KR101971931B1 - Method for Producing D-Chiro-Inositol From Myo-Inositol Using In Vitro Enzyme Reaction - Google Patents

Abstract

The present invention relates to a method for producing D-chiro-inositol by reacting a substrate, myo-inositol, with an enzyme reaction buffer comprising inositol dehydrogenase and inosose isomerase, or a fusion protein of these enzymes . According to the method of the present invention, D-chiro-inositol is produced in an extracellular enzyme reaction buffer, so that D-chiro-inositol can be produced at a low cost and a high yield as compared with a fermentation method using recombinant cells. In addition, D-chiro-inositol can be isolated in high purity from a reaction buffer in which the mixture of myo-inositol and D-chiro-inositol is mixed through a simple process such as addition of an organic solvent, separation of supernatant and drying. Further, according to the present invention, the myo-inositol isolated from D-cairo-inositol has an advantage that it can be reused as a substrate.

Description

Inositol from Myo-Inositol Using In-Vitro Enzyme Reaction Method for Producing D-Chiro-Inositol from Myo-

The present invention relates to a method for producing D-chiro-inositol from myo-inositol using an enzyme. More particularly, the present invention relates to a method for producing a D-chylo-inositol derivative by reacting a substrate, myo-inositol, with two enzymes inositol dehydrogenase and inosose isomerase, Inositol. ≪ / RTI >

D-chiro-inositol (cis-1,2,4-trans-3,5,6-cyclohexol) is a myo-inositol, cis- trans-4,6-cyclohexanehexol) in which the hydroxyl group 3 is epimerized (Fig. 1). D-cairo-inositol is a major component of inositol phosphoglycans (IPG) and has been reported to be an important mediator of insulin signaling and is known to be effective in the treatment of type 2 diabetes. D-cairo-inositol is found mainly in eukaryotes and is biosynthesized by epimerization of myo-inositol. D-chiro-inositol is mainly produced by hydrochloric acid hydrolysis of D-pinitol or kasugamycin (US Pat No. 5827896, US Pat No. 5091596, US Pat No. 5463142, US Pat No. 5714643). However, the raw materials, such as pinitol and casgalmycin, are expensive and organic synthesis methods (US Pat No. 5406005, WO 96/25381) are known, but the separation of by-products is not easy and economic efficiency is low. D-cairo-inositol is produced from myo-inositol by fermentation methods using transformed cells expressing myo-inositol dehydrogenase and inosose isomerase It is known to be able to. However, the fermentation method using the transformed cells has a low production yield, and it has been known that there is a problem in refining D-cairo-inositol from a fermentation broth.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

As a result of efforts to solve the above problems and to develop a method for efficiently producing D-chiro-inositol from myo-inositol, the inventors of the present invention have found that when a substrate of myo-inositol is replaced with inositol dehydrogenase and inoso- The present inventors completed the present invention by confirming that D-chiro-inositol can be successfully produced by reacting two enzymes of malase, or fusion protein type thereof, extracellularly.

Accordingly, an object of the present invention is to provide a method for producing D-chiro-inositol from myo-inositol using an enzyme reaction method.

According to one aspect of the present invention, the present invention provides a method for producing D-chiro-inositol from myo-inositol using an enzymatic reaction method comprising the steps of: (a) (i) reacting inositol dihydro (Ii) a Nicotinamide Adenine Dinucleotide (NAD) as a coenzyme, and (iii) a fusion protein comprising a myo-inositol as a substrate, Preparing an enzyme reaction buffer; And (b) reacting the enzyme reaction buffer to produce D-chiro-inositol.

Hereinafter, the present invention will be described in detail with reference to the respective steps.

Step (a): (i) inositol dehydrogenase and inosose Isomerase , or a fusion protein of these two enzymes, (ii) NAD ( Nicotinamide Adenine Dinucleotide ), and (iii) preparing an enzyme reaction buffer solution containing the myo-inositol as a substrate

The term " inositol dehydrogenase " or " myo-inositol dehydrogenase " is used herein to refer to the reaction of the myo-inositol with NAD ⁺ Inositol (2-keto-myo-inositol), or an enzyme that catalyzes a reverse reaction thereof. In the present specification, the name of a gene coding for "inositol dehydrogenase" is described as "iolG".

[Reaction Scheme 1]

Myo-inositol + NAD ^+? 2-keto-myo-inositol + NADH + H ⁺

Inositol dehydrogenases are known as Aerobacter aerogenes [Berman T, Magasanik B (1966). J. Biol. Chem. 241 (4): 800-806; LARNER J, JACKSON WT, GRAVES DJ, STAMER JR (1956). Arch. Biochem. Biophys. 60 (2): 352-363) and yeast Cryptococcus melibiosum [Vidal-Leiria M, van Uden N (1973). Biochim. Biophys. Acta. 293 (2): 295-303]. The inositol dehydrogenase which can be used in the present invention is preferably selected from the group consisting of Agrobacterium tumefaciens, Bacillus subtilis, Corynebacterium glutamicum, It is separated from Pantoea ananatis. More preferably, the inositol dehydrogenase used in the present invention is an inositol dehydrogenase derived from Corynebacterium glutamicum. Even more preferably, the inositol dehydrogenase of the present invention has the amino acid sequence of SEQ ID NO: 1.

As used herein, the term " inosose isomerase " refers to 2-keto-myo-inositol as 1-keto-D-chiro -inositol) or an enzyme that catalyzes the reverse reaction of the isomerization reaction. In the present specification, the name of the gene coding for "inosource isomerase" is described as "iolI".

[Reaction Scheme 2]

2-keto-myo-inositol < RTI ID = 0.0 > 1-keto-D-

The inosource isomerase that can be used in the present invention is preferably selected from the group consisting of Agrobacterium tumefaciens, Bacillus subtilis, Corynebacterium glutamicum, It is separated from Pantoea ananatis. More preferably, the inosome isomerase of the present invention is an inosano isomerase derived from panto-aananatis. Even more preferably, the inosource isomerase of the present invention has the amino acid sequence of SEQ ID NO: 2.

The 1-keto-D-chiro-inositol produced by Reaction Scheme 2 produces D-chiro-inositol, which is the final product in the present invention, according to the following Reaction Scheme 3. The reaction of Scheme 3 below is catalyzed by the enzyme " inositol dehydrogenase " described above.

[Reaction Scheme 3]

1-keto-D-chiro-inositol + NADH + H ^+? D-chiro-inositol + NAD ⁺

In the present invention, the above-described two enzymes, that is, enzymes in which inositol dehydrogenase and inosome isomerase are fused can be used. Enzyme reaction efficiency and thermal stability can be increased when fused form of enzyme is used.

In the present invention, in the fusion enzyme, inositol dehydrogenase and inosose isomerase can be linked through a peptide linker.

As used herein, the term " peptide linker " refers to a relatively short peptide used to link different proteins. The contents of peptide linkers are described in detail in the article " Toon H. Evers et al., 2006, J. Christopher Anderson et al., 2010 ", the disclosures of which are incorporated herein by reference.

In general, a variety of linkers are known between domains and domains, or enzymes and enzymes, and can be divided into flexible linkers and rigid linkers. Flexible linkers consist of relatively short peptides, rich in amino acids, such as glycine and alanine, which are freely rotatable. When expressed, the two domains or enzymes rotate freely to lessen the affects of their respective protein conformation do. On the other hand, as a solid linker, it is made up of an amino acid that forms alpha-helix on a three-dimensional structure, and when it is actually expressed, it has an alpha-helix structure.

According to one embodiment of the present invention, the linker of the present invention can use a flexible linker and a rigid linker, preferably a flexible linker.

According to one embodiment of the present invention, the peptide linker of the present invention is composed of 2 to 24 amino acids, more preferably 4 to 12 amino acids.

According to one specific embodiment of the present invention, in the present invention, "GGSGGSGGGSG" peptide is used as the long linker, "GGSGGGSG" peptide is used as the middle linker, and "GGSG" peptide is used as the short linker.

And the myo-inositol dehydrogenase and inososomal isomerase are bound to the N-terminal and C-terminal of the peptide linker, respectively. For example, inositol dehydrogenase may be coupled to the N-terminus of the linker and the inosomal isomerase may be conjugated to the C-terminus. Alternatively, the inosome isomerase may be coupled to the N-terminus of the linker, The inositol dehydrogenase can be bound to the C-terminus.

According to one embodiment of the present invention, the fusion enzyme is a form in which the C-terminus of the inositol dehydrogenase is linked to the N-terminus of the inosomal isomerase.

The initial reaction rate increases as the concentration of the inositol dehydrogenase and the inosome isomerase or the fused form of the enzyme increases in the enzyme reaction buffer of the present invention. Thus, by increasing the concentration of the enzyme in the enzyme reaction buffer, the rate of production of D-chiro-inositol can be increased. The concentration of the inositol dehydrogenase, inosome isomerase, or the fusion enzyme thereof in the enzyme reaction buffer is not particularly limited, and a suitable concentration can be selected by those skilled in the art in consideration of the production efficiency and the manufacturing cost. According to one embodiment of the present invention, the concentration of the inositol dehydrogenase, inosome isomerase, or the fusion enzyme thereof may be selected within the range of 100-500 占 퐂 / ml.

The concentration of NAD in the enzyme reaction buffer of the present invention is not particularly limited, but can be determined by selecting an appropriate concentration within the range of 0.1 to 2.0 mM. According to one embodiment of the present invention, the initial reaction rate increases with an increase in NAD concentration up to 0.5 mM, but the reaction rate does not increase with increasing NAD concentration at a concentration above 0.5 mM.

