CN111944796A

CN111944796A - D-mannose isomerase and application thereof

Info

Publication number: CN111944796A
Application number: CN202010811740.9A
Authority: CN
Inventors: 金鹏; 杜琪珍; 王圆圆
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2020-11-17
Anticipated expiration: 2040-08-13
Also published as: CN111944796B

Abstract

The invention discloses a D-mannose isomerase and application thereof, belonging to the technical field of bioengineering. The method adopts D-mannose isomerase coding THMI from thermophilic bacteria Thermobifida halotolerans, the THMI is recombined to an escherichia coli expression system to realize high-efficiency activity expression, the THMI enzyme of the recombinant expression realizes 36% mannose conversion under the conditions of a substrate D-fructose of 500g/L, a reaction temperature of 65 ℃ and a pH value of 9, meanwhile, the recombinant strain carries out whole-cell catalysis on fructose, and 31% mannose conversion is realized under the conditions of a substrate D-fructose of 500g/L, a reaction temperature of 60 ℃ and a pH value of 9. The invention lays a certain foundation for efficiently preparing mannose and is suitable for industrial production and application.

Description

D-mannose isomerase and application thereof

Technical Field

The invention relates to a D-mannose isomerase and application thereof, belonging to the technical field of biological engineering.

Background

D-mannose is a natural bioactive monosaccharide, is a common food nutritional dietary supplement, can be particularly used as a nutritional dietary supplement affecting carbohydrate nutrients, and has a great contribution to human beings. D-mannose is a monomer unit in polysaccharide mannan, is an important component of many glycoproteins, and thus can be widely found in various foods in theory. The D-mannose has prebiotic effect, and can promote anti-inflammatory and anti-inflammatory cytokine expression, and as supplement, the D-mannose can reduce Salmonella typhimurium contamination, prevent urinary tract infection, and treat phosphomannose isomerase deficiency. D-mannose is widely used as a basic material for synthesizing vitamins, immunostimulating drugs and antitumor drugs. In general, D-mannose is a dietary supplement affecting the contribution of sugar nutrients to human health, and should be widely used in food products in view of its beneficial effects on human health.

In nature, D-mannose is usually present as a component of cellulose, hemicellulose, or mannan in various cell wall structures, which are produced by breakdown of hemicellulose by extracellular xylanases. At present, the D-mannose synthesis method mainly comprises an extraction separation method, a chemical synthesis method and an enzymatic preparation method, wherein the extraction separation method is used for extracting and separating mannose from plants, and the method has the disadvantages of complex process and low yield and purity; the chemical synthesis method is to obtain D-mannose by catalyzing isomerization of glucose, and the method usually needs complicated purification steps, easily causes solvent residue and then leads to generation of byproducts; the method for preparing D-mannose by the enzyme method takes D-fructose or D-glucose as a substrate and obtains the D-mannose by using biological enzyme catalysis, and the method has more and more important position in the preparation of the D-mannose, and has the advantages of mild reaction condition, less by-products, easy separation and purification, no environmental pollution, low cost and the like.

There are three major classes of D-mannose producing enzymes currently in use, which are divided into cellobiose-2-epimerase (EC 5.1.3.11), D-xylose isomerase (EC 5.3.1.15) and D-mannose isomerase (MIase, EC 5.3.1.7). The cellobiose-2-epimerase production can catalyze D-fructose or D-glucose as a substrate to produce D-mannose; and D-xylose isomerase and D-mannose isomerase catalyze the substrate D-fructose to generate D-mannose. A large number of three isomerases have been reported and characterized, however, most of them have a D-mannose conversion rate of less than 25%, a low substrate concentration and a long reaction time (several hours to 14 days). The highest invertase reported in the literature is derived from a mesophilic enzyme of the m. smegmatis strain, which achieves 35% conversion at low substrate concentration (6 uM). Therefore, these enzymes are limited by the conversion rate and the substrate concentration, and the scale-up production is difficult to realize. Therefore, the method for efficiently preparing the D-mannose under the high substrate concentration is obtained, and has good application value and commercial development significance.

Disclosure of Invention

In order to efficiently prepare D-mannose at a high substrate concentration, the invention provides a D-mannose isomerase, and the amino acid sequence of the D-mannose isomerase is shown as SEQ ID No. 1.

