CN108048439B - Preparation method and application of mutant trehalose synthase - Google Patents
Preparation method and application of mutant trehalose synthase Download PDFInfo
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Abstract
The invention relates to a preparation method and application of mutant trehalose synthase. The nucleotide sequence of the expression gene of the trehalose synthase mutant V407M is shown in SEQ ID NO. 2; the nucleotide sequence of the expression gene of the trehalose synthase mutant K490L is shown in SEQ ID NO. 3; the nucleotide sequence of the expression gene of the trehalose synthase mutant V407M/K490L is shown in SEQ ID NO. 4. The mutant is simple to prepare, high in yield and high in purity, the trehalose yield is improved, and under the same condition, the conversion rates of trehalose produced by the mutants V407M, K490L and V407M/K490L reach 68%, 65% and 70% respectively, and are obviously improved compared with 50% of original enzyme.
Description
Technical Field
The invention relates to a preparation method and application of mutant trehalose synthase, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Trehalose is a non-reducing disaccharide naturally occurring in nature, is an isomer of maltose, and is composed of two molecules of glucose linked by α, α -1, 1-glycosidic bonds. Trehalose is found in bacteria, fungi, insects and some invertebrates and has its primary function as an energy storage material and a constituent of cell walls. With the continuous and deep research, trehalose is widely applied in the fields of food, medicine, cosmetics and agriculture.
With the continuous exploration of trehalose application, the functions and effects of trehalose are recognized, which leads to the increasing market demand of trehalose, and the market price of trehalose is still higher than that of other disaccharides such as sucrose and maltose at present, but because of the complexity of the process, the limitation of raw materials, and the practicability of industrial production, the yield of trehalose is difficult to meet the market demand, and thus the production process still needs to be continuously innovated and improved. With the continuous development of biotechnology, the means for obtaining trehalose are also continuously improved, mainly including microbial extraction, microbial fermentation (including immobilized cell method and permeabilized cell conversion), two-enzyme method, trehalose synthase conversion and direct conversion of engineering bacteria. The methods developed to date for realizing industrial production of trehalose mainly comprise a double-enzyme method and a trehalose synthase method.
Trehalose synthase (trehalose synthase) is capable of converting alpha, alpha-1, 4-glycosidically linked maltose directly into alpha, alpha-1, 1-glycosidically linked trehalose, the conversion reaction does not require the presence of phosphate, does not require the consumption of energetic materials, and the enzyme has a strong substrate specificity. At present, the preparation process of maltose is mature, the preparation cost is low, and the purity of maltose solution is high, so that the conversion process has the advantages of raw materials and is convenient for the application of trehalose production. The trehalose synthase conversion method has the advantages of one-step conversion, simple process and convenient regulation, so the method is currently considered to be the optimal method for industrial production of trehalose.
Chinese patent document CN104789539A (application No. 201510210023.X) discloses a mutant of trehalose synthase and a preparation method and application thereof, wherein an amino acid sequence of Thermobifida fusca YX (accession No. WP _011291031.1) trehalose synthase is taken as a parent, and the conversion rates of the mutant in trehalose production respectively reach 69.7%, 70.5%, 70.3%, 69.6%, 70.4%, 70.9%, 72.3% and 73.7%, which are slightly higher than the conversion rate of a wild enzyme by 62.2%. Chinese patent document CN105524936A (application No. 201610073828.9) discloses a mutant trehalose synthase, an expression gene and an application thereof, wherein the amino acid sequence of the trehalose synthase of Pseudomonas stutzeri Qlu3 is used as a parent, and the conversion rate of the mutant trehalose is improved to 74.7% from 71.5% of a wild type.
Disclosure of Invention
Aiming at the problem of low conversion rate of the existing trehalose synthase, the invention provides a preparation method and application of mutant trehalose synthase. The mutant trehalose synthase has improved conversion rate of converting maltose into trehalose. The mutants comprise substitutions containing one or two active amino acid residues relative to the trehalose synthase activity of Pseudomonas putida P06 (accession number AE 015451.1).
