Recombinant engineering bacterium, immobilized cell, preparation method and application
By expressing Thermotoga maritima MSB8-derived sucrose convertase in E. coli and immobilizing cells, the problems of low enzyme purity and high production cost in sucrose hydrolysis method are solved, and efficient and stable production of sucrose hydrolyzed syrup is achieved, reducing production costs.
Patent Information
- Application Number
- CN202411447152.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-16
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, the sucrose hydrolysis method has a high temperature reaction, resulting in dark color, poor stability, low enzyme purity and high price, which limits the wide application of sucrose converting enzymes in the fields of food processing and renewable resources.
Escherichia coli was used as the chassis strain to express the sucrose convertase from Thermotoga maritima MSB8, and enhance expression by SUMO or SSO tags, immobilized cells were prepared, and cross-linked and fixed in combination with inorganic soil and flocculant, simplifying the enzyme production process.
The efficient expression and immobilization of sucrose converting enzymes is achieved, the separation and purification steps of enzymes are simplified, the quality of sucrose hydrolyzed syrup and the utilization rate of enzymes are improved, the production cost is reduced, and the continuity and efficiency of sucrose hydrolysis rate are ensured.
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Figure CN119242545A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a recombinant engineering bacterium, an immobilized cell, a preparation method and an application. Background Art
[0002] Sucrose is a disaccharide, which is composed of α-D-glucose and β-D-fructose through α-1,2 glycosidic bonds. It is the most commonly used sweetener in the food industry. At the same time, sucrose, as a sugar product of plant photosynthesis, is a very abundant biomass resource. Sucrose can be hydrolyzed into glucose and fructose. Sucrose hydrolysis is an important way to obtain glucose and fructose. At the same time, the monosaccharides hydrolyzed from it can be used as an important carbon source in the fermentation industry. In the fermentation industry, glucose and fructose can be used as important industrial raw materials to produce high-value-added chemical products such as organic acids, amino acids and bioethanol. For example, the patent (ZL200910091614.4) uses glucose and a small amount of sucrose and fructose in sucrose hydrolyzate as carbon sources, adds inorganic and organic nitrogen sources, and various nutrients to ferment and produce citric acid.
[0003] The main methods for preparing sucrose hydrolyzed syrup in industry are divided into acid hydrolysis and enzyme hydrolysis. Among them, acid hydrolysis is a common method for sucrose hydrolysis. This method mainly uses 0.1-0.3% inorganic acid or organic acid to hydrolyze sucrose at a temperature above 70°C. The temperature of sucrose hydrolysis in this method is relatively high, and the long-term high-temperature reaction makes the syrup easy to produce furfural compounds, making the obtained hydrolyzed syrup darker in color and less stable. At the same time, the pH of the sucrose hydrolyzed syrup obtained by the acid hydrolysis method is usually around 2.0-3.0. After the sucrose hydrolysis is completed, alkali needs to be used for neutralization. Therefore, organic acid salts or ash substances are inevitably introduced into the hydrolyzed syrup, which changes the original flavor of the syrup, is not conducive to compounding in the subsequent use process, and limits its application in related products.
[0004] Sucrose invertase (EC3,2,1,26), also known as β-D-fructofuranosidase hydrolase, can specifically catalyze the hydrolysis of sucrose into glucose and fructose. The sucrose hydrolyzed syrup prepared by hydrolysis with sucrose invertase is bright yellow in color and has a strong sweet sucrose flavor, and its quality is better than that of acid hydrolyzed syrup. In recent years, with the improvement of people's living standards and the emphasis on renewable resources, sucrose invertase has received more and more attention, but it has not been widely used in China. This is because the current commercial sucrose invertase has low purity and high price, and does not have competitive advantages in industrial production, which seriously limits its wide application in the field of food processing and renewable resources. Therefore, it is very important to develop recombinant engineering strains with high efficiency expression of sucrose invertase, prepare immobilized cells that can be easily obtained, simplify the production process of enzymes, reduce the cost of enzyme production, and realize the recycling of sucrose invertase to reduce the industrial production cost of sucrose hydrolyzed syrup produced by enzymatic hydrolysis. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a recombinant engineering bacterium, an immobilized cell, a preparation method and an application, so as to improve the enzyme activity, improve the quality of sucrose hydrolyzed syrup and reduce the production cost.
