CN114703211A

CN114703211A - Sucrose isomerase activity inclusion body fused with coiled coil structure domain

Info

Publication number: CN114703211A
Application number: CN202210142279.1A
Authority: CN
Inventors: 王从纲; 高向红; 庞焦; 马志宇; 李明玉; 李宪臻
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-02-16
Filing date: 2022-02-16
Publication date: 2022-07-05

Abstract

The invention discloses a sucrose isomerase activity inclusion body fused with a coiled coil domain, belonging to the field of biological engineering. The invention takes SI derived from Klebsiella sp.LX3 as a research object, Tdot is respectively fused at the N end or the C end of the SI from which a natural signal peptide sequence is removed, a recombinant expression vector is constructed and transferred into Escherichia coli E.coli BL21(DE3) for induced expression, and enzymatic property characterization is carried out after active inclusion bodies are further separated and purified, thereby providing a new strategy for heterologous expression and efficient utilization of recombinant sucrose isomerase.

Description

Sucrose isomerase activity inclusion body fused with coiled coil structure domain

Technical Field

The invention relates to a sucrose isomerase activity inclusion body fused with a coiled coil structure domain, belonging to the field of biological engineering.

Background

Isomaltulose (alpha-D-glucopyranosyl-1, 6-fructose) is an isomer of sucrose, naturally present in honey, sugarcane juice, and has a sweetness of about 50% of sucrose. At present, isomaltulose is used mainly in the food industry as a substitute for sucrose in the production of health products, diabetic foods and sports drinks. In addition, isomaltulose is also used as a raw material for producing some surfactants and high molecular polymers in the chemical field, and thus is a functional sugar with good development prospects. At present, Sucrose Isomerase (SI) is mainly used for catalyzing Sucrose to be converted into isomaltulose industrially, and compared with a chemical method for synthesis, the method has the advantages of being mild in reaction conditions, environment-friendly in preparation process, high in reaction specificity and the like. Since the application of isomaltulose is more and more widespread and is more popular with consumers, the market demand of isomaltulose is increasing year by year, but the production is mainly monopolized by foreign companies, and the market price per ton is about 1.5 ten thousand yuan, so that the utilization rate of sucrose isomerase is increased, and the production cost of isomaltulose is reduced.

Various sucrose isomerase-producing bacteria have been found, including Erwinia rhapontici (Erwinia rhapontici), Serratia vulgaris (Serratia plymuthica), Polyporus frondosa (Pantoea dispersa), and Klebsiella pneumoniae (Klebsiella sp). However, the wild bacteria have low enzyme production level generally and are difficult to meet the requirements of industrial production, such as the fermentation enzyme activity of Erwinia rhapontici NX-5 is 1.3U/mL, and KlebsThe fermentation enzyme activity of iella sp.LX3 is 15.12U/mL. Therefore, researchers currently prepare recombinant sucrose isomerase mainly through heterologous expression means and immobilize it on a water-insoluble carrier to prepare immobilized enzyme so as to improve the utilization rate. Such as mesoporous TiO modified by epsilon-poly-L-lysine₂And the sponge prepared from epsilon-poly-L-lysine and gelatin is used for immobilizing the sucrose isomerase, and the enzyme activity recovery rates are respectively 93.3% and 84.5%; the chitosan-glutaraldehyde crosslinking method, the sodium alginate-sodium carboxymethylcellulose mixed embedding method and the cross-linked enzyme aggregate method are used for immobilizing the sucrose isomerase, so that the enzyme activity recovery rates of 70.3%, 32.5% and 30.9% are respectively realized. In summary, although sucrose isomerase is successfully prepared into immobilized enzyme by the traditional adsorption method, binding method, crosslinking method and embedding method, the pure enzyme needs to be separated and purified by chromatographic technology first, and then the water-insoluble carrier and the pure enzyme are used for carrying out immobilization reaction, and the separation and purification, carrier preparation and chemical reagents related to the immobilization reaction in the process all increase the production cost of the immobilized enzyme, and are not beneficial to the large-scale production and application of the immobilized sucrose isomerase. In recent years, researches show that a specific tag sequence and enzyme protein are used for fusion expression, so that the specific tag sequence can be induced to form an Inclusion body (CatIBs, hereinafter referred to as active Inclusion body) with catalytic activity in cells, the expression quantity is large, the separation and purification are easy, especially the expression and immobilization processes are carried out synchronously, no additional carrier material and immobilization reaction are needed, the novel self-immobilized enzyme is a novel self-immobilized enzyme, the view that the past 'the Inclusion body is a completely inactive protein aggregate' is theoretically changed, and the novel enzyme heterologous expression and immobilization method has better industrial application potential.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a sucrose isomerase activity inclusion body fused with a coiled coil domain. The SI derived from Klebsiella sp.LX3 is taken as a research object, and the invention has the advantages of higher sucrose conversion rate (99.5%), isomaltulose serving as a main product and the like. Tdot is respectively fused at the N end or the C end of SI from which a natural signal peptide sequence is removed, a recombinant expression vector is constructed and transferred into Escherichia coli E.coli BL21(DE3) for induced expression, and enzymatic property characterization is carried out after active inclusion bodies are further separated and purified, so that a new strategy is provided for heterologous expression and efficient utilization of the recombinant sucrose isomerase.

The invention provides a gene, the nucleotide sequence of which is shown as SEQ ID NO. 1.

The invention also provides a gene, and the nucleotide sequence is shown as SEQ ID NO. 2.

The invention also provides a fusion protein containing Tdot-SI and encoded by the gene with the nucleotide sequence shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 3.

The invention also provides a fusion protein containing SI-Tdot and encoded by the gene with the nucleotide sequence shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 4.

The invention also provides a recombinant engineering bacterium containing the gene with the nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a recombinant engineering bacterium containing the gene with the nucleotide sequence shown as SEQ ID NO. 2.

The invention also provides a sucrose isomerase active inclusion body which is obtained by inducing fusion protein expression by recombinant engineering bacteria containing a gene with a nucleotide sequence shown as SEQ ID NO.1 or recombinant engineering bacteria containing a gene with a nucleotide sequence shown as SEQ ID NO.2, and crushing, separating and purifying thalli.

