WO2018230952A1 - Novel polypeptide having turanose production activity and method for producing turanose using same - Google Patents

Abstract

The present application relates to a polypeptide having an activity of converting sucrose into turanose, a mutant polypeptide thereof, a polynucleotide encoding the polypeptide or the mutant polypeptide, a vector comprising the polynucleotide, and a recombinant microorganism comprising the polynucleotide or the vector, and a method for producing turanose using the same.

Description

Novel polypeptide having turanose production activity and method for producing turanose using same

The present application relates to a polypeptide having an activity of converting sucrose to turanose, a variant polypeptide thereof, and a method for producing turanose using the same.

Turanose is an isomer of sucrose, α-D-glucopyranosyl- (1 → 3) -αD-fructopira containing glucose and fructose units linked via α (1 → 3) bonds. North [α-D-glucopyranosyl- (1 → 3) -α-D-fructopyranose].

Turanos have a sweetness of about half (about 0.5) of sucrose sweetness (about 1), can be easily crystallized, has high solubility and low cariogenicity, and can be applied to food as a sugar substitute. Turanose is also of interest in the pharmaceutical and diagnostic fields as it is an inhibitor of α-glucosidase useful for the diagnosis of Pompe's disease. However, turanose is present in the honey in very small amounts of 0% to 3%, so an alternative method for industrial production is required.

Conventionally, a method for converting sucrose to turanose using an enzyme derived from Neisseria polysaccharea has been proposed (Wang et al, 2012, Food Chemistry , 132, 773-779; Korean Patent No. 10-1177218). Ho), because it is not a heat-resistant enzyme, there was a problem that is difficult to apply to industrial production.

The present inventors have diligently researched to develop a polypeptide having an activity of converting into turanose having heat resistance, and as a result, the polypeptide of the present application was found to have activity of converting sucrose into turanose while having heat resistance. The present application was completed by obtaining a variant polypeptide with further improved turanose conversion activity.

One object of the present application is to provide a polypeptide having the activity of converting sucrose to turanose comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1.

Another purpose of this application is

A variant polypeptide comprising one or more amino acid variations in the amino acid sequence of SEQ ID NO: 1, wherein said amino acid variant is one Glycine (G: Glycine) amino acid residue from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 It is to provide a variant polypeptide having the activity of converting sucrose to turanose, including those substituted with the above other amino acids.

Another object of the present application is to provide a polynucleotide encoding the polypeptide or variant polypeptide.

Another object of the present application is to provide a vector comprising a polynucleotide of the present application.

Another object of the present application is to provide a recombinant microorganism comprising the polynucleotide or the vector.

Another object of the present application to a polypeptide comprising an amino acid sequence having an amino acid sequence of at least 85% homology with the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the polypeptide, or a composition for producing a turanose comprising a culture of the microorganism To provide.

Another object of the present application is to contact a sucrose to a polypeptide consisting of a sequence consisting of an amino acid sequence having at least 85% identity with an amino acid sequence of SEQ ID NO: 1, a microorganism expressing the polypeptide, or a culture of the microorganism, To provide a method for producing turanose, comprising the step of converting sucrose to turanose.

The present application has the advantage of enabling the industrial production of turanose by newly proposing a heat resistant polypeptide and a variant polypeptide thereof having the activity of converting sucrose to turanose.

1A to 1D show the results of confirming the turanose production activity of MRF, MRF + G392T, MRF + G392E, and MRF + G392A, respectively.

2 is a result of activity evaluation according to the temperature of MRF, MRF + G392T, MRF + G392E and MRF + G392A.

3A and 3B show the results of activity evaluation according to pH of MRF and MRF + G392A, respectively.

In the following, the present application will be described in more detail.

In addition, each description and embodiment disclosed in this application can be applied also to each other description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of the present application. In addition, the scope of this application is not limited by the specific description described below.

In addition, one of ordinary skill in the art can recognize or ascertain a number of equivalents to certain aspects of the present application described in this application using conventional experiments only. Also, such equivalents are intended to be included in this application.

One aspect of the present application for achieving the object of the present application provides a polypeptide having the activity of converting sucrose to turanos comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1 It is.

The polypeptide of the present application may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% homology or identity thereto. Specifically, the polypeptide may include SEQ ID NO: 1 and polypeptides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1. Can be. An amino acid sequence having homology or identity may exclude sequences having 100% identity in the above categories or may be sequences having less than 100% identity. In addition, if the polypeptide has such homology or identity and has an amino acid sequence that exhibits an efficacy corresponding to the polypeptide (ie, the activity of converting sucrose to turanose), the amino acid sequence of which some sequences are deleted, modified, substituted or added It is apparent that a polypeptide having a can be used as a polypeptide of the present application.