A trehalose synthase mutant V407M, characterized in that, on the basis of the wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID No.1, valine (Val) at position 407 is mutated to methionine (Met).
The nucleotide sequence of the expression gene of the trehalose synthase mutant V407M is shown in SEQ ID NO. 2.
A recombinant expression vector comprises an expression gene of the trehalose synthase mutant V407M.
Preferably, according to the invention, the expression vector is a pET-15b plasmid vector.
A recombinant cell comprising the above recombinant expression vector or the expression gene of the above trehalose synthase mutant V407M.
Preferably, according to the invention, the host cell is E.coli BL21(DE 3).
The application of the trehalose synthase mutant V407M in preparing trehalose.
A trehalose synthase mutant K490L, characterized in that, on the basis of the wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID No.1, lysine (Lys) at position 490 is mutated to leucine (Leu).
The nucleotide sequence of the expression gene of the trehalose synthase mutant K490L is shown in SEQ ID NO. 3.
A recombinant expression vector comprises an expression gene of the trehalose synthase mutant K490L.
A recombinant cell comprising the expression gene of the recombinant expression vector or the trehalose synthase mutant K490L.
The application of the trehalose synthase mutant K490L in preparing trehalose.
A trehalose synthase mutant V407M/K490L is characterized in that, on the basis of a wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID NO.1, valine (Val) at position 407 is mutated into methionine (Met) and lysine (Lys) at position 490 is mutated into leucine (Leu).
The nucleotide sequence of the expression gene of the trehalose synthase mutant V407M/K490L is shown as SEQ ID NO. 4.
A recombinant expression vector contains the expression gene of the trehalose synthase mutant V407M/K490L.
A recombinant cell, which contains the recombinant expression vector or the expression gene of the trehalose synthase mutant V407M/K490L.
The application of the trehalose synthase mutant V407M/K490L in preparing trehalose.
Advantageous effects
The invention takes the predicted three-dimensional structure of the trehalose synthase of Pseudomonas putida P06 as the basis, carries out site-directed mutagenesis of key amino acid on the active center of the trehalose synthase to obtain the trehalose synthase mutant. The mutant is simple to prepare, high in yield and high in purity, the trehalose yield is improved, and under the same condition, the conversion rates of trehalose produced by the mutants V407M, K490L and V407M/K490L reach 68%, 65% and 70% respectively, and are obviously improved compared with 50% of original enzyme.
Drawings
FIG. 1 is a diagram showing the results of PCR amplification and agarose gel electrophoresis;
in the figure, M is DNA marker, and lanes 1, 2 and 3 are mutant plasmids V407M, K490L, V407M/K490L respectively;
FIG. 2 is a SDS-PAGE result of trehalose synthase after nickel column purification;
in the figure, M is a standard molecular weight Marker, and lanes 1, 2 and 3 are mutant enzymes V407M, K490L, V407M/K490L respectively;
FIG. 3 is a graph showing the optimum reaction temperature profile and temperature stability of purified trehalose synthase mutants;
wherein, FIG. 3a is the optimum reaction temperature curve of purified trehalose synthase mutant, and FIG. 3b is the temperature stability curve of purified trehalose synthase mutant;
FIG. 4 is a pH curve and pH stability profile of the purified trehalose synthase mutant in the optimum reaction;
wherein, FIG. 4a is the pH curve of the optimum reaction of the purified trehalose synthase mutant, and FIG. 4b is the pH stability curve of the purified trehalose synthase mutant.
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.
Detection method
HPLC determination method: chromatographic column Inertsil NH2(4.6 mm. times.250 mm, 5 μm); mobile phase (acetonitrile: water ═ 3: 1); flow rate (1.0 mL/min);
a detector: a differential refractive detector; the sample volume is 10 mu L; the column temperature was 40 ℃.