The concentration of the myo-inositol in the enzyme reaction buffer of the present invention is not particularly limited, but it is preferable to select an appropriate value within the range of 5 - 20% (w / v).

The enzyme reaction buffer of the present invention may further include Tris-HCl, MgSO _4, and MnSO _4.

Step (b): reacting the enzyme reaction buffer to produce D-chiro-inositol

In the present invention, the reaction temperature of the enzyme reaction is not particularly limited, but a suitable temperature may be selected within the range of 25 - 65 캜. More preferably from 30 to 60 占 폚, even more preferably from 35 to 55 占 폚, and most preferably from 37 to 55 占 폚.

The method of the present invention further comprises the step of purifying D-chiro-inositol with high purity by adding an organic solvent to the enzyme reaction buffer after completion of the enzyme reaction.

According to a preferred embodiment of the present invention, the present invention further comprises, after the step (b), a further step of: (c) adding an organic solvent to the enzyme reaction buffer after the reaction has been completed To produce an inositol precipitate; (d) separating the supernatant from the resulting inositol precipitate; And (e) drying the separated supernatant to obtain an inositol powder.

Step (c): an organic solvent is added to the enzyme reaction buffer after the reaction is completed to produce an inositol precipitate

Since the final equilibrium concentration of the product D-chiro-inositol in the enzyme reaction is about 14%, the myo-inositol and the D-chiro-inositol exist in a ratio of about 86:14 in the enzyme reaction buffer. The difference in solubility of myo-inositol and D-chiro-inositol in an aqueous solution with water as a solvent is shown in Fig. The method of the present invention is based on the difference in solubility between the myo-inositol and the D-chiro-inositol in the reaction buffer containing the reaction product of the myo-inositol and the product, D-chiro-inositol, Highly purified. To this end, in the method of the present invention, the organic solvent is first added to the enzyme reaction buffer which has been reacted. The addition of the organic solvent induces precipitation of myo-inositol and D-chiro-inositol. On the other hand, the addition of the organic solvent increases the sedimentation rate of both myo-inositol and D-chiro-inositol, but significantly increases the sedimentation rate of myo-inositol as compared to D-chiro-inositol (see FIG. 18). Therefore, the concentration of D-chiro-inositol in the solution can be increased by adding an organic solvent to the enzyme reaction buffer which has been reacted to precipitate the larger amount of the myo-inositol than the D-chiro-inositol.

The organic solvent usable in the present invention is not particularly limited as long as it has properties capable of inducing the precipitation of myo-inositol to a greater extent than the precipitation of D-cairo-inositol. For example, alcohols, acetone, ethyl acetate, butyl acetate, 1, 3-butylene glycol or ethers are preferred. The alcohol is preferably an alcohol having from 2 to 6 carbon atoms. The organic solvent may more preferably be ethanol, isopropanol or acetone.

The amount of the organic solvent to be added to the enzyme reaction buffer is not particularly limited and may be appropriately selected within the range of 0.1 to 9 times (v / v) of the enzyme reaction buffer. Preferably, the organic solvent is used in an amount of 0.1 to 6 times (v / v), more preferably 0.1 to 4 times (v / v), more preferably 0.1 to 2 times (v / v), and most preferably 0.1 - 1.5 times (v / v).

In the present invention, the inositol precipitate induced by the addition of the organic solvent contains both of the myo-inositol and the D-chiro-inositol, but since the precipitation rate of the myo-inositol is higher, have.

Step (d): separating the supernatant from the resulting inositol precipitate

The supernatant is separated from the inositol precipitate produced through step (c). The inositol precipitate is a precipitate of myo-inositol and D-chiro-inositol, but the amount of myo-inositol is larger than that of D-chiro-inositol, since the myo-inositol exhibits a larger sedimentation rate. Due to the large precipitation of myo-inositol, the concentration of myo-inositol is lowered in the supernatant, and the concentration (purity) of D-chiro-inositol is relatively increased. Separation of the supernatant from the inositol precipitate is carried out by filtration or centrifugation, preferably by filtration.

Step (e): The separated supernatant is dried to obtain an inositol powder

The supernatant separated in the step (d) is dried to obtain an inositol powder. The drying of the supernatant can be carried out while heating the supernatant or in a vacuum. The inositol dried product obtained by drying the supernatant was mixed with myo-inositol and D-chiro-inositol, but the purity of D-cairo-inositol was increased as compared with the enzyme reaction buffer in which the reaction of step (b) have.

According to a preferred embodiment of the present invention, the present invention further provides, after step (e), the following step: (f) the inositol powder obtained in step (e) To prepare an aqueous solution; (g) adding an organic solvent to the aqueous solution to produce an inositol precipitate; (h) separating the supernatant from the resulting inositol precipitate; And (i) drying the separated supernatant to obtain an inositol powder.

Step (f): dissolving the inositol powder obtained in the step (e) in water to prepare an aqueous solution

The powder of inositol obtained in the above step (e) is dissolved in water to prepare an aqueous solution in which inositol is dissolved. The water is preferably distilled water. Preferably, the inositol powder is dissolved in water at a low concentration of not more than 20% (w / v), more preferably not more than 18% (w / v) ) Or less.

Step (g): adding an organic solvent to the aqueous solution to produce an inositol precipitate

An organic solvent is added to the inositol-dissolved aqueous solution obtained in the step (f) to produce a precipitate of inositol. The organic solvent is the same as that described in the step (c). The organic solvent is not particularly limited as long as it has properties capable of inducing the precipitation of the myo-inositol to a greater extent than that of D-cairo-inositol, and examples thereof include alcohols, acetone, ethyl acetate, butyl acetate, Ranging glycol or ether is preferred. The alcohol is preferably an alcohol having 2 to 6 carbon atoms, more preferably ethanol, isopropanol or acetone. The amount of the organic solvent added to the aqueous solution may be suitably selected in the range of 0.1 to 10 times (v / v) relative to the aqueous solution. Preferably, the organic solvent is added in an amount of 1.5 to 9 times (v / v) to the aqueous solution, most preferably in an amount of 9 times (v / v). The inositol precipitate is a precipitate of myo-inositol and D-chiro-inositol, but the amount of myo-inositol is larger than that of D-chiro-inositol, since the myo-inositol exhibits a larger sedimentation rate.

Step (h): separating the supernatant from the resulting inositol precipitate

The supernatant is separated from the inositol precipitate produced through step (g). Separation of the supernatant from the inositol precipitate is carried out by filtration or centrifugation, preferably by filtration. Since the inositol precipitate contains a large amount of myo-inositol, the purity of D-cairo-inositol is increased in the supernatant.

Step (i): The separated supernatant is dried to obtain an inositol powder

The supernatant separated in step (h) is dried to obtain an inositol powder. The drying of the supernatant may be carried out while heating the supernatant, or in a vacuum. The dried inositol obtained by drying the supernatant is mixed with myo-inositol and D-chiro-inositol, but the purity of D-chiro-inositol is increased.

According to another preferred embodiment of the present invention, the present invention further provides, after said step (e), a further step of: (f) applying the inositol powder obtained in step (c) Dissolving in water to prepare an aqueous solution and producing an inositol precipitate; (g) separating the supernatant from the inositol precipitate produced in the aqueous solution; And (h) drying the supernatant to obtain an inositol powder.

Step (f) ': dissolving the inositol powder obtained in the step (e) in water to prepare an aqueous solution and producing an inositol precipitate

The powder of inositol obtained in the above step (e) is dissolved in water to prepare an aqueous solution in which inositol is dissolved. The water is preferably distilled water. Preferably, the inositol powder is dissolved in water at a high concentration of greater than 20% (w / v), more preferably at a concentration of greater than 20% (w / v) to less than 80% (w / v) More preferably at a concentration of 30% (w / v) to 70% (w / v) or less. According to one embodiment of the present invention, the solubility of myo-inositol in water is at most about 15% and the solubility of D-chiro-inositol is at most about 55% in water. Therefore, the inositol powder Is dissolved in water at a high concentration of 70%, the myo-inositol will dissolve at a maximum concentration of 15%, and the remaining undissolved myo-inositol is precipitated. Since D-chiro-inositol has a solubility of up to 55%, all D-chiro-inositol present in the powder is soluble in water.

Step (g): separating the supernatant from the inositol precipitate produced in the aqueous solution

The supernatant is separated from the precipitate of inositol produced in step (f). Separation of the supernatant from the inositol precipitate is carried out by filtration or centrifugation, preferably by filtration. Most of the inositol precipitates will contain myo-inositol, and the purity of D-cairo-inositol in the supernatant is increased.

Step (h): Step of drying the supernatant to obtain an inositol powder .

The supernatant separated in step (g) is dried to obtain an inositol powder. The drying of the supernatant may be carried out while heating the supernatant, or in a vacuum. The dried inositol obtained by drying the supernatant is mixed with myo-inositol and D-chiro-inositol, but the purity of D-chiro-inositol is increased. According to one embodiment, when the inositol powder is dissolved in water at a high concentration of about 70% (w / v), the purity of D-chiro-inositol in the dried powder is about 73% .

According to another aspect of the present invention, there is provided a fusion protein comprising (a) a fusion protein of inositol dehydrogenase and inosose isomerase, or inositol dehydrogenase and inososomal isomerase; And (b) an enzyme reaction composition for producing D-chiro-inositol from a myo-inositol containing NAD (Nicotinamide Adenine Dinucleotide) as an active ingredient.

