CN112725304B - Low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof - Google Patents
Low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering and protein modification, in particular to a low-temperature inulinase mutant MutAP122EK5 and application thereof, wherein the amino acid sequence of the mutant MutAP122EK5 is 5 amino acids obtained by replacing AAPLP from 122 th site to 126 th site of a wild inulinase InuAMN8 with EEDRK, and the sequence of the MutAP122EK5 is shown as SEQ ID No. 1. Compared with a wild enzyme InuAMN8, the mutant enzyme MutAP122EK5 has improved low-temperature activity and reduced thermal stability, and is beneficial to the application in the biotechnology field under the requirement of low-temperature environment. The low-temperature exoinulase mutant MutAP122EK5 can be applied to the industries of food, wine brewing, washing and the like.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to a protein modification technology, and particularly relates to a low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof.
Background
Inulin is mainly present in roots or stems of plants such as jerusalem artichoke, chicory, dandelion, burdock, artichoke and the like, and is a renewable resource with rich sources. The jerusalem artichoke is low in price and high in yield, more importantly, the jerusalem artichoke belongs to non-grain crops, and the characteristics enable the effective utilization of the jerusalem artichoke to be the focus of attention.
Inulin is hydrolyzed by exoinulase to obtain fructose syrup with sugar content up to 95%. The sweetness of the fructose is 1.5-2.0 times that of the sucrose, and the fructose has low calorie and good flavor and can be used as a natural sweetener to replace the sucrose; the fructose metabolism is not restricted by insulin, and can be eaten by diabetic patients; the fructose can be used for producing bioethanol, 2, 3-butanediol and the like after being fermented by yeast and the like. Therefore, the inulase exonuclease can be applied to industries such as food, wine brewing and bioenergy (Singh RS et al. International Journal of Biological Macromolecules, 2017, 96: 312 to 322.).
The low-temperature enzyme has high catalytic activity at low temperature, and can be applied to the field of biotechnology under the requirement of low-temperature environment, such as fermentation temperature of sake and wine is generally less than 25 ℃, and aquaculture environment, washing and sewage treatment are generally carried out at low temperature. In addition, treatment at low temperature (if juice is clear) can prevent Microbial contamination, nutrient loss and food quality degradation, and conversion of medium temperature or high temperature treatment to low temperature treatment can also serve to reduce energy consumption (Cavicchiali et al, microbiological Biotechnology, 2011, 4 (4): 449 to 460.). Therefore, the improvement of the catalytic activity of the enzyme at low temperature is beneficial to the application of the enzyme in the industries of food, wine making, washing and the like.
The enzyme which is more sensitive to heat is easier to denature, the enzyme is easy to degrade due to thermal denaturation, the catalytic reaction of the enzyme is easy to control due to the characteristic, the enzyme can be inactivated by simple heat treatment, the operation is simple and effective, the use of the enzyme is safer, and the enzyme has application value in the industries of food, wine making, washing and the like. Therefore, the obtained heat-sensitive mutant enzyme is beneficial to the application of the enzyme in the industries of food, wine brewing, washing and the like.
Disclosure of Invention
The invention aims to provide a low-temperature exoinulase mutant MutAP122EK5 which can be applied to the industries of food, wine brewing, washing and the like.
In order to achieve the technical aim, the invention is specifically realized by the following technical scheme:
a low-temperature inulinase mutant MutAP122EK5, wherein the amino acid sequence of the mutant MutAP122EK5 is shown in SEQ ID NO. 1. Compared with the sequence AGC01505 (SEQ ID NO. 3) recorded in GenBank, 5 amino acids of MutAP122EK5 are different from the sequence, namely, the 122 th to 126 th amino acids of AGC01505 are AAPLP, and the 122 th to 126 th amino acids of MutAP122EK5 are EEDRK.
The optimal temperature of the mutant MutAP122EK5 is 30 ℃, the mutant MutAP122EK5 has 67 percent of enzyme activity and 18 percent of enzyme activity at 40 ℃ and 50 ℃, the temperature is increased from 0 ℃ to 20 ℃, and the activity of the MutAP122EK5 is increased from 22 percent to 76 percent; after the treatment at 50 ℃ for 10-60min, more than 73% of enzyme activity of MutAP122EK5 remains; after being treated at 55 ℃ for 10-60min, the enzyme activity of MutAP122EK5 is reduced from 54% to 7%.
