WO2009145576A2 - Aldohexose epimerase and enzymatic preparation method of aldohexose epimer using the same - Google Patents

Abstract

The present invention relates to an aldohexose epimerase, an enzymatic preparation method of aldohexose epimer using the same, and a screening method of aldohexose epimerase.

Description

Aldohexose epimerase and enzymatic preparation method of aldohexose epimer using the same

The present invention relates to aldohexose epimerase, an enzymatic preparation method of aldohexose epimer using the same, and a screening method of aldohexose epimerase. More specifically, the present invention treats an aldohexose epimer by treating an aldohexose such as glucose with an epimerase such as a modified aldohexose epimerase obtained by directional molecular evolution of the wild type UDP-galactose 4-epimerase. It relates to a method for producing enzymatically and a method for screening epimerase enzyme by measuring the activity of aldohexose epimerase.

Recently, due to the rapid increase in international oil prices and exhaustion of oil resources, efforts have been made to replace finite fossil raw materials with cheaper and infinite bioenergy. In particular, a combination of high demand for biodiesel and bioethanol, particularly in Europe, the shortage of raw materials due to the expansion of the Chinese economy, and the US policy to convert up to 30% of strategic reserve oil into bioethanol, and competition for weaponization of food and energy. Overall grain prices are on the rise. Therefore, there is a need for a technique capable of producing relatively high value added monosaccharides such as galactose and tagatose from cheap raw materials such as glucose.

As one of the sugar conversion techniques, isomerization enzymes of sugars derived from various kinds of microorganisms are actively studied for industrial use. For example, xylose isomerase mainly used in the preparation of high fructose syrup and arabinose isomerase capable of producing tagatose, which is emerging as a functional substitute sweetener, are typical. The industrial potential of these enzymes is that they can catalyze the interconversion of sugars in addition to the native substrates of xylose and arabinose. However, as a result of natural evolution over a long period of time, general properties of enzymes, namely limited substrate specificity and affinity, optimal conditions, etc., are known to be a problem that must be overcome in terms of industrial applications of enzymes.

Examples of aldohexose epimerases present in nature include UDP-galactose-4-epimerase (EC 5.1.3.2), an epimerizing enzyme of E. coli-derived nucleic acid active substrates. In the case of the enzyme, the catalytic site directly involved in the enzymatic activity has a space to recognize UDP to recognize UDP-galactose or UDP-glucose as a substrate and to convert the recognized substrate into UDP-glucose or UDP-galactose. Catalyzes the merization reaction. This natural aldohex epimerase uses only UDP-aldohexose, which is an aldohexose activated form of UDP, as a substrate. Thus, in order to produce aldohexose using the substrate, aldohexose is used as a substrate. There is an additional disadvantage that the step of converting to active form and converting UDP-aldohexose which is a product by epimerization back to aldohexose are additionally required.

Therefore, if an epimerase enzyme is produced that recognizes aldohexose itself, which is not UDP-active, as a substrate and produces epimer aldohexose, food and pharmaceuticals can be efficiently and inexpensively reduced by drastically reducing the additional steps described above. Alohexose used as a raw material can be obtained. However, until now, epimerase enzymes capable of epimerizing aldohexose itself have not been developed, and in particular, there is no known method for enzymatically preparing galactose, which is a raw material of tagatose, a high value added saccharide from glucose. not.

The general method of preparing aldohexose having high added value has a problem of complicated process such as using expensive raw materials or obtaining specific mutants recognized by enzymes present in nature. Therefore, the present inventors have provided an economical method for producing high value-added epimer type aldohexose from low-cost aldohexose as a raw material, for example, an enzymatic method for producing galactose from glucose, thereby providing high-value sugars at low cost. It is intended to enable efficient production.

The present invention does not go through a cumbersome process such as converting an inexpensive aldohexose raw material such as glucose into a UDP-active form, and converts the raw material itself into a relatively high value added monosaccharide such as galactose by treating the raw material with an epimerase enzyme. Provided is a method for enzymatic preparation of a hexose epimer.

