CN114277049A - Genetic engineering bacterium for heterologous expression of sucrose isomerase and application thereof - Google Patents
Genetic engineering bacterium for heterologous expression of sucrose isomerase and application thereof Download PDFInfo
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Abstract
The invention discloses a genetically engineered bacterium for heterologous expression of sucrose isomerase and application thereof, belonging to the technical field of industrial biology. The invention constructs the recombinant bacillus subtilis carrying the Pgrac100 promoter, 13 different signal peptides and sucrose isomerase genes derived from Klebsiella sp.LX3, and determines that the extracellular enzyme activity of the recombinant bacteria containing the WapA signal peptide is the highest through comparison, the extracellular enzyme activity of shake flask culture reaches 23.03U/mL, the recombinant bacillus subtilis is further fermented in a 5L fermentation tank, the extracellular enzyme activity is improved to 124.97U/mL, and the purified recombinant sucrose isomerase has excellent enzymological properties. The invention provides an effective strategy for the basic research of the high-efficiency heterologous expression and the structural function research of the sucrose isomerase, and lays a foundation for the industrial large-scale preparation and application of the sucrose isomerase.
Description
Technical Field
The invention belongs to the technical field of industrial biology, and particularly relates to a genetic engineering bacterium capable of heterologously expressing sucrose isomerase, and application of the genetic engineering bacterium in heterologously expressing sucrose isomerase.
Background
Isomaltulose (α -D-glucopyranosyl-1, 6-D-fructose) is a reducing disaccharide present in honey, sugarcane juice, which as an isomer of sucrose has similar physicochemical properties as sucrose. The sweetness of the isomaltulose is about 50 percent of that of the cane sugar, however, the isomaltulose has a health-care function, so the isomaltulose can be used as a substitute of the cane sugar and has great application potential in the production of sugar-free foods, sugar-free health-care products and sugar-free medicines. Because the natural isomaltulose in honey and sugarcane juice has low content and the isomaltulose is difficult to synthesize by a chemical method, the isomaltulose is mainly prepared by a sucrose isomerase catalytic sucrose conversion method at home and abroad at present.
Sucrose isomerase originates mainly from bacteria, and it has been found that sucrose isomerase-producing bacteria mainly have: erwinia rhapontici (Erwinia rhapontici), Protaminobacter rubrum (Protamobacter rubrum), Serratia pusilvestris (Serratia plymuthica), Polyporus versicolor (Pantoea dispersa), and Klebsiella pneumoniae (Klebsiella sp). At present, many reports have been made on the production of sucrose isomerase by using wild bacteria for fermentation, for example, the enzyme activity reaches 15.12U/mL by optimizing the culture conditions of Klebsiella sp.LX 3. The enzyme activity of the wild Erwinia rhapontici NX-5 strain fermentation enzyme production is 1.3U/mL. Through the optimization of the culture medium, the enzyme activity of Erwinia sp.D12 fermentation enzyme production reaches 10.84U/mL. The level of sucrose isomerase produced by wild fungi is low, and the requirement of industrial scale production and application cannot be met, and in order to improve the expression level of sucrose isomerase and promote the large-scale application of sucrose isomerase in the food industry, many research works adopting heterologous expression strategies are carried out in recent years, wherein most of the research works adopt escherichia coli as a host to carry out heterologous expression of sucrose isomerase. The sucrose isomerase from Erwinia rhapontici NX-5 is induced and expressed in Escherichia coli at low temperature by using lactose as an inducer, the enzyme activity of the sucrose isomerase reaches 15U/mL, and an enzyme production fermentation culture medium of the Escherichia coli is optimized by a response surface method, so that the enzyme activity reaches 29.1U/mL. Meanwhile, some research works expressed sucrose isomerase recombinantly in food-safe strains, and sucrose isomerase derived from Pantoea dispersa was successfully displayed on the surface of yarrowia lipolytica cells, achieving a conversion rate of isomaltulose of 93%. FMB-1 sucrose isomerase was successfully displayed on the surface of s.cerevisiae cells with 6.4-7.4% conversion at substrate sucrose concentrations in the range of 50-250 mM. Sucrose isomerase derived from Enterobacter sp.FMB-1 was recombinantly expressed and secreted extracellularly using Lactococcus lactis MG1363 as the host, with a conversion rate of 72%. The sucrose isomerase gene derived from Erwinia rhapontici NX-5 realizes intracellular expression in the Bacillus subtilis WB800, and the whole-cell enzyme activity reaches 5.2U/mL through fermentation tank amplification and culture condition optimization. Because the enzyme yield of the existing genetic engineering bacteria for heterologously expressing sucrose isomerase needs to be improved, the production and use cost of the sucrose isomerase is caused, the industrial production cost of isomaltulose is high, and the development of related industries is seriously restricted, the construction of the genetic engineering bacteria for expressing the recombinant sucrose isomerase at a high level has urgent and objective requirements for promoting the industrial application of the sucrose isomerase.