The invention provides a coding gene mutAP122EK5 of a low-temperature inulinase mutant mutAP122EK5, and the nucleotide sequence of the coding gene mutAP122EK5 is shown in SEQ ID NO. 2.
Another object of the present invention is to provide a recombinant vector comprising the gene mutAP122EK5 encoding a mutant inulinase at low temperature.
Another purpose of the invention is to provide a recombinant bacterium containing a low-temperature inulase mutant encoding gene mutAP122EK5.
In addition, the application of the low-temperature exoinulase mutant MutAP122EK5 in the preparation of foods, wines and washing products is also in the protection scope of the invention.
The preparation method of the low-temperature inulase exonuclease mutant MutAP122EK5 specifically comprises the following steps:
1) Connecting a wild exoinulase gene inuAMN8 (SEQ ID NO. 4) with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing the inuAMN8;
2) Designing mutation primers 5 'CGAAGAGACCGAAAGGCCGGCAGGCGCAGTCG 3' and 5 'CCTTTCGGCTTCTCGTCACTGTAGGCACTGGTGTAAATG 3' by taking the plasmid pEasy-E1-inuAMN8 as a template, and obtaining a recombinant expression plasmid pEasy-E1-mutAP122EK5 containing mutAP122EK5 through PCR amplification;
3) Transforming the recombinant expression plasmid pEasy-E1-mutAP122EK5 into escherichia coli BL21 (DE 3) to obtain a recombinant strain containing the mutAP122EK5;
4) Culturing the recombinant strain, and inducing the expression of a recombinant exoinulase mutant MutAP122EK5;
5) Recovering and purifying the expressed recombinant exoinulase mutant MutAP122EK5;
6) And (4) measuring the activity.
The invention has the beneficial effects that:
compared with the wild enzyme InuAMN8, the mutant enzyme MutAP122EK5 has changed thermal activity and thermal stability, and the mutant enzyme MutAP122EK5 has higher activity at low temperature and reduced thermal stability. The optimum temperature of the wild enzyme InuAMN8 is 35 ℃, the enzyme activity is 94% and 40% at 40 ℃ and 50 ℃, the temperature is increased from 0 ℃ to 20 ℃, and the activity of the InuAMN8 is increased from 15% to 58%; the optimum temperature of the mutant enzyme MutAP122EK5 is 30 ℃, the mutant enzyme has 67 percent of enzyme activity and 18 percent of enzyme activity at 40 ℃ and 50 ℃, the temperature is increased from 0 ℃ to 20 ℃, and the activity of the MutAP122EK5 is increased from 22 percent to 76 percent; after the treatment at 55 ℃ for 10-60min, the enzyme activity of a wild enzyme InuAMN8 is reduced from 70% to 17%, and the enzyme activity of a mutant enzyme MutAP122EK5 is reduced from 54% to 7%. The low-temperature exoinulase mutant MutAP122EK5 can be applied to the industries of food, wine brewing, washing and the like.
Drawings
FIG. 1 is an SDS-PAGE analysis of the wild-type enzyme InuAMN8 and the mutant enzyme MutAP122EK5, where M: a protein Marker;
FIG. 2 shows the thermal activity of the purified wild-type enzyme InuAMN8 and the mutant enzyme MutAP122EK5;
FIG. 3 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5 at 50 ℃;
FIG. 4 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5 at 55 ℃.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Experimental materials and reagents in the following examples of the invention:
1. bacterial strain and carrier: escherichia coliEscherichia coli BL21 (DE 3) and expression vector pEasy-E1 are available from Beijing Quanji Biotech, inc.; arthrobacter (A), (B) and (C)Arthrobactersp.) is offered by university of Yunnan.
2. Enzymes and other biochemical reagents: nickel-NTA Agarose is purchased from QIAGEN company, DNA polymerase, dNTP and Mut Express II Fast Mutagenesis Kit is purchased from Nanjing Novosa company, inulin is purchased from Alfa Aesar company, bacterial genome DNA extraction Kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd, and other reagents are made in China (all can be purchased from common biochemical reagent company).
3. Culture medium
LB medium: peptone 10 g, yeast extract 5g, naCl 10 g, distilled water to 1000 mL, pH natural (about 7). On the basis of the solid medium, 2.0% (w/v) agar was added.
Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
EXAMPLE 1 construction and transformation of expression vector for wild enzyme InuAMN8
1) Extracting Arthrobacter genome DNA: centrifuging the liquid culture for 2d to obtain thallus, adding 1mL of lysozyme, treating at 37 deg.C for 60min, extracting Arthrobacter genome DNA according to the instruction of bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.), and standing at-20 deg.C for use.