Specifically, the preparation method of the inclusion body with sucrose isomerase activity comprises the following steps: a coiled-coil domain Tdot is fused at the N or C end of sucrose isomerase SI to construct a recombinant expression vector, and the recombinant expression vector is transferred into Escherichia coli E.coli BL21(DE3) for induced expression to prepare an active inclusion body.

The preparation method of the sucrose isomerase activity inclusion body of the recombinant engineering bacteria containing the gene with the nucleotide sequence shown as SEQ ID NO.1 specifically comprises the following steps:

(1) constructing a recombinant expression vector pET-24a-Tdot-SI containing a coding gene of Tdot-SI fusion protein, and transferring the recombinant expression vector into an expression host cell to obtain recombinant engineering bacteria;

(2) culturing the recombinant engineering bacteria, and inducing the expression of the fusion protein;

(3) collecting bacterial cells, carrying out cell disruption, separating cell disruption supernatant and a sediment sample containing the inclusion body, and washing and purifying the sediment sample by buffer solution to obtain the active inclusion body.

The preparation method of the sucrose isomerase activity inclusion body of the recombinant engineering bacteria containing the gene with the nucleotide sequence shown as SEQ ID NO.2 specifically comprises the following steps:

(1) constructing a recombinant expression vector pET-24b-SI-Tdot containing an encoding gene of SI-Tdot fusion protein, transferring the recombinant expression vector into an expression host cell to obtain recombinant engineering bacteria;

Preferably, the sucrose isomerase SI is derived from Klebsiella sp.LX3, and the nucleotide sequence of the sucrose isomerase SI is shown as SEQ ID NO. 5.

Preferably, the Tdot gene is derived from Staphylothermus marinus (Staphylothermus marinus), and the nucleotide sequence of the Tdot gene is shown as SEQ ID NO. 6.

By way of example and not limitation, the expression host cell is preferably E.coli BL21(DE 3).

The invention also provides application of the inclusion body with sucrose isomerase activity in catalyzing conversion of sucrose into isomaltulose.

Further, the inclusion body of the sucrose isomerase catalyzes sucrose to be converted into isomaltulose, the pH value is 4.5-6.5, and the temperature is 30-45 ℃.

The present invention expresses the tetrameric coiled-coil domain of the cell-surface protein tetrabrachion (Tdot) derived from the cell surface protein tetrabrachion in fusion with SI and studies the enzymatic properties of the recombinase. Respectively fusing the N/C terminals of Tdot and SI to construct a recombinant expression vector, transferring the recombinant expression vector into E.coli BL21(DE3) for induced expression, and displaying that Tdot-SI and SI-Tdot are expressed as inclusion bodies with catalytic activity, while SI inclusion bodies without fused Tdot have no catalytic activity. Further, active inclusion bodies are purified by centrifugation and washing with a buffer solution, and the study on the enzymology property shows that the optimum reaction temperature of Tdot-SI and SI-Tdot active inclusion bodies is 40 ℃, and the optimum reaction pH is 5.5 and 5.0 respectively. The kinetic constants Km of Tdot-SI active inclusion bodies are 103.9 +/-9.5 mmol/L, kcat/Km is 0.06 +/-0.002L/(mmol.s), the kinetic constants Km of SI-Tdot active inclusion bodies are 54.4 +/-6.6 mmol/L, and kcat/Km is 0.03 +/-0.002L/(mmol.s). The results of the product specificity studies show that as the reaction temperature increases, the isomaltulose content of the product does not change significantly, whereas the trehalulose content of the product decreases as the conversion reaction temperature increases, while the monosaccharide content increases as the conversion reaction temperature increases. The sucrose isomerase active inclusion body fused with the coiled coil structure domain is successfully prepared by a fusion expression technology, has the advantages of synchronous realization of expression and immobilization as a novel self-immobilized enzyme, and provides a new strategy for realizing large-scale preparation and high-efficiency utilization of recombinant sucrose isomerase.

Drawings

FIG. 1 is colony PCR identification of transformed recombinant plasmid engineering bacteria; lane M is 250bp DNA Ladder Marker, and Lane 1 is pET-24 a-Tdot-SI; lane 2 is pET-24 b-SI-Tdot.

FIG. 2 is an SDS-PAGE analysis of recombinant 6 XHis-SI/SI-6 XHis; lane M is Protein Marker; lanes 1-4 are pET24b-6 XHis-SI before induction, after induction, supernatant, and pellet; lanes 5-8 show pET24b-SI-6 XHis, pre-induction, post-induction, supernatant, and pellet.

FIG. 3 is an SDS-PAGE analysis of recombinant Tdot-SI/SI-Tdot; lane M is Protein Marker; lanes 1-4 are inclusion bodies before, after, supernatant, and washing pET24a-Tdot-SI induction; lanes 5-8 are inclusion bodies before, after, supernatant, and wash of pET24b-SI-Tdot induction.

FIG. 4 is a graph of the effect of temperature on Tdot-SI and SI-Tdot enzyme activity.

FIG. 5 is a graph of the effect of pH on Tdot-SI and SI-Tdot enzyme activity.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Sucrose isomerase SI (NCBI access number AAK82938) is derived from Klebsiella sp.LX3, and the nucleotide sequence of the sucrose isomerase SI gene is shown in SEQ ID NO. 5. The plasmid pET-28a-SI carrying the SI gene of sucrose isomerase can be synthesized by conventional means in the art. Plasmids pET24b-6 XHis-SI (nucleotide sequence shown in SEQ ID NO. 13) and pET24b-SI-6 XHis (nucleotide sequence shown in SEQ ID NO. 14) fused with histidine tags at the N/C terminal respectively can be synthesized by the conventional method in the field.

The nucleotide sequence of Tdot is shown in SEQ ID NO.6, and the plasmid pET-24a-Tdot carrying Tdot gene is synthesized by Suzhou Hongxi Biotechnology GmbH.

Kanamycin, Isopropyl-beta-D-thiogalactopyranoside (IPTG), Triton-X100, a SanPrep column type plasmid DNA small extraction kit, a SanPrep column type DNA gel recovery kit and a BCA protein quantitative detection kit are all purchased from the company Limited in the Biotechnology engineering (Shanghai).

HS DNA Polymerase, QuickCutTM BamH I and QuickCutTM Xho I, Dpn I, T4 DNA ligase, DNA and protein relative molecular weight standards were purchased from Takara Bio Inc.

Gene sequencing work was performed by Jilin province, Kuumei, Biotechnology, Inc.

Isomaltulose was purchased from Sigma.