[0006] The present invention provides a recombinant engineering bacterium, Escherichia coli Escherichia coli The invention is a chassis strain, wherein the chassis strain contains an expression vector of a target gene expressing sucrose invertase (sucrose invertase (TmINVase) derived from strain Thermotoga maritima MSB8 (accession number: WP_004081649.1), whose amino acid sequence and gene sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), or the chassis strain is inserted with a target gene expressing sucrose invertase, wherein the target gene is connected with a solubilizing protein SUMO tag or an SSO tag, wherein the SUMO tag is located at the N-terminus of the target gene, and the SSO tag is located at the N-terminus or the C-terminus of the target gene.
[0007] Optionally, the SUMO tag is located at the N-terminus of the target gene.
[0008] Optionally, the sequence of the SUMO tag is SEQ ID NO:6, and the sequence of the SSO tag is SEQ ID NO:8.
[0009] Optionally, the sequence of the target gene expressing sucrose invertase is SEQ ID NO: 2.
[0010] Optionally, the expression vector is an Escherichia coli expression vector pET series.
[0011] The embodiment of the present invention provides a method for preparing the recombinant engineered bacteria, comprising the following steps: Connect the tag between the two restriction sites of the expression vector to obtain an expression vector containing the tag; Connect the target gene between the two restriction sites of the expression vector to obtain a recombinant plasmid; Enzyme digestion of the expression vector and recombinant plasmid containing the tag to obtain the tag and the target gene; Connect the tag and the target gene to obtain a connection product, and transfer the connection product into competent cells of the chassis strain to obtain a recombinant engineered bacterium; When the tag is a SUMO tag, the two restriction enzyme sites are Nde Ⅰ and BamH Ⅰ restriction enzyme sites; When the tag is an SSO tag, the two restriction enzyme cleavage sites are Nde Ⅰ and BamH Ⅰ restriction enzyme cleavage sites, or Hind Ⅲ and Xho I restriction enzyme cleavage sites.
[0012] An embodiment of the present invention provides an immobilized cell, comprising a bacterial suspension containing the recombinant engineered bacteria, inorganic soil, a flocculant and a cross-linking agent, wherein the weight ratio of the bacterial suspension volume to the inorganic soil is 100 ml: 1-20 g, the weight ratio of the bacterial suspension volume to the flocculant is 100 ml: 0.05-1 g, and the volume ratio of the bacterial suspension to the cross-linking agent is 100: 0.05-0.5.
[0013] Optionally, the concentration of the bacterial suspension is 100-200 g / L; the inorganic soil is diatomaceous earth, montmorillonite, bentonite or kaolin (preferably diatomaceous earth); the flocculant is polyethyleneimine, polyacrylamide or chitosan, and the molecular weight of the polyethyleneimine is 600-70000 (preferably polyethyleneimine); the cross-linking agent is glutaraldehyde, N,N-methylenebisacrylamide or trihydroxymethyl phosphine (preferably glutaraldehyde).
[0014] The embodiment of the present invention provides a method for preparing the immobilized cells, which comprises uniformly mixing a bacterial suspension and an inorganic soil, adding a flocculant for flocculation, and then adding a cross-linking agent to obtain the immobilized cells.
[0015] The preparation method of the bacterial suspension is as follows: the recombinant engineered bacteria are inoculated into a liquid LB medium containing ampicillin resistance, cultured at 37°C and 220 r / min overnight, and then inoculated into a new liquid LB medium containing ampicillin resistance at an inoculum size of 2% the next day, cultured at 37°C and 220 r / min for about 2 h, and when the OD600 value of the bacterial solution reaches 0.6-0.8, IPTG with a final concentration of 0.5-1 mmol / L is added as an inducer, and after cultured on a shaker at 16°C and 220 r / min for 16-18 h, the bacterial cells are collected by centrifugation at 6000 rpm for 10 min, the bacterial cells are washed twice with physiological saline, and then the bacterial cells are resuspended with physiological saline to obtain a bacterial suspension.
