CN109402152B - Method for expression preparation of UDP-glucose-4-epimerase - Google Patents
Method for expression preparation of UDP-glucose-4-epimerase Download PDFInfo
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- CN109402152B CN109402152B CN201811331222.6A CN201811331222A CN109402152B CN 109402152 B CN109402152 B CN 109402152B CN 201811331222 A CN201811331222 A CN 201811331222A CN 109402152 B CN109402152 B CN 109402152B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
- C12Y501/03002—UDP-glucose 4-epimerase (5.1.3.2), i.e. UDP-galactose 4-epimerase
Abstract
The invention relates to a method for preparing UDP-glucose-4-epimerase by high-efficiency expression, which is to construct a recombinant expression vector by UDP-glucose-4-epimerase (GalE) gene and a vector for bacillus subtilis. And then transforming the recombinant expression vector into bacillus subtilis to construct recombinant engineering bacteria. Inducing and culturing the recombinant engineering bacteria in a liquid culture medium, centrifuging the bacteria liquid, and taking supernatant. The method has the advantages of high UDP-glucose-4-epimerase yield, purer protein, easy recovery and purification, simple production operation, convenience for industrial large-scale production of GalE, improvement of yield, time and labor conservation and cost conservation. Especially provides safety guarantee for the application of the enzyme in food industry, and has great significance.
Description
Technical Field
The invention relates to a recombinant engineering bacterium capable of efficiently expressing UDP-glucose-4-epimerase and a method for preparing UDP-glucose-4-epimerase by improved efficient expression, belonging to the technical field of genetic engineering.
Background
UDP-glucose-4-epimerase (GalE), a key enzyme involved in galactose metabolism in organisms, is commonly involved in galactose metabolism together with galactokinase (galctokinase), 1-phosphogalactose uridyltransferase (galac-tose-1-p-uridyltransferase), a process known as the Leloir pathway. UDP-glucose 4-epimerase catalyzes galactose metabolism as the last and critical step in Leloir, and is accomplished by the conversion between UDP-galactose and UDP-glucose, coupling glycolysis and the TCA cycle. In addition, the bacillus subtilis is considered as probiotic bacteria in animal intestinal tracts, has high safety, can be applied to industries such as food, medicines and the like, and has non-pathogenicity; the exogenous DNA is convenient to obtain, the plasmid can be used as a cloning vector of a heterologous gene, and the phage can also be applied to the cloning vector, so that the exogenous DNA has strong hereditary property; the secretion system of the protein is complete, the secreted exogenous protein can cross over a cell membrane and be directly released outside the cell, and the protein has strong protein secretion capacity, so that the recovery and purification processes of the protein are simplified, and the protein is suitable for industrial production; the strain can reach high strain density by culturing in a simpler culture medium, and is suitable for industrial production. However, in the prior art, the yield of UDP-glucose 4-epimerase extracted from wild bacteria is low, separation and refining are difficult, and the method is not suitable for large-batch preparation. UDP-glucose-4-epimerase is expressed by escherichia coli, but is expressed intracellularly in the escherichia coli, inclusion bodies are easily formed, meanwhile, the acquisition of enzyme protein needs to crush cells, the content of impure protein is high, the separation and the refining are difficult, the operation is complicated and time-consuming, and the protein expression amount is not high.
Disclosure of Invention
In order to solve the technical problems, the invention aims to construct a recombinant expression vector and a recombinant engineering bacterium for efficiently expressing UDP-glucose-4-epimerase by adopting a genetic engineering technology.
The invention also aims to provide a method for producing UDP-glucose-4-epimerase by utilizing recombinant engineering bacteria, which has high yield, relatively pure protein, easy recovery and purification and simple production operation.
The technical scheme adopted by the invention is as follows: a recombinant expression vector for efficiently expressing UDP-glucose-4-epimerase is a recombinant expression vector constructed by connecting UDP-glucose-4-epimerase (GalE) genes with an expression vector; the expression vector is a vector for bacillus subtilis or a shuttle vector for escherichia coli-bacillus subtilis.
Furthermore, the recombinant expression vector for efficiently expressing UDP-glucose-4-epimerase is a pHT series vector, a shuttle vector pMA5 or a shuttle vector pWB980 and the like. But are not limited to these vectors, and all vectors for Bacillus subtilis or shuttle vectors for Escherichia coli-Bacillus subtilis can be used.
