 sugar column.
Detailed Description
The inventors of the present invention demonstrated the probiotic effect of agarotriose by producing agarotriose from agarose, which is the main carbohydrate constituting red algae, via endo-type β -agarase and neoagarobiose hydrolase reactions, isolating and purifying only agarotriose among enzymatic reaction products using a size-exclusion chromatography (size-exclusion chromatography) technique, and testing the fermentation ability of probiotic bifidobacteria to agarotriose. In addition, since neoagarobiose produced by fermentation of agarotriose by bifidobacterium can be degraded into galactose and 3, 6-AHG by neoagarobiose hydrolase from bacteroides vulgatus (bacteroides) as an intestinal microorganism, and AHG is a bioactive substance having colon cancer preventive and anti-inflammatory effects, it was confirmed that agarotriose can be expected to have not only probiotic activity but also anticancer and anti-inflammatory biological activity such as metabolism via intestinal microorganisms.
Accordingly, the present invention provides a pharmaceutical composition comprising: one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose; common Bacteroides (Bacteroides plexius) strains; and a Bifidobacterium (Bifidobacterium) strain.
The usual Bacteroides (Bacteroides plexiius) strain may comprise the usual Bacteroides (Bacteroides plexiius) DSM17135 strain.
The Bifidobacterium (Bifidobacterium) strain may comprise Bifidobacterium longum subsp.sp.infantis ATCC 17930, Bifidobacterium longum subsp.infantis ATCC15702, Bifidobacterium bifidum (B.bifidum) DSM20082, Bifidobacterium bifidum (B.kashiwanensis) DSM21854, etc.
The pharmaceutical composition of the present invention is characterized in that the substrate is finally degraded into 3, 6-AHG by usual Bacteroides (bacilli) and Bifidobacterium (Bifidobacterium) strains.
More specifically, 3, 6-AHG was prepared by the method of identifying the amino acid sequence of SEQ ID NO: 1 (beta-agarase) and SEQ ID NO: 2 (neo-agarobiose hydrolase) and the sequence of SEQ ID NO: 3 or 4 (beta-galactosidase) from the substrate.
Beta-agarase is an enzyme derived from the usual Bacteroides species (Bacteroides plebeius) DSM17135, and agar, agarose or neoagarohexaose are used as substrates to degrade the substrates into neoagarotetraose and neoagarobiose, and can be represented by SEQ ID NO: 1 is shown.
Beta-agarase can be transcribed and transformed not only via regions preceding and following the coding region of the enzyme but also via DNA fragments which are associated with the production of polypeptides comprising intermediate sequences between the individual coding fragments. In addition, a protein having a hydrolytic activity of agar, agarose, or neoagarohexaose, which is a variant protein having one or more of substitution, deletion, transposition, addition, or the like of an enzyme, is also included in the scope of the enzyme of the present invention, and preferably includes a protein having a sequence similar to SEQ ID NO: 1, has a sequence identity of 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 93% or more than 93%, 94% or more than 94%, 95% or more than 95%, 96% or more than 96%, 97% or more than 97%, 98% or more than 98%, and 99% or more than 99%.
The beta-agarase may be isolated and purified from the supernatant of the culture of Bacteroides vulgaris (Bacteroides plebeius) DSM17135, and may be produced and isolated from strains other than Bacteroides vulgaris (Bacteroides plebeius) DSM17135 using genetic engineering recombinant technology, artificial chemical synthesis methods, and the like. When using a recombinant technique of genetic engineering, the β -agarase may be replaced by a supernatant or a supernatant fluid of a culture product of transformed escherichia coli, but the technique is not particularly limited thereto. According to a specific exemplary embodiment, β -agarase may be obtained from escherichia coli transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding β -agarase or a culture thereof.
Neoagarobiose hydrolase is an enzyme derived from the usual Bacteroides species (Bacteroides plebeius) DSM17135, and neoagarobiose or neoagarobiose is used as a substrate to degrade the substrate into agarotriose, galactose or 3, 6-anhydro-L-galactose, and can be represented by SEQ ID NO: 2 in the sequence listing.
Neoagarobiose hydrolase can be transcribed and transformed not only via regions preceding and following the coding region of neoagarobiose hydrolase but also via DNA fragments associated with the production of polypeptides comprising intervening sequences between the individual coding fragments. In addition, a protein having a hydrolytic activity of agar, agarose, or neoagarohexaose, which is a variant protein having one or more of substitution, deletion, transposition, addition, or the like of an enzyme, is also included in the scope of the enzyme of the present invention, and preferably includes a protein having a hydrolytic activity equivalent to SEQ ID NO: 2, has a sequence identity of 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 93% or more than 93%, 94% or more than 94%, 95% or more than 95%, 96% or more than 96%, 97% or more than 97%, 98% or more than 98%, and 99% or more than 99%.
The neoagarobiose hydrolase may be isolated and purified from the supernatant of the culture of Bacteroides (Bacteroides plexius) DSM17135, and may be produced and isolated from a strain other than Bacteroides (Bacteroides plexius) DSM17135 using genetic engineering recombinant technology, artificial chemical synthesis methods, or the like. When using a recombinant technique of genetic engineering, the neoagarobiose hydrolase may be replaced by a supernatant or supernatant fluid of a culture product of transformed E.coli, but the technique is not particularly limited thereto. According to specific exemplary embodiments, the neoagarobiose hydrolase may be obtained from escherichia coli transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding the neoagarobiose hydrolase, or a culture thereof.
β -galactosidase is an enzyme derived from Bifidobacterium longum subsp.infantus ATCC15697 and degrades agarotriose into neoagarobiose and galactose, is a protein produced from Blon _2016, Blon _2334 genes, and can be represented by SEQ ID NO: 3 or 4.
Beta-galactosidase can be transcribed and transformed not only via regions preceding and following the coding region of the enzyme but also via DNA fragments associated with the production of polypeptides comprising intermediate sequences between the individual coding fragments. In addition, a protein having a hydrolytic activity of agarotriose as a protein variant having one or more of substitution, deletion, transposition, addition, etc. of the enzyme is also included in the scope of the enzyme of the present invention, and preferably includes a protein having a sequence similar to SEQ ID NO: 3 or 4, or a sequence having a sequence identity of 80% or more than 80%, 85% or more than 85%, 90% or more than 90%, 93% or more than 93%, 94% or more than 94%, 95% or more than 95%, 96% or more than 96%, 97% or more than 97%, 98% or more than 98%, and 99% or more than 99%.
Beta-galactosidase can be isolated and purified from the supernatant of a culture of Bifidobacterium longum subsp. infantis ATCC15697, and can be produced and isolated from strains other than Bifidobacterium longum subsp. infantis ATCC15697 using genetic engineering recombination techniques, artificial chemical synthesis methods, and the like. When using a genetically engineered recombination technique, the β -galactosidase can be replaced by a supernatant or supernatant fluid of a culture product of transformed escherichia coli, but the technique is not particularly limited thereto. According to a specific exemplary embodiment, β -agarase may be obtained from escherichia coli transformed with a recombinant vector comprising a nucleic acid sequence of a gene encoding β -agarase or a culture thereof.
In the present specification, "protein" and "polypeptide" are used interchangeably.
