CN106282221B - Construction method and application of genetic engineering bacteria for secretory expression of trehalose synthase - Google Patents
Construction method and application of genetic engineering bacteria for secretory expression of trehalose synthase Download PDFInfo
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Abstract
The invention relates to a construction method and application of a genetic engineering bacterium for secretory expression of trehalose synthase. The construction method comprises the following steps: (1) PCR amplifying an NprE signal peptide sequence and a trehalose synthase sequence ps TreS; (2) preparing an NprE-ps TreS fragment; (3) after double enzyme digestion, the NprE-ps TreS fragment is connected with pHT01 plasmid which is also subjected to double enzyme digestion, and is transferred into Escherichia coli DH5 alpha, so that recombinant plasmid pHT01-NprE-ps TreS is prepared; (4) transferring the recombinant plasmid into bacillus subtilis by an electrotransformation method, and screening by a chloramphenicol plate to obtain the gene engineering bacteria for secreting and expressing trehalose synthase. The invention adds a signal peptide fragment NprE into a recombinant vector for the first time, and the signal can secrete the target protein trehalose synthase to the outside of cells of recombinant engineering bacteria for expression, thereby greatly simplifying the subsequent steps of enzyme separation and purification.
Description
Technical Field
The invention relates to a construction method and application of genetic engineering bacteria for secretory expression of trehalose synthase, belonging to the technical field of biotechnology.
background
trehalose (trehalase) is a non-reducing disaccharide composed of two pyranosyl glucose molecules linked via an α -1, 1-glycosidic bond, and is widely found in organisms such as bacteria, yeast, filamentous fungi, plants, insects, and invertebrates. The research shows that the compound has stable property and has very important biological significance to organisms. It is mainly characterized by that it is a storage material of biological energy source and carbon source, and is a stabilizing agent and protective agent of protein and biological membrane molecule in the harsh environment of dehydration, high temp., oxygen free radical and low temp., and is a signal sensing compound and growth regulating factor, and also is one of the components of some bacterial cell walls, so that the trehalose has the reputation of "life sugar" in the scientific field. Trehalose has a magical protective effect on organisms because trehalose can form a unique protective film on the cell surface under severe environmental conditions such as high temperature, high cold, high osmotic pressure, drying and dehydration and the like, so that protein molecules are effectively protected from being inactivated without denaturation, and the life process and biological characteristics of the organisms are maintained. The unique functional characteristic enables the trehalose to be used as an excellent activity protective agent for protein drugs, enzymes, vaccines and other biological products, also be an important component of cosmetics for keeping cell activity and moisture, and be used as a unique food ingredient for preventing food deterioration, keeping fresh flavor of food and improving food quality. Therefore, the trehalose can be widely applied to the pharmaceutical industry, the cosmetic industry and the food industry, and has attractive development and application prospects and great economic benefits.
in view of the wide and important application value of trehalose, research on finding efficient, convenient and low-cost production methods of trehalose is widely regarded. The prior trehalose production methods mainly comprise a yeast extraction method, a fermentation method and an enzymatic synthesis method. The trehalose produced by the enzyme method has the characteristics of high specificity, rapidness, mildness and the like, and becomes a hot spot for researching and developing trehalose industrial production and is one of feasible ways with short-term effect.