Furthermore, the above recombinant expression vector for efficiently expressing UDP-glucose-4-epimerase is pHT43, pHT304 or pHT 01.
Furthermore, the recombinant expression vector for efficiently expressing the UDP-glucose-4-epimerase contains or does not contain a protein purification tag.
Furthermore, in the recombinant expression vector for efficiently expressing UDP-glucose-4-epimerase, the protein purification tag is a histidine (His-tag) tag.
A recombinant engineering bacterium for efficiently expressing UDP-glucose-4-epimerase is a recombinant engineering bacterium constructed by transforming the recombinant expression vector for efficiently expressing UDP-glucose-4-epimerase into a host bacterium; the host bacterium is bacillus subtilis.
Further, the recombinant engineering bacterium for efficiently expressing the UDP-glucose-4-epimerase is converted into electricity.
A method for preparing UDP-glucose-4-epimerase by high-efficiency expression. The method comprises the following steps: inoculating the recombinant engineering bacteria for efficiently expressing UDP-glucose-4-epimerase into a liquid culture medium at an inoculation amount of 1-10%, and culturing at 60-200rpm to OD600When the concentration is 0.3-1.0, IPTG is added to the final concentration of 0.1-1.5mM, then induction culture is carried out for 9-36 hours under the conditions of 20-40 ℃ and 80-220rpm, the bacterial liquid is centrifuged, and the supernatant is taken to recover protein.
Further, the liquid culture medium is LB liquid culture medium containing antibiotics.
The invention has the beneficial effects that:
1. the expression strain used in the present invention is Bacillus subtilis, which is certified as GRAS (general Recognized as safe) and can be safely used in food industry. It has high application value, simple and fast culture, powerful protein secreting capacity, no pathogenicity, excellent fermentation foundation and excellent production technology. GalE is an extracellular secretion protein in the expression system, and the protein is relatively pure, easy to recover and purify, simple in production operation and time-saving.
2. The invention adopts the genetic engineering technology to connect UDP-glucose-4-epimerase (GalE) gene with an expression vector to construct recombinant expression plasmid. Then, the recombinant strain is transformed into a host bacterium of bacillus subtilis to construct a recombinant engineering bacterium for efficiently expressing GalE. After the engineering bacteria are cultured in a liquid culture medium, the bacterial liquid is centrifuged, and the supernatant is the enzyme liquid containing GalE. The yield and the production efficiency are obviously higher than the expression level of the homologous gene in wild strains, and the method is simple and easy to operate and low in cost.
3. The invention provides convenience for the industrial large-scale production of GalE, improves the yield, saves time and labor and saves the cost. Especially provides safety guarantee for the application of the enzyme in food industry, and has great significance.
Drawings
FIG. 1 is a schematic diagram of the construction of the recombinant expression vector pHT43-GalE of example 1.
FIG. 2 shows the results of double digestion in example 1.
FIG. 3 is an SDS-PAGE electrophoresis of GalE constructed in example 1; m is Marker; 1: crude enzyme solution.
FIG. 4 is an SDS-PAGE electrophoresis of the GalE purified constructed in example 1; m is Marker; 1: enzyme solution.
Detailed Description
Example 1
(I) construction of recombinant expression vector pHT43-GalE
UDP-glucose-4-epimerase (GalE) gene (shown in SEQ ID NO: 1) is derived from Bifidobacterium longum JCM 1217, is connected with a cloning vector pMD19 after PCR amplification and purification, and constructs a recombinant plasmid pMD 19-GalE.
The recombinant plasmid pMD19-GalE and the expression vector pHT43 are subjected to double enzyme digestion by taking XbaI and SmaI as enzyme digestion sites respectively, and are connected at 16 ℃ overnight to obtain the recombinant expression vector pHT 43-GalE.
Transforming the expression vector pHT43-GalE into Bacillus subtilis WB800N, coating an LB plate containing chloramphenicol (5ug/mL) resistance, culturing overnight at 37 ℃, picking a transformant, extracting a recombinant plasmid and carrying out double enzyme digestion verification. As shown in FIG. 2, two fragments are present after enzyme digestion, and the size is respectively about 8000bp (expression vector pHT43) and 6611bp (UDP-glucose-4-epimerase), namely the connection is successful.
