CN108048439A - A kind of preparation method and application of saltant type trehalose synthase - Google Patents

A kind of preparation method and application of saltant type trehalose synthase Download PDF

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CN108048439A
CN108048439A CN201711157105.8A CN201711157105A CN108048439A CN 108048439 A CN108048439 A CN 108048439A CN 201711157105 A CN201711157105 A CN 201711157105A CN 108048439 A CN108048439 A CN 108048439A
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leu
trehalose synthase
mutant
trehalose
ala
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CN108048439B (en
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王腾飞
刘洪玲
王瑞明
吕鑫
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Qilu University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y504/00—Intramolecular transferases (5.4)
    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99016—Maltose alpha-D-glucosyltransferase (5.4.99.16)

Abstract

The present invention relates to a kind of preparation method and applications of saltant type trehalose synthase.The expressing gene of trehalose synthase mutant V407M, nucleotide sequence is as shown in SEQ ID NO.2；The expressing gene of trehalose synthase mutant K490L, nucleotide sequence is as shown in SEQ ID NO.3；The expressing gene of trehalose synthase mutant V407M/K490L, nucleotide sequence is as shown in SEQ ID NO.4.The mutant is prepared simply, and yield is big, and purity is high, realize the raising of seaweed candy output, under the same terms, the conversion ratio of mutant V407M, K490L, V407M/K490L production trehalose has respectively reached 68%, 65%, 70%, and 50% compared with protoenzyme increases significantly.

