WO2014168302A1

WO2014168302A1 - D-psicose epimerase, and psicose production method using same

Info

Publication number: WO2014168302A1
Application number: PCT/KR2013/008585
Authority: WO
Inventors: 김민정; 최정윤; 허진솔; 김혜정; 박종진; 이강표
Original assignee: 주식회사 삼양제넥스
Priority date: 2013-04-09
Filing date: 2013-09-25
Publication date: 2014-10-16
Also published as: KR101318422B1

Abstract

The present invention relates to psicose 3-epimerase of SEQ ID NO:1, a composition for producing psicose containing the epimerase, and the psicose production method using the epimerase.

Description

[Specifications

[Name of invention]

Cycos Epimerase. And Psycho production method using the same [Technical field]

The present invention relates to a cosmos 3-epimerase enzyme, a composition for producing a cosmos comprising the enzyme, and a method for producing a cosmos by the enzyme.

[Technique to become background of invention]

Psicose is the third carbon epimer of fructose (D-fructose), which is comparable in intensity (70%) and type of sweetness compared to fructose. However, unlike fructose, it is useful as a diet low calorie sweetener because it is hardly metabolized when absorbed into the body. In addition, Psychos has a function of inhibiting blood glucose in the body, so it can be used for foods for diabetics or food and drink for receiving diabetics. It can be used for functional foods.

For this reason, Psychos is in the limelight as a diet sweetener, but it belongs to a rare sugar that is extremely rare in nature _. On the door. Application to the food industry requires the development of technologies for the efficient mass production of psychoses.

In the conventional method of producing a psychos, most of the methods are mainly manufactured through a chemical synthesis process. However, this method has the disadvantage of having very small amounts of psychocos, expensive and by-products during molasses or glucose isomerization reactions. In order to solve such a problem, a method of producing sacose by an enzymatic method based on fructose has been actively studied. However, according to the existing enzymatic method, the enzymes producing the psychos are alkaline The pH is often optimal under conditions, but the reaction under alkaline conditions is not suitable for industrialization because it induces browning of sugars. Existing enzymes also suffered from poor reaction rates due to slow reaction rates, leading to higher manufacturing costs.

Therefore, there is a need for the development of a new D-psicose 3-epimerase enzyme suitable for industrial application of the reaction yield such as the production yield of Pycos, and the temperature, pH and reaction rate. ,

[Content of invention]

【Problem to solve】 ^

One embodiment of the present invention provides a protein having an amino acid sequence of SEQ ID NO: 1 and having an enzymatic activity for converting fructose into a psychose.

Another example provides a polynucleotide encoding the protein. Another example provides a recombinant vector comprising a polynucleotide encoding the protein.

Another example provides a recombinant strain comprising said recombinant vector.

Another iii provides a composition for producing a psychos for producing a psychos from fructose comprising the protein having the amino acid sequence of SEQ ID NO: 1 and at least one selected from the group consisting of the recombinant strain.

Another example is the composition for preparing the psychos. It provides a method of producing cycos from fructose by reacting with fructose.

[Brief Description of Drawings]

1 is a cleavage map of a recombinant vector for expressing the D—Cycos 3-epimerase protein of one embodiment. .

FIG. 2 is a graph showing the activity of the D-psicose 3-epimerase of one embodiment according to the added metal group. Figure 3 is a graph showing the activity according to the temperature of the D- psychocos 3-epimerase in one embodiment. _ᅵ ^'' Figure 4 is a graph showing the pH-dependent activity of the D- _psychocos 3-epimerase of one embodiment.

FIG. 5 is a graph showing the pH-dependent activity of D-psicose 3 ′ epimerase in one example in PIPES buffer. FIG.

FIG. 6 is a graph showing the time-dependent activity of the acecos 3-epimerase of an example at 50 ^° C. FIG.

FIG. 7 is a graph showing the efficiency of psychos production (cycose conversion) from high fructose fructose using the acecos 3-epimerase of one embodiment. FIG. 8 is a graph showing the results obtained by HPLC measuring psychocosylated productivity from high fructose using a cycos 3-epimerase of one embodiment.

FIG. 9 is a graph showing the efficiency of psychos production (cycose conversion) from high fructose using a recombinant strain containing D-psicose 3 ′ epimerase of one embodiment.

10 is derived from crosstridium sdndens ^ 入 \

The amino acid sequence of the protein (SEQ ID NO: 1) is shown. [Measures of problem]

The inventors of the present invention are non-pathogenic Crosstridium, Luminococos, Treponema, Paracocos, Citrusella. And a study of polynucleotides whose functions are not known among the genetic information of microorganisms included in cynorizobium, Agrobacterium tumefaciens or Rhodobacter spheroides-derived ketose 3-epimerase Catalytic residues are identical and the total amino acid sequence is 40-60% Homologous ketose enzymes were screened.