Other chemicals were purchased from chemical reagents ltd, miuiou, department of tianjin.

LB liquid medium: contains 10g/L tryptone, 5g/L yeast powder and 10g/L NaCl, and has the pH value of 7.2; the LB solid medium was LB liquid medium to which 1.5% agar was added.

LLB medium: contains 10g/L tryptone, 5g/L yeast powder and 5g/L NaCl, and has a pH value of 7.2.

Buffer a Buffer: na (Na)₂HPO₄·12H₂O：16.937g、NaH₂PO₄2H 2O: 0.62g of the extract is dissolved in 750mL of deionized water, the pH value is adjusted to 8.0, 50mL of glycerol is added, and the volume is adjusted to 1L by using the deionized water.

Buffer B Buffer: 0.5mL of Triton-X100 was dissolved in 100mL of Buffer A Buffer.

Buffer C Buffer: na (Na)₂HPO₄·12H₂O：16.937g、NaH₂PO₄·2H₂O: 0.62g of the suspension is dissolved in 750mL of deionized water with the concentration of 450-.

EXAMPLE 1 construction of recombinant expression vectors

The pET-24a-Tdot-SI expression vector is constructed by using the inner primers in the table 1 and adopting an enzyme digestion connection method: taking pET-28a-SI preserved in a laboratory as a template, carrying SI coding gene which does not contain natural signal peptide, amplifying by using a primer P1/P2 to obtain an SI gene fragment containing a restriction enzyme cutting site, carrying out double restriction on the SI gene fragment and pET-24a-Tdot by using QuickCutTM BamH I and QuickCutTM Xho I, carrying out electrophoresis separation on restriction enzyme products by using 1% agarose gel, recovering a linearized vector and a target gene fragment by using a DNA gel recovery kit, and continuing carrying out ligation reaction by using T4 DNA ligase. The ligation reaction products were transformed into E.coli DH10B competent cells by heat shock and plated on LB solid plates containing 50. mu.g/mL kanamycin. The single colony is picked and colony PCR identification is carried out by using the primer P5/P6, and the result is shown in FIG. 1(A), and the amplification product has a single band at the position of 1500-2250bp and is consistent with the size of the expected amplification sequence 2055 bp. And continuing culturing the positive monoclonal antibody which is verified to be correct, extracting plasmids, then carrying out gene sequencing, and marking the recombinant expression vector which is correctly sequenced as pET-24 a-Tdot-SI.

The cloning technology of the Restriction free-cloning (RF) is adopted to construct a pET-24b-SI-Tdot expression vector: and carrying out a first round of PCR amplification reaction by using pET-24a-Tdot as a template and using a primer P3/P4, separating a PCR amplification product by using 1% agarose gel electrophoresis, recovering by using a DNA gel recovery kit, using the PCR amplification product as a long primer of a second round of PCR reaction, and continuously carrying out a second round of PCR reaction by using pET24b-SI-6 × His as a template. After the reaction was completed, Dpn I was added to decompose the template plasmid, and the reaction product was transformed into E.coli DH10B competent cells by electric shock and plated on LB solid plates containing 50. mu.g/mL kanamycin. The single colony is picked and subjected to colony PCR identification by using the primer P5/P6, and the result is shown in FIG. 1(B), and the amplification product has a single band at the position of 1500-2250bp and is consistent with the expected amplification sequence 2049bp in size. And continuing culturing the positive monoclonal antibody which is verified to be correct, extracting plasmids, then carrying out gene sequencing, and marking the recombinant expression vector which is correctly sequenced as pET-24 b-SI-Tdot.

TABLE 1 primer sequences used in the present invention

Example 2 inducible expression and isolation and purification of recombinant genes in E.coli

The constructed recombinant expression vectors pET-24a-Tdot-SI and pET-24b-SI-Tdot and the recombinant expression vectors pET24b-6 XHis-SI and pET24b-SI-6 XHis are respectively transformed into E.coli BL21(DE3) competent cells, the positive monoclonals are selected and inoculated into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and the liquid is cultured at 37 ℃ and 200r/min for 14h to obtain seed liquid. Inoculating the seed liquid into LLB liquid culture medium containing 50 μ g/mL kanamycin at 1% (v/v), culturing at 37 deg.C and 200r/min until OD600nm reaches 0.6-0.8, adding inducer, wherein recombinant engineering bacteria E.coli BL21(DE3)/pET-24a-Tdot-SI is added with IPTG with final concentration of 0.8mmol/L, E.coli BL21(DE3)/pET-24b-SI-Tdot is added with IPTG with final concentration of 0.1mmol/L, E.coli BL21(DE3)/pET24b-6 × His-SI and E.coli BL21(DE3)/pET24b-SI-6 × His is added with IPTG with final concentration of 0.5mmol/L, and culturing the bacterial liquid with IPTG added with the above liquid at 16 deg.C and 200r/min for inducing expression for 20 h.

And (3) separating and purifying the active inclusion bodies by using a buffer solution cleaning method: the bacterial liquid after induction expression is collected by centrifugation and is subjected to ultrasonic disruption in an ice water bath after being resuspended by Buffer A (the power is 500W, the disruption time is 3s, the interval is 5s, and the disruption time is 20 min). And centrifuging the crushed whole bacteria sample at 10000r/min for 15min to separate a soluble supernatant and a precipitate component containing the inclusion body. And washing the precipitate for 2 times and 3 times by using a Buffer B and a Buffer C in sequence, centrifuging the washed inclusion body sample for 15min at 10000r/min for recycling, and fully suspending the washed inclusion body sample by using the Buffer C to obtain the active inclusion body enzyme solution. And (3) adding a small amount of active inclusion body enzyme solution into an isometric 10% SDS solution for treatment for 5min to completely dissolve the inclusion bodies, and then determining the protein concentration by using a BCA protein quantitative detection kit. The cell disruption supernatant and the washed active inclusion body sample were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

As shown in FIG. 2, SDS-PAGE analysis of recombinant 6 XHis-SI and SI-6 XHis was performed. After induction, a target band which is consistent with the theoretical relative molecular mass of 6 XHis-SI and SI-6 XHis of 68.14kDa appears at 66.4kDa, and is mainly expressed in the form of inclusion bodies, and enzyme activity detection finds that the inclusion body precipitate has no catalytic activity, which shows that recombinant SI protein is mainly expressed in the form of inactive inclusion bodies no matter histidine tags are fused at the N/C end.