The invention also provides a recombinant escherichia coli for producing the D-mannan isomerase, wherein the recombinant escherichia coli takes escherichia coli as a host to express the D-mannose isomerase with an amino acid sequence shown as SEQ ID No. 1.

In one embodiment of the invention, the recombinant escherichia coli takes pET21a as an expression vector and E coli BL21(DE3) as a host.

The invention provides a method for preparing D-mannose, which takes D-mannose isomerase with an amino acid sequence shown as SEQ ID No.1 as a catalyst, takes D-fructose as a substrate, and adds the D-mannose isomerase with the amino acid sequence shown as SEQ ID No.1 into a reaction system containing the D-fructose for catalysis to obtain the D-mannose.

In one embodiment of the present invention, the nucleotide sequence of the D-mannose isomerase is shown as SEQ ID NO. 2.

In one embodiment of the present invention, the D-mannose isomerase is added in an amount of not less than 80U/mL.

In one embodiment of the present invention, the concentration of D-fructose in the reaction system is 1 to 700 g/L.

In one embodiment of the present invention, the concentration of D-fructose in the reaction system is 300-600 g/L.

In one embodiment of the present invention, in the reaction system, the reaction temperature is 20 to 80 ℃ and the pH is 2 to 11.

In one embodiment of the present invention, the reaction temperature in the reaction system is 65 ℃ and the pH is 9.

The invention also provides a method for producing D-mannose by whole-cell transformation, which takes the recombinant escherichia coli as a catalyst and the D-fructose as a substrate, and adds the recombinant escherichia coli into a reaction system containing the D-fructose for transformation to obtain the D-mannose.

In one embodiment of the present invention, the recombinant Escherichia coli is added to a solution of D-fructose with a concentration of 100-600g/L to an OD600 of 0.1-25, and then transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

In one embodiment of the present invention, the recombinant Escherichia coli is added to a D-fructose solution with a concentration of 300-600g/L until the OD600 is 10, and then the mixture is transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

The invention also provides the application of the D-mannose isomerase, the gene coding the D-mannose isomerase, the recombinant plasmid containing the gene coding the D-mannose isomerase, the host cell carrying the gene coding the D-mannose isomerase, or the method for preparing D-mannose in preparing D-mannose or products containing D-mannose.

Has the advantages that:

(1) the thermophilic D-mannose isomerase THMI expressed by the recombinant expression method realizes the high-efficiency active expression of the D-mannose isomerase by carrying out heterologous expression on the thermophilic D-mannose isomerase coding gene THMI derived from the thermophilic bacteria Thermobifida halolerans, is easy to ferment in a large scale and separate and purify products due to the strong protein secretion capacity of the escherichia coli, and realizes the high-efficiency conversion of 36% by using a high-concentration D-fructose substrate, which is the highest conversion rate reported at present. The method has simple operation process and high product purity, and is easy to realize industrial production of mannose.

(2) The invention utilizes the escherichia coli to produce mannose isomerase, and compared with other mannose isomerase, the mannose isomerase has great application advantages, high temperature resistance and high conversion rate. In addition, the host escherichia coli used in the process has stronger growth capacity, the extracellular secretion impurities are less, and the product is easy to separate and purify; secondly, high-concentration fructose is used as a reaction substrate, and mannose can be efficiently produced through catalysis.

Drawings

FIG. 1: protein expression was analyzed by SDS-PAGE.

FIG. 2: effect of different temperatures on the activity of D-mannose isomerase.

FIG. 3: effect of different pH values on the activity of D-mannose isomerase.

FIG. 4: effect of different substrate concentrations on the activity of D-mannose isomerase.

FIG. 5: the enzyme method is used for catalyzing the conversion rate of a high-concentration substrate D-fructose.

FIG. 6: effect of different final cell concentrations on the conversion of D-fructose catalyzed by whole cells.

FIG. 7: effect of different substrate concentrations on the conversion of D-fructose catalyzed by whole cells.

Detailed Description

The pET21a plasmid and E.coli BL21(DE3) referred to in the examples below were purchased from Stratagene, La Jolla, Calif., USA.

The media involved in the following examples are as follows:

LB liquid medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 100 mu g/mL of ampicillin concentration.