In the present invention, the fact that a polypeptide has a specific proportion of sequence identity (e.g., 80%, 85%, 90%, 95%, or 99%) with another sequence means that when two sequences are aligned, the proportions of amino acid residues in the compared sequences are the same as each other. Alignment and percent homology or identity can be determined using those documents described in any suitable software program known in the art for public disclosure (e.g., the literature [ modern molecular BIOLOGY laboratory guidelines [ CURRENT PROTOCOLS inner search BIOLOGY, edition 1987, section 7.7.18 of the supplement 30) ] examples of preferred programs include the GCG accumulation program, FASTA (pilson et al, 1988, american academy of sciences journal 85: 2444-2448) and BLAST (BLAST handbook, attshul et al, american national center for biotechnology information, national library of medicine (NCIB NLMNIH), maryland, bessel, attshul et al, 1997, NAR 25: 33893402.) another preferred alignment program is align plus (and software, PA), and preferably another alignment program using basic parameters is the set of computer programs available in the educational program,
As used herein, the term "recombinant" when used in conjunction with a cell, nucleic acid, protein or vector indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of the original nucleic acid or protein, or that the cell is derived from a cell so modified. That is, for example, the recombinant cell expresses a gene that is not found within the original (non-recombinant) form of the cell, or alternatively, the recombinant cell expresses the original gene that is abnormally expressed or not expressed at all when expressed.
In the present specification, "nucleic acid" encompasses single-or double-stranded DNA and RNA as well as chemical variants thereof. "nucleic acid" and "polynucleotide" are used interchangeably in this application. Due to the degeneracy of the genetic code, one or more codons may be used in order to encode a particular amino acid, and the present invention encompasses polynucleotides encoding particular amino acid sequences.
The term "introduction" of a nucleic acid sequence into a cell refers to "transfection" or "transformation" and includes reference to the integration of the nucleic acid sequence into a eukaryotic or prokaryotic cell, and in this case the nucleic acid sequence is integrated into the genome of the cell (e.g., chromosome, plastid, chromosome, or mitochondrial DNA) and thereby converted into an autonomous replicon or transiently expressed.
The pharmaceutical composition of the present invention metabolizes agar, agarose, neoagarohexaose or agarotriose into 3, 6-AHG having anti-inflammatory and anti-cancer activities, and thus can be used for preventing or treating cancer or inflammatory diseases.
As used herein, the term "preventing" refers to all actions that inhibit or delay the onset of cancer or inflammatory disease by administering to a subject a pharmaceutical composition of the invention.
As used herein, the term "treating" refers to all actions that ameliorate or beneficially modify the symptoms of cancer or inflammatory disease by administering to a subject a pharmaceutical composition of the present invention.
As used herein, 'effective amount' refers to an amount of a compound capable of exhibiting an anti-cancer effect or inhibiting inflammation.
The cancer may be colon cancer, cervical cancer, breast cancer, gastric cancer, liver cancer, etc.
As used herein, 'anti-inflammatory effect' or 'anti-inflammatory activity' refers to inhibition of inflammation, and inflammation is one of the defensive responses of living tissue to a certain stimulus, and refers to complex injury involving three conditions: tissue degeneration, circulatory disorders and exudation and tissue proliferation. More specifically, inflammation is part of innate immunity, and human innate immunity recognizes cell surface patterns that are specifically present in pathogens. Phagocytes recognize cells with such surfaces as foreign bodies and attack pathogens. If the pathogen breaks through the physical barriers of the body, an inflammatory response occurs. The inflammatory response is a non-specific defensive action that creates an adverse environment for microorganisms that have invaded the wound site. In the inflammatory response, when a wound appears or an external pathogenic agent enters the body, leukocytes responsible for the immune response aggregate and express cytokines in the initial stage. Thus, the expression level of intracellular cytokines is an indicator of the activation of the inflammatory response.
Inflammatory diseases include general inflammatory symptoms such as edema, and may include inflammatory bowel disease, peritonitis, osteomyelitis, cellulitis, pancreatitis, traumatic shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, juvenile ankylosing spondylitis, reactive arthropathy, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, lyme disease, arthritis associated with' vasculitis syndrome ', polyarteritis nodosa, allergic vasculitis, graves' disease, inflammatory bowel disease, polymyalgia rheumatica, articular chondrocyte arteritis, calcium crystal deposition arthropathy, pseudogout, non-articular rheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow), neurotic arthropathy (also known as ' summer arthropathy '), hemarthrosis (hemarthrosic), allergic purpura, hypertrophic osteoarthropathy, multicentrical reticulocyte hyperplasia, scoliosis (scoliosis), hemochromatosis, hemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinemia, familial mediterranean fever (familial athereranean lesion), behcet's disease, systemic lupus erythematosus, regression fever, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ dysfunction syndrome, chronic obstructive pulmonary disease (chronic obstructive pulmonary disease), rheumatoid arthritis (chronic obstructive pulmonary disease), acute pulmonary injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease (chronic obstructive pulmonary disease), chronic obstructive pulmonary disease, and acute respiratory disease, Bronchopulmonary dysplasia (bronchus-pulmonary dysplasia), type 2 diabetes, arteriosclerosis, senile dementia of the alzheimer type, familial coldness-type autoinflammatory syndrome, Muckle-Wells syndrome, neonatal multiple system inflammatory disease (neonatal multiple system inflammation syndrome), chronic infant neurogenic skin joint syndrome (neonatal neurological cutaneous joint syndrome), adult Still disease (adult-in Still's disease), contact dermatitis, hydatidiform mole (hydatidiform mole), PAPA syndrome, acne and pyknosis (pyknosis of pyenothritis, pyknosis, synovitis vulgaris, hyperimmune cycle syndrome), cryptoperiod-related autoimmune diseases (cryptoperiod syndrome, etc.).
The pharmaceutical compositions of the invention may further comprise a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical field, and specific examples thereof include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum protein), buffer substances (e.g., various phosphoric acids, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, wool, and the like, but are not limited thereto.
In addition, the pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the aforementioned ingredients.
As one aspect, the pharmaceutical compositions of the present invention may be formulated and used in a variety of dosage forms suitable for oral or parenteral administration.
Non-limiting examples of formulations for oral administration include troches (troches), lozenges (lozenge), tablets, aqueous suspensions, oil suspensions, prepared powders, granules, emulsions, hard gelatin capsules, soft gelatin capsules, syrups, elixirs and the like.
To formulate the pharmaceutical compositions of the present invention for oral administration, binders such as lactose, sucrose, sorbitol, mannitol, starch, Gelatin starch (Amylopectin), Cellulose (Cellulose) or Gelatin (Gelatin); excipients, such as dicalcium phosphate (dicalcium phosphate); disintegrants, for example corn starch or sweet potato starch; lubricants such as Magnesium stearate (Magnesium stearate), Calcium stearate (Calcium stearate), sodium stearyl fumarate (sodium stearyl fumarate), or Polyethylene glycol wax (Polyethylene glycol wax), and the like, and sweetening agents, flavoring agents, syrups, and the like may also be used.
Further, in the case of capsules, a liquid carrier such as fatty oil may be further used in addition to the above-mentioned substances.
Non-limiting examples of formulations for parenteral administration include injections, suppositories, breath powders, aerosol sprays, oral sprays, mouth cleansers, toothpastes, ointments, powders for application, oils, creams and the like.
For the preparation of the pharmaceutical composition of the present invention for parenteral administration, a sterile aqueous solution, a nonaqueous solvent, a suspension, an emulsion, a lyophilized preparation, a medicament for external use, and the like may be used, and as the nonaqueous solvent and the suspension, propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethyl oleate, and the like may be used.
