KR20180132408A - Psicose epimerase and method of psicose using the same - Google Patents

Abstract

Provided are a psicose 3-epimerase, a polynucleotide encoding the enzyme, a strain producing the enzymes, and a use thereof in producing psicose. One example of the present invention provides a psicose epimerase protein of a Microbacterium sp. strain. The enzyme protein may be a protein having activities of converting fructose into psicose.

Description

[0001] The present invention relates to a method for producing epidermal enzymes,

The present invention relates to a psicose epimerase, a strain expressing the enzyme protein, and a use of the enzyme protein or strain in the manufacture of psicose.

Psycosis is the epimer of carbon 3 of D-fructose, which has a sweetness equivalent to 70% of fructose. However, unlike fructose, it is rarely metabolized when absorbed in the body, and it can be used for various functional foods such as glucose control, prevention of cavities and functional sugar which inhibits lipogenesis in liver.

Sugar alcohols, which are widely used as sweeteners, have side effects such as diarrhea when consumed over a certain amount, but there is no known side effect. Therefore, although the interest as a sweetener of psicose is increasing, it belongs to a rare saccharide which is a monosaccharide rarely present in the natural world. Therefore, it is necessary to develop a technique for efficiently producing psicose to be applied to the food industry.

Conventional production of the cyclosaccharide has a chemical method of producing the cyclosporin from the fructose using the catalytic action of the molybdate ion, and the most recently known effective method is the synthesis of the cyclic epimerase from Agrobacterium tumefaciens And the production method by a biological method such as the production of the sauces by the seeds. The production of syngas by the chemical method is problematic in that very small amounts of synuclein are present in the molasses treatment or glucose isomerization reaction, the cost is high and byproducts are generated, and the biological method is also low in yield and high in production cost There is a problem.

Accordingly, there is a demand for a method capable of producing a psicose with a high yield and stability, showing a temperature and a pH suitable for industrialization.

An example of the present invention provides an enzyme protein that converts fructose into a cytosine and a polynucleotide that encodes the enzyme protein.

Another example provides a recombinant vector comprising the polynucleotide and a recombinant strain comprising the recombinant vector.

Another example provides a composition for the manufacture of a scent comprising at least one selected from the group consisting of the enzyme protein, a strain expressing the enzyme protein, a culture of the strain, and a disruption of the strain.

Another example provides a method for producing a scikos using at least one selected from the group consisting of a base enzyme protein, a strain expressing the enzyme protein, a culture of the strain, and a disruption of the strain.

The present inventors have identified polynucleotides encoding enzymes capable of converting fructose into a cytosine among all genes possessed by a microbacterium strain having high activity of converting fructose into a cytosine, To prepare a scicom epimerase having the characteristics of changing enzyme activity and / or reaction conditions. Furthermore, the present inventors have developed a use of producing fructose from fructose by using at least one selected from the group consisting of the wild-type enzyme, the mutant enzyme or a strain expressing the same, the culture of the strain, and the disruption of the strain . Therefore, the inventors of the present invention have found that a microorganism belonging to the microbacterium belonging to the genus of wild-type Cicosch epimerase protein, a mutant of the protein, a gene of the wild-type enzyme and the mutant enzyme, and a strain expressing the enzyme protein or enzyme protein, Water, and a disruption of the strain. The present invention provides a use of producing at least one fructose from fructose. The strain may be a strain isolated from nature that produces a wild-type enzyme or a mutant strain that induces mutation, or a recombinant strain into which a gene encoding the wild-type enzyme or the mutant enzyme is introduced.

An example of the present invention is an enzyme derived from a strain of microbacterium genus including a wild type enzyme and its mutant type enzyme and specifically includes an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of 34, 64, 75, 105, 108, 109, 155, 177, and 209, wherein the amino acid sequence is substituted with at least one amino acid selected from the group consisting of amino acids located at positions 1,

A further embodiment of the present invention relates to a polynucleotide encoding said enzyme protein, a recombinant vector comprising said polynucleotide, and a recombinant strain comprising said recombinant vector.

Another embodiment of the present invention relates to a method for producing a protein comprising the steps of: (a) culturing a host cell comprising an enzyme protein, a polynucleotide encoding the protein, a strain expressing the enzyme protein, a culture of the strain, And to a composition for the manufacture of a cosmetic composition for producing a cosmetic composition from a fructose-containing substrate. The strain may be a strain isolated from nature that produces a wild-type enzyme or a mutant strain that induces mutation, or a recombinant strain into which a gene encoding the wild-type enzyme or the mutant enzyme is introduced.

Another embodiment of the present invention is a method for producing a fructose-containing substrate, comprising the steps of: (a) cultivating at least one selected from the group consisting of an enzyme protein, a polynucleotide encoding the protein, a strain expressing the enzyme protein, ≪ RTI ID = 0.0 > a < / RTI > The strain may be a strain isolated from nature that produces a wild-type enzyme or a mutant strain that induces mutation, or a recombinant strain into which a gene encoding the wild-type enzyme or the mutant enzyme is introduced.

An example of the present invention is also a process for the production of psicose, which comprises a psicose epimerase having a pI value in the range of 4.0 to 5.1, more preferably a pI value in the range of 4.5 to 5.1, A composition and a method for producing cyucose by reacting with a fructose-containing substrate using the enzyme.

Hereinafter, the present invention will be described in more detail.

An example of the present invention provides a microcosm of Escherichia coli strains. The enzyme protein may be a protein having an activity of converting fructose into a cytosine.

For example, the protein or enzyme may be at least one selected from the group consisting of microbacterium sp., Preferably Microbacterium foliorum, Microbacterium oxydans, and Microbacterium phyllosphaerae.

The wild-type psicose epimerase protein obtained from the microbacterium belonging to the genus Microbacterium may contain the amino acid sequence of SEQ ID NO: 1 and may be a wild type psicose epimerase obtained from microbacterium sp. An amino acid sequence having at least 93%, at least 95%, at least 98%, or at least 99% sequence identity with the amino acid sequence of the enzyme protein.

A further embodiment of the present invention relates to a method for the production of a microorganism having an activity of converting a fructose into a cyclosu (for example, D-cicosco-3-epimerizing activity) in a wild type scythogenic epimerase protein obtained from a microbacterium genus strain 1, wherein at least one of the amino acids of SEQ ID NO: 1 is substituted, inserted and / or deleted, and the like. The mutant enzyme protein may have properties such as high enzyme activity, high heat stability, and / or high enzymatic activity at a relatively low pH and / or relatively low temperature range.

The enzyme mutant according to the present invention comprises an amino acid sequence selected from the group consisting of 34 isoleucine (I), 64 leucine (L), 75 serine (S), 105 valine (I) from the N-terminus of the amino acid sequence having SEQ ID NO: 1 in the cyclic epimerase V), 108 valine (S), 109 alanine (A), 155 valine (V), 177 histidine (H), 207 histidine (H), and 209 serine Or one or more amino acids may be substituted.

For example, in the amino acid sequence of SEQ ID NO: 1, the amino acid substitutable at at least one amino acid position is selected from the group consisting of Pro, Val, Leu, Iso, Thr, Ser, Tyr, Trp, Phe, Asp, Glu, Met and Cys And may be an amino acid selected from the group consisting of Pro, Leu, Iso, Phe, Asp, Met and Cys.