As shown in FIG. 3, SDS-PAGE analysis of recombinant Tdot-SI and SI-Tdot is performed. After induction, a target band which is consistent with the theoretical relative molecular mass of Tdot-SI and SI-Tdot and has 73.18kDa appears on the whole bacteria sample above the 66.4kDa protein relative molecular mass standard, which indicates that the fusion protein is successfully induced and expressed. The expressed fusion proteins Tdot-SI and SI-Tdot still mainly exist in a precipitate sample in the form of inclusion bodies, and the inclusion bodies are subjected to enzyme activity detection to find that the fusion proteins have high catalytic activity, which indicates that the fusion Tdot label induces the SI to be expressed as an active inclusion body.

Example 4 determination of active Inclusion body enzyme Activity

(1) Determination of optimum reaction temperature

Taking 100 mu L of Tdot-SI and SI-Tdot activity inclusion body enzyme liquid with the concentration of 4mg/mL, respectively mixing with 400 mu L of pH 6.0 disodium hydrogen phosphate-citric acid buffer solution containing 4% of cane sugar, respectively reacting at 30, 35, 40, 45, 50 and 55 ℃ at 200r/min for 2h, and then treating a reaction sample in boiling water with the temperature of 100 ℃ for 15min for enzyme inactivation. Then, the reaction product after enzyme deactivation is centrifuged for 15min at 10000r/min, and the obtained supernatant is filtered by a 0.22 mu m filter membrane and then is subjected to HPLC detection. The HPLC detection conditions are as follows: the reaction product was detected using a Hypersil APS-2, 4.6X 250mm, 5 μm amino column, with a differential detector, mobile phase acetonitrile: water (80: 20 v/v), column temperature 35 ℃, sample size 10 μ L, flow rate 1.0 mL/min. And calculating the concentration of the isomaltulose in the reaction product and calculating the enzyme activity according to a labeling curve fitted by the isomaltulose standard substance and the corresponding peak area. Definition of enzyme activity units: the amount of enzyme required to release 1. mu. moL of isomaltulose per minute using sucrose as substrate was 1 enzyme activity unit (U). Definition of relative enzyme activity: the highest enzyme activity of the same group is 100%, and the calculated ratio is the relative enzyme activity.

The results are shown in fig. 4, the Tdot-SI and SI-Tdot active inclusion bodies have the highest enzyme activity at 40 ℃, the enzyme activity increases and increases with the temperature within the range of 30-40 ℃, the both have the enzyme activity of more than 80% within the range of 35-45 ℃, and the enzyme activities of the Tdot-SI and SI-Tdot active inclusion bodies are rapidly reduced after the temperature is higher than 45 ℃.

(2) Determination of optimum reaction pH

Mixing Tdot-SI and SI-Tdot activity inclusion body enzyme liquid with 4% sucrose pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 disodium hydrogen phosphate-citric acid buffer solution, performing enzyme catalysis reaction at 40 deg.C, quantitatively detecting isomaltulose in the product, calculating enzyme activity, and measuring enzyme activity to determine optimum reaction pH.

As shown in FIG. 5, the enzyme activities of Tdot-SI and SI-Tdot activity inclusion bodies were increased with increasing pH in the range of pH 4.0 to 5.0, and had the highest enzyme activities at pH 5.5 and pH 5.0, respectively. In addition, both have more than 80% catalytic activity in the pH range of 5.0-6.0, and the enzyme activity rapidly decreases after pH above 6.5.

(3) Determination of the kinetic constants of the enzymatic reaction

Tdot-SI and SI-Tdot active inclusion body enzyme solutions are respectively mixed with sucrose solutions with different concentrations (10, 20, 40, 50, 100, 200, 250, 400, 500 and 800mmol/L), catalytic reaction is carried out under the optimal reaction conditions (Tdot-SI: 40 ℃, pH 5.5; SI-Tdot: 40 ℃, pH 5), and then the generation amount of the product isomaltulose is detected by HPLC and the reaction speed is calculated. Then nonlinear fitting is carried out by utilizing GraphPad Prism 5.0 based on a Michaelis-Menten equation to obtain kinetic constants Km and Vmax, and kcat/Km is calculated.

The specific activity of Tdot-SI and SI-Tdot active inclusion bodies is 622.6 +/-17.5U/g and 252.6 +/-17.1U/g respectively under the optimal reaction condition. Further detecting the conversion reaction product of the active inclusion body under sucrose substrates with different concentrations, calculating to obtain the catalytic reaction speed, obtaining a kinetic constant through nonlinear fitting, and obtaining the result shown in Table 2, wherein the K of Tdot-SI_m103.9. + -. 9.5mmol/L, k_cat/K_mK of 0.06 + -0.002L/(mmols), SI-Tdot_mIs 54.4 +/-6.6 mmol/L, k_cat/K_mThe activity inclusion body formed by fusing the Tdot label with the N/C end of the SI has different kinetic parameters on substrate sucrose, and the SI-Tdot has stronger binding capacity with the substrate sucrose, but the catalytic efficiency is slightly lower than that of the Tdot-SI.

TABLE 2 kinetic parameters of Tdot-SI/SI-Tdot

Research work has been reported on the preparation of immobilized sucrose isomerase, such as mesoporous TiO modified with epsilon-poly-L-lysine₂The specific activity of immobilized SI is 39.41U/g, Km is 204.92mmol/L [ Wu L, Liu Y, Chi B, et al, an innovative method for immobilizing a sugar isomer on epsilon-poly-L-lysine modified meso-TiO 2[ J].Food chemistry,2015,187:182-188.]Sucrose Isomerase was immobilized using a Sponge made of epsilon-poly-L-lysine and gelatin with a specific activity of 71.58U/g and a Km of 245.71mmol/L, and was not significantly changed from the unimmobilized free enzyme Km of 259.71mmol/L [ Wu L, Qiu J, Wu S, et al, Bioinspired Production of Antibacterial Sucrose isomer-Sponge for the Synthesis of Isomaltulose [ J L].Advanced Synthesis&Catalysis,2016,358(24):4030-4040.]. The invention synchronously expresses and fixes SI in cellsThe active inclusion body is prepared by the customization, so that a carrier material and an additional immobilization reaction are not needed, and the specific activity per unit mass is better, and the application potential is better. Meanwhile, the invention discovers that the active inclusion bodies formed by fusing the Tdot label at the N/C terminal of the SI show different kinetic parameters to the substrate sucrose, which indicates that the formed active inclusion bodies influence the combination with the substrate and the catalytic process due to different microstructures.