Further, more specifically, when the pharmaceutical composition of the present invention is formulated into an injection, the pharmaceutical composition of the present invention may be mixed with a stabilizer or a buffer in water to prepare a solution or a suspension, which is then formulated into a unit dosage form such as an ampoule (ampoule) or a vial (via). Further, when the pharmaceutical composition of the present invention is formulated into an aerosol, a propellant or the like may be mixed with an additive to disperse the water dispersible concentrate or the wet powder.
In addition, when the pharmaceutical composition of the present invention is formulated into ointments, creams, and the like, the pharmaceutical composition may be formulated using animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, and the like as a carrier.
The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may vary according to the formulation method, administration mode, administration schedule and/or administration route, etc., and may vary according to various factors including the type and degree of reaction to be achieved through administration of the pharmaceutical composition of the present invention, the type, age, body weight and general health of the subject to which the composition is administered, the symptoms or severity of the disease, sex, diet, excretion, drugs used simultaneously or at different times in the corresponding subject, the components of other compositions, etc., and similar factors well known in the medical field, and the effective dose of the desired treatment may be easily determined and prescribed by one of ordinary skill in the art. The pharmaceutical composition of the present invention may be administered once or several times daily. Thus, the dosage is not intended to limit the scope of the invention in any way.
The route of administration and mode of administration of the pharmaceutical composition of the present invention may be independent of each other, the method of administration is not particularly limited, and the route of administration and mode of administration may follow any route of administration and mode of administration as long as they enable the pharmaceutical composition to reach the targeted corresponding site. The pharmaceutical compositions may be administered orally or parenterally.
Parenteral administration may use, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, subcutaneous administration, etc., and a method for applying or spraying a pharmaceutical composition onto a disease site or inhaling a pharmaceutical composition may also be used, but the method is not limited thereto.
The pharmaceutical compositions of the present invention may preferably be administered orally or by injection.
The present invention also provides a method for treating cancer or an inflammatory disease, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition.
As used herein, the term "individual" refers to all animals including mammals, including rats, livestock, humans, and the like.
In the method for treating cancer or inflammatory diseases of the present invention, the description of the dose, administration route, administration mode, etc. of the pharmaceutical composition is the same as that described above for the pharmaceutical composition. In addition, the type of cancer or inflammatory disease is also the same as described above for the pharmaceutical composition.
The present invention also provides a food composition comprising: one or more substrates selected from the group consisting of agar, agarose, neoagarohexaose, and agarotriose; common Bacteroides (Bacteroides plexius) strains; and a Bifidobacterium (Bifidobacterium) strain.
The food composition may be prepared as a food preparation prepared by encapsulation, powdering, suspension, etc.
Since the food formulation can be taken daily, the food formulation can be expected to prevent or alleviate cancer or inflammatory diseases and is very useful.
The type of food is not particularly limited and includes, for example, dairy products, health foods in a typical sense, and the like.
The present invention also provides a method for preparing agarotriose, which comprises:
reacting the reaction product with SEQ ID NO: 2 or 6, wherein the reaction product is prepared by reacting any one of agar, agarose or neoagarohexaose with the sequence of SEQ ID NO: 1 or 5 by beta-agarase reaction; and
the agarotriose was purified from the resulting product via a size exclusion column.
In the case of the conventional process (a of fig. 13) in which the enzymatic hydrolysis is performed after the weak acid pretreatment, a large amount of salt is generated in the neutralization process, and when a low concentration neutral buffer is used, the pretreatment reaction needs to be performed at a high temperature (170 ℃), thereby making a high temperature and high pressure reactor necessary. In addition, the production yield of agarotriose is rather low by focusing on improving the production yield of 3, 6-AHG, and in particular, acetic acid used for pretreatment generates an unpleasant odor. For these reasons, there may be problems when agarotriose is used as a probiotic substance. Therefore, the method for preparing agarotriose of the present invention solves the above problems by the following method.
First, a pretreatment process is omitted by using β -agarase, which generally produces neoagarotetraose as a precursor of agarotriose during its production, and high yields of agarotriose, 3, 6-AHG and D-Gal are obtained via two-step enzymatic reactions (that is, endo- β -agarase, neoagarobiose hydrolase) under mild conditions.
Next, during the purification of agarotriose, monosaccharide and trisaccharide were separated using Size exclusion chromatography technique via Size exclusion Bio P2 Gel column (Size exclusion Bio-P2 Gel column) using the difference in polymerization degree, thereby obtaining high purity agarotriose (B of fig. 13). Since this purification process uses water as a mobile phase, does not use organic solvents harmful to the human body, and there is little loss of agarotriose during the purification process, the purification process has an advantage in that purified high-yield agarotriose can be obtained.
Beta-agarase degrades any one of the substrates agar, agarose or neoagarohexaose into neoagarobiose and neoagarotetraose, which are neoagaro-oligosaccharides, and the following can be used, which are derived from the above ordinary bacteroides (Bacteroidesplebeius) DSM17135 strain, SEQ ID NO: 1, or the beta-agarase of SEQ ID NO: 5 using agar or agarose as a substrate to degrade the substrate into neoagarotetraose and neoagarohexaose.
The thermostable agarase can be derived from polysaccharide-degrading bacteria (Saccharomyces degradans)2-40TBut is not particularly limited thereto.
Thermostable agarase can be extracted from polysaccharide degrading bacteria (Saccharomyces degradans)2-40TSeparating and purifying the culture product supernatant, and using genetic engineering recombination technology, artificial chemical synthesis method, etc. to remove polysaccharide from polysaccharide-degrading bacteria (Saccharomyces degradans)2-40TProduction and isolation in other strains. When using a genetic engineering recombination technique, the heat-resistant agarase may be replaced by a supernatant or supernatant fluid of a culture product of transformed escherichia coli, but the technique is not particularly limited thereto.
The reaction of any one substrate of agar, agarose or neoagarohexaose with β -agarase may be carried out at a temperature of 30 to 60 ℃ for 5 minutes to 12 hours at 0 to 200 rpm.
Neoagarobiose hydrolase degrades neoagarobiose and neoagarotetraose into 3, 6-AHG, D-Gal and agarotriose, and the peptide of SEQ ID NO: 2, or 2-40 neoagarobiose hydrolase derived from polysaccharide degrading bacteria (Saccharomyces degradans) can be usedTα -neoagarobiose hydrolase of SEQ ID NO. 6.
Polysaccharide degrading bacteria (Saccharomyces degradans)2-40TThe derivative α -neoagarobiose hydrolase can be derived from polysaccharide degrading bacteria (Saccharomyces degradans)2-40TThe culture product of (a) is isolated and purified from the supernatant or supernatant fluid, and can be isolated and purified from polysaccharide-removing degrading bacteria (Saccharomyces degradans)2 to 40 using genetic engineering recombination techniques, artificial chemical synthesis methods, or the likeTProduction and purification in other strains.
The reaction of the reaction product of β -agarase with neoagarobiose hydrolase may be carried out at a temperature of 25 ℃ to 45 ℃ for 30 minutes to 12 hours at 0rpm to 200 rpm.
After obtaining the
Hereinafter, the present invention will be described in more detail via examples according to the present invention, but the scope of the present invention is not limited by the examples set forth below.