Calculation formula of trehalose synthase conversion rate:
wherein m istrehalose、mglucose、mmaltoseRespectively represent the mass of trehalose, glucose and maltose in the transformation system.
Example 1: preparation of trehalose synthase mutant
(1) Single mutation
Two mutant enzymes of trehalose synthase from Pseudomonas putida P06V 407M, K490L: according to the gene sequence of trehalose synthase of Pseudomonas putida P06, primers for introducing V407M and K490L mutations are designed and synthesized respectively, the trehalose synthase is subjected to site-directed mutagenesis, a DNA coding sequence is determined, and the DNA coding sequence is introduced into Escherichia coli for expression, so that the single-mutation trehalose synthase is obtained. Site-directed mutagenesis of single mutations V407M, K490L: utilizing a Quickchange site-directed mutagenesis kit, taking a constructed expression vector pET-15b-TreS plasmid as a template:
site-directed mutagenesis primers for introducing the V407M mutation were:
a forward primer: 5' -aaccatgacgagctgaccatggagctggtgcacttctgg-3' (the mutated base is underlined)
Reverse primer: 5' -ccagaagtgcaccagctccatggtcagctcgtcatggtt-3' (the mutated base is underlined)
The site-directed mutagenesis primers for introducing the K490L mutation were:
a forward primer: 5' -agcggatatcgaactgatcttgaaggtgcacctgctgctgg-3' (the mutated base is underlined)
Reverse primer: 5' -ccagcagcaggtgcaccttcaagatcagttcgatatccgct-3' (the mutated base is underlined)
The PCR reaction systems are as follows:
max Master Mix 25. mu.L, 2. mu.L each of upstream and downstream primers, 1. mu.L of template DNA, ddH2The content of O is filled to 50 mu L.
The PCR reaction program is: 3min at 95 ℃; at 95 ℃ for 15s and 68 ℃ for 8min, and 30 cycles; 10min at 72 ℃. After the reaction, 5. mu.L of the product was detected by 1% agarose gel electrophoresis, and the detection results are shown in lanes 1 and 2 in FIG. 1.
The obtained PCR amplification product is digested for 30min by using Dpn I endonuclease, and the template plasmid is cut up by being sensitive to Dpn I and subjected to dam methylation modification. E.coli BL21(DE3) super competent cells were transformed with the enzyme-digested product, the transformed solution was spread on LB solid medium containing 50. mu.g/mL ampicillin, cultured overnight at 37 ℃, single clones were randomly picked up and placed in a glycerol tube containing 1mL of LB medium (containing 100. mu.g/mL ampicillin), cultured overnight, and the sequence was verified to be correct.
(2) Double mutation
Double mutant enzyme of trehalose synthase from Pseudomonas putida P06V 407M/K490L: the 490 th lysine (Lys) in the gene of the single mutant enzyme V407M was mutated to leucine (Leu) and was designated V407M/K490L. The preparation method of the double mutant enzyme comprises the steps of respectively designing and synthesizing primers for introducing K490L mutation by using a single mutant enzyme V407M encoding gene as a template, carrying out site-directed mutation on the single mutant V407M, determining a sequence, and introducing the sequence into escherichia coli for expression to obtain the double mutant trehalose synthase. Site-directed mutagenesis of double mutation V407M/K490L: utilizing a Quickchange site-directed mutagenesis kit, taking a constructed expression vector pET-15b-TreS/V407M plasmid as a template:
the site-directed mutagenesis primers for introducing the K490L mutation were:
a forward primer: 5' -agcggatatcgaactgatcttgaaggtgcacctgctgctgg-3' (the mutated base is underlined)
Reverse primer: 5' -ccagcagcaggtgcaccttcaagatcagttcgatatccgct-3' (the mutated base is underlined)
The PCR reaction system, reaction conditions and method for sequencing mutant genes are the same as the single mutant method, wherein the PCR amplification detection result is shown in lane 3 in FIG. 1.