Preferably, the mutant enzyme according to the invention is an enzyme protein which comprises the following substituted amino acids in the amino acid sequence of SEQ ID NO: 1:

The amino acid 34 is Leu, Ser or Tyr,

The amino acid 64 is Ile, Ser or Tyr,

The amino acid 75 is Pro, Trp or Phe,

The amino acid at position 105 is Pro, Trp or Phe,

The amino acid 108 is Asp or Glu,

The amino acid 109 is Phe, Trp, Met, Val, Leu or Ile,

The amino acid 155 is Leu, Ile, Met, Phe or Trp,

177 amino acid is Met, Val, Leu, Ile or Phe,

The amino acid 209 is Cys, Met or Thr.

Accordingly, the wild-type enzyme and the mutant enzyme according to the present invention may be an enzyme protein comprising the amino acid sequence shown in SEQ ID NO: 1.

The amino acid 34 is Ile, Leu, Ser or Tyr,

The amino acid 64 is Leu, Ile, Ser or Tyr,

The amino acid 75 is Ser, Pro, Trp or Phe,

The amino acid at position 105 is Val, Pro, Trp or Phe,

The amino acid at position 108 is Ser, Asp or Glu,

109 amino acids are Ala, Phe, Trp, Met, Val, Leu or Ile,

The amino acid at position 155 is Val, Leu, Ile, Met, Phe or Trp,

177 amino acid is His, Met, Val, Leu, Ile or Phe,

The amino acid 209 is Ser, Cys, Met or Thr.

More preferably, the wild-type enzyme and the mutant enzyme according to the present invention may be an enzyme protein comprising the amino acid sequence of SEQ ID NO: 1 in the following positions:

The amino acid 34 is Ile or Leu,

The amino acid 64 is Leu or Ile,

The amino acid 75 is Ser or Pro,

The amino acid at position 105 is Val or Pro,

The amino acid at position 108 is Ser or Asp,

The amino acid 109 is Ala or Phe,

The amino acid 155 is Val or Leu,

The amino acid 177 is His or Met,

The amino acid 209 is Ser or Cys.

The wild-type enzyme protein having the amino acid sequence of SEQ ID NO: 1 and the mutant enzyme thereof according to the present invention may have a pI value of 5.1 or less, such as 5.05 or less, 5.0 or less, 4.99 or less, 4.99 or less, 4.90 or less, May be 4.0 or more, preferably 4.5 or more. Since the pI value of the enzyme protein is low, it is advantageous because the pH condition can be stably maintained at a relatively low value in the conversion reaction for producing the cucus from the fructose-containing starting material, and more preferably for the immobilization reaction.

In one embodiment of the present invention, the mutant enzyme protein may have a relative activity of more than 100, for example, not less than 102, not less than 103, or not less than 105 based on the conversion activity of the wild-type enzyme protein of 100, Or more. The upper limit of the enzyme conversion activity may vary depending on the enzyme reaction conditions, and may be 800 or less, for example. The relative conversion activity of the enzyme protein can be measured, for example, by adding the enzyme to a 40% (w / v) D-fructose substrate at a concentration of 0.3 unit / ml and then adding 50 mM PIPES buffer (pH 7.0) And then measuring the reaction.

In an embodiment of the present invention, the mutant enzyme protein may have a half-life of 120 to 180 as a measure of thermal stability, and the wild enzyme is 90 minutes long or has a much longer stability than other known enzymes . For example, 50 mM fructose was used as a substrate and reacted in a solution containing 1 mM CoCl ₂ and 0.3 unit / ml of enzyme at pH 6.0 and 60 ° C for 10 minutes to measure enzyme activity. This excellent thermal stability contributes to the stability of the enzyme, which has the advantage of increasing productivity in mass production.

An example of the present invention provides a wild-type enzyme according to the present invention and a polynucleotide encoding the mutant enzyme. Another example provides a recombinant vector comprising a polynucleotide encoding said protein. Another example provides a recombinant strain comprising the recombinant vector, i.e., transformed with the recombinant vector. The recombinant strain may express the protein.

The polynucleotide may be used as such, or in the form of a recombinant vector comprising the polynucleotide. The recombinant vector means a recombinant nucleic acid molecule capable of delivering a target polynucleotide operably linked thereto, and the target polynucleotide may be operably linked to one or more transcriptional regulatory elements such as a promoter and a transcription termination factor . In addition, the polynucleotide may be operably linked to, for example, an inducible element and a temperature sensitive element. The chemical inducible element may be selected from the group consisting of lac operon, T7 promoter, trc promoter, and the like. The T7 promoter is derived from the virus T7 phage and includes a T7 terminator with the promoter.

The recombinant vector can be constructed as a vector for cloning or as a vector for expression by methods well known in the art. The recombinant vector may be any vector that has been used for gene recombination. For example, the recombinant vector may be a plasmid expression vector, a virus expression vector (for example, replication defective retrovirus, adenovirus, and adeno-associated virus) A viral vector, and the like, but the present invention is not limited thereto. For example, the recombinant vector may be one selected from the group consisting of pET, pBR, pTrc, pLex, pUC vector and the like suitable for expression in E. coli.

The host cell capable of transforming with the recombinant vector may be selected from all microorganisms having an expression system capable of expressing (overexpressing) the protein, for example, E. coli. Examples of the E. coli include, but are not limited to, BL21, JM109, K-12, LE392, RR1, DH5α or W3110. For example, BL21 (DE3) may be used. In addition, as the host cells, there may be mentioned Bacillus subtilis such as Bacillus subtilis, Bacillus subtilis, Corynebacterium sp. Such as Corynebacterium glutamicum, Salmonella spp. Such as Salmonella typhimurium, A strain selected from the group consisting of Enterobacteriaceae such as Marsex and various Pseudomonas species and strains may be used.

Methods for transforming the host cell with the recombinant vector can be selected and used without particular restriction on all transformation methods known in the art. For example, fusion of bacterial protoplasts, electroporation, projectile bombardment, Infection using a vector, and the like.

As described above, the enzyme protein is an enzyme protein that is excellent in the ability to convert fructose into a cytosine. Accordingly, the wild-type enzyme and the mutant enzyme according to the present invention or a strain expressing the enzyme can be usefully applied to the manufacture of the psicose.

Thus, another example of the present invention relates to a recombinant vector comprising the enzyme protein, a polynucleotide encoding the protein, a recombinant vector comprising the polynucleotide, a recombinant strain comprising the recombinant vector, a culture of the recombinant strain, And a disintegrating agent of the present invention.

The composition for making Saikosu may be a preparation of psicose from fructose using fructose as a substrate. The culture contains an enzyme protein produced from the recombinant strain, and may include the recombinant strain or may be in a cell-free form containing no strain. The lysate refers to 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 this specification, unless otherwise stated, the recombinant strains used in the manufacture of a scikos means at least one selected from the group consisting of the strains of the strains, the cultures of the strains and the lysates of the strains. do.

Another example is a polynucleotide encoding the wild-type enzyme and the mutant enzyme according to the present invention, a polynucleotide encoding the protein, a recombinant vector containing the polynucleotide, a recombinant strain containing the recombinant vector, a culture of the recombinant strain, And a disruption product of the recombinant strain (hereinafter referred to as " enzyme protein "). The above-mentioned method of producing a sacca comprises the step of reacting the enzyme protein and the like with fructose. In one embodiment, the step of reacting the enzyme protein or the like with fructose may be performed by contacting the protein with fructose. In one embodiment, the step of contacting the enzyme protein or the like with fructose may be carried out, for example, by mixing the enzyme protein or the like with fructose or contacting fructose to the carrier to which the enzyme protein or the like is immobilized . In another example, the step of reacting the enzyme protein with fructose may be performed by culturing the cells of the recombinant strain in a culture medium containing fructose. Thus, by reacting the enzyme protein or the like with fructose, it is possible to convert fructose into cyclosaccharide and produce cyclosaccharide from fructose.