Modes for carrying out the invention
< example 1> experiment for degrading agarose by beta-agarase
The enzymatic reaction is performed to produce agar-derived oligosaccharides at different degrees of polymerization, comprising agarotriose from agarose, which is the main carbohydrate constituting red algae. First, 1% (w/v) concentration of agarose was used as a substrate to perform an enzymatic reaction of Aga16B, which Aga16B is a polysaccharide-degrading bacterium (s.degradans) 2-40T-derived endo-agarase. In this case, the enzymatic reaction was carried out at 50 ℃ and 200rpm for 2 hours.
As a result of the enzymatic reaction, neoagarotetraose (DP4) and neoagarohexaose (DP6) were produced (B of fig. 2).
Agarotriose (DP3), agaropentaose (DP5) and 3, 6-AHG were produced via the enzymatic reaction of the next enzyme, polysaccharide degrading bacteria (s. degradans)2-40T derived neoagarobiose hydrolase (SdNABH) and the reaction product as substrate. The SdNABH enzymatic reaction is carried out at 30 ℃ and 200rpm for 2 hours.
In addition, disaccharide neoagarobiose (DP2) was produced via an enzymatic reaction of Aga50D, polysaccharide degrading bacteria (s.degradans) 2-40T-derived exoagarase, and Aga16B reaction product as a substrate. The Aga50D enzymatic reaction was carried out at 30 ℃ and 200rpm for 2 hours.
Next, the enzymatic reaction conditions of BpGH16A (BACPLE-01670) as an endo-type β -agarase derived from the intestinal microorganism, Bacteroides plexius, DSM17135 were as follows: enzyme loading: 8 mg of BpGH16A per g of agarose; buffer solution: 20 mM Tris-HCl (pH 7.0); and reaction temperature and time: 40 ℃ and 2 hours.
The enzymatic reaction conditions for the neoagarobiose hydrolase BpGH117(BACPLE _01671) were as follows: enzyme loading: 4 mg of BpGH117 per gram of neoagarobiose; buffer solution: 20 mM Tris-HCl (pH 7.0); and reaction temperature and time: 40 ℃ and 2 hours.
As shown in C of fig. 2, agarotriose and 3, 6-AHG can be produced from agarose via a combination of reactions that degrade related enzymes via agar derived from the gut microorganism Bacteroides vulgatus (Bacteroides plebeius) DSM 17135. As a result of analyzing the reaction product by TLC (thin layer chromatography), neoagarotetraose (DP4) was produced as a main product (lane 2) from an agarose substrate (lane 1) via a BpGH16A (BACPLE _01670) enzymatic reaction. Thereafter, agarotriose and 3, 6-AHG were produced via a BpGH117 (BACPLE-01671) enzymatic reaction (lane 2). Even when the two enzymes were reacted simultaneously, agarotriose and 3, 6-AHG were produced mainly in the same manner as when the two enzymes were reacted sequentially (lane 3).
< example 2> recombinant and enzymatic reaction experiments of endo-type beta-agarases BpGH16A (BACPLE _01670) and BpGH50(BACPLE _01683) derived from Bacteroides vulgaris (Bacteroides plebeius) DSM17135 with Neoagarobiose hydrolase BpGH117(BACPLE _01671)
As shown in fig. 3, in the case of the ordinary Bacteroides (Bacteroides plebeius) DSM 17135-derived GH50 family enzyme BpGH50(BACPLE _01683), no β -agarase activity was exhibited.
< example 3> experiment for production of agarotriose and AHG from agarose substrate by enzymatic reaction of endo-type beta-agarase BpGH16A (BACPLE-01670) derived from usual Bacteroides (Bacteroides plebeius) DSM17135 with neoagarobiose hydrolase BpGH117 (BACPLE-01671)
Oligosaccharides, neoagarobiose and 3, 6-AHG at different degrees of polymerization produced via reaction of the respective purified recombinant enzymes Aga16B, Aga50D and SdNABH were purified by size exclusion column chromatography. In this case, Sephadex G-10 was used as column resin for size exclusion column chromatography.
Agarotriose and 3, 6-AHG were produced from agarose via reaction of endo-beta-agarase BpGH16A (BACPLE _01670) with neoagarobiose hydrolase BpGH117(BACPLE _01671) as shown in fig. 4.
< example 4> experiment for culturing probiotic microorganism Bifidobacterium longum subsp.infantis ATCC15697 strain by using biogrid C with correspondingly purified sugar as carbon source
To demonstrate the probiotic effect of agar-derived sugars, each purified sugar containing agarotriose was used as a single carbon source to monitor cell growth of Bifidobacterium longum infantis (Bifidobacterium longum subsp. In this case, as the culture composition, 10 g/L of bactopeptone, 5 g/L of yeast extract, 2 g/L of K were used2HPO4Acid anhydride, 5 g/l sodium acetate acid anhydride, 2 g/l citric acid ternary NH40.2 g/l magnesium sulfate heptahydrate, 0.05 g/l manganese sulfate, 1 ml/l tween 80 (polysorbate 80), 0.5 g/l cysteine, and 5 g/l each purified sugar, and culturing was performed at 37 ℃.
As shown in fig. 5, it was confirmed that Bifidobacterium longum infantis ATCC15697 strain selectively fermented only agarotriose among various purified sugars.
< example 5> analysis of agarotriose fermentation curve of Bifidobacterium longum subsp
To monitor the fermentation product, bifidobacterium longum subspecies infantis ATCC15697 strain was cultured under test tube conditions.
As shown in fig. 6, Bifidobacterium longum infantis ATCC15697 degraded agarotriose to galactose and neoagarobiose under a carbon source at a concentration of 5 g/l agarotriose, and galactose was fermented in cells to produce acetic acid. Neoagarobiose is secreted and accumulates extracellularly and is no longer degraded in the cell.
< example 6> degradation experiment of agarotriose and neoagarobiose using crude enzyme solution of Bifidobacterium longum subsp
To confirm the metabolic pathway of agarotriose, crude enzyme solutions of bifidobacterium longum subsp. For this purpose, cells and culture medium were separated by centrifuging (14,000rpm, 5 minutes, 4 ℃) a culture solution of Bifidobacterium longum subsp. Crude extracellular enzyme was obtained by ammonium sulfate precipitation of the supernatant. In addition, for the cell-free extract containing crude intracellular enzymes, crude supernatant enzymes were obtained by resuspending the cells in 20 mM Tris-HCl buffer, then lysing the cells by sonication and centrifuging the lysate. During the crude enzyme experiment, 2 mg/ml of crude enzyme and 2 mg/ml of agarotriose were used as substrates to conduct the reaction in 20 mmol of Tris-HCl buffer (pH7.0) at 30 ℃ and 200rpm for 2 hours.
As shown in fig. 7, it was confirmed that β -galactosidase reaction degrading agarotriose into galactose and neoagarobiose occurred in the cell-free extract containing crude intracellular enzyme. In addition, it was confirmed that the activity of the crude enzyme on neoagarobiose was not exhibited.
< example 7> production of recombinant proteins and enzymatic reaction experiments of four beta-galactosidase-encoding genes (Blon _2016, Blon _2123, Blon _2334, and Blon _2416) of Bifidobacterium longum subsp
To confirm which enzyme gene has the activity of degrading agarotriose, the enzyme activity was tested by overexpressing four β -galactosidases in escherichia coli and purifying each enzyme protein after cloning all four β -galactosidases of Bifidobacterium longum subsp.
As shown in FIG. 8, the protein expression activities of two enzyme genes, Blon _2016 and Blon _2334, were confirmed.