As a result, it was the first to identify a polynucleotide that encodes an enzyme capable of converting fructose into a psychocos among the entire genes of Crosstridium synth.

In addition, the present invention was completed by synthesizing a polynucleotide sequence optimized for efficient expression of proteins, inserting it into a recombinant vector, and expressing it to obtain an enzyme having an activity of producing ^' cos.

That is, the present invention confirms that a protein whose function is not known in the prior art has D-psychcos 3 epimerase activity and can be prepared in a high yield _: acosicose 3-epimerase of the protein. It is to provide a use as. Hereinafter, the present invention will be described in more detail.

First, in one embodiment of the present invention, a protein derived from the crosstridium synth Clostridkm ^, and having a activity of producing cycos from fructose, is provided. .

For example, the protein may have an amino acid sequence of SEQ ID NO: 1 and have activity to produce sacos from fructose.

Although the protein sequence and biochemical functional properties of the protein which encodes it are not known in a microorganism, the protein is epitaxial at the third carbon position of fructose in the process of screening for an enzyme having an activity of producing a psychosis from fructose. It was confirmed that the mercury reaction caused the activity to switch the cycle between fructose. Thus, the protein may be an enzyme protein (eg, D-psicose 3-epimerase) that has the activity of producing a psychos from fructose by epimerizing the carbon position 3 of fructose. _.

The protein may be one having an amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and converts fructose into a psychos as described above. As long as activity is maintained, some of the amino acids of SEQ ID NO: 1 may have been substituted, inserted and / or deleted. for example. The protein may include an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% homology with the amino acid sequence of SEQ ID NO: 1. .

The protein may be one having a molecular weight of 30 to 37 kDa, such as 30 to 35 kDa, measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE). The optimal silver of the protein may be 40 to 65 ^° C, specifically 50 to 65 ^° C (see Figure 3). The optimum degree of silver may be, for example, a result of progress of the reaction for 5 minutes in the presence of pH 7.0, ImM Co ²⁺ , but is not limited thereto. In addition, the optimal pH of the protein is pH 6 to 9, pH 7 to 9. pH 7 to 8.5. Or pH 7-8 (see FIGS. 4 and 5). The optimum pH may be, for example, a result of progress of the reaction for 5 minutes in the presence of 60 ^° C., ImM Co ²⁺ , but is not limited thereto.

The enzyme protein having the amino acid sequence of SEQ ID NO: 1 may have the following properties:

(a) molecular weight: 30 to 37 kDa, or 30 to 35 kDa; .

(b) optimum active temperature: 40 to 65 ^° C., or 50 to 65 ^° C .; And

(c) Activity optimum pH: pH 6 to 9, H 6.8 to 9.0, pH 7 to 9, pH 6.8 to 8.5, pH 7 to 8.5, pH 6.8 to ^8.0, or pH 7 to .8.

Another example of the present invention provides a polynucleotide encoding a protein having the amino acid sequence of Singe SEQ ID NO: 1. Another example provides a recombinant vector comprising a polynucleotide encoding the protein. Another example provides a recombinant strain transformed with a recombinant vector expressing the protein.

The polynucleotide encoding the enzyme protein, for example, with respect to the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or the base sequence It may be one having a sequence having substantial identity. Said substantial identity is such that the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is aligned with any other sequence as much as possible, and the sequence is analyzed to determine that the other sequence is the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 And more than 70%, more than 90%. Or 98% or more sequence homology.

One of ordinary skill in the art will recognize the same in a range having substantially the same homology by substituting, adding or deleting one or more bases of the nucleotide sequence of the polynucleotide using genetic recombination techniques known in the art. It will be readily appreciated that polynucleotides encoding enzymatic proteins with activity can be prepared. Such homology comparisons can be performed by calculating homology between two or more sequences as a percentage (%) using commercially available computer programs.

The polynucleotide is itself. Or in the form of a recombinant vector comprising the polynucleotide. The recombinant vector refers to a recombinant nucleic acid molecule capable of delivering an operably linked target polynucleotide, wherein the target polyline nucleotide is a promoter. And one or more transcriptional regulatory elements consisting of a transcription terminator column. Further, the polynucleotides are, for ^'example, chemical inducible element (inducible element) and the temperature. It may be operatively connected with a temperature sensitive element or the like. 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 promoter and a T7 terminator. ^'Through methods well known in the art, the recombinant vectors can be constructed as a vector for expression or for cloning vectors (Francois Baneyx, current Opinion Biotechnology 1999, 10: 411-421). The recombinant vector may be used as long as the vector has been used for gene recombination, for example. It may be selected from the group consisting of plasmid expression vector, viral expression vector (e.g., replication-defective retrovirus, adenovirus, and adeno-associated virus) and viral vectors capable of performing their equivalent functions, but are not limited thereto. no. for example. The recombinant vector may be prepared from one selected from the group consisting of ^" pET. PBR, pTrc, pLex. PUC vector 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 enzyme protein, for example, E. coli. E. coli BL21, JM109. K-12. LE392. RR1, DH5a or W3110 and the like can be exemplified, for example, BL2KDE3) strain can be used, but is not limited thereto. In addition, as the host cell, Bacillus subtilis, Bacillus strains such as Bacillus thuringiensis, Corynebacteria strains such as Corynebacterium glutamicum, Salmonella strains such as Salmonella typhimurium, Other Ceratia Strains selected from the group consisting of enterobacteria and strains such as Marsesons and various Pseudomonas species may be used.

