WO2020067788A1 - Novel fructose-4-epimerase and tagatose production method using same - Google Patents

Abstract

The present application relates to a fructose-4-epimerase variant having tagatose conversion activity and to a tagatose production method using same.

Description

New fructose-4-epimerase and method for preparing tagatose using the same

The present application relates to a fructose-4-epimerase enzyme variant having improved conversion activity or stability, and a method for preparing tagatose using the same.

Tagatose has a natural sweet taste that is almost indistinguishable from sugar, and its physical properties are similar to sugar. Tagatose is a natural sweetener that is present in small amounts in foods such as milk, cheese, cacao, sweet fruits such as apples and tangerines, and has a calorie of 1.5 kcal / g, 1/3 of sugar and GI (Glycemic index, blood sugar). The index is 3, which is 5% of sugar, but it has a sweetness similar to the taste of sugar and has various health functionalities, so it can be used as an alternative sweetener that can satisfy both health and taste at the same time.

Conventionally known methods of producing tagatose include chemical (catalytic reaction) method and biological (isomerization reaction) method mainly using galactose as a raw material (see Korean Patent Registration No. 10-0964091). In order to economically obtain galactose, which is a raw material for the reactions, studies have been conducted on various basic raw materials containing galactose and a method for producing tagatose by obtaining galactose from it. A typical basic raw material for obtaining galactose is lactose, but the instability of the price of lactose or lactose-containing products in accordance with crude oil and lactose production, demand and supply, etc. in the international market exists. There is a limit to stable supply and demand. Therefore, there is a need for a new method for producing tagatose using generalized general sugars (sugar, glucose, fructose, etc.).

The present inventors have discovered a novel variant protein comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the variant protein has conversion activity like the wild type of SEQ ID NO: 1, or conversion activity or stability compared to wild type This application was completed by confirming this improvement and increasing the production capacity of tagatose.

Another object of the present application is to provide a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

Another object of the present application is to provide a polynucleotide encoding the fructose-4-epimerase enzyme variant.

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

Another object of the present application is to provide a microorganism comprising the above variant.

One object of the present application is fructose-4-epimerase enzyme variant; Microorganisms expressing this; Or it is to provide a composition for producing tagatose containing one or more cultures of the microorganism.

Another object of the present application is the microorganism; Its culture; Or to provide a tagatose production method comprising the step of reacting fructose in the presence of fructose-4-epimerase derived from them.

The fructose-4-epimerase enzyme variant of the present application is industrially capable of producing tagatose having excellent properties, and has a high economical effect by converting the fructose, a common sugar, to tagatose.

1 is a HPLC chromatography result showing that the tagatose-diphosphate aldolase enzyme (CJ_KO_F4E) prepared in one embodiment of the present application has fructose-4-epimerase activity.

Figure 2 is a graph measuring the thermal stability of the single mutant strains prepared in an embodiment of the present application over time at 60 ℃.

Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not considered to be limited by the specific descriptions described below.

One aspect of the present application for achieving the above object provides a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of the fructose-4-epimerase.

Another aspect of the present application for achieving the above object provides a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

The term "fructose-4-epimerization enzyme" in the present application is an enzyme having fructose-4-epimerization activity that epimerizes the 4th carbon position of fructose to convert fructose to tagatose. For the purpose of the present application, if tagatose can be produced using fructose as a substrate, it can be included without limitation, and can be used in combination with 'D-fructose C4-epimerase.' For example, in the known database Kyoto Encyclopedia of Genes and Genomes (KEGG), EC 4.1.2.40 tagatose diphosphate aldolase or tagatose-diphosphate aldolase class II accessory protein (Tagatose-bisphosphate aldolase or tagatose bisphosphate class) II accessory protein) can be included as fructose-4-epimerase if it has the activity of converting fructose to tagatose as a substrate. The tagatose-diphosphoric acid aldolase was previously prepared with D-tagatose 1,6-bisphosphate as a substrate as shown in [Scheme 1] and glycerone phosphate. It is known as an enzyme that produces D-glyceraldehyde 3-phosphate.

[Scheme 1]

D-tagatose 1,6-diphosphate <=> glycerone phosphate + D-glyceraldehyde 3-phosphate

For example, if tagatose-6-phosphate kinase (EC 2.7.1.144) has the activity of converting fructose to tagatose as a substrate, it may be included as a fructose-4-epimerase. The tagatose-6-phosphate kinase was previously ADP and D-tagatose 6-phosphate (D-tagatose 6-phosphate) as a substrate, as shown in [Scheme 2], ADP and D-tagatose 1,6- It is known as an enzyme that produces D-tagatose 1,6-bisphosphate.

