CN112481245B - Recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof - Google Patents

Abstract

The invention provides a recombinant D-tagatose 3 epimerase DTE-CM as well as a construction method and application thereof, belonging to the technical field of bioengineering; in the invention, a gene engineering means is utilized to construct and recombine D-tagatose 3 epimerase, and a nucleotide sequence of the D-tagatose 3 epimerase is subjected to codon optimization, so that the recombined D-tagatose 3 epimerase has excellent enzymological characteristics, is a good source enzyme for preparing high-added-value rare sugar D-psicose, and is more beneficial to the industrial production of the D-psicose.

Description

Recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant D-tagatose 3 epimerase DTE-CM as well as a construction method and application thereof.

Background

D-psicose, also called D-ribose-2-hexulose, is a C-3 epimer of D-fructose, not only has the characteristic of low calorific value, but also has good application in the fields of blood sugar reduction, hyperlipidemia resistance, inflammation diminishing and nerve protection. In addition, D-psicose is also active against active oxygen. Therefore, D-psicose can be applied to the healthcare industry as a therapeutic agent.

However, D-psicose is contained in a small amount in nature and is currently synthesized mainly by chemical and biological methods. In the chemical method, D-psicose is synthesized by hydrogenation, addition reaction, ferrier rearrangement and the like, but a catalyst is required to be added in the synthesis process. The cost is high, the process is complex, the side reactions are more, the environment is not friendly, and the large-scale production is not facilitated. Biological methods produce D-psicose by conversion using enzymatic methods, such as the family of D-tagatose 3 epimerases (DTEase). The enzymatic synthesis of D-psicose can effectively overcome the defects of the chemical method, but because an acidic environment is needed to inhibit the browning effect of saccharides and reduce the formation of byproducts when the D-psicose is produced, DTEase family enzymes in the prior art are mostly high in enzyme activity at alkaline and low temperature, so that the application of the DTEase family enzymes in the biological production of the D-psicose is greatly limited.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a recombinant D-tagatose 3 epimerase, a construction method and application thereof. In the invention, a gene engineering means is utilized to construct and recombine D-tagatose 3 epimerase, and a nucleotide sequence of the D-tagatose 3 epimerase is subjected to codon optimization, so that the recombined D-tagatose 3 epimerase has excellent enzymological characteristics, is a good source enzyme for preparing high-added-value rare sugar D-psicose, and is more beneficial to the industrial production of the D-psicose.

The invention firstly provides a recombinant D-tagatose 3 epimerase DTE-CM, wherein the nucleotide sequence of the gene for coding the recombinant D-tagatose 3 epimerase is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2.

Further, the gene for coding the recombinant D-tagatose 3 epimerase is subjected to codon optimization, and the optimized nucleotide sequence is shown as SEQ ID No. 3.

The invention also provides a construction method of the recombinant D-tagatose 3 epimerase DTE-CM, which specifically comprises the following steps:

(1) Construction of recombinant D-tagatose 3 epimerase transformants:

target gene after PCR amplification and synthesis of optimized sequencedte-CmThen, a target gene is integrated on a multiple cloning site of an expression vector pANY1 by utilizing a seamless cloning technology, and the target gene is transferred into a competence E by utilizing a chemical conversion method.coliRosetta cells, screening positive clones and successfully sequencing to obtain recombinant D-tagatose 3 epimerase transformantsE.coliRosetta / pANY1-cm-dte。

(2) Expression and purification of recombinant D-tagatose 3 epimerase DTE-CM:

and (3) obtaining the E.coli Rosettathe/pANY 1-Cm-dte is cultured in LB culture medium, then inducer IPTG is added, the culture is carried out overnight at low temperature and low speed, then the collected cells are ultrasonically crushed to release recombinant protein, and Ni is utilized ²⁺ Purification on NTI column to obtain purified recombinant D-taggerSugar 3 epimerase DTE-CM.

Further, in the step (1), the reaction parameters of the PCR are: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 30s; annealing at 58 ℃ 30s; extension at 72 ℃ for 45s; final extension at 72 ℃ for 5 min;34 cycles.

Further, in the step (2), theE.coliRosetta / pANY1-cm-dteCulturing in LB medium at 37 deg.C under shaking at 200 rpm until OD is 0.6-0.8.

Further, in the step (2), the final concentration of the IPTG in the culture medium is 1mM.

Further, in the step (2), the low-temperature and low-speed culture overnight is a culture overnight at 150 rpm and 25 ℃.