Another aspect of the present application is a variant polypeptide comprising at least one amino acid variation in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid variation is the 392th glycine from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (G: Glycine) It is to provide a variant polypeptide having the activity of converting sucrose to turanose, wherein the amino acid residue is substituted with one or more other amino acids.

The variant polypeptide is sucrose, in which the 392th glycine amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine (T: Threonine), glutamic acid (E: Glutamic acid) or alanine (A: Alanine) It may be a variant polypeptide having the activity of converting to, but is not limited thereto. Such variant polypeptides have enhanced activity in converting sucrose to turanose as compared to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "polypeptide having the activity of converting sucrose to turanose" refers to a variant polypeptide having the activity of converting sucrose to turanose, a turanose producing variant polypeptide, a variant polypeptide that produces turanose It can be used in combination with a variant amylosucrase, amylosucrase variant and the like.

Specifically, the variant polypeptide may be composed of the amino acid sequence of SEQ ID NO: 3, 5 or 7. In addition, the variant polypeptide may include, but is not limited to, an amino acid sequence of SEQ ID NO: 3, 5, or 7 or an amino acid sequence having at least 80% homology or identity thereto. Specifically, the variant polypeptide of the present application has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 3, 5 or 7 And polypeptides. In addition, as long as the polypeptide has an amino acid sequence having such homology or identity and exhibits a potency corresponding to the polypeptide, it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the present application. .

As used herein, the term “variant polypeptide” refers to a culture or individual that exhibits one stable phenotypic change, genetically or non-genetically, and specifically in the present application, one or more amino acid sequences derived from Corynebacterium glutamicum It refers to a variant polypeptide whose amino acid is mutated and its activity is weakened compared to wild type, so that the carbon flow is efficiently balanced. For the purposes of the present application, the variant polypeptide is a polypeptide having an activity of converting sucrose to turanose, a variant polypeptide having activity of converting sucrose to turanose, a variant amylosucrase, amylosucra It can be used interchangeably as the first variant.

In addition, the 'variant' may be used interchangeably with terms such as variant, mutated protein, and variant polypeptide, and in English, it may be used as variant, modification, modified protein, modified polypeptide, mutant, mutein, divergent, etc. As long as the term is used in a mutated sense, it is not limited thereto.

In addition, even if it is described as 'a protein or polypeptide having an amino acid sequence described by a specific sequence number' in the present application, if the sequence having the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the sequence number, some sequences are deleted, It is apparent that proteins having modified, substituted or added amino acid sequences can also be used in the present application. For example, in the case of having the same or corresponding activity as that of the variant polypeptide, in addition to the specific 392th mutation that confers a specific activity, a meaningless sequence before or after the amino acid sequence of the corresponding SEQ ID number or a naturally occurring mutation, or its It is not intended to exclude latent mutations, and to have such sequence additions or mutations is obviously within the scope of the present application.

The term “homology” refers to the degree of relevance to two given amino acid sequences or base sequences and can be expressed as a percentage. In this specification, homologous sequences thereof having the same or similar activity as a given amino acid sequence or base sequence are designated as "% homology".

In addition, "identity" refers to the degree of agreement between amino acid or nucleotide sequences, and in some cases determined by the match between strings of such sequences.

The terms "homology" and "identity" are often used interchangeably.

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and the default gap penalty established by the program used may be used together. Substantially, homologous or identical polynucleotides or polypeptides generally have a medium stringency or along at least about 50%, 60%, 70%, 80% or 90% of all or the full-length of the target polynucleotide or polypeptide. Will hybrid at high stringency. Also contemplated are polynucleotides containing degenerate codons instead of codons in the hybridizing polynucleotides.

Any two polynucleotide or polypeptide sequences are at least for example 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 Whether having% or 99% homology or identity is described, for example, in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: can be determined using known computer algorithms such as the " FASTA " program using default parameters such as at 2444. Or in the needle-only program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later) Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as can be determined, can be determined. (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); including 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, homology or identity can be determined using BLAST, or ClustalW, of the National Biotechnology Information Database Center.

The homology or identity of a polynucleotide or polypeptide is described, for example, in Smith and Waterman, Adv. Appl. As known in Math (1981) 2: 482, for example, Needleman et al. (1970), J Mol Biol. 48: 443, and can be determined by comparing the sequence information using a GAP computer program. In summary, the GAP program defines the total number of symbols in the shorter of the two sequences, divided by the number of similarly arranged symbols (ie, nucleotides or amino acids). The default parameters for the GAP program are (1) a binary comparison matrix (containing 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. As disclosed by 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 gap opening penalty 10, gap extension penalty 0.5); And (3) no penalty for the end gap. Thus, as used herein, the term “homology” or “identity” refers to a comparison between polypeptides or polynucleotides.