The method of transforming the host cell with the recombinant vector can be selected and used without particular limitation for all transformation methods known in the art, such as fusion of bacterial protoplasts, electroporation, projectile bombardment, and viruses. It may be selected such as an infection using a bag.

In one example, the recombinant strain may be Escherichia co //-pETCDPE-SYG32 Accession No .: KCCM11406P).

As described above, the protein having the amino acid sequence of SEQ ID NO: 1 is an enzyme having excellent psychocosylation ability to convert fructose to psychocos Protein. therefore . The protein having the amino acid sequence of SEQ ID NO: 1 or a recombinant strain expressing the same may be usefully used for the preparation of a psychos.

therefore. Another example of the present invention, a protein having the amino acid sequence of SEQ ID NO: 1, a recombinant strain expressing the protein. Culture of said recombinant strain. And lysates of the recombinant strain _. Provided is a composition for producing a psychosis comprising at least one selected from the group. The composition for producing a psychos may be to prepare a psychos from fructose using fructose as a substrate. The culture includes an enzyme protein produced from the recombinant strain, and may be in a cell-free form including the recombinant strain or without the strain. The lysate refers to a lysate obtained by crushing the recombinant strain or a supernatant obtained by centrifuging the lysate. It includes an enzyme protein produced from the recombinant strain. In the present specification, unless otherwise stated, the recombinant strain used for the preparation of the psychic means .1 or more species selected from the group consisting of the cells of the strain, the culture of the strain and the lysate of the strain. Used.

Another example is a protein having an amino acid sequence of SEQ ID NO: 1, recombinant strain expressing the protein. Provided are a method for producing a psychos using the culture of the recombinant strain and at least one selected from the group consisting of lysates of the recombinant strain (hereinafter, 'enzyme protein and the like'). Sai ^' course production method includes the step of reacting the enzyme protein and the like with fructose. In one embodiment, the step of reacting the enzyme protein and the like with fructose may be performed by contacting the elevated protein with fructose. In one embodiment, contacting the enzyme protein with fructose may be performed, for example, by mixing the enzyme protein with fructose or contacting fructose with a carrier to which the enzyme protein or the like is immobilized. have. In another example, the step of reacting the enzyme protein with fructose comprises culturing the cells of the recombinant strain in a culture medium containing fructose. Can be performed by a step. As such, by reacting the enzyme protein and the like with fructose, the fructose can be converted into the psychos to produce the psychos from the fructose. .

In the above method for producing a psychos, the amount of enzyme protein used is 0.001 mg / ml to 1.0 mg / ml, 0.005 mg / ml to l.Omg / ml, 0.01 mg / ml to l.Omg / ml, 0.01 mg / ml to 0.1 mg / ml, or 0.05 mg / ml to aimg / ml. If the amount of the enzyme is lower than the concentration, the cycle conversion efficiency may be lowered. If the concentration is higher than the concentration, the economic range in the industry is lowered, so the range is appropriate.

In the above method for producing sacose, the concentration of fructose used as a substrate is 40 to 75% (w / v), for example, 50 to 75% (w / v), based on the total reaction water, Can be. If the concentration of fructose is lower than the above range, the economy becomes low, and if it is above the above range, fructose does not dissolve well. The concentration of fructose is preferably in the above range. The fructose can be used in the form of a solution dissolved in buffer or water (such as distilled water).

Considering the optimum conditions of the enzyme protein, the reaction is a condition of pH 6-9, pH 6.8-9.0, pH 7-9, pH 6.8-8.5, pH 7-8.5, pH 6.8-8.0, or pH 7-8 It can be performed under. Also,. The reaction may be carried out at 30 ° C subphase, for example under temperature conditions of 40 ^° C or more. If the temperature exceeds 80 ^° C browning of fructose as a substrate may occur, the reaction is 30 to 80 ^° C, 40 to 75 ^° C, 30 to 70 ^° C, 40 to 65 ^° C, or 50 to 65 ^° C It can be carried out under the conditions of. In addition, although it depends on the enzyme concentration, in general, the longer the half-unggan interval, the higher the cycle change. For example, taking into account the thermal stability of the enzyme (eg, thermal stability at 50 ^° C.), the reaction time may be at least 1 hour, such as at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours. Good to do. ^{'If the} reaction time exceeds 8 hours, As the rate of increase is small or rather declining. The reaction time should not exceed 8 hours. Therefore, the reaction time is 1 to 8 hours. 1 to 6 hours, 1 to 4 hours, 1 to 2 hours, 2 to 8 hours. Or 2 to 6 hours. The above conditions were selected as conditions under which fructose-to-sycos conversion efficiency is maximized.