[Scheme 2]

ATP + D-tagatose 6-phosphate <=> ADP + D-tagatose 1,6-diphosphate

The activity of the fructose-4-epimerization enzyme is 0.01% or more, specifically 0.1% or more, and more specifically 0.3% or more, as the substrate conversion rate from fructose to tagatose (conversion rate = tagatose weight / initial fructose weight * 100). May be More specifically, the conversion rate may be in the range of 0.01% to 100%, and in the range of 0.1% to 50%.

The fructose-4-epimerase, tagatose-diphosphate aldolase, tagatose-6-phosphate kinase of the present application may be a heat-resistant microorganism-derived enzyme or a variant thereof, for example, Kosmotoga olearia ), Thermanaerothrix daxensis , Rhodothermus profundi , Rhodothermus marinus , Limnochorda pilosa , Caldithrix abyssi , Caldilinea aerophila , Thermoanaerobacter thermohydrosulfuricus , Acidobacteriales bacterium , Caldicellulosiruptor kronotskyensis Thermo aerobacterium thermosaccharolyticum , or Pseudoalteromonas sp. H103, a derived enzyme or a variant thereof, but is not limited thereto. Specifically, Kosmotoga olearia ( Kosmotoga olearia ) (SEQ ID NO: 1), Thermosaka loliticum ( Thermoanaerobacterium thermosaccharolyticum ) (SEQ ID NO: 3), Pseudoalteromonas sp. H103 (SEQ ID NO: 5), Thermanaerothrix daxensis (SEQ ID NO: 7), Acidobacteriales bacterium (SEQ ID NO: 9), Rhodothermus profundi (SEQ ID NO: 9) No. 11), Rhodothermus marinus (SEQ ID NO: 13), Limnochorda pilosa (SEQ ID NO: 15), Caldithrix abyssi (SEQ ID NO: 17), Caldicellulos Derived from Syrupter Chronoskiensis (SEQ ID NO: 19), Caldilinea aerophila (SEQ ID NO: 21), or Thermoanaerobacter thermohydrosulfuricus (SEQ ID NO: 23) It may be an enzyme or a variant thereof, but is not limited thereto.

Specifically, the fructose-4-epimerase, tagatose-diphosphate aldolase, or tagatose-6-phosphate kinase has SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 , 21, 23 amino acid sequence or an amino acid sequence having 70% or more homology or identity therewith, but is not limited thereto. More specifically, the fructose-4-epimerase of the present application is at least 60%, 70% with the amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 , 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity. In addition, if the amino acid sequence having such homology or identity and exhibiting an efficacy corresponding to the protein, it is obvious that an auxiliary 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.

In the present application, SEQ ID NO: 1 refers to an amino acid sequence having fructose-4-epimerase activity. The SEQ ID NO: 1 can be obtained from the known database of NCBI GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, Cosmoto may be derived from Kosmotoga olearia , and more specifically, may be a polypeptide / protein including the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. In addition, a sequence having the same activity as the amino acid sequence may be included without limitation. In addition, it may include the amino acid sequence of SEQ ID NO: 1 or more than 70% homology (homology) or identity (identity) with it, but is not limited thereto. Specifically, the amino acid sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1 and SEQ ID NO: 1 Amino acid sequence. In addition, if the amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein, 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.

That is, even though it is described as 'a protein having an amino acid sequence described with a specific sequence number' in the present application, if it has the same or corresponding activity as a protein consisting of the amino acid sequence of the corresponding sequence number, some sequences are deleted, modified, It is apparent that proteins having a substituted, conservative or added amino acid sequence can also be used in the present application. For example, in the case of having the same or corresponding activity as the mutant protein, a sequence that does not change the function of the protein before and after the amino acid sequence is added, a naturally occurring mutation, a potential mutation, or a preservation thereof. It does not exclude the red permutation, and it is obvious that even within such a sequence addition or mutation, it is within the scope of the present application.

In the present application, the term "tagatose" is a kind of ketohexose among monosaccharides and is used interchangeably with "D-tagatose".

As used herein, the term “fructose-4-epimerase enzyme variant” refers to a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having fructose-4-epimerase activity.

Specifically, the amino acid substitution is from the N-terminal i) 52th, 136th, 197th, 317th, and 320th selected from the group consisting of amino acids at one or more positions are substituted with other amino acids, or ii) 414th And amino acids substituted with glutamic acid (E).

The 'N position' of the present application may include an N position and an amino acid position corresponding to the N position (Correspoding). Specifically, an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence may be included. The specific amino acid sequence may be any one of the amino acid sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

The amino acid position corresponding to the N-position or the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence is Needle Program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et. al., 2000, Trends Genet. 16: 276-277), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), specifically version 5.0.0 or later. The parameters used may be a gap open penalty of 10, a gap extension penalty of 0.5 and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of amino acid residues corresponding to the amino acid position corresponding to the N-position or any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence is not limited to multiple sequences using MUSCLE (MUSCLE) using their respective default parameters. comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: 1899-1900) and EMBOSS EMMA (1.83 or later) using ClustalW (Thompson et al., 1994, Nucleic Acids) Research 22: 4673-4680]) Used it may be determined by the alignment of a multi-polypeptide sequence.