In the above-mentioned method of producing the sciucose, the amount of the protein used for efficient production of the sciatica can be appropriately selected in consideration of the cyclosuction efficiency, for example, 0.001 mg / ml to 2.0 mg / ml on the basis of the total reactant .

The concentration of fructose used as a substrate for efficient production of the psicose can be selected in consideration of solubility and conversion yield, etc. Fructose can be used by dissolving an isomerization product or fructose in a solvent, For example, 10 (w / v)% or more, 20 (w / v)% or more, 30 (w / v)% or more or 40 (w / v)% or more based on the total reactants.

Given the optimal conditions of the enzyme protein, the reaction may be carried out at a pH of at least 5, such as pH 4.5 to pH 8, pH 5 to pH 8, or pH 5.5 to pH 7. The reaction may be carried out at a temperature of 30 DEG C or higher, or 40 DEG C or higher, for example, 40 to 85 DEG C or 40 to 65 DEG C. The time of the enzyme reaction can be selected as a condition for maximizing the conversion efficiency of fructose to psicose.

When the recombinant strain is used, the cell concentration of the strain to be used may be selected in consideration of the conversion activity of the psicose. For example, 1 mg (dcw: dry cell weight) / ml, for example, 1 to 50 mg (dcw) / ml.

The enzyme protein having the above-mentioned cyclosporin-converting enzyme activity may have a metalloenzyme characteristic that activation is controlled by a metal ion. The reaction by the enzyme protein can be carried out in the presence of metal ions to enhance the yield of the production of the psicose. The metal ion that can contribute to the yield of the psicose production may be at least one selected from the group consisting of copper ions, manganese ions, calcium ions, magnesium ions, zinc ions, nickel ions, cobalt ions, iron ions, aluminum ions, And may be at least one selected from the group consisting of manganese ions and magnesium ions. The metal ion concentration suitable for the reaction can be selected in consideration of the effect of increasing the yield of the production of the psicose. For example, the addition amount of the metal ion can be 0.1 mM or more. The metal ion may be added to the substrate fructose or may be added to the mixture of the enzyme protein and the fructose.

In another preferred embodiment, the method for producing Saikos comprises the steps of culturing and recovering a strain expressing the wild-type enzyme and the mutant enzyme protein according to the present invention; And reacting the strain or an enzyme protein separated from the strain with fructose. The step of culturing the strain may be carried out under culture conditions and culture conditions that are easily selected by those skilled in the art depending on the characteristics of the strain (host cell) to be used.

Another embodiment of the present invention is a process for the production of cyclosporin, which comprises a cyclosporidase having a pI value in the range of 4.0 to 5.1, more preferably a pI value in the range of 4.5 to 5.1, And a method for producing cicos from a fructose-containing substrate using the above-mentioned cyclic epimerase. When the above-mentioned cyclic epimerase has a pI value in the above-mentioned range, the optimal activity of the enzyme can be maintained in the step of immobilizing the strain or enzyme for the mass production of the scarcos and the conversion of the scarcos using the same, Can be maximized.

The above-mentioned cyclic epimerase may be derived from a strain of Mycobacterium sp., Preferably a wild type enzyme having the amino acid sequence of SEQ ID NO: 1 according to the present invention and a mutant type enzyme derived therefrom, The enzymes are as described above.

The above-mentioned psicose epimerase may be provided in at least one selected from the group consisting of an enzyme protein, a strain expressing the enzyme protein, a culture of the strain, and a disruption of the strain. The above-mentioned psicose epimerase may be provided by immobilizing an enzyme protein or a strain expressing an enzyme protein on a carrier, which may be alginic acid or a salt thereof.

Saikos obtained from fructose by the method of the present invention can be purified by conventional methods, and such crystals belong to a technique common to those skilled in the art. For example, by one or more methods selected from the group consisting of centrifugation, filtration, crystallization, ion exchange chromatography, and combinations thereof.

The novel scorpion epimerase protein according to the present invention is an enzyme having an activity to produce a scorch, which is excellent in heat stability under industrially applicable conditions, has a long half-life, and can be mass produced from fructose at high yield It is possible. Therefore, the D-psicose 3-epimerase of the present invention and the method for producing psicose using the same are expected to be usefully used in a variety of foods, medicines, and cosmetic materials industries.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing a chromatogram obtained by high-performance liquid chromatography (HPLC) of the production of psicose from high-concentration fructose in an embodiment of the present invention.
2 is an SDS-PAGE diagram showing the purification process of the wild-type enzyme protein according to the present invention.
FIG. 3 is a graph showing the activity of wild-type enzyme protein according to the present invention according to the kind of metal ions added.
FIG. 4 is a graph showing the temperature-dependent activity of the wild-type enzyme protein according to the present invention.
FIG. 5 is a graph showing the pH-dependent activity of the wild-type enzyme protein according to the present invention (Mcilvaine buffer, Glycine-NaOH buffer).
6 is a graph for confirming the thermal stability of the wild-type enzyme protein according to the present invention at an enzyme temperature of 60 ° C.
FIG. 7 is a graph showing the activity of the wild-type enzyme protein according to the present invention at 60 ° C. over time.
FIG. 8 is a graph showing the production efficiency (cyclosity conversion rate) of the cosmetic composition from the high concentration fructose using the wild-type enzyme protein according to the present invention.
FIG. 9 is a homology modeling result using a SWISS-MODEL for producing a mutant enzyme protein according to the present invention.
10 is a protein tertiary structure for the wild-type enzyme protein (MDPE enzyme) according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  One: Saikos  production Mycobacterium  Strain isolation and characterization

1-1: Fructose Saikos  Isolation of microorganisms to convert

(KH ₂ PO ₄ 2.4 g / L, K ₂ HPO ₄ 5.6 g / L, (NH ₄ ) ₂ SO ₄ ), which was added with 1% (w / v) 2.6 g / L, MgSO ₄ 7H ₂ O 0.1 g / L, yeast extract 1 g / L) was used.

1 g of each food was selected and added to the MSP broth, followed by incubation at 30 ° C for 24 hours to effect enrichment of the food (for example, broccoli, ginseng, edible flowers, etc.). Then, 100 microliter of culture solution was taken on agar medium and cultured at 30? Until colonies were confirmed. Colonies different in shape and size from the colonies formed in the agar medium were selected, inoculated into MSP broth, incubated at 30 ° C for 24 hours with shaking, and centrifuged to collect only the cells. The recovered cells were suspended in 100 microliter of 50 mM PIPES (piperazine-N, N'-bis (2-ethanesulfonic acid)) buffer solution (pH 7.0) and disrupted by using an ultrasonic processor (ColepParmer) Lt; / RTI > The digested lysate was centrifuged at 12,000 rpm for 10 minutes at 4 ° C, and the supernatant was recovered and used as a crude enzyme. The enzyme solution was reacted with 10 mM fructose and 10 mM sucrose for 12 hours at 30 ° C. .

Thin Layer Chromatography (TLC) analysis was performed to confirm that the reaction mixture was converted into fructose. The thin layer chromatography analysis was carried out for 10 minutes using a mobile phase developing solvent in which silica gel (Silica gel 60F254 (Merck, Germany)) having a width of 20 cm and a length of 10 cm and acetonitrile and water were mixed 85:15 by volume Respectively.