In addition, in the above method for producing a psychos, when the recombinant strain is used, the cell concentration of the strain used is 0.1 mg (dcw: dry cell weight) / ml or more, for example, 0.1 to IGOmgWcw Vml, 0.1 based on the total reaction product. To 50 mg (dc) / ml 0.1 to 10 mg (dc \ v) / ml, 1 to 100 mg (dcw) / ml,

50 mg (dcw) / ml 1 to 10 mg (dcw) / ml, 2 to 100 mg (dcw) / ml _T

50 mg (dcw) / ml 2 to 10 mg (clcw) / ml. 3 to 100 mg (dcw) / ml

50 mg (dcw) / ml or from 3 to 10 mg (dc \ v) / ml _. Can be.

The enzymatic protein having the cyclase converting enzyme activity may be one having a metalloenzyme property in which activation is controlled by metal ions. By performing the reaction by the enzyme protein in the presence of metal, it is possible to enhance the yield of the production of the cycle. The metal ions that may contribute to the sacos production yield may be at least one selected from the group consisting of copper ions, manganese ions, calcium silver, magnesium silver, zinc silver, nickel silver, cobalt ions, iron silver, aluminum ions, and the like. It may be at least one selected from the group consisting of manganese ions, iron ions, and cobalt silver. When the amount of the metal silver added is less than 0.1 mM, since the effect of improving the yield of the cycle course production is insignificant, the amount of the metal ion added may be at least O.lmM. On the other hand, if the amount of the metal ion exceeds 5mM because the effect is insignificant compared to the excess amount, the amount of the metal ions can ^"be less than 5mM. For example, the amount of the metal ions to 5mM O.lmM For example, 0.1 to 2 mM, or 0.5 mM to 1.5 mM. Accordingly, the composition for preparing the psychos may further include the above-described metal ions, and the type and content of the metal ions are as described above. In addition, the method of producing a psychos may further include the step of adding metal silver and the type and content of metal ions are as described above. In one embodiment, the metal ion may be added to the fructose as a substrate, or may be added to a mixture of the enzyme protein and the fructose. In another embodiment, the metal ion is added to the carrier to which the enzyme protein or the like is immobilized (before fructose addition). The enzyme protein or the like may be added to the mixture of the immobilized carrier and fructose (after fructose addition), or may be added in the form of fructose and the combination at the time of fructose addition, or respectively. When using a recombinant strain, the metal ion may be added to the culture or cultured in a culture medium to which the metal ion is added.

The carrier may create an environment in which the activity of the immobilized enzyme protein can be maintained for a long time, and may be any known carrier that can be used for enzyme immobilization. For example, sodium alginate may be used as the carrier. Sodium alginate is a natural colloidal polysaccharide that is abundant in algae cell walls. It contains mannuronic acid (β- mannuronic acid) and gluronic acid (α—b-gluronic ackl), which are randomly selected. It is formed by forming a -1,4 bond, it may be advantageous to stably fix the strain or enzyme to show a good psychocos yield. In one embodiment, a sodium alginate solution (e.g. aqueous sodium alginate solution) at a concentration of 1.5 to 4.0% (w / v), such as sodium alginate at a concentration of about 2.5% The solution can be used for immobilization of the strain.

In one embodiment, the method for producing the psychocos, the Pseudomonas 3 'epimerase protein having an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence of at least 70% homology with the amino acid sequence of SEQ ID NO: 1 It may include the step of reacting with fructose. Said psychos production method Adding and / or purifying the Pseudo-3 epimerase enzyme protein having an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence of at least 70% homology with the amino acid sequence of SEQ ID NO: 1 before the reaction step It can be included as. ^'

In another embodiment, the method of producing the psycos may include the step of reacting with a fructose a recombinant strain expressing a protein having an amino acid sequence of SEQ ID NO: 1 or an enzyme protein isolated from the system combination strain with fructose. . The psicose production method may include the further step of culturing, and recovering the recombinant strain expressing a protein of amino acid sequence of SEQ ID NO: 1 in the previous step the ^banum.