When other polypeptides deviate from the mature polypeptide of a specific amino acid sequence, conventional sequence-based comparisons are unable to detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615) , Other pairwise sequence comparison algorithms can be used. Greater susceptibility in sequence-based searches can be achieved using a search program that uses probabilistic representation of a polypeptide family (profile) to search the database. For example, the PSI-BLAST program can produce profiles and detect distant homologs through an iterative database search process (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has more than one indication in the protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815); McGuffin and Jones, 2003, Bioinformatics 19: 874-881) are structured for the query sequence. Information from various sources (PSI-BLAST, secondary structure prediction, structure alignment profile and solvation potential) is used as input to the neural network to predict folding. Similarly, Gough et al., 2000, J. Mol. Biol. 313: 903-919] can be used to align sequences of unknown structures with superfamily models present in the SCOP database. These alignments can in turn be used to generate models of homology, similarity, or identity to polypeptides, which can be evaluated for accuracy using various tools developed for that purpose.

The 'other amino acid' of i) is not limited as long as it is other amino acids except for the amino acid corresponding to each position. 'Amino acids' are classified into four types according to the nature of the side chain: acidic, basic, polar (hydrophilic) and non-polar (hydrophobic).

In the amino acid sequence of SEQ ID NO: 1, the amino acid at each position is a non-polar amino acid, glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine ( F), tryptophan (W), and proline (P); Polar amino acids serine (S), threonine (T), cysteine (C), tyrosine (Y), asphaltic acid (D), and glutamine (Q); Acidic amino acids asparagine (N), and glutamic acid (E); It may be a protein substituted with one or more amino acids selected from the group consisting of basic amino acids lysine (K), arginine (R), and histidine (H), but is not limited thereto.

Specifically, the amino acid at position 52 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, substituted with methionine (M), serine (S), threonine (T), or leucine (L). It can be. The amino acid at position 136 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, may be substituted with phenylalanine (F), tryptophan (W), proline (P), or tyrosine (Y). The amino acid at position 197 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, may be substituted with alanine (A) or serine (S). The amino acid at position 317 may be substituted with a non-polar amino acid or a polar amino acid, and more specifically, may be substituted with phenylalanine (F) or tyrosine (Y).

The fructose-4-epimerase variant has the recited sequence listed above for conservative substitution and / or modification of one or more amino acids other than the amino acid at a specific position being replaced by another amino acid. sequence), but may include a polypeptide in which the functions or properties of the protein are maintained.

The term "conservative substitution" in this application means to replace one amino acid with another amino acid having similar structural and / or chemical properties. The variant may retain one or more biological activities, but may have one or more conservative substitutions, for example. Conservative substitutions have little or no effect on the activity of the resulting polypeptide.

In addition, a variant type in which one or more amino acids other than the amino acid at the specific position described above may be modified may include deletion or addition of amino acids having minimal influence on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated with a signal (or leader) sequence of the protein N-terminal that is involved in the translation of a protein co-translationally or post-translationally. In addition, the polypeptide may be conjugated with other sequences or linkers to identify, purify, or synthesize the polypeptide.

In addition, the variant may be at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 70%, 80%, 85%, 90%, other than the mutation of SEQ ID NO. Amino acids having at least 99% homology or identity. The variation of SEQ ID NO: 1 is as described above, and the homology or identity thereof may have homology or identity at positions other than the aforementioned variation.

For the purposes of the present application, the fructose-4-epimerase enzyme variant is characterized by improved stability compared to the wild type.

The term "stability" means an enzyme having high heat resistance and having thermal stability.

Specifically, the fructose-4-epimerase enzyme variant of SEQ ID NO: 1 is characterized by improved thermal stability compared to the wild type of SEQ ID NO: 1.

As an example, the fructose-4-epimerase enzyme variant of the present application may be an enzyme having high heat resistance. Specifically, the fructose-4-epimerase enzyme variant of the present application exhibits 50% to 100%, 60% to 100%, 70% to 100%, or 75% to 100% of the maximum activity at 50 ° C to 70 ° C. Can be represented. More specifically, the fructose-4-epimerase enzyme variant of the present application is 80% to 100% or 85% to maximal activity at 55 ° C to 60 ° C, 60 ° C to 70 ° C, 55 ° C, 60 ° C, or 70 ° C 100% of activity.

The fructose-4-epimerase enzyme variant is, for example, as described in Tables 3 to 4, but is not limited thereto.

Another aspect of the present application is to provide a polynucleotide encoding the fructose-4-epimerase enzyme variant, or a vector comprising the polynucleotide.