In addition, the present invention recognizes aldohexose itself, such as glucose, which is not UDP-active, as a substrate by directional molecular evolution of UDP-galactose 4-epimerase, and as its epimer having higher utility by epimerization of the substrate. Provided are novel aldohexose epimerases that can be converted.

The present invention also measures the amount of NADH produced after reacting a microbial cell lysate of interest with a reaction solution comprising glucose, NAD ⁺ , and β-galactose dehydrogenase, and measuring the amount of wild type UDP-galactose 4- Selecting microorganisms having aldohexose epimerase activity as compared to the amount of NADH produced in the reaction by lysate of control cells with epimerase gene, and obtaining aldohexose epimerase therefrom. Provided are aldohexose epimerase screening methods and aldohexose epimerases selected therefrom.

According to the present invention, it is possible to provide a simple method for enzymatic preparation of an aldohexose epimer which can convert aldohexose into a relatively high value aldohexose epimer. The present invention also provides an aldohexose epimerase that performs epimerization of aldohexose, such as aldohexose epimerase, for example, by directional molecular evolution, thereby enzymatic preparation of epimeric aldohexose. It can be useful for. In addition, the present invention provides a method for screening aldohexose epimerase, so that it is possible to efficiently select industrially useful aldohex epimerase.

FIG. 1 schematically shows the evolution of UDP-galactose 4-epimerase (epimerase A) to aldohexose epimerase (epimerase B).

FIG. 2 is an HPLC result showing the conversion efficiency of galactose from glucose by aldohexose epimerase selected according to the example. (a) is an HPLC chromatogram of glucose and galactose standards, and (b) is an HPLC chromatogram showing peaks of galactose detected in the reaction solution by aldohexose epimerase selected according to an embodiment of the present invention.

3 is a graph showing the pH-dependent activity of aldohexose epimerase selected according to an embodiment of the present invention.

Figure 4 is a graph showing the activity according to the temperature of aldohexose epimerase selected in accordance with an embodiment of the present invention.

As used herein, “UDP-galactose 4-epimerase” is understood to refer to an enzyme that recognizes UDP-galactose or UDP-glucose as a substrate and catalyzes epimerization between them.

As used herein, "aldohexose" refers to any D-aldohexose or L-aldohex that is naturally present or synthesized, such as glucose, allose, altrose, mannose, gulose, idose, galactose, talos. It is interpreted to include a source.

In the present specification, "aldohexose epimerase" refers to an aldohexose such as glucose, allose, altrose, mannose, gulose, idose, galactose, and talos as a substrate to be subjected to epimerization at any carbon position. Enzymes that can be converted into its epimeric type aldohexose by the present invention include all mutated or naturally occurring enzymes, including aldohexose 3-epimerase, aldohexose 4-epimerase and the like. It is interpreted as. The substrate of the aldohexose epimerase may be D-aldohexose or L-aldohexose.

One aspect of the present invention is to provide a method for enzymatic preparation of an aldohexose epimer, comprising the step of obtaining an aldohexose epimer by subjecting the aldohexose to an epimerase enzyme to epimerize it.

According to the invention, the aldohexose substrate treated with the epimerase enzyme is D-aldohexose or L- selected from the group consisting of glucose, allose, altrose, mannose, gulose, idose, galactose and talos. Aldohexose, preferably glucose, most preferably D-glucose.

According to the present invention, high value-added monosaccharides such as galactose can be prepared by a simple epimerase enzyme treatment process without converting aldohexose such as glucose to UDP-active type.

According to the present invention, aldohexose epimers can be prepared by treating aldohexose with an epimerase enzyme to C-3 epimerization or C-4 epimerization, preferably C-4 epimerization.