Example 6 product specificity analysis

Mixing 100 μ L of Tdot-SI active inclusion body enzyme solution with concentration of 4mg/mL and 400 μ L of disodium hydrogen phosphate-citric acid buffer solution with pH 5.5 containing 4% sucrose; mu.L of SI-Tdot active inclusion body enzyme solution with the concentration of 4mg/mL is mixed with 400 mu.L of disodium hydrogen phosphate-citric acid buffer solution with the pH value of 5.0 and containing 4 percent of sucrose. And (3) reacting the active inclusion body conversion reaction system at 30, 35 and 40 ℃ for 12h at 200r/min respectively, then treating an enzyme conversion sample by boiling water at 100 ℃ for 15min for inactivating enzyme, centrifuging at 10000r/min for 15min to obtain reaction product supernatant, filtering by using a 0.22 mu m filter membrane, and detecting the conversion reaction product by using HPLC.

As shown in Table 3, the isomaltulose content of the conversion reaction products did not change significantly in the current temperature range of Tdot-SI and SI-Tdot active inclusion bodies, while the trehalulose content of the products generally showed a decreasing trend with increasing conversion reaction temperature, while the monosaccharide (fructose and glucose) content increased with increasing temperature.

TABLE 3 analysis of the composition of the catalytic product of sucrose isomerase fused with Tdot tag at different temperatures

The invention successfully expresses sucrose isomerase as an active inclusion body in Escherichia coli E.coli BL21(DE3) by using a coiled coil domain Tdot as a fusion label through a fusion expression technology, realizes the synchronous expression and immobilization, is used as a novel self-immobilized enzyme, does not need a carrier material and a chemical reagent for a traditional immobilization reaction, has potential industrial application value, provides a new strategy for realizing the efficient preparation and utilization of the recombinant sucrose isomerase, and lays a foundation for further design and optimization.

SEQUENCE LISTING

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcaccat ccttgaatca ggatattcac gttcaaaagg aaagtgaata tcctgcatgg 60

tggaaagaag ctgtttttta tcagatctat cctcgctcat ttaaagacac caatgatgat 120

ggcattggcg atattcgcgg tattattgaa aagctggact atctgaaatc gctcggtatt 180

gacgctatct ggatcaatcc ccattacgac tctccgaaca ccgataacgg ctatgacatc 240

agtaattatc gtcagataat gaaagagtat ggcacaatgg aggattttga tagccttgtt 300

gccgaaatga aaaaacgaaa tatgcgctta atgatcgacg tggtcattaa ccataccagt 360

gatcaacacc cgtggtttat tcagagtaaa agcgataaaa acaaccctta tcgtgactat 420

tatttctggc gtgacggaaa agataatcag ccacctaata attacccctc atttttcggc 480

ggctcggcat ggcaaaaaga tgcaaagtca ggacagtact atttacacta ttttgccaga 540

cagcaacctg atctcaactg ggataacccg aaagtacgtg aggatcttta cgcaatgctc 600

cgcttctggc tggataaagg cgtttcaggc atgcgatttg atacggtggc aacttattcc 660

aaaatcccgg gatttcccaa tctgacacct gaacaacaga aaaattttgc tgaacaatac 720

accatggggc ctaatattca tcgatacatt caggaaatga accggaaagt tctgtcccgg 780

tatgatgtgg ccaccgcggg tgaaattttt ggcgtcccgc tggatcgttc gtcgcagttt 840

tttgatcgcc gccgacatga gctgaatatg gcgtttatgt ttgacctcat tcgtctcgat 900

cgcgacagca atgaacgctg gcgtcacaag tcgtggtcgc tctctcagtt ccgccagatc 960

atcagcaaaa tggatgtcac ggtcggaaag tatggctgga acacgttctt cttagataac 1020

catgacaacc cccgtgcggt atctcacttc ggggatgaca ggccgcaatg gcgggaggcg 1080

tcggctaagg cactggcgac gattaccctc actcagcggg cgacgccgtt tatttatcag 1140

ggttcagagc tgggaatgac taattatccc ttcaggcaac tcaacgaatt tgacgacatc 1200

gaggtcaaag gtttctggca ggattatgtc cagagtggaa aagtcacggc cacagagttt 1260

ctcgataatg tgcgcctgac gagccgcgat aacagcagaa cacctttcca gtggaatgac 1320

accctgaatg ctggttttac tcgcggaaag ccgtggtttc acatcaaccc aaactatgtg 1380

gagatcaacg ccgaacgcga agaaacccgc gaagattcag tgctgaatta ctataaaaaa 1440

atgattcagc tacgccacca tatccctgct ctggtatatg gcgcctatca ggatcttaat 1500

ccacaggaca ataccgttta tgcctatacc cgaacgctgg gtaacgagcg ttatctggtc 1560

gtggtgaact ttaaggagta cccggtccgc tatactctcc cggctaatga tgccatcgag 1620

gaagtggtca ttgatactca gcagcaggcg gctgcgccgc acagcacatc cctgtcattg 1680

agcccctggc aggcaggtgt gtataagctg cggggatcca tcattaacga aactgccgat 1740

gacatcgttt atcgcctgac agtcattatc gatgatcgct acgaatcgct gaaaaacctg 1800

attaccttac gtgcagatcg cttggagatg atcatcaatg acaatgtgtc caccattctc 1860

gcgagcatt 1869

<210> 3

<211> 623

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ile Ile Asn Glu Thr Ala Asp Asp Ile Val Tyr Arg Leu Thr Val