In addition, a variant polypeptide having a homologous or homologous polypeptide having a amino acid sequence in which the 392th amino acid is replaced with another amino acid from the N-terminus in the amino acid sequence of SEQ ID NO. 1 by codon degeneracy. Obviously, polynucleotides that can be translated into can also be included. In addition, probes which can be prepared from known gene sequences, for example, hydride under stringent conditions with complementary sequences for all or part of the nucleotide sequence, wherein the 392th amino acid in the amino acid sequence of SEQ ID NO: 1 is threonine Any polynucleotide sequence encoding a variant polypeptide having the activity of a variant polypeptide consisting of an amino acid sequence selected from the group substituted with (T: Threonine), Glutamic acid (E: Glutamic acid) or Alanine (A: Alanine) May be included.

Polypeptides of the present application or variant polypeptides of the present application may have heat resistance.

As used herein, the term "heat resistance" means that the thermal stability of the variant polypeptide is increased. Specifically, by the heat resistance, the enzyme reaction is possible at high temperature, and the solubility of the substrate may be increased in the high temperature reaction process. As the substrate solubility increases, it is possible to use a high concentration of the substrate, and the productivity may be improved by shortening the reaction time by increasing the diffusion rate or the reaction rate of the material. In addition, process contamination due to external microorganisms can be minimized.

In addition, after the mass expression of the polypeptide in GRAS (generally recognized as safety) microorganisms can be used as a sterile microbial cells using heat resistance properties. Specifically, not only can the microorganisms be effectively sterilized by heat treatment at a high temperature, but also if the polypeptide is to be separated and used, the proteins derived from the recombinant microorganism can be selectively denatured and removed, thereby improving the purification process of the polypeptide. There are advantages to it.

Specifically, the polypeptide or variant polypeptide may maintain at least 75% of its activity before the heat treatment when heat treated at 50 ° C. to 70 ° C. for 0.5 hour to 24 hours. More specifically, the polypeptide can maintain at least 100% of the activity before the heat treatment at 50 ℃ to 70 ℃ heat treatment for 0.5 hours to 24 hours. In addition, the variant polypeptide can maintain at least 85% of the activity before the heat treatment at 50 ℃ to 60 ℃ heat treatment for 0.5 hours to 24 hours. In addition, the variant polypeptide can maintain at least 85% of the activity before the heat treatment at 50 ℃ to 70 ℃ heat treatment for 0.5 hours to 9 hours. More specifically, the variant polypeptide wherein the glycine (G) amino acid residue 392 from the N- terminal of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 mutated to threonine (T) is 0.5 hours to 50 ℃ to 70 ℃ 95% or more of the activity before the heat treatment at 24 hours, or 0.5% to 9 hours at 50 ℃ to 70 ℃ heat treatment can maintain at least 98% of the activity before the heat treatment. In addition, the variant polypeptide wherein the glycine (G) amino acid residue 392 is mutated to glutamic acid (E) from the N-terminal end of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 at 50 ℃ to 60 ℃ 0.5 hour to 24 hours heat treatment 85% or more of the activity before the heat treatment, at least 85% of the activity before the heat treatment at 50 ° C. to 70 ° C. for 0.5 hours to 9 hours, or 0.5 to 24 hours of the heat treatment at 50 ° C. to 60 ° C., prior to the heat treatment. It can maintain at least 90% activity. In addition, the variant polypeptide mutated glycine (G) amino acid residue No. 392 to alanine (A) from the N-terminal end of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 at 50 ℃ to 60 ℃ 0.5 hour to 24 hours heat treatment At least 100% of the activity before the heat treatment, at least 90% of the activity before the heat treatment at 50 ° C. to 70 ° C. for 0.5 hours to 9 hours, or at the time of the heat treatment at 50 ° C. to 70 ° C. for 0.5 hours to 3 hours. It can maintain at least 100% activity.

The polypeptide of the present application is Meiothermus lupus. rufus ) may be derived from, but is not limited thereto.

Another aspect of the present application is to provide a polynucleotide encoding the polypeptide or variant polypeptide.

As used herein, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are long chained by covalent bonds, and are DNA or RNA strands of a predetermined length or more, and more specifically, the polypeptide or variant. By polynucleotide fragment encoding a polypeptide.

Polynucleotides of the present application may include base sequences that encode amino acid sequences of polypeptides or variant polypeptides of the present application due to genetic code degeneracy.