In the enzymatic preparation of aldohexose epimers, the epimerase enzyme treatment is carried out at pH 7 to 10 and at a temperature of 30 to 50 ° C., preferably at pH 8 to 10 and at a temperature of 35 to 40 ° C., most preferably. Is carried out at pH 8.6 and at a temperature of 37 ° C. If the pH is lower than 7 or higher than 10 during enzyme treatment, enzyme degeneration of epimerase may be induced, thereby lowering enzyme activity. In addition, if the temperature is lower than 30 ℃ the activity of the enzyme is too low, there is a problem that the process is inefficient, if it is higher than 50 ℃ there is a problem that the protein structure of the enzyme is rapidly changed to show little enzyme activity.

The epimerase enzyme according to the present invention is not particularly limited as long as it is an enzyme capable of converting aldohexose into its epimer, and includes all naturally occurring enzymes or enzymes mutated by aromatic molecular evolution. Preferably, the aldohexose epimerase obtained by directional molecular evolution of the wild type UDP-galactose 4-epimerase.

Another aspect of the present invention is to provide an aldohexose epimerase which performs epimerization of aldohexose, preferably epimerization at the carbon position 4.

The aldohexose epimerase according to the present invention is not particularly limited as long as it is an enzyme that performs epimerization of aldohexose, and includes enzymes naturally modified by enzymes or aromatic molecular evolution. Preferably, the aldohexose epimerase obtained by directional molecular evolution of the wild type UDP-galactose 4-epimerase, more preferably aldohexose epimerase having the amino acid sequence of SEQ ID NO: 3.

In the present invention, the method of directing directional molecular evolution of wild-type UDP-galactose 4-epimerase is not particularly limited, for example, to a mutagen such as error-prone PCR, chemicals or UV. Treatment, use of mutagenic host cells, or DNA shuffling.

Another aspect of the invention comprises the steps of (a) culturing and lysing the microorganism of interest to obtain a microbial cell lysate; And (b) reacting the cell lysate with a reaction solution comprising glucose, NAD ⁺ , and β-galactose dehydrogenase, and measuring the amount of NADH produced, which is then determined using wild type UDP-galactose 4-epimerase. Screening of aldohexose epimerase comprising selecting a microorganism having aldohexose epimerase activity and obtaining aldohexose epimerase therefrom compared to the amount of NADH produced by the control cell lysate having Provide a method.

In the screening method of aldohexose epimerase according to the present invention, step (a) is a step of obtaining a cell lysate of the microorganism of interest. The microorganism of interest here is not particularly limited as long as the microorganism is expected to have aldohexose epimerase activity, and the microorganism into which the mutated UDP-galactose 4-epimerase gene is introduced or UDP-galactose 4-epi according to the present invention. It may be a microorganism obtained by mutating a microorganism having a merase gene. The gene encoding the mutated UDP-galactose 4-epimerase may be one obtained by artificial mutation of the wild type UDP-galactose 4-epimerase gene or as a result of natural mutation. For example, wild-type UDP-galactose 4-epimerase genes can be used as a template to perform error-prone PCR, treatment with chemicals or mutagens such as UV, DNA shuffling, or the like. The mutated UDP-galactose 4-epimerase gene can be obtained. In addition, the mutated UDP-galactose 4-epimerase gene is obtained in vitro and then introduced into the microorganism by a conventional method such as transformation, or a mutant material treatment or homology to a microorganism having a wild-type gene. It may be obtained in microbial cells by applying a technique such as recombination.

In addition, the mutated UDP-galactose 4-epimerase gene may be transformed into a microorganism in the form of a single or a plurality of mutant genes. When a plurality of variant genes are introduced into a plurality of microorganisms, a microbial population comprising a plurality of different variant genes may be formed. The microorganism includes prokaryotic and eukaryotic cells, preferably prokaryotic cells. One example of the microorganism is E. coli.

In one embodiment of the invention, the microorganism to be screened is transfected with a UDP-galactose 4-epimerase variant gene library obtained by mutation-induced PCR of a gene encoding a wild type UDP-galactose 4-epimerase into a template. Converted microorganisms, preferably E. coli.

At this time, the UDP-galactose epimerase variant library is composed of variants of wild type UDP-galactose 4-epimerase. The wild type UDP-galactose 4-epimerase may have a nucleotide sequence of SEQ ID NO: 2 derived from E. coli K-12 W3110.