In the present application, the term, "polynucleotide" is a polymer of nucleotides in which a nucleotide monomer (monomer) is long chained by a covalent bond, a DNA or RNA strand of a certain length or more, and more specifically, the mutant protein. Means a polynucleotide fragment that encodes.

The polynucleotide encoding the fructose-4-epimerase enzyme variant of the present application may be included without limitation as long as it is a polynucleotide sequence encoding the fructose-4-epimerase enzyme variant of the present application. For example, the polynucleotide encoding the fructose-4-epimerase enzyme variant of the present application may be a polynucleotide sequence encoding the amino acid sequence, but is not limited thereto. The polynucleotide may be variously modified in the coding region within a range that does not change the amino acid sequence of the protein due to the degeneracy of the codon or in consideration of the codon preferred in the organism to express the protein. . Therefore, it is obvious that a polynucleotide that can be translated into a polypeptide composed of the amino acid sequence or a polypeptide having homology or identity thereto by codon degeneracy may also be included.

Also, a probe that can be prepared from a known gene sequence, for example, a sequence that hybridizes under strict conditions with complementary sequences for all or part of the base sequence, and encodes the fructose-4-epimerase enzyme variant. It can be included without limitation.

The term “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., Homology). For example, genes with high homology or identity, 70% or more, 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, more specifically In the case of hybridization between genes having at least 97%, particularly specifically at least 99% homology or identity, and not hybridization between genes with less homology or identity, or washing of conventional southern hybridization At salt concentrations and temperatures corresponding to the conditions 60 ° C, 1 X SSC, 0.1% SDS, specifically 60 ° C, 0.1 X SSC, 0.1% SDS, more specifically 68 ° C, 0.1 X SSC, 0.1% SDS, Conditions for washing once, specifically 2 to 3 times can be enumerated.

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

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. Further, the Tm value may be 60 ° C, 63 ° C or 65 ° C, but is not limited thereto, and may be appropriately adjusted by a person skilled in the art according to the purpose.

The appropriate stringency to hybridize a polynucleotide depends on the length and degree of complementarity of the polynucleotide, and variables are well known in the art (see Sambrook et al., Supra, 9.50-9.51, 11.7-11.8).

In the present application, the term 'homology' or 'identity' refers to the degree of correlation with 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.

The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and default gap penalties established by the program used can be used together. Substantially, homologous or identical sequences are generally at least about 50%, 60%, 70%, 80% of the entire or full-length sequence in medium or high stringent conditions. Or it can be hybridized to 90% or more. Hybridization also contemplates polynucleotides containing degenerate codons instead of codons in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity, see, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], and can be determined 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), It can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). (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, homology, similarity, or identity can be determined using BLAST, or ClustalW from the National Center for Biotechnology Information.

The homology, similarity or identity of a polynucleotide or polypeptide is, for example, Smith and Waterman, Adv. Appl. As known in Math (1981) 2: 482, for example, Needleman et al. (1970), J Mol Biol. 48: 443 can be determined by comparing 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 aligned symbols (ie, nucleotides or amino acids). The default parameters for the GAP program are (1) Binary Comparison Matrix (contains 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 gap open 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 relevance between sequences.

As used herein, the term "vector" is a DNA preparation containing a nucleotide sequence of a polynucleotide encoding the target variant protein operably linked to a suitable regulatory sequence so that the target variant protein can be expressed in a suitable host. Means The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence to regulate such transcription, a suitable mRNA ribosome binding site sequence, and a sequence that regulates 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.

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

For example, a polynucleotide encoding a target mutant protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosomal insertion. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for checking whether the chromosome is inserted may be further included. Selection markers are used to select cells transformed with a vector, that is, to confirm whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface variant proteins. Markers to give can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit different expression traits, so that the transformed cells can be selected.

As another aspect of the present application, the present application is to provide a microorganism that produces tagatose, including the mutated protein or a polynucleotide encoding the mutated protein. Specifically, the microorganism containing the mutant protein and / or the polynucleotide encoding the mutant protein may be a microorganism prepared by transformation with a vector containing the polynucleotide encoding the mutant protein, but is not limited thereto. .

The term "transformation" in the present application means that a vector containing a polynucleotide encoding a target protein is introduced into a host cell so that the protein encoded by the polynucleotide in the host cell can be expressed. The transformed polynucleotide may include all of them, whether they can be inserted into the host cell chromosome or located outside the chromosome, as long as it 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 as long as it can be expressed in any form. 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 elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form and may be operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" in the above means that the promoter sequence and the gene sequence to initiate and mediate the transcription of the polynucleotide encoding the target variant protein of the present application are functionally linked.

