KR102312806B1 - Amylases having resistance to starch decomposition activity derived from Bifidobacterium genus and uses thereof - Google Patents

Abstract

The present invention relates to amylases exhibiting resistant starch decomposition activity derived from Bifidobacterium adolescentis and to a use thereof. A composition for decomposing resistant starch according to the present invention contains alpha-amylase having resistance starch decomposition activity, and thus can be provided for use in decomposing resistant starch in various industrial fields.

Description

Amylases having resistance to starch decomposition activity derived from Bifidobacterium genus and uses thereof

The present invention relates to amylases exhibiting resistant starch degrading activity derived from Bifidobacterium adolescentis and uses thereof.

Resistance starch (RS) is a type of starch that is resistant to digestive enzymes and is known to have beneficial effects on human health, such as lowering blood sugar and improving insulin sensitivity because it is not broken down into glucose in the small intestine after ingestion. In addition, RS, which has reached the large intestine in its intact form, is fermented by the gut microbiota and transformed into short chain fatty acids (SCFAs), which are key signaling molecules between the gut microbiota and host health (Bird A, et al. (2010) ) Resistant starch, large bowel fermentation and a broader perspective of prebiotics and probiotics. Beneficial microbes 1(4):423-431). SCFAs are small fatty acids with less than 6 carbons and include acetic acid, propionic acid and butyric acid. SCFAs are known to be a major source of energy for colon cells and play a variety of physiological roles in human health, such as reducing the risk of gastrointestinal disorders, cardiovascular disease and cancer. Recently, as RS serves as a source of SCFAs in the colon, interest in the prebiotic use of RS has increased. Accordingly, further studies focusing on the gut microbiota and RS metabolism utilizing intestinal RS are required.

On the other hand, Bifidobacterium is known as probiotics (probiotics, probiotics) as symbiotic bacteria in the human gut. Although it is known that Bifidobacterium is advantageous in degrading starch through a number of studies, there are few studies on starch degrading enzymes derived from Bifidobacterium. In particular, RS degrading enzymes derived from Bifidobacterium have been reported. none.

Under this background, the present inventors have completed the present invention by identifying a resistant starch degrading enzyme derived from Bifidobacterium adolescentis and confirming its resistant starch degrading activity.

An object of the present invention is to provide a composition for decomposing resistant starch, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.

Another object of the present invention is to provide a recombinant microorganism having a resistant starch degrading activity, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.

Another object of the present invention is any one or more of a recombinant microorganism having a resistant starch degrading activity or a culture medium comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3. To provide a method for decomposing resistant starch, comprising adding to a sample containing resistant starch.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides a composition for decomposing resistant starch, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.

In the present invention, the term "α-amylase" is an enzyme that cleaves glucose chains such as starch or glycogen without regularity from the inside, and acts only on α-1, 4-linkage of glucose. α-amylase is present in saliva and pancreatic juice as well as malt, mold, and bacteria.

For the purposes of the present invention, the α-amylase may be an enzyme having an activity to degrade resistant starch, but is not limited thereto.

In the present invention, the term "resistance starch (RS)" is a type of starch resistant to digestive enzymes, and is not broken down into glucose in the small intestine after ingestion, thereby lowering blood sugar and improving insulin sensitivity. is known to give In addition, resistant starch, which has reached the colon in its complete form, is a major energy source for colon cells by intestinal microbes, and short chain fatty acids (short chain fatty acids) known to play various physiological roles in human health, such as reducing the risk of gastrointestinal disorders, cardiovascular disease and cancer. fatty acids (SCFAs). Examples of the resistant starch include granular starch such as high amylose corn starch (HAS) and potato starch, starch that is aged and crystallized after gelatinization (retrograded starch) or chemically modified starch (chemically modified starch). modified starch), but is not limited thereto.

For the purposes of the present invention, the resistant starch may be high amylose corn starch, but is not limited thereto.

The present invention is significant in first identifying a protein having a resistant starch degrading activity from Bifidobacterium adolescentis and confirming its activity.

In the present invention, SEQ ID NOs: 1 to 3 refer to amino acid sequences having α-amylase activity that degrades resistant starch. The polypeptide or protein comprising the amino acid sequence of SEQ ID NOs: 1 to 3 may be used in combination with a polypeptide or protein having the amino acid sequence of SEQ ID NOs: 1 to 3, or a polypeptide or protein comprising the amino acid sequence of SEQ ID NOs: 1 to 3 .

The amino acid sequence of SEQ ID NOs: 1 to 3 may be derived from Bifidobacterium adolescentis , and more specifically, the resistance encoded by the gene derived from Bifidobacterium adolcentis P2P3 deposited as accession number KACC92235P. It may be an amino acid sequence of a protein having α-amylase activity for decomposing starch, but is not limited thereto, and a sequence having the same activity as the amino acid may be included without limitation. The amino acid sequence of SEQ ID NOs: 1 to 3 can be obtained from a known database, National Institutes of Health (NIH GenBank). In addition, although the protein having α-amylase activity for decomposing resistant starch in the present invention is defined as a protein including the amino acid sequence of SEQ ID NOs: 1 to 3, meaningless sequences before and after the amino acid sequence of SEQ ID NOs: 1 to 3 are added or The resistance starch of the present invention does not exclude naturally occurring mutations or latent mutations thereof, and if they have the same or corresponding activities as the protein comprising the amino acid sequence of SEQ ID NOs: 1 to 3 It is apparent to those skilled in the art that it corresponds to a protein having α-amylase activity that degrades the α-amylase. As a specific example, the protein having α-amylase activity for decomposing resistant starch of the present invention is the amino acid sequence of SEQ ID NOs: 1 to 3 or 80%, 85%, 90%, 95%, 96%, 97%, 98 %, or a protein consisting of an amino acid sequence having at least 99% homology or identity. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein, a protein consisting of an amino acid sequence in which some sequence is deleted, modified, substituted or added is also included within the scope of the protein subject to mutation of the present invention. is self-evident

That is, even if it is described as 'protein or polypeptide consisting of the amino acid sequence described in a specific SEQ ID NO:' or 'protein or polypeptide consisting of the amino acid sequence described in the specific SEQ ID NO' in the present invention, the polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO: It is apparent that a protein consisting of an amino acid sequence in which some sequences are deleted, modified, substituted or added may also be used in the present invention, provided they have the same or corresponding activity. For example, it is apparent that a 'polypeptide consisting of the amino acid sequence of SEQ ID NO: 1' may belong to a 'polypeptide consisting of the amino acid sequence of SEQ ID NO: 1' if it has the same or corresponding activity.

In the present invention, the gene encoding the amino acid sequence of SEQ ID NOs: 1 to 3 may be derived from the genus Bifidobacterium , specifically, Bifidobacterium adolescentis It may be derived from, and more Specifically, it may be derived from Bifidobacterium adolcentis P2P3 deposited with accession number KACC92235P, but is not limited thereto.

The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 may be a sequence including the nucleotide sequence of SEQ ID NO: 4, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 is a sequence including the nucleotide sequence of SEQ ID NO: 5 may be, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 may be a sequence including the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto. The polynucleotide comprising the base sequence of SEQ ID NOs: 4 to 6 may be used in combination with the polynucleotide having the base sequence of SEQ ID NOs: 4 to 6 and the polynucleotide comprising the base sequence of SEQ ID NOs: 4 to 6.

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.

Specifically, the polynucleotide of the present invention may contain a variety of coding regions within a range that does not change the amino acid sequence of the polypeptide due to codon degeneracy or considering codons preferred in the organism to express the polypeptide. Deformation can be made. Specifically, as long as it is a polynucleotide sequence encoding α-amylase that degrades resistant starch consisting of the amino acid sequence of SEQ ID NO: 1, it may be included without limitation.

In addition, α that degrades resistant starch consisting of the amino acid sequence of SEQ ID NO: 1 by hydridation under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the nucleotide sequence. - Any sequence encoding a protein having amylase activity may be included without limitation. The "stringent condition" refers to a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, 40% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, particularly specifically 99% or more homology between genes with high homology or identity, or Genes with the same identity are hybridized and genes with homology or identity lower than that are not hybridized, or washing conditions of normal southern hybridization at 60 ° C., 1ХSSC, 0.1% SDS, specifically At a salt concentration and temperature equivalent to 60° C., 0.1ХSSC, 0.1% SDS, more specifically 68° C., 0.1ХSSC, 0.1% SDS, the conditions of washing once, specifically 2 to 3 times can be listed. have.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present invention may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, and the variables are well known in the art (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring). Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

In the present invention, the term 'homology' or 'identity' refers to a degree related to two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially, homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art. , can be determined by methods well known to those skilled in the art (eg, J. Sambrook et al., supra).

In one embodiment, B. adolescentis P2P3 is high amylose by reducing sugar generated by decomposition of corn starch granules were confirmed to grow (Fig. 1), B. adolescentis P2P3 after inoculation until 36 hours of B. adolescentis P2P3 The log phase continued. During the log phase, reducing sugars produced by B. adolescentis P2P3 were produced at a concentration of 26.8 μg/mL at 12 hours and then gradually decreased after 30 hours. On the other hand, it was confirmed that B. adolescentis DSM 20083, a strain homologous to B. adolescentis P2P3, did not grow in HACS granulation medium.

From the above results, it was confirmed that there is a strain that does not show resistant starch degradation, particularly high amylose corn starch granulating activity, even in homologous strains of Bifidobacterium species known to have starch degradation activity, which indicates that HACS granulation activity is B .adultis P2P3 It was suggested that it is a specific effect.

In addition, B. adolescentis P2P3 is essentially expected to have resistant starch-degrading enzymes [RS (resistant starch)-degrading enzymes, RSD], and in another embodiment of the present invention, B. adolescentis P2P3, which is RS-degrading bifidobacterium and a gene encoding an amylolytic enzyme in the genome of B. adolescentis DSM 20083, a non-RS-degrading bifidobacterium that does not degrade resistant starch.

As a result, In the genome of B. adolescentis DSM 20083, a non-RS-degrading bifidobacterium, unverified, Three predicted RSDs designated as hypothetical proteins (P2P3-RSD1: CV760_07855 (SEQ ID NO: 4), P2P3-RSD2: CV760_07940 (SEQ ID NO: 5), P2P3-RSD3: CV760_07945 (SEQ ID NO: 6)) were identified in the genome of B. adolescentis P2P3. confirmed (Table 1), and the above results suggested that three predicted RSDs found only in the RS-degrading strain B. adolescentis P2P3 were responsible for the degradation of RS granules.

In another embodiment of the present invention, it was confirmed that the three expected RSDs were α-amylase, and as a result of confirming their HACS granule hydrolysis properties, P2P3-RSD1/2/3 showed activity against high amylose corn starch granules ( Table 4). In particular, in the genome of B. adolescentis DSM 20083, a non-RS-degrading Bifidobacterium, identical to P2P3-RSD1/2/3, unverified, Another predicted RSDs designated as hypothetical proteins (P2P3-RSD4) showed activity on soluble starch but not on high amylose corn starch granules.

From the above results, it was confirmed that there is an amylase that does not exhibit HACS granulolytic activity even with amylase derived from isogeneic strains, suggesting that HACS granulolytic activity is a P2P3-RSD1/2/3 specific effect.

α-amylase, that is, P2P3-RSD1/2/3, consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 according to the present invention, is pH 4 to 9 and at a temperature in the range of 30 ° C to 55 ° C. It may exhibit activity, and may exhibit optimal activity at pH 5 to 6 and a temperature in the range of 45° C. to 50° C., but is not limited thereto.

In addition, the P2P3-RSD1/2/3 interacts with soluble starch, amylose, γ-cyclodextrins (CD), maltotriose (G3) or maltopentaose (G5) to form G2, Can produce primary end products of G3/G1, G2/G1, G2 and G3, but is not limited thereto.

Another aspect of the present invention provides a recombinant microorganism having a resistant starch degrading activity, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.

The terms used herein are the same as described above.

In the present invention, the term "recombinant microorganism having a resistant starch decomposition activity" includes all microorganisms in which genetic modification has occurred, either naturally or artificially, as an external gene is inserted or the activity of an intrinsic gene is enhanced or inactivated. As a result, a specific mechanism is weakened or strengthened, and a gene is introduced for a desired resistance starch decomposition activity, a genetic mutation occurs, or the activity of a specific protein is enhanced or weakened.

The gene encoding α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 is introduced into a microorganism by a conventional method known in the art, and α that degrades resistant starch in the microorganism -amylase can be expressed.

As used herein, the term "to be/are expressed" refers to a state in which a target protein is introduced into a microorganism or modified to be expressed in the microorganism. For the purposes of the present invention, the "target protein" may be α-amylase that degrades the aforementioned resistant starch.

As used herein, the term “introduction of protein” refers to introduction of protein activity into a microorganism without specific protein activity. This can also be expressed as the enhancement of protein activity in microorganisms without specific protein activity.