Trehalose synthase (EC5.4.99.16, Trehalose synthase, TreS) is an intramolecular transglucosidase that requires only a single reaction step to convert the alpha-1, 4 glycosidic bond of maltose to the alpha-1, 1 glycosidic bond to produce Trehalose. The enzyme reaction process is short, easy to regulate and control, does not need to consume high-energy substances, does not need to coexist with phosphate, and can obtain the trehalose by one-step reaction of one enzyme, so that the trehalose synthase conversion method is a method suitable for industrial production of trehalose, has good application prospect, and is widely concerned. Up to now, all exogenous expression trehalose synthase genetic engineering bacteria reported at home and abroad are genetic engineering strains constructed by escherichia coli, and are only expressed intracellularly. However, Bacillus subtilis is a food-safe microorganism and has a strong ability to secrete proteins, as compared to Escherichia coli. The invention constructs trehalose synthase gene on a bacillus subtilis secretion expression vector by means of genetic engineering, and converts the secretion expression vector into bacillus subtilis by an electrical conversion method, thereby obtaining a genetic engineering bacterium capable of secreting and expressing trehalose synthase and applying the genetic engineering bacterium to production of food and pharmaceutical grade trehalose.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a construction method and application of a genetic engineering bacterium for secretory expression of trehalose synthase. The invention utilizes the signal peptide NprE which is screened in the early stage and can secrete and express trehalose, constructs a vector which can secrete and express trehalose synthase on the basis of a commercial vector pHT01, and transforms the secretory expression vector into bacillus subtilis by a high-efficiency electrotransformation method, thereby obtaining the genetically engineered bacterium which can secrete and express the trehalose synthase.
the technical scheme of the invention is as follows:
The nucleotide sequence of the recombinant expression vector for efficiently secreting and expressing trehalose synthase by using the bacillus subtilis is shown in SEQ ID No. 1.
compared with the Bacillus subtilis expression vector pHT01 vector reported in the literature, the recombinant expression vector is added with a signal peptide fragment NprE of neutral protease, and the signal can secrete the target protein trehalose synthase to the outside of cells for expression. After the trehalose synthase is secreted out of cells, a relatively pure protease liquid is obtained by carrying out ammonium sulfate precipitation on fermentation liquor.
A construction method of genetic engineering bacteria for secretory expression of trehalose synthase comprises the following steps:
(1) Taking the genome of the bacillus subtilis 168 as a template, and carrying out PCR amplification on an NprE signal peptide sequence in the Sec pathway; performing PCR amplification on a trehalose synthase sequence psTreS by taking a genome of Pseudomonas stutzeri as a template;
The PCR primer sequences for the NprE signal peptide sequence were as follows:
NprE-F：5’-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3’；SEQ ID NO.3
NprE-R：5’-GAATGCTCATactagtAGCCTGAACACC-3’；SEQ ID NO.4
the sequence of the PCR primer of the trehalose synthase sequence ps TreS is as follows:
ps TreS-F：5’-actagtATGAGCATTCCAGA-3’；SEQ ID NO.5
ps TreS-R：5’-tctagaTTAGATCACCGGGGATGC-3’；SEQ ID NO.6
(2) Fusing the signal peptide sequence NprE prepared in the step (1) with a trehalose synthase sequence ps TreS from pseudomonas stutzeri by adopting overlapping PCR to prepare an NprE-ps TreS fragment; the gene sequence of the fusion gene fragment of the NprE-ps TreS is shown in SEQ ID NO. 2;
(3) carrying out double enzyme digestion on the NprE-ps TreS fragment prepared in the step (2) by BamHI and XbaI, connecting the NprE-ps TreS fragment with pHT01 plasmid subjected to double enzyme digestion by BamHI and XbaI, and transferring the fragment into Escherichia coli DH5 alpha to prepare recombinant plasmid pHT01-NprE-ps TreS;
(4) transferring the recombinant plasmid prepared in the step (3) into bacillus subtilis by an electrotransformation method, and screening by a chloramphenicol plate to prepare the gene engineering bacteria for secreting and expressing trehalose synthase.
preferably, in step (1), the reaction system for PCR amplification of NprE signal peptide is as follows:
2 XPfu mix 25 uL, 10 umol/L upstream primer (NprE-F)1 uL, 10 umol/L downstream primer (NprE-R)1 uL, template 1 uL, make up 50 uL with ddH 2 O;
The PCR reaction procedure was as follows:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min.