The Bacillus subtilis is a gram-positive bacterium, has clear genetic background, is used as a food safety level microorganism, not only has mature gene operation means and diversified carriers, but also has strong capability of secreting protein to the extracellular space, is easy for large-scale fermentation culture, is an important industrial enzyme preparation production strain, and is particularly suitable for the production of enzyme preparations in the food industry. Signal peptides are short peptides usually present at the N-terminus of a newly synthesized peptide chain, typically comprising 15-30 amino acids, which facilitate transport of secreted proteins across membranes and are hydrolyzed after transport is complete. The signal peptide can be divided into a positively charged N-terminal region, mainly comprising the middle hydrophobic region where neutral amino acids are able to form an alpha helix, and a C-terminal region including the cleavage site of the signal peptide, depending on the type of amino acids in the sequence. The selection of an appropriate signal peptide has an important effect on successful high-level secretory expression of a foreign protein in Bacillus subtilis. Therefore, the problem to be solved at present is to select a proper signal peptide to construct a high-efficiency secretory expression system and to establish a proper fermentation process by using bacillus subtilis as a host.
Disclosure of Invention
The invention aims to provide a genetically engineered bacterium for heterologously expressing sucrose isomerase and a method for preparing recombinant sucrose isomerase by fermentation culture by using the genetically engineered bacterium. The invention can effectively improve the heterologous expression level of the sucrose isomerase, has simple experimental operation method and stable expression level, is suitable for a bacillus subtilis expression system, and thus solves the problems that the existing sucrose isomerase production wild bacteria and recombinant gene engineering bacteria have low expression level and are difficult to secrete and express, the production and use cost of the sucrose isomerase is high, the large-scale production and application are difficult, and the like.
The technical scheme of the invention for solving the technical problems is as follows:
a recombinant expression vector comprising Pgrac100 promoter, WapA signal peptide and sucrose isomerase gene.
Further, in the above technical scheme, the expression vector is a pHT254 expression vector, the pHT254 expression vector includes an exogenous gene expression cassette, and the exogenous gene expression cassette includes a Pgrac100 promoter, a WapA signal peptide, a sucrose isomerase gene, and a transcription termination sequence in order from 5 'to 3'.
The pHT254 expression vector comprises the following functional elements: pHT254 plasmid skeleton, exogenous gene expression box, screening marker gene expression box.
The exogenous gene expression cassette sequentially comprises a Pgrac100 promoter, a WapA signal peptide, a sucrose isomerase gene and a transcription termination sequence from 5 'to 3'.
Further, in the above technical scheme, the sucrose isomerase gene comprises a sucrose isomerase gene derived from Klebsiella sp.lx3.
Furthermore, in the above technical scheme, the nucleotide sequence of the Pgrac100 promoter is shown as SEQ ID No.1, and the nucleotide sequence of the WapA signal peptide is shown as SEQ ID No. 2.