The strains which were confirmed to be converted to fructose by the above TLC analysis were selected and inoculated into MS broth supplemented with 0.1% (w / v) course, shake cultured at 30 ° C for 24 hours, Respectively. The recovered cells were washed with 0.85% (w / v) NaCl, suspended in 50 mM PIPES buffer (pH 7.0) containing 400 g / L fructose and 1 mM manganese ion, and reacted at 70 ° C for 1 hour.

Then, the reaction product was centrifuged, and the supernatant was recovered and subjected to high performance liquid chromatography (HPLC) analysis. The liquid chromatographic analysis was performed using HPLC (Agilent, USA) RID (Refractive Index Detector, Agilent 1260 RID) equipped with Aminex HPX-87C column (BIO-RAD). The mobile phase solvent was water, and the temperature was 80 ° C and the flow rate was 0.6 mL / min. The results obtained are shown in Fig. 1, and one strain which produced the most of the psicose among the strains of the species was finally selected.

The nucleotide sequence of 16S ribosomal RNA was identified to identify isolated strains. The nucleotide sequence (5 '-> 3') of the 16S ribosomal RNA of the isolate was confirmed to be 99.5% identical to that of Microbacterium foliorum DSM12966 and designated as Microbacterium foliorum SYG27B. The above strain was deposited with the Korean Society for Microbiological Research on September 24, 2015, and received the accession number KCCM11774P.

1-2: Cell reaction  Optimum temperature used

The strains isolated from the above were reacted with cells and substrates under various temperature conditions, and the conversion activity thereof was compared.

The cell concentration of the strain isolated in Example 1 was 5 mg (dcw) / mL in a 50 mM PIPES buffer (pH 7.0) containing 400 g / L fructose and 1 mM manganese metal ion. The reaction was carried out for 1 hour while changing the temperature. After completion of the reaction, the yield of cyclosaccharide was measured by HPLC analysis in the same manner as in Example 1, and the results are shown in Table 1 below.

Reaction temperature (캜) Relative activity (%) 50 54 60 66 I65 86 70 97 75 100 80 88

As shown in Table 1, the relative activity of Microbacterium foliorum increased with increasing reaction temperature up to 70 ° C, and the relative activity decreased at 80 ° C.

1-3: Saikos  productivity

The activity of the strain Microbacterium foliorum SYG27B isolated in Example 1-1 was confirmed at a cell concentration of 20 mg / mL, a fructose concentration of 400 g / L, a temperature of 70 DEG C and a pH of 7.0. The reaction was carried out for 12 hours, and the productivity of the cyclosaccharide was determined at regular intervals through HPLC analysis. The results are shown in Table 2 below.

Reaction time (hr) Microbacterium foliorum
Relative activity (%) One 8.4 2 13.9 4 18.5 6 20.2 8 24.8 10 26.4 12 27.1 14 27.1

As can be seen from Table 2, the conversion rate of the microbacterium foliorum SYG27B increased with the passage of time. Especially, the conversion rate of the microbacterium foliorum was about 27% after 12 hours of reaction at 70 ° C, Was found to be about 75 g / L.

Example  2: Saikos  Isolation of Converting Enzyme

The isolated microbial cells exhibiting cyclosporin conversion activity in Example 1-1 were suspended in a 50 mM PIPES buffer solution (pH 7.0) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and then, using an ultrasonic processor (ColepParmer) 0.0 > 4 C < / RTI > for 40 minutes. The digested solution was centrifuged at 13,000 rpm for 20 minutes at 4 DEG C, and the supernatant was recovered and used as a coenzyme. The coenzyme solid ammonium sulfate saturation of 50 to 60% _{[(NH 4) 2 SO 4} ] was added and the precipitate, collected by centrifugation for at 4 ℃ to 13,000 rpm 20 min, and then the protein 50 mM PIPES buffer solution (pH 7.0) for 12 hours to obtain partially purified enzyme.

The partially purified enzyme was heat-treated at 60 ° C for 10 minutes, centrifuged at 8,000 rpm for 20 minutes at 4 ° C, and the supernatant was recovered and equilibrated with 50 mM Tris-HCl (pH 8.0) trap Q ion exchange chromatography column (Amersham Biosciences, Uppsala, Sweden). The binding protein was eluted with a concentration gradient up to 1.0 M using NaCl, and the activity of each fraction was measured and the active fractions were collected and concentrated.

The samples separated by Hi-trap Q were equilibrated in 10 mM sodium phosphate (pH 6.8) buffer and then subjected to Biogel hydroxyapatite column (Bio-Rad, Hercules, CA, USA). The bound protein was eluted with a concentration gradient of 10-100 mM sodium phosphate (pH 6.8), and the activity of each fraction was measured and the active fractions were collected and concentrated.

Samples separated by a Biogel hydroxyapatite column were equilibrated with 50 mM sodium phosphate (pH 7.0) containing 1.5 M ammonium sulfate, adsorbed on a Hi-trap phenyl column (Amersham Biosciences, Uppsala, Sweden) To obtain fractions.

SDS-PAGE was used to confirm the degree of purification by each purification step. SDS-PAGE was performed with 12% polyacrylamide gel and 20 mA. The results obtained are shown in Fig. As shown in FIG. 2, it was confirmed that the purified protein had a molecular weight of about 30 kDa.

The purified protein was reacted at a concentration of 0.004 mg / ml for 50 minutes at a temperature of 55 캜 and at a temperature of 55 캜 for 10 minutes, and the obtained product was subjected to HPLC analysis in the same manner as described in Example 1. As a result of HPLC analysis (HPLC conditions are the same as in Example 1), it can be confirmed that the purified protein has an activity of producing psicose from fructose. Thus, it can be seen that the purified protein has the activity of D-psicose epimerase which produces psicose from fructose.

The amino acid sequence of the purified enzyme protein was analyzed by the E-MASS.Co method, and the resulting amino acid sequence was shown in SEQ ID NO: 1.

Example  3: Production and purification of enzyme

3-1: Enzyme Production

(SEQ ID NO: 2) encoding the amino acid sequence of SEQ ID NO: 1 derived from the microbacterium foliorum obtained in Example 2 was submitted to Bioneer.Co. Korea to synthesize the polynucleotide.

The synthesized polynucleotide was inserted into the same restriction enzyme site of the expression vector pET21a (Novagen) using restriction enzymes Not I and Nde I to prepare a recombinant vector pET21a / psicose epimerase (pET-MDPE). E. coli BL21 (DE3) (invitrogen) was transformed with the recombinant vector pET-MDPE by the heat shock method (Sambrook and Russell: Molecular Cloning.) to prepare a recombinant strain. The prepared recombinant strain was inoculated in a 5 ml LB-ampicilline medium (Difco) and cultured with shaking at 37 ° C and 200 rpm until the absorbance (OD) at 600 nm reached 1.5, and then inoculated into 500 ml LB-ampicilline medium Followed by seed culture in a shaking incubator at 37 ° C. When the absorbance at 600 nm of the culture was 0.5, 1 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added to induce overexpression of the target enzyme. From the induction of induction of the overexpression, the culture conditions were changed to 16 캜 and 150 rpm and maintained for 16 hours. The overexpression-induced culture was centrifuged at 4000 rpm for 20 minutes in a centrifuge to collect only the cells. The recovered cells were washed twice with 0.85% (w / v) NaCl and used for enzyme purification described below.