Further, in the step (2), the Ni ²⁺ The NTI column was 1.0X10.0 mm.

The invention also provides application of the recombinant D-tagatose 3 epimerase in preparation of D-psicose by taking D-fructose as a substrate.

Further, the application comprises the following steps: adding purified recombinant D-tagatose 3 epimerase DTE-CM and Ni to D-fructose solution ²⁺ Reacting at 50-60 ℃, heating to 100 ℃, centrifuging to obtain supernatant, and obtaining mixed liquor containing the product D-psicose.

Compared with the prior art, the invention has the beneficial effects that:

(1) In the invention, build upE.coliRosetta / pANY1-cm-dteThe recombinants realize the high-efficiency expression of the D-tagatose 3 epimerase DTE-CM in escherichia coli, and obtain a large amount of pure D-tagatose 3 epimerase DTE-CM.

(2) The recombinant enzyme D-tagatose 3 epimerase DTE-CM constructed by the invention is derived fromChristensenella minutaThe intestinal bacteria related to human obesity are safe in source, and the enzyme has excellent enzymological characteristics and can show high catalytic activity in an acidic pH environment. In addition, the enzyme can effectively solve the problem of preparation in the prior art when D-fructose is used as a substrate to prepare D-psicoseThe defects of browning effect, more byproducts and the like in the preparation process provide a good enzyme source for industrialization of producing D-psicose by a biological method.

(3) The recombinant D-tagatose 3 epimerase DTE-CM disclosed by the invention can be used for efficiently converting D-fructose to produce D-psicose in an environment with pH of 6.0 and 50 ℃ and containing 1mM Ni2+, and the conversion rate is 30%. The highest conversion rate under acidic condition among enzymes with the same function is reported so far.

Drawings

FIG. 1 shows the reversible reaction of D-fructose and D-tagatose catalyzed by D-tagatose family enzymes.

FIG. 2 shows a gene of interestdte-Cm pCR amplification results.

FIG. 3 shows the expression and purification of DTE-CM.

FIG. 4 shows the result of the evolutionary tree analysis of DTE-CM.

FIG. 5 shows the results of amino acid comparison analysis of DTE-CM.

FIG. 6 shows the result of HPLC detection of DTE-CM reactant.

FIG. 7 shows the ionic species (A) and Ni ²⁺ (B) Ion concentration versus enzymatic properties.

FIG. 8 is a graph showing the effect of temperature and pH on the enzymatic activity and stability of DTE-CM; wherein, A is the relative enzyme activity of the DTE-CM at different temperatures, B is the stability test result of the DTE-CM at different temperatures, C is the relative enzyme activity of the DTE-CM at different pH values, and D is the stability test result of the DTE-CM at different pH values.

FIG. 9 shows the results of the conversion of DTE-CM with different concentrations of the substrate D-fructose.

Detailed Description

The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.

In the present invention, the active unit of the recombinant D-tagatose 3 epimerase DTE-CM involved in the examples is defined as: catalyzing an amount of enzyme that generates 1 μmol of D-psicose per minute at pH6.0 and 50 ℃. The measurement method is as follows: 1mM Ni in 1mL of the reaction system ²⁺ 50g/L D fructose50mM sodium phosphate buffer solution with pH6.0 and DTE-CM with final concentration of 0.5 mu M, and continuously reacting for 10 min at 50 ℃. Then, the reaction was terminated by boiling water bath for 10 minutes. The reaction sample was centrifuged at 13000rmp for 4min, and the supernatant was collected through a 0.22 μm filter and assayed for the amount of D-psicose produced by HPLC.

HPLC conditions: the Ishiking optical differential detector RID-250, the chromatographic column of Agilent HPLX-Ca (300X 7.7 mm) and pure water are used as mobile phases, the detection temperature is 84 ℃, and the flow rate is 0.6ml/min.

The formulations of the buffer solutions involved in the purification section of the examples are as follows:

lysis buffer: 50mM phosphate buffer, 150 mM NaCl, 1mM DTT (dithiothreitol), pH 7.0;

and (3) an equilibrium buffer: 50mM phosphate buffer, 500 mM NaCl, 1mM DTT, pH 7.0;

washing buffer solution: 50mM phosphate buffer, 500 mM NaCl, 50mM imidazole, 1mM DTT, pH 7.0;

elution buffer: 50mM phosphate buffer, 150 mM NaCl, 300 mM imidazole, 1mM DTT, pH 7.0.

