KR20180027962A - D-Fructose-4-epimerase and production of tagatose using the same - Google Patents

Abstract

The present invention relates to a D-fructose 4-epimerase enzyme protein; a gene, a recombinant protein and a host cell encoding the same; and a method of producing tagatose from fructose using the enzyme protein or cell. After epimerizing a position of fourth carbon of fructose, screening a gene capable of converting tagatose in fructose, and then developing D-fructose 4-epimerase, the tagatose can be produced from the fructose.

Description

D-Fructose-4-epimerase and production of tagatose using the same

The present invention relates to a method for producing tagatose from fructose using D-Fructose-4-epimerase.

D-tagatose (D-tagatose) is one of the rare saccharides rarely found in nature, and has a similar sweetness that is almost indistinguishable from sugar. Tagatose is not metabolized and is not used as calories. It does not have the diarrheic properties of sugar alcohols, so it can be used variously as food substitute for sugar. Functionally, it has been reported that it has a synbiotic function that helps blood sugar suppression and the proliferation of some lactic acid bacteria functionally superior to the human body, and thus contributes to the function of the rectum. It has a characteristic of non-insoluble and low calorie, Lt; RTI ID = 0.0 > a < / RTI > sweetener.

Known methods for producing tagatose include the use of a galactose-based chemical method (using a Ca (OH) ₂ catalyst) and a biological method (using L-arabinose isomerase (EC 5.3.1.5) have. The price of lactose, which is a raw material used in the production of tagatos industrially, is very liquid and expensive, which is a stumbling block to industrialization because it forms a high price when entering the tagatos market.

Therefore, the most important problem in tagatos industrialization is to start with raw materials such as low-priced glucose or fructose, which can be easily obtained from the natural world.

The most important enzyme that produces tagatose from fructose is the 4-epimerase, and known sugar-4-epimerases include UDP glucose 4-epimerase, a nucleic acid active form, and L-ribulose 5 - phosphate 4-epimerase.

UDP glucose 4-epimerase is an enzyme that converts UDP glucose to UDP fructose, which is converted from fructose to tagatose, but its conversion rate is extremely low, which is difficult to apply industrially. This is because the catalytic reaction is difficult to occur when monosaccharide fructose enters the substrate attachment region because the substrate attachment region is wide in the protein structure of the UDP glucose 4-epimerase. The enzymatic modification using the molecular evolution method showed about 3 times increase in activity, but the activity suitable for industrialization was not reached.

Therefore, it is necessary to develop an epimerizing enzyme that produces tagatose at a high yield using fructose as a raw material which is inexpensive and readily available.

The present invention makes it possible to prepare tagatose from fructose by epimerizing the fourth carbon position of fructose, screening a gene capable of converting tagatose in fructose and then developing D-fructose 4 epimerase.

An example of the present invention provides a fructose 4-epimerase protein. The enzyme protein has an activity of converting fructose to tagatose by epimerizing the 4-carbon position of fructose.

Another example provides a polynucleotide encoding the enzyme protein, a recombinant vector comprising the polynucleotide, or a recombinant strain transformed with the recombinant vector.

Another example is a method for producing tagatose comprising at least one selected from the group consisting of the enzyme protein, a polynucleotide encoding the enzyme protein, a recombinant vector comprising the polynucleotide, and a recombinant strain transformed with the recombinant vector Lt; / RTI >

Another example provides a method for producing tagatose comprising the step of reacting the tagatose producing composition with fructose.

The present invention relates to a novel D-fructose 4-epimerase, which is an enzyme having an activity of producing tagatose by epimerizing the 4th carbon position of fructose. By using the fructose, which is a universal raw material, This makes it possible to produce tagatose with a high yield.

Therefore, in the present invention, it is possible to provide a method for producing tagatose, which is economical and has a high yield, by reducing the manufacturing cost by using fructose, which is a universal raw material, rather than lactose having a large price fluctuation.

Since it is widely known that fructose can be industrially produced from glucose or sugar in general, the raw materials for the process presented in the present invention include fructose in the form of whole or partially containing fructose As shown in FIG. That is, the present invention includes the production of tagatose from starch, sugar or sugar via enzymatic conversion.

Further, the present invention makes it possible to produce tagatose from fructose, which is an inexpensive raw material, by developing an enzyme having an activity of converting epimer of the fourth carbon position of fructose and converting it into tagatose. The present invention can produce tagatose using fructose efficiently, and efficiently produce tagatose, which is currently regarded as an important food material.

In a specific example, the fructose 4-epimerase according to the present invention is a fructose 4-epimerase having a homology of 70% or more with the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 derived from Thermotoga hypogea Lt; / RTI > sequence. The enzyme protein according to the present invention may be an enzyme having an activity of producing tagatose by epimerizing the fourth carbon position of fructose.

An enzyme protein having an amino acid sequence that is 70% or more homologous to the amino acid sequence of SEQ ID NO: 1 is an enzyme protein having at least one characteristic selected from the group consisting of the following characteristics:

(a) a monomer having a molecular weight of 50 to 60 kDa,

(b) an optimum temperature of 50 to 90 DEG C,

(c) an optimum pH of 6.0 to 9.0. And

(d) Increased enzyme activity by cobalt (Co), calcium (Ca), manganese (Mn), or nickel (Ni) ions.

The enzyme protein may be a wild type enzyme or a mutant enzyme in which the thermostat is derived from Thermogoga hypogea or a mutant strain thereof. For example, the enzyme protein may have an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, and a polynucleotide encoding the amino acid sequence may be synthesized and used.

More specifically, the enzyme protein may comprise the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4. In addition, the enzyme protein may be a protein having an amino acid sequence in which part of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 is substituted with another amino acid, or an amino acid sequence of SEQ ID NO: Inserted or deleted. For example, the enzyme protein may have an amino acid sequence that is at least about 70%, at least about 80%, at least about 80% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4 while maintaining the function of converting fructose into tagatose by epimerizing the 4- , About 90% or more, about 95% or more, about 98% or more, or about 99% or more homology with the amino acid sequence.