Introduction of the protein may be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same/similar activity as the protein, or a codon-optimized mutant polynucleotide thereof into a host cell. The foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell. The introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to produce a protein and increase its activity.

Enhancing the activity of the introduced protein,

1) increase the copy number of the polynucleotide encoding the protein,

2) modification of the expression control sequence to increase the expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome to enhance the activity of the protein, or

4) It may be carried out by a method of deforming to be strengthened by a combination thereof, but is not limited thereto.

1) The increase in the number of copies of the polynucleotide is not particularly limited thereto, but may be performed in a form operably linked to a vector or inserted into a chromosome in a host cell. Specifically, the polynucleotide encoding the protein of the present disclosure is operably linked to a vector capable of replicating and functioning independently of the host and introduced into a host cell, or inserting the polynucleotide into a chromosome in the host cell. The polynucleotide is operably linked to a capable vector and introduced into a host cell, thereby increasing the number of copies of the polynucleotide in the chromosome of the host cell.

Next, 2) modification of the expression control sequence so as to increase the expression of the polynucleotide, but is not particularly limited thereto, deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence to further enhance the activity of the expression control sequence, or these It can be carried out by inducing a mutation in the sequence with a combination of , or by replacing it with a nucleic acid sequence having a stronger activity. The expression control sequence is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, and the like.

A strong heterologous promoter may be linked to the upper portion of the polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include lysCP1 promoter (International Patent Publication No. 2009-096689), spl1 promoter, spl7 promoter, spl13 promoter (Registered in Korea) Patent No. 10-1783170) EF-Tu promoter, groEL promoter, CJ7 promoter (Korean Patent No. 10-0620092 and International Patent Publication No. 2006-065095), aceA or aceB promoter, etc., but are not limited thereto.

In addition, 3) the modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but the expression control sequence by deletion, insertion, non-conservative or conservative substitution of the nucleic acid sequence or a combination thereof to further enhance the activity of the polynucleotide sequence. It can be carried out by inducing a mutation in the phase, or by replacing it with an improved polynucleotide sequence to have stronger activity.

Finally, the method of modifying to be enhanced by a combination of the above methods includes increasing the number of copies of the polynucleotide encoding the introduced protein, modifying the expression control sequence to increase its expression, and adding the polynucleotide sequence inserted into the chromosome. Modification and modification of a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide thereof may be applied together.

As used herein, the term “vector” refers to a DNA preparation containing a target polynucleotide sequence operably linked to a suitable regulatory sequence, so that a target gene can be introduced in a suitable host. Such regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself. For example, a target polynucleotide in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.

The vector of the present invention is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pET, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

As used herein, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form, as long as it can be expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" of the present invention means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present invention.

The method for transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell, and can be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.

For the purpose of the present invention, the recombinant microorganism having the resistant starch decomposing activity may be a microorganism having an improved resistant starch degrading ability than the parent strain or unmodified microorganism before transformation. The 'unmodified microorganism' does not exclude strains containing mutations that can occur naturally in microorganisms, and either the native microorganism itself, the wild type microorganism itself, or the expression level of the gene involved in the resistance starch degradation pathway is regulated. It may be a microorganism before it, or it may be a microorganism before the gene encoding any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3, which does not exist internally, is introduced.

In the present invention, the microorganism may include known microorganisms without limitation, and a gene encoding any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 is introduced to express α-amylase therefrom, and , so long as it can exhibit resistant starch decomposition activity is not particularly limited.

Another aspect of the present invention is any one or more of a recombinant microorganism having a resistant starch degradation activity or a culture medium comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 It provides a method for decomposing resistant starch, comprising adding to a sample containing resistant starch.

The terms used herein are the same as described above.

In the present invention, the term "cultivation" means growing the recombinant microorganism under appropriately controlled environmental conditions. The culturing process of the present invention can be made according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be batch, continuous, and fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material mixed with nutrients required for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganism of the present invention may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms, but a suitable carbon source, nitrogen source, phosphorus, inorganic It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids and/or vitamins.

In the present invention, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or appropriate precursors may be included. These components or precursors may be added to the medium in a batch or continuous manner, but is not limited thereto.

In addition, during the culture of the microorganism, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, but not limited thereto. does not

The temperature of the medium may be 20 °C to 45 °C, specifically 25 °C to 40 °C, but is not limited thereto. The culture period may be continued until a desired strain amount is obtained, specifically, 10 hours to 160 hours, more specifically 12 hours to 36 hours, but is not limited thereto.

In one embodiment of the present invention, B. adolescentis P2P3 expressing α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 is obtained by using HACS granules in a medium containing HACS granules as the sole carbon source. It was grown by reducing the sugar produced by decomposition (FIG. 1). Accordingly, a recombinant microorganism or a culture medium comprising α-amylase consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 expresses α-amylase having a resistant starch degrading activity, or the recombinant Since it contains α-amylase having a resistant starch decomposing activity derived from a microorganism, it can exhibit a resistant starch decomposing activity.

The composition for decomposing resistant starch according to the present invention contains α-amylase having an activity for decomposing resistant starch, and may be provided for use in decomposing resistant starch in various industrial fields.

1 is a diagram showing cell growth and reducing sugar production during culturing of Bifidobacterium with non-gelatinized high amylose corn starch granules. Blue line indicates cell growth; The red line is the optical density of the reducing sugar.
2 is a scanning electron microscope image of HACS granules after reaction with an expected resistant starch degrading enzyme. a) without enzyme, b) P2P3-RSD1, c) P2P3-RSD2, d) P2P3-RSD3.
3 is a diagram showing the reducing sugar produced by the expected resistant starch degrading enzyme. Black, light gray and dark gray are RSD1, RSD2 and RSD3, respectively. Figure 3a is a reaction of water-soluble starch, Figure 3b is a reaction of high amylose corn starch granules.
Figure 5 is a view confirming the purified predicted resistant starch degrading enzyme. Figure 5 is a diagram showing the multi-domain structure of the predicted resistant starch degrading enzyme derived from Bifidobacterium [RS (resistant starch)-degrading enzymes, RSD]. RSD: expected resistant starch degrading enzyme; SP: signal peptide; CBM: carbohydrate binding domain; BID: bacterial Ig-like domain; SLH, S-layer homology domain. The numbers are the start and stop positions of amino acids.
Figure 6 is a diagram showing the multiple alignment of the evolution tree of CSR of a) seven GH 13 trait conserved regions (CSR) and b) RSD. RSD: Expected resistant starch degrading enzyme. The red, green and blue inverted triangles are the catalytic triad (Asp, Glu and Asp), conserved histidine residues and the constant residue arginine residues, respectively.
7 is a diagram showing the multiple alignment of a) CBM25 or b) CBM26 in the expected resistant starch degrading enzyme. Bh_amy: malto hexaose-forming amylase from Bacillus halodurans C-125. Residues important for carbohydrate binding in the CBM25 and CBM26 modules are shown in yellow (stacking interactions) and red (hydrogen bond forming residues).
8 is a diagram showing the evolution tree of CBM25 and CBM26 in the expected resistant starch degrading enzyme. . RSD: Expected resistant starch degrading enzyme.
9 is an optimal pH analysis result of α-amylase that degrades resistant starch. a) P2P3-RSD1, b) P2P3-RSD2, c) P2P3-RSD3,
10 is an optimal temperature analysis result of α-amylase that degrades resistant starch.
11 is a diagram showing the reaction results of α-amylase resistant starch decomposition with various α-glucosidic substrates. a) enzyme-free reaction, b) reaction with P2P3-RSD1, c) reaction with P2P3-RSD2, d) reaction with P2P3-RSD3, M: G1 to G7 standard, 1: soluble starch, 2: amylose, 3 : pullulan, 4: α-cyclodextrin, 5: β-cyclodextrin, 6: γ-cyclodextrin, 7: G2-β-cyclodextrin, 8: G3, 9: G5.

Hereinafter, the present invention will be described in more detail by way of Examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these examples, and it will be apparent to those of ordinary skill in the art to which the present invention pertains.

Example 1. Bifidobacterium adolcentis in high amylose corn starch (HACS) granules ( Bifidobacterium adolescentis ) P2P3 culture

B. adolescentis P2P3 (KACC92235P) was isolated from feces of healthy Korean adults. To confirm the growth of B. adolescentis P2P3 on HACS, 2 mL of seed broth was mixed with sterile 0.5% non-gelatinized HACS [70% (w/w) amylose and 30% (w/w) w) amylopectin] containing granules of CM medium (Chopped meat broth, MBcell, Korea) was inoculated into 20 mL. Samples were incubated at 37° C. under anaerobic conditions with slow mixing, and cultures were obtained every 6 hours. As a control group, B. adolescentis DSM 20083, an allogeneic strain, was used.

Cell growth and reducing sugars in B. adolescentis P2P3 and B. adolescentis DSM 20083 were measured at 600 nm and 550 nm with an iMark microplate reader (BioRad, Hercules, CA, USA) using optical densitometry and dinitrosalicylic acid (DNS) methods, respectively. .

Specifically, the DNS method is pre-incubated with a substrate solution containing soluble starch at a final concentration of 0.5% in 50 mM sodium citrate buffer (pH 5) at 35 °C for 5 min, and then 10 µL of each strain culture solution is added to 90 µL of The reaction was initiated by addition to the substrate solution, and the reaction was carried out for 10 minutes.

As a result, it was confirmed that B. adolescentis P2P3 was grown using HACS granules as the sole carbon source, but B. adolescentis DSM 20083 did not ( FIG. 1 ). Specifically, the log phase of B. adolescentis P2P3 continued until 36 h after inoculation. During the log phase, reducing sugars were produced by B. adolescentis P2P3 at a concentration of 26.8 μg/mL at 12 hours and then gradually decreased after 30 hours.

From the above results, it was confirmed that there are strains that do not exhibit RS degradation, particularly HACS granulolytic activity, even in isogeneic strains of Bifidobacterium species known to have starch-degrading activity, which indicates that HACS granulolytic activity is B. adolescentis P2P3 It was suggested that it is a specific effect.

Based on these results, B. adolescentis P2P3 was confirmed to grow, by reducing sugar generated by decomposing the HACS granules, whereby B. adolescentis P2P3 is essentially resistant starch degrading enzyme [(resistant starch) RS -degrading enzymes , RSD ] was expected to have

Accordingly, in the following examples, B. adolescentis P2P3, which is an RS-decomposing bifidobacterium that decomposes RS, and B. adolescentis DSM 20083, a non-RS-decomposable bifidobacterium that does not decompose RS, in the examples below. Genes encoding amylolytic enzymes in the genome were compared.

Example 2. Comparative gene analysis of amylo-degrading enzyme derived from RS-degrading Bifidobacterium

Protein annotation analysis in Bifidobacterium was performed using the NCBI Prokaryotic Genome Annotation Pipeline. Comparative genetic analysis A protein-to-protein comparative study was performed using NCBI-BLAST+ 2.9.0, and the proteins were compared using both the databases of GenBank (GCA) and RefSeq (GCF).

As a result, in the genome of B. adolescentis P2P3, glycogen phosphorylase, 4-α-glucanotransferase, and glycogen debranching enzyme showing activity on α-glucan substrates debranching enzyme), α-amylase, α-1, 4-glucan branching enzyme, glycosidase, pullulanase, α- Encodes amylolytic enzymes such as 1,4-glucan-maltose-1-phosphate maltosyltransferase (α-1,4-glucan-maltose-1-phosphate maltosyltransferase) and α-glucosidase gene was found.

In addition, glycoside hydrolase family 13 (GH 13) α-amylase, designated as a hypothetical protein in the Prokaryotic Genome Annotation Pipeline (PGAP), as analyzed using other protein annotation engines (RAST and interproscan). Four predicted RSDs (CV760_07855, CV760_07940, CV760_07945 and CV760_08790) were further identified, containing an extra starch binding domain with a catalytic domain of α-amylase.

Finally, a total of 19 enzymes were assumed to be enzymes that directly or indirectly participate in the degradation of RS granules and saccharides released from RS, which are the same as the protein to protein alignment results shown in Table 1 below.