LB solid medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride, 100 mu g/mL of ampicillin concentration and 15g/L of agar.

D-mannose content determination:

d-mannose was quantitatively analyzed by high performance liquid chromatography HPLC. HPLC is Shimadzu LC20A system (Shimadzu, Japan) consisting of two LC-10A pumps, a SIL-10Avp autosampler, an evaporative light scattering detector and a Luna Amino 5-. mu.m 100A Amino column (250X 4.60mm) (Phenomenex). The mobile phase is acetonitrile/water (80: 20), the sample injection amount is 10ul, and the constant flow rate is 1.0 mL/min; column temperature: 30 ℃, detection wavelength: 250nm, D-mannose standard as quantitative standard sample.

D-mannose isomerase enzyme activity assay:

for precise determination of the enzyme activity, the reaction volume was 1mL, containing 50mM Tris-HCl buffer (pH 9.0), 500g/L D-fructose and appropriate amount of purified enzyme and addition of 1mM Na⁺And uniformly mixing the reaction system, and placing the mixture in a water bath at 65 ℃ for 30 minutes. One unit of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol D-mannose per minute at 60 ℃ and pH 9.0. Enzyme specific activity is defined as the number of units of enzyme contained per mg of pure enzyme protein.

Example 1: acquisition of D-mannose isomerase and construction of recombinant escherichia coli

1. Obtaining N-acyl-D-glucopyranosamine 2-epimerase (N-acetyl-D-glucosamine 2-epimerase) with a nucleotide sequence shown as SEQ ID NO.2 from an NCBI genome database; N-acyl-D-glucopyranosamine 2-epimerase (N-acetyl-D-glucosamine 2-epimerase) is analyzed by NCBI/Basic Local Alignment Search Tool, the homology of the enzyme and D-mannose isomerase is less than 50%, and the enzyme coded by the sequence has a far-away evolutionary relationship with a D-mannose isomerase family.

2. Artificially synthesizing a target gene (the nucleotide sequence is shown as SEQ ID NO. 2), designing a recombinant primer, and connecting the target gene and pET21a plasmid after restriction enzyme digestion by restriction enzymes NdeI and XhoI to obtain a connection product; transforming E.coli JM109 with the ligation product to obtain a transformation product, coating the transformation product on an ampicillin-containing LB solid medium, performing inverted culture in a 37 ℃ constant temperature incubator for 8-12 h to obtain a transformant, selecting the transformant, inoculating the transformant to an ampicillin-containing LB liquid medium, performing shake flask culture at 37 ℃ and 120-180 rpm for 8-12 h, extracting plasmids, performing enzyme digestion verification and sequencing verification, and obtaining the recombinant plasmid pET21 a-target gene after verification is correct.

And (3) primer F: 5'-CGGCATATGAACCCGTGGACCACCCGGACCGCGCAC-3' (SEQ ID NO.3) primer R: 5'-CCGCTCGAGGCCGTCTGCGGCCAGTGCCCCGGCCAG-3' (SEQ ID NO.4)

Transforming a recombinant plasmid pET21 a-target gene to Escherichia coli E.coli BL21(DE3), carrying out inverted culture in a constant temperature incubator at 37 ℃ for 8-12 h on an LB solid culture medium containing ampicillin to obtain a transformant, selecting the transformant, inoculating the transformant to an LB liquid culture medium containing ampicillin, carrying out shake-flask culture at 37 ℃ and 120-180 rpm for 8-12 h, extracting a plasmid, and carrying out enzyme digestion verification and sequencing verification to verify the correctness, namely the recombinant Escherichia coli E.coli BL21(DE3)/pET21 a-target gene.