In another embodiment of the present invention, the nucleic acid molecule encoding the fructose 4-epimerase comprises a nucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 1, such as a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 It is a nucleic acid molecule. SEQ ID NO: 2 is a nucleotide sequence in which the wild-type thermo-motif encodes the fructose 4-epimerase of Thermotoga hypogea, and SEQ ID NO: 3 is a nucleotide sequence optimized for E. coli encoding the enzyme protein of SEQ ID NO:

The polynucleotide of the nucleic acid molecule is also interpreted to include a sequence that exhibits substantial identity to the nucleotide sequence described above. As used herein, the term "substantial identity" means that the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5 is aligned with the sequence of any other amino acid sequence as much as possible and is determined using algorithms commonly used in the art Refers to having a nucleotide sequence having about 90% or more, about 95% or more, or about 98% or more homology when analyzing an aligned sequence.

The polynucleotide may be used as such, or may be used in the form of a recombinant vector containing the polynucleotide.

A further aspect of the invention provides a recombinant vector comprising a nucleic acid molecule encoding a fructose 4-epimerase. Another example provides a recombinant strain transformed with the recombinant vector.

The recombinant vector means a recombinant nucleic acid molecule comprising the desired polynucleotide and an expression regulatory element necessary for expressing the polynucleotide operably linked in a particular host cell, wherein the expression regulatory element comprises a transcriptional and translational terminator ), Transcriptional and translational initiation sequences, and / or promoters useful for the modulation of the expression of a particular target nucleic acid. The polynucleotide may also be operably linked to, for example, an inducible element and / or a temperature sensitive element. The chemical inducible element may be at least one selected from the group consisting of lac operon, T7 promoter, trc promoter, tac promoter, pL promoter and the like. The T7 promoter is derived from the virus T7 phage and includes a T7 terminator with the promoter.

The vector system may be constructed as a vector for cloning or as a vector for expression via a variety of methods well known in the art (Francois Baneyx, current Opinion Biotechnology 1999, 10: 411-421).

Such a vector includes, for example, a plasmid or a virus-derived vector. Plasmid is a circular double-stranded DNA chain to which additional DNA can be ligated. The vectors used in the present invention include, for example, plasmid expression vectors, virus expression vectors (for example, replication defective retroviruses, adenoviruses, and adeno-associated viruses) and viral vectors capable of performing equivalent functions thereto, It is not. For example, pET, pBR, pTrc, pLex, pUC, pKK vectors and the like suitable for expression in E. coli can be used, but they can be used without particular limitation as long as they can efficiently express the enzyme protein.

In one embodiment, the recombinant vector more preferably comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, and may have the structure shown in the cleavage map of Fig.

The recombinant strain is a cell obtained by transforming a host cell with the above-mentioned recombinant vector and comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, and capable of expressing the same. "Transformation" as used herein refers to molecular biology, in which a DNA chain or plasmid having a different type of gene from that of the original cell has penetrated into the cell and binds to DNA originally present in the cell, Phenomenon.

The host microorganism (host cell) capable of stably and continuously cloning and / or expressing the recombinant vector is not particularly limited as long as it is capable of overexpressing the enzyme protein of the active form. Any microorganism known in the art Can be used. For example, the host cell may be E. coli, Bacillus sp., Corynebacterium sp., Or Salmonella sp., Serratia sp. Or Pseudomonas sp.

Specific host cells include various E. coli strains such as E. coli JM109, E. coli BL21, E. coli ER2566, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776 and E. coli W3110, Such as Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, Corynebacterium such as Corynebacterium glutamicum, Salmonella subspecies such as Salmonella typhimurium, Other serratia marcesensus and various Pseudomonas spp. And a microorganism selected from the group consisting of Enterobacteriaceae and strains.

As a method for transforming using the above vector, there is no particular limitation, and methods known in the art such as fusion of bacterial protoplasts, electroporation, projectile bombardment, infection using a viral vector, etc. can be used .

Another example comprises the enzyme protein, a polynucleotide encoding the enzyme protein, a recombinant vector comprising the polynucleotide, a recombinant strain transformed with the recombinant vector, a culture of the recombinant strain, and a lysate of the recombinant strain The present invention provides a composition for producing tagatose comprising at least one selected from the group consisting of The composition for producing tagatose has an activity of converting fructose into tagatose, more specifically, an activity of converting the 4-carbon position of fructose into an epimer and converting it into tagatose. The culture of the recombinant strain may be a cell-free culture containing cells or a cell-free culture, or a concentrate or a dried product of the culture. The lysate of the recombinant strain means a lysate obtained by disrupting the recombinant strain or a supernatant obtained by centrifuging the lysate, and comprises an enzyme protein produced from the recombinant strain.

In addition, the enzyme protein having the conversion activity from fructose to tagatose may have a metalloenzyme property that activation is controlled by a metal ion. Therefore, the composition for producing tagatose can further increase the yield of tagatose by further including a metal ion. The metal ions that can contribute to the yield of tagatose production may be at least one selected from the group consisting of cobalt (Co), calcium (Ca), manganese (Mn), and nickel (Ni) ions. The content of the metal ion may be 0.1 mM to 5 mM, 0.1 to 2 mM, 0.5 to 1.5 mM, or about 1 mM. The above range is set considering the effect of increasing the activity of the enzyme protein.

Another example provides a method for producing tagatose using the composition for producing tagatose. More specifically, the production method includes a step of reacting the tagatose-producing composition with fructose, and further comprising preparing the composition for producing tagatose prior to the reaction step.

In the method for producing tagatose using the composition for producing tagatose, the enzyme protein, the recombinant vector, the strain and the like are as described above.