In addition, if the polynucleotide encoding a variant polypeptide having the activity of converting sucrose of the present application to Turanose, if the polynucleotide sequence encoding a variant polypeptide having the activity of converting sucrose of the present application to Turanose May be included without limitation. Specifically, the polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in the organism in which the polypeptide is to be expressed. This can be done. Any amino acid sequence 392 in the amino acid sequence of SEQ ID 1 may be included without limitation as long as it is a polynucleotide sequence encoding a variant polypeptide substituted with another amino acid. For example, a variant polypeptide of the present application may be, but is not limited to, a polynucleotide sequence encoding the variant polypeptide. In addition, probes which can be prepared from known gene sequences, for example, the hydride under stringent conditions with complementary sequences for all or part of the nucleotide sequence, and the 392th amino acid in the amino acid sequence of SEQ ID NO. Any sequence encoding a protein having the activity of a substituted variant polypeptide can be included without limitation.

Specifically, the polynucleotide of the present application is at least 80%, at least 85%, at least 90% of the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8, or the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 Polynucleotide consisting of a base sequence having at least%, at least 95%, at least 97%, at least 99% homology or identity, but is not limited thereto.

In addition, a probe can be prepared from a known gene sequence, for example, a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with complementary sequences to all or part of the nucleotide sequence encoding the polypeptide. Any polypeptide having the activity of converting the cross to turanose can be included without limitation. By "stringent conditions" are meant conditions that enable specific hybridization between polynucleotides. Such conditions are described specifically in the literature (eg, J. Sambrook et al., Homology). For example, genes with high homology or identity are hybridized with genes having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% homology or identity. 60 ° C., 1 × SSC, 0.1% SDS, specifically 60 ° C., 0.1 × SSC, 0.1% SDS, more specifically, conditions that do not hybridize between genes of low homology or identity, or washing conditions of conventional Southern hybridization Examples thereof include the conditions of washing once, specifically, two to three times at a salt concentration and temperature corresponding to 68 ° C., 0.1 × SSC, and 0.1% SDS.

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

Specifically, polynucleotides having homology or identity can be detected using hybridization conditions comprising a hybridization step at a Tm value of 55 ° C. and using the conditions described above. 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.

Proper stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).

Polypeptides or variant polypeptides consisting of the amino acid sequence of SEQ ID NO: 1, 3, 5 or 7 of the present application may be encoded by the polynucleotide sequence of SEQ ID NO: 2, 4, 6 or 8, respectively.

Another aspect of the present application is to provide a vector comprising a polynucleotide of the present application.

As used herein, the term "vector" refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding said target protein operably linked to a suitable regulatory sequence such that the target protein can be expressed in a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent of the host genome and integrated into the genome itself.

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

For example, a polynucleotide encoding a desired variant polypeptide in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, such as, but not limited to, homologous recombination. The method may further include a selection marker for checking whether the chromosome is inserted. The selection marker is for selecting cells transformed with the vector, i.e., confirming the insertion of the nucleic acid molecule of interest, and selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface variant polypeptides. Markers that impart a may be used. In an environment in which a selective agent is treated, only cells expressing a selection marker survive or exhibit different expression traits, so that transformed cells can be selected. As another aspect of the present application, the present application is to provide a microorganism that produces a purine nucleotide comprising the variant polypeptide, or comprising a polynucleotide encoding the variant polypeptide. Specifically, a microorganism comprising a variant polypeptide and / or a polynucleotide encoding the variant polypeptide may be a microorganism prepared by transformation with a vector comprising a polynucleotide encoding a variant polypeptide, but is not limited thereto. .

In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding a target protein of the present application. Operable linkages can be prepared using known genetic recombination techniques, and site-specific DNA cleavage and ligation can be made using, but are not limited to, cleavage and ligation enzymes in the art.

Another aspect of the present application is to provide a recombinant microorganism comprising a polynucleotide of the present application or a vector of the present application.

In the recombinant microorganism of the present application, the recombination may be achieved by transformation. The term "transformation" in the present application means introducing a vector comprising a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide in the host cell can be expressed. The transformed polynucleotides may include all of them, as long as they can be expressed in the host cell, either inserted into the chromosome of the host cell or located outside the chromosome. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. The expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell, but is not limited thereto. The transformation method may include any method of introducing a polynucleotide into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (Ca (H ₂ PO ₄ ) ₂ , CaHPO ₄ , or Ca ₃ (PO ₄ ) ₂ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, Polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method and the like, but is not limited thereto.