According to one embodiment of the present invention, the composition further comprises (c) a mie-inositol as a substrate.

According to another embodiment of the invention, the composition comprises a Tris-HCl, MgSO _4, MnSO _4, and more.

According to another embodiment of the present invention, the inositol dehydrogenase in the composition is an enzyme derived from C. Glutamicum. More preferably, the inositol dehydrogenase comprises the amino acid sequence of SEQ ID NO: 1.

According to another embodiment of the present invention, the inosome isomerase in the composition is an enzyme derived from P. ananatis. More preferably, the inosomal isomerase comprises the amino acid sequence of SEQ ID NO: 2.

According to another embodiment of the present invention, the fusion protein is a form in which the C-terminus of the inositol dehydrogenase is linked to the N-terminus of the inosomal isomerase.

The features and advantages of the present invention are summarized as follows.

(I) The method of the present invention is a method for producing D-chiro-inositol by reacting a substrate, myo-inositol, with a reaction buffer in the presence of inositol dehydrogenase and inosose isomerase, .

(Ii) The present invention includes a step of separating D-chiro-inositol from myo-inositol and purifying it with high purity by adding an organic solvent to the enzyme reaction buffer which has been reacted.

(Iii) The method of the present invention produces D-chiro-inositol by reaction in an enzyme reaction buffer, and thus D-ciro-inositol can be produced at a low cost and a high yield as compared with a fermentation method using recombinant cells.

(Iv) The method of the present invention can be carried out by a simple process such as addition of an organic solvent, supernatant liquid separation and drying to obtain D-cairo-inositol from a reaction buffer in which miio-inositol and D-cairo- Can be separated.

(V) According to the method of the present invention, the myo-inositol isolated from D-cairo-inositol can be reused as a substrate.

According to the method of the present invention, D-chiro-inositol is produced in an extracellular enzyme reaction buffer, so that D-chiro-inositol can be produced at a low cost and a high yield as compared with a fermentation method using recombinant cells. In addition, D-chiro-inositol can be isolated in high purity from a reaction buffer in which the mixture of myo-inositol and D-chiro-inositol is mixed through a simple process such as addition of an organic solvent, separation of supernatant and drying. Further, according to the present invention, the myo-inositol isolated from D-cairo-inositol has an advantage that it can be reused as a substrate.

Figure 1 shows the structure of the stereoisomers myo-inositol and D-chiro-inositol.
FIG. 2 shows the results of a recombinant plasmid containing pET15b-CP (N-terminal His-Tag), pET15b-PI (N-terminal His-Tag), pET21b- SDS-PAGE results confirming the expression pattern of the E. coli protein introduced with CP (C-terminal His-Tag) and pET21b-PI (C-terminal His-Tag).
Figure 3 shows HPLC chromatograms of myo-inositol and D-chiro-inositol and their stereoisomers or derivatives.
FIG. 4 shows the results of an enzyme reaction using myo-inositol dehydrogenase and inosource isomerase with His-Tag at the N-terminus and C-terminus. The white bars represent myo-inositol and the black bars represent D-cairo-inositol.
FIG. 5 shows the result of purification of myo-inositol dehydrogenase (Cgiep) and inosose isomerase (Paiol I) using His-Tag at the C-terminus by SDS-PAGE.
FIG. 6 shows the results of the production of D-chiro-inositol from myo-inositol using purified enzyme myo-inositol dehydrogenase and inosource isomerase. 5% (w / v) myo-inositol was added as a substrate and the ratio (conversion ratio) of the myo-inositol to the produced D-chiro-inositol was shown. The reaction equilibrium concentration of D-chiro-inositol to 50 g / L (5% (w / v)) myo-inositol is about 14% level of the existing myo-inositol, i.e. 7 g / L.
7 shows the results of D-chiro-inositol production according to the concentration of myo-inositol. Panel A is the yield of D-cairo-inositol by concentration of myo-inositol over time and Panel B is the conversion rate of D-cairo-inositol by concentration of myo-inositol over time. The concentration of the added myo-inositol is: 0%, 5%, 10%, 15%, and 20%.
Fig. 8 shows the production results of D-cairo-inositol according to the concentration of NAD as a coenzyme. Panel A shows the results of measuring the conversion of D-cairo-inositol by NAD concentration over time. The concentration of NAD is: 0 mM, O: 0.1 mM,?: 0.2 mM,?: 0.5 mM,?: 0.7 mM,?: 1 mM,?: 1.5 mM,: 2 mM. Panel B is the measurement result of initial reaction rate according to NAD concentration.
9 shows the results of D-chiro-inositol production according to enzyme concentration. Panel A shows the results of measuring the conversion of D-cairo-inositol by enzyme concentration over time. 0.5 mg / L;?: 0.1 mg / L;?: 0.2 mg / L;?: 0.3 mg / Panel B is the measurement result of the initial reaction rate according to the enzyme concentration.
FIG. 10 shows the result of measurement of the conversion reaction of myo-inositol to D-chiro-inositol according to the reaction temperature. Panel A shows the conversion of D-cairo-inositol by reaction temperature over time. 50 ° C, 55 ° C, 65 ° C, and 25 ° C, respectively. Panel B is the result of measuring the initial reaction rate with temperature.
Fig. 11 shows the results of testing the thermal stability of the enzyme. Panel A is the result of an enzyme reaction after exposure to a specific temperature. : 4 ° C,: 37 ° C, ▲: 42 ° C, ▽: 47 ° C, ◆: 50 ° C, ◁: 55 ° C. Panel B is the result of measuring the enzyme activity ratio with exposure time. : 4 DEG C,?: 37 DEG C,?: 42 DEG C,?: 47 DEG C,?: 50 DEG C,?
Fig. 12 is a graph showing the results of the expression of pCOLAD-1CPFLLPI, pCOLAD-1CPFMLPI, pCOLAD-1CPFSLPI and pCOLAD-1CPFSLPI in which a fusion protein in which the N-terminus and the C- terminus of the inositol dehydrogenase and the inosome isomerase, respectively, , a recombinant plasmid pACYCD-StiolT1-StiolT2 (F2) containing a myo-inositol transporter derived from S. typhimurium already constructed with pCOLAD-2PIFLLCP, pCOLAD-2PIFMLCP and pCOLAD- (DE3) with E. coli BL21 (see, for example, EP-A-10-2012-0107278) to prepare a recombinant strain, and this strain was cultured in the presence of myo-inositol. The cell concentration and the amount of D-chiro-inositol produced were measured at the time of incubation.
Fig. 13 shows the expression (left) and the purification result (right) for the three fusion protein enzymes to which the His-tag was applied at the N-terminus and the C-terminus, respectively.
Fig. 14 is a graph showing the relationship between the vector pACYCD-StiolT1-StiolT2 (F2) containing the myo-inositol transporter and the vector pACYCD-StiolT1-StiolT2 (F2) containing the three fusion gene genes having His- Escherichia coli BL21 (DE3) was introduced together to measure the productivity of D-chiro-inositol. : PET21b-CPFLLPI,?: PET21b-CPFMLPI,?: PET21b-CPFSLPI,?: PET15b-CPFLLPI,?: PET15b-CPFMLPI,?: PET15b-CPFSLPI.
FIG. 15 shows the result of measurement of conversion of myo-inositol to D-chiro-inositol according to the reaction temperature of the fusion protein enzyme CPFMLPI. Panel A shows the conversion of D-cairo-inositol by reaction temperature over time. : 25 ° C, O: 30 ° C, ▲: 37 ° C, ▽: 42 ° C, ◆: 47 ° C, ◁: 50 ° C, ▶: 55 ° C, ☆: 65 ° C. Panel B is the result of measuring the initial reaction rate with temperature.
Figure 16 shows the result of testing the thermal stability of the fusion enzyme CPFMLPI. Panel A is the result of an enzyme reaction after exposure to a specific temperature. ∘: 50 ° C, ◁: 55 ° C, ▶: 65 ° C. Panel B is the result of measuring the enzyme activity ratio with exposure time. ∘: 50 ° C, ◁: 55 ° C, ▶: 65 ° C.
17 shows the solubilities of myo-inositol and D-chiro-inositol in distilled water at 25 ° C. ■: solubility of myo-inositol, ○: solubility of D-cairo-inositol.
18 shows the results of measurement of concentrations of myo-inositol and D-chiro-inositol in the supernatant after ethanol was added to the enzyme reaction buffer in which the myo-inositol and D-chiro-inositol were dissolved to precipitate the inositol. ■: concentration of myo-inositol, ○: concentration of cairo-inositol.
FIG. 19 shows the sedimentation rates of myo-inositol and D-chiro-inositol when ethanol was added to an enzyme reaction buffer in which myo-inositol and D-chiro-inositol were dissolved. ■: sedimentation rate of myo-inositol, ○: sedimentation rate of cairo-inositol.
20 shows the results of measuring the purity of D-chiro-inositol in the inositol powder obtained by precipitating inositol by adding ethanol to the enzyme reaction buffer in which the myo-inositol and D-chiro-inositol are dissolved, and drying the supernatant Show the results.
Fig. 21 shows dissolution characteristics of each inositol when the dried powder (mixed powder of myo-inositol and D-chiro-inositol) of the supernatant obtained by adding primary ethanol was re-dissolved in water. Panel A shows the concentrations of myo-inositol and D-chiro-inositol in the supernatant of aqueous solution. Panel B shows the solubilities of myo-inositol and D-chiro-inositol in the supernatant of aqueous solution. Panel C shows the sedimentation rates of myo-inositol and D-chiro-inositol in the supernatant of aqueous solution. Panel D shows the purity of D-chiro-inositol in the supernatant of aqueous solution. ■: myo-inositol, ○: D-cairo-inositol.
22 shows the results of precipitation and purification in the case where various amounts of ethanol were added to an aqueous solution containing about 12% of dried powder (mixed powder of myo-inositol and D-chiro-inositol) of the supernatant obtained by adding primary ethanol This is the result of measuring the characteristics. Panel A is the result of measuring the concentrations of myo-inositol and D-chiro-inositol in the supernatant. Panel B shows the results of measuring the sedimentation rates of myo-inositol and D-chiro-inositol. Panel C shows the purity of D-chiro-inositol in the supernatant. ■: myo-inositol, ○: D-cairo-inositol.
23 shows the precipitation characteristics of myo-inositol and D-chiro-inositol when various organic solvents were added to an enzyme reaction buffer solution in which the ratio of myo-inositol and D-chiro-inositol was dissolved in a weight ratio of about 13: . Panel A is the sedimentation characteristics when ethanol, isopropanol and acetone are added. Here, the white bars indicate the concentration of myo-inositol and the gray bars show the concentration of D-cairo-inositol. Panel B shows the relative precipitation characteristics of isopropanol and acetone for ethanol. Here the white bar is D-chiro-inositol and the gray bar represents myo-inositol.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Cloning of Inosidol Dehydrogenase and Inosource Isomerase Gene In order to produce D-chiro-inositol from myo-inositol through the continuous reaction of Scheme 1 to Scheme 3, The inositol dehydrogenase gene iolG, which catalyzes the reaction, is synthesized from Corynebacterium glutamicum and the inosose isomerase gene iolI catalyzed in Scheme 2 with pantoheana And amplified from Nantis (Pantoea ananatis).