NO. B. adolescentis P2P3
(Locus_tag) B. adoelscentis DSM 20083
Cover/identity (protein_ID) One Glycogen phosphorylase
(CV760_00120) Glycogen/starch/α-glucan family phosphorylase
100% / 98% (WP_011742583.1) 2 4-α-Glucanotransferase
(CV760_01720) 4-α-Glucanotransferase
100% / 99% (WP_041777242.1) 3 Glycogen debranching enzyme GlgX
(CV760_01735) Probable glycogen operon protein GlgX
82% / 87% (BAF39092.1) 4 α-Amylase
(CV760_02460) α-Amylase
100% / 99% (WP_050731445.1) 5 1,4-α-Glucan branching enzyme
(CV760_04015) 1,4-α-Glucan branching enzyme
100% / 99% (WP_011743089.1) 6 Glycogen debranching enzyme GlgX
(CV760_04100) Glycogen debranching enzyme GlgX
99% / 100% (WP_003809530.1) 7 Glycosidase
(CV760_04240) Putative α-amylase/Glycosidase
65% / 99% (BAF39488.1) 8 Type I pullulanase
(CV760_04245) Type I pullulanase
100% / 80% (WP_011743123.1) 9 α-Amylase
(CV760_05710) α-Amylase
100% / 99% (WP_011743335.1) 10 Starch-binding protein
(CV760_07855) - 11 Starch-binding protein
(CV760_07940) - 12 Starch-binding protein
(CV760_07945) - 13 α-1,4-Glucan-maltose-1-phosphate maltosyltransferase
(CV760_08165) α-1,4-Glucan-maltose-1-phosphate maltosyltransferase
100% / 98% (WP_011743724.1) 14 Pullulanase
(CV760_08770) Pullulanase precursor
82% / 84% (BAF40340.1) 15 α-Amylase
(CV760_08790) - 16 4-α-Glucanotransferase
(CV760_08795) 4-α-Glucanotransferase
100% / 99% (WP_011743858.1) 17 α-Amylase
(CV760_08800) α-Amylase
100% / 100% (WP_003811282.1) 18 α-Glucosidase
(CV760_08880) α-Glucosidase
100% / 97% (WP_011743867.1) 19 α-Glucosidase
(CV760_09045) α-Glucosidase
100% / 99% (WP_011743891.1)

In Table 1 above, in the case of B. adolescentis DSM 20083, a non-RS-degrading strain, the four expected RSDs (CV760_07855, CV760_07940, CV760_07945 and CV760_08790) were not found, but the remaining 15 enzymes were all identified.

These results suggested that the four predicted RSDs found only in the RS-degrading strain B. adolescentis P2P3 were responsible for the degradation of RS granules. The four predicted RSDs were named P2P3-RSD1/2/3/4 or P-RSD1/2/3/4, respectively (corresponding to CV760_07855, CV760_07940, CV760_07945 and CV760_08790, respectively).

On the other hand, as shown in Table 1 above, most of the 15 enzymes exhibited 97% or more of query covers and identity, indicating that these enzymes have functional similarities in general Bifidobacterium species. have.

The whole genomes used for comparative analysis in this example were CP024959.1 ( B. adolescentis P2P3) and AP009256.1 ( B. adolescentis DSM20083), and the corresponding sequences were obtained from the NCBI genome database (https://www.ncbi.nlm. nih.gov/genome/). Genetic information for the 19 enzymes disclosed in Table 1 is shown in Table 2 below.

NO. Gene ID Start Stop Length Strand Function One CV760_00120 26,833 29,289 2,457 + Glycogen phosphorylase 2 CV760_01720 418,320 420,482 2,163 + 4-α-Glucanotransferase 3 CV760_01735 421,966 424,413 2,448 - Glycogen debranching enzyme GlgX 4 CV760_02460 575,946 577,703 1,758 + α-Amylase 5 CV760_04015 906,233 908,488 2,256 - α-1, 4-Glucan branching enzyme 6 CV760_04100 926,415 928,559 2,145 + Glycogen debranching enzyme GlgX 7 CV760_04240 960,030 961,667 1,638 + Glycosidase 8 CV760_04245 961,987 967,281 5,295 + Type I pullulanase 9 CV760_05710 1,288,451 1,290,226 1,776 + α-Amylase 10 CV760_07855 1,820,096 1,823,419 3,324 + Starch-binding protein 11 CV760_07940 1,841,220 1,844,879 3,660 - Starch-binding protein 12 CV760_07945 1,844,964 1,849,244 4,281 - Starch-binding protein 13 CV760_08165 1,904,172 1,906,430 2,259 + α-1,4-Glucan-maltose-1-phosphate maltosyltransferase 14 CV760_08770 2,070,230 2,072,284 2,055 - Pullulanase 15 CV760_08790 2,076,325 2,077,956 1,632 + α-Amylase 16 CV760_08795 2,078,243 2,080,411 2,169 + 4-α-Glucanotransferase 17 CV760_08800 2,080,528 2,082,276 1,749 - α-Amylase 18 CV760_08880 2,098,964 2,100,784 1,821 - α-Glucosidase 19 CV760_09045 2,147,285 2,149,810 2,526 + α-Glucosidase

Example 3. Expression and purification of predicted RSDs derived from RS-degraded Bifidobacterium

Confirmed in Example 2 To express and purify the four predicted RSDs, P2P3-RSD1/2/3/4 (CV760_07855 (SEQ ID NO: 4), CV760_07940 (SEQ ID NO: 5), CV760_07945 (SEQ ID NO: 6) and CV760_08790 (SEQ ID NO: 7)), Genomic DNA of B. adolescentis P2P3 was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Four genes encoding each of the four predicted RSDs were amplified by PCR using primer sets. The primers used here are shown in Table 3 below.

strain RSD_accession Primers Forward Revers B. adolescentis P2P3 RSD1_CV760_07855 atgagcgagggcgccac (SEQ ID NO: 8) tttgttggttatggtgcagtttggtg (SEQ ID NO: 9) RSD2_CV760_07940 gccacccgtgacagctacg (SEQ ID NO: 10) tttgttagttactgcgcagtttggtgt (SEQ ID NO: 11) RSD3_CV760_07945 atgacccgtgcagagcg (SEQ ID NO: 12) tttgttggttatggtgcagtttggtg (SEQ ID NO: 13) RSD4_CV760_08790 atgctttcccccgcgaaaca (SEQ ID NO: 14) ctcggcttcgagaattgcaaagc (SEQ ID NO: 15)

PCR was performed using KOD polymerase (Toyobo, Tokyo, Japan) under the following conditions: initial denaturation at 95 °C for 5 minutes; 25 cycles of 95° C. for 30 sec, 60° C. for 30 sec, 72° C. for 1 min/kb; followed by a final extension step at 72 °C for 7 min. Each amplified gene fragment was fused with a pET-21a vector (Novagen, Darmstadt, Germany) using an infusion cloning kit (Takara) according to the manufacturer's instructions. Each vector (pET-RSD), each containing genes encoding the four predicted RSDs, was transformed into Escherichia coli DH5α (Escherichia coli DH5α., Takara, Shiga, Japan), followed by E. coli BL21 codon plus (DE3) for expression. -RP ( E. coli BL21 CodonPlus (DE3)-RP, Stratagene, La Jolla, CA, USA) was transformed.

Each E. coli BL21 codon plus (DE3)-RP, each carrying four pET-RSDs, was treated with LB (Luria-Bertani) medium (Difco Laboratories) containing 100 μg/mL of ampicillin and 34 μg/mL of chloramphenicol. Inc., USA) was incubated at 37 °C with 250 mL. When the optical density reached 0.5 at 600 nm, 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the cell culture to induce protein expression. After continuous incubation of the cells at 20°C for 18 hours, the grown cells were harvested by centrifugation at 4,000×g for 20 minutes. Cells were resuspended in 20 mL of lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 10 mM imidazole, pH 7.5) and sonicator (Sonifier 450, Branson, USA) at 4 °C. destroyed The disrupted cells were removed by centrifugation at 12,000 x g for 20 minutes, and crude enzymes were passed through a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen, Valencia, CA, USA). Non-target proteins were washed with wash buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 20 mM imidazole, pH 7.5) and recombinant RSD was washed in elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 250 mM imidazole, pH 7.5). eluted. The purified enzyme was concentrated in 50 mM sodium acetate (pH 5) using ultrafiltration (50,000 MW cut-off membrane; Amicon, USA).

The final purified protein was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using a 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel. Protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, MA, USA, USA), and bovine serum albumin was used as a standard.

Example 4. HACS granule hydrolysis properties and reducing sugar analysis of predicted RSDs derived from RS-degraded Bifidobacterium

4-1. HACS granule hydrolysis characterization

To confirm the hydrolysis properties of HACS granules of P2P3-RSD1/2/3/4, 2 µg of each of P2P3-RSD1/2/3/4 purified in Example 3 was mixed with 1,000 µL of 50 mM sodium citrate buffer (pH 5). It was reacted with 20 mg of soluble starch (SS) dissolved in a solution at 40° C. for 10 minutes or with 20 mg of HACS granules at 40° C. for 24 hours, and the degree of their activity was measured. The structure of the HACS granules after the reaction was completed was additionally confirmed by scanning electron microscopy.

As a result, as shown in Table 4 below, P2P3-RSD1/2/3/4 in soluble starch exhibited specific activity at 1.35/12.7/6.86/0.85 μmole/h·mg, respectively. In HACS granules, P2P3-RSD1/2/3 showed specific activity at 0.10/0.62/0.12 μmole/h·mg, respectively, whereas P2P3-RSD4 did not show activity against HACS granules. Among P2P3-RSD1/2/3, P2P3-RSD2 showed the highest activity at 0.62 μmole/h·mg in HACS granules.

strains RSDs Activity levels to starch (μmole/h mg) Solubble High amylose corn B. adolescentis P2P3 RSD1 (CV760_07855) 1.35 ± 0.32 0.10 ± 0.18 RSD2 (CV760_07940) 12.70 ± 0.22 0.62 ± 0.10 RSD3 (CV760_07945) 6.86 ± 0.37 0.12 ± 0.08 RSD4 (CV760_08790) 0.85 ± 0.11 ^* ND

^* Not detected

In addition, as a result of observing the HACS granules with a scanning electron microscope, the structure of each HACS granule changed to a porous structure when reacted to the saturation point, and it was confirmed that the structure of the HACS granules reacted with P2P3-RSD2 was significantly destroyed (Fig. 2). ).

From the above results, it was confirmed that there is an amylase that does not exhibit HACS granulolytic activity even with amylase derived from isogeneic strains, suggesting that HACS granulolytic activity is a P2P3-RSD1/2/3 specific effect.

4-2. reducing sugar analysis

Among the P2P3-RSDs purified in Example 3, the activity of P2P3-RSD1/2/3, except for P2P3-RSD4, which did not show activity on HACS granules, was evaluated for the degree of sugar reduction using the DNS (Dinitrosalicylic acid) method of Example 1. measured accordingly. One unit of P2P3-RSDs was defined as the amount of enzyme producing 1 μmol of reducing sugar per minute under assay conditions using maltose standards.

As a result, 80 μg/mL or more of reducing sugar was generated from the soluble starch reacted with P2P3-RSD1/2/3 ( FIG. 3A ). While HACS granules reacted with P2P3-RSD2 produced reducing sugars of 80 μg/mL, P2P3-RSD1/3 produced reducing sugars of 14/22 μg/mL, respectively (FIG. 3b). Therefore, it was confirmed that P2P3-RSD2 showed stronger activity against HACS granules than P2P3-RSD1/3.

From these results, among the predicted RSDs present in B. adolescentis P2P3, P2P3-RSD1/2/3 have RS-degrading activity, and among them, the enzyme named P2P3-RSD2 is the most potent enzyme for HACS granules, and maltoin in HACS granules. It was confirmed that it is a key enzyme for producing oligosaccharides.

Meanwhile, as a result of theoretically calculating the molecular weight (MW) and isoelectric point (pI) of P2P3-RSD1/2/3 purified in Example 3 using ExPASy-ProtParam, the mature protein of P2P3-RSD1/2/3 is each Molecular weights of 113/125/156 kDa and theoretical pI values of 5.19/6.17/6.04, respectively, were shown. In addition, the SDS-PAGE analysis result of Example 3 was consistent with the theoretical size (FIG. 4).

Example 5. In silico analysis of predicted RSDs derived from Bifidobacterium

For in silico analysis, the conserved domains of four predicted RSDs from B. adolescentis P2P3 were identified by NCBI-CD search. Predictions of signal peptides (Sec- and Tat- secretion) of each gene were CBS-SignalP and CBS-TatP. was performed using Multiple sequence alignments were performed using the EBI-Clustal Omega, and conserved regions were visually corrected using GeneDoc software with quantitation mode (http://www.psc.edu/biomed/genedoc). Evolution trees were calculated using MEGA7 software as a minimal evolutionary statistical method applied to bootstrap replicates of 1,000 data sets.

As a result, the full-length coding sequences (CDS) of P2P3-RSD1/2/3/4 consisted of 3,324/3,660/4,278/1,632 bp (CV760_07855, CV760_07940, CV760_07945 and CV760_08790, respectively), and the CDS G+ It was confirmed that the C content values were 60.8/56.6/61.0/59.5%, respectively. In addition, it was found that the coding gene of P2P3-RSD1/2/3/4 can be translated into 1,107/1,219/1,426/543 amino acids, respectively.

As a result of domain analysis in the four predicted RSDs, the cell wall anchoring domain [specifically, bacterial immunoglobulin- It was found that there is a domain presumed to be a Bacterial immunoglobulin-like domain (BID) and an additional S-layer homology domain (SLH)] (FIG. 5).