The culturing step of the recombinant strain may be performed under medium and culture conditions which are easily selected by those skilled in the art according to the characteristics of the strain (host cell) used. For example, the culture may be continuous, semi-continuous, or batch culture, but is not limited to these methods. The medium may be any host cell, including E. coli, and any cell that may contain or contain cell contents. Culture media, solutions _, solids, semisolids or rigid supports;

It may be selected from the group consisting of 2YT medium, LB medium, SOB medium, TB medium and the like.

Such recombinant strains and cultures can generally be obtained using conditions suitable for culturing host cells (eg, E. coli). For example, the recombinant strain may be cultured by shaking culture under conditions of 35 ° C to 37 ^° C and 150 to 250 rpm. In addition, an inducer commonly used in the art may be added to induce overexpression of the enzyme protein. For example, when the inducing element is a lac operon or trc promoter, it may be Lactose or IPTG (isopropyl β-Dl-thiogalactopiopyranoside), and the induction period may be an appropriate time of culture by a person skilled in the art. Can be done on. The recombinant strain may be obtained by performing centrifugation and / or filtration of the obtained culture, and further, fractionating the supernatant or the supernatant obtained by homogenizing and centrifuging the obtained strain. Purification by chromatography can be used to obtain enzyme proteins. For example, a suspension of the recovered strains in 50mM phosphate buffer and then homogenized and then centrifuged, only the supernatant to recover the 20mM.250mM already enzyme protein at a concentration of dachul after absorption in the ^'-N.TA nickel column (Qiagen) Can be.

The step of reacting with fructose can be carried out under optimal activation conditions of the enzyme protein. Such as pH 6-9, H 6.8-9.0, pH 7-9, pH 6.8-8.5, pH 7-8.5, pH 6.8-8.0, or pH 7-8, and / or 40 ^° C.-65 ^° C. or 50 ^° It may be made under a temperature condition of C to 65 ^° C. The fructose may be used at a concentration of 40 to 75% (w / v), such as 50 to 75% (w / v), based on the total counterungmul. By using fructose in the above range, it is possible to produce psychocos more economically and efficiently.

In the case of using a recombinant strain, preferably, the recovered cells may be washed two or more times with, for example, 0.85% (w / v) NaCl or the like before reacting with fructose.

Also preferably, the step of reacting with fructose may be performed by further adding ions of at least one metal selected from the group consisting of manganese and cobalt. The metal ions may be added in the range of O.lmM to 5 mM, such as 0.1 to 2 mM, or 0.5 mM to 1.5 mM. If it is less than the above range, the effect of increasing the activity cannot be sufficiently obtained, and if it exceeds the above range, the effect of increasing the activity obtained compared to the amount used is not preferable.

Psychoses obtained from fructose by the method of the present invention can be purified by conventional methods, and such crystals are well known 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.

[Effect of the Invention]-The novel cosmos gepymerase according to the present invention possesses the activity of producing a psychose by epimerizing the third carbon position of fructose. As an enzyme, it is useful for the industrial use for the production of psychos, because it is possible to mass-produce cyclose from fructose in high yield under conditions of excellent thermal stability, long half-life and industrially applicable conditions. The D-psicose 3-epimerase of the present invention and the method of producing a psychos using the same are expected to be usefully used as well as in the functional sugar industry, as well as health food materials, pharmaceuticals and cosmetic materials using the same. [Specific contents to carry out invention]

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and various modifications or changes are possible to those skilled in the art within the spirit and scope of the present invention. Accordingly, the appended claims should be construed broadly according to the spirit and scope of the present invention.

Example 1 Preparation of Recombinant Strain Producing D-Pycos 3-Epimerase Enzyme Sequence Number Derived from Crosstridium Syndance sc ^ ci? / 7s)

A polynucleotide encoding the amino acid sequence of 1, wherein a polynucleotide (SEQ ID NO: 2) having a modification to the nucleotide sequence of the original coding polynucleotide (SEQ ID NO: 3) is optimized for E. coli to be used as an expression strain. Korea) to synthesize.

The synthesized polynucleotide was inserted into the same restriction enzyme site of pET21a (Novagen), which is an expression vector using restriction enzymes Ndel and XhoKNEB). Recombinant vector pET21a / Phycose epimerase (pET-CDPE) was prepared. The recombinant vector pET-CDPE prepared above is shown in FIG. 1.

E. coli BL21 (DE3) (invitrogen) was transformed with the recombinant vector pET-CDPE prepared above by a heat shock method (Sambrook and Russell: Molecular Cloning.) to prepare a recombinant strain.

After inoculating the prepared recombinant strain in 5ml LB-ampicilline medium (Difco) and shaking culture at 37 ^° C, 200rpm until the absorbance (OD) at 600nm reaches 1.5, it was again in 500ml LB-ampicilline medium After inoculation, species cultured in a shaker at 37 ^° C. When the absorbance was 0.5 at 600 nm of this culture, IPM (isopropyl-l-thio-β-D-galactopyranoside) of ImM was added to induce overexpression of the target enzyme. From the time of induction of overexpression, the culture conditions were switched to 16 ^* 0 and 150rpm and maintained for 16 hours.