< example 8> experiment of fermentation ability of other strains belonging to Bifidobacterium longum subsp
To confirm whether agarotriose has a probiotic effect on probiotic microorganisms other than Bifidobacterium longum subsp. infantis ATCC15697 strain, Bifidobacterium longum subsp. infantis ATCC 17930, Bifidobacterium longum subsp. infantis ATCC15702, Bifidobacterium bifidum (b.bifidum) DSM20082, and Bifidobacterium bailii (b.kashiwanoheense) DSM21854, culture was performed under agarotriose monocarbon source conditions.
As shown in fig. 9 and 10, all four probiotic microorganisms metabolize agarotriose and degrade agarotriose into galactose and neoagarobiose in the same manner as Bifidobacterium longum subsp.
< example 9> stability test of agarotriose to artificial gastric juice
To test whether agarotriose can reach the intestinal tract without degradation, agarotriose was reacted in artificial gastric fluid each time, and then TLC and HPLC were used to confirm whether agarotriose was degraded.
As shown in fig. 11, it was confirmed that 80% or more than 80% of agarotriose remained after the reaction at 37 ℃ for 3 hours.
According to the results, it was first confirmed that agarotriose is a novel red alga-derived probiotic and a substance selectively fermented by the probiotic. Agarotriose can be produced by the enzymatic action of agarase and NABH from marine-derived polysaccharide-degrading bacteria (s. degradans)2-40T or enteromicrobially-derived Bacteroides vulgatus (Bacteroides plexius) (fig. 12). Agarotriose is transported into cells by ABC transporter-associated genes of probiotic bifidobacteria and is degraded into galactose and neoagarobiose by intracellular β -galactosidase, and galactose is used for acetic acid fermentation. In addition, neoagarobiose can be degraded in the intestinal tract to galactose and 3, 6-AHG by neoagarobiose hydrolase from the intestinal microorganism, Bacteroides distabilis (Bacteroides spobeius). Since AHG is referred to as a bioactive substance having a colon cancer preventing effect and an anti-inflammatory function, agarotriose can exhibit not only probiotic activity but also colon cancer preventing and anti-inflammatory bioactivity due to AHG produced via metabolism by intestinal microorganisms bifidobacterium and Bacteroides vulgaris (Bacteroides plexius) (fig. 12).
< example 10> production of BACPLE _01670 and NABH recombinase
The β -agarase hydrolase BACPLE _01670 gene derived from Bacteroides (Bacteroides plexius) was introduced into E.coli (E.coli) BL21(DE3) using pET21a vector. For preculture (pre-culture) of the gene-introduced recombinant E.coli, the recombinant E.coli was cultured in a50 ml conical tube at 37 ℃ for 9 hours in 10 ml of LB liquid medium containing 100. mu.g/ml of ampicillin (ampicillin). Thereafter, after inoculating 10 ml of the preculture solution into 1 liter of the main culture solution having the same medium composition, when an Optical density value (Optical density spectrum) shows a growth to metaphase index step (OD 0.4 to 0.6), 0.1 mmol of isopropyl- β -D-thiogalactoside (IPTG) was added to the main culture solution, and the intracellular protein was expressed by induction at 16 ℃ for 16 hours. Thereafter, the cell culture solution was transferred to a 500 ml test tube and centrifuged at 10,000rpm at 4 ℃ for 30 minutes, and then cells were obtained. To prevent protein denaturation, cells collected in 30 ml of Tris buffer (20 mmol Tris-HCl, pH7.0) were released again and lysed using a sonicator (sonicator) (cell lysis). Thereafter, the cells were centrifuged at 16,000rpm at 4 ℃ for 1 hour. Proteins were purified using a HisTrap column (5 ml, ge healthcare group), and the size of each purified protein was confirmed using SDS-PAGE gel. The salt (imidazole) used for protein purification was removed using a desalting column. The concentration of recombinant protein with salt removed was quantified by BCA assay.
Next, polysaccharide-degrading bacteria (Saccharomyces degradans)2 to 40 were inoculated using pET21a vectorTThe derived α -neoagarobiose hydrolase NABH gene was introduced into e.coli (e.coli) BL21(DE3) and recombinant proteins were prepared as described above.
< example 11> enzymatic reaction of BACPLE _01670 with NABH
During the BACPLE-01670 enzymatic reaction, 1% (w/v) concentration of agarose was used as a substrate, and the reaction was carried out in 20 mM Tris-HCl buffer (pH7.0) at 50 ℃ and 100rpm for 10 hours.
The NABH enzymatic reaction was carried out using the BACPLE-01670 enzymatic reaction products neoagarotetraose and neoagarobiose as substrates, and the enzymatic reaction was carried out at 37 ℃ and 100rpm for 10 hours.
The reaction products after the enzymatic reaction in each step were analyzed by TLC. For TLC analysis conditions, 1. mu.l of the enzymatic reaction product was loaded onto a silica gel plate as stationary phase, and n-butanol at 3: 1 (v/v/v): ethanol: water was eluted for 1 hour as a mobile phase solvent, and then developed using 10% sulfuric acid in ethanol and 0.2% 1, 3-dihydroxynaphthalene in ethanol.
As shown in a of fig. 14, agarose was degraded into neoagaroose via the enzymatic reaction of endo-type β -agarase BACPLE _01670, and in this case, the main products were neoagarobiose and neoagarotetraose corresponding to DP2 and DP4 as Degrees of Polymerization (DP). Thereafter, trisaccharide agarotriose and 3, 6-AHG were produced from DP4, and D-galactose and 3, 6-AHG were produced from DP2, via an enzymatic reaction of α -agarase neoagarobiose hydrolase (NABH).
< example 12> HPLC analysis of BACPLE _01670 enzymatic reaction product
The substances produced during the BACPLE _01670 enzymatic reaction were neoagarotetraose (DP4) and neoagarobiose (DP2) (B of fig. 14). Among them, the precursor for making agarotriose is neoagarotetraose, and the more DP4 product, the more agarotriose can be obtained. To determine the yield of DP4 relative to BACPLE _01670, the yield was calculated using an HPLC KS-802 size exclusion column.
As shown in fig. 15, with respect to the reaction product of BACPLE _01670, results of 0.795 g of neoagarobiose (0.795 g of neoagarobiose per gram of agarose) and 0.205 g of neoagarobiose (0.205 g of neoagarobiose per gram of agarose) were obtained with respect to 1 g of agarose.
< example 13> separation of DP3 (agarotriose) and DP1(3, 6-AHG, D-Gal) using a size exclusion column (Bio-gel P2 column)
The final reaction products obtained in examples 10 and 11 were 3, 6-AHG and D-Gal and agarotriose, and the 3, 6-AHG and D-Gal therein were separated from the agarotriose using a sugar separation column. The machine AKTAprime (general electric medical group) was used. For the mobile phase used for separation of sugars, the column was stabilized for 10 minutes by flowing three times treated Distilled water (Distilled water) at a flow rate of 0.3 ml/min, and then 1 ml of a solution flowing through the column after injection of 2 ml of the reaction product was transferred with a 2 ml eppendorf tube and analyzed via TLC. TLC analysis conditions were the same as those in example 11.
As shown in FIG. 16, it was confirmed that DP3 (agarotriose) and DP1(3, 6-AHG, D-Gal) were separated as a result of analyzing the partially collected samples by TLC.