Example 2: shake flask fermentation for producing enzyme

The method comprises the following specific steps:

(1) inoculating a single colony of the recombinant Escherichia coli E.coli BL21(DE3)/pET21 a-target gene obtained in example 1 into a test tube of 5mL LB liquid culture medium containing 100ug/mL ampicillin, and placing the test tube at 37 ℃ and under the condition of 200rpm for shaking culture for 12-14h to obtain a seed solution;

(2) transferring 0.5mL of the seed solution obtained in the step (1) into a shake flask containing 50mL of fresh LB liquid medium (containing 100ug/mL of ampicillin), and placing the shake flask at 37 ℃ and under the condition of 200rpm for shake culture; culturing until OD600 value is 0.4-0.5, adding IPTG with final concentration of 0.1mM into the shake flask, placing the shake flask at 15 deg.C and 200rpm for low temperature induction expression of protein, and culturing for 24h to obtain fermentation liquor;

(3) centrifuging the fermentation liquor obtained in the step (2) for 30min at 5000rpm, filtering to obtain thalli, adding 12.5mL of 50mM PBS (pH 7.0) into each 100mL of thalli obtained from the bacterial liquid, and resuspending the thalli to obtain a mixed liquor;

(4) carrying out ultrasonic treatment on the mixed solution obtained in the step (3) for 7.5min, crushing bacterial cells by adopting ultrasonic waves to obtain a cell crushing solution, carrying out SDS-polyacrylamide gel protein electrophoresis verification on the obtained cell crushing solution, and analyzing the expression quantity and purity of the target protein, wherein the result is shown in figure 1;

(5) centrifuging the cell disruption solution (10000rpm, 4 ℃) for 10min, filtering, separating to obtain cell disruption solution sediment and cell disruption solution supernatant, respectively performing SDS-polyacrylamide gel protein electrophoresis verification on the cell disruption solution sediment and the cell disruption solution supernatant, and analyzing the expression quantity and the purity of the target protein, wherein the result is shown in figure 1;

using the method of example 1-2, except that pET21a was not linked to the target gene, a cell disruption solution of pET21a/BL21(DE3) was prepared; performing SDS-polyacrylamide gel protein electrophoresis verification on the obtained cell disruption solution, and analyzing the expression amount and purity of the target protein, wherein the result is shown in figure 1;

as shown in fig. 1: taking a cell disruption solution of pET21a/BL21(DE3) under the same culture conditions as a control, wherein a protein band is obviously added at a position of about 45.5kDa in a whole cell disruption solution and a supernatant of a fine cell disruption solution of E.coli BL21(DE3)/pET21 a-target genes, and no obvious band is found at the position by cell disruption solution precipitation;

further carrying out D-fructose catalytic reaction on supernatant of cell disruption solution of E.coli BL21(DE3)/pET21 a-target gene, wherein the reaction system is 1mL, the reaction system contains 100g/L of D-fructose, 100 mu L of supernatant of cell disruption solution has pH of 9.0, uniformly mixing, carrying out reaction for 1h at 60 ℃, inactivating enzyme by boiling water bath after the reaction is finished, and appropriately diluting for next step HPLC determination;

the results, as detected by HPLC, show: coli BL21(DE3)/pET21 a-target gene cell disruption liquid supernatant and D-mannose standard substance have absorption peaks at the wavelength of 250 nm; and preliminarily judging that the target protein is D-mannose isomerase by combining the electrophoresis verification result of the SDS-polyacrylamide gel protein.

(6) D-mannose isomerase biopsy is carried out on the E.coli BL21(DE3)/pET21 a-target gene cell disruption solution sediment obtained in the step (5) and the cell disruption solution supernatant, and the result shows that no enzyme activity is detected in the cell disruption solution sediment; the activity of the D-mannose isomerase in the supernatant of the cell disruption solution is 812U/mL.

From the results, it was found that the mannose-converting enzyme activity of the supernatant of the E.coli BL21(DE3)/pET21 a-target gene cell disruption solution was 812U/mL, and it was found that N-acyl-D-glucosamine2-epimerase (N-acetyl-D-glucosamine 2-epimerase) and D-mannose isomerase had very low homology, but they did have the enzyme activity of D-mannose isomerase, and thus N-acyl-D-glucosamine2-epimerase (N-acetyl-D-glucosamine 2-epimerase) was used as D-mannose isomerase.

Example 3: purification of D-mannose isomerase

The method comprises the following specific steps:

(1) the supernatant of the cell disruption solution of E.coli BL21(DE3)/pET21 a-objective gene obtained in step (5) of example 2 was filtered through a 0.45 μm cellulose acetate membrane to obtain a filtrate.