According to the present invention, in the step (1), the reaction system for PCR amplification of the trehalose synthase sequence ps TreS is as follows:
2 XPfu mix 25 uL, 10 umol/L upstream primer (ps TreS-F)1 uL, 10 umol/L downstream primer (ps TreS-R)1 uL, template 1 uL, make up 50 uL with ddH 2 O;
The PCR reaction procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 4min, and 30 cycles; extension at 72 ℃ for 10 min.
Preferably, in step (2), the primary amplification system of the overlap PCR is 25 μ l:
trehalose synthase sequence ps TreS 4. mu.L, signal peptide fragment NprE 4. mu.L, 2 XPfu mix 12.5. mu.L, ddH 2 O4.5. mu.L;
The primary amplification procedure for the overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, and extension at 72 ℃ for 30s, for 5 cycles; extending for 10min at 72 ℃;
the complementary amplification system of the overlapping PCR is 25 mu L:
2 muL of upstream primer (NprE-F), 2 muL of downstream primer (ps TreS-R), 12.5 muL of 2 x Pfu mix, 8.5 muL of ddH 2 O;
the complementary amplification procedure for overlapping PCR is as follows:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 5min, 30 cycles; extension at 72 ℃ for 10 min.
Preferably, the bacillus subtilis in the step (4) is bacillus subtilis WB 800N. Bacillus subtilis WB800N was obtained from Hangzhou Bao Sai Bio Inc.
preferably, in step (4), the conditions for the electrical conversion are as follows:
Competent cells were electrotransformed for 5ms at 2100V.
the application of the gene engineering bacteria for secreting and expressing trehalose synthase in the preparation of trehalose synthase.
The application comprises the following steps:
Carrying out amplification culture on the gene engineering bacteria for secreting and expressing trehalose synthase in a TB culture medium until the growth logarithmic phase, adding IPTG (isopropyl-beta-D-thiogalactoside) into the TB culture medium to the final concentration of 1 mu M, inducing for 12h at 37 ℃, centrifuging, taking supernate, and concentrating protein to obtain the trehalose synthase.
Preferably according to the invention, the centrifugation conditions are 4 ℃, 10000rpm, 20 min.
preferably according to the invention, the protein is concentrated to a membrane concentration; more preferably, the membrane is concentrated with an ultrafiltration membrane having a pore size of 0.2 μm.
the TB culture medium is a culture medium which is conventional in the field and comprises the following components per liter:
Peptone 12g, yeast extract 24g, KH 2 PO 4 2.31.31 g, K 2 HPO 4 12.54.54 g, and glycerol 4 ml.
advantageous effects
1. according to the invention, a signal peptide fragment NprE is added into a recombinant vector for the first time, and the signal can secrete a target protein trehalose synthase to the outside of cells of recombinant engineering bacteria for expression, so that the subsequent steps of enzyme separation and purification are greatly simplified;
2. The engineering bacteria directly secrete exogenous protein into a culture medium, the target enzyme solution can be obtained without cell disruption, and the adopted bacillus subtilis is a food safety strain, has clear genetic background and perfect and accurate genetic operation system, can ensure that a final expression system is in a complete food-grade position, achieves the food safety-grade standard, and lays a foundation for the industrial production of downstream trehalose.
3. The invention adopts high-copy free recombinant plasmid to express trehalose synthase gene, which can improve the copy number of the trehalose synthase of target gene, thereby improving the expression quantity of exogenous gene and realizing the secretory expression of the trehalose synthase in bacillus subtilis.
drawings
FIG. 1 is a schematic diagram of pHT01-NprE-ps TreS vector construction;
FIG. 2a is an agarose gel electrophoresis of the NprE signal peptide;
FIG. 2b is an agarose gel electrophoresis image of the trehalose synthase sequence ps TreS from Pseudomonas stutzeri;
FIG. 3 is an agarose gel electrophoresis of the NprE-ps TreS fusion fragment;
FIG. 4 is an agarose gel electrophoresis of pHT01 plasmid after BamHI/Xba I double digestion;
Detailed Description
the technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.