Example 2: fermentation induction and purification of mutant enzymes
The mutants were inoculated into 50mL of LB liquid medium (containing 100. mu.g/mL ampicillin), cultured at 37 ℃ at 200r/min to OD600When the concentration reaches 2.5-3.0, adding inducer lactose to the final concentration of 4g/L, and inducing for 8 hours at 27 ℃ under the condition of 200 r/min. The fermentation liquor is centrifuged for 10min at the temperature of 4 ℃ and the speed of 8000r/min, 5mL of 10mM PBS (pH8.0) is used for resuspending the thalli, the mutated enzyme is extracted by an ultrasonic crusher, and the crushing conditions are as follows: 300W, working time of 5s, pause of 5s and whole process of 15 min. The disruption solution was centrifuged at 8000r/min for 10min at 4 ℃ to collect the supernatant, which was then sterilized by filtration through a 0.22 μm filter.
Nickel column affinity chromatography: pouring the collected supernatant of the crude enzyme solution into a regenerated nickel column; after the supernatant was washed out, the column was washed 10 times in volume with wash buffer (25mM PBS, pH8.0,100mM NaCl,15mM imidazole) to remove non-specifically adsorbed proteins, and finally the target protein was eluted with elution buffer (25mM PBS, pH8.0,100mM NaCl,250mM imidazole), and the purified protein was collected, concentrated and desalted using an ultrafiltration tube, and the concentrated and desalted protein was subjected to SDS-PAGE as shown in FIG. 2.
Example 3: method for determining conversion rate of trehalose synthase mutant
5mL of a maltose solution (pH8.0) with a substrate conversion concentration of 300g/L of the crude enzyme solution is taken to react for 8h under the condition of 25 ℃, and the reaction is stopped by boiling water bath for 15 min. Centrifuging at 13000r/min for 15min at room temperature, taking the supernatant, and detecting the contents of trehalose, glucose and maltose in the transformation system by using High Performance Liquid Chromatography (HPLC). The same mutant was repeated 3 times, and the difference between the measured values was less than 5%, and the average value was taken, and the measured results are shown in Table 1.
TABLE 1 content of glucose, maltose and trehalose in the original enzyme and mutant transformation systems
Compared with the original enzyme, the mutant enzyme obtained by expressing the mutant can find that the mutant realizes the improvement of the conversion rate of preparing the trehalose by the trehalose synthase. The conversion rates of the mutant enzymes V407M, K490L and V407M/K490L for producing trehalose reach 68%, 65% and 70% respectively, and the original enzyme is 50%.
Example 4: determination of enzymatic Properties of trehalose synthase mutants
Analysis of optimum temperature and thermal stability of mutant enzyme
Respectively converting at different temperatures (20-65 ℃) and pH8.0 for 1h, determining the optimal reaction temperature of the original enzyme and the mutant enzyme by taking the highest enzyme activity as 100%, and determining the result as shown in figure 3 (a). Respectively preserving the heat at different temperatures (20-65 ℃) for 60min, then converting the mixture for 1h at 25 ℃ and pH8.0, determining the residual enzyme activity, and comparing the heat stability of the original enzyme and the mutant enzyme, wherein the determination result is shown in figure 3 (b).
Analysis of optimum pH and pH tolerance of mutant enzyme
Respectively converting at 25 ℃ for 1h under different pH (4.0-10) conditions, determining the optimum reaction pH of the original enzyme and the mutant enzyme by taking the highest enzyme activity as 100%, and determining the result as shown in figure 4 (a). Respectively reacting for 1h at 25 ℃ and pH8.0 after keeping the temperature at 25 ℃ for 60min under the condition of pH (4.0-10), calculating the residual enzyme activity, comparing the pH stability of the original enzyme and the mutant enzyme, and determining the result as shown in figure 4 (b).