Another fructose-4-epimerase variant of the present application, a polynucleotide encoding the fructose-4-epimerase variant, or a vector comprising the polynucleotide, and a fructose-4-epimerase Providing microorganisms to produce.

The term "microorganism including fructose-4-epimerase enzyme variant" used in the present application may mean a microorganism that has been recombined so that the fructose-4-epimerase enzyme variant of the present application is expressed. For example, a host capable of expressing the variant by transforming it with a vector comprising a polynucleotide encoding a fructose-4-epimerase variant, or a vector comprising a polynucleotide encoding a fructose-4-epimerase variant Cell or microorganism. Specifically for the purpose of the present application, the microorganism is a microorganism expressing a fructose-4-epimerase enzyme variant comprising one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid substitutions are at one or more positions from the N-terminus. It may be a microorganism that expresses a variant protein having one or more amino acids substituted and having fructose-4-epimerase activity, but is not limited thereto.

The fructose-4-epimerase enzyme variant of the present application transforms the DNA expressing the enzyme or variant thereof of the present application into a strain such as E. coli , and then cultivates it to obtain a culture, and the culture is obtained. It may be obtained by crushing and purifying through a column or the like. The transformation strains include, but are not limited to, Escherichia coli , Corynebacterum glutamicum , Aspergillus oryzae , or Bacillus subtilis . Does not.

If the microorganism of the present application is a microorganism capable of producing the fructose-4-epimerase of the present application, including the nucleic acid of the present application or the recombinant vector of the present application, both prokaryotic and eukaryotic microorganisms may be included. For example, Escherichia genus, Erwinia genus, Serratia genus, Providencia genus, Corynebacterium genus and Brevibacterium genus Belonging to the microorganism strain may be included, but is not limited thereto.

The microorganism of the present application may include all microorganisms capable of expressing the fructose-4-epimerase of the present application by various known methods in addition to the introduction of the nucleic acid or vector.

The culture of the microorganism of the present application may be prepared by culturing a microorganism expressing the fructose-4-epimerase of the present application in a medium.

In the above method, "culturing" means growing the microorganism under appropriately controlled environmental conditions. The process of culturing the microorganism is not particularly limited, but may be performed by a known batch culture method, a continuous culture method, a fed-batch culture method, or the like. In this case, the culture conditions are not particularly limited, but using a basic compound (e.g. sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (e.g. phosphoric acid or sulfuric acid) to a proper pH (e.g. pH 5 to 9, specifically Can adjust pH 6 to 8, most specifically pH 6.8), and maintain aerobic conditions by introducing oxygen or an oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45 ° C, specifically 25 to 40 ° C, and cultured for about 10 to 160 hours, but is not limited thereto.

In addition, the culture medium used is sugar and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) Oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) can be used individually or in combination. , But is not limited to this. Nitrogen sources include nitrogen-containing organic compounds (e.g. 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) may be used individually or in combination, but is not limited thereto. As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt may be used individually or in combination, but are not limited thereto. Additionally, the medium may contain other metal salts (eg, magnesium sulfate or iron sulfate), essential growth-promoting substances such as amino acids and vitamins.

As another aspect of the present application, the fructose-4-epimerase enzyme variant; Microorganisms expressing this; Or it provides a composition for producing tagatose comprising a culture of the microorganism.

The composition for producing tagatose of the present application may further include fructose.

In addition, the composition for tagatose production of the present application may further include any suitable excipients commonly used in the composition for tagatose production. Such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers or isotonic agents, but are not limited thereto.

The composition for producing tagatose of the present application may further include a metal ion or a metal salt. In one embodiment, the metal of the metal ion or metal salt may be a metal containing a divalent cation. Specifically, the metal of the present application may be nickel (Ni), iron (Fe), cobalt (Co), magnesium (Mg), or manganese (Mn). More specifically, the metal salt MgSO ₄ , FeSO ₄ , NiSO ₄ , NiCl ₂ , MgCl ₂ , CoSO ₄ , MnCl ₂ or MnSO ₄ .

As another aspect of the present application, the fructose-4-epimerase enzyme variant; Microorganisms comprising the fructose-4-epimerase variant; Or it provides a method for producing tagatose comprising the step of converting fructose into tagatose by contacting the culture thereof with fructose.

Specifically, it relates to a method for preparing tagatose from fructose using a fructose-4-epimerase enzyme variant as a fructose-4-epimerase.

For example, the contact of the present application may be performed at pH 5.0 to pH 9.0 conditions, at 30 ° C to 80 ° C temperature conditions, and / or for 0.5 to 48 hours.