[Reaction Scheme 1]

Myo-inositol + NAD ^+? 2-keto-myo-inositol + NADH + H ⁺

[Reaction Scheme 2]

2-keto-myo-inositol < RTI ID = 0.0 > 1-keto-D-

[Reaction Scheme 3]

1-keto-D-chiro-inositol + NADH + H ^+? D-chiro-inositol + NAD ⁺

The amplified gene was inserted into pET-15b (Novogen) and pET-21b (Novogen), which are plasmid vectors having T7 promoter and His-Tag, to prepare recombinant plasmids for protein expression. pET-15b is a vector for expressing a protein fused with a His-tag at the N-terminus, and pET-21b is a plasmid vector for expressing a protein fused with a His-tag at the C-terminus. Inositol dehydrogenase used CGIep (NCgl2957 or GI: 19554252), which is an iolG homologue of Corynebacterium glutamicum, and inosource isomerase was a pantoeaananthis (P. Paiol I (PANA_3268, GI: 291618821), which is an iol I of ananatis, was cloned. More specifically, Cgiep was amplified from genomic DNA of Corynebacterium glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) using ETCP-F and ETCP-R primers, BamHI and inserted into the same site of pET-15b to prepare pET15b-CP. The amplified Cgiep gene was further digested with NdeI and HindIII and inserted into the same site of pET-21b to prepare pET21b-CP.

In the case of inosomal isomalase, PaiolI was amplified from the genomic DNA of Pantoea ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) using ETPI-F and ETPI-R primers, And BamHI and inserted into pET-15b digested with the same enzyme to construct pET15b-PI. The amplified PaiolI was digested with restriction enzymes NdeI and XhoI and inserted into the same region of pET-21b to produce pET21b-PI Respectively. The sequences of the above genes, primers, and recombinant plasmids are shown in Table 1.

Example  2: Inositol Dihydrogenase and Innosource Isomerase  Identification and purification of enzyme

The recombinant plasmids pET15b-CP, pET21b-CP, pET15b-PI and pET21b-PI constructed in Example 1 were introduced into Escherichia coli BL21 (DE3) star (Invitrogen) to prepare recombinant strains. Each recombinant strain was inoculated on LB medium containing 100 mg / L ampicillin and seeded to OD 3 at 37 ° C. This was inoculated in a 300 mL baffled Erlenmeyer flask containing 50 mL of LB medium containing 100 mg / L of ampicillin at an initial OD of 0.1 and incubated at 37 ° C to OD 0.6, followed by the addition of 1 mM IPTG Lt; RTI ID = 0.0 > 37 C < / RTI > for about 6 hours. SDS-PAGE was performed to confirm protein expression of the cultured recombinant strain, and the results are shown in Fig. PET15b-CP, pET15b-PI, and pET21b-PI plasmids to which N-terminal His-Tag and C-terminal His-Tag were respectively applied to myo-inositol dehydrogenase and inosource isomerase The expression was confirmed to be normal.

Purification of the protein was performed using a nickel-filled resin through a method known in the art. In more detail, 50 mL of the culture was centrifuged to obtain cells, which were resuspended in 8 mL of binding buffer (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, pH 8.0). 10 mg of lysozyme was added to the resuspension, left for 30 minutes on ice, and disrupted by an ultrasonic crusher (10 seconds of crushing, 3 times of 10 seconds of cooling). The crushed suspension was immediately cooled in liquid nitrogen and rapidly thawed in a constant temperature water bath at 37 ° C. The above-mentioned crushing-cooling-thawing was repeated three times to obtain a highly viscous cell lysate, which was passed through an 18-gauge syringe about 5-10 times to lower the viscosity. The cell lysate was centrifuged and the supernatant was applied to a nickel-filled column. The cell lysate supernatant obtained above was added to a nickel packing column equilibrated with the binding buffer and slowly shaken in a refrigerated state for about 1 hour. After shaking, the supernatant was removed by gentle centrifugation and then washed 4 times with washing buffer (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 20 mM imidazole, pH 8.0) and finally 4 mL of elution buffer (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 250 mM imidazole, pH 8.0). The enzyme myo-inositol dehydrogenase Cgiep has a molecular weight of 36,222.73, 27.607 pM per 1 μg, and PaiolI, an inosolic isomerase, has a molecular weight of 30,417.05 and 32.876 pM per 1 μg.

Example  3: Measurement of the reaction of the purified enzyme and selection of recombinant plasmids

Enzymatic reactions were carried out to confirm the activity of inositol dehydrogenase and inosource isomerase using N-terminal His-Tag and C-terminal His-Tag, respectively. The enzyme reaction conditions are as follows. The reaction was carried out in a total volume of 200 μl. The reaction mixture contained 50 mM Tris-HCl (pH 8.0), 1 mM MgSO ₄ , 0.1 mM MnSO ₄ , 0.5 mM NAD, 5% (w / v) -Inositol, 0.25 mg / mL (6.5 nM) inositol dehydrogenase (Cgiep), 0.25 mg / mL (7.7 nM) inosose isomerase (Paiol I). Therefore, the concentration of the enzyme mixture was about 0.5 mg / mL, which was about 14.2 nM. The reaction solution was allowed to react at 37 ° C for about 15 hours. After completion of the reaction, the reaction was stopped by boiling for 10 minutes. The reaction was terminated by centrifugation, diluted 5-fold with water, and analyzed by HPLC. The pretreatment reaction solution was applied to HPLC (Shimadzu LC10Avp) and analyzed using Kromasil 5NH2 column (4.6 mm x 250 mm), mobile phase 75% acetonitrile, column temperature 40 ° C and RI detector. HPLC chromatograms of each of these isomers, including myo-inositol and D-chiro-inositol, are shown in FIG. The amounts of myo-inositol and D-chiro-inositol measured in the reaction solution are shown in FIG. About 5% of D-chiro-inositol was produced when N-terminal His-Tag was applied and about 9% of D-ciro-inositol was produced when C-terminal His- The enzyme conversion was excellent. Therefore, the enzyme reaction was carried out using pET21b-CP and pET21b-PI to which His-Tag was applied at the C-terminus.

FIG. 5 shows the results of SDS-PAGE analysis of inositol dehydrogenase and inosose isomerase purified from Escherichia coli into which pET21b-CP and pET21b-PI were introduced as described above. The concentration of each protein was 0.86 mg / mL (about 22.8 nM) for Cgiep and 0.64 mg / mL (about 20.32 nM) for PaiolI.

Using the two enzymes purified by the above method, the reaction was carried out for about 24 hours while confirming the reaction results over time. The reaction conditions are as described above, and the results of the reaction are shown in FIG. The substrate, myo-inositol, was rapidly converted to D-chiro-inositol after the start of the reaction. After about 5 hours, the reaction rate gradually decreased and reached about 14% of the reaction equilibrium concentration in about 10 hours. Thereafter, the concentration of D-chiro-inositol, which is a reaction product, did not significantly increase.

Example  4: Productivity of D-cairo-inositol according to the concentration of myo-inositol, which is a substrate

As described in Example 3, in the present invention, since the reaction of myo-inositol to D-chiro-inositol is a reaction involving physicochemical reaction equilibrium, the production amount of D-cairo- Depending on the quantity. Therefore, the reaction rate was tested according to the concentration of the substrate, myo-inositol. For this, the maximum solubility of myo-inositol was investigated under standard reaction conditions and it was found to be about 20% (w / v) maximum at 37 ℃. Based on this, the enzyme reaction was performed at 5% to 20% of the concentration of myo-inositol. The results are shown in Fig. It was confirmed that the reaction rate did not decrease even when the concentration of the substrate, myo-inositol, was increased (Panel A of FIG. 7), but the time to reach the equilibrium of the reaction was delayed as much as the substrate concentration was high. When the concentration of myo-inositol was 5%, the reaction equilibrium reached about 10 hours, whereas the addition of 20% myo-inositol required 24 hours to reach the equilibrium reaction equilibrium.

