A sucrose isomerase active inclusion body fused with coiled-coil domain

Abstract

The invention discloses a sucrose isomerase active inclusion body fused with a coiled-coil domain, and belongs to the field of bioengineering. The invention takes SI derived from Klebsiella sp.LX3 as the research object, respectively fuses TdoT to the N or C end of SI with its own natural signal peptide sequence removed, constructs a recombinant expression vector and transfers it into Escherichia coli E.coli BL21 (DE3) for induced expression, further separates and purifies the active inclusion body, and characterizes the enzymatic properties, thereby providing a new strategy for heterologous expression and efficient utilization of recombinant sucrose isomerase.

Description

Technical Field

[0001] The invention relates to a sucrose isomerase active inclusion body fused with a coiled coil domain, and belongs to the field of bioengineering. Background Art

[0002] Isomaltulose (α-D-pyranoglucopyranosyl-1,6-fructose, Isomaltulose) is an isomer of sucrose, naturally present in honey and sugarcane juice, and its sweetness is about 50% of sucrose. At present, isomaltulose is mainly used in the food industry as a substitute for sucrose in the production of health products, diabetic food and sports drinks. In addition, isomaltulose is also used as a raw material for the production of some surfactants and high molecular polymers in the chemical industry, so it is a functional sugar with good development prospects. At present, the industry mainly uses sucrose isomerase (SI) to catalyze the conversion of sucrose into isomaltulose, which has great advantages over chemical synthesis, such as mild reaction conditions, green and environmentally friendly preparation process, and high reaction specificity. As isomaltulose is used more and more widely and becomes more and more popular among consumers, the market demand for isomaltulose is increasing year by year. However, its production is mainly monopolized by foreign companies, and the market price is about 15,000 yuan per ton. Therefore, increasing the utilization rate of sucrose isomerase and thus reducing the production cost of isomaltulose has received increasing attention.

[0003] A variety of bacteria that produce sucrose isomerase have been found, including Erwinia rhapontici, Serratia plymuthica, Pantoea dispersa, and Klebsiella sp. However, the enzyme production level of wild fungi is generally low, which is difficult to meet the requirements of industrial production. For example, the fermentation enzyme activity of Erwinia rhapontici NX-5 is 1.3U / mL, and the fermentation enzyme activity of Klebsiella sp.LX3 is 15.12U / mL. Therefore, researchers currently mainly prepare recombinant sucrose isomerase by heterologous expression and immobilize it on a water-insoluble carrier to prepare an immobilized enzyme to improve utilization. For example, using mesoporous TiO2 modified with ε-poly-L-lysine 2The sponge prepared by ε-poly-L-lysine and gelatin immobilized sucrose isomerase, with an enzyme activity recovery rate of 93.3% and 84.5% respectively; the chitosan-glutaraldehyde crosslinking method, the sodium alginate-sodium carboxymethyl cellulose mixed embedding method and the crosslinked enzyme aggregate method were used to immobilize sucrose isomerase, and the enzyme activity recovery rates were 70.3%, 32.5% and 30.9% respectively. In summary, although sucrose isomerase was successfully prepared into immobilized enzyme by traditional adsorption method, binding method, crosslinking method and embedding method, it is necessary to first separate and purify the pure enzyme by chromatography technology, and then use water-insoluble carriers to carry out immobilization reaction with the pure enzyme. The separation and purification, preparation of carriers and chemical reagents related to the immobilization reaction in this process all increase the production cost of the immobilized enzyme, which is not conducive to the large-scale production and application of immobilized sucrose isomerase. Recent studies have found that the use of specific tag sequences and enzyme protein fusion expression can induce it to form catalytically active inclusion bodies (Catalytically-active Inclusion Bodies, CatIBs, hereinafter referred to as active inclusion bodies) in cells. The expression level is large, and it is easy to separate and purify. In particular, the expression and immobilization processes are carried out simultaneously, without the need for additional carrier materials and immobilization reactions. It is a new type of self-immobilized enzyme, which not only theoretically changes the past view that "inclusion bodies are completely inactive protein aggregates", but also has good industrial application potential as a new enzyme heterologous expression and immobilization method. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an active inclusion body of sucrose isomerase fused with a coiled-coil domain. The present invention takes SI derived from Klebsiella sp.LX3 as the research object, which has the advantages of high sucrose conversion rate (99.5%) and isomaltulose as the main product. TdoT is fused to the N or C terminus of SI with its own natural signal peptide sequence removed, and a recombinant expression vector is constructed and transferred into Escherichia coli E.coli BL21 (DE3) for induced expression. The active inclusion body is further separated and purified, and the enzymatic properties are characterized, thereby providing a new strategy for the heterologous expression and efficient utilization of recombinant sucrose isomerase.

[0005] The present invention provides a gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0006] The present invention also provides a gene, the nucleotide sequence of which is shown as SEQ ID NO.2.