More specifically, the production method comprises the steps of: preparing an enzyme protein having an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; And reacting the enzyme protein with fructose.

In another embodiment, when a recombinant strain is used, the production method comprises culturing the recombinant strain; And a step of reacting the culture (cell-containing or cell-free) obtained through the above-mentioned culture step or the cell or cell lysate recovered from the culture with fructose.

The step of culturing the recombinant strain may be carried out under a culturing method, a culture medium and / or a culturing condition which is easily selected by a person skilled in the art depending on the kind and / or the characteristic of a cell to be used. The culture may be performed by any culture method known in the art, for example, batch, continuous, fed-batch culture, and the like, but is not limited thereto.

The medium used for the above cultivation includes any host cell including Escherichia coli and any culture medium, solution, solid, semi-solid or rigid support capable of supporting or containing cell contents, such as 2YT medium, LB medium , SOB medium, TB medium, and the like, but the present invention is not limited thereto. The culture conditions may generally be conditions suitable for culturing the host cell (e.g., E. coli).

For example, the culture may be carried out by adding the culture broth (e.g., 50 ml LB (1 ml) to the culture broth (for example, a culture obtained by inoculating one colony into a 15 ml test tube containing 3 ml of LB-ampicillin liquid medium and culturing at 37 DEG C and 250 rpm for 16 hours) (V / v) in a 250 ml flask containing ampicillin liquid medium), cultured under the conditions of 35 ° C to 37 ° C and 150 to 250 rpm and cultured at 0.5 to 0.8 at 600 nm, And further cultured under the conditions of 16 ° C to 30 ° C and 150 to 250 rpm to induce protein overexpression. The inducer may be one or more selected from the group consisting of isopropyl-beta-D-1-thiogalactopyranoside (IPTG), lactose, and the like. The addition amount of the inducing agent may be 0.01 to 1 mM, 0.05 to 0.5 mM, or about 0.1 mM.

The cells may be obtained by performing centrifugation, filtration, or the like on the culture of the recombinant strain.

The cell lysate may be obtained by homogenizing the recovered cells and centrifuging to obtain a supernatant, fractionating the supernatant, and / or performing chromatography on the supernatant or fraction to isolate the enzyme protein ≪ / RTI > and / or purification. In one embodiment, the recovered cells are suspended in a predetermined buffer solution (for example, a 50 mM phosphate buffer solution containing about 10 mM concentration of imidazole), disrupted and centrifuged, and then only the supernatant is collected, , Ni-NTA column (Qiagen)), and the weakly adsorbed E. coli-derived protein was washed away using imidazole of about 20 mM, and the desired enzyme protein was recovered using imidazole of about 200 mM concentration have.

The step of reacting the composition for producing tagatose with fructose may be performed by contacting the composition for tagatose production with fructose. In one embodiment, the step of contacting the composition for producing tagatose with fructose may include, for example, mixing the composition for producing tagatose with fructose or contacting fructose to the carrier to which the composition for tagatose production is immobilized Lt; / RTI > In another example, the step of reacting the composition for producing tagatose with fructose may be performed by culturing the cells of the recombinant strain in a culture medium containing fructose. As described above, the composition for producing tagatose can be reacted with fructose to convert fructose into tagatose to produce tagatose from fructose.

The step of reacting with the fructose may preferably be carried out under the activation conditions of the enzyme protein. For example, the step of reacting with the fructose may be carried out at a pH of from 6 to 9, at a pH of from 6.5 to 8.5, or at a pH of about 7.0, and also at a temperature of from 50 to 90 ?, from 55 to 85 ?. When the cells recovered from the culture of the recombinant strain are used, the recovered cells may be treated with, for example, 0.1 to 1.5% (w / v) or 0.5 to 1% (w / v) For example, 0.85% (w / v) NaCl or the like. The fructose is a substrate of the enzyme protein. For efficient reaction, the amount of fructose used in the reaction step is 1 to 60% (w / v), for example, 5 to 60% (w / v) ) ≪ / RTI > If the concentration of fructose is lower than the above range, the economical efficiency is lowered. If the concentration of fructose is higher than the above range, the fructose may not dissolve well and rather the enzyme reaction may be inhibited. The fructose may be used in the form of a solution dissolved in a buffer solution or water (e.g., distilled water).

For efficient tagatose production, the amount of the enzyme protein used in the step of reacting with the fructose is preferably 0.001 mg / ml to 1.0 mg / ml, 0.005 mg / ml to 1.0 mg / ml, 0.01 mg / ml to 1.0 mg / ml, 0.01 mg / ml to 0.1 mg / ml, or 0.05 mg / ml to 0.1 mg / ml. When the amount of the enzyme protein used is lower than the above-mentioned concentration, the conversion efficiency of the psicose may be lowered. If the concentration of the enzyme protein is higher than the above-mentioned concentration, the economical efficiency in the industry is lowered. When the recombinant strain is used, the cell concentration of the used strain is 0.1 mg (dcw: dry cell weight) / ml or more, for example, 0.1 to 100 mg (dcw) / ml, 0.1 to 50 mg (dcw) ml, 1 to 100 mg (dcw) / ml, 1 to 50 mg (dcw) / ml, 1 to 10 mg (dcw) / ml, 2 to 100 mg (Dcw) / ml, 2 to 10 mg (dcw) / ml, 3 to 100 mg (dcw) / ml 3 to 50 mg (dcw) / ml, or 3 to 10 mg (dcw) / ml.

In addition, the enzyme protein having the conversion activity from fructose to tagatose may have a metalloenzyme property that activation is controlled by a metal ion. The production of tagatose can be improved by performing the reaction with the enzyme protein in the presence of metal ions. Thus, the step of reacting with the fructose may further comprise adding a metal ion. The metal ions that can contribute to the production yield of tagatose are at least one selected from the group consisting of cobalt (Co), calcium (Ca), manganese (Mn), and nickel (Ni) ions.