Trehalose synthase enzyme activity definition: the amount of enzyme required to convert maltose to 1. mu. mol of trehalose per hour was defined as 1 enzyme activity unit (U).
Comparative example
The 232 th aspartic acid (Asp) which is also near the active center is mutated into leucine (Leu) according to the method, the conversion rate and the enzymological properties of the mutant enzyme are detected according to the same method, the result shows that the conversion rate of the mutant D232L is not increased compared with the original enzyme, and the research on the enzymological properties shows that the mutant D232L is not greatly different from the original enzyme.
SEQUENCE LISTING
<110> university of Qilu Industrial science
<120> preparation method and application of mutant trehalose synthase
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aagcgcctgt cggagatcgg cgtacagggc ctgcacaccg gcccgatcaa actgtccggt 300
ggcatccgcg gccgcgaact cacccccagc gtggacggca acttcgaccg catcagcttc 360
gacatcgacc cactgtacgg cagcgagcag gaactgatcc agatgagccg catggccgct 420
gcgcacaatg ccgtgaccat cgacgacctg atcccctcgc acaccggcaa gggcgccgac 480
ttccgcctgg ccgagctcgc ccatggcccc tacccggggc tgtaccacat ggtcgagatc 540
cgcgaagaag actgggcgct gctgcccgag gtgcccgccg ggcgcgatgc ggtcaatctg 600
ctgccagctc agtgtgacga gctgaaggcg cgccattaca tcgttggcca gctgcaacgg 660
gtaatcttct tcgagccggg cgtgaaggaa accgactgga gcgccacgcc gccgatcaca 720
ggcgtcgacg gcaagacccg ccgctgggtg tacctgcatt acttcaagga aggccagccc 780
tcgctgaact ggctggaccc taccttcgcc gcccaacaga tgatcattgg tgacgcactg 840
cacgcgctgg actgcctggg tgcacgcggc ctgcgcctgg acgccaacgg ctttctcggc 900
gtggaaaccc gcgccagcgg caccgcctgg tcggaaagcc acccgctgtc gctcgtcggc 960
aaccagctga tcggtggcat gatccgcaag gccggcggtt tcagcttcca ggagctgaac 1020
ctgaccctcg atgacattgc gcagatgtcc aagggtggtg ccgacctgtc ctacgatttc 1080
attacccggc cggcctacca gcatgcgctg ctgacgggcg acaccgagtt cctgcgcctg 1140
atgctcaagg agatgcacgc cttcggcatc gacccggcct cgctcatcca tgccctgcaa 1200
aaccatgacg agctgaccgt ggagctggtg cacttctgga cactgcacgc gcacgatatg 1260
tacctgtaca agggccaaac cctgcctggc agcatcctgc gcgaacatat tcgcgaagag 1320
atctacgaac ggctgtcggg ggaacatgcg ccgtacaacc tgcgcttcgt gaccaacggc 1380
attgcctgca ccaccgccag cctgatcgct gctgcactgg gtattcgcga cctcgaacag 1440
attggtgtag cggatatcga actgatcttg aaggtgcacc tgctgctggt catgtacaac 1500
gccatgcagc cgggggtggt cgccttgtcc ggctgggacc tggtcggtgc cctgcccttg 1560
cccgccgaag cggttgccga acgcatgctc gatggcgata cccgctggat tcaccggggc 1620
ggctatgacc tggccgggct tgacccacag gcagaggctt ctgtgcgggg catgccgcgt 1680
gcccgggcgc tatacggcag cctggacagg cagctggacg agagtgattc atttgcctgc 1740
aaggtgaaga aactgctggc tgtgcgccag gcctacggca tcgccaccag ccgtcaggtg 1800
ctggtacctg aggtgagcag cccggggctg ctggtgatgg tgcatgagct gccagccggg 1860
cgcggtatcc agatcactgc gctgaacttc ggccaggacg cgattgccga ggaactgctg 1920