The overexpression-induced culture was centrifuged in a centrifuge 4000rpm for 20 minutes to recover only the cells. The recovered cells were washed twice with 0.85% (w / v) NaCl and used for the following enzyme purification.

The prepared recombinant strain was named Escherichia co //-pETCDPE_SYG321 and deposited on March 29, 2013 with the Korea Microorganism Conservation Center and received accession number KCCM11406P. Example 2. Purification and Characterization of D-Pycos 3-Epimerase

2-1. Purification of D-Pycos 3-Epimerase

The cells recovered in Example 1 were suspended in lysis buffer (50 mM Tris_HCl 300 mM NaCl pH8.0, 10 mM imidazol) and then disrupted at 4 ^° C. for 20 minutes using an ultrasonic processor (ColpParmer). The lysate was centrifuged at 13,000 rpm for 20 minutes to collect only the supernatant, and then placed in a Ni—NTA column (Ni— NTA Superflow. Qiagen) previously equilibrated with lysis buffer. After application, the buffer solution containing 20 mM imidazol and 250 mM imidazol in 50 mM Tris_HCl 300 mM NaCl pH8.0 was sequentially poured. The 50 mM Tris_HCl 300 mM NaCl pH8.0, 250 mM imidazol by the process of blowing the target protein was isolated and purified.

The isolated purified protein of interest (50mM buffer for enzyme activity measurement)

PIPES pH7.5) was used for the experiment. In addition, the isolated (partially) purified protein of Pseudomonas epimerase was found to have a monomer size of about 32.8 kDa through SDS-PAGE. -2-2. Metal Grain Constituent Analysis of D-Pycos 3-Epimerase

Tagatose or Pycos 3-epimerase known in the art is known to have metal ion requirements. It was examined whether metal ions also affect the D-psicose epimerase obtained in Example 2-1.

.The metal silver is CuCl ₂ , MnCl ₂ , CaCl ₂ in the protein (enzyme) purified in Example 2-1. Enzyme activity was measured by treating 1 mM of ZnSO ₄ , MgSO ₄ , NiSO ₄ , or CoCl ₂ , respectively. The enzyme activity was measured by measuring 50 mM fructose and 0.3 unit / ml of enzyme in the presence of metal ions at 60 ^° C. for 5 minutes in 50 mM PIPES buffer solution (pH7,0). After the reaction was heated for 5 minutes at 100 ^° C to stop the enzyme activity. The enzyme activity was defined as lunit by the amount of 50 mM fructose and 50 mM PIPES buffer pH7.0 containing ImM Co ²⁺ , reacted with the enzyme at 60 ^° C. to produce an imol (micromole) cyclase per minute as lunit. . As a control group (Non), a metal ion was not treated.

The enzymatic activity was calculated by dividing the amount of psychos produced (mM) by the amount of enzyme used and reaction time, and the amount of psychos was analyzed by HPLC. The HPLC analysis was performed at 8CTC using a 87C (BIO-AD) column, flowing 100% (v / v) of water at a flow rate of 0.6 ml / min into the mobile phase, Refractive Psychoproductivity was analyzed by index detector (Agilent 1260 TID). ᅳ

Enzyme activity in the case of treating each metal silver measured as described above is shown in FIG. 2 compared to the enzyme activity in the control group. As shown in FIG. 2, the enzyme of Example 2-1 was found to increase in activity by the addition of manganese ions, iron silver and cobalt ions, indicating that the metal ions were required.

2-3.Analysis of Psychoepimerase by Activity

In order to confirm the activity according to the pH and temperature change of the enzyme purified in Example 2-1, the enzyme and fructose substrate were reacted at various pH and silver and the enzyme activity was confirmed. At this time, for activity measurement, refer to the method described in Example 2.2. Pi—I 4 to 9 and temperature using 50 mM fructose and enzyme 0.3 unit / ml

The reaction took place for 5 minutes in the range of 30-80 ^° C., after which the reaction was stopped by heating in lOCrc for 5 minutes.

First, the pH was changed to 7 and the temperature was changed in the range of 30 to 80 ^° C., and the result of measuring enzyme activity in the same manner as in Example 2-2 is shown in FIG. 3. The relative activity of the Y axis in FIG. 3 means a relative value when the best enzyme activity is 100.

As confirmed in Figure 3, the enzyme showed a high activity in the range of 40 to 65 ° C, specifically 50 to 65 ^° C, it can be seen that the maximum activity at 60 ° C. in particular.