Further, in the above technical scheme, the nucleotide sequence of the sucrose isomerase gene derived from Klebsiella sp.LX3 is shown as SEQ ID No. 3.
A genetically engineered bacterium containing the recombinant expression vector.
Further, in the above technical scheme, the genetically engineered bacteria comprise bacillus subtilis WB 800N.
An application of the genetic engineering bacteria is used for extracellular production of sucrose isomerase.
An application of the genetic engineering bacteria is used for catalyzing sucrose to produce isomaltulose.
Further, in the technical scheme, the temperature of the gene engineering bacteria for catalyzing sucrose to produce isomaltulose is 35-50 ℃, and the pH is 4.5-7.5.
The construction of the genetic engineering bacteria comprises the following steps:
A. the pHT254 expression vector containing the WapA signal peptide and the sucrose isomerase gene is constructed by the PCR technology and the RF cloning technology, and the recombinant expression vector is stored in escherichia coli.
B. Extracting recombinant expression vector, transferring into Bacillus subtilis WB800N by electric transformation method, and constructing recombinant engineering bacteria.
The application of the genetic engineering bacteria comprises the following steps:
the genetically engineered bacterium is applied to fermentation production of recombinant sucrose isomerase, after shake flask fermentation culture is carried out for 24 hours, the activity of the recombinant sucrose isomerase in extracellular fermentation liquid is detected, wherein the extracellular enzyme activity of the engineered bacterium with the WapA signal peptide is the highest and reaches 23.03U/mL, and the engineered bacterium with the WapA signal peptide is further cultured and induced to express in a 5L fermentation tank by utilizing 2L of culture medium, so that the activity of the extracellular enzyme is improved to 124.97U/mL.
The purified recombinant sucrose isomerase has excellent enzymological properties, the optimal reaction temperature is 45 ℃, the stability is realized within the range of 35-50 ℃, the optimal reaction pH is 5.5, and the stability is realized within the range of pH 4.5-7.5.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the optimal signal peptide WapA is obtained by screening 13 signal peptides from different sources, and the efficient secretory expression of the sucrose isomerase gene in the bacillus subtilis is realized by using the strong promoter PGrac100 and the signal peptide WapA for the first time. Compared with the method for expressing the sucrose isomerase by using escherichia coli as a host, the method can directly secrete the soluble sucrose isomerase into an extracellular culture medium, does not need cell disruption, can convert sucrose into isomaltulose by using a crude enzyme solution, simplifies the production and use processes of the sucrose isomerase, can simplify the separation and purification steps of the sucrose isomerase to obtain pure enzyme, and saves a large amount of labor, material resources and time cost. The extracellular enzyme activity is improved to 124.97U/mL through the amplification culture of a fermentation tank, thereby laying a foundation for further industrial amplification production of sucrose isomerase and promoting the heterologous high-efficiency expression and industrial production of the sucrose isomerase.
Drawings
FIG. 1 shows the extracellular enzyme activities of sucrose isomerase carrying 13 different signal peptides secreted and expressed by Bacillus subtilis WB 800N.
FIG. 2 shows the enzymatic properties of the purified recombinant sucrose isomerase. A: the optimum reaction temperature; b: the optimum reaction pH value; c, temperature stability; and D, pH stability.
FIG. 3 shows the results of amplifying the enzyme production in the 5L fermenter.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The reagents or raw materials used in the present invention are commercially available unless otherwise specified.
The experimental method of the present invention, in which the specific conditions are not specified, is generally performed according to conventional conditions, such as "molecular cloning experimental guidelines: the conditions described in 4 th edition "or according to the conditions recommended by the reagent manufacturer.