(II) recombinant engineering bacteria
The constructed recombinant expression vector pHT43-GalE is transformed by adopting an electric shock transformation method, bacillus subtilis WB800N competent cells are mixed with the recombinant expression vector pHT43-GalE plasmid, and the mixture is added into an electric shock cup for ice bath (1-10min) and then is subjected to electric shock under the conditions of electric shock: 22KV/cm, and obtaining the recombinant engineering bacteria containing the recombinant expression vector pHT 43-GalE.
Taking recombinant engineering bacteria containing a recombinant expression vector pHT43-GalE, adding 1ml of electric shock recovery liquid, incubating at 37 ℃ and 100rpm for 3h, coating the electric shock recovery liquid on a chloramphenicol solid basal medium LB plate, and observing positive colonies within 12-24 h.
Preparation of (tri) GalE and condition optimization
The method comprises the following steps: inoculating the recombinant engineering bacteria containing the recombinant expression vector into LB liquid culture medium containing chloramphenicol in an inoculation amount of 1%, culturing at 180rpm until the initial bacteria density OD600 is 0.8, adding IPTG to the final concentration of 0.1-1.5mM, inducing and culturing at 20-40 ℃ and 120rpm for 9-36 hours, centrifuging the bacterial liquid, and obtaining the supernatant which is the enzyme liquid containing GalE. The factors were set as in table 1 and the orthogonality test was performed as in table 2.
TABLE 1
| Level of | A (Induction time h) | B (induction temperature ℃) | C (IPTG concentration mM) |
| 1 | 9 | 30 | 0.5 |
| 2 | 18 | 35 | 1 |
| 3 | 27 | 40 | 1.5 |
TABLE 2
According to the results of tables 1 and 2, the factor primary and secondary sequence is A, B, C, and the optimal process scheme is A2B2C2. Further to A2B2C2The verification result shows that the activity of GalE reaches 295U/mL. As can be seen, the optimum process conditions were IPTG addition to a final concentration of 1mM, an induction temperature of 35 ℃ and an induction culture time of 18 hours.
(IV) comparative example
The method is the same as the steps (I) and (III), and is different from the method in that the host bacterium bacillus subtilis WB800N in the step (II) is changed into escherichia coli, and the enzyme is produced by fermentation under the optimal process conditions (OD600 is 0.8, IPTG is 1mM to the final concentration, the induction temperature is 35 ℃, and the induction culture is carried out for 18 hours). The results are shown in Table 3
TABLE 3
| Total protein | Large intestine | Subtilis spike |
| GalE | 55mg | 113mg |
As can be seen from Table 3, the yield of GalE was only 55mg using E.coli as the host strain, whereas the yield of GalE was as high as 113mg using B.subtilis. Therefore, the method of the invention can highly express UDP-glucose-4-epimerase.
Example 2
(I) construction of recombinant expression vector pHT43-GalE
The recombinant plasmid pMD19-GalE prepared in example 1 and the expression vector pHT43 were double-digested with XbaI and SmaI as the cleavage sites, respectively, and ligated at 16 ℃ overnight to obtain a recombinant expression vector pHT 43-GalE.
The recombinant expression vector pHT43-GalE is transformed into Bacillus subtilis WB800, an LB plate containing chloramphenicol (5ug/mL) resistance is coated, the mixture is cultured overnight at 37 ℃, a transformant is picked, and a recombinant plasmid is extracted and verified by double enzyme digestion. As shown in FIG. 2, two fragments are present after enzyme digestion, and the size is respectively about 8000bp (expression vector pHT43) and 6611bp (UDP-glucose-4-epimerase), namely the connection is successful.
(II) recombinant engineering bacteria
Adopting an electric shock transformation method for the constructed recombinant expression vector pHT43-GalE, mixing 60ul of bacillus subtilis WB800 electrotransformation competent cells with 1ul (50ng/ul) of recombinant expression vector pHT43-GalE plasmids, adding into an electric shock cup, carrying out electric shock after 5min of ice bath, and carrying out electric shock under the electric shock condition: 22KV/cm, and obtaining the recombinant engineering bacteria containing the recombinant expression vector pHT 43-GalE.
Taking recombinant engineering bacteria containing a recombinant expression vector pHT43-GalE, adding 1ml of electric shock recovery liquid, incubating at 37 ℃ and 100rpm for 3h, coating the electric shock recovery liquid on a chloramphenicol solid basal medium LB plate, and observing positive colonies within 12-24 h.