Specifically, the contacting of the present application may be performed under pH 6.0 to pH 9.0 conditions or pH 7.0 to pH 9.0 conditions. In addition, the contact of the present application is 35 ℃ to 80 ℃, 40 ℃ to 80 ℃, 45 ℃ to 80 ℃, 50 ℃ to 80 ℃, 55 ℃ to 80 ℃, 60 ℃ to 80 ℃, 30 ℃ to 70 ℃, 35 ℃ to 70 ℃, 40 ℃ to 70 ℃, 45 ℃ to 70 ℃, 50 ℃ to 70 ℃, 55 ℃ to 70 ℃, 60 ℃ to 70 ℃, 30 ℃ to 65 ℃, 35 ℃ to 65 ℃, 40 ℃ to 65 ℃, 45 ℃ ~ 65 ℃, 50 ℃ ~ 65 ℃, 55 ℃ ~ 65 ℃, 30 ℃ -60 ℃, 35 ℃ -60 ℃, 40 ℃ -60 ℃, 45 ℃ -60 ℃, 50 ℃ -60 ℃ Or it may be carried out at a temperature of 55 ℃ to 60 ℃. In addition, the contact of the present application is from 0.5 hour to 36 hours, from 0.5 hour to 24 hours, from 0.5 hour to 12 hours, from 0.5 hour to 6 hours, from 1 hour to 48 hours, from 1 hour to 36 hours, 1 For hours to 24 hours, for 1 hour to 12 hours, for 1 hour to 6 hours, for 3 hours to 48 hours, for 3 hours to 36 hours, for 3 hours to 24 hours, for 3 hours to 12 hours, for 3 hours For 6 hours, 6 hours to 48 hours, 6 hours to 36 hours, 6 hours to 24 hours, 6 hours to 12 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to It can be carried out for 24 hours, 18 hours to 48 hours, 18 hours to 36 hours, or 18 hours to 30 hours.

In addition, the contact of the present application can be performed in the presence of a metal ion or metal salt. The metal ions or metal salts that can be used are as in the above-described embodiments.

The manufacturing method of the present application may further include separating and / or purifying the prepared tagatose. The separation and / or purification may use methods conventionally used in the technical field of the present application. Non-limiting examples include dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography and fractional crystallization. The purification may be performed by only one method, or two or more methods may be performed together.

In addition, the manufacturing method of the present application may further include a step of performing decolorization and / or desalination before or after the separation and / or purification step. By performing the decolorization and / or desalination, tagatose with better quality can be obtained.

As another example, the manufacturing method of the present application may further include the step of converting to tagatose of the present application, separating and / or purifying, or crystallizing tagatose after decoloring and / or desalting. The crystallization can be performed using a conventional crystallization method. For example, crystallization may be performed using a cooling crystallization method.

In addition, the manufacturing method of the present application may further include the step of concentrating tagatose prior to the crystallization step. The concentration can increase the crystallization efficiency.

As another example, the manufacturing method of the present application comprises contacting unreacted fructose with an enzyme of the present application, a microorganism expressing the enzyme, or a culture of the microorganism after the separation and / or purification of the present application, After the crystallization step, the step of reusing the mother liquor from which the crystals are separated may be further included in the separation and / or purification step, or a combination thereof. Through the additional step, tagatose can be obtained in a higher yield and the amount of fructose discarded can be reduced, which is an economical advantage.

Hereinafter, the present application will be described in more detail by examples. However, these examples are intended to illustrate the present application by way of example, the scope of the present application is not limited by these examples, it will be apparent to those skilled in the art to which this application belongs.

Example 1 Preparation of Recombinant Expression Vector and Transformants Containing Fructose-4-Epimerase Enzyme Wild and Improved

Example 1-1. Preparation of recombinant expression vector containing fructose-4-epimerase wild type gene

To produce fructose-4-epimerase, Cosmoto obtains fructose-4-epimerase gene information derived from Kosmotoga olearia to produce E. coli expression vectors and transforming microorganisms (transformants) Did. It was confirmed that the sequence can be used as a fructose-4-epimerase that converts fructose to tagatose (FIG. 1).

Specifically, the gene was registered in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Cosmoto selected the fructose-4-epimerase gene sequence for the Oelia gene sequence, and Cosmoto was the amino acid sequence of Oelia ( Based on SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2), the recombinant expression vector pBT7-C-His-KO was inserted into pBT7-C-His, which is an E. coli expression-capable vector, and synthesized by Bionica.

Example 1-2. Production of fructose-4-epimerase-enhancing library and screening of variants with improved activity

A variant library of fructose-4-epimerase was constructed by random mutation using fructose-4-epimerase gene derived from Kosmotoga olearia as a template. Specifically, using the Diversify random mutagenesis kit (ClonTech), a random mutation was induced to cause 2 to 3 mutations per 1000 base pairs of fructose-4-epimerase gene, and PCR reaction conditions are shown in Table 1 and Table below. It is shown in 2. A gene library encoding a variant of fructose-4-epimerase was constructed, and then inserted into E.coli BL21 (DE3).