However, in the case of P2P3-RSD4, it had only the catalytic domain of GH 13 α-amylase, and as confirmed in Example 4, recombinant P2P3-RSD4 showed typical α-glucosidase activity and did not degrade HACS granules (Table 4). ), P2P3-RSD4 was not further analyzed.

In all three RSDs except P2P3-RSD4 (P2P3-RSD1/2/3), the catalytic domain is a typical α-amylase superfamily (β/α) with an A region in the form of 8 barrel domains and additional B and C regions. included. Seven conserved regions (CR) have been identified, and residues in the catalytic triad that are generally conserved in α-amylases are CR II, CR IV and CR III (two aspartic acids and one glutamic acid), respectively. was present in (Fig. 6a). Two conserved histidine residues involved in the stabilization of the transition state were present in CR I and CR IV. An arginine residue, a constant residue of GH 13, was conserved at position i-2 relative to the catalytic nucleophile Asp at position i (CR II).

Based on these results, it was confirmed that P2P3-RSD1/2/3 is an α-amylase belonging to the GH 13 family. Specifically, P2P3-RSD1/2/3 was classified as belonging to the GH 13_28 subfamily according to the classification of GH 13 α-amylase (Janecek et al. 2014) (FIG. 6b).

In addition, it was confirmed that all of P2P3-RSD1/2/3 consisted of one or more CBM25 and CBM 26 (FIG. 5).

In addition, CBM25 and CBM26 of Bifidobacterium were compared with CBM25 and CBM26 that bind to granular starch in Bacillus halodurans C-125-derived maltohexaose-forming amylase, P2P3_RSD1 Important residues except for Gln ⁷⁰ of CBM26a are present in Bifidobacterium CBM25 and CBM26 ( FIG. 7 ), so these CBMs were expected to bind to granular starch.

The evolutionary tree containing CBM25 and CBM26 characterized in the CAZY database showed that the families CBM25 and CBM26 were divided, and in the same CBM family, the CBMs of RSD from Bifidobacterium were close to each other (Fig. 8).

On the other hand, a novel CBM family 74 that was not discovered by NCBI-CD search was found in P2P3_RSD3. CBM74 is a novel CBM that occurs widely in bacteria and affects binding to starch granules. Thus, the CBMs found in P2P3_RSDs (25, 26 and 74) were confirmed to be conserved CBMs in Bifidobacterium species. α-Amylolytic enzymes acting on starch granules are generally known to include CBM as an auxiliary module. Through these results, it was reconfirmed that P2P3-RSD1/2/3 is RS-degrading α-amylase.

Example 6. Analysis of optimal reaction conditions for α-amylase derived from Bifidobacterium

6-1. Optimal pH analysis

To determine the optimal pH of P2P3-RSD1/2/3, 10 µL of P2P3-RSD1/2/3 (0.2 units) purified in Example 3, 50 mM sodium acetate (pH 4-5), and 50 mM sodium citrate Soluble starch, amylopectin to a final concentration of 0.5% dissolved in (sodium citrate) (pH 5-6), 50 mM sodium phosphate (pH 6-8) or 50 mM Tris-Cl (pH 7-9) buffer , pullulan, amylose, α-CD, β-CD, γ-CD, G2-β-CD, cyclodextrins (CD) at a final concentration of 1 mM, maltodextrins at a final concentration of 1 mM React with 90 μL of phosphorus maltotriose (maltotriose, G3) or maltopentaose (G5) at 45 °C for 10 minutes, and analyze the reaction product through thin layer chromatography (TLC) to determine the activity pattern of the enzyme. Confirmed.

To perform TLC, a reaction mixture (1 mL) of each reactant reacted on various substrates was loaded onto a TLC silica gel 60 F254 TLC plate (Merck; Damstadt, Germany) and the solvent system [isopropanol: ethyl acetate: water (3 : 1 : 1, v/v/v)]. The plate was then dried in a hood and 0.3% (w/v) N-(1-naphthyl)-ethylenediamine (N-(1-naphthyl)-ethylene diamine) and 5% (v/v) H _{2 in methanol} It developed rapidly in _{SO 4 .} The plate was dried and placed in an oven at 110° C. for 10 minutes to visualize the reaction point.

The relative activity was compared with the reducing sugar released using the DNS method of Example 1.

As a result, the α-amylase activity of P2P3-RSD1/2/3 was the best in sodium citrate buffer at pH 5 to 6 ( FIG. 9 ).

6-2. Optimum temperature analysis

In order to confirm the optimum temperature of P2P3-RSD1/2/3, the reaction was performed in the same manner as in Example 6-1 using 50 mM sodium citrate buffer (pH 5) at a temperature ranging from 30°C to 55°C.

The relative activity was compared with the reducing sugar released using the DNS method of Example 1.

As a result, the α-amylase activity of P2P3-RSD1/2/3 was the best at 45°C for P2P3-RSD1/3 and at 50°C for P2P3-RSD2 ( FIG. 10 ).

6-3. Optimal Substrate Analysis

In order to identify the optimal substrate for P2P3-RSD1/2/3, various substrates (soluble starch, amylose, pullulan, α-CD, β-CD, γ-CD, G2-β -CD, G3 and G5), the P2P3-RSD1/2/3 activity pattern was confirmed at the optimal pH and temperature derived above.

As a result, as shown in FIG. 11, P2P3-RSD1/2/3 interacts with soluble starch, amylose, γ-CD, G3 and G5 to form primary end products of G2, G3/G1, G2/G1, G2 and G3, respectively. was created. Specifically, G3 was not a good substrate for P2P3-RSD1, but was completely hydrolyzed to G1 and G2 by P2P3-RSD2. β-CD and G2-β-CD were hydrolyzed to G1 and G2 by P2P3-RSD2, γ-CD was fully hydrolyzed by P2P3-RSD2/3, and pullulan and α-CD were hydrolyzed to P2P3-RSD2. was partially hydrolyzed to G2 by

In addition, γ-CD was hydrolyzed by P2P3-RSD2/3, and neither P2P3-RSD1/2/3 showed activity in G2, so it was confirmed that these enzymes are amylases acting on endo-, P2P3 - Among RSDs, it was confirmed that the activity of P2P3-RSD2 was the best.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Agricultural Biotechnology Research Institute KACC92235P 20180911