Wild type UDP-galactose 4-epimerase (EC 5.1.3.2) has a portion that accommodates UDP at the active site as shown in FIG. 1, but variants selected by directional molecular evolution in accordance with the present invention are UDP Glucose is recognized as a substrate and does not have a recognized site and is converted to galactose.

In the method for screening aldohexose epimerase according to the present invention, step (b) is carried out by adding the microbial cell lysate obtained in step (a) to a reaction solution containing glucose, NAD ⁺ , and β-galactose dehydrogenase. Aldohexose 4-epimerase activity was measured after addition and the amount of NADH produced was measured and compared to the amount of NADH produced in the reaction with the lysate of control cells with wild type UDP-galactose 4-epimerase. It is to select a microorganism having a. When the amount of NADH measured from the microbial cells to be screened in the reaction is increased compared to the control cells, the microbial sample is determined to have aldohexose epimerase activity.

According to the present invention, the activity of aldohexose epimerase is obtained by adding cell lysate to the reaction solution containing glucose, NAD ⁺ , and β-galactose dehydrogenase, thereby converting glucose into galactose and subsequent galactose. It is assessed by measuring the amount of NADH produced through dehydrogenation of. The amount of NADH can be measured via absorbance at 340 nm. In addition, the cell concentration before preparation of the cell lysate used as a source of aldohex epimerase was determined by measurement of absorbance at 600 nm, and the absorbance per unit cell was calculated to estimate specific activity. Can be.

In the method according to the invention, aldohexose epimerase activity measured from microbial cells is higher than control compared to aldohexose epimerase activity measured from control microbial cells with wild type UDP-galactose 4-epimerase. The microbial cells showing activity are selected to have variants of UDP-galactose 4-epimerase evolved into aldohexose epimerase. At this time, by culturing the microorganisms selected to have aldohexose epimerase activity to purify the aldohexose epimerase, or directly to the source of aldohexose epimerase It can also be used.

The present invention also provides aldohexose epimerase obtained by the screening method of the present invention.

In one embodiment of the invention, the aldohexose epimerase may be a glucose epimerase, most preferably glucose 4-epimerase, having a particularly high substrate specificity for glucose.

In one embodiment of the invention, the aldohexose epimerase may have the amino acid sequence of SEQ ID NO: 3. The aldohexose epimerase may be encoded by the nucleotide sequence of SEQ ID NO.

The aldohexose epimerase having the amino acid sequence represented by SEQ ID NO: 3 is mutated to Val of Met No. 1 and Arg of 313 compared to the amino acid sequence of UDP-galactose 4-epimerase of the wild type E. coli strain (SEQ ID NO: 1). A nucleotide sequence of SEQ ID NO: 4, wherein the base sequence of SEQ ID NO: 4 is replaced with A of G and 937 C, relative to the nucleotide sequence (SEQ ID NO: 2) of the gene encoding UDP-galactose 4 epimerase of the wild type E. coli strain Substitution with G is included.

In one embodiment of the invention, the aldohexose epimerase is obtained by molecular evolution of UDP-galactose 4 epimerase and the activity assessed by the production of galactose from glucose is 0.02 of wild type UDP-galactose 4 epimerase. mmole / min. 0.1 to 1 mmole / min. mg protein, compared to less than mg protein.

The invention also provides a recombinant vector comprising a polynucleotide encoding an aldohexose epimerase according to the invention.

In the present invention, "vector" refers to a DNA construct comprising a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles, or potential genomic inserts. Once introduced into a suitable host by transformation, the vector can replicate and function independently of the host genome, or can be integrated into the genome.

The invention also provides a transformed microorganism obtained by introducing a recombinant vector comprising a polynucleotide encoding an aldohexose epimerase into a host cell selected from the group consisting of bacteria, yeast and fungi. In the present invention, the transforming microorganism may preferably be transformed E. coli.