the source of the biological material is as follows:
bacillus subtilis 168 was purchased from hangzhou baosu bio ltd;
bacillus subtilis WB800N was purchased from hangzhou baosui bio ltd;
Shuttle plasmid pHT01 was purchased from Hangzhou Bao Sai Bio Inc.;
pseudomonas stutzeri (Pseudomonas stutzeri) was purchased from China academy of sciences (CGMCC);
example 1: construction of recombinant plasmids
(1) Cloning to obtain signal peptide gene fragment
Using genome of Bacillus subtilis 168 as template, primers were designed for PCR amplification to obtain NprE signal peptide fragment.
The PCR primer sequences are as follows:
NprE-F：5’-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3’
NprE-R：5’-GAATGCTCATactagtAGCCTGAACACC-3’
The PCR reaction system is as follows:
2 XPfu mix 25 uL, 10 umol/L upstream primer (NprE-F)1 uL, 10 umol/L downstream primer (NprE-R)1 uL, template 1 uL, make up 50 uL with ddH 2 O;
The above PCR reaction was performed according to the following procedure:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min.
after the PCR is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a gel cutting product is recovered by using a Shanghai crude gel recovery kit.
(2) Cloning to obtain the trehalose synthase gene of Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3)
primers were designed based on the full-length nucleotide sequence of Pseudomonas stutzeri trehalose synthase disclosed at NCBI and the primer sequences were as follows:
ps TreS-F：5’-actagtATGAGCATTCCAGA-3’
ps TreS-R：5’-tctagaTTAGATCACCGGGGATGC-3’
The PCR amplification is carried out by using the genome of Pseudomonas stutzeri as a template and the primers, and the PCR reaction system is as follows:
2 XPfu mix 25 uL, 10 umol/L upstream primer (ps TreS-F)1 uL, 10 umol/L downstream primer (ps TreS-R)1 uL, template 1 uL, make up 50 uL with ddH 2 O;
The above PCR reaction was performed according to the following procedure:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 4min, and 30 cycles; extension at 72 ℃ for 10 min.
after the PCR is finished, the length of the fragment is analyzed through 1% agarose gel electrophoresis, a target band is cut according to the size of the fragment, and a gel cutting product is recovered by using a Shanghai crude gel recovery kit.
(3) performing overlapping PCR on the NprE signal peptide fragment prepared in the step (1) and the pseudomonas stutzeri trehalose synthase gene fragment prepared in the step (2) to prepare an NprE-ps TreS fragment;
The primary amplification system of the overlapping PCR is 25 mu l:
Trehalose synthase sequence ps TreS 4. mu.L, signal peptide fragment NprE 4. mu.L, 2 XPfu mix 12.5. mu.L, ddH 2 O4.5. mu.L;
the primary amplification procedure for the overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, and extension at 72 ℃ for 30s, for 5 cycles; extending for 10min at 72 ℃;
the complementary amplification system of the overlapping PCR is 25 mu L:
2 muL of upstream primer, 2 muL of downstream primer, 12.5 muL of 2 Xpfu mix and 8.5 muL of ddH 2 O;
The complementary amplification procedure for overlapping PCR is as follows:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 5min, 30 cycles; extension at 72 ℃ for 10 min. Storing at-20 deg.C;
(4) Connecting the NprE-ps TreS fragment prepared in the step (3) with pHT01 plasmid subjected to same double enzyme digestion through BamHI/XbaI double enzyme digestion, and transferring into Escherichia coli DH5 alpha; after successful identification and correct sequencing, the prepared recombinant vector is named pHT01-NprE-ps TreS.
the enzyme cutting system of the PCR product is as follows:
50 mu L of PCR product; 2.0 mu L of BamHI; xba I2.0. mu.L; 10 × buffer K6.0 μ L;
reaction conditions are as follows: react at 37 ℃ for 4 h.