<110> University-Industry Cooperation Group of Kyung Hee University <120> Amylases having resistance to starch decomposition activity derived from Bifidobacterium genus and uses thereof <130> KPA191676-KR <160> 15 <170> KoPatentIn 3.0 <210> 1 <211> 1107 <212> PRT <213> Unknown <220> <223> P2P3-RSD1 <400> 1 Met Lys His Arg Lys Pro Ala Pro Ala Trp His Arg Leu Gly Leu Lys 1 5 10 15 Ile Ser Lys Lys Val Val Val Gly Ile Thr Ala Ala Ala Thr Ala Phe 20 25 30 Gly Gly Leu Ala Ile Ala Ser Thr Ala Ala Gln Ala Ser Thr Asp Arg 35 40 45 Asp Ser Tyr Ala Asp Thr Val Glu Asn Ala Thr Phe Glu Gln Ala Arg 50 55 60 Lys His Tyr Gly Leu Ala Gln Gln Met Ser Glu Gly Ala Thr Leu His 65 70 75 80 Ala Trp Glu Trp Ser Phe Lys Thr Ile Glu Glu Asn Ile Pro Ala Ile 85 90 95 Ala Glu Ala Gly Tyr Thr Ser Val Gln Thr Glu Pro Ile Ser Ala Ile 100 105 110 His Asn Gly Gly Lys Gly Met Ile Phe Thr Glu Asn Trp Tyr Tyr Val 115 120 125 Tyr Gln Pro Thr Asp Thr Thr Ile Gly Asn Trp Val Met Gly Thr Glu 130 135 140 Asp Asp Leu Lys Ser Leu Cys Asn Thr Ala His Lys Tyr Gly Val Arg 145 150 155 160 Ile Ile Val Asp Val Val Ala Asn His Met Thr Ala Thr Trp Gly Ala 165 170 175 Ile Ala Asp Arg Trp Lys Lys Ser Glu Tyr Tyr His His Asp Cys Asn 180 185 190 Asp Gly Asp Val Gln Asp Trp Asn Asn Arg Tyr Gln Val Thr His Cys 195 200 205 Lys Leu Leu Gly Leu Tyr Asp Ile Asn Thr Glu Asn Thr Lys Thr Ala 210 215 220 Asn Met Met His Asp Phe Leu Val Gln Ala Val Asn Asp Gly Val Asp 225 230 235 240 Gly Phe Arg Phe Asp Ala Ala Lys His Ile Glu Leu Pro Asp Glu Tyr 245 250 255 Asn Gly Ser Gln Tyr Trp Asn Ile Ile Leu Asn Asn Gly Ala Gln Phe 260 265 270 Gln Tyr Gly Glu Val Leu Gln Asp Ser Ile Ser Arg Asp Ser Asp Tyr 275 280 285 Ala Lys Leu Phe Ser Ser His Ser Lys Asn Gly Gly Gly Val Thr Ala 290 295 300 Ser Ser Tyr Gly Ser Lys Leu Arg Gly Ala Leu Asn Ser Lys Asn Leu 305 310 315 320 Asn Ala Gly Thr Leu Ser Asp Trp Ser Asn Ser Ala Ser Pro Ser Asn 325 330 335 Leu Val Ser Trp Val Glu Ser His Asp Asn Tyr Ser Asn Ser Asp Arg 340 345 350 Glu Ser Thr Gly Met Ser Glu Trp Gln Met Thr Met Gly Trp Gly Val 355 360 365 Ile Gly Ser Arg Ser Gln Thr Met Pro Leu Tyr Phe Asp Arg Pro Val 370 375 380 Gly Ser Gly Gly Ser Gln Pro Gln Phe Ala Glu Lys Ser Lys Leu Gly 385 390 395 400 Asp Ala Gly Ala Asp Ser Trp Lys Asp Pro Gln Val Val Ala Val Asn 405 410 415 His Phe Arg Asn Thr Met Asn Asn Asn Lys Ala Ser Glu Tyr Leu Arg 420 425 430 Asn Cys Ser Ala Asn Ser Cys Leu Met Ala Glu Arg Tyr Ile Lys Asp 435 440 445 Gly Asn Phe Lys Asn Asp Gly Val Thr Ile Thr Asn Met Gly Asp Thr 450 455 460 Gln Glu Leu Ser Gly Thr Ala Thr Asn Leu Asp Asp Gly Thr Tyr Lys 465 470 475 480 Asp Gln Val Ser Gly Gly Thr Ile Thr Val Ser Gly Gly Lys Ile Thr 485 490 495 Ser Gly Ser Ala Pro Gly Gly Lys Ile Ser Val Phe Phe Thr Asp Asn 500 505 510 Ser Gly Ser Val Ser Ala Thr Pro Gly Asp Ser Ser Phe Lys Thr Asp 515 520 525 Thr Thr Thr Val Thr Leu Asn Ala Asn Asn Val Thr Asp Ala Thr Tyr 530 535 540 Thr Thr Ser Glu Gly Lys Ser Gly Ser Tyr Gln Asp Gly Asp Thr Ile 545 550 555 560 Thr Ile Gly Ala Ser Thr Ala Ile Gly Asp Thr Ile Thr Val Lys Leu 565 570 575 Gln Gly Lys Asp Ala Asp Gly Gln Thr Val Ser Ala Thr Tyr Lys Tyr 580 585 590 Thr Lys Lys Asp Pro Ala Ala Thr Ser Thr Ala Tyr Ala Lys Lys Pro 595 600 605 Ser Ala Trp Ser Asn Leu Tyr Ala Tyr Val Tyr Val Asp Asp Ser Ser 610 615 620 Ala Thr Thr Leu Lys Glu Asn Ala Lys Trp Pro Gly Glu Pro Met Thr 625 630 635 640 Gln Val Ala Ser Gly Asp Thr Cys Gly Lys Asp Gly Glu Tyr Lys Tyr 645 650 655 Glu Ile Pro Asp Asp Leu Glu Gly Ser Asn Thr Arg Ile Ile Phe Asn 660 665 670 Asp Gly Asn Ala Thr Asn Thr Lys Lys Tyr Pro Ala Asp Thr Thr Glu 675 680 685 Gly Glu Asp Ala Ala Gly Leu Lys Ile Asp Gly Asn Tyr Ala Trp Asp 690 695 700 Gly Asn Thr Ser Ser Gly Thr Trp Glu Ala Arg Asn Cys Val Val Ile 705 710 715 720 Pro Thr Lys Ser Val Lys Ile Asp Gln Ser Asp Tyr Thr Thr Asp Leu 725 730 735 Ser Asn Gly Val Ala Thr Lys Gln Leu Thr Ala Thr Thr Asp Pro Lys 740 745 750 Gly Ala Ser Val Ser Trp Ser Ser Ser Asp Lys Asp Val Ala Thr Val 755 760 765 Ser Pro Lys Gly Val Val Thr Pro Lys Lys Ala Gly Lys Ala Thr Ile 770 775 780 Thr Ala Lys Ser Gly Thr Lys Thr Ser Ser Ile Thr Val Thr Val Thr 785 790 795 800 Gly Glu Leu Pro Pro Asp Pro Val Ala Lys Asn Thr Val Tyr Ala Ser 805 810 815 Lys Pro Ser Gly Trp Gly Lys Ile Tyr Ala Tyr Val Tyr Thr Gly Asp 820 825 830 Gly Ala Thr Ala Ala Asn Asn Ala Ala Trp Pro Gly Val Glu Met Thr 835 840 845 Ala Pro Ser Ala Thr Asp Gly Cys Arg Gln Thr Asp Leu Tyr Lys Tyr 850 855 860 Val Val Pro Asp Asp Leu Ala Lys Asp Ala Lys Val Ile Phe Asn Asp 865 870 875 880 Gly Gly Ser Gln Gln Tyr Pro Gly Ser Arg Gln Pro Gly Leu Asp Tyr 885 890 895 Asn Gly Gly Ile Val Gln Trp Asp Gly Ser Ser Ala Ala Leu Ala Ala 900 905 910 Val Glu Cys Glu Thr Thr Ile Pro Val Thr Ser Val Ser Ile Ser Gly 915 920 925 Asp Gly Val Ser Gly Gly Lys Leu Ser Leu Lys Ser Gly Ala Ser Ala 930 935 940 Gln Leu Thr Ala Thr Val Lys Pro Asp Asn Ala Thr Asp Arg Lys Val 945 950 955 960 Thr Trp Thr Ser Ser Asp Ser Ser Val Ala Asn Val Met Gly Thr Gly 965 970 975 Val Val Thr Ala Gly Ser Lys Ala Gly Lys Ala Thr Ile Lys Ala Thr 980 985 990 Ala Gly Gly Lys Ser Ala Ser Val Thr Val Thr Val Thr Asp Val Pro 995 1000 1005 Lys Gln Pro Met Thr Val Trp Tyr Lys Pro Ala Ser Ser Trp Lys Thr 1010 1015 1020 Ala Lys Val His Tyr Lys Ala Asn Gly Lys Trp Thr Gly Ser Ala Gln 1025 1030 1035 1040 Gln Met Thr Ala Ala Cys Gly Gly Trp Tyr Lys Tyr Thr Ile Pro Asp 1045 1050 1055 Thr Ala Gly Gly Gln Val Arg Met Ala Phe Thr Asn Gly Ser Ala Trp 1060 1065 1070 Asp Asn Asn Ser Gly Lys Asp Tyr Met Val Ser Gly Asp Ser Ala Ala 1075 1080 1085 Val Ala Gly Gly Gln Ser Val Pro Asp Val Thr Pro Asn Cys Thr Ile 1090 1095 1100 Thr Asn Lys 1105 <210> 2 <211> 1219 <212> PRT <213> Unknown <220> <223> P2P3-RSD2 <400> 2 Met Met Lys Gln Ser Ala Thr Leu Ala Arg Leu Ser Lys Lys Thr Val 1 5 10 15 Ala Phe Leu Ala Ala Ala Thr Thr Leu Leu Ala Gly Leu Ser Leu Ala 20 25 30 Thr Ala Ala Pro Gln Gln Ala Ser Ala Ala Thr Arg Asp Ser Tyr Ala 35 40 45 Asp Thr Ile Gly Asn Pro Thr Phe Glu Ala Ala Arg Gln Lys Tyr Gly 50 55 60 Leu Pro Lys Asp Met Lys Asp Gly Ala Thr Leu His Ala Phe Glu Trp 65 70 75 80 Ser Phe Lys Thr Ile Met Glu Asn Ile Pro Asp Ile Ala Lys Ala Gly 85 90 95 Tyr Thr Ser Ile Gln Thr Glu Pro Ile Val Lys Ile Lys Asp Asn Arg 100 105 110 Lys Leu Gly Thr Gly Asn Trp Tyr Leu Asn Trp Tyr Tyr Val Tyr Gln 115 120 125 Pro Thr Asp Met Ser Ile Gly Asn Tyr Val Val Gly Ser Glu Asp Glu 130 135 140 Phe Lys Glu Met Cys Lys Leu Ala His Gln Tyr Gly Leu Arg Ile Ile 145 150 155 160 Val Asp Ser Val Ser Asn His Phe Thr Ser Asp Phe Asp Val Ile Glu 165 170 175 Gly Lys Trp Lys Asn Lys Ala Tyr Tyr His Glu Asp Lys Gln Ile Ser 180 185 190 Asp Tyr Asn Asn Arg Glu Asp Cys Thr Gln His Lys Leu Ser Gly Leu 195 200 205 Trp Asp Leu Asn Thr Gln Asp Asp Thr Val Thr Glu Gly Met His Asp 210 215 220 Leu Leu Val Gln Thr Val Val Asp Gly Ala Asp Gly Phe Arg Tyr Asp 225 230 235 240 Ala Ala Lys His Ile Glu Leu Ser Asp Glu Val Phe Gly Gly Lys Gln 245 250 255 Ser His Tyr Trp Asp Thr Ile Leu Gln Asn Gly Ala Gln Tyr Gln Tyr 260 265 270 Gly Glu Val Leu Glu Asp Ala Asn Val Arg Glu Ala Asp Tyr Ala Asp 275 280 285 Leu Phe Asn Lys Ser Ser Val Gly Gly Gly Gly Ile Thr Asp Ser Ser 290 295 300 Tyr Gly His Glu Val Arg Asn Ala Val Gln Phe Lys Asn Leu Gly Ala 305 310 315 320 Ser His Phe Thr Ser His Ser Lys Val Thr Glu Asp Lys Thr Val Asn 325 330 335 Trp Val Glu Ser His Asp Asn Phe Ala Asn Gly Glu Ala Asn Ile Pro 340 345 350 Gln Glu Leu Ser Asp Glu Trp Ile Lys Tyr Gly Trp Ala Gly Val Thr 355 360 365 Ala Gln Lys Asn Gly Met Ser Leu Phe Phe Asp Arg Pro Tyr Lys Asp 370 375 380 Gly Gly Thr Tyr Gly Thr Gly Gly Val Gly Thr Tyr Gly Asn Gly Ser 385 390 395 400 Gly Pro Phe Thr Glu Asn Ser Lys Leu Gly Asp Ala Gly Ser Asp Leu 405 410 415 Trp Lys Asp Pro Glu Val Val Ala Val Asn His Phe Arg Asn Ala Met 420 425 430 Val Gly Glu Ala Ser Asn Val Ser Asn Cys Gly Asp Asp Asn Cys Leu 435 440 445 Met Val Glu Arg Tyr Ala Gly Ser Ala Ala Gln Asp Gly Met Val Val 450 455 460 Ala Asn Ala Asn Gly Ser Asp Lys Asn Leu Ala Gly Gln Ser Thr Lys 465 470 475 480 Leu Ala Asn Gly Thr Tyr Thr Asp Glu Val Thr Gly Ser Thr Ile Thr 485 490 495 Val Ser Gly Gly Lys Val Thr Ser Gly Thr Val Lys Gly Gln Ser Ile 500 505 510 Ala Ala Phe Ser Asn Lys Thr Arg Ser Gly Lys Val Ser Thr Ala Glu 515 520 525 Ala Tyr Pro Asn Lys Gly Thr Ile Pro Gly Glu Ser Lys Thr Ile Thr 530 535 540 Leu Arg Ser Tyr Gln Ser Thr Asn Thr Thr Tyr Ser Thr Ser Asp Gly 545 550 555 560 Gln Ser Gly Ser Phe Lys Asp Gly Asp Thr Ile Glu Ile Gly Ser Lys 565 570 575 Ala Lys Ser Asp Glu Val Val Thr Val Thr Val Lys Gly Thr Gly Ala 580 585 590 Asp Gly Glu Ala Ile Lys His Thr Tyr Ser Tyr Thr Lys Tyr Gly Asp 595 600 605 Pro Thr Tyr Asp Pro Ser Asp Gly Thr Ser Ser Pro Cys Asp Lys Tyr 610 615 620 Cys Met Glu Tyr Met Glu Lys Asn Gly Leu Asp Pro Asp Cys Tyr Ile 625 630 635 640 Asp Ala Lys Glu Pro Cys His Pro Arg Asn Val Thr Val Thr Ala Ile 645 650 655 Lys Val Ser Gly Asp Thr Ser Met Asp Leu Ala Ser Thr Gln Ser Val 660 665 670 Gln Leu Lys Ala Asp Val Thr Thr Asp Pro Ala Gly Lys Arg Pro Ser 675 680 685 Val