The present invention also cultures a microorganism transformed by a recombinant vector comprising a polynucleotide encoding an aldohexose epimerase, and isolates the aldohexose epimerase from the cultured microorganism or culture medium to aldohexose. Provided are methods for producing epimerase.

The present invention is also directed to epidermalization from aldohexose using an aldohexose epimerase selected by the screening method according to the invention or an enzyme capable of epimerizing aldohexose among naturally occurring enzymes. Provided are methods for producing epimeric aldohexose thereof.

In the aldohexose production method according to the present invention, aldohexose epimerase is a form isolated by purification from strain culture selected as having aldohexose epimerase activity by molecular evolution or the cultured strain or strain It may be in the form of a solution of.

In the aldohexose production method according to the invention, the aldohexose epimerase may have an amino acid sequence of SEQ ID NO: 3.

In the aldohexose production method according to the present invention, the epimerization reaction by aldohexose epimerase is carried out at a pH of 7 to 10 and a temperature of 30 to 50 ° C, preferably of a pH of 8 to 10 and a temperature of 35 to 40 ° C. Most preferably at a pH of 8.6 and a temperature of 37 ° C.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention and should not be construed as limiting the invention.

Example 1.Preparation of variant libraries of UDP-galactose 4-epimerase (EC 5.1.3.2) gene

Mutation-induced PCR using the primers of SEQ ID NOS: 5 and 6 and the Random mutagenesis kit (CloneTech Co.) with a template of the gene galE (SEQ ID NO: 2) encoding UDP-galactose 4-epimerase of E. coli K-12 W3110 Was performed to amplify the mutated galE gene at a frequency of about 2.5 mutations / kb. The PCR reaction was performed 25 cycles consisting of 1 minute denaturation at 95 ° C., 30 seconds annealing at 60 ° C. and 1 minute elongation at 72 ° C. The PCR product was treated with the restriction enzymes NcoI and XbaI and ligated to pTrc99A (GenBank U13872, AP Biotech. Co.) treated with the same restriction enzyme to prepare a recombinant vector.

The recombinant vector was mixed with competent E. coli DH5α prepared for transformation, and the vector was introduced by applying a current for 5 seconds under a voltage of 2.5 kV with an electroporator (Gene Pulser, Bio-Rad). Transformed Escherichia coli was prepared and transformed Escherichia coli was selected by smearing in LB medium containing 20 μg / ml ampicillin. Thus, the recombinant vector was introduced into E. coli DH5α to prepare a gene library consisting of variants of the mutated UDP-galactose 4-epimerase gene.

Example 2 Screening of Aldohexose 4-Epimerase

In this example, the activity of aldohexose 4-epimerase was screened for screening for aldohexose 4-epimerase from the UDP-galactose 4-epimerase variant library prepared in Example 1.

Determination of Aldohexose 4-epimerase Activity

E. coli DH5α from the library prepared in Example 1 was plated in LB (Luria Bertani) plate medium containing ampicillin (20 μg / mL) and incubated at 37 ° C. for 16 hours to obtain colonies. The obtained colonies were transferred to a master plate and used for measuring enzymatic activity as follows.

First, 50 μl of LB medium containing 20 μg / ml of ampicillin and 0.1 mM of IPTG (Isopropyl-thiogalactoside) per 96 well plate was added, inoculated with each colony, and incubated for 3 hours in a 37 ° C. incubator, followed by 600 nm. Absorbance (A1) was measured at. Thereafter, 10 µl of cell lysis buffer (Triton-X 100 0.05 mM) was added and allowed to stand at 37 ° C for 15 to 20 minutes. Then 150 μl of Buffer A (20 μl 10 mM glucose + 0.3 mM NAD ⁺ 20 μl + β-galactose dehydrogenase (0.35U) 1 μl + Tris-HCl (50 mM, pH 8.6) 109 μl) was added. Absorbance (A2) was measured at 340 nm. After reacting the reaction solution at 37 ° C. for 20 minutes, absorbance (A3) was measured again at 340 nm, and from this, the activity per cell per unit time of aldohexose 4-epimerase was determined as (A3-A2) / A1 / 20. Calculated in minutes.