the pHT01 plasmid enzyme cutting system is as follows:
50 μ L of pHT01 plasmid; 2.0 mu L of BamHI; xba I2.0. mu.L; 10 XBuffer K6.0 uL
reaction conditions are as follows: react at 37 ℃ for 4 h.
and (3) carrying out electrophoresis on the PCR product and the product obtained after double enzyme digestion of the vector by using 1% agarose gel, recovering by using DNA gel, and then carrying out gel recovery by using a DNA gel recovery kit.
the connecting body is as follows:
pHT01 enzyme digestion plasmid 5.0 μ L; 3.0 mu L of PCR product after enzyme digestion; 10 XT 4buffer 1.0 μ L; T4-DNA Ligase1.0. mu.L
Reaction conditions are as follows: reacting at 16 ℃ for 10 h.
The ligated NprE-ps TreS gene and pHT01 ligation product were transformed and introduced into E.coli DH 5. alpha. competent cells.
example 2: preparation of Bacillus subtilis WB800N electrotransformation competent cell
a single colony of Bacillus subtilis WB800N on the surface of a fresh LB solid medium was picked up and cultured overnight in 5ml of LB medium. 2.5mL of the overnight culture was inoculated into 40mL of a cell growth medium (LB +0.5M sorbitol), and the mixture was shake-cultured at 37 ℃ and 200rpm until the OD600 was 0.85 to 0.95. And (3) carrying out ice-water bath on the bacterial liquid for 10min, then centrifuging the bacterial liquid for 5min at 4 ℃ by 5000g, and collecting thalli. The cells were resuspended in 50mL of pre-chilled electroporation medium (0.5M sorbitol, 0.5M mannitol, 10% glucose), 5000g, centrifuged at 4 ℃ for 5min, the supernatant removed and rinsed 4 times. The washed cells were resuspended in 1ml of an electroporation medium and dispensed into EP tubes, each containing 60. mu.L of cells.
example 3: the recombinant plasmid is transferred into bacillus subtilis WB800N
50ng of plasmid DNA (1-8. mu.L) was added to 60. mu.l of competent cells, incubated on ice for 2min, added to a pre-cooled electric rotor (2mm), and subjected to electrotransformation at 2100V for 5 ms. After the completion of the electric shock, the cups were removed and 1mL of a cell recovery medium (LB +0.5M sorbitol +0.38M mannitol) was immediately added thereto, and the mixture was subjected to shaking recovery culture at 37 ℃ and 200rpm for 3 hours and then spread on a LB plate containing chloramphenicol. Incubate at 37 ℃ overnight. Screening strains resistant to chloramphenicol.
Example 4: culture and identification of positive recombinant bacteria
Inoculating the positive recombinant bacterial colony into an LB liquid culture medium (containing chloramphenicol) for culture overnight, sucking 1mL of bacterial liquid, extracting genome DNA by using a kit provided by Shanghai bioengineering Co., Ltd, and performing PCR amplification by using the obtained genome DNA as a template and ps TreS-F, ps TreS-R, NprE-F and NprE-R as primers respectively.
The primary amplification system of colony PCR is 20 μ L:
1 muL of upstream primer, 1 muL of downstream primer, 2 muL of template, 2 Xpfu mix 10 muL and ddH 2 O6 muL;
The primary amplification procedure for colony PCR is as follows:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 2min for 30s, 30 cycles; extending for 10min at 72 ℃;
agarose gel electrophoresis demonstrated that the exogenous fragments ps TreS and NprE signal peptides were both ligated to the pHT01 plasmid, respectively.
example 5: fermentation of positive recombinant bacteria
the recombinant bacterium pHT01-NprE-ps TreS constructed in the embodiment 4 is inoculated in 50mL LB liquid culture medium (containing chloramphenicol with the concentration of 25 mug/mL), cultured overnight at 37 ℃ at 200rpm, transferred in TB culture medium according to the inoculum size of 1% the next day, cultured for 7h until the logarithmic phase of growth, IPTG is added until the final concentration is 1 mug M, induced for 12h at 37 ℃, the fermentation liquid is taken and centrifuged for 20min at 4 ℃ and 10000rpm, and the supernatant is the crude extracellular enzyme liquid. The enzyme activity is measured.