Preparation of (tri) GalE
The method comprises the following steps: the recombinant engineering bacteria containing the recombinant expression vector pHT43-GalE are inoculated in LB liquid culture medium containing chloramphenicol in an inoculation amount of 5%, cultured at 180rpm until the initial bacteria density OD600 is 0.8, IPTG is added to the final concentration of 1mM, induced and cultured for 18 hours at 35 ℃ and 120rpm, the bacteria liquid is centrifuged, the supernatant is the enzyme liquid containing GalE, and the enzyme activity is 290U/mL.
Taking the enzyme solution for PCR verification. The SDS-PAGE electrophoresis of the enzyme solution is shown in FIG. 3, and the purified results are shown in FIG. 4, and the target 37kDa GalE band appears.
Example 3:
(I) construction of recombinant expression vector pMA5-GalE
The recombinant plasmid pMD19-GalE prepared in example 1 and the shuttle vector pMA5 were double-digested with XbaI and SmaI as cleavage sites, respectively, and ligated at 16 ℃ overnight to obtain the recombinant expression vector pMA 5-GalE.
The recombinant expression vector pMA5-GalE is transformed into bacillus subtilis 168, an LB plate containing ampicillin (100ug/mL) resistance is coated, the culture is carried out overnight at 37 ℃, a transformant is selected, a recombinant plasmid is extracted and double enzyme digestion verification is carried out, and the verified construction is successful.
(II) recombinant engineering bacteria
The constructed recombinant expression vector pMA5-GalE adopts a shock transformation method, bacillus subtilis 168 electrotransformation competent cells are mixed with the recombinant expression vector pMA5-GalE plasmid, and the mixture is added into a shock cup to be subjected to electric shock after ice bath for 5min under the conditions of electric shock: 22KV/cm, and obtaining the recombinant engineering bacteria containing the recombinant expression vector pMA 5-GalE.
The recombinant engineering bacteria containing the recombinant expression vector pMA5-GalE are taken, added with 1ml of electric shock recovery liquid, incubated for 3h at 37 ℃ and 100rpm, and coated on an ampicillin solid basal medium LB plate, and positive colonies can be seen within 12-24 h.
Preparation of (tri) GalE
The method comprises the following steps: the recombinant engineering bacteria containing the recombinant expression vector pMA5-GalE are inoculated into LB liquid culture medium containing ampicillin in an inoculation amount of 10 percent, when the culture is carried out at 180rpm until the initial bacteria density OD600 is 0.8, IPTG is added until the final concentration is 1mM, the induction culture is carried out for 18 hours at 35 ℃ and 120rpm, the bacteria liquid is centrifuged, the supernatant is the enzyme liquid containing the GalE, and the enzyme activity is 293U/mL.
Example 4
(I) construction of recombinant expression vector pHT43-GalE
The recombinant plasmid pMD19-GalE prepared in example 1 and the expression vector pHT43 were double-digested with XbaI and SmaI as the cleavage sites, respectively, and ligated at 16 ℃ overnight to obtain a recombinant expression vector pHT 43-GalE.
Transforming the recombinant expression vector pHT43-GalE into Bacillus subtilis WB600, coating an LB plate containing chloramphenicol (5ug/mL) resistance, culturing overnight at 37 ℃, selecting a transformant, extracting a recombinant plasmid, performing double enzyme digestion verification, and successfully connecting by verification.
(II) recombinant engineering bacteria
Adopting an electric shock transformation method for the constructed recombinant expression vector pHT43-GalE, mixing 60ul of bacillus subtilis WB600 electrotransformation competent cells with 1ul (50ng/ul) of recombinant expression vector pHT43-GalE plasmids, adding into an electric shock cup, carrying out electric shock after 5min of ice bath, and carrying out electric shock under the electric shock condition: 22KV/cm, and obtaining the recombinant engineering bacteria containing the recombinant expression vector pHT 43-GalE.
Taking recombinant engineering bacteria containing a recombinant expression vector pHT43-GalE, adding 1ml of electric shock recovery liquid, incubating at 37 ℃ and 100rpm for 3h, coating the electric shock recovery liquid on a chloramphenicol solid basal medium LB plate, and observing positive colonies within 12-24 h.