Description

A kind of preparation method and application of saltant type trehalose synthase
Technical field
The present invention relates to a kind of preparation method and applications of saltant type trehalose synthase, belong to genetic engineering and enzyme engineering skill Art field.
Background technology
Trehalose is naturally occurring a kind of non-reducing disaccharide in nature, is the isomer of maltose, by two The glucose of molecule is made up of α, α -1,1- glucosides key connections.It is all sent out in bacterium, fungi, insect and some invertebrates Show the presence of trehalose, function primarily as the constituent of ergastic substances and cell membrane.With the continuous depth of research Enter, trehalose is widely used in food, drug, cosmetics and agriculture field.
As trehalose application is constantly explored, the function and effect of trehalose get the nod, this causes the market of trehalose Demand gradually increases, and the market price of trehalose remains above other disaccharide, such as sucrose, maltose, but because technique at present Complexity, the factor of restricted and industrialized production the practicability of raw material etc., the yield of trehalose is difficult to full The sufficient market demand, therefore its production technology still needs constantly innovation and improves.With the continuous development of biotechnology, seaweed The obtaining means of sugar are also constantly improving, and mainly have a microorganism extraction method, microbe fermentation method (including immobilized cell reaction and Permeabilized cells conversion method), two enzymes method, trehalose synthase conversion method and engineering bacteria direct translation method.It is developed so far, trehalose Can realize the method for industrialized production mainly has two enzymes method and trehalose synthase method.
Trehalose synthase (trehalose synthase) can directly turn α, the maltose of α -1,4- glucosides key connections Turn to α, α -1, the trehalose of 1- glucosides key connections, which is not required phosphatic presence, need not consume high energy object Matter, and the Substratspezifitaet of the enzyme is strong.The mature preparation process of maltose at present, manufacturing cost is low, and maltose solution is pure Degree is high, therefore the conversion process has raw material advantage, convenient for the application of trehalose production.Trehalose synthase conversion method passes through a step Conversion, simple for process, convenient for regulation and control, therefore this method is presently believed to be the best practice of trehalose industrialized production.
Chinese patent literature CN104789539A (application number 201510210023.X) discloses a kind of trehalose synthase Mutant and preparation method and application, with Thermobifida fusca YX (accession number WP_011291031.1) trehalose The amino acid sequence of synthase is parent, conversion ratio of the mutant when producing trehalose has respectively reached 69.7%, 70.5%, 70.3%th, 69.6%, 70.4%, 70.9%, 72.3%, 73.7%, the conversion ratio 62.2% of slightly above wild enzyme.It is Chinese special Sharp document CN105524936A (application number 201610073828.9) discloses a kind of trehalose synthase of mutation and its expression base Cause and application, using the amino acid sequence of Pseudomonas stutzeri (Pseudomonas stutzeri Qlu3) trehalose synthase as parent This, which is increased to 74.7% by the 71.5% of wild type.
The content of the invention
For existing trehalose synthase conversion ratio it is relatively low the problem of, the present invention provides a kind of saltant type trehalose synthases Preparation method and application.The conversion ratio of the trehalose synthase conversion maltose generation trehalose of the mutation improves.The mutant It lives including containing one or two compared with Pseudomonas putida P06 (accession number AE015451.1) trehalose synthase The substitution of acidic amino acid residue.
A kind of trehalose synthase mutant V407M, which is characterized in that in amino acid sequence as shown in SEQ ID NO.1 On the basis of wild type trehalose synthase, the 407th valine (Val) sports methionine (Met).
The expressing gene of above-mentioned trehalose synthase mutant V407M, nucleotide sequence is as shown in SEQ ID NO.2.
A kind of recombinant expression carrier, the expression vector include the expressing gene of above-mentioned trehalose synthase mutant V407M.
Preferred according to the present invention, the expression vector is pET-15b plasmid vectors.
A kind of recombinant cell, the recombinant cell include above-mentioned recombinant expression carrier or above-mentioned trehalose synthase mutant The expressing gene of V407M.
Preferred according to the present invention, the host cell is e. coli bl21 (DE3).
Applications of the above-mentioned trehalose synthase mutant V407M in trehalose is prepared.
A kind of trehalose synthase mutant K490L, which is characterized in that in amino acid sequence as shown in SEQ ID NO.1 On the basis of wild type trehalose synthase, the 490th lysine (Lys) sports leucine (Leu).
The expressing gene of above-mentioned trehalose synthase mutant K490L, nucleotide sequence is as shown in SEQ ID NO.3.
A kind of recombinant expression carrier, the expression vector include the expressing gene of above-mentioned trehalose synthase mutant K490L.
A kind of recombinant cell, the recombinant cell include above-mentioned recombinant expression carrier or above-mentioned trehalose synthase mutant The expressing gene of K490L.
Applications of the above-mentioned trehalose synthase mutant K490L in trehalose is prepared.
A kind of trehalose synthase mutant V407M/K490L, which is characterized in that in amino acid sequence such as SEQ ID NO.1 On the basis of shown wild type trehalose synthase, the 407th valine (Val) sports methionine (Met) and the 490th Lysine (Lys) sports leucine (Leu).
The expressing gene of above-mentioned trehalose synthase mutant V407M/K490L, nucleotide sequence such as SEQ ID NO.4 institutes Show.
A kind of recombinant expression carrier, the expression vector include the table of above-mentioned trehalose synthase mutant V407M/K490L Up to gene.
A kind of recombinant cell, the recombinant cell include above-mentioned recombinant expression carrier or above-mentioned trehalose synthase mutant The expressing gene of V407M/K490L.
Applications of the above-mentioned trehalose synthase mutant V407M/K490L in trehalose is prepared.