[0007] The present invention also provides a fusion protein containing TdoT-SI encoded by a gene whose nucleotide sequence is shown as SEQ ID NO.1, and whose amino acid sequence is shown as SEQ ID NO.3.

[0008] The present invention also provides a fusion protein containing SI-TdoT encoded by a gene whose nucleotide sequence is shown as SEQ ID NO.2, and whose amino acid sequence is shown as SEQ ID NO.4.

[0009] The present invention also provides a recombinant engineering bacterium containing a gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0010] The present invention also provides a recombinant engineering bacterium containing a gene with a nucleotide sequence as shown in SEQ ID NO.2.

[0011] The present invention also provides a sucrose isomerase active inclusion body, which is obtained by inducing fusion protein expression in a recombinant engineering bacterium containing a gene with a nucleotide sequence such as SEQ ID NO.1 or a recombinant engineering bacterium containing a gene with a nucleotide sequence such as SEQ ID NO.2, and then breaking, separating and purifying the bacterium.

[0012] Specifically, the method for preparing the sucrose isomerase active inclusion body comprises the following steps: using the coiled-coil domain TdoT to fuse to the N or C terminus of sucrose isomerase SI, constructing a recombinant expression vector and transferring it into Escherichia coli BL21 (DE3) for induced expression to prepare active inclusion bodies.

[0013] The method for preparing the sucrose isomerase activity inclusion body of the recombinant engineering bacteria containing the gene with the nucleotide sequence as shown in SEQ ID NO.1 specifically comprises the following steps:

[0014] (1) constructing a recombinant expression vector pET-24a-TdoT-SI containing a gene encoding a TdoT-SI fusion protein, and transferring the recombinant expression vector into an expression host cell to obtain a recombinant engineered bacterium;

[0015] (2) culturing the recombinant engineered bacteria to induce expression of the fusion protein;

[0016] (3) Collecting bacterial cells and disrupting the cells, separating the cell disruption supernatant and the precipitate sample containing the inclusion bodies, and washing the precipitate sample with a buffer solution to purify the active inclusion bodies.

[0017] The method for preparing the sucrose isomerase activity inclusion body of the recombinant engineering bacteria containing the gene with the nucleotide sequence as shown in SEQ ID NO.2 specifically comprises the following steps:

[0018] (1) constructing a recombinant expression vector pET-24b-SI-TdoT containing a gene encoding the SI-TdoT fusion protein, and transferring the recombinant expression vector into an expression host cell to obtain a recombinant engineered bacterium;

[0019] (2) culturing the recombinant engineered bacteria to induce expression of the fusion protein;

[0020] (3) Collecting bacterial cells and disrupting the cells, separating the cell disruption supernatant and the precipitate sample containing the inclusion bodies, and washing the precipitate sample with a buffer solution to purify the active inclusion bodies.

[0021] Preferably, the sucrose isomerase SI is derived from Klebsiella sp.LX3, and its nucleotide sequence is shown in SEQ ID NO.5.

[0022] Preferably, the TdoT gene is from Staphylothermus marinus, and its nucleotide sequence is shown in SEQ ID NO.6.

[0023] By way of example but not limitation, the expression host cell is preferably E. coli BL21 (DE3).

[0024] The present invention also provides an application of sucrose isomerase active inclusion bodies in catalyzing the conversion of sucrose into isomaltulose.

[0025] Furthermore, the pH of the sucrose isomerase active inclusion body catalyzing the conversion of sucrose into isomaltulose is 4.5-6.5 and the temperature is 30-45°C.

[0026] The present invention fuses the tetrameric coiled-coil domain of the cell-surface protein tetrabrachion (TdoT) derived from the cell surface protein tetrabrachion with SI for expression and studies the enzymatic properties of the recombinant enzyme. TdoT is fused to the N / C termini of SI respectively, a recombinant expression vector is constructed, and the vector is transferred into Escherichia coli E.coli BL21 (DE3) for induced expression. The results show that TdoT-SI and SI-TdoT are expressed as inclusion bodies with catalytic activity, while SI inclusion bodies without TdoT fusion have no catalytic activity. The active inclusion bodies are further purified by centrifugation and buffer washing. Enzymatic property studies show that the optimal reaction temperature of the active inclusion bodies of TdoT-SI and SI-TdoT is 40°C, and the optimal reaction pH is 5.5 and 5.0, respectively. The kinetic constant Km of the active inclusion body of TdoT-SI is 103.9±9.5mmol / L, and kcat / Km is 0.06±0.002L / (mmol·s). The kinetic constant Km of the active inclusion body of SI-TdoT is 54.4±6.6mmol / L, and kcat / Km is 0.03±0.002L / (mmol·s). The results of the product specificity study showed that with the increase of the reaction temperature, the content of isomaltulose in the product did not change significantly, while the trehalulose in the product decreased with the increase of the conversion reaction temperature, and the content of monosaccharides increased with the increase of the conversion reaction temperature. The present invention successfully prepared the active inclusion body of sucrose isomerase fused with the coiled-coil domain through fusion expression technology. As a new type of self-immobilized enzyme, it has the advantage of simultaneous expression and immobilization, and provides a new strategy for the large-scale preparation and efficient utilization of recombinant sucrose isomerase. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the colony PCR identification of transformed recombinant plasmid engineering bacteria; Lane M is 250bp DNA Ladder Marker, Lane 1 is pET-24a-TdoT-SI; Lane 2 is pET-24b-SI-TdoT.