Example  5: Coenzyme NAD D-CYRO-INOSITOL PRODUCTIVITY

The productivity of D-chiro-inositol according to the concentration of NAD, which is a coenzyme necessary for the reaction of myo-inositol dehydrogenase, was examined. The substrate, myo-inositol, was added in an amount of 10%, and the enzyme was added at a concentration of 250 μg / mL (14.2 nM as a mixed enzyme) to each of the myo-inositol dehydrogenase and inosone isomerase. The production rate of D-cairo-inositol was confirmed by adding various concentrations of coenzyme NAD ranging from 0.1 mM to 2 mM. The reaction results are shown in Fig. The initial reaction rate increased sharply with increasing NAD concentration to a certain level, but did not significantly increase above about 0.5 mM. Panel B of FIG. 8 shows that the enzyme mixture shows a typical state of NAD-dependent enzyme, and the optimum concentration of NAD is 0.5 mM per 10% (555 mM) of the substrate concentration.

Example  6: D-cairo-inositol productivity according to the concentration of added enzyme

The productivity of D-chiro-inositol according to the addition concentration of the myo-inositol dehydrogenase and the inosource isomerase was examined. The substrate, myo-inositol, was adjusted to 10% and NAD was added at 1 mM. The mixed enzyme concentration was 100 ㎍ / mL from 100 ㎍ / mL (about 5.7 nM) to 500 ㎍ / mL (about 28.4 nM) to confirm the production rate of D-chiro-inositol according to the enzyme concentration. The reaction results are shown in Fig. The higher the concentration of the enzyme, the higher the reaction rate (panel A of FIG. 9), which is generally the result of the reaction kinetics that the reaction rate is proportional to the enzyme concentration when the substrate concentration is sufficient Panel B).

Example  7: D-cairo-inositol productivity by reaction temperature

The productivity of D-chiro-inositol according to the enzyme reaction temperature was examined. Enzymes were administered at 250 ㎍ / mL and coenzyme at 1 mM, and the substrate, myo-inositol, was 10%. The productivity of D-chiro-inositol was confirmed by reaction at reaction temperatures of 25 ° C, 30 ° C, 37 ° C, 42 ° C, 47 ° C, 50 ° C, 55 ° C and 65 ° C. The results are shown in FIG. The reaction rate increased with increasing reaction temperature up to 55 ℃, and the reaction rate decreased sharply at 65 ℃. This indicates that the optimum reaction temperature is about 50 ° C, following the Arrhenius equation, a state equation between a typical enzyme and the reaction temperature. Considering that the optimal reaction temperature of the enzyme is about 37 ° C, it is a relatively thermostable enzyme mixture.

Example  8: Mio-inositol Dihydrogenase  And Innosource Isomerase Thermal stability

The heat stability of the myo-inositol dehydrogenase and inosource isomerase was confirmed. The enzyme was allowed to stand at a certain temperature for a certain time and reacted for a predetermined time to measure the productivity and the reaction rate of D-cairo-inositol. The enzymes exposed for 2, 6, 12, and 24 hours at 4, 37, 42, 47, 50, and 55 ℃ were used as the control. The results are shown in FIG. It was found that the reaction was stable even after 24 hours of exposure to about 42 ℃, and the activity rate was increased when exposed to more than 12 hours at 47 ℃. Also, at higher temperatures, even if exposed to heat for a short period of time, the effective rate increased sharply. As shown in Example 7, the enzyme mixture of the present invention appears to be relatively stable against heat as compared with a general enzyme.

Example  9: Inositol Dihydrogenase and Innosource Isomerase  Preparation of recombinant vectors for expression of fusion proteins

In this example, two enzyme inositol-dehydrogenases and inosource isomerase were linked using a flexible linker consisting of glycine and serine. The length of the linker was varied in order to find the conditions that would have the least effect on each enzyme, and for the same reason, a linker was constructed to connect the N-terminal and the C-terminal of each enzyme. For linkers, we refer to three sequences of flexible linkers of different lengths, glycine and serine, from the Registry of Standard Biological Parts (http://partsregistry.org) (see Table 2).

First, three recombinant plasmids for expressing fusion proteins were prepared by linking the C-terminus of Cgiep and the N-terminus of PaiolI with linkers of different lengths.

In detail, for the expression of fusion proteins linked by a long-length linker, Corynebacterium glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC003450) was prepared using primers CPFLL-F and CPFLL- ), The Cgiep gene was amplified from the genomic DNA of P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) using the PCR product containing the Cgiep and the CPFLLPI-R primer PaiolI was amplified from the DNA of the genome to amplify the gene fused with the long linker of Cgiep and PaiolI. The amplified gene was inserted into pCOLADuet-1 (Novogen) digested with restriction enzymes BspHI and SalI and digested with NcoI and SalI to construct pCOLAD-1CPFLLPI.

Genomic DNA of Corynebacterium glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) was amplified using primers CPFLL-F and CPFML-R for the expression of fusion proteins with a medium-length linker Cgiep gene was amplified and PCR products containing this Cgiep and CPFLLPI-R primer were used to amplify Paiol I from the genomic DNA of P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) Amplified and Cgiep and PaiolI amplified the fused gene to a medium length linker. The amplified gene was inserted into pCOLADuet-1 digested with restriction enzymes BspHI and SalI and digested with NcoI and SalI to prepare pCOLAD-1CPFMLPI.

Genomic DNA of Corynebacterium glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) was amplified using primers CPFLL-F and CPFSL-R for expression of fusion proteins with short- Cgiep gene was amplified and PCR products containing this Cgiep and CPFLLPI-R primer were used to amplify Paiol I from the genomic DNA of P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) Amplified to amplify the gene fused to CGIep and PaiolI short linkers. The amplified gene was inserted into pCOLADuet-1 digested with restriction enzymes BspHI and SalI and digested with NcoI and SalI to construct pCOLAD-1CPFSLPI.

Three recombinant plasmids for expression of fusion proteins were constructed by linking the C-terminus of PaiolI and the N-terminus of Cgiep with linkers of different lengths.

In detail, P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) was amplified using primers PIFLL-F and PIFLL-R for expression of fusion proteins linked by a long- And the PaiolI gene was amplified from the genomic DNA of P. glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) using the Paiol I-containing PCR product and PIFLLCP-R primer Cgiep was amplified from the genomic DNA to amplify the genes linked by long linkers of PaiolI and Cgiep. The amplified gene was digested with restriction enzymes NdeI and BglII and inserted into pCOLADuet-1 digested with the same restriction enzymes to produce pCOLAD-2PIFLLCP. For the expression of the fusion protein linked by a medium length linker, the genomic DNA of P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) was amplified using primers PIFLL-F and PIFML- PaiolI gene was amplified and the PCR product containing the Paiol I and the PIFLLCP-R primer were used to amplify the genomic DNA of C. glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) To amplify the genes linked by the intermediate length linker between PaiolI and Cgiep. The amplified gene was digested with restriction enzymes NdeI and BglII and inserted into pCOLADuet-1 digested with the same restriction enzymes to produce pCOLAD-2PIFMLCP.

Genomic DNA of P. ananatis LMG 20103 (taxid: 706191, GenBank NID: CP001875) was amplified using primers PIFLL-F and PIFSL-R for the expression of fusion proteins linked by short- PaiolI gene was amplified and the PCR product containing the Paiol I and the PIFLLCP-R primer were used to amplify the genomic DNA of C. glutamicum ATCC 13032 (taxid: 196627; GenBank NID: NC_003450) To amplify the genes linked by PaiolI and Cgiep with short linkers. The amplified gene was digested with restriction enzymes NdeI and BglII and inserted into pCOLADuet-1 digested with the same restriction enzyme to prepare pCOLAD-2PIFSLCP. The information on the above genes is shown in Table 2, and the primers used are shown in Table 3.

Example  10: Inositol Dihydrogenase and Innosource Isomerase  Production of D-chiro-inositol from myo-inositol through expression of fusion protein

The recombinant plasmid prepared in Example 9 was introduced into Escherichia coli to confirm the productivity of D-chiro-inositol. In detail, a microorganism belonging to the genus Pseudomonas spp. Containing the myo-inositol transporter derived from S. typhimurium already constructed with pCOLAD-1CPFLLPI, pCOLAD-1CPFMLPI, pCOLAD-1CPFSLPI, pCOLAD-2PIFLLCP, pCOLAD-2PIFMLCP, The recombinant strain was transformed into E. coli BL21 (DE3) together with the recombinant plasmid pACYCD-StiolT1-StiolT2 (F2) (see Korean Patent Application No. 10-2012-0107278). The recombinant strains were cultured in 5 mL of a glass tube with a diameter of 25 mm and a height of 150 mm. The culture conditions were as follows: 37 占 폚, 250 rpm condition using TB medium containing 15% (w / v) myo-inositol, 50 mg / L chloramphenicol, 50 mg / L kanamycin, 0.5% lactose For 24 hours. For the analysis of myo-inositol and D-chiro-inositol, the culture broth was centrifuged, and 1 mL of the culture supernatant was boiled for 10 minutes and then centrifuged again to obtain 100 μL of the supernatant, which was then mixed with 900 μL of water. The pretreatment culture supernatant was analyzed by HPLC (Shimadzu LC10Avp), and the analysis conditions were the same as in Example 3. The result of the D-chiro-inositol productivity of each fusion protein is shown in Fig. 12, when the C-terminus of Cgiep and the N-terminus of PaiolI were linked, it was found that the productivity of D-cairo-inositol was superior to that of C-terminus of PiolI and C-terminus I could.