Reaction solution composition	Addition amount (μl)
PCR Grade Water	36
10X TITANIUM Taq Buffer	5
MnSO4 (8 mM)	4
dGTP (2 mM)	One
50X Diversify dNTP Mix	One
Primer mix	One
Template DNA	One
TITANIUM Taq Polym.	One

step	Temperature ( ℃)	Time (seconds)	cycle
Initial Denaturation	94	30	One
Denaturation	94	30	25
Annealing / Extension	68	60
Final Extension	68	60	One
soak	4	-

E.coli BL21 (DE3) with pBT7-C-His plasmid containing the variant gene of the fructose-4-epimerase, containing 0.2 mL of LB liquid medium containing ampicillin antibiotic Inoculated into a deep well rack, and cultured for more than 16 hours in a 37 ° C shake incubator. The culture solution obtained as a result of the seed culture was inoculated in a culture deep well rack containing a liquid medium containing LB and a protein expression regulator, lactose, to perform the main culture. The seed culture and main culture were carried out at a stirring speed of 180 rpm and 37 ° C. Next, the culture solution was centrifuged at 4 ° C for 20 minutes at 4,000 rpm, and then the cells were recovered to conduct an activity test.

In order to perform high-speed screening of active modified mutants in a large amount in a randomized mutant library, a colorimetric method capable of specifically quantifying D-fructose was used. Specifically, after mixing the 70% polylinic acid solution (folin-ciocalteu reagent, SIGMA-ALDRICH) and the substrate reaction completion ratio at a ratio of 15: 1, react at 80 ° C for 5 minutes and measure at 900nm to obtain an OD value. When comparing the relative activity with the wild-type enzyme (SEQ ID NO: 1), variants with activity (D-tagatose conversion from D-fructose) were selected. As a result of sequencing the 10 selected colonies and confirming the nucleotide sequence, it was confirmed that the total 6 regions (52th, 136th, 197th, 317th, 320th, 414th) were mutated.

Example 2. Mutagenase production and stability improvement Mutagen screening

The single-site saturation mutagenesis library of six target sites (52th, 136th, 197th, 317th, 320th, and 414th) selected in Example 1-2 was prepared and stable. The improved mutation sites and amino acids were screened. After mutating enzymes by integrating information from the selected improvement site, we developed mutases with improved stability of fructose 4-epimerization conversion reaction.

Example 2-1. Saturation mutagenesis

Saturation for the construction of a mutant library using the recombinant expression vector pBT7-C-His-KO (expressing a recombinase bound by 6xHis-tag to the wild-type C-terminus) designed for expression of wild type E. coli BL21 (DE3) of the wild type enzyme gene It was used as a template for mutagenesis. Inverse PCR-based saturation mutagenesis was used in consideration of variation in diversity and yield of variants (2014. Anal. Biochem. 449: 90-98), and the screening scale of the prepared mutant library was minimized (introduced during saturation) In order to minimize the number of codons), terminating codons were excluded, and NDT / VMA / ATG / TGG mixed primers with rare codons of E. coli (2012. Biotechniques 52: 149-158) were designed and used. . Specifically, a primer was prepared by using a total length of 33 bp with 15 bp of the front base, 3 bp of the substitution base, and 15 bp of the rear base of each site. PCR conditions were denatured at 94 ° C. for 2 minutes, then 30 ° C. denaturation at 30 ° C., annealing at 60 ° C. for 30 seconds, and extension at 72 ° C. for 10 minutes was repeated 30 times, followed by extension reaction at 72 ° C. for 60 minutes. After the saturation mutant library was prepared for each selected amino acid site, the mutant strains for each library were randomly selected (<variation 11) and the nucleotide sequence was analyzed to evaluate the amino acid variation distribution. Based on the analysis results, a screening scale of 90% or more of the sequence coverage for each library was set (2003. Nucleic Acids Res. 15; 31: e30)

Through the saturation mutation, candidate strains with excellent characteristics were prepared and sequenced to confirm displacement sites. Through this, a total of 11 variants were obtained (Table 3).