The host cell or microorganism of the present application may be any microorganism capable of producing turanose from sucrose, including the polynucleotide of the present application or the vector of the present application. Specifically, for example, Escherichia (Escherichia) genus, Serratia marcescens (Serratia), An air Winiah (Erwinia) genus, Enterobacter bacteria (Enterobacteria) genus, Providencia (Providencia) genus, Salmonella (Salmonella) genus Streptomyces ( Streptomyces) genus Pseudomonas (Pseudomonas) genus Brevibacterium (Brevibacterium) in or Corynebacterium (Corynebacterium), and may contain the microorganism strain, such as in, specifically, Escherichia (Escherichia) in or Corynebacterium (Corynebacterium) may be in microorganisms, more specific examples of Escherichia coli (Escherichia coli ) or Corynebacterium glutamicum ( Corynebacterium) glutamicum ), but is not limited thereto. Recombinant microorganism of the present application is a microorganism capable of expressing a polypeptide or a mutant polypeptide having the activity of converting sucrose of the present application to turanose by various known methods, in addition to the introduction of the polynucleotide of the present application or the vector of the present application. It can contain everything. In one embodiment, recombinant microorganisms of the present application are CJ_Mrf_AS (KCCM11939P), E. coli BL21 (DE3) / MRF + G392T (KCCM12113P), E. coli BL21 (DE3) / MRF + G392E (KCCM12112P) and E. coli BL21 (DE3) / MRF + G392A (KCCM12111P)

As another aspect of the present application, the present application provides a polypeptide comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the polypeptide, or a culture comprising the microorganism. It is to provide a composition for producing lanose. Specifically, the composition for producing turanose may comprise a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

As still another aspect of the present application, the present application provides a variant polypeptide comprising at least one amino acid variation in the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the variant polypeptide, or a culture comprising the microorganism. A composition for producing lanose, wherein the amino acid variation comprises glycan (G) amino acid residue 392 substituted at least one other amino acid from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the composition for producing turanose To provide. The composition is not limited thereto as long as it is involved in the production of turanose.

In addition, the composition may further include sucrose, but is not limited thereto. The composition of the present application may further comprise any suitable excipient commonly used in the composition for producing the turanose. Such excipients may be, for example, but not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents.

As another aspect of the present application, the present application is directed to a polypeptide comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1, sucrose to a microorganism expressing the polypeptide, or a culture of the microorganism. By contacting, converting the sucrose to turanose, to provide a method for producing turanose. Specifically, the method for producing turanose may include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

As another aspect of the present application, the present application is a variant polypeptide comprising one or more amino acid variations in the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the variant polypeptide, or a culture comprising the microorganism A composition for producing lanose, wherein the amino acid variation comprises glycan (G) amino acid residue 392 substituted at least one other amino acid from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the composition for producing turanose To provide. The composition is not limited thereto as long as it is involved in the production of turanose.

In addition, the contact may be carried out at pH 5.0 to 9.0 conditions, 40 ℃ to 80 ℃ temperature conditions, and / or 0.5 hours to 24 hours, but is not limited thereto. Specifically, the contact of the present application may be performed at pH 6.0 to pH 9.0, pH 7.0 to pH 9.0, pH 5.0 to pH 8.0, pH 6.0 to pH 8.0, pH 7.0 to pH 8.0. In addition, the contact of the present application is 45 ℃ to 80 ℃, 50 ℃ to 80 ℃, 55 ℃ to 80 ℃, 60 ℃ to 80 ℃, 40 ℃ to 75 ℃, 45 ℃ to 75 ℃, 50 ℃ to 75 ℃, 55 ℃ to 75 ℃, 60 ℃ to 75 ℃, 40 ℃ to 70 ℃, 45 ℃ to 70 ℃, 50 ℃ to 70 ℃, 55 ℃ to 70 ℃, 60 ℃ to 70 ℃, 40 ℃ to 65 ℃, 45 ℃ to It may be carried out at 65 ℃, 50 ℃ to 65 ℃, 55 ℃ to 65 ℃, 40 ℃ to 60 ℃, 45 ℃ to 60 ℃ or 50 ℃ to 60 ℃ temperature conditions. In addition, the contact of the present application is 0.5 hours to 24 hours, 0.5 hours to 12 hours, 0.5 hours to 6 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 6 hours, 3 For hours to 24 hours, for 3 hours to 12 hours, for 3 hours to 6 hours, for 6 hours to 48 hours, for 6 hours to 36 hours, for 6 hours to 24 hours.

In the composition or the method, the culture of the microorganism may be prepared from the step of culturing the microorganism expressing the polypeptide or variant polypeptide of the present application in the medium.