Example  11: Inositol Dihydrogenase and Innosource Isomerase  Construction of Recombinant Plasmids for His-tag Purification of Fusion Enzymes

The N-termini of CPFLLPI, CPFMLPI and CPFSLPI, which are the three kinds of genes having the C-terminus of the inositol dehydrogenase and the N-terminus of the inosome isomerase bound to each other and having different lengths of the linker, A recombinant plasmid was constructed by inserting a gene encoding a fusion enzyme into the plasmid vectors pET15b and pET21b to apply the His-tag to the C-terminus, respectively. pET-15b can be a His-tag at the N-terminus and pET-21b is a plasmid vector to which His-tag can be applied at the C-terminus. In detail, CPFLLPI was amplified using primers ETCP-F1 and ETPI-R1 as a template of pCOLAD-1CPFLLPI plasmid, digested with NdeI and BamHI, and inserted into the same site of pET-15b to prepare pET15b-CPFLLPI . The above-amplified CPFLLPI was further cut into NdeI and SalI and inserted into the same site of pET-21b to prepare pET21b-CPFLLPI. CPFMLPI was amplified from the pCOLAD-CPFMLPI plasmid using the primers ETCP-F1 and ETPI-R1 for the gene CPFMLPI, and then cut into NdeI and BamHI and inserted into the same site of pET-15b to construct pET15b-CPFMLPI. The amplified CPFMLPI was further digested with NdeI and SalI and inserted into the same site of pET-21b to prepare pET21b-CPFMLPI. CPFSLPI was amplified from the pCOLAD-CPFSLPI plasmid using the primers ETCP-F1 and ETPI-R1, and then cut into NdeI and BamHI and inserted into the same site of pET-15b to construct pET15b-CPFSLPI . The amplified CPFSLPI was also digested with NdeI and SalI and inserted into the same region of pET-21b to construct pET21b-CPFSLPI. The primers and the gene information used to construct the six recombinant plasmids are shown in Table 4 below.

Example  12: His - tag Expression, Purification and Activity Measurement of the Fusion Enzyme Applied

PET15b-CPFLLPI, pET21b-CPFLLPI, pET21b-CPFMLPI, and pET21b-CPFSLPI, which were constructed in Example 11, were introduced into Escherichia coli BL21 (DE3) in the presence of the recombinant plasmids pET15b-CPFLLPI, pET15b- To construct a recombinant E. coli. Fusion enzyme expression and purification were carried out in the same manner as in Example 2. The results of expression and purification are shown in Fig. The reaction mixture was diluted with 50 mM Tris-HCl (pH 8.0), 1 mM MgSO ₄ , 0.1 mM MnSO ₄ , 0.5 mM NAD, 5% (w / v) ) (278 mM) of myo-inositol and 0.5 mg / mL (about 7.2 nM) of fusion enzyme. The reaction solution was allowed to react at 37 ° C for about 15 hours. After completion of the reaction, the reaction was stopped by boiling for 10 minutes. After stopping the reaction, the reaction solution was centrifuged, diluted 5-fold with water, and analyzed by HPLC. The HPLC analysis method was as described in Example 3. As a result of the reaction using the purified fusion protein enzyme, the myo-inositol was almost not converted to D-chiro-inositol.

Example  13: His - tag Of the fusion enzyme, in vivo ) Active measurement

When the His-tag was applied to the fusion enzyme that showed activity in Example 10, the N-terminal and C-terminal did not have conversion activity to D-chiro-inositol. Therefore, in vivo ). The recombinant plasmids pCOLAD-StiolT1-StiolT2 (F2) having the myo-inositol transporter were used as the recombinant plasmids pET15b-CPFLLPI, pET15b-CPFMLPI, pET15b-CPFSLPI, pET21b-CPFLLPI, pET21b- (Refer to Korean Patent Application No. 10-2012-0107278) to construct a recombinant strain, and cultured and analyzed in the same manner as in Example 10. The results are shown in FIG. Unlike the case where the fusion enzyme was purified and reacted, 6 recombinant plasmids were introduced into the cells, and as a result, it was confirmed that D-cairo-inositol was produced normally in all experimental groups. In particular, pET15b-CPFMLPI It was confirmed that it was produced quickly. Therefore, when the His-tag fusion enzyme purified in Example 12 was used for the in vitro reaction, the fact that the D-chiro-inositol was not produced was not the result of the application of the His-tag, Respectively.

Example  14: by dialysis Imidazole  Removed CPFMLPI  Determination of reaction pattern according to reaction temperature of fusion enzyme

In the present invention, an imidazole was excessively introduced during the purification process, and the imidazole was dialyzed to remove the imidazole from the purified enzyme in anticipation of affecting the activity of the enzyme. Dialysis was performed only for pET15b-CPFMLPI that produced D-cairo-inositol most rapidly in the results of Example 13 (see Fig. 14). The dialyzed solution was dialyzed against an enzyme solution (50 mM Tris-HCl, pH = 8.0, 1 mM MgSO ₄ , pH 8.0) except for the substrate and NAD, after the final eluted enzyme solution was added to the spectral dialysis membrane (Spectra / Por 1 dialysis tubing, 6-8K MWCO) , 0.1 mM MnSO4), stirred, and incubated for about 24-48 hours with 6-8 hours replacement of the enzyme reaction buffer. The enzyme was removed from the imidazole by dialysis and the reaction rate was measured by the reaction temperature. Fusion enzyme was administered at 0.5 mg / mL and coenzyme at 1 mM, and the substrate, myo-inositol, was 10%. The productivity of D-chiro-inositol was confirmed by reaction at reaction temperatures of 25 ° C, 30 ° C, 37 ° C, 42 ° C, 47 ° C, 50 ° C, 55 ° C and 65 ° C. The results are shown in Fig. Similar to the case of using unfused single enzyme Cgipe and PaiolI, the reaction rate increased as the reaction temperature was increased up to 55 ° C, and the reaction rate was greatly decreased at 65 ° C (panel A of FIG. This follows the Arrhenius equation of state equation between typical enzyme and reaction temperature, indicating that the optimal reaction temperature is about 50 ° C to 55 ° C (panel B in Figure 15). The reaction rates were similar to those of the single enzymes Cgipe and PaiolI, and were slightly more thermally stable at the same temperature, but no significant difference was observed. As a result, when the fusion was performed using the linker, the reaction rate was similar to that of using the respective enzymes. Therefore, it was judged that fusion enzyme was effective in the enzyme reaction method.

Example  15: fusion enzyme CPFMLPI of Thermal stability  Research

The heat stability of the fusion enzyme CPFMLPI was confirmed. The enzyme was allowed to stand at a certain temperature for a certain time and reacted for a predetermined time to measure the productivity and the reaction rate of D-cairo-inositol. The reaction was carried out at 4, 37, 42, 47, 50, 55 and 65 ℃ for 2, 6, 12 and 24 hours, respectively. The reaction was carried out for about 7 hours using the enzyme exposed during the reaction, and the results are shown in FIG. The reaction was relatively stable even after 24 hours of exposure to about 55 ° C, and the activity rate remained below 20%. It was rapidly inactivated after exposure at 65 ° C. Thus, the fusion enzyme CPFMLPI appears to be stable even after prolonged exposure to heat at a temperature of 10 ° C or higher, compared with the use of the existing enzyme Cgiep and PaiolI. That is, it means that the enzyme stability is increased when the enzyme is fused more than when each enzyme is used, and it is expected that the use efficiency of the purified enzyme can be increased when it is applied in actual production. In addition, the fusion enzyme CPFMLPI is expected to be very efficient because it can reduce the time and cost in half in the expression and purification processes when using the two existing enzymes separately.

In the above examples, the reaction rate of the purified enzyme was 54.6 mM / hr for the single enzyme and 71.2 mM / hr for the fusion enzyme, based on the weight of the enzyme (500 μg) added at 37 ° C and 1 mM NAD The enzyme activity was 3.7 mM / nM Enzymes / hour for the enzyme, and 9.9 mM / nM Enzymes / hour for the fusion enzyme, based on the molar concentration of the enzyme. Enzyme was found to be high.

Example  16: Solubility measurement of myo-inositol and D-chiro-inositol

The solubilities of myo-inositol and D-chiro-inositol were measured. First, the myo-inositol and the D-chiro-inositol were dissolved in the primary distilled water at a room temperature (about 25 ° C.) to a concentration of 5% (w / v) to 80% (w / v). The undissolved portions of each solution were separated by centrifugation, and the supernatant fraction in which the inositol was completely dissolved was analyzed for each concentration by HPLC. The analytical conditions were as follows: Kromasil 5NH2 column (4.6 mm X 250 mm), mobile phase 75% acetonitrile, column temperature 40 ° C, RI detector using HPLC (Shimadzu LC10Avp). The results are shown in Fig. The solubility of myo-inositol was about 15% (w / v) at 25 ° C and about 55% (w / v) for D-chiro-inositol, It was confirmed that the solubility difference of inositol was very large.