Advantageous effect
The present invention is based on the prediction three-dimensional structure of Pseudomonas putida P06 trehalose synthases, to its activity Center carries out the rite-directed mutagenesis of key amino acid, obtains trehalose synthase mutant.The mutant is prepared simply, and yield is big, Purity is high, realizes the raising of seaweed candy output, under the same terms, mutant V407M, K490L, V407M/K490L production seaweed The conversion ratio of sugar has respectively reached 68%, 65%, 70%, and 50% compared with protoenzyme increases significantly.
Description of the drawings
Fig. 1 verifies agarose gel electrophoresis result figure for PCR amplification；
In figure, M is DNA marker, and swimming lane 1,2,3 is respectively mutant plasmid V407M, K490L, V407M/K490L；
Fig. 2 is trehalose synthase SDS-PAGE electrophoresis result figures after ni-sepharose purification；
In figure, M is standard molecular weight Marker, and swimming lane 1,2,3 is respectively mutant enzyme V407M, K490L, V407M/ K490L；
Fig. 3 is the optimal reactive temperature curve of trehalose synthase mutant and temperature stability graph after purification；
Wherein, Fig. 3 a are the optimal reactive temperature curve of trehalose synthase mutant after purification, and Fig. 3 b are seaweed after purification The temperature stabilization linearity curve of sugared synthase mutant；
Fig. 4 is the optimal reaction pH curve of trehalose synthase mutant and pH stability curve figures after purification；
Wherein, Fig. 4 a are the optimal reaction pH curves of trehalose synthase mutant after purification, and Fig. 4 b are trehalose after purification The pH stability curves of synthase mutant.
Specific embodiment
Technical scheme is further elaborated with reference to embodiment, but institute's protection domain of the present invention is not limited to This.
Detection method
HPLC assay methods：Chromatographic column Inertsil NH2 (4.6mm × 250mm, 5 μm)；Mobile phase (acetonitrile：Water=3： 1)；Flow velocity (1.0mL/min)；
Detector：Differential refraction detector；10 μ L of sample size；40 DEG C of column temperature.
Trehalose synthase conversion ratio calculation formula：
Wherein, mtrehalose、mglucose、mmaltoseThe matter of trehalose in transformation system, glucose, maltose is represented respectively Amount.
Embodiment 1：The preparation of trehalose synthase mutant
(1) single mutation
From two kinds of mutant enzymes V407M, K490L of the trehalose synthase of Pseudomonas putida P06：Root According to the gene order of the trehalose synthase of Pseudomonas putida P06, separately design and synthesize introducing V407M, K490L The primer of mutation carries out rite-directed mutagenesis to trehalose synthase, measures DNA encoding sequence, and imports in Escherichia coli and expressed, Obtain single mutation trehalose synthase.The rite-directed mutagenesis of single mutation V407M, K490L：Utilize Quickchange rite-directed mutagenesis reagents Box, using the expression vector pET-15b-TreS plasmids built as template：
Introducing the rite-directed mutagenesis primer that V407M is mutated is：
Forward primer：5'-aaccatgacgagctgaccatgGagctggtgcacttctgg-3'(underscores are mutation alkali Base)
Reverse primer：5'-ccagaagtgcaccagctccatGgtcagctcgtcatggtt-3'(underscores are mutation alkali Base)
Introducing the rite-directed mutagenesis primer that K490L is mutated is：
Forward primer：5'-agcggatatcgaactgatcttgAaggtgcacctgctgctgg-3'(underscores are mutation Base)
Reverse primer：5'-ccagcagcaggtgcaccttcaaGatcagttcgatatccgct-3'(underscores are mutation Base)
PCR reaction systems are：25 μ L of Max Master Mix, each 2 μ L of upstream and downstream primer, template DNA 1 μ L, ddH2O polishings are to 50 μ L.
PCR response procedures are：95℃3min；95 DEG C of 15s, 68 DEG C of 8min, 30 Xun Huans；72℃10min.Reaction terminates Afterwards, 5 μ L products is taken to be detected with 1% agarose gel electrophoresis, testing result is as shown in the swimming lane 1,2 in Fig. 1.
By obtained pcr amplification product using Dpn I endonuclease digestion 30min, template plasmid is methylated modification through dam It is sensitive to Dpn I and be chopped into.Digestion products Transformed E .coli BL21 (DE3) super competent cell, conversion fluid are coated on LB solid mediums containing 50 μ g/mL ampicillins, 37 DEG C are incubated overnight, random picking monoclonal to the culture mediums of LB containing 1mL In the glycerol tube of (containing 100 μ g/mL ampicillins), it is incubated overnight, sequence verification sequence is correct.
(2) double mutation
The double-mutant enzyme V407M/K490L of the trehalose synthase of Pseudomonas putida P06：By single mutant The 490th lysine (Lys) in enzyme V407M genes is mutated into leucine (Leu), and is named as V407M/K490L.Double mutation The preparation method of body enzyme using single-mutant enzyme V407M encoding genes as template, separately designs and synthesizes introducing K490L mutation Primer carries out rite-directed mutagenesis to single mutant V407M, measures sequence, and imports in Escherichia coli and expressed, and obtains double mutation Trehalose synthase.The rite-directed mutagenesis of double mutation V407M/K490L：Using Quickchange site-directed mutagenesis kits, to build Expression vector pET-15b-TreS/V407M plasmids be template：
Introducing the rite-directed mutagenesis primer that K490L is mutated is：
Forward primer：5'-agcggatatcgaactgatcttgAaggtgcacctgctgctgg-3'(underscores are mutation Base)
Reverse primer：5'-ccagcagcaggtgcaccttcaaGatcagttcgatatccgct-3'(underscores are mutation Base)
The sequencing approach of PCR reaction systems, reaction condition and mutator with single mutant method, wherein, PCR amplification Testing result is as shown in the swimming lane 3 in Fig. 1.
Embodiment 2：The fermentation inducement of mutant enzyme and purifying
Mutant is inoculated in 50mL LB fluid nutrient mediums (containing 100 μ g/mL ampicillins), 37 DEG C, 200r/min is trained It supports to OD600When reaching 2.5~3.0, addition derivant lactose 27 DEG C, induces 8h to final concentration 4g/L under the conditions of 200r/min. Zymotic fluid centrifuges 10min in 4 DEG C under the conditions of 8000r/min, 5mL 10mM PBS (pH8.0) are resuspended thalline, utilize ultrasonication Instrument extracts mutant enzyme, broken condition：300W, working time 5s, interval 5s, whole 15min.Broken 4 DEG C of liquid, 8000r/min from Heart 10min, collects supernatant, 0.22 μm of membrane filtration degerming of supernatant.