As used herein, the term "culture" means growing the microorganisms under appropriately controlled environmental conditions. Cultivation of the present application can be carried out according to the appropriate medium and culture conditions known in the art. Such culture can be easily adjusted and used by those skilled in the art according to the strain selected. Specifically, the culture of the present application may be carried out by a known batch culture method, continuous culture method, fed-batch culture method, but is not limited thereto. At this time, the culture conditions are not particularly limited thereto, but using a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid), an appropriate pH (eg, pH 5 to pH 9, specific) PH 6 to pH 8, most specifically pH 6.8) can be adjusted. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation, and in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas is injected into the culture, or anaerobic and microaerobic conditions are maintained. To maintain, it can be injected with no gas or with nitrogen, hydrogen or carbon dioxide gas. The culture temperature may be maintained at 20 ℃ to 45 ℃, specifically 25 ℃ to 40 ℃, can be incubated for about 0.5 hours to 160 hours, but is not limited thereto. Polypeptides or variant polypeptides of the present application produced by the culture may be secreted into the medium or remain in cells.

In addition, the culture medium used may include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) may be used individually or in combination. This is not restrictive. Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination, but is not limited thereto. As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like may be used individually or in combination, but is not limited thereto. The medium may also contain essential growth-promoting substances such as other metal salts (eg magnesium sulfate or iron sulfate), amino acids and vitamins.

The preparation method of the present application may further comprise the step of separating and / or purifying the manufactured turanose. The separation and / or purification may use a method commonly used in the art of the present application. Non-limiting examples include dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography, fractional crystallization, and the like. The purification may be carried out in only one method, or may be performed in combination of two or more methods.

In addition, the production method of the present application may further comprise the step of performing the decolorization and / or desalting before or after the separation and / or purification step. By carrying out the above-mentioned decolorization and / or desalting, it is possible to obtain turanose with better quality.

In another embodiment, the preparation method of the present application may further comprise the step of crystallizing the turanose after the step of converting, separating and / or purifying, or decolorizing and / or desalting into the turanose of the present application. have. The crystallization can be carried out using a conventionally used crystallization method. For example, crystallization may be performed using a cooling crystallization method.

In another embodiment, the production method of the present application may further comprise the step of concentrating the turanose before the step of crystallization. The concentration can increase the crystallization efficiency.

In another embodiment, the preparation method of the present application is a microorganism or microorganism expressing the unreacted sucrose polypeptide or variant polypeptide, the polypeptide or variant polypeptide of the present application after the separation and / or purification step of the present application The method may further include contacting the culture of the mother solution, reusing the mother liquor from which the crystals are separated after the crystallization of the present application to the separation and / or purification steps, or a combination thereof. Through this additional step, turanose can be obtained in higher yield, and the amount of discarded sucrose can be reduced, which is an economic advantage.

Mutations were used (2014. Anal. Biochem . 449: 90-98). PCR was performed at 94 ° C for 2 minutes, 94 ° C for 30 seconds, followed by annealing at 60 ° C for 30 seconds, followed by extension at 72 ° C for 10 minutes, and repeated 30 times at 72 ° C for 60 minutes. The primer was prepared by using a primer of 33bp consisting of the base 15bp of the substitution site, the base 3bp (RMG) of the substitution site, 15bp of the back base of the substitution site, the primer sequence is as described in Table 2 below.

SEQ ID NO:	primer	Sequence (5'-3 ')
11	Forward direction	TGCCACGACGACATCRMGTGGGCCATCTCCGAC
12	Reverse	GTCGGAGATGGCCCACKYGATGTCGTCGTGGCA

1-3. Recombinant Microbial Manufacturing

Each recombinant vector prepared in Examples 1-1 and 1-2 was transformed into E. coli BL21 (DE3) (invitrogen) by heat shock transformation (Sambrook and Russell: Molecular cloning, 2001) to recombinant microorganisms After the preparation, it was used by storing frozen in 50% glycerol. The recombinant microorganisms are named CJ_Mrf_AS, E. coli BL21 (DE3) / MRF + G392T, E. coli BL21 (DE3) / MRF + G392E, and E. coli BL21 (DE3) / MRF + G392A, respectively, and are under the international treaty of Budapest. Deposited to the Korean Culture Center of Microorganisms (KCCM), Deposit No. KCCM11939P (deposited on November 22, 2016), KCCM12113P (deposited on September 13, 2017), KCCM12112P (September 13, 2017) Daily deposit) and KCCM12111P (deposited September 13, 2017).