[0028] Figure 2 This is the SDS-PAGE analysis of recombinant 6×His-SI / SI-6×His; Lane M is Protein Marker; Lanes 1 to 4 are pET24b-6×His-SI before and after induction, supernatant, and precipitate; Lanes 5 to 8 are pET24b-SI-6×His before and after induction, supernatant, and precipitate.

[0029] Figure 3This is the SDS-PAGE analysis of recombinant TdoT-SI / SI-TdoT; Lane M is Protein Marker; Lanes 1 to 4 are pET24a-TdoT-SI before induction, after induction, supernatant, and inclusion bodies after washing; Lanes 5 to 8 are pET24b-SI-TdoT before induction, after induction, supernatant, and inclusion bodies after washing.

[0030] Figure 4 It is the effect of temperature on the enzyme activity of TdoT-SI and SI-TdoT.

[0031] Figure 5 The effect of pH on the enzyme activity of TdoT-SI and SI-TdoT. DETAILED DESCRIPTION

[0032] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0033] Sucrose isomerase SI (NCBI accession 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 sucrose isomerase SI gene can be synthesized by conventional means in the art. The plasmids pET24b-6×His-SI (nucleotide sequence shown in SEQ ID NO.13) and pET24b-SI-6×His (nucleotide sequence shown in SEQ ID NO.14) fused with histidine tags at the N / C termini can be synthesized by conventional means in the art.

[0034] The nucleotide sequence of TdoT is shown in SEQ ID NO.6. The plasmid pET-24a-TdoT carrying the TdoT gene was synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0035] Kanamycin, isopropyl-beta-D-thiogalactopyranoside (IPTG), Triton-X100, SanPrep column plasmid DNA mini-extraction kit, SanPrep column DNA gel recovery kit, and BCA protein quantitative detection kit were all purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0036] 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 Biotechnology (Dalian) Co., Ltd.

[0037] The gene sequencing work was completed by Jilin Kumei Biotechnology Co., Ltd.

[0038] Isomaltulose was purchased from Sigma.

[0039] Other chemical reagents were purchased from Tianjin Kemeiou Chemical Reagent Co., Ltd.

[0040] LB liquid medium: contains 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl, pH 7.2; LB solid medium is prepared by adding 1.5% agar to LB liquid medium.

[0041] LLB medium: contains 10 g / L tryptone, 5 g / L yeast powder, 5 g / L NaCl, pH 7.2.

[0042] Buffer A: Na 2 HPO 4 12H 2 O: 16.937 g, NaH 2 PO 4 2H2O: 0.62 g, dissolved in 450-750 mL deionized water, adjusted to pH 8.0, added 50 mL glycerol, and made up to 1 L with deionized water.

[0043] Buffer B: Dissolve 0.5 mL Triton-X100 in 100 mL Buffer A.

[0044] Buffer C: Na 2 HPO 4 12H 2 O: 16.937 g, NaH 2 PO 4 ·2H 2 O: 0.62 g, dissolved in 450-750 mL deionized water, adjusted to pH 8.0, and then made up to 1 L with deionized water.

[0045] Example 1 Construction of recombinant expression vector

[0046] The pET-24a-TdoT-SI expression vector was constructed by restriction digestion and ligation using the primers in Table 1: pET-28a-SI preserved in the laboratory was used as a template, which carries the SI coding gene without the natural signal peptide. The SI gene fragment containing the restriction site was amplified using primers P1 / P2, and it was double-digested with pET-24a-TdoT using QuickCutTM BamH I and QuickCutTM Xho I. The digestion products were separated by 1% agarose gel electrophoresis, and the linearized vector and the target gene fragment were recovered using a DNA gel recovery kit, and the ligation reaction was continued using T4 DNA ligase. The ligation reaction product was transformed into E. coli DH10B competent cells using heat shock and spread on LB solid plates containing 50μg / mL kanamycin. Single colonies were picked for colony PCR identification using primers P5 / P6. The results are shown in the figure. Figure 1 As shown in (A), the amplified product has a single band at 1500-2250bp, which is consistent with the expected amplified sequence size of 2055bp. The positive single clone that was verified correctly was further cultured and the plasmid was extracted for gene sequencing. The recombinant expression vector with correct sequencing was marked as pET-24a-TdoT-SI.