Thr Trp Thr Ser Ser Asp Thr Ser Val Ala Thr Val Asp Ser Lys 690 695 700 Gly Lys Val Ser Gly Leu Lys Ala Gly Thr Val Lys Ile Thr Ala Thr 705 710 715 720 Ala Gly Thr Asn Lys Lys Ser Asp Ser Ile Thr Ile Thr Ile Thr Gly 725 730 735 Val Lys Pro Glu Val Ser Asn Val Ile Tyr Ala Thr Lys Pro Ser Gly 740 745 750 Trp Ser Lys Ile Tyr Ala Tyr Val Tyr Asn Asp Thr Thr Ser Lys Asn 755 760 765 Asn Gly Asn Trp Pro Gly Val Glu Met Thr Gln Leu Thr Ala Asp Asp 770 775 780 Ser Cys Ala Lys Ala Gly Thr Tyr Lys Tyr Glu Val Ser Asp Leu Gly 785 790 795 800 Glu Gly Lys Tyr Arg Val Ile Phe Thr Asn Gly Ser Gly Ala Gln Thr 805 810 815 Pro Gly Gly Asn Gln Pro Gly Ile Glu Phe Ser Gly Lys Val Ser Trp 820 825 830 Asp Gly Ser Ser Ala Ala Val Ser Ala Val Asn Cys Asn Thr Pro Gln 835 840 845 Pro Val Pro Val Ser Ser Val Ser Ile Ser Gly Ser Gly Val Ser Asn 850 855 860 Gly Lys Leu Ser Leu Lys Ser Gly Ala Ser Val Gln Leu Thr Ala Thr 865 870 875 880 Val Thr Pro Ser Asn Ala Thr Asp Lys Thr Val Ser Trp Thr Ser Ser 885 890 895 Asn Ser Ser Val Ala Lys Val Ser Asp Gly Lys Ile Thr Ala Val Lys 900 905 910 Gly Gly Thr Ala Thr Ile Thr Ala Thr Ala Gly Gly Lys Thr Ala Ser 915 920 925 Val Ala Val Thr Val Ala Asp Asn Pro Val Pro Val Thr Ser Val Ser 930 935 940 Ile Ser Gly Asp Gly Val Ser Gly Gly Lys Leu Ser Leu Lys Ser Gly 945 950 955 960 Ala Ser Ala Gln Leu Thr Ala Thr Val Lys Pro Asp Asn Ala Thr Asp 965 970 975 Arg Lys Val Thr Trp Thr Ser Ser Asp Ser Ser Val Ala Asn Val Met 980 985 990 Gly Thr Gly Val Val Thr Ala Gly Ser Lys Ala Gly Lys Ala Thr Ile 995 1000 1005 Lys Ala Thr Ala Gly Gly Lys Ser Ala Ser Val Thr Val Thr Val Thr 1010 1015 1020 Asp Val Pro Lys Gln Pro Met Thr Val Trp Tyr Lys Pro Ala Ser Ser 1025 1030 1035 1040 Trp Lys Thr Ala Lys Val His Tyr Lys Ala Asn Gly Lys Trp Thr Gly 1045 1050 1055 Ser Ala Gln Gln Met Thr Leu Tyr Arg Asp Gly Trp Tyr Lys Tyr Thr 1060 1065 1070 Ile Pro Asp Thr Ala Gly Gly Gln Val Arg Met Ala Phe Thr Asn Gly 1075 1080 1085 Ser Ala Trp Asp Asn Asn Ser Gly Lys Asp Tyr Phe Ala Ser Gly Ser 1090 1095 1100 Val Val Ser Val Ser Gly Gly Lys Met Ser Ser Lys Ala Pro Ser Phe 1105 1110 1115 1120 Gly Thr Pro Met Thr Val Trp Tyr Lys Pro Ala Ser Ser Trp Lys Thr 1125 1130 1135 Ala Lys Val His Tyr Lys Ala Asn Gly Lys Trp Thr Gly Ser Ala Gln 1140 1145 1150 Gln Met Thr Ala Ala Cys Gly Gly Trp Tyr Lys Tyr Thr Ile Pro Asp 1155 1160 1165 Thr Ala Gly Gly Gln Val Arg Met Ala Phe Thr Asn Gly Ser Ala Trp 1170 1175 1180 Asp Asn Asn Ser Gly Lys Asp Tyr Met Val Ser Gly Asp Ser Ala Ala 1185 1190 1195 1200 Val Ala Gly Gly Gln Ser Val Pro Asp Val Thr Pro Asn Cys Ala Val 1205 1210 1215 Thr Asn Lys <210> 3 <211> 1426 <212> PRT <213> Unknown <220> <223> P2P3-RSD3 <400> 3 Met Thr Arg Ala Glu Arg Arg Arg Ala Ala Arg Lys Ala Arg Ala Ala 1 5 10 15 Lys Thr Trp Lys Lys Val Ala Ala Gly Thr Val Ala Val Ala Thr Leu 20 25 30 Phe Gly Gly Met Gly Val Ala Ser Thr Ala Leu Ala Ala Asp Arg Asp 35 40 45 Ser Tyr Gln Asp Thr Ile Gly Asn Ser Ser Phe Glu Ala Ala Arg Asn 50 55 60 Gln Tyr Gly Leu Thr Lys His Met Lys Asn Gly Ala Ile Leu His Ala 65 70 75 80 Trp Met Trp Ser Phe Lys Thr Ile Thr Gln His Met Pro Glu Ile Ala 85 90 95 Ala Ala Gly Tyr Thr Ser Val Gln Thr Glu Pro Met Ser Lys Ile Lys 100 105 110 Glu Val Ala Ala Asn Gly Lys Lys Phe Thr Glu Asn Trp Tyr Tyr Val 115 120 125 Tyr Gln Pro Ala Asn Thr Ser Ile Gly Asn Phe Val Val Gly Thr Glu 130 135 140 Ala Asp Leu Lys Glu Met Thr Ala Thr Ala His Lys Tyr Gly Val Arg 145 150 155 160 Val Ile Val Asp Val Val Ala Asn His Phe Thr Ser Asp Trp Asn Ala 165 170 175 Ile Asp Pro Asp Trp Gln Asn Lys Glu Tyr Phe His Lys Arg Thr Gly 180 185 190 Cys Asp Gly Pro Asn Gly Glu Ile Asn Asp Tyr Ser Asn Arg Tyr Lys 195 200 205 Val Thr Gln Cys His Leu Leu Gly Leu Trp Asp Leu Asn Thr Gln Asn 210 215 220 Gln Ala Val Ala Asp Arg Met Gln Lys Phe Leu Lys Thr Ala Val Ala 225 230 235 240 Asp Gly Val Asp Gly Phe Arg Tyr Asp Ala Ala Lys His Val Glu Leu 245 250 255 Pro Thr Glu Val Phe Asp Asn Lys Gln Ser Asn Tyr Trp Asn Thr Ile 260 265 270 Leu Lys Asn Gly Ser Gln Phe Gln Tyr Gly Glu Val Leu Gln Gly Asp 275 280 285 Ser Gly Leu Asp Tyr Lys Ala Tyr Ala Asn Met Phe Arg Asp Asn Ser 290 295 300 Ser Asp Gly Gly Gly Asn Thr Ala Ser Asn Tyr Gly Lys Thr Ile Arg 305 310 315 320 Ala Ala Val Gly Ser Asn Asn Leu Asp Val Lys Met Val Lys Asn Ile 325 330 335 Asp Thr Gly Gly Ala Ser Glu Asp Gln Leu Val Thr Trp Val Glu Ser 340 345 350 His Asp Asn Tyr Ala Asn Gly Asp Lys Glu Ser Thr Gly Leu Thr Asp 355 360 365 Tyr Gln Ile Met Met Gly Trp Ala Val Val Gly Ser Arg Arg Ala Gly 370 375 380 Ala Pro Leu Tyr Phe Asn Arg Pro Lys Gly Ser Gly Gly Thr Asn Pro 385 390 395 400 Gln Phe Ala Glu Gln Ser Gln Leu Gly Asp Ala Gly Asp Asp Met Trp 405 410 415 Lys Asn Lys Ser Val Ala Ala Val Asn His Phe Arg Asn Ala Met Asp 420 425 430 Gly Lys Gly Glu Asn Leu Gln Asn Cys Gly Asp Lys Ser Cys Leu Met 435 440 445 Ile Glu Arg Ser Thr Ser Asp Gly Ile Gln Asn Asp Gly Val Val Ile 450 455 460 Ala Asn Met Gly Gly Asp Lys Ser Leu Ser Gly Met Glu Thr Thr Leu 465 470 475 480 Asp Asp Gly Thr Tyr Pro Asp Glu Val Asn Gly Gly Gln Leu Val Val 485 490 495 Ser Asn His Lys Ile Val Ser Gly Thr Ala Lys Gly Gly Ala Val Ser 500 505 510 Val Phe Tyr Val Lys Gly Glu Ser Asp Pro Asn Val Ser Val Glu Ala 515 520 525 Ala Ser Lys Glu Phe Ser Ser Asp Asn Val Lys Val Thr Leu Arg Ala 530 535 540 Gln Asp Ala Asp Asn Leu Lys Tyr Thr Thr Thr Glu Gly Glu Ser Gly 545 550 555 560 Ser Phe Thr Asp Gly Lys Val Ile Ser Val Gly Lys Thr Leu Ser Ile 565 570 575 Gly Glu Thr Ala Thr Val Lys Val Thr Gly Thr Ala Ala Lys Asp Gly 580 585 590 Lys Lys Val Lys Lys Gly Gln Ala Leu Ser Ala Ser Val Thr Val Lys 595 600 605 Lys Val Glu Val Pro Lys Gln Asn Leu Ala Ala Gln Tyr Ser Thr Asn 610 615 620 Lys Val Gly Met Gly Val Lys Lys Thr Ile Asn Phe Asn Ala Gly Lys 625 630 635 640 Asp Ala Ser Ile Ala Asp Trp Asp Ser Ser Met Leu Ile Ala Gln Gly 645 650 655 Ala Ala Asn Asp Asp Pro Arg Val Tyr Arg Pro Asn Ser Met Tyr Glu 660 665 670 Val Pro Ile Asp Leu Tyr Ala Leu Tyr Gly Ala Tyr Asp Asp Asp Asn 675 680 685 Leu Tyr Leu Met Trp Glu Met Thr Asn Val Gln Asp Val Val Asp Thr 690 695 700 Gly Asp Asp Tyr Pro Leu Ser Gln Gly His Leu Trp Gln Thr Gln Asn 705 710 715 720 Leu Pro Phe His Ile Ala Ile Asp Thr Lys Asp Asp Ser Thr Cys Ile 725 730 735 Gly Asn Asn Gly Gly Leu Gln Thr Gly Gly Ser Leu Trp Ala Ser Asn 740 745 750 Ile Thr Trp Gly Gly Glu Gln Lys Leu Asn Asn Val Val Thr Ile Ser 755 760 765 Thr Asn Gly Ser Asn Gly Pro Trp Ile Tyr Lys Gly Asp Glu Thr Gly 770 775 780 Leu Asn Ser Lys Ala Ala Tyr Gly Pro Ala Ala Asn Ala Ala Thr Asn 785 790 795 800 Thr Lys Lys Ser Asn Ile Lys Phe Gly Tyr Gly Asn Gly Ile Leu Ser 805 810 815 Lys Asp Val Ile Gly Ile Asp Gly Gly Trp Gly Glu Asn Asn Gly Arg 820 825 830 Phe Ile Gly Asp Met Lys Ala Glu Asn Gln Asn Lys Ala Lys Trp Val 835 840 845 Asn Phe Asn Glu Lys Gly His Asn Ser Ala Gln Met Asp Asp His Tyr 850 855 860 Glu Ile Ala Ile Pro Leu Glu Glu Ile Gly Thr Thr Ala Asp Arg Ile 865 870 875 880 Ala Ser Asn Gly Ile Gly Ile Glu Leu Ala Ala Thr Phe Gly Leu Ser 885 890 895 Ala Met Asp Ser Leu Pro Tyr Asp Leu Ala Met Asn Asp Asn Ala Asp 900 905 910 Leu Pro Asp Thr Gly Ser Gln Val Asn Asn Ser Phe Glu Lys Ser Asp 915 920 925 Asp Asp Met Phe Ser Val Lys Met Ala Asn Ile Gly Gly Glu Glu Pro 930 935 940 Pro Val Glu Thr Lys Ser Val Arg Ile Asp Gln Ser Asp Tyr Ser Val 945 950 955 960 Asp Leu Ser Asp Gly Val Thr Thr Lys Gln Leu Thr Ala Thr Thr Asp 965 970 975 Pro Lys Gly Ala Ser Val Ser Trp Ser Ser Ser Asp Lys Asp Val Ala 980 985 990 Thr Val Ser Pro Lys Gly Val Val Thr Pro Lys Lys Ala Gly Lys Ala 995 1000 1005 Thr Ile Thr Ala Lys Ser Gly Thr Lys Thr Ser Ser Ile Thr Val Thr 1010 1015 1020 Val Thr Gly Glu Leu Pro Pro Asp Pro Val Ala Lys Asn Thr Val Tyr 1025 1030 1035 1040 Ala Ser Lys Pro Ser Gly Trp Gly Lys Ile Tyr Ala Tyr Val Tyr Thr 1045 1050 1055 Gly Asp Gly Ala Thr Ala Ala Asn Asn Ala Ala Trp Pro Gly Val Glu 1060 1065 1070 Met Thr Ala Pro Ser Ala Thr Asp Gly Cys Arg Gln Thr Asp Leu Tyr 1075 1080 1085 Lys Tyr Val Val Pro Asp Asp Leu Ala Lys Asp Ala Lys Val Ile Phe 1090 1095 1100 Asn Asp Gly Gly Ser Gln Gln Tyr Pro Gly Ser Arg Gln Pro Gly Leu 1105 1110 1115 1120 Asp Tyr Asn Gly Gly Ile Val Gln Trp Asp Gly Ser Ser Ala Ala Leu 1125 1130 1135 Ala Ala Val Glu Cys Glu Thr Thr Ile Pro Val Thr Ser Val Ser Ile 1140 1145 1150 Ser Gly Asp Gly Val Ser Gly Gly Lys Leu Ser Leu Lys Ser Gly Ala 1155 1160 1165 Ser Ala Gln Leu Thr Ala Thr Val Lys Pro Asp Asn Ala Thr Asp Arg 1170 1175 1180 Lys Val Thr Trp Thr Ser Ser Asp Ser Ser Val Ala Asn Val Met Gly 1185 1190 1195 1200 Thr Gly Val Val Thr Ala Gly Ser Lys Ala Gly Lys Ala Thr Ile Lys 1205 1210 1215 Ala Thr Ala Gly Gly Lys Ser Ala Ser Val Thr Val Thr Val Thr Asp 1220 1225 1230 Val Pro Lys Gln Pro Met Thr Val Trp Tyr Lys Pro Ala Ser Ser Trp 1235 1240 1245 Lys Thr Ala Lys Val His Tyr Lys Ala Asn Gly Lys Trp Thr Gly Ser 1250 1255 1260 Ala Gln Gln Met Thr Leu Tyr Arg Asp Gly Trp Tyr Lys Tyr Thr Ile 1265 1270 1275 1280 Pro Asp Thr Ala Gly Gly Gln Val Arg Met Ala Phe Thr Asn Gly Ser 1285 1290 1295 Ala Trp Asp Asn Asn Ser Gly Lys Asp Tyr Phe Ala Ser Gly