[0016] After the cross-linking is completed, the immobilized cell filter cake is obtained by vacuum filtration, the filter cake is squeezed into long strips (using a rotary granulator), and then the long strips of immobilized cells are cut into short and uniform particles (using a spherical shot blasting machine); the particles are boiled and dried to obtain immobilized cells, wherein the boiling and drying temperature is controlled at 60-80°C.
[0017] The embodiment of the present invention provides an application of the recombinant engineered bacteria or the immobilized cells, which is used to hydrolyze sucrose to prepare a hydrolyzed fructose-glucose solution; the amount of sucrose added is 50-700 g / L, a buffer solution with a pH value of 4.0-7.0 is added to the hydrolysis system, and the temperature is 50-75°C (preferably 60°C). The sucrose can be pure sucrose or sugarcane juice or sweet sorghum stem juice containing sucrose, which is hydrolyzed to obtain glucose and fructose to prepare a hydrolyzed fructose-glucose solution.
[0018] The beneficial effect of the present invention is that the recombinant engineered bacteria of the present invention use a recombinant plasmid with a solubilizing tag as a vector backbone, a sucrose invertase (TmINVase) gene (derived from the strain Thermotoga maritima MSB8) as a target gene, and Escherichia coli (BL21) as an expression host, and successfully achieve the efficient expression of TmINVase sucrose invertase. After shake flask fermentation, the enzyme activity of the sucrose invertase of the recombinant engineered bacteria can reach 9733.2 U / g wet cells.
[0019] Compared with whole-cell catalysis, the recombinant engineered bacteria immobilized cells of the present invention not only simplify the production and preparation process of the enzyme, but also overcome the difficulty of separating the enzyme from the product, which is beneficial to the separation and purification of sucrose hydrolyzed syrup. At the same time, the immobilized cells realize the reuse of sucrose invertase. After 10 batches of continuous catalysis, the sucrose hydrolysis rate can still be maintained at 100%, which greatly improves the utilization rate of sucrose invertase recombinant engineered bacteria cells and reduces the production cost of sucrose hydrolyzed syrup prepared by enzymatic hydrolysis.
[0020] The invention simplifies the separation and purification steps of the enzymes required in the production of sucrose hydrolyzed syrup, improves the enzyme recovery rate, and realizes the recycling of the enzymes. The method of the invention has the advantages of easy product separation, simple production process, low cost, etc., and has great application prospects in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 : Example 3 Effect of repeated use of immobilized cells of recombinant engineering bacteria on the hydrolysis of sucrose.
[0022] Figure 2 : Example 3 Effect of repeated use of recombinant engineered bacterial cells on sucrose hydrolysis. DETAILED DESCRIPTION
[0023] The present invention is described below by specific embodiments. The embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and principles of the present invention, but these modifications or replacements all fall within the scope of protection of the present invention.
[0024] The detection methods involved in the following embodiments are as follows: Sucrose invertase activity detection method: 50 g / L sucrose was used as substrate, 0.5% (v / v) crude enzyme solution was added, oscillated and mixed, reacted at 60 °C, pH 5.0 for 10 min, and then boiled for 10 min to inactivate. The reaction product was centrifuged and passed through a membrane, diluted to a certain concentration and then detected by HPLC. The detection conditions were: chromatograph: Agilent1260; detector: differential refractive index detector; chromatographic column: Speax Carbomix Pb-NP (10 μm, 7.8×300 mm); column temperature 65 °C; mobile phase: water; flow rate 0.5 mL / min.
[0025] Under the above conditions, the amount of enzyme required to hydrolyze 1 μmol of sucrose per unit time (min) is defined as 1 U.