[0047] The pET-24b-SI-TdoT expression vector was constructed using Restriction free-cloning (RF) cloning technology: pET-24a-TdoT was used as a template and primers P3 / P4 were used for the first round of PCR amplification reaction. The PCR amplification product was separated by 1% agarose gel electrophoresis and recovered using a DNA gel recovery kit. It was used as the long primer for the second round of PCR reaction, and the second round of PCR reaction was continued using pET24b-SI-6×His as a template. After the reaction was completed, DpnⅠ was added to decompose the template plasmid, and the reaction product was electroporated into E.coli DH10B competent cells and coated on LB solid plates containing 50μg / mL kanamycin. Single colonies were picked and colony PCR was performed using primers P5 / P6. The results are shown as follows: Figure 1 As shown in (B), the amplified product has a single band at 1500-2250bp, which is consistent with the expected amplified sequence size of 2049bp. The positive single clone that was verified correctly was further cultured and the plasmid was extracted for gene sequencing. The recombinant expression vector with correct sequencing was marked as pET-24b-SI-TdoT.

[0048] Table 1 Primer sequences used in the present invention

[0049]

[0050] Example 2 Induced expression and separation and purification of recombinant genes in Escherichia coli

[0051] The constructed recombinant expression vectors pET-24a-TdoT-SI and pET-24b-SI-TdoT, as well as the recombinant expression vectors pET24b-6×His-SI and pET24b-SI-6×His were transformed into E. coli BL21 (DE3) competent cells, respectively. Positive single clones were picked and inoculated into 5 mL of LB liquid culture medium containing 50 μg / mL kanamycin, and cultured at 37°C, 200 r / min for 14 h to obtain seed solution. The seed solution was inoculated into LLB liquid culture medium containing 50 μg / mL kanamycin at 1% (v / v), and the inducer was added when the OD600nm reached 0.6-0.8 at 37°C and 200 r / min. The recombinant engineered bacteria E. coli BL21 (DE3) / pET-24a-TdoT-SI was added with IPTG at a final concentration of 0.8 mmol / L, E. coli BL21 (DE3) / pET-24b-SI-TdoT was added with IPTG at a final concentration of 0.1 mmol / L, and E. coli BL21 (DE3) / pET24b-6×His-SI and E. coli BL21 (DE3) / pET24b-SI-6×His were added with IPTG at a final concentration of 0.5 mmol / L. The above bacterial solution added with IPTG was continued to be cultured at 16°C and 200 r / min for 20 hours to induce expression.

[0052] Active inclusion bodies were isolated and purified by buffer washing method: the bacterial solution after induction expression was collected by centrifugation and resuspended with Buffer A, and then ultrasonically broken in an ice water bath (power 500W, breaking for 3s, interval 5s, breaking for 20min). The whole bacterial sample after breaking was centrifuged at 10000r / min for 15min to separate the soluble supernatant and the precipitate containing inclusion bodies. The precipitate was washed with Buffer B and Buffer C for 2 and 3 times respectively, and the washed inclusion body sample was recovered by centrifugation at 10000r / min for 15min. Finally, the washed inclusion body sample was fully suspended with Buffer C to obtain the active inclusion body enzyme solution. A small amount of active inclusion body enzyme solution was added to an equal volume of 10% SDS solution for 5min to completely dissolve the inclusion body, and then the protein concentration was determined using the BCA protein quantitative detection kit. The cell supernatant and the washed active inclusion body sample were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0053] like Figure 2The figure shows the SDS-PAGE analysis of recombinant 6×His-SI and SI-6×His. After induction, a target band appeared at 66.4 kDa, which was consistent with the theoretical molecular weight of 6×His-SI and SI-6×His of 68.14 kDa, and was mainly expressed in the form of inclusion bodies. Enzyme activity detection showed that the inclusion body precipitation had no catalytic activity, indicating that regardless of the N / C-terminal fusion histidine tag, the recombinant SI protein was mainly expressed in the form of inactive inclusion bodies.

[0054] like Figure 3 The figure shows the SDS-PAGE analysis of recombinant TdoT-SI and SI-TdoT. After induction, the whole bacterial sample showed a target band above the protein relative molecular mass standard of 66.4kDa, which was consistent with the theoretical relative molecular mass of TdoT-SI and SI-TdoT of 73.18kDa, indicating that the fusion protein was successfully induced to express. The expressed fusion proteins TdoT-SI and SI-TdoT still mainly exist in the precipitation sample in the form of inclusion bodies. The inclusion bodies were tested for enzyme activity and found to have high catalytic activity, indicating that the fusion TdoT tag induced SI expression as active inclusion bodies.