< example 14> yield and purity of agarotriose in agarose by enzymatic saccharification quantitative analysis by HPLC and separation by size exclusion column
After confirming the agarotriose obtained by the sugar separation column in example 13 by TLC, a fraction having high purity of agarotriose was collected.
As shown in fig. 17, it was confirmed that 0.4 g of agarotriose (0.4 g of NAB per gram of agarose) was obtained from 1 g of agarose, and that about 90.2% of the samples were agarotriose.
INDUSTRIAL APPLICABILITY
The present invention can be used as an anti-cancer or anti-inflammatory agent in the food and pharmaceutical fields based on the probiotic properties of agarotriose.
<110> university school labor cooperation group of Korean university
<120> method for producing agarotriose and use thereof as probiotic
<130>X18U13C0184
<150>KR10-2017-0153350
<151>2017-11-16
<160>6
<170>KopatentIn 2.0
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<213> Bacteroides vulgaris DSM17135
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<213> Bacteroides vulgaris DSM17135
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<210>3
<211>691
<212>PRT
<213> Bifidobacterium longum subspecies infantis ATCC15697
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His Tyr Lys Asp Asn Pro Tyr Val Val Ser Trp His Val Ser Asn Glu
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Asp Ala Trp Gly Thr Ala Phe Trp Ala Gln Arg Met Asn Asn Phe Ser
195 200 205
Glu Ile Ile Pro Pro Arg Phe Ile Gly Asp Gly Asn Phe Met Asn Pro
210 215 220
Gly Lys Leu Leu Asp Trp Lys Arg Phe Ser Ser Asp Ala Leu Leu Asp
225 230 235 240
Phe Tyr Lys Ala Glu Arg Asp Ala Leu Leu Glu Ile Ala Pro Lys Pro
245 250 255
Gln Thr Thr Asn Phe Met Val Ser Ala Gly Cys Thr Val Leu Asp Tyr
260 265 270
Asp Lys Trp Gly His Asp Val Asp Phe Val Ser Asn Asp His Tyr Phe
275 280 285
Ser Pro Gly Glu Ala His Phe Asp Glu Met Ala Tyr Ala Ala Cys Leu
290 295 300
Thr Asp Gly Ile Ala Arg Lys Asn Pro Trp Phe Leu Met Glu His Ser
305 310 315 320
Thr Ser Ala Val Asn Trp Arg Pro Thr Asn Tyr Arg Leu Glu Pro Gly
325 330 335
Glu Leu Val Arg Asp Ser Leu Ala His Leu Ala Met Gly Ala Asp Ala
340 345 350
Ile Cys Tyr Phe Gln Trp Arg Gln Ser Lys Ala Gly Ala Glu Lys Trp
355 360 365
His Ser Ala Met Val Pro His Ala Gly Pro Asp Ser Gln Ile Phe Arg
370 375 380
Asp Val Cys Glu Leu Gly Ala Asp Leu Asn Lys Leu Ala Asp Glu Gly
385 390 395 400
Leu Leu Ser Thr Lys Leu Val Lys Ser Lys Val Ala Ile Val Phe Asp
405 410 415
Tyr Glu Ser Gln Trp Ala Thr Glu His Thr Ala Thr Pro Thr Gln Glu
420 425 430
Val Arg His Trp Thr Glu Pro Leu Asp Trp Phe Arg Ala Leu Ala Asp
435 440 445
Asn Gly Leu Thr Ala Asp Val Val Pro Val Arg Gly Pro Trp Asp Glu
450 455 460
Tyr Glu Ala Val Val Leu Pro Ser Leu Ala Ile Leu Ser Glu Gln Thr
465 470 475 480
Thr Arg Arg Val Arg Glu Tyr Val Ala Asn Gly Gly Lys Leu Phe Val
485 490 495
Thr Tyr Tyr Thr Gly Leu Val Asp Asp Arg Asp His Val Trp Leu Gly
500 505 510
Gly Tyr Pro Gly Ser Ile Arg Asp Val Val Gly Val Arg Val Glu Glu
515 520 525
Phe Ala Pro Met Gly Thr Asp Ala Pro Gly Thr Met Asp His Leu Asp
530 535 540
Leu Asp Asn Gly Thr Val Ala His Asp Phe Ala Asp Val Ile Thr Ser
545 550 555 560
Val Ala Asp Thr Ala His Val Val Ala Ser Phe Lys Ala Asp Lys Trp
565 570 575
Thr Gly Phe Asp Gly Ala Pro Ala Ile Thr Val Asn Asp Phe Gly Asp
580 585 590
Gly Lys Ala Ala Tyr Val Gly Ala Arg Leu Gly Arg Glu Gly Leu Ala
595 600 605
Lys Ser Leu Pro Ala Leu Leu Glu Glu Leu Gly Ile Glu Thr Ser Ala
610 615 620
Glu Asp Asp Arg Gly Glu Val Leu Arg Val Glu Arg Ala Asp Glu Thr
625 630 635 640
Gly Glu Asn His Phe Val Phe Leu Phe Asn Arg Thr His Asp Val Ala
645 650 655
Val Val Asp Val Glu Gly Glu Pro Leu Val Ala Ser Leu Ala Gln Val
660 665 670
Asn Glu Ser Glu His Thr Ala Ala Ile Gln Pro Asn Gly Val Leu Val
675 680 685
Val Lys Leu
690
<210>4
<211>1023
<212>PRT
<213> Bifidobacterium longum subspecies infantis ATCC15697
<400>4
Met Thr Asp Val Thr His Val Asp Arg Ala Ser Gln Ala Trp Leu Thr
1 5 10 15
Asp Pro Thr Val Phe Glu Val Asn Arg Thr Pro Ala His Ser Ser His
20 25 30
Lys Trp Tyr Ala Arg Asp Pro Gln Ser Gly Gln Trp Ser Asp Leu Lys
35 40 45
Gln Ser Leu Asp Gly Glu Trp Arg Val Glu Val Val Gln Ala Ala Asp
50 55 60
Ile Asn Leu Glu Glu Glu Pro Ala Thr Ala Glu Ser Phe Asp Asp Ser
65 70 75 80
Ser Phe Glu Arg Ile Gln Val Pro Gly His Leu Gln Thr Ala Gly Leu
85 90 95
Met Asn His Lys Tyr Val Asn Val Gln Tyr Pro Trp Asp Gly His Glu
100 105 110
Asn Pro Leu Glu Pro Asn Ile Pro Glu Asn Asn His Val Ala Leu Tyr
115 120 125