1 5 10 15

Ile Ile Asp Asp Arg Tyr Glu Ser Leu Lys Asn Leu Ile Thr Leu Arg

20 25 30

Ala Asp Arg Leu Glu Met Ile Ile Asn Asp Asn Val Ser Thr Ile Leu

35 40 45

Ala Ser Ile Gly Ser Ala Pro Ser Leu Asn Gln Asp Ile His Val Gln

50 55 60

Lys Glu Ser Glu Tyr Pro Ala Trp Trp Lys Glu Ala Val Phe Tyr Gln

65 70 75 80

Ile Tyr Pro Arg Ser Phe Lys Asp Thr Asn Asp Asp Gly Ile Gly Asp

85 90 95

Ile Arg Gly Ile Ile Glu Lys Leu Asp Tyr Leu Lys Ser Leu Gly Ile

100 105 110

Asp Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn

115 120 125

Gly Tyr Asp Ile Ser Asn Tyr Arg Gln Ile Met Lys Glu Tyr Gly Thr

130 135 140

Met Glu Asp Phe Asp Ser Leu Val Ala Glu Met Lys Lys Arg Asn Met

145 150 155 160

Arg Leu Met Ile Asp Val Val Ile Asn His Thr Ser Asp Gln His Pro

165 170 175

Trp Phe Ile Gln Ser Lys Ser Asp Lys Asn Asn Pro Tyr Arg Asp Tyr

180 185 190

Tyr Phe Trp Arg Asp Gly Lys Asp Asn Gln Pro Pro Asn Asn Tyr Pro

195 200 205

Ser Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Ala Lys Ser Gly Gln

210 215 220

Tyr Tyr Leu His Tyr Phe Ala Arg Gln Gln Pro Asp Leu Asn Trp Asp

225 230 235 240

Asn Pro Lys Val Arg Glu Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu

245 250 255

Asp Lys Gly Val Ser Gly Met Arg Phe Asp Thr Val Ala Thr Tyr Ser

260 265 270

Lys Ile Pro Gly Phe Pro Asn Leu Thr Pro Glu Gln Gln Lys Asn Phe

275 280 285

Ala Glu Gln Tyr Thr Met Gly Pro Asn Ile His Arg Tyr Ile Gln Glu

290 295 300

Met Asn Arg Lys Val Leu Ser Arg Tyr Asp Val Ala Thr Ala Gly Glu

305 310 315 320

Ile Phe Gly Val Pro Leu Asp Arg Ser Ser Gln Phe Phe Asp Arg Arg

325 330 335

Arg His Glu Leu Asn Met Ala Phe Met Phe Asp Leu Ile Arg Leu Asp

340 345 350

Arg Asp Ser Asn Glu Arg Trp Arg His Lys Ser Trp Ser Leu Ser Gln

355 360 365

Phe Arg Gln Ile Ile Ser Lys Met Asp Val Thr Val Gly Lys Tyr Gly

370 375 380

Trp Asn Thr Phe Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser

385 390 395 400

His Phe Gly Asp Asp Arg Pro Gln Trp Arg Glu Ala Ser Ala Lys Ala

405 410 415

Leu Ala Thr Ile Thr Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln

420 425 430

Gly Ser Glu Leu Gly Met Thr Asn Tyr Pro Phe Arg Gln Leu Asn Glu

435 440 445

Phe Asp Asp Ile Glu Val Lys Gly Phe Trp Gln Asp Tyr Val Gln Ser

450 455 460

Gly Lys Val Thr Ala Thr Glu Phe Leu Asp Asn Val Arg Leu Thr Ser

465 470 475 480

Arg Asp Asn Ser Arg Thr Pro Phe Gln Trp Asn Asp Thr Leu Asn Ala

485 490 495

Gly Phe Thr Arg Gly Lys Pro Trp Phe His Ile Asn Pro Asn Tyr Val

500 505 510

Glu Ile Asn Ala Glu Arg Glu Glu Thr Arg Glu Asp Ser Val Leu Asn

515 520 525

Tyr Tyr Lys Lys Met Ile Gln Leu Arg His His Ile Pro Ala Leu Val

530 535 540

Tyr Gly Ala Tyr Gln Asp Leu Asn Pro Gln Asp Asn Thr Val Tyr Ala

545 550 555 560

Tyr Thr Arg Thr Leu Gly Asn Glu Arg Tyr Leu Val Val Val Asn Phe

565 570 575

Lys Glu Tyr Pro Val Arg Tyr Thr Leu Pro Ala Asn Asp Ala Ile Glu

580 585 590

Glu Val Val Ile Asp Thr Gln Gln Gln Ala Ala Ala Pro His Ser Thr

595 600 605

Ser Leu Ser Leu Ser Pro Trp Gln Ala Gly Val Tyr Lys Leu Arg

610 615 620

<210> 4

<211> 623

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Pro Ser Leu Asn Gln Asp Ile His Val Gln Lys Glu Ser Glu

1 5 10 15

Tyr Pro Ala Trp Trp Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg

20 25 30

Ser Phe Lys Asp Thr Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile

35 40 45

Ile Glu Lys Leu Asp Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp

50 55 60

Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile

65 70 75 80

Ser Asn Tyr Arg Gln Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe

85 90 95

Asp Ser Leu Val Ala Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile

100 105 110

Asp Val Val Ile Asn His Thr Ser Asp Gln His Pro Trp Phe Ile Gln

115 120 125

Ser Lys Ser Asp Lys Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg

130 135 140

Asp Gly Lys Asp Asn Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly

145 150 155 160

Gly Ser Ala Trp Gln Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His

165 170 175

Tyr Phe Ala Arg Gln Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val

180 185 190

Arg Glu Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val

195 200 205

Ser Gly Met Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly

210 215 220

Phe Pro Asn Leu Thr Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr

225 230 235 240

Thr Met Gly Pro Asn Ile His Arg Tyr Ile Gln Glu Met Asn Arg Lys

245 250 255

Val Leu Ser Arg Tyr Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val

260 265 270

Pro Leu Asp Arg Ser Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu

275 280 285

Asn Met Ala Phe Met Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn

290 295 300

Glu Arg Trp Arg His Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile

305 310 315 320

Ile Ser Lys Met Asp Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe

325 330 335

Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp

340 345 350

Asp Arg Pro Gln Trp Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile

355 360 365

Thr Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu

370 375 380

Gly Met Thr Asn Tyr Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile

385 390 395 400

Glu Val Lys Gly Phe Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr

405 410 415

Ala Thr Glu Phe Leu Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser

420 425 430

Arg Thr Pro Phe Gln Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg

435 440 445

Gly Lys Pro Trp Phe His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala

450 455 460

Glu Arg Glu Glu Thr Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys

465 470 475 480

Met Ile Gln Leu Arg His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr

485 490 495

Gln Asp Leu Asn Pro Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr

500 505 510

Leu Gly Asn Glu Arg Tyr Leu Val Val Val Asn Phe Lys Glu Tyr Pro

515 520 525

Val Arg Tyr Thr Leu Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile

530 535 540

Asp Thr Gln Gln Gln Ala Ala Ala Pro His Ser Thr Ser Leu Ser Leu

545 550 555 560

Ser Pro Trp Gln Ala Gly Val Tyr Lys Leu Arg Gly Ser Ile Ile Asn

565 570 575

Glu Thr Ala Asp Asp Ile Val Tyr Arg Leu Thr Val Ile Ile Asp Asp

580 585 590

Arg Tyr Glu Ser Leu Lys Asn Leu Ile Thr Leu Arg Ala Asp Arg Leu

595 600 605

Glu Met Ile Ile Asn Asp Asn Val Ser Thr Ile Leu Ala Ser Ile

610 615 620

<210> 5

<211> 1713

<212> DNA

<213> Sucrose Isomerase (SI)