Example 1: recombinantsE.coliRosetta / pANY1-cm-dteConstruction of

(1) Published as Genbank databaseChristensenella minutaPutative encoding of D-tagatose 3 epimerasedteGene sequence (NCBI accession number: NZ _ CP 029256.1) SEQ NO:1, in combination with the codon preference of Escherichia coli, in order to makedte-cmCan better express, optimize the original sequence, and optimize the gene sequence SEQ NO:3, respectively. The optimized sequence is submitted to Suzhou Jin Wei Intelligent science and technology limited for gene synthesis.

(2) According to the sequence of the multiple cloning site on the vector pANY1 and the characteristics of the homologous arm, the upstream Primer F: 5-ACATCAGCCGTAGGATCCATGAAATACGGTTTATTGTACCATTATT-3' and downstream primer R: 5-GGGGACGTCGACTCTAGACTAGTGATGATGATGGTGATGCG-3' (the underlined part is the same sequence as the pANY1 plasmid) and is assigned to the science and technology of Jin Wei, suzhouPrimers were synthesized by Limited.

(3) Synthesized by the companydte-CmThe gene is used as a template, and the NEB Q5 high-fidelity polymerase PCR is utilized to amplify the target gene. The PCR reaction conditions are as follows: pre-denaturation: 98. 3 min, denaturation: 98. c, 30s, anneal: 58. deg.c, 30s, extension: 72. c, 45s, final extension: 72. at 5min, 34 cycles.

(4) The obtained PCR product was detected by 1% agarose gel electrophoresis to obtain an electrophoretic band of about 0.9 Kb, and as shown in FIG. 2, the PCR product was purified by a product purification kit to obtain a purified dte gene.

(5) Carrying out double enzyme digestion linearization on the pANY1 plasmid by BamH1 and Xba1, purifying by using a cutting glue to obtain a linearized plasmid, and constructing recombinant plasmids of the dte gene and the linearized plasmid in the step (3) according to a method of a seamless cloning kit; coli, screening positive clones successfully transformed by PCR verification, plasmid extraction verification and sequencing verification, storing and naming asE.coli Rosetta / pANY1-cm-dte。

Example 2: induced expression and purification of recombinant D-tagatose 3 differential isomerase DTE-CM

The recombinants areE.coliRosetta /pANY1-cm-dteInoculating into LB culture medium (yeast powder 10 g/L, tryptone 20 g/L, sodium chloride 20 g/L), culturing at 37 deg.C and 200 rpm under shaking to OD ₆₀₀ To 0.6-0.8, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 1mM for induction, inducing and expressing DTE-CM at low speed of 150 rpm overnight at 25 ℃, detecting the expression of the DTE-CM by SDS-PAGE, and taking the recombinant bacteria without IPTG for induction as a blank control, as shown in figure 3.

To purify the DTE-CM, the centrifuged cells were washed twice with 0.85% NaCl solution and resuspended in lysis buffer and sonicated using a sonicator for 10 min at 400w for 1.5s with 2.5s pauses, centrifuged at 4 ℃ and 8000 g for 30min to remove cell debris, and the supernatant was filtered through a 0.22 μm filter. Loading the filtrate to Ni ²⁺ On an NTI column and equilibrated with 5 column volumes of equilibration buffer.Unbound protein was eluted from the column with 5 column volumes of wash buffer. The recombinant DTE-CM was then eluted from the column using 10 column volumes of elution buffer. DTE-CM was desalted and concentrated using an Amicon Ultra 15 ultrafilter tube and 50mM pH 7.0 sodium phosphate buffer. Protein purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

FIG. 3 shows the result of DTE-CM expression and purification, where M is protein marker, 1 is the recombinant E.coli cell protein glue pattern without IPTG induction, and 2 is the recombinant E.coli cell protein glue pattern with IPTG induction; and 3 is a purified DTE-CM pure enzyme protein glue picture. As can be seen from the figure, a single pure protein band of 33kDa was obtained. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard. Then adding the purified pure enzyme sample into glycerol with the final concentration of 15 percent to be stored at the low temperature of 80 ℃.

Comparing the amino acid sequence of DTE-CM with the successfully identified D-tagatose 3 epimerase family to construct phylogenetic tree, as shown in FIG. 5, DTE-CM and the gene from DTE-CMR. cellulolyticumAnd C. bolteaethe enzyme homology is 42.9% at most; as shown in FIG. 6, the active site of recombinant D-tagatose 3 epimerase has strict conservation by amino acid sequence alignment, in which the metal binding sites Glu150, asp183, his209 and Glu244 of DTE-CM are completely conserved, indicating that the catalytic mechanism of D-tagatose 3 epimerase family is locally the same.