[0026] Example 1: Construction of recombinant engineering bacteria for sucrose invertase (1) Construction of the sucrose invertase expression vector backbone According to the amino acid sequences of the SUMO tag (as shown in SEQ ID NO:5) and the SSO tag (as shown in SEQ ID NO:7), the nucleic acid sequences as shown in SEQ ID NO:6 (the sequence of the SUMO tag used in the present invention) and SEQ ID NO:8 (the sequence of the SSO tag used in the present invention) were obtained after codon optimization. The nucleic acid sequences were fully synthesized, and two restriction enzyme sites, Nde Ⅰ and BamH Ⅰ, or two restriction enzyme sites, Hind Ⅲ and Xho I, were introduced into the 5' end and 3' end of the above gene nucleotide sequence, respectively. The synthesized genes were ligated between the restriction sites Nde I and BamH I or between Hind Ⅲ and Xho I of the Escherichia coli expression vector pET-22b(+), and expression vectors SUMO-pET22b, SSO-pET22b, pET22b-SUMO and pET22b-SSO were obtained, respectively, so as to optimize the screening of the gene position of the SUMO tag and the SUMO tag.
[0027] (2) Construction of recombinant engineering bacteria for sucrose invertase According to the amino acid sequence of sucrose invertase (TmINVase) derived from strain Thermotoga maritima MSB8 (accession number: WP_004081649.1) (as shown in SEQ ID NO:1), a nucleic acid sequence as shown in SEQ ID NO:2 was obtained after codon optimization. The nucleic acid sequence was fully synthesized and ligated between the restriction sites Nde I and BamH I of the Escherichia coli expression vector pET-22b(+) to obtain the recombinant plasmid pET-22b-TmINVase. The recombinant plasmid pET-22b-TmINVase was transformed into Escherichia coli BL21 (DE3) competent cells by chemical transformation to obtain the sucrose invertase recombinant engineering strain TmINVase-BL21.
[0028] (3) Primers were designed using the fully synthesized pET-22b-TmINVase plasmid as a template (the upstream and downstream primers are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively). The BamH I and Hind III restriction sites were introduced through the primers for PCR amplification. The total volume of the reaction system was 50 μL. The components of the reaction system are shown in Table 1.
[0029] Table 1 PCR amplification reaction system
[0030] The PCR reaction program was as follows: 98°C, 1 min (pre-denaturation); 98°C, 15 s (denaturation); 68°C, 15 s (annealing); 72°C, 20 s (extension); 30 cycles; 72°C, 5 min (final extension); 4°C, ∞ (storage).
[0031] After the PCR amplification reaction was completed, the PCR product was double-digested to recover the target gene fragment, and the SUMO-pET22b or SSO-pET22b vector fragment was double-digested at the same time. The total volume of the double-digestion system of the target gene and the vector fragment was 50 μL. The components of the reaction system refer to Table 2.
[0032] Table 2 Double restriction enzyme digestion system of target gene and vector
[0033] Reaction conditions: 37°C for 1.5 h.
[0034] After the double restriction enzyme digestion reaction, the target gene fragment and the SUMO-pET22b and SSO-pET22b vector fragments were recovered and connected using ligase Solution I. The total volume of the target gene and vector connection system was 8 μL, and the components of the reaction system were referred to Table 3.
[0035] Table 3 Solution Ⅰ Connection system
[0036] Reaction conditions: 16°C, overnight reaction.
[0037] After the ligation reaction between the target gene and the vector was completed, the ligation product was transferred into the competent cells of E. coli JM109 by chemical transformation, and the transformants were screened by ampicillin resistance plate. The transformants were picked and the recombinant plasmid was extracted and sequenced after double enzyme digestion verification. The total volume of the recombinant plasmid double enzyme digestion verification system was 10 μL, and the components of the reaction system were referred to Table 4.
[0038] Table 4 Recombinant plasmid double restriction enzyme digestion verification system
[0039] Reaction conditions: 37°C for 1.5 h.