The sucrose isomerase enzyme biopsy method comprises the following steps:
enzyme-catalyzed reaction: taking 100 mu L of enzyme solution which is diluted moderately, adding 400 mu L of disodium hydrogen phosphate-citric acid buffer solution containing 4 wt% of sucrose, mixing uniformly, reacting for 15min at a specific temperature, then treating a sample in boiling water at 100 ℃ for 15min for inactivating enzyme, centrifuging at 10000rpm for 15min to obtain supernatant, filtering with a 0.22 mu m filter membrane, and carrying out HPLC detection. The concentration of isomaltulose in the reaction product was calculated according to a standard curve and the enzyme activity was calculated. The relative enzyme activity is defined as the ratio calculated by taking the measured highest enzyme activity as 100 percent as the relative enzyme activity.
And (3) enzyme activity determination: isomaltulose standard solutions of different concentrations were prepared, filtered through a 0.22 μm filter and subjected to HPLC detection. Detection conditions of HPLC: hypersil APS-2, 4.6X 250mm, 5 μm amino column was used as a chromatographic column, and detection was carried out by a differential detector, and the mobile phase was an aqueous solution containing 80% acetonitrile. The column temperature was 35 ℃ and the amount of sample was 10. mu.L, the flow rate was 1.0 mL/min. And (4) fitting a standard curve according to the peak areas of the isomaltulose standards with different concentrations. After the enzyme catalysis reaction product is analyzed by HPLC, the concentration of isomaltulose in the reaction product is calculated according to a standard curve, and the enzyme activity is calculated.
Definition of enzyme activity units: the amount of enzyme required to release 1. mu. mol isomaltulose per minute using sucrose as substrate was 1 enzyme activity unit (U).
Example 1 Signal peptide screening to increase extracellular enzymatic Activity of recombinant sucrose isomerase
The recombinant expression vector containing the Pgrac100 promoter, different signal peptides and sucrose isomerase genes is constructed by a PCR technology and an RF cloning technology. In the present invention, 13 signal peptides selected were WapA, Bpr, LipA, Yjfa, Mpr, SacB, YvgO, AmyQ, YfhK, AmyE, WprA, Vpr, and AprE, respectively, and the nucleotide sequences of these signal peptides are shown in SEQ ID No.2(WapA) and SEQ ID Nos. 4 to 15 (other 12 signal peptides). And respectively transforming the recombinant expression vectors carrying different signal peptide fusion sucrose isomerase genes into bacillus subtilis WB800N to construct different recombinant engineering bacteria.
The recombinant genetic engineering bacteria can utilize different signal peptides to secrete the soluble sucrose isomerase into an extracellular culture medium, so that extracellular supernatant of the recombinant genetic engineering bacteria can be directly taken to convert sucrose and the activity of the sucrose isomerase can be detected. Activating each constructed recombinant gene engineering bacterium in a 2 XYT culture medium for 12-14h at 37 ℃, then connecting the seed liquid in a 2 XYT culture medium in a final volume of 2% (v/v) at 37 ℃ for amplification culture until OD600nm reaches 0.8-1.0, adding an inducer IPTG with a final concentration of 1mM for expression at 30 ℃ for 24h, and taking extracellular supernatant for enzyme activity determination of sucrose isomerase. The result of the extracellular enzyme biopsy of each recombinant gene engineering bacterium is shown in figure 1, and the enzyme activity of sucrose isomerase in the extracellular supernatant of the recombinant bacterium containing the WapA signal peptide is the highest and reaches 23.03U/mL.