ttgaccgggt tcacacctgg gccggtggtc gacatgatca acgagacggt cgaaggcgat 1980
ttgaccgagg acgggcgcct gatggtgaac ctggacccgt acgaggcgct gtgcctgcgg 2040
atcgtcaaca gcagcgggca tgtttga 2067
<210> 4
<211> 2067
<212> DNA
<213> Artificial Synthesis
<400> 4
atgacccagc ccgacccgtc atacgtcaaa tggctcgaag accgcgccat gctcaaggcc 60
tcccaggacc gggccagcct gtactcaggc cagtcgcgcc tgtggcagca accctatgcc 120
gaggcccagc cccgccgcgc caccgaaatc gcctcggtgt ggctgacggt ctaccccgac 180
gccatcatcg cgcccgaggg ttgctcggtg ctcggtgccc tggcccacga agcgttgtgg 240
aagcgcctgt cggagatcgg cgtacagggc ctgcacaccg gcccgatcaa actgtccggt 300
ggcatccgcg gccgcgaact cacccccagc gtggacggca acttcgaccg catcagcttc 360
gacatcgacc cactgtacgg cagcgagcag gaactgatcc agatgagccg catggccgct 420
gcgcacaatg ccgtgaccat cgacgacctg atcccctcgc acaccggcaa gggcgccgac 480
ttccgcctgg ccgagctcgc ccatggcccc tacccggggc tgtaccacat ggtcgagatc 540
cgcgaagaag actgggcgct gctgcccgag gtgcccgccg ggcgcgatgc ggtcaatctg 600
ctgccagctc agtgtgacga gctgaaggcg cgccattaca tcgttggcca gctgcaacgg 660
gtaatcttct tcgagccggg cgtgaaggaa accgactgga gcgccacgcc gccgatcaca 720
ggcgtcgacg gcaagacccg ccgctgggtg tacctgcatt acttcaagga aggccagccc 780
tcgctgaact ggctggaccc taccttcgcc gcccaacaga tgatcattgg tgacgcactg 840
cacgcgctgg actgcctggg tgcacgcggc ctgcgcctgg acgccaacgg ctttctcggc 900
gtggaaaccc gcgccagcgg caccgcctgg tcggaaagcc acccgctgtc gctcgtcggc 960
aaccagctga tcggtggcat gatccgcaag gccggcggtt tcagcttcca ggagctgaac 1020
ctgaccctcg atgacattgc gcagatgtcc aagggtggtg ccgacctgtc ctacgatttc 1080
attacccggc cggcctacca gcatgcgctg ctgacgggcg acaccgagtt cctgcgcctg 1140
atgctcaagg agatgcacgc cttcggcatc gacccggcct cgctcatcca tgccctgcaa 1200
aaccatgacg agctgaccat ggagctggtg cacttctgga cactgcacgc gcacgatatg 1260
tacctgtaca agggccaaac cctgcctggc agcatcctgc gcgaacatat tcgcgaagag 1320
atctacgaac ggctgtcggg ggaacatgcg ccgtacaacc tgcgcttcgt gaccaacggc 1380
attgcctgca ccaccgccag cctgatcgct gctgcactgg gtattcgcga cctcgaacag 1440
attggtgtag cggatatcga actgatcttg aaggtgcacc tgctgctggt catgtacaac 1500
gccatgcagc cgggggtggt cgccttgtcc ggctgggacc tggtcggtgc cctgcccttg 1560
cccgccgaag cggttgccga acgcatgctc gatggcgata cccgctggat tcaccggggc 1620
ggctatgacc tggccgggct tgacccacag gcagaggctt ctgtgcgggg catgccgcgt 1680
gcccgggcgc tatacggcag cctggacagg cagctggacg agagtgattc atttgcctgc 1740
aaggtgaaga aactgctggc tgtgcgccag gcctacggca tcgccaccag ccgtcaggtg 1800
ctggtacctg aggtgagcag cccggggctg ctggtgatgg tgcatgagct gccagccggg 1860
cgcggtatcc agatcactgc gctgaacttc ggccaggacg cgattgccga ggaactgctg 1920
ttgaccgggt tcacacctgg gccggtggtc gacatgatca acgagacggt cgaaggcgat 1980
ttgaccgagg acgggcgcct gatggtgaac ctggacccgt acgaggcgct gtgcctgcgg 2040
atcgtcaaca gcagcgggca tgtttga 2067
<210> 5
<211> 39
<212> DNA
<213> Artificial Synthesis
<400> 5
aaccatgacg agctgaccat ggagctggtg cacttctgg 39
<210> 6
<211> 39
<212> DNA
<213> Artificial Synthesis
<400> 6
ccagaagtgc accagctcca tggtcagctc gtcatggtt 39