[0040] After correct sequencing, the sucrose invertase recombinant expression vectors SUMO-TmINVase-pET22b and SSO-TmINVase-pET22b were obtained respectively, and the above recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) competent cells (i.e., chassis strains, which can be purchased) by chemical transformation to obtain sucrose invertase recombinant engineering strains SUMO-TmINVase-BL21 and SSO-TmINVase-BL21.
[0041] The recombinant plasmid pET-22b-TmINVase and the expression vector pET22b-SUMO and pET22b-SSO vector fragments were double-digested respectively. The total volume of the double-digestion system was 50 μL. The components of the reaction system were referred to Table 5.
[0042] Table 5 Vector double enzyme digestion system
[0043] Reaction conditions: 37°C for 1.5 h.
[0044] After the double enzyme digestion reaction, the target gene fragment TmINVase and the vector fragments pET22b-SUMO and pET22b-SSO were recovered and connected using ligase Solution I, respectively. The total volume of the connection system of the target gene and the vector was 8 μL. The components of the reaction system refer to Table 6.
[0045] Table 6 Solution Ⅰ Connection system
[0046] Reaction conditions: 16°C, overnight reaction.
[0047] After the ligation reaction between the target gene and the vector is completed, the ligation product is transferred into E. coli JM109 competent cells by chemical transformation, and the transformants are screened by ampicillin resistance plates. The transformants are picked and the recombinant plasmid is extracted and sequenced after double enzyme digestion verification.
[0048] After correct sequencing, the sucrose invertase recombinant expression vectors TmINVase-SUMO-pET22b and TmINVase-SSO-pET22b were obtained respectively, and the above recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) competent cells by chemical transformation to obtain sucrose invertase recombinant engineering strains TmINVase-SUMO-BL21 and TmINVase-SSO-BL21.
[0049] (3) Shake flask fermentation of sucrose invertase recombinant engineering bacteria The five sucrose invertase recombinant engineered bacteria obtained in step (2) were inoculated into LB medium at 37°C and 200rpm shaking overnight to obtain seed solution; the seed solution was inoculated into fresh LB medium at a volume ratio of 2% and cultured at 37°C until the OD600 value was 0.6-0.8, then cooled to 16°C, and IPTG with a final concentration of 1.0mM was added for induction for 16-18 hours to obtain fermentation liquid. The fermentation liquid was centrifuged at 6000 rpm for 10 min to collect bacterial cells, and the bacterial cells were washed twice with physiological saline, and then resuspended with physiological saline (bacterial concentration was 250 g / L), and ultrasonically disrupted on ice (2 s, 3s interval, power 400 W), and crude enzyme solution was obtained after cell disruption. The obtained crude enzyme solution was subjected to sucrase enzyme activity detection. The sucrase enzyme activities of five sucrase invertase recombinant engineering bacteria are shown in Table 7, among which the recombinant engineering bacteria SUMO-TmINVase-BL21 had the highest enzyme activity, reaching 9733.2 U / g wet cells.
[0050] Table 7 Sucrose invertase recombinant engineering bacteria enzyme activity
[0051] Example 2: Preparation of immobilized cells of recombinant engineering bacteria for sucrose invertase The concentration of the resuspended bacterial solution of the recombinant engineered bacteria SUMO-TmINVase-BL21 cells in step (3) of Example 1 was adjusted to 150 g / L, 10% diatomaceous earth was added to the bacterial suspension, magnetically stirred for 30 min, and after stirring evenly, 0.2% (w / v) polyethyleneimine aqueous solution with a molecular weight of 70,000 was added, magnetically stirred for 30 min, and flocculated at room temperature. Then, 0.1% (v / v) glutaraldehyde aqueous solution was added, magnetically stirred for 30 min, and then allowed to stand for crosslinking at room temperature for 2 h. After vacuum filtration, a filter cake was obtained, which was extruded into a long strip with a particle size of 3.0 mm using a rotary granulator, and then the long strip was cut into particles of uniform length by a spherical shot blasting machine. The obtained immobilized cells were dried by boiling at 65°C to obtain immobilized cells.