Example 2 characterization of the enzymatic Properties of recombinant sucrose isomerase
Carrying out an enzyme catalysis reaction in a pH 6.0 disodium hydrogen phosphate-citric acid buffer solution containing 4 wt% of sucrose at 20-60 ℃, wherein the optimum reaction temperature of the purified sucrose isomerase is 45 ℃, and the enzyme activity of the purified sucrose isomerase is higher than 90% within the range of 35-50 ℃, as shown in figure 2A. Carrying out an enzyme catalysis reaction in a disodium hydrogen phosphate-citric acid buffer solution with the pH value of 4.0-8.0 and containing 4 wt% of sucrose at the temperature of 45 ℃, wherein the optimum reaction pH value of the purified sucrose isomerase is 5.5, and the enzyme activity in the pH value range of 5.0-6.5 is higher than 86%, as shown in figure 2B. Detecting the temperature stability of sucrose isomerase, respectively incubating the purified sucrose isomerase at 35-60 ℃ for 30min, and then detecting the enzyme activity under the optimal reaction condition, wherein the result is shown in figure 2C, the relative enzyme activity after incubation at 35-50 ℃ for 30min is higher than 97%, when the temperature is further increased, the temperature stability of the enzyme is rapidly reduced, and only 1% of the enzyme activity is remained after incubation at 55 ℃. And (3) detecting the pH stability of the sucrose isomerase, incubating the purified sucrose isomerase at 25 ℃ for 24h at pH 4.0-8.0 respectively, and then detecting the enzyme activity under the optimal reaction condition, wherein the result is shown in figure 2D, more than 95% of the enzyme activity of the sucrose isomerase can be reserved after incubation at pH 4.5-7.5, which shows that the sucrose isomerase has better stability in the pH range.
Example 3 fermentation level of recombinant Bacillus subtilis containing WapA Signal peptide in 5L fermentor
Further examine the effect of recombinant engineering bacteria containing WapA signal peptide on enzyme production by fermentation in a 5L fermentation tank using 2L fermentation medium. Streaking the recombinant engineering bacteria on LB solid plate containing chloramphenicol with the final concentration of 5 mug/mL, carrying out inverted culture at 37 ℃ for 12-14h, selecting a single colony to be inoculated in 2 XYT culture medium containing chloramphenicol with the final concentration of 5 mug/mL, carrying out activated culture in a shaking table at 37 ℃ for 8h, inoculating 2mL of activated bacterial liquid into 200mL of 2 XYT culture medium containing chloramphenicol with the final concentration of 5 mug/mL, and carrying out expanded culture in the shaking table at 37 ℃ for 8 h. 2L of medium was charged into a 5L fermenter, the fermentation medium consisting of: 8g/L glycerol, 8.5g/L soytone, 8.5g/L beef extract, pH 7.0, containing a final concentration of 5. mu.g/mL chloramphenicol. Inoculating 200mL of the bacterial liquid subjected to amplification culture into a fermentation tank, maintaining the pH value to be 7.0 by using ammonia water and phosphoric acid in the fermentation process, controlling the dissolved oxygen to be 30% in the fermentation process, controlling the initial fermentation temperature to be 37 ℃, and starting feeding after dissolved oxygen rebounds, wherein the feeding culture medium comprises: 150g/L of glucose, 100g/L of soybean peptone and 50g/L of beef extract. After feeding, the fermentation temperature was adjusted to 30 ℃ after further cultivation until OD600nm reached about 12, IPTG was then added to a final concentration of 1mM, and the fermentation was continued for inducible expression with extracellular enzyme activity up to 124.97U/mL (FIG. 3), which was about 5.4 times the shake flask level.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