3-2: Enzyme purification

The cells recovered in Example 3-1 were suspended in lysis buffer (50 mM Tris-HCl and 300 mM NaCl at pH 7.4, 10 mM imidazole) and disrupted at 4 ° C for 20 minutes using an ultrasonic processor (ColepParmer) . After the supernatant was collected by centrifugation at 13,000 rpm for 20 minutes, the supernatant was applied to a Ni-NTA column (NiNTA Superflow. Qiagen) previously equilibrated with a lysis buffer. Then, 20 mM imidazol was added to 50 mM Tris-HCl and 300 mM NaCl And a buffer solution containing 300 mM imidazole were sequentially flowed. The target protein was isolated and purified by flowing the 50 mM Tris-HCl and 300 mM NaCl at pH 8.0 and 300 mM imidazole. The separated and purified target protein was converted into a buffer solution for measurement of enzyme activity (50 mM PIPES pH 7.0) and then used in the experiment. In addition, it was confirmed by SDS-PAGE that the size of the monocyte was about 32.8 kDa in the case of the isolated (partially) purified target protein, Sacchos epimerase.

3-3: Metals of enzymes Composition  analysis

Enzyme activity was measured by treating the protein (enzyme) purified in Example 3-1 with 1 mM each of metal ions CuCl ₂ , MnCl ₂ , CaCl ₂ , ZnSO ₄ , MgSO ₄ , NiSO ₄ or CoCl ₂ . The enzymatic activity was measured by reacting 50 mM fructose and 0.3 unit / ml of enzyme in the presence of the metal ion in 50 mM Micalavine buffer solution (pH 6.0) for 5 minutes at 60 ° C. After the reaction, the enzyme activity was stopped by heating at 100 ° C for 5 minutes. The enzyme activity was defined as 1 unit in which a 50 mM Mclavine buffer solution (pH 6.0) containing 50 mM fructose and 1 mM Co ^{2 +} as a substrate was reacted with an enzyme at 60 ° C. to produce a 1-micromole course per minute. As a control (Non), those not treated with metal ions were used.

The enzyme activity was calculated by dividing the amount of produced scorch (mM) by the amount of enzyme used and the reaction time, and the amount of scorch was analyzed by HPLC. The HPLC analysis was carried out using a 87C (BIO-RAD) column at 80 ° C with 100% (v / v) water flowing at a flow rate of 0.6 ml / min to the mobile phase. The refractive index detector (Agilent 1260 TID) And the productivity of psychosomes was analyzed.

The measured enzyme activity in the case of treating each metal ion is shown in FIG. 3 in comparison with the enzyme activity in the control group. As shown in FIG. 3, the activity of the enzyme of Example 3-1 was increased by addition of manganese ion and cobalt ion, indicating that the enzyme had a metal ion requirement.

3-4: Analysis of enzyme activity by temperature and pH

In order to confirm the activity of the protein (enzyme) purified in Example 3-1 according to pH and temperature changes, enzyme and fructose substrate were reacted at various pH and temperature, and enzyme activity was confirmed.

The activity measurement was carried out for 10 minutes at a pH of 5 to 9.5 and a temperature of 40 to 80 ° C using 50 mM fructose and 0.3 unit / ml of enzyme with reference to the method described in Example 3-3, The reaction was stopped by heating at 100 DEG C for 10 minutes. The enzyme activity was measured in the same manner as in Example 3-3, except that the pH was changed to 7 and the temperature was changed in the range of 40 to 80 ° C. The result is shown in FIG. The relative activity of the Y-axis in FIG. 4 means a relative value when the best enzyme activity is taken as 100. As can be seen in FIG. 4, the enzyme exhibited an activity of 80% or more at 40 to 65 ° C, specifically in the range of 50 to 65 ° C, and showed maximum activity at 60 ° C.

50 mM sodium citrate pH 5-6.5, 50 mM Tris-HCl pH 7-9, and 50 mM Tris-HCl at 60 ° C to examine the activity depending on the pH change, with reference to the method described in Example 2.2. Enzyme activity was measured using a buffer solution of mM glycine NaOH pH 9.5. The results obtained are shown in Fig. As can be seen from FIG. 5, the enzyme activity was 80-100 at pH 5.5 or higher, and the enzyme activity was particularly high at pH 6.0.

3-5: Analysis of thermal stability of enzyme

In order to confirm the stability of the Saikoso-3 epimerase according to the temperature change, the enzyme purified in the above-mentioned Example 3-1 was heat-treated at 60 DEG C for a predetermined time (180 minutes), and 50 mM fructose was used as a substrate The enzyme activity was measured by reacting in 50 mM Mcilvaine buffer containing 1 mM CoCl ₂ and 0.3 unit / ml of enzyme for 10 minutes at pH 6.0 and 60 ° C. Enzyme activity was stopped by heating at 100 占 폚 for 5 minutes. The results obtained according to the heat treatment time are shown in Fig.

As shown in Fig. 6, the half-life of the enzyme purified in Example 3-1 was calculated to be 90 minutes at 60 占 폚. It is confirmed that this is much higher than that of most of the previously reported cyclosporin 3-epimerases, and that the cyclosporin 3-epimerase according to the present invention has excellent thermal stability at a temperature at which the reaction is industrially easy .

3-6: Enzyme Analysis of specific activity

In order to confirm the reaction rate of the Saikoso-3 epimerase with time, the enzyme purified in Example 3 was reacted with 50 mM fructose as a substrate, 50 mM of 1 mM CoCl ₂ and 0.3 unit / ml of the enzyme mM Mcilvaine buffer solution at pH 6.0 and 60 ° C at 0, 1, 2, and 5 minutes intervals, and the enzyme activity was stopped by heating at 100 ° C for 5 minutes.

The results obtained are shown in FIG. 7, and the specific activity was found to be about 150 U / mg. Reversible is the psicose mole unit in which 1 mg of enzyme is produced per minute.

3-6: Enzyme-induced Saikos  production

In order to produce a high concentration of the cytosine, the concentration of D-psicose 3-epimerase purified in Example 3-1 was adjusted to 50 mg / ml in a concentration of 50 mM Mcilvaine buffer solution pH 6.0 and 1 mM CoCl ₂ (400 g / L) fructose. In the same manner as in Example 2-2, the yield of the produced psicose was measured and the conversion rate from fructose to psicose was measured. Since the used fructose was high concentration, the sample solution was diluted 20 times with each time, and HPLC analysis was carried out. The results obtained are shown in Fig. As shown in FIG. 8, at a concentration of 0.1 mg / ml, the final yield of cyclospores was 118.6 g / L and the conversion rate of cyclosaccharides was about 30%.

3-7: Enzyme pI  Measure

Experiments were performed by isoelectric focusing (two-dimensional electrophoresis) of proteins in order to measure the pI value of the D-psicose 3-epimerase purified in Example 3-1.

As a result, the pI value of the isolated wild-type enzyme was 4.88. The pI value of this enzyme was found to be 5.41 for pI = 5.41 of Clostrium cellulolyticum, 6.2 for pI = 5.88 of Agrobacterium tumefaciens, PI of mercaptase enzyme = 5.93, Ruminococcus sp. Was lower than that of psicarboxylase (pI = 5.24).

Example  4: Preparation of mutant enzyme protein

4-1: Three-dimensional structure Homology modelling

In order to prepare a mutant enzyme of a scissor epimerase having the amino acid sequence of SEQ ID NO: 1, first, an NCBI website (BLAST) tool and other biologically-derived enzymes and array alignment tools used, a sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html) was used to compare enzymes from other organisms.

In addition, the homology modeling of the three-dimensional structure of the enzyme protein can be carried out using a protein three-dimensional homology model through SWISS-MODEL (http://www.expasy.org/swissmod/SWISS-MODEL.html) (Singh, PK et al, (2012), Indian journal of microbiology 52 (3): 373-380). Template The protein structure was selected as a template with a protein structure very similar to that of the epimerase through a template search of a protein data bank (PDB) in a SWISS-MODEL server. The homology modeling results using the SWISS-MODEL for preparing the mutant enzyme protein are shown in FIG. 9, and the wild-type protein structure is shown in FIG.