[0055] Example 4 Determination of active inclusion body enzyme activity

[0056] (1) Determination of the optimal reaction temperature

[0057] 100 μL of 4 mg / mL TdoT-SI and SI-TdoT active inclusion body enzyme solution was mixed with 400 μL of pH 6.0 sodium dihydrogen phosphate-citrate buffer containing 4% sucrose, and reacted at 30, 35, 40, 45, 50, and 55°C at 200 r / min for 2 h, and then the reaction sample was treated in 100°C boiling water for 15 min to inactivate the enzyme. After that, the reaction product after inactivation was centrifuged at 10000 r / min for 15 min, and the supernatant obtained was filtered with a 0.22 μm filter membrane and then detected by HPLC. The HPLC detection conditions were: the reaction product was detected using a Hypersil APS-2, 4.6×250 mm, 5 μm amino column, a differential detector, acetonitrile: water (v / v=80:20) as the mobile phase, column temperature 35°C, injection volume 10 μL, flow rate 1.0 mL / min. The concentration of isomaltulose in the reaction product and the enzyme activity were calculated based on the labeled curve fitted by the isomaltulose standard and its corresponding peak area. Definition of enzyme activity unit: With sucrose as substrate, the amount of enzyme required to release 1 μmol isomaltulose per minute is 1 enzyme activity unit (U). Definition of relative enzyme activity: With the highest enzyme activity in the same group as 100%, the calculated ratio is the relative enzyme activity.

[0058] The results are as follows Figure 4As shown, both TdoT-SI and SI-TdoT active inclusion bodies have the highest enzyme activity at 40°C, and the enzyme activity increases with the temperature in the range of 30-40°C. In the range of 35-45°C, both have more than 80% enzyme activity. When the temperature is higher than 45°C, the enzyme activity of TdoT-SI and SI-TdoT active inclusion bodies decreases rapidly.

[0059] (2) Determination of the optimal reaction pH

[0060] The active inclusion body enzyme solutions of TdoT-SI and SI-TdoT were mixed with sodium dihydrogen phosphate-citrate buffer solutions containing 4% sucrose at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The enzyme catalyzed reaction was carried out at 40°C, and the isomaltulose in the product was quantitatively detected and the enzyme activity was calculated. The enzyme activity was measured to determine the optimal reaction pH.

[0061] The results are as follows Figure 5 As shown in the figure, within the pH range of 4.0-5.0, the enzyme activity of TdoT-SI and SI-TdoT active inclusion bodies increased with the increase of pH, and had the highest enzyme activity at pH 5.5 and pH 5.0, respectively. In addition, within the pH range of 5.0-6.0, both had more than 80% catalytic activity, and the enzyme activity dropped rapidly after the pH was higher than 6.5.

[0062] (3) Determination of enzyme-catalyzed reaction kinetic constants

[0063] The active inclusion body enzyme solutions of TdoT-SI and SI-TdoT were mixed with sucrose solutions of different concentrations (10, 20, 40, 50, 100, 200, 250, 400, 500, 800 mmol / L) respectively, and the catalytic reaction was carried out under the optimal reaction conditions (TdoT-SI: 40℃, pH=5.5; SI-TdoT: 40℃, pH=5). The amount of product isomaltulose produced was detected by HPLC and the reaction rate was calculated. Then, GraphPad Prism 5.0 was used to perform nonlinear fitting based on the Michaelis-Menten equation to obtain the kinetic constants Km and Vmax, and kcat / Km was calculated.

[0064] Under the optimal reaction conditions, the specific activities of TdoT-SI and SI-TdoT active inclusion bodies were 622.6±17.5U / g and 252.6±17.1U / g, respectively. The conversion reaction products of active inclusion bodies were further detected under different concentrations of sucrose substrate and the catalytic reaction rate was calculated. The kinetic constants were obtained by nonlinear fitting. The results are shown in Table 2. The K of TdoT-SI m 103.9±9.5mmol / L, kcat / K m The K of SI-TdoT is 0.06±0.002L / (mmols). m 54.4±6.6mmol / L, k cat / K m It was 0.03±0.002L / (mmols), indicating that the active inclusion bodies formed by TdoT tag fusion at the N / C terminus of SI had different kinetic parameters for the substrate sucrose. SI-TdoT had a stronger binding ability with the substrate sucrose, but its catalytic efficiency was slightly lower than that of TdoT-SI.

[0065] Table 2 Kinetic parameters of TdoT-SI / SI-TdoT

[0066]

[0067] Studies on the preparation of immobilized sucrose isomerase have been reported, such as using mesoporous TiO2 modified with ε-poly-L-lysine. 2 The specific activity of immobilized SI was 39.41 U / g and Km was 204.92 mmol / L [Wu L, Liu Y, Chi B, et al. An innovative method for immobilizing sucrose isomerase on ε-poly-l-lysinemodified mesoporous TiO2 [J]. Food chemistry, 2015, 187: 182-188.], and the specific activity of sucrose isomerase immobilized on a sponge prepared by ε-poly-L-lysine and gelatin was 71.58 U / g and Km was 245.71 mmol / L, which was not significantly different from the Km of the unimmobilized free enzyme of 259.71 mmol / L [Wu L, Qiu J, Wu S, et al. Bioinspired Production of Antibacterial Sucrose Isomerase-Sponge for the Synthesis of Isomaltulose [J]. Advanced Synthesis & Catalysis, 2016, 358 (24): 4030-4040.]. The present invention prepares active inclusion bodies by synchronously expressing and immobilizing SI in cells, which not only does not require carrier materials and additional immobilization reactions, but also has good specific activity per unit mass, showing good application potential. At the same time, the present invention found that the active inclusion bodies formed by fusion of TdoT tags at the N / C termini of SI showed different kinetic parameters for the substrate sucrose, indicating that the formed active inclusion bodies affect the binding and catalytic process with the substrate due to the different microstructures.