The TB culture medium is a culture medium which is conventional in the field and comprises the following components per liter:
Peptone 12g, yeast extract 24g, KH 2 PO 4 2.31.31 g, K 2 HPO 4 12.54.54 g, and glycerol 4 ml.
the LB liquid culture medium comprises the following components per liter:
10g of tryptone, 5g of yeast extract, 10g of NaCl and pH 7.0.
example 6: detection of trehalose synthase enzyme Activity
to the reaction system were added 500. mu.L of a 60% maltose solution and 500. mu.L of a crude extracellular enzyme solution. The reaction was carried out at 37 ℃ for 12 h. Determining the conversion rate of trehalose by a high-pressure liquid phase method, wherein an amino column is adopted in the determination process; the column temperature is 40 ℃, the mobile phase adopts a mixed solution of acetonitrile and water, and the ratio of the acetonitrile to the water is 3: 1; the flow rate is 1 mL/min; the detector is a differential detector; the detection time is 25 min. The conversion was calculated according to the following formula.
and fitting a curve by using software according to the maltose peak area, the trehalose peak area and the glucose peak area in the high performance liquid chromatography result to calculate the mass of the three, wherein m3 is the mass converted into trehalose, m2 is the mass converted into glucose, and m1 is the mass of the residual maltose.
the result shows that the trehalose synthase gene engineering bacteria constructed by the invention can realize the secretory expression of the trehalose synthase in bacillus subtilis. The conversion rate of converting maltose in the crude extracellular enzyme solution into trehalose can reach 72 percent. Has wide industrial application prospect.
Comparative example 1
Trehalose synthase derived from Pseudomonas stutzeri was cloned into a shuttle plasmid pHT43 containing AmyQ signal peptide, and its intracellular expression in bacillus subtilis was successfully achieved but its extracellular expression was not achieved by inducing its expression with IPTG as an inducer.
comparative example 2
According to the disclosure in Chinese patent publication CN103215300A (application No. 201310174692.7), an integrated recombinant Bacillus subtilis was constructed by integrating a trehalose synthase expression element into the chromosome of Bacillus subtilis. The results showed that the conversion of total maltose to trehalose reached 63.1%.
the trehalose synthase gene is cloned into a shuttle plasmid pHT01, and a recombinant plasmid is transformed into bacillus subtilis WB800N by an electrotransformation method, wherein the plasmid can be stably inherited in bacillus subtilis, and the dosage of a target gene carried by the plasmid is correspondingly increased due to higher copy number of the plasmid, so that the trehalose synthase genetic engineering bacteria constructed by the invention can realize the secretory expression of trehalose synthase in bacillus subtilis, and the transformation rate of converting maltose into trehalose is up to 72%. Compared with the production of trehalose synthase by integrated recombinant bacillus subtilis, the method for producing trehalose synthase by the method is more advantageous.