Preparation of (tri) GalE
The method comprises the following steps: the recombinant engineering bacteria containing the recombinant expression vector pHT43-GalE are inoculated in LB liquid culture medium containing chloramphenicol in an inoculation amount of 7%, cultured at 180rpm until the initial bacteria density OD600 is 0.8, IPTG is added to the final concentration of 1mM, induced and cultured for 18 hours at 35 ℃ and 120rpm, the bacteria liquid is centrifuged, the supernatant is the enzyme liquid containing GalE, and the enzyme activity is 291U/mL.
Example 5
(I) construction of recombinant expression vector pWB980-GalE
The recombinant plasmid pMD19-GalE prepared in example 1 and shuttle vector pWB980 were double-digested with XbaI and SmaI as cleavage sites, respectively, and ligated overnight at 16 ℃ to obtain recombinant expression vector pWB 980-GalE.
Transforming the recombinant expression vector pWB980-GalE into Bacillus subtilis WB800N, coating an LB plate containing kanamycin resistance (50ug/mL), culturing overnight at 37 ℃, selecting a transformant, extracting a recombinant plasmid, performing double enzyme digestion verification, and successfully connecting by verification.
(II) recombinant engineering bacteria
Adopting an electric shock transformation method for the constructed recombinant expression vector pWB980-GalE, mixing bacillus subtilis WB600 electrotransformation competent cells and the recombinant expression vector pWB980-GalE plasmid, adding the mixture into an electric shock cup, carrying out ice bath for 5min, and then carrying out electric shock under the electric shock conditions: 22KV/cm, and obtaining the recombinant engineering bacteria containing the recombinant expression vector pWB 980-GalE.
Taking recombinant engineering bacteria containing a recombinant expression vector pWB980-GalE, adding 1ml of electric shock recovery liquid, incubating at 37 ℃ and 100rpm for 3h, coating on a kanamycin solid basal medium LB plate, and observing positive bacterial colonies within 12-24 h.
Preparation of (tri) GalE
The method comprises the following steps: inoculating recombinant engineering bacteria containing a recombinant expression vector pWB980-GalE into an LB liquid culture medium containing kanamycin in an inoculation amount of 3%, culturing at 180rpm until the initial bacteria density OD600 is 0.8, performing induction culture at 35 ℃ and 120rpm for 18 hours, centrifuging a bacterial liquid, and obtaining a supernatant, namely an enzyme liquid containing GalE, wherein the enzyme activity is 290U/mL.
Sequence listing
<110> Shenyang agriculture university
<120> method for preparing UDP-glucose-4-epimerase by high-efficiency expression
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6611
<212> DNA
<213> UDP-glucose-4-epimerase gene (UDP-glucose 4-epimerase, GalEAB303839)
<400> 1
aggccggcct cgccgctggt gcggtcaagg gctgacgcgt taagcggaca gcccggtggg 60
ctgtctgtag cgtcggcccg agcgaggcgg tagccgagcg aaggcagcat tgagtctcgc 120
cgtaggcgag tccttaattg caggggccga aggcgacgcg ttaagcggac agcctaagac 180
caataattcc cgccgtccgg tgcctcgtgc tactccttcc ggcgccggac ggcttctcaa 240
caataagtag cacgcaacca taccgatcca gagccttcct gattcttgtc atgagattgt 300