Variation position
D136	F	P	W	Y
L320	F
C52	M	T	S	L
V197	A	S
T317	Y	F
S414	E

Example 2-2. Stability improvement variant enzyme production

In order to evaluate the relative activity of fructose 4-epimerase to a mutagenase for a single site with improved stability and a mutagenase for a single site to which they are combined, the saturation mutant library gene prepared in 3-1 above was E. After transformation into coli BL21 (DE3), each transformed microorganism was inoculated into a culture tube containing 5 mL of LB liquid medium containing ampicillin antibiotic, and the absorbance at 600 nm was 2.0. Seed culture was carried out in a shaker at ℃ ℃. This culture was performed by inoculating the culture solution obtained as a result of the seed culture in a culture flask containing a liquid medium containing LB and a protein expression regulator, lactose. The seed culture and main culture were carried out at a stirring speed of 180 rpm and 37 ° C. Next, the culture medium was centrifuged at 4 ° C for 20 minutes at 8,000 rpm, and the cells were recovered. The recovered cells were washed twice with 50 mM Tris-HCl (pH8.0) buffer solution, and resuspended in 50 mM NaH2PO4 (pH 8.0) buffer solution containing 10 mM imidazole and 300 mM NaCl. Did. The resuspended cells were crushed using a sonicator, centrifuged at 4 ° C for 20 minutes at 13,000 rpm, and only the supernatant was taken. The supernatant was purified using His-taq affinity chromatography, and a 50 mM NaH2PO4 (pH 8.0) buffer solution containing 20 mM imidazole and 300 mM NaCl was flowed in a 10-fold amount of a filler to generate a non-specific binding protein. Removed. Subsequently, 50 mM NaH2PO4 (pH8.0) buffer solution containing 250 mM imidazole and 300 mM NaCl was further flown and purified by elution, followed by dialysis with 50 mM Tris-HCl (pH 8.0) buffer solution to analyze enzyme properties. Each purified enzyme was obtained for the sake of.

Example 3. Comparative evaluation of stability improvement variant enzyme properties

To measure the fructose-4-epimerization activity of the recombinant mutants obtained in Example 2-2, 50 mM Tris-HCl (pH 8.0), 3 mM MnSO4 and 5 mg / mL each were added to 30% by weight fructose. And reacted at 60 ° C for 2 hours. In addition, in order to evaluate the heat stability of fructose-4-epimerization of the recombinant mutants obtained, each 5 mg / mL was placed at 60 ° C for 3 hours, 24 hours, 48 hours, and then left on ice for 5 minutes. Each modified enzyme exposed to heat was added to a substrate solution (50 mM Tris-HCl (pH 8.0), 3 mM MnSO4 in 30 wt% fructose) to a final 2 mg / ml concentration, and reacted at 60 ° C for 2 hours.

As a result, the fructose-4-epimerization stability of all mutant strains of the present application was higher than that of the wild type (FIG. 2). In addition, the results of measuring the relative activity compared to the wild type for some of the recombinant mutant enzymes are shown in Table 4. As a result, it was found that the relative activity increased or was similar to that of the wild type.

	Relative activity (%)
KO	100
C52S	360
D136Y	98
T317Y	100
T317F	100
L320F	98
S414E	97

The present inventors transformed into E.coil BL21 (DE3) strains to transform E.coil BL21 (DE3) / CJ_KO_F4E_M4 (D136F), E.coil BL21 (DE3) / CJ_KO_F4E_M6 (L320F), E.coil BL21 (DE3) / CJ_KO_F4E_M7 (S414E) Transformants (transformed microorganisms), respectively, were named, and the transformants were deposited under the Budapest Treaty to the Korea Microorganism Conservation Center (KCCM), an international depository organization, on September 19, 2018, with accession number KCCM12323P ( E.coil BL21 (DE3) / CJ_KO_F4E_M4), KCCM12325P ( E.coil BL21 (DE3) / CJ_KO_F4E_M6), KCCM12326P ( E.coil BL21 (DE3) / CJ_KO_F4E_M7), respectively.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. 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 including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof.

Claims (8)

1. From the N-terminus of the fructose-4-epimerase comprising the amino acid sequence of SEQ ID NO: 1) at one or more positions selected from the group consisting of 52), 136, 197, 317, and 320 Fructose-4-epimerase enzyme variant, wherein the corresponding amino acid is replaced with another amino acid, or ii) the amino acid corresponding to position 414 is substituted with glutamic acid (E).
2. According to claim 1, wherein the other amino acids of i) alanine (A), leucine (L), methionine (M), threonine (T), asparagine (N), proline (P), serine (S), Fructose-4-epimerase enzyme variant selected from the group consisting of tryptophan (W), phenylalanine (F), tyrosine (Y), and asphaltic acid (D).
3. A polynucleotide encoding the fructose-4-epimerase enzyme variant of claim 1 or 2.
4. A vector comprising the polynucleotide of claim 3.
5. The fructose-4-epimerase enzyme variant of claim 1 or 2; A polynucleotide encoding the fructose-4-epimerase enzyme variant; Or a microorganism comprising a vector comprising the polynucleotide.
6. The fructose-4-epimerase enzyme variant of claim 1 or 2; Microorganisms expressing this; Or a composition for producing tagatose containing a culture of the microorganism.
7. The composition for tagatose production according to claim 6, wherein the composition further comprises fructose.
8. The fructose-4-epimerase enzyme variant of claim 1 or 2; Microorganisms comprising the fructose-4-epimerase variant; Or contacting the culture of fructose, tagatose production method comprising the step of converting fructose to tagatose.

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