Example  17: Primary ethanol treatment for the separation of myo-inositol

The enzymatic reaction buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM MgSO ₄ , 0.1 mM MnSO ₄ , 0.5 mM NAD 12.9% (w / v) ) D-chiro-inositol. Ethanol was added to the enzyme reaction buffer so that the ratio of the reaction buffer and ethanol was 10: 0, 9: 1 (0.11), 8: 2 (0.25), 7: 3 (0.43) (4 times), 1: 9 (9 times), and the mixture was mixed thoroughly at room temperature (About 25 < 0 > C) for 1 hour. After centrifugation at 3,500 rpm for 30 minutes, the supernatant was taken and the concentration of myo-inositol and D-chiro-inositol was analyzed by HPLC.

The results of the concentration analysis are shown in Figs. 18, 19, and 20. 18 shows the concentrations of the myo-inositol and the D-chiro-inositol remaining in the supernatant obtained by removing precipitates formed after adding ethanol to the enzyme reaction buffer after the reaction was completed. In the case of myo-inositol, the concentration rapidly decreased according to the amount of ethanol added, and when about one-fold of ethanol was added, about 99 g / L was precipitated from the initial 126 g / L concentration to about 27 g / L of myo-inositol. In contrast, the reduction of D-chiro-inositol is very low, and when approximately one-fold of ethanol is added, it decreases to 15 g / L at the initial concentration of 17 g / L, indicating that only about 2 g / L is precipitated.

The result shown in FIG. 18 is converted into the sedimentation rate and shown in FIG. The precipitation rate of myo - inositol increased rapidly with the addition of ethanol. When 1 - fold ethanol was added, the precipitation rate of 70 - 80% was comparatively similar under all reaction conditions. It was confirmed that the myo-inositol precipitated. In contrast, the precipitation rate of D-cairo-inositol was about 15% or less until about 1-fold ethanol was added, and the precipitation rate of D-cairo-inositol was remarkably increased when ethanol was added twice or more.

The purity of D-cairo-inositol was calculated based on the experimental results shown in Figs. 18 and 19 and is shown in Fig. The purity of D-chiro-inositol was about 35-40% when ethanol was added at a ratio of 1: 1, and when the amount of ethanol was more than that, the purity increased but the loss ratio of D-chiro-inositol increased .

In conclusion, about 80% of myo-inositol precipitates when about 1-fold of ethanol is added. At this time, the settling rate of D-chiro-inositol is below 15% The purity of D-chiro-inositol was increased to about 30-40%. On the other hand, when the amount of ethanol added exceeds 1, the precipitation rate (loss ratio) of D-chiro-inositol rapidly increases. Therefore, when ethanol is added in an amount of about one-fold, Can be separated and removed by precipitation.

Example  18: After primary ethanol treatment Supernatant  Concentrated drying and reuse

(1: 1) ethanol was mixed with the reaction buffer after the enzyme reaction was completed, and the precipitate was removed. The supernatant obtained by heating was heated to 80 ° C, concentrated under vacuum, and dried . The dried powder of inositol (mixed powder of myo-inositol and D-chiro-inositol) obtained after drying was dissolved in distilled water at various concentrations of 15% to 70% to prepare a mixture of myo-inositol and D-cairo- Were observed.

The results of analysis of dissolution characteristics of myo-inositol and D-cairo-inositol are shown in Fig. 21 shows the concentration or solubility of each inositol in the supernatant, and it was confirmed that the solubility of myo-inositol did not increase more than about 15% as shown in Example 16 above. On the other hand, since D-cairo-inositol has a solubility of about 55%, it was confirmed that all the D-cairo-inositol was dissolved even when the dry powder was dissolved at a concentration of 70%. The dissolution characteristics of the myo-inositol and the D-cairo-inositol as solubility and sedimentation rate are shown in Panel B and Panel C of FIG. 21, respectively. The solubility of the myo - inositol began to decrease from about 20% solution, and it decreased continuously as the solution concentration increased. The precipitation rate increased with increasing solution concentration. In the case of D-chiro-inositol, the solubility was maintained at 100% until the concentration of the solution was 70%, indicating that the sedimentation rate was 0% and that no precipitation occurred at all. In conclusion, when the maximum 70% solution was prepared, only 15% of the myo-inositol was dissolved and the purity of the D-chiro-inositol was about 73% since D-chiro-inositol was completely dissolved Panel D).

Example  19: Purification characteristics when secondary ethanol was treated with a mixed solution of low concentration of myo-inositol and D-cairo-inositol

The same amount of ethanol as in Example 17 was added to the reaction buffer, and the supernatant was removed from the supernatant. The supernatant was heated to 80 ° C, concentrated under vacuum, and dried . An experiment was conducted in which a mixed dried product of myo-inositol and D-cairo-inositol was prepared as a solution having a low concentration of 15% or less and ethanol was added thereto. After the first ethanol treatment, the total amount of inositol was about 11.5%, and the concentrations of the myo-inositol were 75 g / L and D-Cairo - Inositol was 40 g / L. Ethanol was added to the mixed solution up to 19 times, and precipitation and separation patterns were confirmed. The results are shown in Fig. 22 shows the concentration of the supernatant after the addition of ethanol. In the case of the myo-inositol, no significant change was observed until about one-fold increase in the amount of ethanol, but when 1.5-fold increase in ethanol was added, - Inositol was found to precipitate. On the contrary, D-cairo-inositol was found to precipitate slightly in 1.5 times of ethanol, and the sedimentation did not increase much after that. Panel B of FIG. 22 shows the sedimentation rate. In the case of the myo-inositol, about 9% of ethanol was added to precipitate more than 90% of the precipitate, and the precipitation rate of D-chiro-inositol was about 20%. Therefore, the addition of about 9 times the amount of ethanol can reduce the D-chiro-inositol loss rate to 20% or less while removing most of the myo-inositol, and purify the D-cairo-inositol purity to 90% or more See Figure 22, panel C).

Example  20: Selective precipitation of myo-inositol and D-chiro-inositol by an organic solvent other than ethanol