Example 2: Preparation of Recombinant Polypeptides and Variant Polypeptides

5 ml LB of the recombinant microorganisms CJ_Mrf_AS, E. coli BL21 (DE3) / MRF + G392T, E. coli BL21 (DE3) / MRF + G392E and E. coli BL21 (DE3) / MRF + G392A, respectively prepared in Example 1 Inoculated in the liquid medium and spawn culture until the absorbance at 600 nm was 2.0. The culture after seed culture was inoculated into LB liquid medium to carry out the main culture, and when the absorbance at 600 nm reached 2.0, 0.5 mM IPTG was added to induce recombinant polypeptide and mutant polypeptide expression. The seed culture and the stirring speed of the main culture were 200 rpm, and the incubation temperature was maintained at 37 ° C. After the incubation, the culture medium was recovered by centrifuging the culture solution at 8,000 × g for 20 minutes at 4 ° C., and the recovered cells were washed twice with 50 mM phosphate buffer (pH 7.5), and then suspended in the same buffer solution. The cells were disrupted using an ultrasonic cell crusher. The cell lysate was centrifuged at 13,000 × g for 20 minutes at 4 ° C., and then only the supernatant was taken, using His-tag affinity chromatography, purified polypeptides from each recombinant microorganism, MRF and purified variant polypeptides MRF + G392T, MRF + G392E and MRF + G392A were obtained. The purified polypeptides were dialyzed with 50 mM phosphate buffer (pH 7.5) and used for activity analysis.

Example  3: of recombinant polypeptides and variant polypeptides Turanos  Conversion activity analysis

In order to confirm the Turanose conversion activity of each polypeptide prepared in Example 2, 0.1 unit / ml of each purified polypeptide was added to a 50 mM potassium phosphate (pH 7.5) buffer solution containing 1M sucrose. After the reaction at 60 ° C. for 5 hours, the reaction was stopped on ice and the resulting turanose was analyzed by HPLC. HPLC analysis was performed using a SUGAR SP0810 (Shodex) column at 80 ° C. while flowing water into the mobile phase at a flow rate of 0.6 ml / min, and turanose was detected with a differential index detector.

As a result, it was confirmed that MRF, MRF + G392T, MRF + G392E and MRF + G392A all have the activity of converting sucrose to turanos. Specifically, MRF produced 60 g / l, MRF + G392T produced 100.7 g / l, MRF + G392E produced 100.3 g / l, and MRF + G392A produced 123.6 g / l of Turanose. All of them had better turanose conversion activity than wild type MRF. In particular, it was confirmed that MRF + G392A has at least 200% conversion activity of the wild type under the same reaction conditions (Table 3 and FIGS. 1A-1D).

Strain name	Turanos (g / L)	% Conversion	% Conversion activity
MRF	60	17.5	100
MRF + G392T	100.7	29.4	168
MRF + G392E	100.3	29.3	167
MRF + G392A	123.6	36.1	206

Example 4 Turanose Conversion Activity Assay According to Reaction Conditions

4-1. Activity analysis by temperature

To assess activity over temperature, each purified polypeptide was added to a 50 mM potassium phosphate (pH 7.5) buffer containing 10% (w / v) sucrose, followed by 40 [deg.] C., 45 [deg.] C., 50 After reacting at 1 ° C., 55 ° C., 60 ° C., 65 ° C., 70 ° C., and 75 ° C. for 1 hour, turanose was analyzed by HPLC in the same manner as in Example 3.

As a result, MRF showed maximum activity at 55 ° C, MRF + G392T, MRF + G392E and MRF + G392A at 65 ° C. In addition, MRF at 40 ℃ to 75 ℃, MRF + G392T at 60 ℃ to 75 ℃, MRF + G392E at 45 ℃ to 75 ℃, MRF + G392A shows at least 80% of the maximum activity at 40 ℃ to 75 ℃ It was confirmed (Table 4 and Figure 2).

Relative activity with temperature

division	MRF	MRF + G392T	MRF + G392E	MRF + G392A
40 ℃	88.0	81.6	71.5	61.1
45 ℃	91.5	88.2	81.9	71.8
50 ℃	92.4	92.0	83.8	73.9
55 ℃	100.0	96.7	84.0	77.2
60 ℃	99.8	98.4	91.0	87.5
65 ℃	98.4	100.0	100.0	100.0
70 ℃	86.3	90.8	98.0	96.9
75 ℃	82.7	72.6	93.5	96.2

4-2. Activity analysis according to pH

50 mM sodium acetate (SA: pH 5-6) buffer and 50 mM potassium-phosphate buffer (KPB: potassium phosphate) containing 10% (w / v) sucrose to assess pH-dependent activity buffer, pH 6-8), and purified MRF and MRf + G392A (0.1 unit / ml), respectively, were reacted at 55 ° C. for 1 hour and analyzed by HPLC in the same manner as in Example 3 It was.

As a result, it showed high activity at pH 7 to pH 8, especially at pH 7.5. In addition, it was confirmed that the maximum activity in the potassium-phosphate buffer among the buffers corresponding to the respective pH (Figs. 3a and 3b).