4-2: Analysis of Alanine Scanning Mutants and Docking Binding

Based on the analysis of the amino acid sequence between the homologous genes performed in Example 4-1 and the analysis of the tertiary structural model of the active site, the selected amino acids were substituted with alanine, and these recombinant mutant enzymes were produced in E. coli, Characteristics were analyzed. After the alanine scanning mutation analysis, amino acids predicted to be functionally important were selected through simulation of the redesigned active site structure and D-fructose to improve the unit activity of the D-psicose C3-epimerization reaction. The site was designed. The amino acid sites (catalytic metal ion binding residues and dedoping / protonation related catalytic residues) that are completely lost activity through the alanine scanning mutation were excluded from the target site for activity improvement.

4-3: Iterative saturation for rapid direct evolution of functional enzymes mutagenesis  ( ISM ))

For expression of the wild-type enzyme, a recombinant expression vector prepared for expression of E. coli BL21 (DE3) (a wild-type enzyme gene was introduced into the Not I and Nde I restriction sites of pET21a and the wild-type C- Recombinant enzyme) was used as a template for the saturation mutagenesis method for constructing the mutant library.

Inverse PCR-based saturation mutagenesis was used (2014. Anal. Biochem. 449: 90-98) in consideration of diversity distribution variability and mutant yield, minimizing the screening scale of the prepared mutant library NNK, NTB, DBK, NRT, NDT, VMA, ATG and TGG mixed primers with minimal codons of E. coli were minimized (2012.Biotechniques 52: 149-158, 2007. Nature Protocols Vol.2, No. 4, 891-903). The mutant primers for the Saikoso-3-epimerase according to the sequence positions are shown in Table 3 below.

SEQ ID NO: Mutation location direction Primer 4 I34L Forward GCGGGTTTCGACCTG NDT GAATTCCCGCTGATGGAC 5 I34L Reverse GTCCATCAGCGGGAATTC CAG CAGGTCGAAACCCGC 6 L64I Forward GCGGTTTCTGCGTCT NDT GGTCTGTCTGGTGCGAC 7 L64I Reverse GTCGCACCAGACAGACC AAT AGACGCAGAAACCGC 8 S75P Forward CGACCGACGTTACCTCT HCN GACCCGGCGGTTG 9 S75P Reverse CAACCGCCGGGTC CGG AGAGGTAACGTCGGTCG 10 V105P Forward GTCGTCACTTCTGCGGT NCN ATCTACTCTGCGATGCAGAAATAC 11 V105P Reverse GTATTTCTGCATCGCAGAGTAGAT CGG ACCGCAGAAGTGACGAC 12 S108D Forward CACTTCTGCGGTGTTATCTAC NAN GCGATGCAGAAATACATGG 13 S108D Reverse CCATGTATTTCTGCATCGC ATC GTAGATAACACCGCAGAAGTG 14 A109F Forward CTGCGGTGTTATCTACTCT NTN ATGCAGAAATACATGGACCC 15 A109F Reverse GGGTCCATGTATTTCTGCATA AAA GAGTAGATAACACCGCAG 16 V155L Forward GTTAACCGTTACGAAACCAAC HCN CTGAACACCGCGCG 17 V155L Reverse CGCGCGGTGTTCAG CAA GTTGGTTTCGTAACGGTTAAC 18 H177M Forward CGTCCGAACCTGGGTATC ATG CTGGACACCTACCACATG 19 H177M Reverse CATGTGGTAGGTGTCCAG CAT GATACCCAGGTTCGGACG 20 S209C Forward GTTACGTTCACATCGGTGAA NDT CACCGTGGTTACCTGG 21 S209C Reverse CCAGGTAACCACGGTG GCA TTCACCGATGTGAACGTAAC

In the primer sequences shown in Table 3, n is a, t, g or c, d is a, t or g, and h is a, t or c.

In detail, 15bp to 20bp of the base in front of each displacement site and 3bp (3) to 3bp (NDT, NCN, NAN, NTN, DBK and ATG) to replace the displacement site, 15bp to 20bp of the back base, To prepare a mixed primer. The PCR conditions were denaturation at 94 ° C for 2 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension at 72 ° C for 10 minutes, and extension at 72 ° C for 60 minutes. After producing the mutant library by mutation site, mutant strains were randomly selected (<mutation 11) and nucleotide sequence was analyzed to evaluate the amino acid mutation distribution. Based on the results of the analysis, screening scales of 80% or more of sequence coverage for each library were set (2003. Nucleic Acids Res. 15; 31: e30).

4-4: Highly active mutant enzyme screening

In order to quantitatively screen a large amount of mutant enzyme in the saturated mutation library obtained in Example 4-4, a colorimetric method capable of specifically quantifying D-fructose was used.

Specifically, for the D-fructose dehydrogenase assay (SIGMA-ALDRICH, assay kit), the culture medium and the substrate reaction were mixed at a ratio of 9: 1 and reacted at 70 ° C for 1 hour. The reaction was carried out for 20 minutes, and then analyzed by an ELISA reader at 600 nm and compared with the OD value. Mutation in which activity (D-fructose conversion D-cyclos production) was increased in wild type enzyme (SEQ ID NO: Site 9 mutants were firstly selected and their genes were analyzed for amino acid variation information after sequencing. Specifically, the amino acid at the mutation site was substituted with various amino acids, and the activity of converting the mutant enzyme into the cytosine was analyzed. The results are shown in Table 4 below.

enzyme Relative activity (%) Wild type (WT) 100 I34L 135 I34S 105 I34Y 110 L64I 129 L64S 108

The first selected mutant enzymes were reacted with fructose using a purified (His-tag affinity chromatography) enzyme solution, and the reaction products were analyzed by HPLC (column: Bio-Rad Aminex-87C, column analysis temperature 80 ° C, mobile phase H ₂ O, flow rate: 0.6 ml / min, Refractive Index Detector) was used to finally select nine mutant enzymes with increased D-fructose-converting D-fucose-producing activity relative to wild type enzymes.

The mutated amino acid positions and substituted amino acid types of the nine selected mutant enzymes are shown in Table 5 below. The amino acid positions shown in Table 5 below represent the amino acid positions from the N-terminal in the amino acid sequence of SEQ ID NO: 1.

Amino acid position Original Variant Relative activity (%) 34 Isoleucine (I) Leucine (L) 135 64 Leucine (L) Isoleucine (I) 129 155 Valine (V) Leucine (L) 150 75 Serine (S) Proline (P) 110 105 Valine (V) Proline (P) 105 209 Serine (S) Cysteine (C) 110 177 Histidine (H) Methionine (M) 150 108 Serine (S) Aspartate (D) 125 109 Alanine (A) Phenylalnine (F) 135

Example  5: Activity and Characterization of Mutant Enzymes

In order to evaluate the relative activity of the mutant enzyme against the wild-type enzyme, each mutant enzyme obtained in Example 4 was expressed in Escherichia coli BL21 (DE3) and purified (His-tag affinity chromatography) in the same manner as in Example 3 .