<400> 5

atggcaccat ccttgaatca ggatattcac gttcaaaagg aaagtgaata tcctgcatgg 60

tggaaagaag ctgtttttta tcagatctat cctcgctcat ttaaagacac caatgatgat 120

ggcattggcg atattcgcgg tattattgaa aagctggact atctgaaatc gctcggtatt 180

gacgctatct ggatcaatcc ccattacgac tctccgaaca ccgataacgg ctatgacatc 240

agtaattatc gtcagataat gaaagagtat ggcacaatgg aggattttga tagccttgtt 300

gccgaaatga aaaaacgaaa tatgcgctta atgatcgacg tggtcattaa ccataccagt 360

gatcaacacc cgtggtttat tcagagtaaa agcgataaaa acaaccctta tcgtgactat 420

tatttctggc gtgacggaaa agataatcag ccacctaata attacccctc atttttcggc 480

ggctcggcat ggcaaaaaga tgcaaagtca ggacagtact atttacacta ttttgccaga 540

cagcaacctg atctcaactg ggataacccg aaagtacgtg aggatcttta cgcaatgctc 600

cgcttctggc tggataaagg cgtttcaggc atgcgatttg atacggtggc aacttattcc 660

aaaatcccgg gatttcccaa tctgacacct gaacaacaga aaaattttgc tgaacaatac 720

accatggggc ctaatattca tcgatacatt caggaaatga accggaaagt tctgtcccgg 780

tatgatgtgg ccaccgcggg tgaaattttt ggcgtcccgc tggatcgttc gtcgcagttt 840

tttgatcgcc gccgacatga gctgaatatg gcgtttatgt ttgacctcat tcgtctcgat 900

cgcgacagca atgaacgctg gcgtcacaag tcgtggtcgc tctctcagtt ccgccagatc 960

atcagcaaaa tggatgtcac ggtcggaaag tatggctgga acacgttctt cttagataac 1020

catgacaacc cccgtgcggt atctcacttc ggggatgaca ggccgcaatg gcgggaggcg 1080

tcggctaagg cactggcgac gattaccctc actcagcggg cgacgccgtt tatttatcag 1140

ggttcagagc tgggaatgac taattatccc ttcaggcaac tcaacgaatt tgacgacatc 1200

gaggtcaaag gtttctggca ggattatgtc cagagtggaa aagtcacggc cacagagttt 1260

ctcgataatg tgcgcctgac gagccgcgat aacagcagaa cacctttcca gtggaatgac 1320

accctgaatg ctggttttac tcgcggaaag ccgtggtttc acatcaaccc aaactatgtg 1380

gagatcaacg ccgaacgcga agaaacccgc gaagattcag tgctgaatta ctataaaaaa 1440

atgattcagc tacgccacca tatccctgct ctggtatatg gcgcctatca ggatcttaat 1500

ccacaggaca ataccgttta tgcctatacc cgaacgctgg gtaacgagcg ttatctggtc 1560

gtggtgaact ttaaggagta cccggtccgc tatactctcc cggctaatga tgccatcgag 1620

gaagtggtca ttgatactca gcagcaggcg gctgcgccgc acagcacatc cctgtcattg 1680

agcccctggc aggcaggtgt gtataagctg cgg 1713

<210> 6

<211> 153

<212> DNA

<213> coiled coil Domain (Tdot)

<400> 6

atgatcatta acgaaactgc cgatgacatc gtttatcgcc tgacagtcat tatcgatgat 60

cgctacgaat cgctgaaaaa cctgattacc ttacgtgcag atcgcttgga gatgatcatc 120

aatgacaatg tgtccaccat tctcgcgagc att 153

<210> 7

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgcggatccg caccatcctt gaatcaggat attcacg 37

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgcggatccg caccatcctt gaatcaggat attcacg 37

<210> 9

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caggcaggtg tgtataagct gcggggatcc atcatcatta acgaaactgc cgatg 55

<210> 10

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcagtggtgg tggtggtggt gttaaatgct cgcgagaatg gtggac 46

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

taatacgact cactataggg 20

<210> 12

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctagttatt gctcagcgg 19

<210> 13

<211> 6968

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatataca tatgcatcat catcatcatc acgcaccatc 5100