<210> 7
<211> 41
<212> DNA
<213> Artificial Synthesis
<400> 7
agcggatatc gaactgatct tgaaggtgca cctgctgctg g 41
<210> 8
<211> 41
<212> DNA
<213> Artificial Synthesis
<400> 8
ccagcagcag gtgcaccttc aagatcagtt cgatatccgc t 41
Claims (11)
1. A trehalose synthase mutant V407M, characterized in that, on the basis of the wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID No.1, valine (Val) at position 407 is mutated to methionine (Met).
2. An expression gene of a trehalose synthase mutant V407M, the nucleotide sequence is shown in SEQ ID NO. 2.
3. A trehalose synthase mutant K490L, characterized in that, on the basis of the wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID No.1, lysine (Lys) at position 490 is mutated to leucine (Leu).
4. An expression gene of a trehalose synthase mutant K490L, the nucleotide sequence is shown in SEQ ID NO. 3.
5. A trehalose synthase mutant V407M/K490L is characterized in that, on the basis of a wild-type trehalose synthase of which the amino acid sequence is shown as SEQ ID NO.1, valine (Val) at position 407 is mutated into methionine (Met) and lysine (Lys) at position 490 is mutated into leucine (Leu).
6. An expression gene of a trehalose synthase mutant V407M/K490L, the nucleotide sequence is shown in SEQ ID NO. 4.
7. A recombinant expression vector comprising the gene of claim 2, claim 4 or claim 6.
8. The expression vector of claim 7, wherein the expression vector is a pET-15b plasmid vector.
9. A recombinant cell comprising the recombinant expression vector of claim 7 or the expressed gene of claim 2, claim 4 or claim 6.
10. The recombinant cell of claim 9, wherein the host cell is e.coli BL21(DE 3).
11. Use of a mutant trehalose synthase according to claim 1 or claim 3 or claim 5 for the preparation of trehalose.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1563372A (en) * | 2004-04-08 | 2005-01-12 | 广西大学 | Synthetase gene for T.fusca mycose and method for preparing mycose |
| CN104805104A (en) * | 2015-05-19 | 2015-07-29 | 齐鲁工业大学 | Trehalose synthase and coding gene and application thereof |
| CN106434586A (en) * | 2016-10-08 | 2017-02-22 | 南京工业大学 | Trehalose synthase mutant and gene thereof |
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| CN1563372A (en) * | 2004-04-08 | 2005-01-12 | 广西大学 | Synthetase gene for T.fusca mycose and method for preparing mycose |
| CN104805104A (en) * | 2015-05-19 | 2015-07-29 | 齐鲁工业大学 | Trehalose synthase and coding gene and application thereof |
| CN106434586A (en) * | 2016-10-08 | 2017-02-22 | 南京工业大学 | Trehalose synthase mutant and gene thereof |
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| Saturation mutagenesis and self-inducible expression of trehalose synthase in Bacillus subtilis;Hongling Liu等;《Biotechnol Prog》;20190506;第35卷(第4期);e2826 * |
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