[0052] Example 3: Preparation of sucrose hydrolyzate syrup In a 1 L reaction system, sucrose and immobilized cells were added to a final concentration of 500 g / L, respectively, to make the bacterial concentration 2.5 g / L, and the reaction was carried out in a 60°C water bath shaker. During the reaction, sucrose hydrolysis was detected by high performance liquid chromatography. After the reaction, the immobilized cells were collected by simple filtration, washed with pure water, and then the next batch of reactions was carried out. The experimental results are shown in Figure 2. Figure 1 As shown, the results showed that when the immobilized cells of the recombinant engineered bacteria continuously catalyzed the reaction, the initial sucrose hydrolysis rate could reach 100%, that is, sucrose was completely hydrolyzed into glucose and fructose. After 10 batches of continuous catalysis, the sucrose hydrolysis rate could still be maintained at 100%.
[0053] In contrast to the immobilized cells, sucrose with a final concentration of 500 g / L and 10 mL of the crude enzyme solution from step (3) of Example 1 were added to a 1 L reaction system, and the reaction was carried out in a 60°C water bath shaker. During the reaction, sucrose hydrolysis was detected by high performance liquid chromatography. After the reaction was completed, the precipitated cells were obtained by centrifugation and washed with pure water before the next batch of reactions. The experimental results are shown in Figure 2. Figure 2 As shown, the results showed that when the crude enzyme solution of the recombinant engineered bacteria continuously catalyzed the reaction, the initial sucrose hydrolysis rate could reach 100%, but after two batches of continuous catalysis, the sucrose hydrolysis rate remained only 40%.
[0054] Furthermore, in order to expand the application scope of the immobilized cells, the present invention uses sucrose in concentrated sugarcane juice and concentrated sweet sorghum stem juice as a substrate to prepare sucrose hydrolyzed syrup, wherein the total sugar concentration of the concentrated sugarcane juice and the concentrated sweet sorghum stem juice is about 55%. The immobilized cells are added to 1 L of concentrated sugarcane juice or concentrated sweet sorghum stem juice, respectively, so that the bacterial concentration is 2.5 g / L, and the reaction is carried out under a 60°C water bath shaker. During the reaction, high performance liquid chromatography is used to detect the sucrose hydrolysis, and the results are shown in Tables 8 and 9: The hydrolysis rate of the recombinant engineered bacteria immobilized cells to the sucrose in the concentrated sugarcane juice and the concentrated sweet sorghum stem juice is 100%, that is, the sucrose in the concentrated sugarcane juice and the concentrated sweet sorghum stem juice can be completely hydrolyzed into glucose and fructose.