The addition amount of the metal ion may be in the range of 0.1 mM to 5 mM, 0.1 to 2 mM, 0.5 to 1.5 mM, or about 1 mM. If it is below the above range, it can be selected in consideration of the effect of increasing the activity of the enzyme protein.

In one embodiment, the metal ion may be added to the substrate fructose or to a mixture of the tagatose production composition and fructose. In another embodiment, the metal ion may be added to the carrier to which the tagatose production composition is immobilized (before addition of the fructose), or the composition for producing the tagatose may be added to the mixture of the carrier immobilized with the fructose Or in the form of a mixture of fructose and fructose upon addition of fructose, respectively. When a recombinant strain is used, the metal ion may be added to the culture, or the culture may be performed in a culture medium to which the metal ion is added.

In addition, the method for producing tagatose may further include recovering (separating) and / or purifying tagatose (converted from fructose) produced after the step of reacting with the fructose. The step of recovering (separating) and / or purifying the tagatose is not particularly limited and can be carried out by any method known in the art. For example, the step of recovering (separating) and / or purifying the tagatose can be carried out by one or more methods selected from the group consisting of centrifugation, filtration, crystallization, ion exchange chromatography, and combinations thereof.

On the other hand, the fructose used as the substrate may be obtained from sugar decomposed by a conversion enzyme, or obtained from liquid fructose, but not limited thereto, from the viewpoint of economy.

The present invention makes it possible to produce tagatose from fructose, which is an inexpensive raw material, by developing an enzyme having an activity of converting epimer of the 4th carbon position of fructose into tagatose.

FIG. 1 is a graph showing the results obtained by inserting the D-fructose 4-epimerase encoding polynucleotide according to an embodiment of the present invention into the same restriction enzyme site of pET21a (Novagen) as an expression vector using restriction enzymes NdeI and XhoI (NEB) It is a cleavage map of the recombinant vector pET-THTE.
FIG. 2 is a photograph showing the result of electrophoresis of D-fructose 4-epimerase produced according to an embodiment of the present invention by SDS-PAGE.
FIG. 3 shows the relative activity of D-fructose 4-epimerase according to the metal ion according to an embodiment of the present invention.
FIG. 4 shows the result of HPLC analysis of the reaction of THTE mutant enzyme with standard analysis results of fructose and tagatose according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by way of specific examples. However, it should be understood that the present invention is not limited to the following embodiments, and various changes and modifications can be made within the spirit and scope of the present invention. Accordingly, the appended claims should be construed broadly to conform to the spirit and scope of the present invention.

Example  1. D- Fructose  4- Epimerization Production of recombinant strains producing enzymes

A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 derived from Thermotoga hypogea was synthesized by requesting Bioneer.Co. Korea to optimize the Escherichia coli to be used as an expression strain, The nucleotide sequence is shown in SEQ ID NO: 3.

The synthesized polynucleotide was inserted into the same restriction enzyme site of the expression vector pET21a (Novagen) using restriction enzymes NdeI and XhoI (NEB) to prepare a recombinant vector pET-THTE (FIG. 1). The recombinant vector was transformed into Escherichia coli BL21 (DE3) (invitrogen) by heat shock method (Sambrook and Russell: Molecular Cloning.) To prepare a recombinant strain.

The transformed recombinant strains were inoculated into 5 ml of LB-ampicilline medium (Difco) and incubated at 37 ° C with shaking at 200 rpm until the absorbance (OD) at 600 nm reached 1.5, and then inoculated into 500 ml LB-ampicilline medium , And seeded in a shaking incubator at 37 ° C. When the absorbance at 600 nm of the culture was 0.5, 0.5 mM IPTG was added to induce overexpression of the target enzyme. At this time, culture conditions were changed to 16 ° C and 150 rpm from the time of induction of overexpression and maintained for 16 hours.

Then, the cells were centrifuged at 4000 rpm for 20 minutes to collect only the cells. The recovered cells were washed twice with 0.85% (w / v) NaCl and used for enzyme purification.

Example  2. D- Fructose  4- Epimerization  Enzyme purification and characterization

2-1. D-Fructose 4- Epimerization  Purification of enzymes

The cells recovered in Example 1 were suspended in lysis buffer (50 mM Tris-HCl 300 mM NaCl pH 8.0, 10 mM imidazole) and disrupted at 4 ° C for 20 minutes using an ultrasonic processor (ColepParmer). The supernatant was centrifuged at 13,000 rpm for 20 minutes to collect the supernatant, and then applied to a Ni-NTA column (Ni-NTA Superflow. Qiagen) previously equilibrated with lysis buffer. Then, the supernatant was resuspended in a 50 mM Tris-HCl 300 mM NaCl pH 8.0 And a buffer solution containing 250 mM imidazole was sequentially flowed. The target protein was purified by flowing the final step of 50 mM Tris-HCl 300 mM NaCl pH 8.0 and 250 mM imidazole.

The eluted protein was converted to a buffer solution (50 mM Potassium phosphate pH 8.0) for enzyme activity measurement and then used in the experiment. The partially purified D-fructose 4-epimerase was found to have a monomer size of about 56.1 kDa through SDS-PAGE (FIG. 2). When 1 L of the strain was cultured, about 35 mg of the purified enzyme was obtained, and the purity seen on SDS-PAGE was about 95% or more.

2-2. D-Fructose 4- Epimerization  Enzyme activity confirmation

To confirm the activity of the enzyme obtained in Example 2-1, 0.1 mg / ml of the enzyme purified in Example 2-1, 1 wt% fructose, 0.01 mM NiSO₄ Or CoCl₂The reaction was carried out in a salt buffer solution (pH 8.0) at a reaction temperature of 60 ° C for 3 hours. Concentration of tagatose converted by the enzyme and conversion rate converted to tagatose from fructose were calculated to give a conversion of 4.4% for a fructose concentration of 1% by weight. The fructose and the product, tagatose, were quantified using HPLC. The column used was Shodex Sugar SP0810, the column temperature was 80 ° C, and water was flowed at 0.5 ml / min to the mobile phase.