[0068] Example 6 Product Specificity Analysis

[0069] 100 μL of 4 mg / mL TdoT-SI active inclusion body enzyme solution was mixed with 400 μL of 4% sucrose pH 5.5 disodium hydrogen phosphate-citric acid buffer; 100 μL of 4 mg / mL SI-TdoT active inclusion body enzyme solution was mixed with 400 μL of 4% sucrose pH 5.0 disodium hydrogen phosphate-citric acid buffer. The above active inclusion body conversion reaction system was reacted at 30, 35, and 40°C at 200 r / min for 12 hours, and then the enzyme conversion sample was treated with 100°C boiling water for 15 minutes to inactivate the enzyme, and then centrifuged at 10000 r / min for 15 minutes to obtain the reaction product supernatant, and filtered with a 0.22 μm filter membrane and detected by HPLC.

[0070] The results are shown in Table 3. Within the current temperature range, the isomaltulose content in the conversion reaction products of TdoT-SI and SI-TdoT active inclusion bodies did not change significantly, while the trehalulose content in the products generally showed a decreasing trend with the increase of the conversion reaction temperature. At the same time, the content of monosaccharides (fructose and glucose) increased with the increase of temperature.

[0071] Table 3 Analysis of the composition of sucrose isomerase catalyzed by TdoT tag at different temperatures

[0072]