Arg Arg Lys Phe Thr Val Ser Ala Pro Val Ala Asn Ala Lys Gln Ala
130 135 140
Gly Gly Ser Val Ser Ile Val Phe His Gly Met Ala Thr Ala Ile Tyr
145 150 155 160
Val Trp Val Asn Gly Ala Phe Val Gly Tyr Gly Glu Asp Gly Phe Thr
165 170 175
Pro Asn Glu Phe Asp Ile Thr Gly Leu Leu His Asp Gly Glu Asn Val
180 185 190
Val Ala Val Ala Cys Tyr Glu Tyr Ser Ser Ala Ser Trp Leu Glu Asp
195 200 205
Gln Asp Phe Trp Arg Leu His Gly Leu Phe Arg Ser Val Glu Leu Ala
210 215 220
Ala Arg Pro His Val His Ile Glu Asn Thr Gln Ile Glu Ala Asp Trp
225 230 235 240
Asp Pro Glu Ala Gly Thr Ala Ser Leu Asp Ala Ala Leu Thr Val Leu
245250 255
Asn Ala Thr Asp Ala Ala Thr Val Arg Ala Thr Leu Lys Asp Ala Asp
260 265 270
Gly Asn Thr Val Trp Gln Thr Thr Gly Asp Ala Glu Ala Gln Thr Ala
275 280 285
Leu Ser Ser Gly Pro Leu Gln Gly Ile Glu Pro Trp Ser Ala Glu Ser
290 295 300
Pro Thr Leu Tyr Glu Leu Asp Val Asp Val Ile Asp Gln Ala Gly Asp
305 310 315 320
Val Ile Glu Cys Thr Ser Gln Lys Val Gly Phe Arg Arg Phe Arg Ile
325 330 335
Glu Asp Gly Ile Leu Thr Ile Asn Gly Lys Arg Ile Val Phe Lys Gly
340 345 350
Ala Asp Arg His Glu Phe Asp Ala Glu Arg Gly Arg Ala Ile Thr Glu
355 360 365
Gln Asp Met Ile Asp Asp Val Val Phe Cys Lys Arg His Asn Ile Asn
370 375 380
Ser Ile Arg Thr Ser His Tyr Pro Asn Gln Glu Arg Trp Tyr Glu Leu
385 390 395 400
Cys Asp Glu Tyr Gly Ile Tyr Leu Ile Asp Glu Thr Asn Leu Glu Ala
405410 415
His Gly Ser Trp Ser Leu Pro Gly Asp Val Leu Thr Glu Asp Thr Ile
420 425 430
Val Pro Gly Ser Lys Arg Glu Trp Glu Gly Ala Cys Val Asp Arg Val
435 440 445
Asn Ser Met Met Arg Arg Asp Tyr Asn His Pro Ser Val Leu Ile Trp
450 455 460
Ser Leu Gly Asn Glu Ser Tyr Val Gly Asp Val Phe Arg Ala Met Tyr
465 470 475 480
Lys His Val His Asp Ile Asp Pro Asn Arg Pro Val His Tyr Glu Gly
485 490 495
Val Thr His Asn Arg Asp Tyr Asp Asp Val Thr Asp Ile Glu Thr Arg
500 505 510
Met Tyr Ser His Ala Asp Glu Ile Glu Lys Tyr Leu Lys Asp Asp Pro
515 520 525
Lys Lys Pro Tyr Leu Ser Cys Glu Tyr Met His Ala Met Gly Asn Ser
530 535 540
Val Gly Asn Met Asp Glu Tyr Thr Ala Leu Glu Arg Tyr Pro Lys Tyr
545 550 555 560
Gln Gly Gly Phe Ile Trp Asp Phe Ile Asp Gln Ala Ile Tyr Ala Thr
565 570 575
Gln Pro Asp Gly Thr Arg Ser Leu Arg Tyr Gly Gly Asp Phe Gly Asp
580 585 590
Arg Pro Ser Asp Tyr Glu Phe Ser Gly Asp Gly Leu Leu Phe Ala Asp
595 600 605
Arg Lys Pro Ser Pro Lys Ala Gln Glu Val Lys Gln Leu Tyr Ser Asn
610 615 620
Val His Ile Asp Val Thr Lys Asp Ser Val Ser Val Lys Asn Asp Asn
625 630 635 640
Leu Phe Thr Ala Thr Gly Asp Tyr Val Phe Val Leu Ser Val Leu Ala
645 650 655
Asp Gly Lys Pro Val Trp Gln Ser Thr Arg Arg Phe Asp Val Pro Ala
660 665 670
Gly Glu Thr Arg Thr Phe Asp Val Ala Trp Pro Val Ala Ala Tyr Arg
675 680 685
Ala Asp Ala Arg Glu Leu Val Leu Gln Val Ser Gln Arg Leu Ala Lys
690 695 700
Ala Thr Asp Trp Ala Glu Ser Gly Tyr Glu Leu Ala Phe Gly Gln Ala
705 710 715 720
Val Val Pro Ala Asp Ala Thr Ala Thr Pro Asp Thr Lys Pro Ala Asp
725 730735
Gly Thr Ile Thr Val Gly Arg Trp Asn Ala Gly Val Arg Gly Ala Gly
740 745 750
Arg Glu Val Leu Leu Ser Arg Thr Gln Gly Gly Met Val Ser Tyr Thr
755 760 765
Phe Ala Gly Asn Glu Phe Val Leu Arg Arg Pro Ala Ile Thr Thr Phe
770 775 780
Arg Pro Leu Thr Asp Asn Asp Arg Gly Ala Gly His Gly Phe Glu Arg
785 790 795 800
Val Gln Trp Leu Gly Ala Gly Arg Tyr Ala Arg Cys Val Asp Asn Val
805 810 815
Leu Glu Gln Ile Asp Asp Ser Thr Leu Lys Gly Thr Tyr Thr Tyr Glu
820 825 830
Leu Ala Thr Ala Gln Arg Thr Lys Val Thr Val Ser Tyr Thr Ala His
835 840 845
Thr Asp Gly Arg Val Asn Leu His Val Glu Tyr Pro Gly Glu Gln Gly
850 855 860
Asp Leu Pro Thr Ile Pro Ala Phe Gly Ile Glu Trp Thr Leu Pro Val
865 870 875 880
Gln Tyr Thr Asn Leu Arg Phe Phe Gly Thr Gly Pro Glu Glu Thr Tyr
885 890895
Leu Asp Arg Lys His Ala Lys Leu Gly Val Trp Asn Thr Asn Ala Phe
900 905 910
Ala Asp His Ala Pro Tyr Leu Met Pro Gln Glu Thr Gly Asn His Glu
915 920 925
Asp Val Arg Trp Ala Glu Ile Thr Asp Asp His Gly His Gly Met Arg
930 935 940
Val Ser Arg Ala Asp Gly Ala Ala Pro Phe Ala Val Ser Leu Leu Pro
945 950 955 960
Tyr Ser Ser Phe Met Leu Glu Glu Ala Gln His Gln Asp Glu Leu Pro
965 970 975
Lys Pro Lys His Met Phe Leu Arg Val Leu Ala Ala Gln Met Gly Val
980 985 990
Gly Gly Asp Asp Ser Trp Met Ser Pro Val His Pro Gln Tyr His Ile
995 1000 1005
Pro Ala Asp Lys Pro Ile Ser Leu Asp Val Asp Leu Glu Leu Ile
1010 1015 1020
<210>5
<211>598
<212>PRT
<213> polysaccharide-degrading bacterium 2-40
<400>5
Met Lys Thr Thr Lys Cys Ala Leu Ala Ala Leu Phe Phe Ser Thr Pro
1 5 10 15
Leu Met Ala Ala Asp Trp Asp Gly Ile Pro Val Pro Ala Asp Pro Gly
20 25 30