The D-Furctose 4-epimerase enzyme, known to date, tends to have a lower conversion as the substrate concentration increases. To confirm the maximum conversion rate, the enzyme content was tested up to 0.1 mg / ml-0.5 mg / ml, Regardless, the conversion rate was about 4.4% final .

2-3. D-Fructose 4- Epimerization  Enzyme activity confirmation

The influence of metal ions on the enzyme obtained in Example 2-1 was also examined.

The enzyme purified in Example 2-1 was treated with 1 mM each of CuCl ₂ , MnCl ₂ , CaCl ₂ , ZnSO ₄ , NiSO ₄ and CoCl ₂ , and the activity was compared with the enzyme (control: Non) As shown in Fig. As shown in FIG. 3, the activity of the enzyme of Example 2 was markedly increased by at least 10 times as compared with that of the control group, especially by the addition of nickel ion and cobalt ion. The results of the experiment are shown in FIG. 3, and the relative activity (%) in FIG. 3 is expressed as a percentage in terms of conversion rate when metal is added based on conversion rate 100 when no metal is added.

Example  3. Variations D- Fructose  4- Epimerization  Enzyme production

Improved activity of fructose 4-epimerase A random mutation was performed using the gene derived from T. hypogea as a template in order to construct an improved strain. Specifically, a random mutation was induced to T. hypogea using a Diversify random mutagenesis kit of ClonTech Inc., and another amplified vector was prepared by inserting the amplified gene into the pET-21a vector through PCR reaction under the conditions shown in Table 1 below , And then transformed into E. coli strain BL21 (DE3). The subsequent procedure was carried out in the same manner as in Example 1 and Example 2. [ The nucleotide sequence of the mutant with increased activity from the mutant library was analyzed and confirmed.

The amino acid sequence of the variant fructose 4-epimerase obtained from the mutant is shown in SEQ ID NO: 4, and the polynucleotide encoding the enzyme is shown in SEQ ID NO:

Example  4. Identification of Mutant Enzyme Activity

In order to confirm the activity of the mutant enzyme obtained in Example 3, the reaction was carried out at a reaction temperature of 60 ° C. for 3 hours in a 1 wt% fructose, 0.01 mM NiSO ₄ or CoSO ₄ salt buffer solution (pH 8.0). Concentration of tagatose converted by the enzyme and conversion rate converted from tagatose to tagatose are shown in Table 2 below. The fructose and the product, tagatose, were quantified using HPLC. The column used was Shodex Sugar SP0810, the column temperature was 80 ° C, and water was flowed at 0.5 ml / min to the mobile phase. The improved enzyme exhibited a conversion rate twice as high as before the improvement (Fig. 4).

Using the purified enzyme, 1% Fructose, 0.01 mM NiSO ₄ Or a CoSO ₄ salt buffer solution (pH 8.0) and reacted at a reaction temperature of 60 ° C for 3 hours, the wild-type enzyme (amino acid sequence of SEQ ID NO: 1) was converted to the tagatose at a conversion rate of 4.4% Amino acid sequence) was 10.5%, which markedly increased the conversion of the enzyme.