From the results of the experiments of Examples 16 to 19, it was confirmed that the separation of the myo-inositol and the D-chiro-inositol contained in the enzyme reaction buffer can be carried out using ethanol because of their large solubility differences. Separation was attempted using isopropanol which is a three carbon alcohol and acetone which is a ketone in order to confirm whether this separation is possible by other organic solvents other than ethanol having two carbon atoms. In the preparation of the mixed solution of myo-inositol and D-chiro-inositol, the ratio of the myo-inositol and the D-chiro-inositol was about 13: 2, which is the normal concentration reached to the reaction equilibrium. Condition), so that the total concentration was about 15%. The same volume of ethanol, isopropanol and acetone was added to the enzyme reaction buffer to which the inositol was added. The mixture was shaken at room temperature for 1 hour, centrifuged, and the supernatant was analyzed by HPLC. The results are shown in Fig. Panel A of FIG. 23 shows the concentration of inositol present in the actual supernatant. In the case of myo - inositol, ethanol and isopropanol were not significantly different, but acetone showed a significant decrease. In the case of D-chiro-inositol, ethanol and isopropanol were similar, whereas acetone showed the same concentration as that of the organic solvent. That is, there was no significant difference in the precipitation rate between the myo-inositol and the D-chiro-inositol when compared to ethanol, and the acetone had a slightly higher sedimentation rate to the myo-inositol and a lower loss rate of the D-chiro-inositol see. This is easily seen in Panel B of FIG. 23, which shows relative values for the precipitation rate of ethanol. That is, isopropanol had no significant difference in the values of 1.0 and 0.9 between myo-inositol and D-chiro-inositol with respect to ethanol, and the sedimentation rate of myo-inositol was about 1.1 higher than that of acetone with respect to ethanol. And the level was about 0.1. Therefore, it has been confirmed that an organic solvent other than ethanol can be used for the separation of myo-inositol and D-chiro-inositol like ethanol, and the separation characteristics of acetone are more advantageous than that of ethanol.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Solgent Co. Ltd. <120> Method for Producing D-Chiro-Inositol From Myo-Inositol Using In          Vitro Enzyme Reaction <160> 21 <170> Kopatentin 1.71 <210> 1 <211> 335 <212> PRT <213> Corynebacterium glutamicum <400> 1 Met Thr Leu Arg Ile Ala Leu Phe Gly Ala Gly Arg Ile Gly His Val   1 5 10 15 His Ala Ala Asn Ile Ala Ala Asn Pro Asp Leu Glu Leu Val Val Ile              20 25 30 Ala Asp Pro Phe Ile Glu Gly Ala Gln Arg Leu Ala Glu Ala Asn Gly          35 40 45 Ala Glu Ala Val Ala Ser Pro Asp Glu Val Phe Ala Arg Asp Asp Ile      50 55 60 Asp Gly Ile Val Ile Gly Ser Pro Thr Ser Thr His Val Asp Leu Ile  65 70 75 80 Thr Arg Ala Val Glu Arg Gly Ile Pro Ala Leu Cys Glu Lys Pro Ile                  85 90 95 Asp Leu Asp Ile Glu Met Val Arg Ala Cys Lys Glu Lys Ile Gly Asp             100 105 110 Gly Ala Ser Lys Val Met Leu Gly Phe Asn Arg Arg Phe Asp Pro Ser         115 120 125 Phe Ala Ala Ile Asn Ala Arg Val Ala Asn Gln Glu Ile Gly Asn Leu     130 135 140 Glu Gln Leu Val Ile Ile Ser Arg Asp Pro Ala Pro Ala Pro Lys Asp 145 150 155 160 Tyr Ile Ala Gly Ser Gly Gly Ile Phe Arg Asp Met Thr Ile His Asp                 165 170 175 Leu Asp Met Ala Arg Phe Phe Val Pro Asn Ile Val Glu Val Thr Ala             180 185 190 Thr Gly Ala Asn Val Phe Ser Gln Glu Ile Ala Glu Phe Asn Asp Tyr         195 200 205 Asp Gln Val Ile Val Thr Leu Arg Gly Ser Lys Gly Glu Leu Ile Asn     210 215 220 Ile Val Asn Ser Arg His Cys Ser Tyr Gly Tyr Asp Gln Arg Leu Glu 225 230 235 240 Ala Phe Gly Ser Lys Gly Met Leu Ala Ala Asp Asn Ile Arg Pro Thr                 245 250 255 Thr Val Arg Lys His Asn Ala Glu Ser Thr Glu Gln Ala Asp Pro Ile             260 265 270 Phe Asn Phe Phe Leu Glu Arg Tyr Asp Ala Ala Tyr Lys Ala Glu Leu         275 280 285 Ala Thr Phe Ala Gln Gly Ile Arg Asp Gly Gln Gly Phe Ser Pro Asn     290 295 300 Phe Glu Asp Gly Val Ile Ala Leu Glu Leu Ala Asn Ala Cys Leu Glu 305 310 315 320 Ser Ala Gln Thr Gly Arg Thr Val Thr Leu Asn Pro Ala Asn Val                 325 330 335 <210> 2 <211> 273 <212> PRT <213> Pantoea ananatis <400> 2 Met Thr Ile Ala Ala Glu Arg Phe Cys Ile Asn Arg Lys Ile Ala Pro   1 5 10 15 Ser Leu Ser Ile Glu Ala Phe Phe Arg Leu Val Asn Gly Leu Gly Leu              20 25 30 Asn Lys Val Glu Leu Arg Asn Asp Leu Pro Ser Gly Asn Val Met Asp          35 40 45 Asn Leu Ser Ala Ala Gln Val Cys Ala Leu Ala Glu Arg Tyr Gly Ile      50 55 60 Asp Ile Ile Thr Ile Asn Ala Val Tyr Pro Phe Asn Gln Arg Thr Glu  65 70 75 80 Gln Val Arg Gln Leu Thr Glu Ser Leu Leu Ala Asp Ala Lys Ala Val                  85 90 95 Gly Ala Arg Ser Leu Val Leu Cys Pro Leu Asn Asp Gly Ser Ser Val             100 105 110 Ala Pro Glu Val Thr Leu Asn Ala Leu Arg Asp Leu Ala Pro Leu Phe         115 120 125 Ala Thr Tyr Gly Ile Thr Gly Leu Val Glu Pro Leu Gly Phe Pro Gln     130 135 140 Ser Ser Leu Arg Ser Ala Gln Ala Gln Lys Leu Ile Arg Asp Ala 145 150 155 160 His Val Pro Phe Lys Leu Leu Ile Asp Thr Phe His His His Leu Tyr                 165 170 175 Pro Asp Ala Asp Ala Glu Phe Ser Gln Val Asp Ile Ala Gln Ile Gly             180 185 190 Leu Val His Leu Ser Gly Val Glu Asp Lys Arg Pro Arg Glu Ser Leu         195 200 205 Thr Asp Ala Glu Arg Ile Met Leu Thr Pro Glu Asp Arg Leu Phe Thr     210 215 220 Cys Arg Gln Val Lys Gln Leu Glu Glu Arg Gly Tyr Lys Gly Ile Tyr 225 230 235 240 Ala Phe Glu Pro Phe Ala Pro Glu Leu Ala Asp Trp Asp Glu Asn Lys                 245 250 255 Ile Gln His Glu Ile Lys Asn Ser Ile Gln Leu Ile Gln His Ser Leu             260 265 270 Lys     <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 3 cgacatatga ctcttcgtat cgcccttttc g 31 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 4 cgaggatcct taaagcttaa cgttggcagg gttgagggtg 40 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 5 cgacatatga ccatcgccgc agaacg 26 <210> 6 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 6 cgaggatcct tactcgagct tcaaggaatg ttgaatcagt tggatgc 47 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 7 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly   1 5 10 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 8 Gly Gly Ser Gly Gly Gly Ser Gly   1 5 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Peptide Linker <400> 9 Gly Gly Ser Gly   One <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 10 cgatcatgac tcttcgtatc gcccttttcg 30 <210> 11 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 11 cgttctgcgg cgatggtcat accagaacca ccaccagaac caccaccaga accaccaacg 60 ttggcagggt tgagggtg 78 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 12 gcagtcgact tacttcaagg aatgttgaat tagttggatg c 41 <210> 13 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 13 cgttctgcgg cgatggtcat accagaacca ccaccagaac caccaacgtt ggcagggttg 60 agggtg 66 <210> 14 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 14 cgttctgcgg cgatggtcat accagaacca ccaacgttgg cagggttgag ggtg 54 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 15 cgtcatatga ccatcgccgc agaacg 26 <210> 16 <211> 89 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 16 cgaaaagggc gatacgaaga gtcataccag aaccaccacc agaaccacca ccagaaccac 60 ccttcaagga atgttgaatc agttggatg 89 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 17 gctagatctt aaacgttggc agggttgagg gtg 33 <210> 18 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 18 cgaaaagggc gatacgaaga gtcataccag aaccaccacc agaaccaccc ttcaaggaat 60 gttgaatcag ttggatg 77 <210> 19 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 19 cgaaaagggc gatacgaaga gtcataccag aaccaccctt caaggaatgt tgaatcagtt 60 ggatg 65 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 20 cgacatatga ctcttcgtat cgcccttttc g 31 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> PCR primer <400> 21 gcaggatcct tagtcgacct tcaaggaatg ttgaattag 39

Claims (23)

A method for producing D-chiro-inositol from myo-inositol using an enzymatic reaction method comprising the steps of:
(a) an enzyme reaction buffer solution containing (i) a fusion protein of inositol dehydrogenase and inosose isomerase, (ii) NAD (nicotinamide adenine dinucleotide) as a coenzyme, and (iii) Preparing; And
(b) reacting the enzyme reaction buffer to produce D-chiro-inositol.
The method according to claim 1, wherein the concentration of the fusion protein of inositol dehydrogenase and inosose isomerase in the enzyme reaction buffer is 100-500 占 퐂 / ml.
2. The method according to claim 1, wherein the concentration of NAD in the enzyme reaction buffer is 0.1 to 2 mM.
The method of claim 1, wherein the concentration of the myo-inositol in the enzyme reaction buffer is 5 - 20% (w / v).
The method according to claim 1, wherein the enzyme reaction temperature in step (b) is 25-65 ° C.
The method of claim 1, wherein the enzyme reaction buffer of step (a) is characterized in that further comprising a Tris-HCl, MgSO _4, and MnSO _4.
The method according to claim 1, wherein the inositol dehydrogenase is an enzyme derived from C. Glutamicum.
The method according to claim 1, wherein the inosome isomerase is an enzyme derived from P. ananatis.
2. The method of claim 1, wherein the fusion protein is a form in which the C-terminus of the inositol dehydrogenase is linked to the N-terminus of the inosomal isomerase.
2. The method of claim 1, further comprising, after step (b), the following steps:
(c) adding an organic solvent to the enzyme reaction buffer to form an inositol precipitate;
(d) separating the supernatant from the resulting inositol precipitate; And
(e) drying the separated supernatant to obtain an inositol powder.
11. The method of claim 10, further comprising, after step (e), the following steps:
(f) dissolving the inositol powder obtained in the step (e) in water to prepare an aqueous solution;
(g) adding an organic solvent to the aqueous solution to produce an inositol precipitate;
(h) separating the supernatant from the resulting inositol precipitate; And
(i) drying the separated supernatant to obtain an inositol powder;
11. The method according to claim 10, wherein in step (f), the inositol powder is dissolved in water at a concentration of 20% (w / v) or less.
The method of claim 10 or 11, wherein the organic solvent in step (c) of claim 10 or in step (g) of claim 11 is selected from the group consisting of alcohols, acetone, ethyl acetate, butyl acetate, Glycol or ether.
The method according to claim 10, wherein the amount of the organic solvent added in step (c) is 0.1 to 9 times (v / v) of the enzyme reaction buffer.
12. The method according to claim 11, wherein the amount of the organic solvent added in step (g) is 1 - 10 times (v / v) relative to the aqueous solution.
11. The method of claim 10, further comprising, after step (e), the following steps:
(f) dissolving the inositol powder obtained in the step (e) in water to prepare an aqueous solution and producing an inositol precipitate;
(g) separating the supernatant from the inositol precipitate produced in the aqueous solution; And
(h) drying the supernatant to obtain an inositol powder;
17. The method of claim 16, wherein in step (f), the inositol powder is dissolved in water at a concentration of greater than 20% (w / v) to less than 70% (w / v).
(a) a fusion protein of inositol dehydrogenase and inosose isomerase; And (b) an enzyme reaction composition for producing D-chiro-inositol from myo-inositol containing NAD (Nicotinamide Adenine Dinucleotide) as an active ingredient.
19. The composition of claim 18, wherein the composition further comprises (c) a mao-inositol as a substrate.
19. The method of claim 18, wherein the composition is a composition further comprising a Tris-HCl, MgSO _4, and MnSO _4.
19. The composition according to claim 18, wherein the inositol dehydrogenase is an enzyme derived from C. Glutamicum.
19. The composition of claim 18, wherein the inosource isomerase is an enzyme derived from P. ananatis.
19. The composition of claim 18, wherein the fusion protein is a form in which the C-terminus of the inositol dehydrogenase is linked to the N-terminus of the inosose isomerase.

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