cggtgaaact gaccgggaag ctccggtgat gttcaattcg ctcttccggc gttgccgtgg 360
ggctactctc aaggatttat acagtcagga tcctgggacg cccgacggtt gtgtcggatt 420
cttgttttat atgcatatat tgtttacaat ttaattatcc aaaaaaataa tcaatgtcct 480
caaaggagag actatgacta gcaccggccg cttcaccttg cctagcgagg agaattttgc 540
ggagaagacc aaggagcttg ccgaactgtg gggagcagac gccattcgta actccgatgg 600
cacgcatctg gatgaggctg tgcttgcctt gggcaagaag atttacaatg cgtatttccc 660
gactcgcgcg cataacgagt ggattacgct gcatatggat gagacgccac aggtgtatct 720
gctgaccgat cgcattctcg ccgaatcgga tacggttgat attccgctga tggaaagctt 780
cttcgctgag cagctgaagc cgaatcgtga tgccgaccct cacaagtatt gggaggttgt 840
agaccgcacc accggcgagg tcgttgattc ggcgaactgg acgcttgacg cggacgagga 900
caccgtgcat gtgagcggcg ttgccgcatg gcatgagtac acggtgagtt tcctggccta 960
catcatctgg gacccggtcg agatgtacaa ccatctgacc aacgactggg gcgacaagga 1020
acacgagatc ccgttcgaca tctaccatcc ggccacccgc aagttcgtgt tcgacacgtt 1080
cgagcagtgg cttaaggaca gcccgcagac cgatgtggtg cgattcacca cgttcttcta 1140
ccagttcacg ctcctgttcg atgaaaagcg ccgcgaaaag gtcgtcgact ggtttggttg 1200
cgcctgcacc gtaagcccgc gtgcgctgga tgatttcgaa gccaagtatg gctaccgcct 1260
gcgcccggag gacttcgtgg acggcggtgc ctacaactcc gcatggcgcg tgcccagaaa 1320
agcccagcgc gactggatcg acttcctctc cggcttcgtg cgcgaaaacg tcaagcagct 1380
cgccgacatg tcccacgccg ccggcaaaga ggcgatgatg ttcctcggcg accagtggat 1440
cggcaccgaa ccgtacaagg acggattcga cgaactcggc ctggacgccg tggtcggttc 1500
catcggcgac ggcaccacca cccgcatgat cgccgatatt ccgggcgtca agtacaccga 1560
aggccgcttc ctgccgtact tcttccccga cacgttctac gagggcaacg atccgagcat 1620
cgaaggcctc gacaactggc gcaaggcccg ccgcgcgatc ctgcgctcgc cgatcagccg 1680
catgggctac ggcggctatc tctccctggc cgccaagttc ccgaagttcg tggacacggt 1740
gacccatatc gccaacgaat tccgcgacat ccacgaccgc accggtgggg tggccgccga 1800
aggtgagctc aacgtggcga ttctgaattc ctggggcaag atgcgctcct ggatggcgtt 1860
caccgtggca cacgccctgc ccaacaagca gacctactcc tactacggca ttctggaatc 1920
cctgtcgggc atgcgcgtga acgtgcggtt catcagcttc gacgacgtac tcgcgcatgg 1980
catcgatagc gatatcgacg taatcatcaa cggcggcccg gtcgacaccg cattcaccgg 2040
cggcgatgtg tggaccaacc cgaagctcgt ggaaaccgtg cgcgcctggg tgcgtggcgg 2100
cggcgcgttc gtgggcgtgg gcgagccgag ctcggcaccg cgcttccaaa cgggccggtt 2160
cttccagctg gccgacgtga ttggcgtgga cgaggagcgc tatcagaccc tttcggtgga 2220
caagtatttc ccgccggtgg tgccggacca cttcatcacc gccgatgtgc cggtcgaccc 2280
ggcggcccgc gaggcttggg agcaggccgg ctaccgcatc ccgctgagcg gctgcggcgg 2340
cggccagtcc atcaagccgt tgggcggcat cgatttcggc gaacccgtgc tcaacacgta 2400
tccggtcaat gaaaacgtga ccctgctgcg cgcggacggc ggccaggtac agctcgcgac 2460
caacgattat ggcaagggcc gcggcgtgta catctccggc ctgccctatt cggcggccaa 2520
cgcccgactg ctggaacgcg tgctgttcta cgcttcccac aacgaagaca agtatgcggc 2580
ctggagctcg tccaatccgg aatgcgaagt ggcacacttc cccgagcaag ggctgtattg 2640
cgtaatcaac aacaccgatc agccgcagaa gaccacggtg actttggccg acggcaccac 2700
cgaggacttc gacctgcctg acagcggtat cgcctggcgt gaagcctgat cccacgtttt 2760
ggagaacgga cccatcatga ccgaaagcaa tgaagtttta ttcggcatcg cctcgcattt 2820
tgcgctggaa ggtgccgtga ccggtatcga accttacgga gacggccaca tcaacaccac 2880
ctatctggtg accacggacg gcccccgcta catcctccag cagatgaaca ccagcatctt 2940