<110> SAMYANG COPORATION <120> Fructose-4-epimerase and production of tagatose using the same <130> DPP20163083 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 490 <212> PRT <213> Thermotoga hypogea <400> 1 Met Ser Asn Leu Phe Ala Glu Phe Gln His Leu Thr Arg Gly Lys Phe   1 5 10 15 Val Pro Tyr Ala Thr Ser Leu Arg Lys Ser Thr Asp Ala Thr Phe Phe              20 25 30 Leu Val Arg Asp Glu Leu Asp Lys Tyr Leu Ile Val Ile Gly Lys Lys          35 40 45 Gly Ile Cys Glu Leu Phe Glu Gly Gln Lys Ile Gly Glu Ile Asp Arg      50 55 60 Gln Asp Val Val Leu Cys Ala Lys Asn Asp Arg Asn Cys Gln Ser Leu  65 70 75 80 Met Ser Leu Phe Pro Ser Leu Lys Pro Gln Ile Cys Asn Ala Lys Leu                  85 90 95 Ser Phe Gly Phe Gly Asp Arg Leu Gly Val Ala Thr Ala Ala His Ala             100 105 110 Gln Cys Val Gln Lys Glu Lys Leu Phe Pro Ile Phe Ala Gln Gln Ser         115 120 125 Val Arg Glu Ile Ser Arg Thr Glu Arg Asn Trp Leu Asp Val Leu His     130 135 140 Ser Ala Val Trp Gly Val Phe Glu Ser Gly Tyr Asp Gly Pro Phe Gly 145 150 155 160 Ala Asp Ala Asp His Val Lys Lys Ile Glu Asp Leu Glu Ser Ala Ala                 165 170 175 Arg Ala Gly Tyr Thr Met Phe Thr Ile Asp Pro Ser Asp His Val Lys             180 185 190 Asp Pro Ala Lys Phe Asp Lys Arg Glu Leu Val Arg Phe Tyr Glu Glu         195 200 205 His Pro Met Arg Arg Thr Leu Glu Met Lys Tyr Ile Gly Lys Ser Phe     210 215 220 Thr Val Leu Gly Glu Lys Leu Thr Phe Asp Glu Glu Asn Phe Ala Glu 225 230 235 240 Val Phe Val Thr Tyr Ile Asp Ala Ile Glu His Val Glu Lys Cys Tyr                 245 250 255 Arg Ala Leu Arg Ala Val Cys Lys Thr Ser Phe Asp Leu Glu Val Ser             260 265 270 Ile Asp Glu Thr Ser Val Pro Thr Thr Ser Leu Ala His Ile Phe Phe         275 280 285 Val Gln Glu Leu Val Arg Arg Gly Val Glu Phe Arg Thr Leu Ala Leu     290 295 300 Arg Phe Pro Gly Glu Trp Gln Lys Gly Ile Asp Tyr Val Gly Asp Ile 305 310 315 320 Asp Leu Phe Ser Glu Asn Leu Asp Lys His Val Ala Ile Val Lys Met                 325 330 335 Phe Thr Gly Tyr Arg Leu Ser Leu His Ser Gly Ser Asp Lys Phe Ser             340 345 350 Val Tyr Pro Ile Leu Ala Glu Lys Thr Asp Arg Thr Ile His Val Lys         355 360 365 Thr Ala Gly Thr Ser Tyr Leu Glu Ala Ile Arg Val Val Ala Lys Phe     370 375 380 Ala Pro Asp Leu Tyr Arg Gln Ile His Lys Tyr Ala Leu Ser Arg Phe 385 390 395 400 Asp Gln Asp Lys Ala Ser Tyr His Val Thr Thr Glu Leu Ser Lys Ile                 405 410 415 Pro Asp Val Asp Lys Leu Glu Asp Ser Glu Leu Pro Ser Leu Leu Asp             420 425 430 Gln Pro Asp Ser Arg Gln Leu Ile His Ile Thr Tyr Gly Ser Val Leu         435 440 445 Thr Ala Lys Lys Glu Gly Arg Ser Leu Phe Lys Asp Arg Ile Met Arg     450 455 460 Val Leu Phe Glu His Glu Ala Glu His Tyr Asp Phe Leu Lys Lys His 465 470 475 480 Leu Gly Lys His Ile Gln Leu Leu Gly Val                 485 490 <210> 2 <211> 1473 <212> DNA <213> Thermotoga hypogea <400> 2 atgtccaatc tgtttgccga gtttcaacac ctcacaaggg gcaaattcgt cccttacgcc 60 acctcgctga gaaaaagcac ggatgccacg ttctttctcg tacgcgacga gctcgacaag 120 tatctgatcg tgatcggcaa gaaggggata tgcgaactgt tcgaaggtca aaagattggt 180 gagatcgatc gacaggacgt tgtcctgtgt gccaaaaacg atagaaactg tcaaagtctc 240 atgagtctgt ttccctcact aaaaccacag atttgcaacg cgaaactttc gtttggtttt 300 ggagacaggc ttggtgtggc tacggccgcg cacgctcagt gtgtgcaaaa agaaaaactg 360 ttccccattt ttgcccagca gtcagttcgg gagatatcac gaacggagag aaactggctc 420 gacgtgttac acagcgccgt atggggtgtg ttcgagagtg gctacgacgg gcccttcgga 480 gcggacgcgg accacgtgaa gaagattgaa gatctggaga gcgcggcacg cgcgggctac 540 accatgttca cgatcgaccc ttcggaccat gtcaaggatc ctgcaaaatt tgacaagaga 600 gaactcgtca ggttctacga agagcatcca atgagacgca cactcgaaat gaaatacatc 660 ggaaagagct tcacggtcct tggagaaaaa ctcacgttcg acgaagaaaa ctttgcggag 720 gtgttcgtga cctacatcga tgcgatcgaa catgtggaga aatgctacag ggcactgcga 780 gcagtgtgca agacgagctt cgatctggag gtctccatag acgagacgtc tgtaccaaca 840 acgtcccttg cacacatctt cttcgtgcag gaactcgtga ggcgcggtgt ggaattccga 900 accttagcgc tgaggttccc gggagagtgg cagaaaggaa ttgactacgt gggtgacatc 960 gatctcttct ctgagaacct tgacaagcac gtcgctatcg tcaaaatgtt cacaggttac 1020 agattgtctc tgcactccgg tagtgacaag ttcagcgtgt accctatctt agctgagaaa 1080 acagatagaa ccatccacgt caagacagct ggaacgagct atctggaagc catcagggtc 1140 gttgcgaagt tcgctccgga tctgtacaga cagatccata aatacgctct ttcaagattt 1200 gaccaagaca aagcttccta ccacgtcacg acggagcttt cgaagatccc agacgttgat 1260 aaacttgaag attcagagct tccgtcctta ctggatcaac ctgacagtag acaattgata 1320 cacataactt acggttccgt tctgactgcg aaaaaagagg gtagatctct cttcaaagat 1380 cgtataatga gagtgttgtt cgaacacgaa gctgagcact acgattttct caaaaagcac 1440 cttggtaaac atatccagct tttgggtgtt tga 1473 <210> 3 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Optimized nucleotide sequence of fructose-4-epimerase <400> 3 atgtctaacc tgttcgcgga attccagcac ctgacccgtg gtaaattcgt tccgtacgcg 60 acctctctgc gtaaatctac cgacgcgacc ttcttcctgg ttcgtgacga actggacaaa 120 tacctgatcg ttatcggtaa aaaaggtatc tgcgaactgt tcgaaggtca gaaaatcggt 180 gaaatcgacc gtcaggacgt tgttctgtgc gcgaaaaacg accgtaactg ccagtctctg 240 atgtctctgt tcccgtctct gaaaccgcag atctgcaacg cgaaactgtc tttcggtttc 300 ggtgaccgtc tgggtgttgc gaccgcggcg cacgcgcagt gcgttcagaa agaaaaactg 360 gt; gacgttctgc actctgcggt ttggggtgtt ttcgaatctg gttacgacgg tccgttcggt 480 gcggacgcgg accacgttaa aaaaatcgaa gacctggaat ctgcggcgcg tgcgggttac 540 accatgttca ccatcgaccc gtctgaccac gttaaagacc cggcgaaatt cgacaaacgt 600 gaactggttc gtttctacga agaacacccg atgcgtcgta ccctggaaat gaaatacatc 660 ggtaaatctt tcaccgttct gggtgaaaaa