Ser Val 1300 1305 1310 Val Ser Val Ser Gly Gly Lys Met Ser Ser Lys Ala Pro Ser Phe Gly 1315 1320 1325 Thr Pro Met Thr Val Trp Tyr Lys Pro Ala Ser Ser Trp Lys Thr Ala 1330 1335 1340 Lys Val His Tyr Lys Ala Asn Gly Lys Trp Thr Gly Ser Ala Gln Gln 1345 1350 1355 1360 Met Thr Ala Ala Cys Gly Gly Trp Tyr Lys Tyr Thr Ile Pro Asp Thr 1365 1370 1375 Ala Gly Gly Gln Val Arg Met Ala Phe Thr Asn Gly Ser Ala Trp Asp 1380 1385 1390 Asn Asn Ser Gly Lys Asp Tyr Met Val Ser Gly Asp Ser Ala Ala Val 1395 1400 1405 Ala Gly Gly Gln Ser Val Pro Asp Val Thr Pro Asn Cys Thr Ile Thr 1410 1415 1420 Asn Lys 1425 <210> 4 <211> 3324 <212> DNA <213> Unknown <220> <223> P2P3-RSD1 <400> 4 atgaaacatc ggaaacccgc accggcctgg cataggctgg ggctgaagat tagcaagaag 60 gtggtggtcg gcatcaccgc tgcggcgacc gccttcggcg gactggcaat cgccagtaca 120 gcggcacagg ccagcaccga tcgtgacagc tacgccgaca ccgttgaaaa cgccacattc 180 gaacaggctc gcaagcatta cggactggcg cagcaaatga gcgagggcgc caccctgcat 240 gcatgggagt ggagcttcaa aaccattgag gaaaacatcc ccgctattgc cgaagccggt 300 tacacctccg tgcagaccga gccgatttcc gctattcata acggcggcaa gggcatgatc 360 ttcaccgaga actggtacta cgtatatcag ccgaccgaca ccaccatcgg caactgggtg 420 atgggtaccg aagatgatct gaagagcctg tgcaacacgg ctcacaagta cggcgtgcgt 480 atcatcgtcg atgtggtagc caaccatatg accgcgactt ggggcgccat cgccgatcgt 540 tggaagaagt ccgagtacta ccaccacgac tgcaatgacg gtgatgtgca ggattggaac 600 aaccgctatc aggtgaccca ctgcaagctg ctcggcctgt atgacatcaa cactgagaac 660 accaaaaccg ccaacatgat gcacgatttc ctcgtgcaag cggtcaacga cggtgtggac 720 gggttccgtt tcgatgcggc caagcacatc gaactgccgg acgagtataa cggctcccag 780 tattggaaca tcattcttaa caatggcgcg cagttccaat acggcgaagt gttgcaggat 840 tccatctccc gcgatagcga ctatgcaaag ctgttctcca gccacagcaa gaacggcggc 900 ggcgtcaccg catcctccta cggcagcaag ctgcgcggag ccctgaattc gaagaacctg 960 aacgccggca ccctgtccga ttggagcaac tccgccagcc cgagcaatct tgtctcttgg 1020 gtggaatcgc atgataacta ttccaatagc gatagggaat ccaccggcat gagcgagtgg 1080 cagatgacca tgggctgggg cgtgatcggt tcccgctccc agaccatgcc gctgtacttc 1140 gaccgcccgg tcggctccgg cggcagccag ccgcagttcg ccgagaaaag caagctcggc 1200 gatgccggtg cggattcttg gaaagatccg caggtcgtgg cggtgaacca cttccgtaat 1260 accatgaaca acaacaaggc ttcggagtat ctgcgcaact gcagcgcgaa ttcctgcctc 1320 atggcggaac gctacatcaa ggacggcaac ttcaagaacg acggcgtgac catcaccaac 1380 atgggtgata cgcaagagtt gtccggcact gccaccaacc ttgacgacgg cacttacaag 1440 gaccaggtct ccggcggtac catcaccgtt tccggcggca agatcacttc cggttccgct 1500 cccggcggca agatcagtgt gttcttcacc gacaattccg gttcggtctc cgcaaccccg 1560 ggcgacagct cgttcaagac ggacaccact accgttaccc tgaatgcgaa caatgtcacc 1620 gatgccactt acaccacttc ggagggcaag tccggctcgt atcaggatgg cgacaccatc 1680 accatcggcg cttccacggc gatcggtgac accatcaccg tgaagctgca gggcaaggat 1740 gcggatggcc aaacggtctc cgcaacatac aagtacacga agaaggatcc ggccgccaca 1800 agcacggctt atgcgaagaa gccgagtgcg tggagcaacc tgtacgccta tgtgtatgta 1860 gacgattcga gcgccaccac tttgaaggaa aatgcgaagt ggccgggcga accgatgaca 1920 caggtcgctt cgggagacac ttgtggcaag gatggcgagt acaagtatga gattcccgac 1980 gatcttgagg gaagcaatac gcgcatcatc ttcaacgatg gcaatgccac aaataccaag 2040 aagtatcctg ccgacaccac cgaaggcgag gatgccgctg gcctcaagat cgacggcaat 2100 tacgcttggg acggcaacac ttcgtccggc acctgggagg cccgcaattg cgtggtcatc 2160 ccgaccaagt ccgtgaagat cgatcagtcg gattacacca cggacctgtc caatggtgtg 2220 gccacgaagc agctgacagc cacgaccgat ccgaagggcg cgtccgtgag ctggagctcg 2280 tccgacaagg acgtggcgac cgtcagcccg aagggcgtgg tcaccccgaa gaaggccggc 2340 aaggccacga tcaccgcgaa gtccgggacc aagacctcgt cgatcaccgt gaccgtgacc 2400 ggtgagcttc cgccggaccc ggtcgcgaag aacaccgtct acgcttcgaa gccctcgggc 2460 tggggcaaga tctacgcgta cgtgtacacc ggcgacggcg cgaccgcggc gaacaacgcg 2520 gcctggccgg gcgtggagat gaccgccccg agcgcgaccg acggctgccg gcagaccgac 2580 ctgtacaagt acgtggtccc ggacgacctc gccaaggacg cgaaggtgat cttcaacgac 2640 ggcggcagcc agcagtatcc gggatcccgc cagccgggcc tggactacaa cggcggcatc 2700 gtgcagtggg acggctcctc cgcggccctg gccgcggtcg agtgcgagac caccatcccg 2760 gtcacgtccg tctcgatctc cggtgatggc gtgagcggcg gcaagctctc gctgaagtcc 2820 ggggcgagcg cgcagctgac cgcgacggtc aagcccgaca acgcgacgga ccgcaaggtc 2880 acatggacga gctcggactc gagcgtcgcg aacgtgatgg gcacgggcgt ggtcaccgcc 2940 ggctcgaagg ccggcaaggc cacgatcaag gcgaccgcgg gcggcaagag cgcgagcgtc 3000 acggtgacgg tcacggacgt gccgaagcag ccgatgacgg tgtggtacaa gccggcgtcg 3060 tcgtggaaga ccgcgaaggt gcattacaag gcgaacggca agtggacggg ctcggcccag 3120 cagatgaccg cggcgtgtgg cggctggtac aagtacacga tcccggacac cgccggcggg 3180 caggtgcgca tggcgttcac gaatggttcc gcgtgggaca acaattccgg caaggactac 3240 atggtgtctg gcgacagtgc cgctgtggcc ggagggcagt ccgttccgga cgtcacacca 3300 aactgcacca taaccaacaa ataa 3324 <210> 5 <211> 3660 <212> DNA <213> Unknown <220> <223> P2P3-RSD2 <400> 5 atgatgaaac aatcagcaac gcttgctcgt ctgagcaaga agaccgtggc gttccttgcc 60 gcggccacca cgctgctcgc gggtctttcg ctcgctacgg ccgcaccgca gcaggccagc 120 gccgccaccc gtgacagcta cgccgatacc atcggcaatc cgacgttcga ggccgcccgt 180 cagaagtacg gcctgccgaa ggacatgaag gacggcgcga ccctgcacgc cttcgaatgg 240 tccttcaaaa ccatcatgga aaacattccg gatatcgcca aggccggtta tacctccatc 300 caaaccgaac caatcgtcaa aatcaaggac aaccgcaagc ttggcaccgg caactggtat 360 ctcaactggt actatgtcta ccagccgacc gacatgtcca tcggcaacta tgtggtcggt 420 tcggaagacg aattcaagga aatgtgcaag ctggcacacc agtacggcct gcgcatcatc 480 gtcgattccg tgtccaacca tttcacttcc gattttgatg tgatcgaagg caagtggaag 540 aacaaagcct actaccatga ggacaagcag atctccgatt acaacaatcg agaggactgc 600 acccagcaca agctctccgg cctgtgggat ctcaacaccc aggatgatac cgtcaccgaa 660 ggcatgcacg atctgctcgt gcagactgtg gtcgacggcg ctgatggctt ccggtatgac 720 gctgccaagc atatcgagct ttccgatgag gtattcggcg gcaagcagtc ccactattgg 780 gacaccatcc tgcagaacgg cgcacagtac cagtacggcg aagtgctgga agacgccaat 840 gtgcgtgaag ccgactatgc cgatctgttc aacaagagct ccgttggcgg tggcggcatc 900 accgactcca gctacggcca tgaggtgcgc aacgccgtgc agttcaagaa tctcggcgca 960 agccatttca cctcgcattc caaggtgacc gaagacaaaa ccgtgaactg ggtggagtcc 1020 catgataact tcgccaacgg cgaggccaac atcccgcagg agctctccga cgaatggatc 1080 aagtacggct gggccggtgt gaccgcgcag aagaacggca tgagcctgtt cttcgaccgc 1140 ccgtacaagg acggcggcac gtatggcact ggtggcgtcg gcacgtatgg taatggcagc 1200 ggtccgttca ccgagaactc caagctcggc gacgccggct ccgacctgtg gaaggatccg 1260 gaagtcgtgg ccgtcaacca cttccgcaac gccatggtcg gcgaagcgtc aaatgtctcc 1320 aactgcggcg acgacaactg cctgatggtc gagcgttatg ccggttccgc ggctcaggac 1380 ggcatggtag tggccaatgc gaacggttcc gacaagaatc ttgccggcca gtccaccaag 1440 ctggccaacg gcacttatac ggatgaagtg accggttcca ccatcaccgt ttccggtggc 1500 aaggttacct ccggcacagt gaagggccag tccatcgcgg cgttttccaa taagacccgt 1560 tccggcaagg tctccaccgc cgaggcatat ccgaacaagg gcaccattcc gggcgaatct 1620 aaaacgatca ctctccggtc ctatcagtcc accaacacca cctactccac ttccgacggt 1680 cagtccggtt ccttcaagga cggcgacacc attgaaatcg gcagcaaggc caagtcggat 1740 gaagtggtga cggtcacggt gaagggcacc ggtgcagacg gcgaagccat caagcacacg 1800 tactcttaca ccaagtatgg cgatcctact tacgacccga gtgacggcac ttccagcccg 1860 tgcgacaaat attgcatgga gtatatggag aagaacggcc tggatccgga ttgctacatt 1920 gacgccaaag agccttgcca tccaaggaat gtgaccgtta ctgccatcaa ggtttccggc 1980 gacacttcca tggatctcgc ttccacgcag agcgtgcagc tcaaggcgga tgtgacaacc 2040 gatccggcgg gcaagcgtcc ttccgtgact tggacttctt ccgacacttc tgttgccacc 2100 gtcgattcca agggcaaggt ttccggcctt aaggcgggaa cggtgaagat taccgccact 2160 gccggcacga acaagaaatc cgactccatc accatcacga tcaccggcgt gaagccggaa 2220 gtctccaacg tgatctacgc caccaagcct tccggctgga gcaagatcta cgcttacgtg 2280 tacaacgaca ccacgtccaa gaacaatggc aactggccgg gcgttgaaat gacccagctg 2340 accgccgacg attcctgtgc gaaggccggc acctacaagt acgaggtatc ggatttgggc 2400 gaaggcaagt accgtgtgat cttcaccaac ggttccggcg cgcagacccc tggtggcaac 2460 cagcccggca tcgaattctc cggcaaagtc agctgggatg gctcttccgc cgcggtgagt 2520 gcggtgaact gcaacacccc gcagccggtt ccggtctctt cagtctcgat ttcgggttcc 2580 ggtgtgagca acggcaagct gagcctgaag tcaggcgcga gcgtgcaatt gacggccacc 2640 gtgaccccgt cgaatgcgac ggacaagaca gtctcttgga ccagctcgaa tagttctgtg 2700 gctaaagtgt ccgacggcaa aatcaccgct gtgaaaggtg gcactgcaac gattacagcg 2760 accgctggcg gtaagacggc ttcggtcgct gttactgtgg ccgataatcc ggttccggtc 2820 acgtccgtct cgatctccgg tgatggcgtg agcggcggca agctctcgct gaagtccggg 2880 gcgagcgcgc agctgaccgc gacggtcaag cccgacaacg cgacggaccg caaggtcaca 2940 tggacgagct cggactcgag cgtcgcgaac gtgatgggca cgggcgtggt caccgccggc 3000 tcgaaggccg gcaaggccac gatcaaggcg accgcgggcg gcaagagcgc gagcgtcacg 3060 gtgacggtca cggacgtgcc gaagcagccg atgacggtgt ggtacaagcc ggcgtcgtcg 3120 tggaagaccg cgaaggtgca ttacaaggcg aacggcaagt ggacgggctc ggcccagcag 3180 atgaccctgt acagggatgg ctggtacaag tacacgatcc cggacaccgc cggcgggcag 3240 gtgcgcatgg cgttcacgaa tggttccgcg tgggacaaca attccggcaa ggactacttc 3300 gcgtccggat cggtcgtttc ggtgtccggc gggaagatgt cctccaaggc cccgtcgttc 3360 ggcacgccga tgacggtgtg gtacaagccg gcgtcgtcgt ggaagaccgc gaaggtgcat 3420 tacaaggcga acggcaagtg gacgggctcg gcccagcaga tgaccgcggc gtgtggcggc 3480 tggtacaagt acacgatccc ggacaccgcc ggcgggcagg tgcgcatggc gttcacgaat 3540 ggttccgcgt gggacaacaa ttccggcaag gactacatgg tgtctggcga cagtgccgct 3600 gtggccggag ggcagtccgt tccggacgtc acaccaaact gcgcagtaac taacaaatga 3660 3660 <210> 