Affinity chromatography：The crude enzyme liquid supernatant of collection is poured into the nickel column regenerated；After supernatant liquid stream is net, with Wash buffer (25mM PBS, pH8.0,100mM NaCl, 15mM imidazoles) rinse 10 times of column volumes, remove non-specific inhale Attached albumen, finally using elution buffer (25mM PBS, pH8.0,100mM NaCl, 250mM imidazoles) by destination protein It elutes, and collects the albumen of purifying, concentration desalination is carried out using super filter tube, the albumen after concentration desalination is taken to carry out SDS- PAGE verifies that verification result is as shown in Figure 2.
Embodiment 3：The conversion ratio assay method of trehalose synthase mutant
The maltose solution (pH8.0) of 5mL crude enzyme liquid conversion of substrate concentration 300g/L is taken, reacts 8h under the conditions of 25 DEG C, is boiled Water-bath 15min terminates reaction.Room temperature 13000r/min centrifuges 15min, and supernatant is taken to utilize high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) trehalose, glucose, maltose contain in detection transformation system Amount.Identical mutation is repeated 3 times, and measured value difference is less than 5%, is averaged, measurement result is as shown in table 1.
Glucose, maltose, the content of trehalose in 1 protoenzyme of table and mutant transformant system
The mutant enzyme that the expression of above-mentioned mutant is obtained is compared with protoenzyme, it is found that mutant realizes trehalose Synthase prepares the raising of trehalose conversion ratio.The conversion ratio point of mutant enzyme V407M, K490L, V407M/K490L production trehalose Do not reach 68%, 65%, 70%, protoenzyme 50%.
Embodiment 4：The zymologic property of trehalose synthase mutant measures
Mutant enzyme optimum temperature and thermal stability analysis
Respectively under different temperatures (20~65 DEG C), pH8.0 conversion 1h, using highest enzyme activity as 100%, measure protoenzyme and The optimal reactive temperature of mutant enzyme, shown in measurement result such as Fig. 3 (a).Respectively 60min is kept the temperature under different temperatures (20~65 DEG C) Afterwards, 25 DEG C, 1h is converted under the conditions of pH8.0, measure remaining enzyme activity, compare the thermal stability of protoenzyme and mutant enzyme, measure knot Shown in fruit such as Fig. 3 (b).
Mutant enzyme optimal pH and the analysis of pH tolerances
Respectively under the conditions of different pH (4.0~10), 25 DEG C conversion 1h, using highest enzyme activity as 100%, measure protoenzyme and Shown in the optimal reaction pH of mutant enzyme, measurement result such as Fig. 4 (a).Respectively under the conditions of pH (4.0~10), 25 DEG C of heat preservation 60min 25 DEG C afterwards, 1h is reacted under the conditions of pH8.0, calculate remaining enzyme activity, compare the pH stability of protoenzyme and mutant enzyme, measurement result As shown in Fig. 4 (b).
Trehalose synthase enzyme activity defines：The enzyme amount that maltose is generated needed for the trehalose of 1 μm of ol is converted per hour to define For 1 enzyme-activity unit (U).
Comparative example
According to the method described above, the be similarly positioned near activated centre the 232nd aspartic acid (Asp) is sported into leucine (Leu), the conversion ratio and zymologic property of mutant enzyme are detected by the same way, and the results show mutant D232L compares protoenzyme Conversion ratio does not increase, and zymologic property research finds that mutant D232L is not much different with protoenzyme.
SEQUENCE LISTING
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<120>A kind of preparation method and application of saltant type trehalose synthase
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Glu Ile Ala Ser Val Trp Leu Thr Val Tyr Pro Asp Ala Ile Ile Ala
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Pro Glu Gly Cys Ser Val Leu Gly Ala Leu Ala His Glu Ala Leu Trp
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Gly Val Asp Gly Lys Thr Arg Arg Trp Val Tyr Leu His Tyr Phe Lys
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Glu Gly Gln Pro Ser Leu Asn Trp Leu Asp Pro Thr Phe Ala Ala Gln
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Gln Met Ile Ile Gly Asp Ala Leu His Ala Leu Asp Cys Leu Gly Ala
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Gly Ala Asp Leu Ser Tyr Asp Phe Ile Thr Arg Pro Ala Tyr Gln His
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Ala Leu Leu Thr Gly Asp Thr Glu Phe Leu Arg Leu Met Leu Lys Glu
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Met His Ala Phe Gly Ile Asp Pro Ala Ser Leu Ile His Ala Leu Gln
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Asn His Asp Glu Leu Thr Val Glu Leu Val His Phe Trp Thr Leu His
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Ala His Asp Met Tyr Leu Tyr Lys Gly Gln Thr Leu Pro Gly Ser Ile
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Ile Gly Val Ala Asp Ile Glu Leu Ile Lys Lys Val His Leu Leu Leu
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Val Met Tyr Asn Ala Met Gln Pro Gly Val Val Ala Leu Ser Gly Trp
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Met Leu Asp Gly Asp Thr Arg Trp Ile His Arg Gly Gly Tyr Asp Leu
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Ala Gly Leu Asp Pro Gln Ala Glu Ala Ser Val Arg Gly Met Pro Arg
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Ala Arg Ala Leu Tyr Gly Ser Leu Asp Arg Gln Leu Asp Glu Ser Asp
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Ser Phe Ala Cys Lys Val Lys Lys Leu Leu Ala Val Arg Gln Ala Tyr
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Gly Leu Leu Val Met Val His Glu Leu Pro Ala Gly Arg Gly Ile Gln
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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 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>It is artificial synthesized
<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>It is artificial synthesized
<400> 5
aaccatgacg agctgaccat ggagctggtg cacttctgg 39
<210> 6
<211> 39
<212> DNA
<213>It is artificial synthesized
<400> 6
ccagaagtgc accagctcca tggtcagctc gtcatggtt 39
<210> 7
<211> 41
<212> DNA
<213>It is artificial synthesized
<400> 7
agcggatatc gaactgatct tgaaggtgca cctgctgctg g 41
<210> 8
<211> 41
<212> DNA
<213>It is artificial synthesized
<400> 8
ccagcagcag gtgcaccttc aagatcagtt cgatatccgc t 41