Claims (10)
1. A construction method of a gene engineering bacterium for secretory expression of trehalose synthase is characterized by comprising the following steps:
(1) Taking the genome of the bacillus subtilis 168 as a template, and carrying out PCR amplification on an NprE signal peptide sequence in the Sec pathway, and taking the genome of pseudomonas stutzeri (Pseudomonas stutzeri) as a template to carry out PCR amplification on a trehalose synthase sequence ps TreS;
The PCR primer sequences for the NprE signal peptide sequence were as follows:
NprE-F：5’-AAAGGAGGAAggatccATGGGTTTAGGTAAGAA-3’；
NprE-R：5’-GAATGCTCATactagtAGCCTGAACACC-3’；
The sequence of the PCR primer of the trehalose synthase sequence ps TreS is as follows:
ps TreS-F：5’-actagtATGAGCATTCCAGA-3’；
ps TreS-R：5’-tctagaTTAGATCACCGGGGATGC-3’；
(2) fusing the signal peptide sequence NprE prepared in the step (1) with a trehalose synthase sequence ps TreS from pseudomonas stutzeri by adopting overlapping PCR to prepare an NprE-ps TreS fragment; the gene sequence of the fusion gene fragment of the NprE-ps TreS is shown as SEQID NO. 2;
(3) Carrying out double enzyme digestion on the NprE-ps TreS fragment prepared in the step (2) by BamHI and XbaI, connecting the NprE-ps TreS fragment with pHT01 plasmid subjected to double enzyme digestion by BamHI and XbaI, and transferring the fragment into Escherichia coli DH5 alpha to prepare recombinant plasmid pHT 01-NprE-psTreS; the nucleotide sequence of the recombinant plasmid pHT01-NprE-ps TreS is shown in SEQ ID NO. 1;
(4) Transferring the recombinant plasmid prepared in the step (3) into bacillus subtilis WB800N by an electrotransformation method, and screening by a chloramphenicol plate to prepare the gene engineering bacteria for secreting and expressing trehalose synthase.
2. the method of claim 1, wherein in step (1), the NprE signal peptide is PCR amplified in the following reaction system:
2 XPfu mix 25 uL, 10 umol/L upstream primer NprE-F1 uL, 10 umol/L downstream primer NprE-R1 uL, template 1 uL, make up 50 uL with ddH 2 O;
the PCR reaction procedure was as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 ℃ for 10 min.
3. The method according to claim 1, wherein in the step (1), the reaction system for PCR amplification of the trehalose synthase sequence ps TreS is as follows:
2 Xpfu mix 25. mu.L, 10. mu. mol/L upstream primer ps TreS-F1. mu.L, 10. mu. mol/L downstream primer ps TreS-R1. mu.L, template 1. mu.L, make up 50. mu.L with ddH 2 O;
the PCR reaction procedure was as follows:
Pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 4min, and 30 cycles; extension at 72 ℃ for 10 min.
4. The method of claim 1, wherein in the step (2), the primary amplification system of the overlap PCR is 25 μ l:
Trehalose synthase sequence ps TreS 4. mu.L, signal peptide fragment NprE 4. mu.L, 2 XPfu mix 12.5. mu.L, ddH 2 O4.5. mu.L;
The primary amplification procedure for the overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, and extension at 72 ℃ for 30s, for 5 cycles; extending for 10min at 72 ℃;
the complementary amplification system of the overlapping PCR is 25 mu L:
2 muL of upstream primer NprE-F, 2 muL of downstream primer ps TreS-R, 12.5 muL of 2 Xpfu mix and 8.5 muL of ddH 2 O;
the complementary amplification procedure for overlapping PCR is as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 5min, 30 cycles; extension at 72 ℃ for 10 min.
5. the constructing method according to claim 1, wherein in the step (4), the conditions for the electrical conversion are as follows:
Competent cells were electrotransformed for 5ms at 2100V.
6. the use of the engineered bacteria of secretion expression of trehalose synthase constructed in claim 1 in the preparation of trehalose synthase.
7. Use according to claim 6, characterized in that the steps are as follows:
Carrying out amplification culture on the gene engineering bacteria for secreting and expressing trehalose synthase in a TB culture medium until the growth logarithmic phase, adding IPTG (isopropyl-beta-D-thiogalactoside) into the TB culture medium to the final concentration of 1 mu M, inducing for 12h at 37 ℃, centrifuging, taking supernate, and concentrating protein to obtain the trehalose synthase.
8. The use of claim 7, wherein the centrifugation conditions are 4 ℃, 10000rpm, 20 min.
9. the use of claim 7, wherein the protein is concentrated to a membrane concentration.
10. the use of claim 9, wherein the membrane is concentrated using an ultrafiltration membrane having a pore size of 0.2 μm.
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