(2) The column was mounted, washed repeatedly several times with distilled water, 50mL of PBS buffer solution of pH 7.0 was added to the column, and allowed to stand for 4 hours. Adding the filtrate obtained in the step (1) into a column, fully and uniformly mixing, stirring by using a glass rod at the temperature of 4 ℃, uniformly mixing the filtrate with Ni-NTA agarose gel, stirring once for 20min, stirring for 8 times, and finishing after 4 h; the supernatant was eluted at a slower flow rate, 50mL of imidazole PBS buffer pH 7.0 at 0mM, 10mM, 20mM, 30mM, 50mM, respectively, was added to remove impure proteins, leaving a single protein, and finally 500mM of imidazole PBS buffer pH 7.0 was added to elute the target protein.

(3) Immediately dialyzing the target protein obtained in the step (2), wherein the dialysis buffer solution is PBS (pH 7.0), replacing the dialysate once for 3h and replacing for 3-4 times, and the whole purification and dialysis processes are carried out at 4 ℃ to obtain the purified D-mannose isomerase.

SDS-PAGE protein electrophoresis detection is carried out on the purified D-mannose isomerase, the result is shown in figure 1, the D-mannose isomerase SDS-polyacrylamide gel protein electrophoresis result shows that the size is 45.5kDa and is consistent with the theoretical molecular size, and the high-efficiency activity expression of the D-mannose isomerase protein is proved, and the protein purification band is single.

Example 4: determination of influencing factors in D-mannose isomerase-catalyzed reaction

The enzymatic determination and analysis of D-mannose isomerase by using D-fructose as a substrate comprises the following specific steps:

(1) effect of temperature on D-mannose isomerase catalyzed reactions

The reaction system is 1mL, wherein the substrate concentration is 500g/L, the purified D-mannose isomerase obtained in example 3 is 0.1mL, the reaction pH is 7.0, the reaction systems are respectively reacted at 20 ℃, 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃ and 80 ℃, the reaction time is 1h, the enzyme activity at each reaction temperature is respectively measured after the reaction is finished, and the result is shown in figure 2, and the optimal reaction temperature of the enzyme is 65 ℃.

(2) Effect of pH on D-mannose isomerase catalyzed reactions

The reaction system is 1mL, wherein the substrate concentration is 500g/L, the pure enzyme is 0.1mL, the reaction temperature is 65 ℃, the reaction system is respectively reacted under the conditions of pH 2.0, pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH9.0, pH 10.0 and pH 11.0, the reaction time is 1h, the enzyme activity under each pH condition is respectively measured after the reaction is finished, and the result is shown in figure 3, and the result shows that the optimum reaction pH of the enzyme is 9.0.

(3) Effect of substrate concentration on D-mannose isomerase catalyzed reactions

The reaction system is 1mL, wherein the pure enzyme is 0.1mL, the reaction temperature is 65 ℃, the reaction pH value is 9.0, the reaction is carried out under the conditions that the concentrations of D-fructose substrates are 0.1g/L, 1g/L, 5g/L, 25g/L, 50g/L, 100g/L, 200g/L, 300g/L, 400g/L, 500g/L, 600g/L and 700g/L respectively, the reaction time is 1h, the enzyme activity under the conditions of the substrate concentrations is respectively measured after the reaction is finished, and the result is shown in FIG. 4, and the result shows that the optimal substrate concentration of the enzyme reacts with 500 g/L.

EXAMPLE 5 enzymatic catalysis of D-fructose to D-mannose

The method comprises the following specific steps:

adding the purified D-mannose isomerase obtained in example 3 into D-fructose solutions with concentrations of 300g/L, 400g/L, 500g/L and 600g/L respectively according to the addition amount of 80U/mL, and reacting for 1h at the temperature of 65 ℃ and the pH of 9.0 to obtain a reaction solution;

detecting the content of D-mannose in the reaction solution, and calculating the mannose conversion rate; the calculation method of the mannose conversion rate comprises the following steps: the conversion (%) (content of produced D-mannose/content of total D-fructose) was 100%, and the results are shown in fig. 5.

As can be seen from FIG. 5, the conversion rates of mannose obtained under the conditions of substrate concentrations of 300g/L, 400g/L, 500g/L and 600g/L were 30%, 32%, 36% and 31%, respectively. This indicates that the THMI enzyme has very high conversion rate, which is obviously higher than the maximum 25% conversion rate of other mannose isomerase currently reported (Zhang et al, 2019, Process Biochemistry 83: 131-.