6 <211> 4281 <212> DNA <213> Unknown <220> <223> P2P3-RSD3 <400> 6 atgacccgtg cagagcgtag gcgcgcggcc cgcaaggctc gtgcggccaa aacctggaag 60 aaagtagcgg cgggaacggt ggcggtcgcc acgctgttcg gcggtatggg tgtggcatcc 120 accgcacttg ccgccgatcg tgacagctac caggacacca tcggaaattc ttctttcgaa 180 gcggcccgta accagtacgg tctgaccaag cacatgaaga acggcgccat cctgcacgca 240 tggatgtggt cgttcaagac catcaccgcag cacatgcctg agatcgccgc ggcgggctac 300 acttccgtgc agaccgagcc tatgagcaag atcaaggaag tcgccgccaa cggcaagaag 360 ttcaccgaga actggtacta cgtgtaccag ccggcgaaca cctccatcgg caacttcgtg 420 gtcggcaccg aggctgacct gaaggaaatg accgccaccg cccataagta tggtgtgcgt 480 gtgatcgtgg atgtggtcgc caaccacttc acctccgatt ggaacgccat cgaccccgat 540 tggcagaaca aggagtactt ccacaagcgt accggctgcg acggccccaa cggtgaaatc 600 aacgattatt ccaaccgtta caaggtgacg cagtgccacc tgcttggcct gtgggatctg 660 aatacgcaga accaggctgt tgccgatcgt atgcagaagt tcctgaagac cgctgtggcg 720 gacggcgttg acggcttccg ctacgatgcg gcaaagcacg tcgagctgcc gaccgaagtc 780 ttcgacaata agcagtcgaa ctactggaac accattctga agaacggctc ccagttccag 840 tacggcgagg tgctgcaggg cgattccggc cttgactaca aggcatacgc caacatgttc 900 cgcgacaatt cctccgatgg cggcggcaac accgcttcca actacggcaa gaccattcgc 960 gccgctgtcg gctcgaacaa ccttgacgtc aagatggtga agaacatcga taccggcggc 1020 gcaagcgaag accagctggt cacctgggtt gagtcccacg ataactacgc caacggcgac 1080 aaggaatcca ccggcctcac cgactatcag atcatgatgg gctgggcggt cgttggttcc 1140 cgtagggccg gcgctccgct gtacttcaac cgtccgaagg gctctggcgg caccaacccg 1200 cagttcgccg agcagtccca gctgggtgac gccggtgacg acatgtggaa gaacaagtcc 1260 gtcgcggcag tcaaccactt ccgtaacgcc atggatggca agggcgagaa cctgcagaac 1320 tgcggtgaca agtcctgcct gatgattgag cgctccacgt ctgatggcat tcagaacgat 1380 ggtgtggtca ttgccaacat gggtggcgac aagagccttt ccggcatgga aaccacactt 1440 gacgatggca cctacccgga tgaggtcaat ggcggccagc ttgtggtctc caaccataag 1500 atcgtttccg gtaccgccaa gggcggcgcg gtcagcgtct tctacgtgaa gggcgaatcc 1560 gatccgaacg tgtccgtgga agcggcttcc aaggaattct cctccgacaa tgtcaaggtc 1620 acgttgcgcg cgcaggatgc cgacaatctg aagtacacca ccaccgaggg cgagagcggc 1680 tccttcactg acggaaaggt catttccgtt ggtaaaactc tttccatcgg cgagaccgct 1740 accgtcaagg tgaccggcac cgccgccaag gatggcaaga aggttaagaa gggtcaggcg 1800 ctgagcgctt ccgtgaccgt gaagaaagtg gaggtcccga agcagaacct tgccgcgcag 1860 tacagtacca acaaggtggg catgggcgtc aagaagacca tcaacttcaa cgccggaaag 1920 gacgcgagca tcgctgactg ggacagctcc atgctgatcg cgcagggcgc cgccaacgac 1980 gatccgcgcg tgtaccgccc gaattccatg tatgaggttc cgatcgatct gtatgcgttg 2040 tatggcgcct acgatgacga caacctctac ctcatgtggg aaatgactaa cgtgcaggat 2100 gtcgtcgaca ctggcgacga ctaccccctg agccaaggcc acctgtggca gacccagaac 2160 ctgccgttcc acatcgccat cgacaccaag gatgacagca cctgcatcgg caacaacggc 2220 ggtctgcaga ccggcggttc cctgtgggcg tccaacatca cttggggtgg cgagcagaag 2280 ctcaataacg tggtgaccat ttccaccaac ggttctaacg gtccgtggat ttacaagggc 2340 gacgagactg gtctgaactc caaggctgcg tatggtccgg ctgctaacgc tgccaccaac 2400 accaagaagt cgaacatcaa gttcggctat ggcaatggca ttctaagcaa ggatgtcatc 2460 ggcatcgacg gtggctgggg tgaaaacaac ggtcgtttca tcggcgatat gaaggccgag 2520 aaccagaaca aggccaagtg ggtcaacttc aacgagaagg gccacaattc cgctcagatg 2580 gatgatcact acgagatcgc catcccgctg gaagagatcg gcaccaccgc tgaccgcatt 2640 gcctcgaacg gtatcggcat cgagctggca gccaccttcg gtctgtccgc catggattcc 2700 ctgccgtacg atctggcaat gaacgacaat gctgacctgc cggacaccgg ttctcaggtg 2760 aataactcct tcgagaagtc cgatgacgac atgttcagcg tgaagatggc caacatcggc 2820 ggcgaagagc ctccggtcga aaccaagtct gtgaggattg atcagtcgga ttacagcgtg 2880 gacctgtccg atggcgtgac cacgaagcag ttgacagcca cgaccgatcc gaagggcgcg 2940 tccgtgagct ggagctcgtc cgacaaggac gtggcgaccg tcagcccgaa gggcgtggtc 3000 accccgaaga aggccggcaa ggccacgatc accgcgaagt ccgggaccaa gacctcgtcg 3060 atcaccgtga ccgtgaccgg tgagcttccg ccggacccgg tcgcgaagaa caccgtctac 3120 gcttcgaagc cctcgggctg gggcaagatc tacgcgtacg tgtacaccgg cgacggcgcg 3180 accgcggcga acaacgcggc ctggccgggc gtggagatga ccgccccgag cgcgaccgac 3240 ggctgccggc agaccgacct gtacaagtac gtggtcccgg acgacctcgc caaggacgcg 3300 aaggtgatct tcaacgacgg cggcagccag cagtatccgg gatcccgcca gccgggcctg 3360 gactacaacg gcggcatcgt gcagtgggac ggctcctccg cggccctggc cgcggtcgag 3420 tgcgagacca ccatcccggt cacgtccgtc tcgatctccg gtgatggcgt gagcggcggc 3480 aagctctcgc tgaagtccgg ggcgagcgcg cagctgaccg cgacggtcaa gcccgacaac 3540 gcgacggacc gcaaggtcac atggacgagc tcggactcga gcgtcgcgaa cgtgatgggc 3600 acgggcgtgg tcaccgccgg ctcgaaggcc ggcaaggcca cgatcaaggc gaccgcgggc 3660 ggcaagagcg cgagcgtcac ggtgacggtc acggacgtgc cgaagcagcc gatgacggtg 3720 tggtacaagc cggcgtcgtc gtggaagacc gcgaaggtgc attacaaggc gaacggcaag 3780 tggacgggct cggcccagca gatgaccctg tacagggatg gctggtacaa gtacacgatc 3840 ccggacaccg ccggcgggca ggtgcgcatg gcgttcacga atggttccgc gtgggacaac 3900 aattccggca aggactactt cgcgtccgga tcggtcgttt cggtgtccgg cgggaagatg 3960 tcctccaagg ccccgtcgtt cggcacgccg atgacggtgt ggtacaagcc ggcgtcgtcg 4020 tggaagaccg cgaaggtgca ttacaaggcg aacggcaagt ggacgggctc ggcccagcag 4080 atgaccgcgg cgtgtggcgg ctggtacaag tacacgatcc cggacaccgc cggcgggcag 4140 gtgcgcatgg cgttcacgaa tggttccgcg tgggacaaca attccggcaa ggactacatg 4200 gtgtctggcg acagtgccgc tgtggccgga gggcagtccg ttccggacgt cacaccaaac 4260 tgcaccataa ccaacaaata a 4281 <210> 7 <211> 1632 <212> DNA <213> Unknown <220> <223> P2P3-RSD4 <400> 7 atgctttccc ccgcgaaaca cccccacccc aaatggcttg ccgacgcaat tttttatgaa 60 atctatccac agtccttcgt cgactccaat ggcgacggca tcggagacat ccccggaatc 120 actttaaaac tggactacat caaggatctc ggatgcaacg cgatctggct caatccctgt 180 ttcgactcgc cattcaaaga cgccggctac gacgtgcgcg actacaaaaa ggtggcatca 240 cgctacggca ccaacgacga tctcattgcg ttgttcgacg ccgcccaccg gcgcgacatg 300 cacgtgatcc tcgatctcgt gccaggccac accagcgagg aacacgagtg gttccaccgc 360 agttgcaagg tcgaacgaaa caattattcc gaccgctaca tctggacaga ctcatggatc 420 tccggtggcg acggactgcc attcatcggc ggcgaatcgc cccgcaacgg tacctacatc 480 ttgaacttct tcaaatgcca gccggcgctc aactacggtt tcgcgcaccc cgaacgtagc 540 tggcagaagc cagcccttgg tccggacgcg aaggccactt gcgacgcgat ggtggacgtc 600 atgcggttct ggctttcccg tggcgcagac ggcttccgcg tggatatggc cgacagcctc 660 gtcaaaaagg acgatgaggg caagccttac accatccgca cctggcagta catgttcgcc 720 cggatccgcc cggccttccc cgaagccgcg ttcgtttcgg aatggggacg gccagaggag 780 tcgatggccg ccggcttcga catggatttc tatctcgatt ggcgttggga cggcgttccc 840 aacggctaca acatgctgtt gcgcaacgtg gacgacccgt tgagccgcaa gggcgacaag 900 agctatttca acgccgattc cggcacgccg atcagcgaat tcctggacga gtacgagccg 960 tgtctgcgcg aagcggaaac ggccgacgga aattttaatt tcatcacctg caaccacgac 1020 accgcacgcg tggcgccgcg actgtcgcag cgcgaaatcg ccctggcata ctgcacgctg 1080 ttcgcgatgc cgggcgtgcc gttcctgtat tacggcgacg aaatcggcat gcgataccgc 1140 gcactgcccg gcaaagaagg cggctacgtg cgcaccggtt cgcgcacacc gatgcagtgg 1200 ggtgcgaacg acgcatccgg agcacttcac gtgtccgatt ctgcagaatc ggacactcgg 1260 gctggcgatg tgaccggtgg cagctacctg cccgccgacc cgtccgccga cgccccgacc 1320 gtcgccgcac agcaggccga cgccgaatca ctgtggcata cggtgcgctc catactccac 1380 gtcaaggggg aatgcgacgc cttgcggtcg ggggctacgt tccatgcaat cgaacgcgat 1440 agagtgttcg ttttcgagcg ttccacccac ctgcagaagc tgatcatagc cgtcaatcca 1500 agccgcgatt gcaaagcttt gcagctacca gatggtagtt atcggacgat tttcagcatc 1560 ggcgaaaacg aaattagcgg cacttcgctg cgtctaggtc tgcaaagctt tgcaattctc 1620 gaagccgagt ag 1632 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> RSD1_F <400> 8 atgagcgagg gcgccac 17 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RSD1_R <400> 9 tttgttggtt atggtgcagt ttggtg 26 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> RSD2_F <400> 10 gccacccgtg acagctacg 19 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> RSD2_R <400> 11 tttgttagtt actgcgcagt ttggtgt 27 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> RSD3_F <400> 12 atgacccgtg cagagcg 17 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> RSD3_R <400> 13 tttgttggtt atggtgcagt ttggtg 26 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RSD4_F <400> 14 atgctttccc ccgcgaaaca 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RSD4_R <400> 15 ctcggcttcg agaattgcaa agc 23

Claims (6)

A composition for decomposing resistant starch, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.
The composition of claim 1, wherein the resistant starch is high amylose corn starch.
According to claim 1, wherein the α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3 is Bifidobacterium adolescentis ( Bifidobacterium adolescentis ) It is derived from, the composition.
The composition of claim 3, wherein the Bifidobacterium adolcentis is Bifidobacterium adolcentis P2P3 deposited with accession number KACC 92235P.
A recombinant microorganism having a resistant starch degrading activity, comprising α-amylase consisting of any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 3.
Any one or more of a recombinant microorganism having a resistant starch degrading activity or a culture medium containing the α-amylase comprising α-amylase consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 is added to a sample containing resistant starch. A method of decomposing resistant starch, comprising adding.

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