Example 5: Thermal Stability Analysis

In order to confirm thermal stability, each purified polypeptide was heat-treated at 50 ° C., 60 ° C. and 70 ° C. for 24 hours, and then the turanose conversion activity was measured. Activity was added to 50 mM potassium phosphate (pH 7.5) buffer containing 10% (w / v) sucrose, and 0.1 unit / ml of each of the heat treated polypeptides was reacted at 55 ° C. for 1 hour. Turanose was analyzed by HPLC in the same manner as in Example 3.

As a result, it was confirmed that MRF, MRF + G392T, MRF + G392A, and MRF + G392E all have thermal stability. In particular, MRF remained active even after 24 hours under all heat treatment conditions (Table 5 and FIG. 4A). MRF + G392T retained 95% or more of its activity after 24 hours under all heat treatment conditions, and MRF + G392A retained its activity after 24 hours under 50 ° C and 60 ° C heat treatment conditions and 24 hours after 70 ° C heat treatment conditions. It was confirmed that the activity was maintained at 80% or more even when (Table 5 and Figures 4b and 4c). MRF + G392E was confirmed to maintain more than 89% of the activity at 24 ℃ or more after 24 hours at 50 ℃ and 60 ℃ heat treatment conditions, 79% or more after 24 hours at 70 ℃ heat treatment conditions (Table 5 and Figure 4d).

	Relative activity (%)
	MRF			MRF + G392T			MRF + G392E			MRF + G392A
	50 ° C	60 ° C	70 ° C	50 ° C	60 ° C	70 ° C	50 ° C	60 ° C	70 ° C	50 ° C	60 ° C	70 ° C
0 h	100	100	100	100	100	100	100	100	100	100	100	100
3 h	112	110	109	98	109	100	108	92	89	102	109	109
6 h	104	106	100	101	111	100	94	93	93	103	105	93
9 h	103	108	101	99	109	102	95	92	89	104	103	95
24 h	111	109	101	107	113	95	102	89	79	103	101	84

From the above description, those skilled in the art will appreciate that the present application can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present application should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present application.

Claims (16)

1. A polypeptide having the activity of converting sucrose to turanose comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1.
2. A variant polypeptide comprising one or more amino acid variations in the amino acid sequence of SEQ ID NO: 1,

   The amino acid variation is an activity of converting sucrose to turanos, wherein the 392th glycine (G: Glycine) amino acid residue is substituted with one or more other amino acids from the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1 Variant polypeptides.
3. The variant polypeptide of claim 2, wherein the other amino acid is Threonine (T :), glutamic acid (E: Glutamic acid), or alanine (A: Alanine).
4. The variant polypeptide of claim 2, wherein the variant polypeptide consists of the amino acid sequence of SEQ ID 3, 5 or 7.
5. A polynucleotide encoding the polypeptide of claim 1 or the variant polypeptide of any one of claims 2 to 4.
6. A vector comprising the polynucleotide of claim 5.
7. A recombinant microorganism comprising the polynucleotide of claim 5.
8. Recombinant microorganism comprising the vector of claim 6.
9. A composition for producing a turanose comprising a polypeptide comprising an amino acid sequence having at least 85% homology with an amino acid sequence of SEQ ID NO: 1, a microorganism expressing the polypeptide, or a culture of the microorganism.
10. The method of claim 9,

    The polypeptide is composed of the amino acid sequence of SEQ ID NO: 1, a composition for producing turanose.
11.  A variant polypeptide comprising at least one amino acid variation in the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the variant polypeptide, or a composition for producing turanose comprising the culture of the microorganism, wherein the amino acid variation is SEQ ID NO: 1 A composition for producing turanose, comprising the substitution of one or more other amino acids for glycine (G) amino acid number 392 from the N-terminus of a polypeptide comprising an amino acid sequence of.
12. The composition of claim 9 or 11, wherein the composition further comprises sucrose.
13. Sucrose is converted into turanose by contacting sucrose with a polypeptide comprising an amino acid sequence having at least 85% homology with the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the polypeptide, or a culture of the microorganism. Including a step, a turanose manufacturing method.
14. The method of claim 13,

    The polypeptide is consisting of the amino acid sequence of SEQ ID NO: 1, method for producing turanose.
15. Converting the sucrose to turanose by contacting sucrose with a variant polypeptide comprising at least one amino acid variation in the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the variant polypeptide, or a culture of the microorganism In the turanose manufacturing method comprising,

    The variation of the amino acid is a turanose production method, wherein the amino acid residue 392 glycine (G) amino acid residue 392 is substituted with one or more other amino acids from the N-terminal of the polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
16. The method of claim 13, wherein the contacting is performed at a pH of 5.0 to 9.0, at a temperature of 40 ° C. to 80 ° C., or for 0.5 to 24 hours.

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