Each mutant enzyme was added to a 40% (w / v) D-fructose substrate at a concentration of 0.3 unit / ml, followed by reaction at 50 ° C in a PIPES buffer (pH 7.0) and 60 ° C for 5 minutes to obtain the amino acid sequence of SEQ ID NO: Relative activity of the mutant enzyme against the activity of the wild type mutant enzyme was measured. The relative activity of the mutant enzymes measured is shown in Table 5. &lt; tb &gt; &lt; TABLE &gt;

In addition, the thermal stability, metal ion requirement, and kinetic parameter analysis for the nine mutant enzymes of Table 5 were performed in substantially the same manner as in Example 3. [ The results of the evaluation of the thermal stability of the mutant enzymes are shown in Table 6 below.

Mutation Time (min) Half life (60 ℃) Half-life (70 ℃) WT 90 30 V34P 130 75 S64C 120 60 S105P 180 90

As used herein, "thermal stability" refers to the ability of an enzyme to retain activity after exposure to elevated temperature. The thermal stability of enzymes such as non-maltogenic exoamylase is measured by half-life. The half-life (t1 / 2) is the time (minutes) during which half of the enzyme activity is inactivated under defined conditions. The half-life value is calculated by measuring the activity of residual interace course.

As a result of evaluating the metal ion requirement of the mutant enzyme, the relative activity of H177M mutant enzyme was about 150 as compared with that of the wild type metal requirement of 100.

For each mutant enzyme obtained in Example 4, the pI value of the enzyme was measured in the same manner as in the experimental method of Example 3-7.

As a result, the pI value of the mutant enzyme S108D was 4.77, and the pI value of this mutant enzyme was found to be 5.41 for pI = 5.41 of the known Clostrium cellulolyticum, 8.1 for the psicose epimerase of Agrobacterium tumefaciens, PI of enzyme was 5.93 and that of psicose epimerase of Ruminococcus sp. Was lower than that of pI = 5.24.

Korea Microorganism Conservation Center (overseas) KCCM11774P 20150924

<110> SAMYANG CORPORATION <120> Psicose epimerase and method of psicose using the same <130> DPP20172153EN <160> 21 <170> KoPatentin 3.0 <210> 1 <211> 289 <212> PRT <213> Artificial Sequence <220> <223> wild type enzyme <400> 1 Met Asn Ile Gly Cys His Gly Leu Val Trp Thr Gly His Phe Asp Ala   1 5 10 15 Asp Gly Ile Arg Leu Ala Ser Glu Gln Thr Lys Ala Ala Gly Phe Asp              20 25 30 Leu Ile Glu Phe Pro Leu Met Asp Pro Phe Thr Phe Asp Val Ala Ala          35 40 45 Ala Lys Glu Ala Leu Glu Glu His Asp Leu Ala Val Ser Ala Ser Leu      50 55 60 Gly Leu Ser Gly Ala Thr Asp Val Thr Ser Ser Asp Pro Ala Val Val  65 70 75 80 Ala Gla Aly Glu Ala Leu Ale Ala Glu                  85 90 95 Leu Gly Gly Arg His Phe Cys Gly Val Ile Tyr Ser Ala Met Gln Lys             100 105 110 Tyr Met Asp Pro Val Thr Thr Glu Gly Leu Glu Ser Ser Arg Arg Thr         115 120 125 Ile Ala Arg Val Ala Asp His Ala Ala Glu Arg Gly Ile Ser Val Ser     130 135 140 Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Leu Asn Thr Ala Arg 145 150 155 160 Gln Ala Leu Ala Tyr Leu Ala Glu Val Asp Arg Pro Asn Leu Gly Ile                 165 170 175 His Leu Asp Thr Tyr His Met Asn Ile Glu Glu Ser Asp Met Phe Ala             180 185 190 Pro Val Leu Asp Ala Ala Pro Ala Leu Arg Tyr Val His Ile Gly Glu         195 200 205 Ser His Arg Gly Tyr Leu Gly Thr Gly Thr Val Asp Phe Asp Asn Phe     210 215 220 Phe Lys Ala Leu Gly Arg Ile Gly Tyr Asp Gly Pro Ile Val Phe Glu 225 230 235 240 Ser Phe Ser Ser Ala Val Val Ala Pro Asp Leu Ser Arg Met Leu Gly                 245 250 255 Ile Trp Arg Asn Leu Trp Thr Asp Asn Val Glu Leu Gly Ala His Ala             260 265 270 Asn Tyr Ile Arg Asp Lys Leu Val Ala Val Asp Ser Ile Arg Leu         275 280 285 His     <210> 2 <211> 870 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding wild type enzyme <400> 2 atgaacatcg gatgccacgg gctcgtctgg accggacact tcgatgccga cggcatccga 60 ctctcggcgg agcagacgaa ggcggcaggc ttcgacctga tcgagttccc gctcatggat 120 ccgttcacgt tcgatgtcgc cgccgcgaag gaggcgctcg aagagcacga cctcgccgtc 180 agcgcctcgc tcggtctctc gggggcgacg gatgtgacca gctctgatcc ggcagtcgtc 240 gccgcgggcg aggcgctgct gatgaaggcc gtagatgtgc tggccgagct gggcggccgg 300 cacttctgcg gagtgatcta cagcgcgatg cagaagtaca tggacccggt gacgacagaa 360 gggctcgaga gcagccgtcg caccatcgcc cgcgtcgcgg atcacgcggc cgagcgaggg 420 atctcggtct cgctcgaggt cgtgaaccgc tatgagacca acgtgctgaa caccgcgcgg 480 caggcactcg cctacctcgc ggaggtcgat cggccgaacc tcggcatcca cctcgacacg 540 taccacatga acatcgagga gtcggatatg ttcgcgccgg tcctcgatgc cgcacccgcc 600 ctccgctacg tgcacatcgg cgagagccac cgcggctatc tcggcaccgg aacggtcgac 660 ttcgacaact tcttcaaggc gctggggcgc atcggctatg acggcccgat cgtgttcgag 720 tcgttctcct cggcggtggt cgcccccgac ctcagccgga tgctcggcat ctggcgcaac 780 ctgtggaccg acaacgtcga gctcggtgcg cacgccaacg cctacatccg cgacaagctc 840 gtcgcggtcg actcgatcag gctgcactga 870 <210> 3 <211> 1466 <212> DNA <213> Artificial Sequence <220> <223> Microbacterium foliorum <400> 3 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac ggtgaacacg gagcttgctc 60 tgtgggatca gtggggaacg ggtgagtaac acgtgagcaa cctacccctg actctgggat 120 aagcgctgga aacggcgtct aatactggat acgagtggcg accgcatggt cagctactgg 180 aaagatttat tggttgggga tgggctcgcg gcctatcagc ttgttggtga ggtaatggct 240 caccaaggcg tcgacgggta gccggcctga gagggtgacc ggccacactg ggactgagac 300 acggcccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgcaagcctg 360 atgcagcaac gccgcgtgag ggatgacggc cttcgggttg taaacctctt ttagcaggga 420 agaagcgaaa gtgacggtac ctgcagaaaa agcgccggct aactacgtgc cagcagccgc 480 ggtaatacgt agggcgcaag cgttatccgg aattattggg cgtaaagagc tcgtaggcgg 540 tttgtcgcgt ctgctgtgaa atccggaggc tcaacctccg gcctgcagtg ggtacgggca 600 gactagagtg cggtagggga gattggaatt cctggtgtag cggtggaatg cgcagatatc 660 aggaggaaca ccgatggcga aggcagatct ctgggccgta actgacgctg aggagcgaaa 720 gggtggggag caaacaggct tagataccct ggtagtccac cccgtaaacg ttgggaacta 780 gttgtggggt ccattccacg gattccgtga cgcagctaac gcattaagtt ccccgcctgg 840 ggagtacggc cgcaaggcta aaactcaaag gaattgacgg ggacccgcac aagcggcgga 900 gcatgcggat taattcgatg caacgcgaag aaccttacca aggcttgaca tatacgagaa 960 cgggccagaa atggtcaact ctttggacac tcgtaaacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg ttctatgttg 1080 ccagcacgta atggtgggaa ctcatgggat actgccgggg tcaactcgga ggaaggtggg 1140 gatgacgtca aatcatcatg ccccttatgt cttgggcttc acgcatgcta caatggccgg 1200 tacaaagggc tgcaataccg cgaggtggag cgaatcccaa aaagccggtc ccagttcgga 1260 ttgaggtctg caactcgacc tcatgaagtc ggagtcgcta gtaatcgcag atcagcaacg 1320 ctgcggtgaa tacgttcccg ggtcttgtac acaccgcccg tcaagtcatg aaagtcggta 1380 acacctgaag ccggtggcct aacccttgtg gagggagccg tcgaaggtgg gatcggtaat 1440 taggactaag tcgtaacaag gtaacc 1466 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme I34L, where n is a, t, g or c          and d is a, t or g <400> 4 gcgggtttcg acctgndtga attcccgctg atggac 36 <210> 5 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme I34L <400> 5 gtccatcagc gggaattcca gcaggtcgaa acccgc 36 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme L64I, where n is a, t, g or c          and d is a, t or g <400> 6 gcggtttctg cgtctndtgg tctgtctggt gcgac 35 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme L64I <400> 7 gtcgcaccag acagaccaat agacgcagaa accgc 35 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme S75P% n is a, t, g or          c, and h is a, t or c. <400> 8 cgaccgacgt tacctcthcn gacccggcgg ttg 33 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme S75P <400> 9 caaccgccgg gtccggagag gtaacgtcgg tcg 33 <210> 10 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme V105P where n is a, t, g or          c <400> 10 gtcgtcactt ctgcggtncn atctactctg cgatgcagaa atac 44 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme V105P <400> 11 gtatttctgc atcgcagagt agatcggacc gcagaagtga cgac 44 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme S108D n is a, t, g or          c <400> 12 cacttctgcg gtgttatcta cnangcgatg cagaaataca tgg 43 <210> 13 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme S108D <400> 13 ccatgtattt ctgcatcgca tcgtagataa caccgcagaa gtg 43 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme A109F where n is a, t, g or          c. <400> 14 ctgcggtgtt atctactctn tnatgcagaa atacatggac cc 42 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme A109F <400> 15 gggtccatgt atttctgcat aaaagagtag ataacaccgc ag 42 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme V155L, n is a, t, g or          c and h is a, t or c <400> 16 gttaaccgtt acgaaaccaa chcnctgaac accgcgcg 38 <210> 17 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme V155L <400> 17 cgcgcggtgt tcagcaagtt ggtttcgtaa cggttaac 38 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme H177M <400> 18 cgtccgaacc tgggtatcat gctggacacc taccacatg 39 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme H177M <400> 19 catgtggtag gtgtccagca tgatacccag gttcggacg 39 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mutated enzyme S209C, n is a, t, g or          c, and d is a, t or g <400> 20 gttacgttca catcggtgaa ndtcaccgtg gttacctgg 39 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mutated enzyme S209C <400> 21 ccaggtaacc acggtggcat tcaccgatgt gaacgtaac 39