[0055] Table 8 Hydrolysis of concentrated sugarcane juice by immobilized cells
[0056] Table 9 Immobilized cell hydrolysis and concentration of sweet sorghum stem juice
[0057] The sequence of this application is as follows: SEQ ID NO:1 MFKPNYHFFP ITGWMNDPNG LIFWKGKYHM FYQYNPRKPE WGNICWGHAV SDDLVHWRHLPVALYPDDET HGVFSGSAVE KDGKMFLVYT YYRDPTHNKG EKETQCVAMS ENGLDFVKYD GNPVISKPPEEGTHAFRDPK VNRSNGEWRM VLGSGKDEKI GRVLLYTSDD LFHWKYEGVI FEDETTKEIE CPDLVRIGEKDILIYSITST NSVLFSMGEL KEGKLNVEKR GLLDHGTDFY AAQTFFGTDR VVVIGWLQSW LRTGLYPTKREGWNGVMSLP RELIEVENL KVKPVDELLA LRKRKVFETA KSGTFLLDVK ENSYEIVCEF SGEIELRMGNESEEVVITKS RDELIVDTTR SGVSGGEVRK STVEDEATNR IRAFLDSCSV EFFFNDSIAF FREE VINYLVKSNQV CLEVFELENI WL。
[0058] SEQ ID NO:2 ATGTTCAAAC CGAACTACCA CTTCTTTCCA ATCACCGGTT GGATGAACGA CCCAAACGGCCTCATCTTCT GGAAAGGCAA GTACCACATG TTCTACCAGT ACAATCCGCG TAAACCGGAA TGGGGTAACATCTGCTGGGG TCACGCTGTT TCCGACGACCTTGGTCGGTT TGTATCCGGATGACGAAACT CACGGTGTGT TCTCTGGTTC TGCAGTTGAG AAAGATGGCA AGATGTTTCT GGTTTACACCTACTACCGTG ACCCTACTCA CAACAAAGGC GAGAAAGAAA CTCAGTGCGT TGCGATGTCT GAGAACGGTCTGGACTTCGT TAAGTACGTCGATCGATCGACCTT GAAGGTACTC ATGCGTTCCGTGACCCAAAG GTGAACCGTT CTAACGGTGA ATGGCGTATG GTTCTGGGTT CTGGTAAAGA CGAGAAAATCGGTCGTGTTC TGCTGTACAC CTCTGACGAC CTGTTCCACT GGAAATACGA GGGTGTAATC TTTGAAGATGAAACCACCAA AGAGATTGAG TGTCCAGACC TGGTTCGTAT CGGTGAGAAA GACATTCTCA TCTACTCCATCACCTCTACC AACTCTGTAC TGTTCTCTAT GGGTGAACTG AAAGAAGGCA AACTGAACGT GGAGAAACGTGGCCTTCTGG ACCACGGTAC TGACTTCTAC GCTGCACAGA CCTTCTTCGG TACTGACCGT GTTGTTGTAATCGGTTGGCT GCAGTCTTGG CTGCGTACTG GTCTGTATCC GACCAAACGT GAAGGTTGGA ACGGTGTTATGTCTCTGCCA CGTGAACTGT ACGTTGAGAA CAACGAGCTG AAAGTCAAAC CGGTTGATGA ACTGCTTGCTCTGCGTAAAC GTAAAGTGTT CGAAACCGCG AAATCTGGTA CGTTTCTGCT GGACGTTAAA GAGAACAGCTACGAAATCGT TTGCGAGTTC TCTGGTGAAA TCGAACTGCG TATGGGTAAC GAATCTGAAG AAGTTGTCATCACCAAATCT CGTGACGAAC TTATCGTTGA CACCACTCGT TCTGGTGTTT CCGGTGGTGA AGTTCGCAAATCTACCGTAG AGGACGAAGC GACTAACCGT ATCCGTGCGT TTCTGGACAG CTGCTCTGTT GAGTTCTTCTTCAACGACTC TATCGCGTTC TCTTTCCGTA TCCATCCGGA GAACGTGTAC AACATCTTGT CCGTGAAATCTAACCAGGTT AAACTGGAAG TATTCGAACT GGAGAACATC TGGCTGTAA。
[0059] SEQ ID NO:3 CGGGATCC ATGTTCAAAC CGAACTACCACTTC。
[0060] SEQ ID NO:4 CCCAAGCTT TTACAGCCAG ATGTTCTCCA GTTC。
[0061] SEQ ID NO:5 MSDSEVNQEA KPEVKPEVKP ETHINLKVSD GSSEIFFKIK KTTPLRRLME AFAKRQGKEMDSLRFLYDGI RIQADQTPED LDMEDNDIIE AHREQIGGS。
[0062] SEQ ID NO:6 ATGTCGGACT CAGAAGTCAA TCAAGAAGCT AAGCCAGAGG TCAAGCCAGA AGTCAAGCCTGAGACTCACA TCAATTTAAA GGTGTCCGAT GGATCTTCAG AGATCTTCTT CAAGATCAAA AAGACCACTCCTTTAAGAAG GCTGATGGAA GCGTTCGCTA AAAGACAGGG TAAGGAAATG GACTCCTTAA GATTCTTGTACGACGGTATT AGAATCCAAG CTGATCAGAC CCCTGAAGAT TTGGACATGG AGAATCCA TATTATTGAGGCTCACAGAG AACAGATTGG TGGATCC。
[0063] SEQ ID NO:7 MATVKFKYKG EEKEVDISK KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLEKQKK。
[0064] SEQ ID NO:8 ATGGCAACAG TAAAGTTCAA GTACAAGGGA GAAGAGAAGG AAGTAGATAT AAGTAAGATAAAGAAGGTAT GGAGAGTAGG CAAAATGATA AGTTTCACCT ATGATGAGGG TGGAGGAAAG ACTGGTAGAGGAGCTGTAAG CGAGAAAGAC GCTCCAAAAG AACTACTACA AATGTTAGAG AAGCAAAAGA AG.