Example 3:

200 μ L of purified D-tagatose 3 differential phase isomerase DTE-CM was added to 50g/L of D-fructose phosphate buffer, and reacted at 6.0,50 ℃ for 30min. The reaction was terminated by boiling a water bath for 10 minutes. 13000rmp for 4min, the supernatant was collected and filtered through a 0.22 μm filter, and HPLC was used to determine whether D-psicose was produced.

As shown in FIG. 4, the DTE-CM reactant had a distinct peak at 22.5min, consistent with the peak time of the D-psicose standard, indicating that D-psicose was produced, which indicates that the recombinant D-tagatose 3 epimerase DTE-CM has the ability to catalyze the production of D-psicose from D-fructose.

Example 4:

in order to investigate the enzymatic properties of D-tagatose 3 epimerase, the present example separately investigated the enzymatic activities of D-tagatose 3 epimerase in different metal ions and concentrations, at different temperatures and under different pH conditions.

(1) Effect of Metal ions on DTE-CM enzymatic Activity:

multiple sets of experiments were set up by mixing 50g/L D-fructose with 50mM pH6.0 sodium phosphate buffer and adding 1mM final Ca separately ²⁺ 、Co ²⁺ 、Mn ²⁺ 、Mg ²⁺ 、Fe ²⁺ 、Ni ²⁺ 、Zn ²⁺ 、Cu ²⁺ And finally, adding 0.5 mu M DTE-CM for mixed reaction, incubating for 30 minutes at 4 ℃, and reacting for 10 min at 50 ℃ to analyze the relative activity of the DTE-CM to metal ions. With Ni ²⁺ The activity measured was expressed as 100% to analyze the activity of DTE-CM in the presence of other metal ions.

FIG. 7A shows the enzymatic activity of DTE-CM in different metal ion environments, and it can be seen that DTE-CM does not have any activity in the absence of metal ions, indicating that DTE-CM is a strict metal ion dependent enzyme, and only has activity in the presence of divalent metal ions, and Cu ²⁺ 、Fe ²⁺ 、Mn ²⁺ Has no promotion effect on enzyme activity, and contains metal ions Zn ²⁺ 、Cu ²⁺ 、Ca ²⁺ 、Co ²⁺ 、Ni ²⁺ 、Mg ²⁺ Can improve the enzyme activity, wherein the Ni2+ has the strongest promoting ability, and the optimal metal ion of DTE-CM is Ni ²⁺ 。

Based on the above experiments, this example discusses Ni in the range of 0-5 mM ²⁺ Effect of ion concentration on enzymatic activity of DTE-CM. FIG. 7B shows different concentrations of Ni ²⁺ The enzyme activity of DTE-CM under ion environment is shown in the figure, and the enzyme activity is along with Ni between 0 and 1Mm ²⁺ The activity is maximum when the concentration is increased and the concentration is 1Mm, and when Ni is added ²⁺ When the ion concentration is 1-5 mM, the enzyme activity tends to be stable and visible, ni ²⁺ The optimum concentration of (3) is 1mM.

(2) Effect of temperature on DTE-CM enzymatic Activity:

multiple sets of experiments were set, and DTE-CM was incubated in 50mm, pH6.0 phosphate buffer, to investigate the enzyme activity of DTE-CM at 30-80 ℃ respectively. As shown in 8A, DTE has the greatest catalytic activity at 50 ℃. FIG. 8B shows that the relative enzyme activity of DTE is relatively stable at 40 deg.C, and when the temperature is higher than 50 deg.C, DTE-CM is easily inactivated. At the same time, the metal ion Ni is investigated ²⁺ Influence on the thermal stability of DTE-CM, ni addition ²⁺ DTE-CM with no addition of Ni ²⁺ Comparison of half-lives at 50 ℃ reveals Ni ²⁺ Can greatly increase the thermal stability.