Claims (22)

The amino acid sequence of SEQ ID NO: 1, or the amino acid at positions 34, 64, 75, 105, 108, 109, 155, 177 and 209 from the N- A &lt; / RTI &gt; amino acid sequence. The enzyme protein according to claim 1, wherein the substituted amino acid is an amino acid selected from the group consisting of Pro, Val, Leu, Iso, Thr, Ser, Tyr, Trp, Phe, Asp, Glu, Met and Cys. The enzyme protein according to claim 1, wherein the substituted amino acid is an amino acid selected from the group consisting of Pro, Leu, Iso, Phe, Asp, Met and Cys. The enzyme protein according to claim 1, wherein the enzyme protein comprises the amino acid sequence of SEQ ID NO: 1,
The amino acid 34 is Leu, Ser or Tyr,
The amino acid 64 is Ile, Ser or Tyr,
The amino acid 75 is Pro, Trp or Phe,
The amino acid at position 105 is Pro, Trp or Phe,
The amino acid 108 is Asp or Glu,
The amino acid 109 is Phe, Trp, Met, Val, Leu or Ile,
The amino acid 155 is Leu, Ile, Met, Phe or Trp,
177 amino acid is Met, Val, Leu, Ile or Phe,
The amino acid 209 is Cys, Met or Thr. The enzyme protein according to claim 1, wherein said enzyme protein comprises an amino acid sequence of SEQ ID NO: 1 in the amino acid sequence shown below:
The amino acid 34 is Ile or Leu,
The amino acid 64 is Leu or Ile,
The amino acid 75 is Ser or Pro,
The amino acid at position 105 is Val or Pro,
The amino acid at position 108 is Ser or Asp,
The amino acid 109 is Ala or Phe,
The amino acid 155 is Val or Leu,
The amino acid 177 is His or Met,
The amino acid 209 is Ser or Cys. 2. The enzyme protein according to claim 1, wherein the enzyme protein comprising the substituted amino acid sequence has an enzyme activity (%) relative to the enzyme protein having the amino acid sequence of SEQ ID NO: 1 of 102 or more. The enzyme protein according to claim 1, wherein the enzyme protein has a pI of 4.0 to 5.1. 8. A polynucleotide encoding an enzyme protein according to any one of claims 1 to 7. 9. A recombinant vector comprising the polynucleotide of claim 8. A recombinant strain comprising the recombinant vector of claim 9. A fructose-containing substrate comprising at least one selected from the group consisting of an enzyme protein according to any one of claims 1 to 7, a strain expressing the enzyme protein, a culture of the strain, and a lysate of the strain To &lt; / RTI &gt; The method according to claim 11, further comprising at least one metal ion selected from the group consisting of copper ion, manganese ion, calcium ion, magnesium ion, zinc ion, nickel ion, cobalt ion, iron ion, aluminum ion, &Lt; / RTI &gt; A method for producing a recombinant bacterium comprising reacting at least one member selected from the group consisting of an enzyme protein according to any one of claims 1 to 7, a strain expressing the enzyme protein, a culture of the strain, and a lysate of the strain with a fructose- &Lt; / RTI &gt; 14. The method according to claim 13, wherein the enzyme protein or strain is immobilized on a carrier. 14. The method of claim 13, wherein the reaction is carried out at a pH of from 4.5 to 8. 14. The method of claim 13, wherein the reaction is carried out at a temperature of 40 to &lt; RTI ID = 0.0 &gt; 85 C. &lt; / RTI &gt; 14. The method of claim 13, wherein the concentration of fructose in the fructose containing substrate is 55 to 99% (w / w).  Wherein the composition comprises psicose epimerase having a pI value in the range of 4.0 to 5.1, and produces psicose from the fructose containing substrate. 19. The composition according to claim 18, wherein the cyclic epimerase is derived from a strain of Mycobacterium sp. 19. The method according to claim 18, wherein the cyclic epimerase is provided as at least one selected from the group consisting of an enzyme protein, a strain expressing the enzyme protein, a culture of the strain, and a disruption of the strain. Composition for production. The composition according to claim 18, wherein the cyclic epimerase is provided by immobilizing a strain expressing an enzyme protein or an enzyme protein on a carrier. 22. The composition according to claim 21, wherein the carrier is alginic acid or a salt thereof.

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