Further, with reference to the method described in Example 2.2, to 60 ^° C., Standing, 50mM sodium acetate pH 4-6, to determine their activity according to the pH _change. Buffer solution of 50 mM sodium citrate pH 5-7, 50 mM piperazine-N, N'-bis (2-ethanesulfonic acid) pH 7.50 mM Tris-HCl pH 7-9, and 50 mM glycine NaOH pH 9-10 The enzyme activity was measured using each of the results. 4 is shown. . ^, As confirmed in FIG. 4. The enzyme activity was particularly high in the pH 7.0-9.0 range, pH7.0. In addition, among the complete solution corresponding to PH 7.0

Enzyme activity was shown to be better in PIPES buffer.

It was shown in Figure 5 by measuring the activity for each pH for the PIPES buffer solution that showed excellent enzyme activity in the test.

As shown in FIG. 5, high activity was exhibited at pH 7.0 or higher, such as pH 7.5. 2-4. Substrate Specificity Analysis of Cycos Epimerase

Substrate at 50 mM concentration. That is, fructose, Psicose, tagatose, mannose. Glucose or xylose was used as a substrate and reacted for 5 minutes under pH 7.5 and 60 ^° C. in 50 mM PIPES buffer solution containing 1 mM CoCl ₂ and 0.3 unit / ml of the enzyme purified in Example _2-1. The degree of activity for each substrate was measured. Enzyme activity was stopped by heating at 100 ^° C for 5 minutes.

The results obtained above are shown in Table 1 below (enzymatic activity of Banung, which is based on Psychos as a substrate, is expressed as a relative value of 100).

Table 1

As shown in Table 1, the enzyme purified in Example 2-1 showed only specific activity among cyclose and fructose among various substrates, and in particular, the substrate specificity of psychose was about 32% higher than that of fructose. Appeared. Known ketose 3-epimerase enzymes are known for In addition, it is confirmed that the enzyme of the present invention has a characteristic that only reacts specifically to psychos and fructose, and thus, the enzyme and the substrate specificity are different from those known to the tagatose and the sorbose. 2-5. Thermal Stability Analysis of Psychos Epimerase

In order to confirm the stability of the Pseudomonas 3-epimerase according to the degree of silver change, the enzyme purified in Example 2-1 above was heated for 5 hours at 5C C, and then 50 mM fructose was used as a substrate. The enzyme activity was measured by reacting 1 mM CoCl ₂ with a 50 mM PIPES complete solution containing 0.3 unit / ml of enzyme under pH 7.5 and 50 ^° C. for 5 minutes. Enzyme activity was stopped by heating at 100 ^° C for 5 minutes. The results obtained according to the heat treatment time are shown in FIG. 6. As shown in Figure 6, the enzyme purified in Example 2-1 did not significantly decrease the activity until 9 hours, the half-life is calculated to about 20 hours _. It was. It is confirmed that the previously reported psychos 3-epimerase enzymes have a half-life of approximately one hour at 50 ^° C., whereas the enzyme of the present invention has excellent thermal stability in the same silver.

2-6.Kinetic Parameter Analysis of Cycos Epimerase

Kinetic parameter analysis was performed to examine the enzyme reaction kinetics of the crosstridium acid ^ CJostrkn ^' um sc // 7t / e; 7s) purified in Example 2-1 and the cosmos epimerase (Zhu Y et.a /. (2012), Kim HJ. et.a / (2006), Mu W. et. 3 / (2011))

Reacting under the conditions of 50 mM PIPES buffer, pH 7.5 and 60 ° C. to which 0.3 unit / ml of enzyme of Example 2-1 and 1 mM C0CI ₂ were added while varying the concentration of fructose as a substrate in the range of 5 to 250 mM. Enzyme activity was measured. Enzyme activity was stopped by heating at KX C for 5 minutes. When the enzyme reaction rate was calculated as a monomer, the Km value of fructose was 56 mM and kcat was 10128.6 mir ^1. The enzyme reaction rate was calculated based on michaelis—mentenequation.

In other words, the catalytic efficiency of fructose of the Pseudomonas 3-epimerase of Example 2-1 was 286.28, which is the highest reported catalytic efficiency among the previously reported Sarcos epimerase. It was found to be about 3.3 times higher than the catalytic efficiency ¾ 85 of the psychic 3 ᅳ epimerase derived from mechasion.

Therefore, the enzyme of the present invention has an advantage in terms of the production rate of psychos because the reaction rate is very fast ^at temperature H (about 50-60 ^° C), which is easy to industrially be expected to have an excellent effect on the industrialization of psychos. Example 3 Psychos Production by D-Pycos 3-Epimerase

3-1.Cycose Production by Bioconversion from High Fructose Fructose

In order to produce a high concentration of cosmos, reaction was performed under the optimization conditions selected in Example 2-3 using the D- cyclose 3-eparmerase purified in Example 2-1. Specifically, using the D- cyclase 3-epimerase enzyme purified in Example 2-1 at a concentration of 0.005 to 0.1 mg / ml at a high concentration under the conditions of 50 ^° C and 50 mM PIPES H 7.5, and cobalt 1 mM It was reacted with (500 g / L) fructose.