ctgaccttcg acgaagaaaa cttcgcggaa 720 gtttcgtta cctacatcga cgcgatcgaa cacgttgaaa aatgctaccg tgcgctgcgt 780 gcggtttgca aaacctcttt cgacctggaa gtttctatcg acgaaacctc tgttccgacc 840 acctctctgg cgcacatctt cttcgttcag gaactggttc gtcgtggtgt tgaattccgt 900 accctggcgc tgcgtttccc gggtgaatgg cagaaaggta tcgactacgt tggtgacatc 960 gacctgttct ctgaaaacct ggacaaacac gttgcgatcg ttaaaatgtt caccggttac 1020 cgtctgtctc tgcactctgg ttctgacaaa ttctctgttt acccgatcct ggcggaaaaa 1080 accgaccgta ccatccacgt taaaaccgcg ggtacctctt acctggaagc gatccgtgtt 1140 gttgcgaaat tcgcgccgga cctgtaccgt cagatccaca aatacgcgct gtctcgtttc 1200 gaccaggaca aagcgtctta ccacgttacc accgaactgt ctaaaatccc ggacgttgac 1260 aaactggaag actctgaact gccgtctctg ctggaccagc cggactctcg tcagctgatc 1320 cacatcacct acggttctgt tctgaccgcg aaaaaagaag gtcgttctct gttcaaagac 1380 cgtatcatgc gtgttctgtt cgaacacgaa gcggaacact acgacttcct gaaaaaacac 1440 ctgggtaaac acatccagct gctgggtgtt 1470 <210> 4 <211> 490 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of mutant fructose-4-epimerase <400> 4 Met Ser Asn Leu Phe Ala Glu Phe Gln His Leu Thr Arg Gly Lys Phe   1 5 10 15 Val Pro Tyr Ala Thr Ser Leu Arg Lys Ser Thr Asp Ala Thr Phe Phe              20 25 30 Leu Val Arg Asp Glu Leu Asp Lys Tyr Leu Ile Val Ile Gly Lys Lys          35 40 45 Gly Ile Cys Glu Leu Phe Glu Gly Gln Lys Ile Gly Glu Ile Asp Arg      50 55 60 Gln Asp Val Val Leu Cys Ala Lys Asn Asp Arg Asn Cys Gln Ser Leu  65 70 75 80 Met Ser Leu Phe Pro Ser Leu Lys Pro Gln Ile Cys Asn Ala Lys Leu                  85 90 95 Ser Phe Gly Phe Gly Asp Arg Leu Gly Val Ala Thr Ala Ala His Ala             100 105 110 Gln Cys Val Gln Lys Glu Lys Leu Phe Pro Ile Phe Ala Gln Gln Asp         115 120 125 Val Arg Glu Ile Ser Arg Thr Glu Arg Asn Trp Leu Asp Val Leu His     130 135 140 Ser Ala Val Trp Gly Val Phe Glu Ser Gly Tyr Asp Gly Pro Phe Gly 145 150 155 160 Ala Asp Ala Asp His Val Lys Lys Ile Glu Asp Leu Glu Ser Ala Ala                 165 170 175 Arg Ala Gly Tyr Thr Met Phe Thr Ile Asp Pro Ser Asp His Val Lys             180 185 190 Asp Pro Ala Lys Phe Asp Lys Arg Glu Leu Val Arg Phe Tyr Glu Glu         195 200 205 His Pro Met Arg Arg Thr Leu Glu Met Lys Tyr Ile Gly Lys Ser Phe     210 215 220 Thr Val Leu Gly Glu Lys Leu Thr Phe Asp Glu Glu Asn Phe Ala Glu 225 230 235 240 Val Phe Val Thr Tyr Ile Asp Ala Ile Glu His Val Glu Lys Cys Tyr                 245 250 255 Arg Ala Leu Arg Ala Val Cys Lys Thr Ser Phe Asp Leu Glu Val Ser             260 265 270 Ile Asp Glu Thr Ser Val Pro Thr Thr Ser Leu Ala His Ile Phe Phe         275 280 285 Val Gln Glu Leu Val Arg Arg Gly Val Glu Phe Arg Thr Leu Ala Leu     290 295 300 Arg Phe Pro Gly Glu Trp Gln Lys Gly Ile Asp Tyr Val Gly Asp Ile 305 310 315 320 Asp Leu Phe Ser Glu Asn Leu Asp Lys His Val Ala Ile Val Lys Met                 325 330 335 Phe Thr Gly Tyr Arg Leu Ser Leu His Ser Gly Ser Asp Lys Phe Ser             340 345 350 Val Tyr Pro Ile Leu Ala Glu Lys Thr Asp Arg Thr Ile His Val Lys         355 360 365 Thr Ala Gly Thr Ser Tyr Leu Glu Ala Ile Arg Val Val Ala Lys Phe     370 375 380 Ala Pro Asp Leu Tyr Arg Gln Ile His Lys Tyr Ala Leu Ser Arg Phe 385 390 395 400 Asp Gln Asp Lys Ala Ser Tyr His Val Thr Thr Glu Leu Ser Lys Ile                 405 410 415 Pro Asp Val Asp Lys Leu Glu Asp Ser Glu Leu Pro Ser Leu Leu Asp             420 425 430 Gln Pro Asp Ser Arg Gln Leu Ile His Ile Thr Tyr Gly Ser Val Leu         435 440 445 Thr Ala Lys Lys Glu Gly Arg Ser Leu Phe Lys Asp Arg Ile Met Arg     450 455 460 Val Leu Phe Glu His Glu Ala Glu His Tyr Asp Phe Leu Lys Lys His 465 470 475 480 Leu Gly Lys His Ile Gln Leu Leu Gly Val                 485 490 <210> 5 <211> 1470 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of mutant fructose-4-epimerase <400> 5 atgtctaacc tgttcgcgga attccagcac ctgacccgtg gtaaattcgt tccgtacgcg 60 acctctctgc gtaaatctac cgacgcgacc ttcttcctgg ttcgtgacga actggacaaa 120 tacctgatcg ttatcggtaa aaaaggtatc tgcgaactgt tcgaaggtca gaaaatcggt 180 gaaatcgacc gtcaggacgt tgttctgtgc gcgaaaaacg accgtaactg ccagtctctg 240 atgtctctgt tcccgtctct gaaaccgcag atctgcaacg cgaaactgtc tttcggtttc 300 ggtgaccgtc tgggtgttgc gaccgcggcg cacgcgcagt gcgttcagaa agaaaaactg 360 ttcccgatct tcgcgcagca ggatgttcgt gaaatctctc gtaccgaacg taactggctg 420 gacgttctgc actctgcggt ttggggtgtt ttcgaatctg gttacgacgg tccgttcggt 480 gcggacgcgg accacgttaa aaaaatcgaa gacctggaat ctgcggcgcg tgcgggttac 540 accatgttca ccatcgaccc gtctgaccac gttaaagacc cggcgaaatt cgacaaacgt 600 gaactggttc gtttctacga agaacacccg atgcgtcgta ccctggaaat gaaatacatc 660 ggtaaatctt tcaccgttct gggtgaaaaa ctgaccttcg acgaagaaaa cttcgcggaa 720 gtttcgtta cctacatcga cgcgatcgaa cacgttgaaa aatgctaccg tgcgctgcgt 780 gcggtttgca aaacctcttt cgacctggaa gtttctatcg acgaaacctc tgttccgacc 840 acctctctgg cgcacatctt cttcgttcag gaactggttc gtcgtggtgt tgaattccgt 900 accctggcgc tgcgtttccc gggtgaatgg cagaaaggta tcgactacgt tggtgacatc 960 gacctgttct ctgaaaacct ggacaaacac gttgcgatcg ttaaaatgtt caccggttac 1020 cgtctgtctc tgcactctgg ttctgacaaa ttctctgttt acccgatcct ggcggaaaaa 1080 accgaccgta ccatccacgt taaaaccgcg ggtacctctt acctggaagc gatccgtgtt 1140 gttgcgaaat tcgcgccgga cctgtaccgt cagatccaca aatacgcgct gtctcgtttc 1200 gaccaggaca aagcgtctta ccacgttacc accgaactgt ctaaaatccc ggacgttgac 1260 aaactggaag actctgaact gccgtctctg ctggaccagc cggactctcg tcagctgatc 1320 cacatcacct acggttctgt tctgaccgcg aaaaaagaag gtcgttctct gttcaaagac 1380 cgtatcatgc gtgttctgtt cgaacacgaa gcggaacact acgacttcct gaaaaaacac 1440 ctgggtaaac acatccagct gctgggtgtt 1470