cttgaatcag gatattcacg ttcaaaagga aagtgaatat cctgcatggt ggaaagaagc 5160

tgttttttat cagatctatc ctcgctcatt taaagacacc aatgatgatg gcattggcga 5220

tattcgcggt attattgaaa agctggacta tctgaaatcg ctcggtattg acgctatctg 5280

gatcaatccc cattacgact ctccgaacac cgataacggc tatgacatca gtaattatcg 5340

tcagataatg aaagagtatg gcacaatgga ggattttgat agccttgttg ccgaaatgaa 5400

aaaacgaaat atgcgcttaa tgatcgacgt ggtcattaac cataccagtg atcaacaccc 5460

gtggtttatt cagagtaaaa gcgataaaaa caacccttat cgtgactatt atttctggcg 5520

tgacggaaaa gataatcagc cacctaataa ttacccctca tttttcggcg gctcggcatg 5580

gcaaaaagat gcaaagtcag gacagtacta tttacactat tttgccagac agcaacctga 5640

tctcaactgg gataacccga aagtacgtga ggatctttac gcaatgctcc gcttctggct 5700

ggataaaggc gtttcaggca tgcgatttga tacggtggca acttattcca aaatcccggg 5760

atttcccaat ctgacacctg aacaacagaa aaattttgct gaacaataca ccatggggcc 5820

taatattcat cgatacattc aggaaatgaa ccggaaagtt ctgtcccggt atgatgtggc 5880

caccgcgggt gaaatttttg gcgtcccgct ggatcgttcg tcgcagtttt ttgatcgccg 5940

ccgacatgag ctgaatatgg cgtttatgtt tgacctcatt cgtctcgatc gcgacagcaa 6000

tgaacgctgg cgtcacaagt cgtggtcgct ctctcagttc cgccagatca tcagcaaaat 6060

ggatgtcacg gtcggaaagt atggctggaa cacgttcttc ttagataacc atgacaaccc 6120

ccgtgcggta tctcacttcg gggatgacag gccgcaatgg cgggaggcgt cggctaaggc 6180

actggcgacg attaccctca ctcagcgggc gacgccgttt atttatcagg gttcagagct 6240

gggaatgact aattatccct tcaggcaact caacgaattt gacgacatcg aggtcaaagg 6300

tttctggcag gattatgtcc agagtggaaa agtcacggcc acagagtttc tcgataatgt 6360

gcgcctgacg agccgcgata acagcagaac acctttccag tggaatgaca ccctgaatgc 6420

tggttttact cgcggaaagc cgtggtttca catcaaccca aactatgtgg agatcaacgc 6480

cgaacgcgaa gaaacccgcg aagattcagt gctgaattac tataaaaaaa tgattcagct 6540

acgccaccat atccctgctc tggtatatgg cgcctatcag gatcttaatc cacaggacaa 6600

taccgtttat gcctataccc gaacgctggg taacgagcgt tatctggtcg tggtgaactt 6660

taaggagtac ccggtccgct atactctccc ggctaatgat gccatcgagg aagtggtcat 6720

tgatactcag cagcaggcgg ctgcgccgca cagcacatcc ctgtcattga gcccctggca 6780

ggcaggtgtg tataagctgc ggtaactcga gcaccaccac caccaccact gagatccggc 6840

tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 6900

ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 6960

atccggat 6968

<210> 14

<211> 6947

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatataca tatggcacca tccttgaatc aggatattca 5100

cgttcaaaag gaaagtgaat atcctgcatg gtggaaagaa gctgtttttt atcagatcta 5160

tcctcgctca tttaaagaca ccaatgatga tggcattggc gatattcgcg gtattattga 5220

aaagctggac tatctgaaat cgctcggtat tgacgctatc tggatcaatc cccattacga 5280

ctctccgaac accgataacg gctatgacat cagtaattat cgtcagataa tgaaagagta 5340

tggcacaatg gaggattttg atagccttgt tgccgaaatg aaaaaacgaa atatgcgctt 5400

aatgatcgac gtggtcatta accataccag tgatcaacac ccgtggttta ttcagagtaa 5460

aagcgataaa aacaaccctt atcgtgacta ttatttctgg cgtgacggaa aagataatca 5520

gccacctaat aattacccct catttttcgg cggctcggca tggcaaaaag atgcaaagtc 5580

aggacagtac tatttacact attttgccag acagcaacct gatctcaact gggataaccc 5640

gaaagtacgt gaggatcttt acgcaatgct ccgcttctgg ctggataaag gcgtttcagg 5700

catgcgattt gatacggtgg caacttattc caaaatcccg ggatttccca atctgacacc 5760

tgaacaacag aaaaattttg ctgaacaata caccatgggg cctaatattc atcgatacat 5820

tcaggaaatg aaccggaaag ttctgtcccg gtatgatgtg gccaccgcgg gtgaaatttt 5880

tggcgtcccg ctggatcgtt cgtcgcagtt ttttgatcgc cgccgacatg agctgaatat 5940

ggcgtttatg tttgacctca ttcgtctcga tcgcgacagc aatgaacgct ggcgtcacaa 6000

gtcgtggtcg ctctctcagt tccgccagat catcagcaaa atggatgtca cggtcggaaa 6060

gtatggctgg aacacgttct tcttagataa ccatgacaac ccccgtgcgg tatctcactt 6120

cggggatgac aggccgcaat ggcgggaggc gtcggctaag gcactggcga cgattaccct 6180

cactcagcgg gcgacgccgt ttatttatca gggttcagag ctgggaatga ctaattatcc 6240

cttcaggcaa ctcaacgaat ttgacgacat cgaggtcaaa ggtttctggc aggattatgt 6300

ccagagtgga aaagtcacgg ccacagagtt tctcgataat gtgcgcctga cgagccgcga 6360

taacagcaga acacctttcc agtggaatga caccctgaat gctggtttta ctcgcggaaa 6420

gccgtggttt cacatcaacc caaactatgt ggagatcaac gccgaacgcg aagaaacccg 6480

cgaagattca gtgctgaatt actataaaaa aatgattcag ctacgccacc atatccctgc 6540

tctggtatat ggcgcctatc aggatcttaa tccacaggac aataccgttt atgcctatac 6600

ccgaacgctg ggtaacgagc gttatctggt cgtggtgaac tttaaggagt acccggtccg 6660

ctatactctc ccggctaatg atgccatcga ggaagtggtc attgatactc agcagcaggc 6720

ggctgcgccg cacagcacat ccctgtcatt gagcccctgg caggcaggtg tgtataagct 6780

gcggctcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 6840

agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 6900

acgggtcttg aggggttttt tgctgaaagg aggaactata tccggat 6947

Claims

1. A gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.

2. A gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 2.

3. The fusion protein encoded by the gene of claim 1 and comprising TdoT-SI, wherein the amino acid sequence is set forth in SEQ ID No. 3.

4. The fusion protein encoded by the gene of claim 2 and comprising SI-Tdot, wherein the amino acid sequence is set forth in SEQ ID No. 4.

5. A recombinant engineered bacterium containing the gene of claim 1.

6. A recombinant engineered bacterium containing the gene of claim 2.

7. An inclusion body of sucrose isomerase activity, which is obtained by inducing the expression of fusion protein by the recombinant engineering bacteria of claim 5 or 6, breaking, separating and purifying the bacteria.

8. Use of an inclusion body of sucrose isomerase activity for catalysing the conversion of sucrose to isomaltulose.

9. The use according to claim 8, wherein the sucrose isomerase activity comprises a sucrose isomerase activity that catalyzes the conversion of sucrose into isomaltulose at a pH of 4.5 to 6.5 and a temperature of 30 to 45 ℃.