SEQUENCE LISTING
<110> constitution of plum wine
<120> genetic engineering bacterium for heterologous expression of sucrose isomerase and application thereof
<130> 2021
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 123
<212> DNA
<213> promoter (Pgrac 100)
<400> 1
aggaggtaag gatcactaga aaatttttta aaaaatctct tgacattgga agggagatat 60
gttattataa gaattgcgga attgtgagcg gataacaatt cccatataaa ggaggaagga 120
tcc 123
<210> 2
<211> 96
<212> DNA
<213> Signal peptide (WapA)
<400> 2
atgaaaaaaa gaaagaggcg aaactttaaa aggttcattg cagcattttt agtgttggct 60
ttaatgattt cattagtgcc agccgatgta ctagca 96
<210> 3
<211> 1710
<212> DNA
<213> sucrose isomerase (Klebsiella sp. LX 3)
<400> 3
gcaccatcct tgaatcagga tattcacgtt caaaaggaaa gtgaatatcc tgcatggtgg 60
aaagaagctg ttttttatca gatctatcct cgctcattta aagacaccaa tgatgatggc 120
attggcgata ttcgcggtat tattgaaaag ctggactatc tgaaatcgct cggtattgac 180
gctatctgga tcaatcccca ttacgactct ccgaacaccg ataacggcta tgacatcagt 240
aattatcgtc agataatgaa agagtatggc acaatggagg attttgatag ccttgttgcc 300
gaaatgaaaa aacgaaatat gcgcttaatg atcgacgtgg tcattaacca taccagtgat 360
caacacccgt ggtttattca gagtaaaagc gataaaaaca acccttatcg tgactattat 420
ttctggcgtg acggaaaaga taatcagcca cctaataatt acccctcatt tttcggcggc 480
tcggcatggc aaaaagatgc aaagtcagga cagtactatt tacactattt tgccagacag 540
caacctgatc tcaactggga taacccgaaa gtacgtgagg atctttacgc aatgctccgc 600
ttctggctgg ataaaggcgt ttcaggcatg cgatttgata cggtggcaac ttattccaaa 660
atcccgggat ttcccaatct gacacctgaa caacagaaaa attttgctga acaatacacc 720
atggggccta atattcatcg atacattcag gaaatgaacc ggaaagttct gtcccggtat 780
gatgtggcca ccgcgggtga aatttttggc gtcccgctgg atcgttcgtc gcagtttttt 840
gatcgccgcc gacatgagct gaatatggcg tttatgtttg acctcattcg tctcgatcgc 900
gacagcaatg aacgctggcg tcacaagtcg tggtcgctct ctcagttccg ccagatcatc 960
agcaaaatgg atgtcacggt cggaaagtat ggctggaaca cgttcttctt agataaccat 1020
gacaaccccc gtgcggtatc tcacttcggg gatgacaggc cgcaatggcg ggaggcgtcg 1080
gctaaggcac tggcgacgat taccctcact cagcgggcga cgccgtttat ttatcagggt 1140
tcagagctgg gaatgactaa ttatcccttc aggcaactca acgaatttga cgacatcgag 1200
gtcaaaggtt tctggcagga ttatgtccag agtggaaaag tcacggccac agagtttctc 1260
gataatgtgc gcctgacgag ccgcgataac agcagaacac ctttccagtg gaatgacacc 1320
ctgaatgctg gttttactcg cggaaagccg tggtttcaca tcaacccaaa ctatgtggag 1380
atcaacgccg aacgcgaaga aacccgcgaa gattcagtgc tgaattacta taaaaaaatg 1440
attcagctac gccaccatat ccctgctctg gtatatggcg cctatcagga tcttaatcca 1500
caggacaata ccgtttatgc ctatacccga acgctgggta acgagcgtta tctggtcgtg 1560
gtgaacttta aggagtaccc ggtccgctat actctcccgg ctaatgatgc catcgaggaa 1620
gtggtcattg atactcagca gcaggcggct gcgccgcaca gcacatccct gtcattgagc 1680
ccctggcagg caggtgtgta taagctgcgg 1710
<210> 4
<211> 90
<212> DNA
<213> Signal peptide (Bpr)
<400> 4
atgaggaaaa aaacgaaaaa cagactcatc agctctgttt taagtacagt tgtcatcagt 60
tcactgctgt ttccgggagc agccggggca 90
<210> 5
<211> 93
<212> DNA
<213> Signal peptide (LipA)
<400> 5
atgaaatttg taaaaagaag gatcattgca cttgtaacaa ttttgatgct gtctgttaca 60
tcgctgtttg cgttgcagcc gtcagcaaaa gcc 93