ccccgatacg gtgaatctga tgcgcaatgt cgaactggtc acctccactc tcaaggctca 3000
gggcaaagag acgctggaca ttgtgcccac cacctcaggc gccacctggg ccgagatcga 3060
tggcggcgca tggcgcgtct acaagttcat cgaacacacc gtgtcctaca acctcgtgcc 3120
gaacccggac gtgttccgcg aagccggcag cgcattcggc gacttccaga acttcctgtc 3180
cgaattcgac gccagccagc tgaccgaaac catcgcccac ttccacgaca ccccgcatcg 3240
tttcgaggac ttcaaggccg ccctcgccgc ggacaagctc ggccgcgccg ccgcatgcca 3300
gccggaaatc gacttctatc tgagtcacgc cgaccagtat gccgtcgtga tggatgggct 3360
cagggacggt tcgattccgc tgcgcgtgac ccacaatgac accaagctca acaacatcct 3420
catggacgcc accaccggca aggcgcgtgc gatcatcgat ctcgacacca tcatgcccgg 3480
ctccatgctg ttcgacttcg gcgattccat acgctttggt gcgtccactg ctctggaaga 3540
cgaaaaggac ctcagcaagg tgcatttcag caccgagctg ttccgcgcct acacggaagg 3600
cttcgtgggc gaactacgcg gcagcatcac cgcgcgcgag gccgaactgc tgccgttcag 3660
cggcaacctg ctcaccatgg aatgcggcat gcgctttctc gccgactact tggaaggcga 3720
tatctacttt gccaccaagt accccgagca taatctggtg cgcacccgca cccagatcaa 3780
actcgtgcag gagatggagc agaaggccag cgaaacccgc gccatcgtag ccgacatcat 3840
ggaggctgcc aggtgagccg tcagccgaac catacccgaa ccagcatgat ggaggcagag 3900
caatgaacga tcagctgacc gaggtatacg ccagcatcga cgccctgatc gattatgcct 3960
tggcccatct tgaccttgac ccacgcaacg cagactggac ccgcaaccaa atcttcgccc 4020
tcttccgcct agattcctac cccggaccca aaaccacaac gtcggcggcg tcagtgtcag 4080
acgtagtcca agacattgtt ggcagccgaa gccaggcccc ctatggcgaa aagacccccg 4140
accccctgct cgccgccttc cgcgccgccg ccacaaccgc cggccttttc aagccggagg 4200
aaggcccggc ctacgccgac accatcatgg gcatcctctc cgccaacccc gctgatctcg 4260
acgaccgatt cctgctcgtc gagcaccggg atggcggcat ggccgccatg caatggttct 4320
acgactactg tgtggccaac aattatgtca agcgtgccca gctcgaccgg aacccgcgct 4380
tcgactcgca cgggctgacg gtgaccatca acctcgccaa acccgaattc aagaacatga 4440
agaaggcggc ggccggcaac gccgtggccg gcggctaccc gaagtgcacg atctgccatg 4500
agaacgaggg cttcgccgga cgtgacaagc gcacgctgcg caccttgccg gtcacgctcg 4560
gcggcgagtc atggttctgg cagttctcgc cctacggcta cttcgaccag cacggcatct 4620
gcgtgaacac cgaccacacg ccgatgcatg tggaccgcga caccttcggc catctgctcg 4680
acttcgtgga ccgtttcccc ggatacttcc tcggatgcaa cgccgcgctg cctcgcatcg 4740
gcggctcggt gctcgcccat gaccattacc agggcggcgg cgaactgctg ccgatgcaca 4800
aggccgccac ttgggccgct ttcacacttg ccgactatcc ggacgccgtg gttgaaatcc 4860
ttgactggcc cggcaccgcc gtgcgcgtgg tgtcgaagag tcgccagagc atcatcgacg 4920
tatccgacat catccgcgag gcatgggtcg gttatgacga cgcggcgaac ggcatcgcca 4980
gccatgacgc ggatggcaac cgccaatccg ccctctcgcc cagcgcgatc atcaccgaac 5040
gcggctatga gatgagtctc atcttccgca acaatgcgat cagcgacgag tacccggaag 5100
gtattttcca tgcacaccca gaatactggc cggtcaagca ggagccgatc ggactgatcg 5160
aggcccaagg cttgttcatt ctgcccggac ggctcgtcga ccagctcggc attgtcgaag 5220
aggcgctcgc cgaaggccgc gatttgcccg atgaagtcag cgaattcagt ctcgaatggg 5280
gcgaactcgc tgaaacgttg gccggaaacc acaaccgcga ggccatccgc caagccgtac 5340
acgacgagct cggcagcgtc tgctaccgca tcctcggcaa cacggccgtc ttcaagcaga 5400
aagccaccac gcaaacattc ctcgaaagcc ttggttttgc cgctaggtga aacaggtcaa 5460
gtgaagtaag ctcaatactg aaggaaaaac cgaaggcaag tccttccaaa ggagggaata 5520
tgactactgt tctggttacg ggcggtgctg gattcatcgc cactcacacc gacatcgaac 5580
tgctcaacaa gggctacgac gttatttccg tagataacta cggcaactcg tcgcccgtgg 5640