[0073] The present invention uses fusion expression technology and coiled-coil domain TdoT as a fusion tag to successfully express sucrose isomerase as active inclusion bodies in Escherichia coli E. coli BL21 (DE3), thereby achieving simultaneous expression and immobilization. As a novel self-immobilized enzyme, the enzyme does not require carrier materials and chemical reagents for traditional immobilization reactions, and has potential industrial application value. At the same time, the present invention also provides a new strategy for achieving efficient preparation and utilization of recombinant sucrose isomerase, laying a foundation for further design and optimization. SEQUENCE LISTING <110> Li Xianzhen <120> A sucrose isomerase active inclusion body fused with coiled-coil domain <130> 2022 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 1869 <212> DNA <213> Artificial Sequence <400> 1 atgatcatta acgaaactgc cgatgacatc gtttatcgcc tgacagtcat tatcgatgat 60 cgctacgaat cgctgaaaaa cctgattacc ttacgtgcag atcgcttgga gatgatcatc 120 aatgacaatg tgtccaccat tctcgcgagc attggatccg caccatcctt gaatcaggat 180 attcacgttc aaaaggaaag tgaatatcct gcatggtgga aagaagctgt tttttatcag 240 atctatcctc gctcatttaa agacaccaat gatgatggca ttggcgatat tcgcggtatt 300 attgaaaagc tggactatct gaaatcgctc ggtattgacg ctatctggat caatccccat 360 tacgactctc cgaacaccga taacggctat gacatcagta attatcgtca gataatgaaa 420 gagtatggca caatggagga ttttgatagc cttgttgccg aaatgaaaaa acgaaatatg 480 cgcttaatga tcgacgtggt cattaaccat accagtgatc aacccgtg gtttattcag 540 agtaaaagcg ataaaaacaa cccttatcgt gactattatt tctggcgtga cggaaaagat 600 aatcagccac ctaataatta cccctcattt ttcggcggct cggcatggca aaaagatgca 660 aagtcaggac agtactattt acactatttt gccagacagc aacctgatct caactgggat 720 aacccgaaag tacgtgagga tctttacgca atgctccgct tctggctgga taaaggcgtt 780 tcaggcatgc gattgatac ggtggcaact tattccaaaa tcccgggatt tcccaatctg 840 acacctgaac aacagaaaaa ttttgctgaa caatacacca tggggcctaa tattcatcga 900 tacattcagg aaatgaaccg gaaagttctg tcccggtatg atgtggccac cgcgggtgaa 960 atttttggcg tcccgctgga tcgttcgtcg cagttttttg atcgccgccg acatgagctg 1020 aatatggcgt ttatgtttga cctcattcgt ctcgatcgcg acagcaatga acgctggcgt 1080 cacaagtcgt ggtcgctctc tcagttccgc cagatcatca gcaaaatgga tgtcacggtc 1140 ggaaagtatg gctggaacac gttcttctta gataaccatg acaacccccg tgcggtatct 1200 cacttcgggg atgacaggcc gcaatggcgg gaggcgtcgg ctaaggcact ggcgacgatt 1260 accctcactc agcgggcgac gccgtttatt tatcagggtt cagagctggg aatgactaat 1320 tatcccttca ggcaactcaa cgaatttgac gacatcgagg tcaaaggttt ctggcaggat 1380 tatgtccaga gtggaaaagt cacggccaca gagtttctcg ataatgtgcg cctgacgagc 1440 cgcgataaca gcagaacacc tttccagtgg aatgacaccc tgaatgctgg ttttactcgc 1500 ggaaagccgt ggtttcacat caacccaaac tatgtggaga tcaacgccga acgcgaagaa 1560 acccgcgaag attcagtgct gaattactat aaaaaaatga ttcagctacg ccaccatatc 1620 cctgctctgg tatatggcgc ctatcaggat cttaatccac aggacaatac cgttatgcc 1680 tatacccgaa cgctgggtaa cgagcgttat ctggtcgtgg tgaactttaa ggagtacccg 1740 gtccgctata ctctcccggc taatgatgcc atcgaggaag tggtcattga tactcagcag 1800 caggcggctg cgccgcacag cacatccctg tcattgagcc cctggcaggc aggtgtgtat 1860 aagctgcgg 1869 <210> 2 <211> 1869 <212> DNA <213> 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 cattacgac 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 aaaatttgc 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 gtggtgaact ttaaggagta cccggtccgc tatactctcc cggctaatga tgccatcgag 1620 gaagtggtca ttgatactca gcagcaggcg gctgcgccgc acagcacatc cctgtcattg 1680 gaagtggtca ttgatactca gcagcaggcg gctgcgccgc acagcacatc cctgtcattg 1680 agcccctggc aggcaggtgt gtataagctg cggggatcca tcattaacga aactgccgat 1740 agcccctggc aggcaggtgt gtataagctg cggggatcca tcattaacga aactgccgat 1740 gacatcgttt atcgcctgac agtcattatc gatgatcgct acgaatcgct gaaaaacctg 1800 gacatcgttt atcgcctgac agtcattatc gatgatcgct acgaatcgct gaaaaacctg 1800 attaccttac gtgcagatcg cttggagatg atcatcaatg acaatgtgtc caccattctc 1860 attaccttac gtgcagatcg cttggagatg atcatcaatg acaatgtgtc caccattctc 1860 gcgagcatt 1869 gcgagcatt 1869 <210> 3<210> 3 <211> 623<211> 623 <212> PRT<212> PRT <213> 人工序列(Artificial Sequence)<213> Artificial Sequence <400> 3<400> 3 Met Ile Ile Asn Glu Thr Ala Asp Asp Ile Val Tyr Arg Leu Thr Val Met Ile Ile Asn Glu Thr Ala Asp Asp Ile Val Tyr Arg Leu Thr Val 1 5 10 15 1 5 10 15 Ile Ile Asp Asp Arg Tyr Glu Ser Leu Lys Asn Leu Ile Thr Leu Arg Ile Ile Asp Asp Arg Tyr Glu Ser Leu Lys Asn Leu Ile Thr Leu Arg 20 25 30 20 25 30 Ala Asp Arg Leu Glu Met Ile Ile Asn Asp Asn Val Ser Thr Ile Leu Ala Asp Arg Leu Glu Met Ile Ile Asn Asp Asn Val Ser Thr Ile Leu 35 40 45 35 40 45 Ala Ser Ile Gly Ser Ala Pro Ser Leu Asn Gln Asp Ile His Val Gln Ala Ser Ile Gly Ser Ala Pro Ser Leu Asn Gln Asp Ile His Val Gln 50 55 60 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 <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 <400> 7 cgcggatccg caccatcctt gaatcaggat attcacg 37 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <400> 8 cgcggatccg caccatcctt gaatcaggat attcacg 37 <210> 9 <211> 55 <212> DNA <213> Artificial Sequence <400> 9 caggcaggtg tgtataagct gcggggatcc atcatcatta acgaaactgc cgatg 55 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <400> 10 tcagtggtgg tggtggtggt gttaaatgct cgcgagaatg gtggac 46 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 taatacgact cactataggg 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <400> 12 gctagttatt gctcagcgg 19 <210> 13 <211> 6968 <212> DNA <213> 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 cccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggtt tttcgccct tgacgttgg agtccacgtt 300 ctttaatagt ggacttgt tccaactgg aaacactc aaccctatct cggtctattc 360 ttttgattta taaggatt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attackttt acatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttattttc taaatacatt caatatgta 540 tccgctcatg aattaatttct tagaaaact catcgagcat caatgaac tgcaatttat 600 tcatatcagg attatcaata ccatatttt gaaaaagccg ttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctgta tcggtctgcg attccgactc 720 gtccaacatc atacaacct attaatttcc cctcgtcaaa ataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gatccggtg agaatggcaa aagtttagc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaccaac 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 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatga catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940. tgcgatgcag atccggaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 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 attaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040 ttttgtttaa cttaagaag 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 5340. 