<210> 6
<211> 84
<212> DNA
<213> Signal peptide (Yjfa)
<400> 6
atgaaaagac tgtttatgaa ggcttcattg gtgttattcg cagtagtatt tgtttttgcc 60
gtcaaaggtg cacccgccaa ggcg 84
<210> 7
<211> 102
<212> DNA
<213> Signal peptide (Mpr)
<400> 7
atgaaattag ttccaagatt cagaaaacaa tggttcgctt acttaacggt tttgtgtttg 60
gctttggcag cagcggtttc ttttggcgta ccggcaaaag cg 102
<210> 8
<211> 87
<212> DNA
<213> Signal peptide (SacB)
<400> 8
atgaacatca aaaagtttgc aaaacaagca acagtattaa cctttactac cgcactgctg 60
gcaggaggcg caactcaagc gtttgcg 87
<210> 9
<211> 78
<212> DNA
<213> Signal peptide (YvgO)
<400> 9
atgaaacgta ttcgtatccc aatgacactt gctcttggtg ccgctttgac aattgcccct 60
ttgtcctttg cttccgct 78
<210> 10
<211> 93
<212> DNA
<213> Signal peptide (AmyQ)
<400> 10
atgattcaaa aacgaaagcg gacagtttcg ttcagacttg tgcttatgtg cacgctgtta 60
tttgtcagtt tgccgattac aaaaacatca gcc 93
<210> 11
<211> 87
<212> DNA
<213> Signal peptide (YfhK)
<400> 11
atgaaaaaga aacaagtaat gctcgcttta acagctgccg caggactggg tttgacagca 60
cttcattccg ctcccgcagc aaaagct 87
<210> 12
<211> 99
<212> DNA
<213> Signal peptide (AmyE)
<400> 12
atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60
tttcatttgg ttctggcagg accggcggct gcgagtgct 99
<210> 13
<211> 93
<212> DNA
<213> Signal peptide (WprA)
<400> 13
atgaaacgca gaaaattcag ctcggttgtg gcggcagtgc ttatttttgc actgattttc 60
agcctttttt ctccgggaac caaagctgca gcg 93
<210> 14
<211> 93
<212> DNA
<213> Signal peptide (Vpr)
<400> 14
atgaaaaagg ggatcattcg ctttctgctt gtaagtttcg tcttattttt tgcgttatcc 60
acaggcatta cgggcgttca ggcagctccg gct 93
<210> 15
<211> 87
<212> DNA
<213> Signal peptide (AprE)
<400> 15
atgagaagca aaaaattgtg gatcagcttg ttgtttgcgt taacgttaat ctttacgatg 60
gcgttcagca acatgtctgc gcaggct 87
Claims (10)
1. A recombinant expression vector comprising a Pgrac100 promoter, a WapA signal peptide and a sucrose isomerase gene.
2. The recombinant expression vector according to claim 1, wherein the expression vector is a pHT254 expression vector, the pHT254 expression vector comprises an exogenous gene expression cassette, and the exogenous gene expression cassette comprises a PGrac100 promoter, a WapA signal peptide, a sucrose isomerase gene and a transcription termination sequence from 5 'to 3'.
3. The recombinant expression vector of claim 1, wherein the sucrose isomerase gene comprises a sucrose isomerase gene derived from Klebsiella sp.lx3.
4. The recombinant expression vector according to claim 1, wherein the nucleotide sequence of the Pgrac100 promoter is shown as SEQ ID No.1, and the nucleotide sequence of the WapA signal peptide is shown as SEQ ID No. 2.
5. The recombinant expression vector according to claim 3, wherein the nucleotide sequence of the sucrose isomerase gene derived from Klebsiella sp.LX3 is shown in SEQ ID No. 3.
6. A genetically engineered bacterium comprising the recombinant expression vector of any one of claims 1 to 5.
7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium comprises Bacillus subtilis WB 800N.
8. Use of the genetically engineered bacterium of claim 6 or 7 for extracellular production of sucrose isomerase.
9. Use of the genetically engineered bacterium of claim 6 or 7 to catalyze the production of isomaltulose from sucrose.
10. The use of claim 9, wherein the temperature of the genetically engineered bacteria catalyzing sucrose to produce isomaltulose is 35-50 ℃ and the pH is 4.5-7.5.
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