cgcttgagcg cgtcgagcaa atcaccggca agccggtcaa gcgttatgac ggcgacgtgc 5700
gcgatgaggc gctcatggaa cgcgtcttcg ctgaaaacaa catcgactgg gtcatccact 5760
ttgccggtct caaggccgtg ggcgagtccg tggccaagcc gatcgaatac tacgacaaca 5820
acctgtactc cacgcttgtg ctgctcaagg tgatgaagaa gcacaacgtc aagaagatca 5880
tcttctcatc ctccgccacc gtgtacggca cgccgaagga actgccgatc accgaggaga 5940
cgccgaccgg cggcaccacc aacccgtacg gcacctccaa gctgttccag gagcagattc 6000
tgcgcgacgt gcatgtggcc gatccgtcct ggaccatcgt gctgctgcgc tacttcaacc 6060
cggtcggcgc gcacgagtcc ggcctgctgg gcgaagaccc gaagggtatt ccggcgaacc 6120
tcaccccgta cgtggccaag gtcgcggtcg gcgagctcaa ggaagtccag gtctacggcg 6180
acgactacga cacgcccgac ggcactggtg tgcgtgacta catccacgtg gtcgacctgg 6240
ccaagggcca cgtggccgtc attgaccaca tcgacaagga aggcgtgttc gtctacaacc 6300
tgggtactgg ccacggctac tccgtgcttg aggttatcaa ggcttacgag aaggccgccg 6360
gtcatccgat tccgtacgcg atcaagccgc gtcgccccgg tgacatcgcc gcctgctacg 6420
ccgacgcttc caaggcggag aaggagcttg gctggaaggc cgagctgacc atcgacgaca 6480
tggccgcctc ctccctcaac tggcagacca agaaccccaa cggtttccgc gacgcggagt 6540
gatatgagca gatgagctcc ctctgatgag ggagctcatc tgtcgtttat cacacaatgc 6600
gcctatcggc g 6611
Claims (4)
1. A method for expressing and preparing UDP-glucose-4-epimerase is characterized by comprising the following steps:
1) connecting the UDP-glucose-4-epimerase gene with an expression vector to construct a recombinant expression vector; the expression vector is pHT series vector, shuttle vector pMA5 or shuttle vector pWB 980; the sequence of the UDP-glucose-4-epimerase gene is shown as SEQ ID NO 1;
2) electrically transforming the recombinant expression vector into host bacteria to construct recombinant engineering bacteria; the host bacterium is Bacillus subtilis (B.) (bacillus subtilis ) WB800N, Bacillus subtilis (B.subtilis)bacillus subtilis ） WB800. Bacillus subtilis (A), (B) and (C)bacillus subtilis ) 168, Bacillus subtilis (B.) (bacillus subtilis ） WB600；
3) Inoculating recombinant engineering bacteria for efficiently expressing UDP-glucose-4-epimerase into a liquid culture medium at an inoculation amount of 1-10%, and culturing at 180rpm to OD600If =0.8, adding IPTG to the final concentration of 1mM, then carrying out induction culture for 18 hours at 35 ℃ and 120rpm, centrifuging the bacterial liquid, and taking the supernatant; the liquid culture medium is LB liquid culture medium containing antibiotics.
2. The method of claim 1, wherein the pHT series vector is pHT43, pHT304 or pHT01 vector.
3. The method of claim 1, wherein the expression vector comprises a protein purification tag.
4. The method of claim 3, wherein the protein purification tag is a histidine (His-tag) tag.
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| CN109295087B (en) * | 2018-11-09 | 2021-08-24 | 沈阳农业大学 | Method for expression preparation of UDP-glucose-hexose-1-phosphate uridyltransferase |
| CN111172174B (en) * | 2020-03-06 | 2022-02-15 | 沈阳农业大学 | Application of OsUGE3 gene in improving rice traits |
| CN112322616B (en) * | 2020-11-25 | 2022-04-05 | 上海市农业科学院 | Expression and purification method of UDP-glucose-4-epimerase from hericium erinaceus |
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