5340. 5340. 5340. 5340. 5340. 5340. 5340. 5340. 5340 tcagataatg aaagagtatg gcacaatgga ggattttgat agccttgttg ccgaaatgaa aaaacgaaat atgcgcttaa tgatcgacgt ggtcattaac cataccagtg atcaacaccc gtggtttatt cagagtaaaa gcgataaaaa caacccttat cgtgactatt atttctggcg tgacggaaaa throwcagc cacctaata ttacccctca tttttcggcg gctcggcatg gcaaaagat gcaaagtcag gacagtacta tttacactat tttgccagac agcaacctga tctcaactgg gataacccga aagtacgtga ggatctttac gcaatgctcc gcttctggct ggataaaggc gtttcaggca tgcgatttga tacggtggca acttattcca aaatcccggg 5760. atttcccaat ctgacacctg grandmother aaattttgct grandmother ccatggggcc taatattcat cgatacattc aggaatgaa ccggaaagtt ctgtcccggt atgatgtggc 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 ggggccta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 6960 atccggat 6968 <210> 14 <211> 6947 <212> DNA <213> Artificial Sequence <400> 14 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg cagcgccct agcgcccgct cctttcgct tcttccttc 120 cttctcgcc acgtcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt tttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggacttgt tccaactgg aaacactc aaccctatct cggtctattc 360 ttttgattta taaggatt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attackttt acatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttattttc taaatacatt caatatgta 540 tccgctcatg aattaatttct tagaaaact catcgagcat caatgaac tgcaatttat 600 tcatatcagg attatcaata ccatatttt gaaaaagccg ttctgtaat gaaggagaaa 660 actcaccgag gcagttccat aggatggcaa gatcctgta tcggtctgcg attccgactc 720 gtccaacatc atacaacct attaatttcc cctcgtcaaa ataaggtta tcaagtgaga 780 aatcaccatg agtgacgact gatccggtg agaatggcaa aagtttagc atttctttcc 840 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaccaac 900 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960 attackacaac aggaatcgaa tgcaccggc gcaggacc tgccagcgca tcacaat 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 taccggggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860. accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggga acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gccttttc ggttcctggc cttttgctgg cttttgctc acatgttctt tcctgcgtta 2160 tccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220 agccgaacga ccgagcgcag cgagtcagtg agcgaggag cggaagagcg cctgatgcgg 2280. tattttctcc ttacgcatct gtgcggtatt tcacaccgca fatherggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 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 attaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040 ttttgtttaa cttaagaag 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 ttattctgg 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. Use of a sucrose isomerase active inclusion body in catalyzing the conversion of sucrose into isomaltulose, It is characterized in that The fusion protein is expressed by inducing recombinant engineering bacteria, and the bacteria are broken, separated and purified; The method for preparing sucrose isomerase activity inclusion bodies of a recombinant engineered bacterium containing a gene with a nucleotide sequence as shown in SEQ ID NO.1 specifically comprises the following steps: (1) constructing a recombinant expression vector pET-24a-TdoT-SI containing a gene encoding a TdoT-SI fusion protein, and transferring the recombinant expression vector into an expression host cell to obtain a recombinant engineered bacterium; (2) culturing the recombinant engineered bacteria to induce expression of the fusion protein; (3) collecting bacterial cells and disrupting the cells, separating the cell disruption supernatant and the precipitate sample containing the inclusion bodies, and washing the precipitate sample with a buffer solution to purify the active inclusion bodies; The method for preparing sucrose isomerase activity inclusion bodies of a recombinant engineered bacterium containing a gene with a nucleotide sequence as shown in SEQ ID NO.2 specifically comprises the following steps: (1) constructing a recombinant expression vector pET-24b-SI-TdoT containing a gene encoding the SI-TdoT fusion protein, and transferring the recombinant expression vector into an expression host cell to obtain a recombinant engineered bacterium; (2) culturing the recombinant engineered bacteria to induce expression of the fusion protein; (3) collecting bacterial cells and disrupting the cells, separating the cell disruption supernatant and the precipitate sample containing the inclusion bodies, and washing the precipitate sample with a buffer solution to purify the active inclusion bodies; The sucrose isomerase SI is derived from Klebsiella sp.LX3, and its nucleotide sequence is shown in SEQ ID NO.5; The TdoT gene is from Staphylothermus marinus, and its nucleotide sequence is shown in SEQ ID NO.6; The pH value at which the sucrose isomerase active inclusion body catalyzes the conversion of sucrose into isomaltulose is 4.5-6.5 and the temperature is 30-45°C.

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