Claims (10)

1. a kind of trehalose synthase mutant V407M, which is characterized in that in open country of the amino acid sequence as shown in SEQ ID NO.1 On the basis of raw type trehalose synthase, the 407th valine (Val) sports methionine (Met).
2. a kind of expressing gene of trehalose synthase mutant V407M, nucleotide sequence is as shown in SEQ ID NO.2.
3. a kind of trehalose synthase mutant K490L, which is characterized in that in open country of the amino acid sequence as shown in SEQ ID NO.1 On the basis of raw type trehalose synthase, the 490th lysine (Lys) sports leucine (Leu).
4. a kind of expressing gene of trehalose synthase mutant K490L, nucleotide sequence is as shown in SEQ ID NO.3.
5. a kind of trehalose synthase mutant V407M/K490L, which is characterized in that in amino acid sequence such as SEQ ID NO.1 institutes On the basis of the wild type trehalose synthase shown, the 407th valine (Val) sports methionine (Met) and the 490th relies Propylhomoserin (Lys) sports leucine (Leu).
6. a kind of expressing gene of trehalose synthase mutant V407M/K490L, nucleotide sequence is as shown in SEQ ID NO.4.
7. a kind of recombinant expression carrier, which is characterized in that the expression vector includes the claims 2 or claim 4 Or the expressing gene described in claim 6.
8. expression vector as claimed in claim 7, which is characterized in that the expression vector is pET-15b plasmid vectors.
9. a kind of recombinant cell, which is characterized in that the recombination expression that the recombinant cell includes described in the claims 7 carries Expressing gene described in body or the claims 2 or claim 4 or claim 6；
Preferably, the host cell is e. coli bl21 (DE3).
10. the trehalose synthase mutant described in the claims 1 or claim 3 or claim 5 is preparing trehalose In application.
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CN114395543A (en) * 2022-01-19 2022-04-26 山东恒仁工贸有限公司 Trehalose synthase mutant and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378631A (en) * 2019-01-28 2020-07-07 江南大学 Trehalose synthase mutant and application thereof in trehalose production
CN111378631B (en) * 2019-01-28 2021-08-24 江南大学 Trehalose synthase mutant and application thereof in trehalose production
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CN114395543B (en) * 2022-01-19 2023-05-30 山东恒仁工贸有限公司 Trehalose synthase mutant and application thereof

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