Aldohexose 4-epimerase activity was measured in 940 colonies as described above, and clones showing higher activity than the control group were selected as compared to the activity obtained from the control group consisting of E. coli DH5α containing wild type galE. . The activity of epimerization from glucose to galactose, ie, aldohexose 4-epimerase, was less than 0.02 mmole / min.mg protein in the control wild-type galE and was higher than the control in the variant library. Dog clones were selected. The highest activity by the selected clones was 1 mmole galactose / min.mg protein.

Example 3 Conversion of Glucose to Galactose by Aldohexose 4-Epimerase Obtained by Directed Molecular Evolution

Colonies selected in Example 2 were suspended in lysis buffer (Triton-X100 0.05 mM) to obtain cell lysates containing crude enzyme. The cell lysate was added to a 30 mM glucose solution in Tris-HCl (pH 8.6) to 10% (v / v), and the prepared reaction solution was placed in a 1 mL vial and maintained at 37 ° C for 30 minutes. Thereafter, the vial was put on ice to stop the reaction. 10 μl of the reaction solution was injected into HPLC (Waters) equipped with an Aminex-87P column (Bio-Rad) and a refractive index (RI) detector. In the HPLC analysis, distilled water, which is a mobile phase, was flowed at a rate of 0.6 ml / min, and the column temperature was maintained at 85 ° C. to obtain a chromatogram as shown in FIG. 2.

As shown in FIG. 2, a peak in which a part of glucose was converted to galactose was detected in a sample of the reaction solution, whereas a peak of galactose was found in the control group using a lysate of a strain having wild-type UDP-galactose 4-epimerase. It was not detected.

Example 4 Characterization of Selected Aldohexose 4-Epimerase

(1) Analysis of genetic information of aldohexose 4-epimerase obtained by directional molecular evolution

Of the clones selected in Example 2, three of the highest activity were selected and the aldohexose 4-epimerase was separated therefrom, and the base sequence and amino acid sequence were analyzed. As a result, the amino acid sequence and base sequence of the aldohexose 4-epimerase isolated from the strain showing the highest activity were identified by SEQ ID NO: 3 and 4, respectively.

The amino acid sequence of SEQ ID NO: 3 confirmed in the present Example is a variation of Met No. 1 to Val and 313 Arg to Gly compared to the amino acid sequence of UDP-galactose 4-epimerase of the wild type E. coli strain (SEQ ID NO: 1) Wherein, the nucleotide sequence of SEQ ID NO: 4 is replaced with G of No. 1 A and G of 937 C with respect to the nucleotide sequence (SEQ ID NO: 2) of the gene encoding UDP-galactose 4 epimerase of the wild type E. coli strain Included.

(2) determination of optimum pH

Three clones showing the highest activity among the clones selected in Example 2 were selected to determine the optimal pH for the activity of the aldohexose 4-epimerase. The measurement of aldohexose 4-epimerase activity was carried out by the method described in Example 2 except that the buffer was changed depending on pH. 50 mM phosphate-NaOH buffer at pH 7-8, 50 mM Tris-HCl at pH 8-9 and 50 mM borate-NaOH buffer at pH 9-10 were used. Specifically, 1 mM of the reaction solution was prepared by adding 50 mM glucose and 10% (v / v) of the enzyme solution derived from the clones selected above to each pH buffer to induce an epimerization reaction, and then in the reaction solution. 100 μl was mixed with 900 μl pH 8.6 Tris-HCl buffer, NAD ⁺ , 1 unit of galactose dehydrogenase to determine the amount of galactose formed in the reaction solution.

As a result, as shown in FIG. 3, clone 1 (black circle) showed a sharp increase in activity up to pH 8 and then increased activity with increasing pH with low slope, and clone 2 (square) and Clone 3 (triangle) showed lower overall activity than clone 1 and showed optimal activity at pH 8.6.