Claims (18)

A D-fructose 4-epimerase protein having at least one characteristic selected from the group consisting of the following characteristics, comprising an amino acid sequence having 70% or more homology with the amino acid sequence of SEQ ID NO: 1:
(a) a monomer having a molecular weight of 50 to 60 kDa,
(b) an optimum temperature of 50 to &lt; RTI ID = 0.0 &gt; 90 C &
(c) an optimum pH of 6.0 to 9.0. The enzyme protein according to claim 1, wherein the enzyme protein is derived from thermotoga hypogea. The enzyme protein according to claim 1, wherein the enzyme protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4. 2. The enzyme protein according to claim 1, wherein the enzyme protein is encoded by a nucleotide sequence consisting of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: The enzyme protein according to claim 1, wherein the enzyme protein is increased in activity by cobalt (Co), calcium (Ca), manganese (Mn), and nickel (Ni) ions. 6. A nucleic acid molecule comprising a polynucleotide sequence encoding an enzyme protein according to any one of claims 1 to 5. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4. 7. The nucleic acid molecule of claim 6, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: A recombinant expression vector comprising a nucleic acid molecule according to claim 6. 10. The recombinant expression vector according to claim 9, wherein the expression vector has a cleavage map of Fig. A recombinant strain transformed with a recombinant expression vector comprising a nucleic acid molecule according to claim 6 and expressing D-fructose 4-epimerase protein. 12. The recombinant strain according to claim 11, wherein the strain is E. coli , a strain belonging to the genus Bacillus, a strain belonging to the genus Corynebacterium or a strain belonging to the genus Salmonella. A method for producing a recombinant microorganism, which comprises culturing an enzyme protein according to any one of claims 1 to 5, a strain expressing the enzyme protein, a culture of the recombinant strain, a disruption product of the recombinant strain and a culture or extract of the recombinant strain A composition for the production of tagatose, which comprises at least one selected from the group consisting of fructose and sugar. 14. The composition of claim 13, wherein the composition for preparing tagatose further comprises at least one metal ion selected from the group consisting of cobalt (Co), calcium (Ca), manganese (Mn), and nickel (Ni) ions. An enzyme protein according to any one of claims 1 to 5, a strain expressing the enzyme protein, a culture of the recombinant strain, a disruption product of the recombinant strain, and an extract of a culture or a lysate of the recombinant strain Wherein the method comprises the step of reacting at least one selected from the group consisting of fructose-containing raw materials with fructose-containing raw materials. 16. The process according to claim 15, wherein the reaction is carried out at a temperature of from 50 to 90 占 폚. And a pH in the range of 6.0 to 9.0. 16. The method of claim 15, wherein the fructose-containing source has a fructose concentration of 1-60% (w / w). 16. The method of claim 15, wherein the reaction is performed with at least one metal ion selected from the group consisting of cobalt (Co), calcium (Ca), manganese (Mn), and nickel (Ni) ions.

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