(3) Effect of pH on DTE-CM enzymatic Activity:

the different buffers used were acetate buffer pH 5.0-5.5, phosphate buffer pH 6.0-7.5 and Tris-HCl buffer 50mM, pH 8.0-9.0. Enzyme activity assays were performed at different pH values. In the pH stability assay, the purified enzyme was incubated at pH 5.0-8.0,4 ℃ for 2h and its activity was measured under standard assay conditions. As a result, DTE-CM showed the highest activity at pH6.0 as shown in FIG. 8C. FIG. 8D shows the relative enzyme activity of DTE-CM treated with 2h at 4 ℃ at different pH values, respectively, and then measured as 100% of the untreated enzyme activity, showing that DTE-CM is relatively stable at different pH values.

Example 5:

under the optimal conditions as given in example 4, D-allose was synthesized enzymatically using a mixture containing 100 g/L, 300 g/L, 500 g/L D-fructose and 5 mM DTE-CM, respectively, and the course of the enzymatic conversion was measured every 20 minutes until the reaction reached equilibrium.

FIG. 9 shows the results of the conversion of DTE-CM with different concentrations of substrate D-fructose, from which it can be seen that three concentrations of D-fructose reach equilibrium after 1.5, 4 and 10 h, respectively, and HPLC analysis shows that D-psicose of 30.2, 90.3 and 156 g/L is obtained, and the conversion rate of D-psicose into D-psicose is calculated, and the result shows 30%. Therefore, the characteristic that the D-tagatose 3 epimerase DTE-CM can efficiently convert and synthesize D-tagatose by taking D-fructose as a substrate is utilized, and the conversion can be completed under an acidic condition, so that the recombinant D-tagatose 3 epimerase can be used for industrial production of D-psicose with a high added value.

Example 6:

in this example, ni was added at pH6.0, 50 ℃ and 1mM ²⁺ The kinetic parameters of DTE-CM were studied under the conditions, and the specificity and kinetic parameters of DTE-CM enzyme were studied with different concentrations (5-400 mM) of D-fructose, D-allose, D-tagatose and D-sorbose as substrates, respectively, and the results are shown in Table 1.

TABLE 1 kinetic parameters and relative viability of DTE-CM under different substrates

Substrate	K m (mM)	K cat (min -1 )	K cat /K m (mM -1 min -1 )	Relative activity (%)
					D-fructose	94.7±1.2	4230±34	45±4.5	26±1.8
D-psicose	53.8±3.4	6572±44	124±6.3	68±0.5
					D-tagatose	34.9±1.6	9318±36	267±3.1	100±0.4
D-sorbose	66.8±2.6	5874±29	89±2.9	40.2±1.2

As can be seen from Table 1, DTE-CM can reversibly isomerize the c-3 position of D-fructose, D-psicose, D-sorbose and D-tagatose as substrates, respectively, but DTE-CM has the highest relative activity to D-tagatose and the lowest relative activity to D-fructose. Analysis of kinetic parameters of DTE-CM on fructose by means of double reciprocal curveK _m , K _cat , K _cat /K _m The values were 94.7 mM, 4230 min, respectively ^-1 And 45 mM ^-1 min ^-1 。

The examples are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any obvious modifications, substitutions or variations can be made by those skilled in the art without departing from the spirit of the present invention.

Sequence listing

<110> university of Jiangsu

<120> recombinant D-tagatose 3 epimerase DTE-CM and construction method and application thereof

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<213> D-tagatose 3 differentially allosteric enzyme (Christensella minuta)

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atgaaatacg gtttattgta ccattattgg tcggacggct ggtcctgcga ttacccgaag 60

accgcggaaa agatcaaaaa ggccgggttc gatgtgatgg agatcggcgg agaccatctg 120

agcaggatga acgcggcgga gatggacgcg ctcaggcggg cggccgtgga ccttggcctc 180

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acgcgggagc atgggatcgc ttacctcacg gacgtgctga agcggatgga cggcatcggc 300

tgcaggatga tgatcggcgc gctgtatacc ttctggccgg cggatttcag cgtcgaaaac 360

gacaaggaac gtgcgtggga ccgttccatc gattcccttc gggaactcgc ccgggccgcg 420

gaggatcttg gctccacctg ttccctcgag atcctgaacc gctatgaggg ttatatcctc 480

aacacctgcg aagaggggct tcggtacctc gagcgtatcg gcagcccaag catgaagctt 540

ttgcttgaca ccttccatat gagcatcgag gaggacagcc ttcccggagc aatccgcctt 600

gcaggcagcc ggctcgggca tatgcacctc ggggaaggca accgcaagct gcccggcctc 660

ggttccctgc cgtgggcgga gatcggccag gccctgcgcg acgcgcattt cgacggtttt 720

gcggtcatcg agcctttcat gcgctacggc gggcagatcg caaaggatat acacttgtgg 780

cacgatttct tccccggcgc tacggaagcg gagatggacc ggatgctggc cgattcactc 840

gccttcctga aacagcattt cgaggcgtag 870

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Met Lys Tyr Gly Leu Leu Tyr His Tyr Trp Ser Asp Gly Trp Ser Cys