Asn Gly Asn Thr Trp Glu Leu Gln Ser Leu Ser Asp Asp Phe Asn Tyr
35 40 45
Ala Ala Pro Ala Asn Gly Lys Ser Thr Thr Phe Tyr Ser Arg Trp Ser
50 55 60
Glu Gly Phe Ile Asn Ala Trp Leu Gly Pro Gly Gln Thr Glu Phe Tyr
65 70 75 80
Gly Pro Asn Ala Ser Val Glu Gly Gly His Leu Ile Ile Lys Ala Thr
85 90 95
Arg Lys Pro Gly Thr Thr Gln Ile Tyr Thr Gly Ala Ile His Ser Asn
100 105 110
Glu Ser Phe Thr Tyr Pro Leu Tyr Leu Glu Ala Arg Thr Lys Ile Thr
115 120 125
Asn Leu Thr Leu Ala Asn Ala Phe Trp Leu Leu Ser Ser Asp Ser Thr
130 135 140
Glu Glu Ile Asp Val Leu Glu Ser Tyr Gly Ser Asp Arg Ala Thr Glu
145 150 155 160
Thr Trp Phe Asp Glu Arg Leu His Leu Ser His His Val Phe Ile Arg
165 170 175
Gln Pro Phe Gln Asp Tyr Gln Pro Lys Asp Ala Gly Ser Trp Tyr Pro
180 185 190
Asn Pro Asp Gly Gly Thr Trp Arg Asp Gln Phe Phe Arg Ile Gly Val
195 200 205
Tyr Trp Ile Asp Pro Trp Thr Leu Glu Tyr Tyr Val Asn Gly Glu Leu
210 215 220
Val Arg Thr Val Ser Gly Pro Glu Met Ile Asp Pro Tyr Gly Tyr Thr
225 230 235 240
Asn Gly Thr Gly Leu Ser Lys Pro Met Gln Val Ile Phe Asp Ala Glu
245 250 255
His Gln Pro Trp Arg Asp Glu Gln Gly Thr Ala Pro Pro Thr Asp Ala
260 265 270
Glu Leu Ala Asp Ser Ser Arg Asn Gln Phe Leu Ile Asp Trp Val Arg
275 280 285
Phe Tyr Lys Pro Val Ala Ser Asn Asn Gly Gly Gly Asp Pro Gly Asn
290 295 300
Gly Gly Thr Pro Gly Asn Gly Gly Ser Gly Asp Thr Val Val Val Glu
305 310 315 320
Met Ala Asn Phe Ser Ala Thr Gly Lys Glu Gly Ser Ala Val Ala Gly
325 330 335
Asp Thr Phe Thr Gly Phe Asn Pro Ser Gly Ala Asn Asn Ile Asn Tyr
340 345 350
Asn Thr Leu Gly Asp Trp Ala Asp Tyr Thr Val Asn Phe Pro Ala Ala
355 360 365
Gly Asn Tyr Thr Val Asn Leu Ile Ala Ala Ser Pro Val Thr Ser Gly
370 375 380
Leu Gly Ala Asp Ile Leu Val Asp Ser Ser Tyr Ala Gly Thr Ile Pro
385 390 395 400
Val Ser Ser Thr Gly Ala Trp Glu Ile Tyr Asn Thr Phe Ser Leu Pro
405 410 415
Ser Ser Ile Tyr Ile Ala Ser Ala Gly Asn His Thr Ile Arg Val Gln
420 425 430
Ser Ser Gly Gly Ser Ala Trp Gln Trp Asn Gly Asp Glu Leu Arg Phe
435 440 445
Thr Gln Thr Asp Ala Asp Thr Gly Thr Asn Pro Pro Ser Thr Ala Ser
450 455 460
Ile Ala Val Glu Ala Glu Asn Phe Asn Ala Val Gly Gly Thr Phe Ser
465 470 475 480
Asp Gly Gln Ala Gln Pro Val Ser Val Tyr Thr Val Asn Gly Asn Thr
485 490 495
Ala Ile Asn Tyr Val Asn Gln Gly Asp Tyr Ala Asp Tyr Thr Ile Ala
500 505 510
Val Ala Gln Ala Gly Asn Tyr Thr Ile Ser Tyr Gln Ala Gly Ser Gly
515 520 525
Val Thr Gly Gly Ser Ile Glu Phe Leu Val Asn Glu Asn Gly Ser Trp
530 535 540
Ala Ser Lys Thr Val Thr Ala Val Pro Asn Gln Gly Trp Asp Asn Phe
545 550 555 560
Gln Pro Leu Asn Gly Gly Ser Val Tyr Leu Ser Ala Gly Thr His Gln
565 570 575
Val Arg Leu His Gly Ala Gly Ser Asn Asn Trp Gln Trp Asn Leu Asp
580 585 590
Lys Phe Thr Leu Ser Asn
595
<210>6
<211>368
<212>PRT
<213> polysaccharide-degrading bacterium 2-40
<400>6
Met Ser Asp Ser Lys Val Asn Lys Lys Leu Ser Lys Ala Ser Leu Arg
1 5 10 15
Ala Ile Glu Arg Gly Tyr Asp Glu Lys Gly Pro Glu Trp Leu Phe Glu
20 25 30
Phe Asp Ile Thr Pro Leu Lys Gly Asp Leu Ala Tyr Glu Glu Gly Val
35 40 45
Ile Arg Arg Asp Pro Ser Ala Val Leu Lys Val Asp Asp Glu Tyr His
50 55 60
Val Trp Tyr Thr Lys Gly Glu Gly Glu Thr Val Gly Phe Gly Ser Asp
65 70 75 80
Asn Pro Glu Asp Lys Val Phe Pro Trp Asp Lys Thr Glu Val Trp His
85 90 95
Ala Thr Ser Lys Asp Lys Ile Thr Trp Lys Glu Ile Gly Pro Ala Ile
100 105 110
Gln Arg Gly Ala Ala Gly Ala Tyr Asp Asp Arg Ala Val Phe Thr Pro
115 120 125
Glu Val Leu Arg His Asn Gly Thr Tyr Tyr Leu Val Tyr Gln Thr Val
130 135 140
Lys Ala Pro Tyr Leu Asn Arg Ser Leu Glu His Ile Ala Ile Ala Tyr
145 150 155 160
Ser Asp Ser Pro Phe Gly Pro Trp Thr Lys Ser Asp Ala Pro Ile Leu
165 170 175
Ser Pro Glu Asn Asp Gly Val Trp Asp Thr Asp Glu Asp Asn Arg Phe
180 185 190
Leu Val Lys Glu Lys Gly Ser Phe Asp Ser His Lys Val His Asp Pro
195 200 205
Cys Leu Met Phe Phe Asn Asn Arg Phe Tyr Leu Tyr Tyr Lys Gly Glu
210 215 220
Thr Met Gly Glu Ser Met Asn Met Gly Gly Arg Glu Ile Lys His Gly
225 230 235 240
Val Ala Ile Ala Asp Ser Pro Leu Gly Pro Tyr Thr Lys Ser Glu Tyr
245 250 255
Asn Pro Ile Thr Asn Ser Gly His Glu Val Ala Val Trp Pro Tyr Lys
260 265 270
Gly Gly Met Ala Thr Met Leu Thr Thr Asp Gly Pro Glu Lys Asn Thr
275 280 285
Cys Gln Trp Ala Glu Asp Gly Ile Asn Phe Asp Ile Met Ser His Ile
290 295 300
Lys Gly Ala Pro Glu Ala Val Gly Phe Phe Arg Pro Glu Ser Asp Ser
305 310 315 320
Asp Asp Pro Ile Ser Gly Ile Glu Trp Gly Leu Ser His Lys Tyr Asp
325 330 335
Ala Ser Trp Asn Trp Asn Tyr Leu Cys Phe Phe Lys Thr Arg Arg Gln
340 345 350
Val Leu Asp Ala Gly Ser Tyr Gln Gln Thr Gly Asp Ser Gly Ala Val
355 360 365