Example 6: whole-cell catalysis D-fructose preparation D-mannose

The method comprises the following specific steps:

the fermentation broth obtained in step (2) of example 2 was centrifuged to collect bacterial cells, the collected bacterial cells were washed twice with 50mM PBS buffer, and then resuspended in pre-cooled 50mM PBS buffer (pH 7.0) at a cell density of OD600 ═ 100, to obtain a resuspended cell broth;

the resuspended cell fluid was added to a D-fructose solution with a concentration of 500g/L until OD600 was 0.1, 1, 5, 10, 25, respectively, and then the mixture was transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

The content of D-mannose in the reaction solution was measured and the conversion of D-mannose was calculated, and as a result, as shown in FIG. 6, the conversion was 4.9%, 13.3%, 21.2%, 31.5%, and 29.8% for OD600 final concentrations of 0.1, 1, 5, 10, and 25, respectively, and the result showed that the optimal reaction condition was achieved for OD600 final concentration of 10.

Example 7: whole-cell catalysis D-fructose preparation D-mannose

The method comprises the following specific steps:

the resuspended cell fluid was added to D-fructose solutions at concentrations of 100g/L, 200g/L, 300g/L, 400g/L, 500g/L and 600g/L, respectively, to an OD600 of 10, and then the mixture was transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

As a result of measuring the content of D-mannose in the reaction solution and calculating the conversion rate of D-mannose, as shown in FIG. 7, the conversion rates of mannose obtained under the conditions of substrate concentrations of 100g/L, 200g/L, 300g/L, 400g/L, 500g/L and 600g/L were 28%, 27.8%, 30.1%, 30%, 31.5% and 31.3%, respectively, and the result showed that the conversion rate of mannose obtained under the condition of substrate concentration of 500g/L was the best.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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<120> D-mannose isomerase and application thereof

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Claims

1. A D-mannose isomerase is characterized in that the amino acid sequence of the D-mannose isomerase is shown as SEQ ID NO. 1.

2. The recombinant escherichia coli for producing the D-mannan isomerase is characterized in that the recombinant escherichia coli takes escherichia coli as a host to express the D-mannose isomerase with an amino acid sequence shown as SEQ ID No. 1.

3. A process for producing D-mannose, which comprises adding the D-mannose isomerase of claim 1 to a reaction system containing D-fructose to catalyze the reaction system using the D-mannose isomerase of claim 1 as a catalyst and D-fructose as a substrate to obtain D-mannose.

4. The method for preparing D-mannose according to claim 3, wherein the amount of the D-mannose isomerase added is not less than 80U/mL.

5. The process for producing D-mannose according to claim 4, wherein the concentration of D-fructose in the reaction system is 1 to 700 g/L.

6. The method for preparing D-mannose according to claim 5, wherein the reaction temperature is 20 to 80 ℃ and the pH is 2 to 11 in the reaction system.

7. A method for producing D-mannose by whole-cell transformation, which is characterized in that the recombinant Escherichia coli of claim 2 is used as a catalyst, D-fructose is used as a substrate, and the recombinant Escherichia coli of claim 2 is added into a reaction system containing the D-fructose for transformation to obtain the D-mannose.

8. The method as claimed in claim 7, wherein the recombinant Escherichia coli of claim 2 is added to a solution of D-fructose having a concentration of 100-600g/L to an OD600 of 0.1-25, and then transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

9. The method as claimed in claim 7 or 8, wherein the recombinant Escherichia coli of claim 2 is added to a D-fructose solution having a concentration of 300-600g/L to an OD600 of 10, and then transformed at pH9.0 and a temperature of 60 ℃ for 1 hour to obtain a reaction solution.

10. Use of the D-mannose isomerase of claim 1, or a gene encoding the D-mannose isomerase of claim 1, or a recombinant plasmid containing the gene encoding the D-mannose isomerase of claim 1, or a host cell carrying the gene encoding the D-mannose isomerase of claim 1, or the method for producing D-mannose of any one of claims 3 to 6, or the method for producing D-mannose of any one of claims 7 to 9, for producing D-mannose or a product containing D-mannose.