 (3) determination of optimum temperature

In clone (1) showing the highest activity in the above (2) was confirmed the optimum temperature for the enzyme activity. The measurement of aldohexose 4-epimerase activity was carried out by the method described in (2) above except that the reaction temperature was changed to 30-50 ° C. As shown in Figure 4, the optimum temperature was 37 ° C.

Galactose, one of relatively high value added monosaccharides, can be prepared by decomposing expensive raw materials, lactose, which are generally components of milk. Theoretically, it is possible to prepare UDP-galactose by using UDP-galactose epimerase by attaching UDP to glucose and then removing UDP. However, this method has a disadvantage in that the process of attaching and detaching the UDP is very cumbersome, and the use of expensive UDP increases the process cost, making it difficult to realize in reality. As such, there is a problem in that a method of preparing aldohexose is complicated, such as using an expensive raw material or obtaining a specific variant that can be recognized by an enzyme present in nature. Accordingly, the present invention provides an economical method for producing high value-added epimeric aldohexose from inexpensive aldohexose, for example, an enzymatic method for producing galactose from glucose, thereby providing high value sugars at low cost. Industrial utility is very high because it can be produced.

Claims (16)

1. A method of enzymatic preparation of an aldohexose epimer, comprising the step of obtaining an aldohexose epimer by subjecting the aldohexose to an epimerase enzyme to epimerize it.
2. The method of claim 1, wherein the aldohexose is an aldohexose selected from the group consisting of glucose, allose, altrose, mannose, gulose, idose, galactose, and talos.
3. The method of claim 1, wherein the aldohexose is D-glucose and the aldohexose epimer is galactose.
4. The method of claim 1, wherein the epimerization is C-4 epimerization of aldohexose.
5. The method of claim 1, wherein the epimerase enzyme treatment is carried out at a pH of 7 to 10 and a temperature of 30 to 50 ℃.
6. The method according to any one of claims 1 to 5, wherein the epimerase enzyme is an aldohexose epimerase obtained by directional molecular evolution of wild type UDP-galactose 4-epimerase. .
7. Aldohexose epimerase, which performs epimerization at the carbon position of aldohexose.
8. 8. The aldohexose epimerase of claim 7, wherein the aldohexose epimerase is obtained by directional molecular evolution of UDP-galactose 4-epimerase.
9. The method of claim 8, wherein the directional molecular evolution is achieved by error-prone PCR, treatment with chemicals or mutagens such as UV, use of mutagenic host cells, or DNA shuffling. Aldohexose epimerase.
10. The aldohexose epimerase according to claim 8, wherein the aldohexose epimerase has an amino acid sequence represented by SEQ ID NO: 3.
11. The method according to any one of claims 7 to 10, wherein the aldohexose is aldohexose selected from the group consisting of glucose, allose, altrose, mannose, gulose, idose, galactose and talos. Characterized by aldohexose epimerase.
12. The aldohexose epimerase according to any one of claims 7 to 10, wherein the aldohexose epimerase epimerizes D-glucose into galactose.
13. (a) culturing and lysing the microorganism of interest to obtain a microbial cell lysate; And

    (b) reacting the cell lysate with a reaction solution comprising glucose, NAD ⁺ , and β-galactose dehydrogenase, and then measuring the amount of NADH produced, and determining the wild type UDP-galactose 4-epimerase. Selecting microorganisms having aldohexose epimerase activity as compared to the amount of NADH produced by the control cell lysates with which the aldohexose epimerase is obtained:

    Screening method of aldohexose epimerase comprising a.
14. The method of claim 13, wherein the microorganism of interest is a microorganism obtained by mutating a microorganism into which a mutated UDP-galactose 4-epimerase gene has been introduced or a microorganism having a UDP-galactose 4-epimerase gene. Way.
15. The method of claim 14, wherein the mutation is an error-prone PCR, treatment with a chemical or mutagenesis such as UV, use of a mutagenesis host cell, or a DNA shuffling. Screening method.
16. An aldohexose epimerase screened according to the method of any one of claims 13-15.

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