In the same manner as in Example 2-2, the yield of the produced reaction was measured to determine the conversion rate of fructose to psychocos. As the fructose used was high, HPLC was performed by diluting the sample sampled at each time by 25 times, and the yield was calculated by substituting the analyzed area (AREA) value for each standard value. Calculated as 50% * 100 per amount / fructose. After ampouling, the calculations show the analysis of fructose and The total amount is 50%.

The obtained result is shown in FIG. As shown in Figure 7, higher enzyme concentrations were shown to increase the Psychoconversion. For example, the final yield of a psychos at an enzyme concentration of 0.1 mg / ml is 152 g / L, with a psychocos conversion of about 30.2%, i.e. a high concentration of fructose at least 30% using an enzyme of the present invention. Produce a course.

On the other hand, by measuring the psicose production by biological conversion of fructose at high concentrations by HPLC as described above were shown in Fig. ^"8 the results. The Y axis of FIG. 8 represents a refractive index. The HPLC analysis was performed using a 87C (BIO-RAD) column at 80 ^° C, flowing 100% (v / v) of water into the mobile phase at a flow rate of 0.6 ml / rain, using a Refractive Index Detector (Agilent 1260 TID). Psychos were detected and analyzed for psychos productivity.

3-2. Psycho Production by Recombinant Strain Reaction with Enzyme

Using the cells of the recombinant strain recovered in Example 1 was tested for the production of the cycle. The reaction was carried out in the same manner as in 3-1, except that the cells of the recombinant strain recovered in Example 1 were used instead of the enzyme of Example 2-1. More ^{specifically, 50 ° C, 50 mM PIPES} pH 7.5 Example recombinant strain of 1 10.9, 5.4 or 2.7 mg (cell dry weight) under conditions of a / mK banung water by volume) and 500 g / L to when each banung ^"Fructose On the basis of the same method as in Example 3-1 was measured for the yield of the psychos and the conversion of the psychos.

The obtained result is shown in FIG. As shown in FIG. 9, the reaction was almost complete after about 1 hour at a condition of fructose concentration of 500 g / L and 5.4 mg (cell dry weight) / ml (reaction volume), wherein 161.9 g / L With the production of Pycos, the conversion rate was about 30.8%. Only enzyme purified from the above result In addition, it can be confirmed that the recombinant strain also has excellent psychocosylation ability.

Claims

Patent Claim

. [Claim 1]

D-psicose 3-epimerase protein having an amino acid sequence of SEQ ID NO: 1.

【Claim ² 】

The D-cyclic 3-epimerase protein of claim 1 having the following properties:

(a) molecular weight: 30 to 37 kDa;

(b) optimum active temperature: 40 to 65 ^° C .; And

(c) optimum active pH: pH 6-9.

[Claim 3]

A polynucleotide encoding the D-psicose 3-epimerase protein of claim 1.

【Claim 4】 _.

The polynucleotide of claim 3 having the nucleotide sequence of SEQ ID NO: 2. 5.

[Claim 5]

A recombinant vector comprising a polynucleotide encoding the D-psicose 3-epimerase protein of claim 1. .

[Claim 6]

The recombinant vector of claim 5, wherein the recombinant vector has a cleavage map of FIG. 1. ^'

[Claim 7]

A recombinant strain transformed with the recombinant vector of claim 5.

[Claim 8]

According to claim 7, wherein the recombinant strain is E. coli transformed, recombinant strain.

[Claim 9]

The recombinant strain according to claim 8, which is Escherichia co // — pETCDPE_SYG321 (Accession Number: KCCM11406P).

[Claim 10]

Claim 1 or claim 2, wherein the recombinant strain of any one of claims 7 to 9, comprising one or more selected from the group consisting of the culture of the recombinant strain and the lysate of the recombinant strain, for producing a psychos Composition.

[Claim 11]

The method of claim 10, further comprising manganese ions, iron ions, cobalt ions, or mixtures thereof. Psychos composition.

[Claim 12]

A method for producing a cycos from fructose comprising the step of reacting the composition for preparing sacocos of claim 10 with fructose;

[Claim 13]

The method of claim 12, wherein the reaction is performed ^at 40 ^' to 65 ^° C.

[Claim 14]

The method of claim 12, wherein the reaction is performed at pH 6.0 to 9.0.

[Claim 15]

13. The method of claim 12, wherein said fructose concentration is between 40 and 75% (w / v).