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Asp Tyr Pro Lys Thr Ala Glu Lys Ile Lys Lys Ala Gly Phe Asp Val

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Met Glu Ile Gly Gly Asp His Leu Ser Arg Met Asn Ala Ala Glu Met

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Asp Ala Leu Arg Arg Ala Ala Val Asp Leu Gly Leu Glu Leu Ser Val

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Asn Ile Gly Pro Ala Arg Asp Gln Asp Leu Ala Ser Glu Asp Lys Ala

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Thr Arg Glu His Gly Ile Ala Tyr Leu Thr Asp Val Leu Lys Arg Met

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Asp Gly Ile Gly Cys Arg Met Met Ile Gly Ala Leu Tyr Thr Phe Trp

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Pro Ala Asp Phe Ser Val Glu Asn Asp Lys Glu Arg Ala Trp Asp Arg

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Ser Ile Asp Ser Leu Arg Glu Leu Ala Arg Ala Ala Glu Asp Leu Gly

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Ser Thr Cys Ser Leu Glu Ile Leu Asn Arg Tyr Glu Gly Tyr Ile Leu

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Asn Thr Cys Glu Glu Gly Leu Arg Tyr Leu Glu Arg Ile Gly Ser Pro

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Ser Met Lys Leu Leu Leu Asp Thr Phe His Met Ser Ile Glu Glu Asp

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His Leu Gly Glu Gly Asn Arg Lys Leu Pro Gly Leu Gly Ser Leu Pro

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Trp Ala Glu Ile Gly Gln Ala Leu Arg Asp Ala His Phe Asp Gly Phe

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Ile His Leu Trp His Asp Phe Phe Pro Gly Ala Thr Glu Ala Glu Met

260 265 270

Asp Arg Met Leu Ala Asp Ser Leu Ala Phe Leu Lys Gln His Phe Glu

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Ala

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<213> Artificial Sequence (Artificial Sequence)

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ggatccatga aatacggttt attgtaccat tattggtcgg acggctggtc ctgcgattac 60

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catctgagcc gcatgaatgc ggcggaaatg gatgcgctgc gtcgtgcggc ggttgatctg 180

ggtctggaac tgagcgttaa catcggtccg gcccgtgatc aagatctggc gagtgaagac 240

aaagcgaccc gcgaacacgg catcgcctat ctgaccgatg tgctgaaacg catggatggc 300

atcggctgcc gcatgatgat cggcgcgctg tataccttct ggccggcgga cttcagcgtg 360

gaaaacgata aagagcgcgc gtgggatcgc agcattgaca gtctgcgtga actggcccgt 420

gcggcggaag atctgggtag tacgtgcagt ctggaaattc tgaaccgcta cgagggctac 480

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aaactgctgc tggacacctt ccacatgagc atcgaggagg atagtctgcc gggcgcgatt 600

cgtctggccg gtagccgtct gggtcatatg catctgggcg aaggcaaccg caaactgccg 660

ggtctcggta gtctcccatg ggcggaaatt ggccaagcgc tgcgtgatgc gcatttcgac 720

ggtttcgcgg tgatcgaacc gtttatgcgc tacggcggcc agattgcgaa ggatatccat 780

ctgtggcacg acttcttccc gggtgccacg gaagcggaga tggaccggat gctggccgat 840

tcactcgcct tcctgaaaca gcatttcgag gcgcatcacc atcatcatca ctagtctaga 900

Claims (2)

1. The application of the recombinant D-tagatose 3 epimerase DTE-CM in the preparation of D-psicose by using D-fructose as a substrate, wherein the nucleotide sequence for coding the recombinant D-tagatose 3 epimerase gene is shown as SEQ ID No. 1, and the amino acid sequence is shown as SEQ ID No. 2.

2. The application according to claim 1, characterized in that it comprises the following steps: adding purified recombinant D-tagatose 3 epimerase DTE-CM and Ni to D-fructose solution ²⁺ Reacting at 50-60 deg.C, heating to 100 deg.CAnd centrifuging to obtain a supernatant to obtain a mixed solution containing the product D-psicose.

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