[0065] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.
[0066] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.
Claims
1. A recombinant engineered bacterium, characterized in that: Escherichia coli Escherichia coli The invention is a chassis strain, wherein the chassis strain contains an expression vector for a target gene expressing sucrose invertase, or the chassis strain is inserted with a target gene expressing sucrose invertase, wherein the target gene is connected with a solubilizing protein SUMO tag or an SSO tag, wherein the SUMO tag is located at the N-terminus of the target gene, and the SSO tag is located at the N-terminus or the C-terminus of the target gene.
2. The recombinant engineered bacterium according to claim 1, characterized in that: The SUMO tag is located at the N-terminus of the target gene.
3. The recombinant engineered bacterium according to claim 1, characterized in that: The sequence of the SUMO tag is SEQ ID NO:6, and the sequence of the SSO tag is SEQ ID NO:
8.
4. The recombinant engineered bacterium according to claim 1, characterized in that: The sequence of the target gene expressing sucrose invertase is SEQ ID NO:
2.
5. The recombinant engineered bacterium according to claim 1, characterized in that: The expression vector is an Escherichia coli expression vector pET series.
6. A method for preparing the recombinant engineered bacteria according to any one of claims 1 to 5, characterized in that: The following steps are included: Connect the tag between the two restriction sites of the expression vector to obtain an expression vector containing the tag; Connect the target gene between the two restriction sites of the expression vector to obtain a recombinant plasmid; Enzyme digestion of the expression vector and recombinant plasmid containing the tag to obtain the tag and the target gene; Connect the tag and the target gene to obtain a connection product, and transfer the connection product into competent cells of the chassis strain to obtain a recombinant engineered bacterium; When the tag is a SUMO tag, the two restriction enzyme sites are Nde Ⅰ and BamH Ⅰ restriction enzyme sites; When the tag is an SSO tag, the two restriction enzyme cleavage sites are Nde Ⅰ and BamH Ⅰ restriction enzyme cleavage sites, or Hind Ⅲ and Xho I restriction enzyme cleavage sites.
7. An immobilized cell, characterized in that: It comprises a bacterial suspension containing the recombinant engineered bacteria according to any one of claims 1 to 5, inorganic soil, a flocculant and a cross-linking agent, wherein the weight ratio of the bacterial suspension volume to the inorganic soil is 100 ml: 1-20 g, the weight ratio of the bacterial suspension volume to the flocculant is 100 ml: 0.05-1 g, and the volume ratio of the bacterial suspension to the cross-linking agent is 100: 0.05-0.
5.
8. The immobilized cells according to claim 7, characterized in that: The concentration of the bacterial suspension is 100-200 g / L; the inorganic soil is diatomaceous earth, montmorillonite, bentonite or kaolin; the flocculant is polyethyleneimine, polyacrylamide or chitosan, and the molecular weight of the polyethyleneimine is 600-70000; the cross-linking agent is glutaraldehyde, N,N-methylenebisacrylamide or trihydroxymethyl phosphine.
9. A method for preparing immobilized cells as claimed in claim 7 or 8, characterized in that: The bacterial suspension and inorganic soil are mixed evenly, a flocculant is added for flocculation, and then a cross-linking agent is added to obtain immobilized cells.
10. A use of the recombinant engineered bacteria according to any one of claims 1 to 5 or the immobilized cells according to any one of claims 7 to 8, characterized in that: Used to hydrolyze sucrose to prepare hydrolyzed fructose solution.