2) According to the exonuclease nucleotide sequence JQ863111 (SEQ ID NO. 4) recorded by GenBank, primers 5 'ATGAATTCATTGACGACGGC 3' and 5 'TCAACGGCCGACGACGTCGA 3' are designed, PCR amplification is carried out by taking Arthrobacter genome DNA as a template, and the PCR reaction parameters are as follows: denaturation at 95 deg.C for 5 min; then, denaturation at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, elongation at 72 ℃ for 1min for 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles. The PCR result obtains the coding gene inuAMN8 of the wild exoinulase inuAMN8. inuAMN8 can also be obtained by gene synthesis based on the inulinase nucleotide sequence JQ 863111.
3) The exoinulase gene inuAMN8 and an expression vector pEasy-E1 are connected to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing the inuAMN8.
4) pEasy-E1-inuAMN8 was transformed into E.coli BL21 (DE 3) by heat shock to obtain a recombinant E.coli strain BL21 (DE 3)/inuAMN 8 comprising inuAMN8.
Example 2 construction and transformation of the expression vector for the mutase MutAP122EK5
1) Designing primers of 5 'CGAAGAGACCGAAAGGCCGGCAGGCGCAGTCG 3' and 5 'CCTTTCGTCTTCGTCACTGTAGGCACTGGTGTAAATG 3', and carrying out PCR amplification by taking a plasmid pEasy-E1-inuAMN8 as a template, wherein the PCR reaction parameters are as follows: denaturation at 95 ℃ for 30 sec; then denaturation at 95 ℃ for 15 sec, annealing at 70 ℃ for 15 sec, extension at 72 ℃ for 3min 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles. The PCR result yielded a recombinant expression linearized plasmid pEasy-E1-mutAP122EK5 containing mutAP122EK5.MutAP122EK5 and pEasy-E1-mutAP122EK5 can also be obtained by gene synthesis.
2) mu.L of the PCR product of the linearized plasmid pEasy-E1-mutAP122EK5 was digested with 1. Mu.L of DpnI enzyme at 37 ℃ for 1h.
3) The digestion products in (2) were placed at 37 ℃ for ligation for 30min using the Mut Express II Fast Mutagenesis Kit.
4) The ligation product in (3) was transformed into E.coli BL21 (DE 3) by heat shock to obtain a recombinant strain BL21 (DE 3)/mutAP 122EK5 comprising mutAP122EK5.
Example 3 preparation of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5
Recombinant strains BL21 (DE 3)/inuAMN 8 and BL21 (DE 3)/mutAP 122EK5 were inoculated to LB (containing 100. Mu.g mL) at an inoculum size of 0.1% respectively −1 Amp) in the culture medium, the mixture was rapidly shaken at 37 ℃ for 16 hours.
Then, the activated bacterial suspension was inoculated to fresh LB (containing 100. Mu.g mL) in an amount of 1% of the inoculum size −1 Amp) is quickly cultured in a culture solution for about 2 to 3 hours (OD 600 reaches 0.6 to 1.0) by shaking, IPTG with the final concentration of 0.7mM is added for induction, and the shaking culture is continued for about 20 hours at the temperature of 20 ℃. Centrifugation was carried out at 12000rpm for 5min to collect the cells. After the cells were suspended in an appropriate amount of pH =7.0 McIlvaine buffer, the cells were disrupted by ultrasonic waves in a low-temperature water bath. The crude enzyme solution concentrated in the cells is centrifuged at 13,000rpm for 10min, and then the supernatant is sucked up and the target protein is respectively subjected to affinity and purification by Nickel-NTA Agarose and 0 to 500mM imidazole.
SDS-PAGE results (FIG. 1) showed that both recombinant InuAMN8 and MutAP122EK5 were purified and the product was a single band.
Example 4 determination of the Properties of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5
1) Activity analysis of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5
The activity determination method adopts a 3, 5-dinitrosalicylic acid (DNS) method: dissolving the substrate inulin in a buffer solution to make the final concentration of the inulin be 0.5% (w/v); the reaction system contains 50 mu L of proper enzyme solution and 450 mu L of substrate; preheating a substrate at a reaction temperature for 5min, adding an enzyme solution, reacting for 10min, adding 750 mu L DNS (Domain name System) to terminate the reaction, boiling in water for 5min, cooling to room temperature, and measuring an OD (optical Density) value at a wavelength of 540 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down the substrate to produce 1. Mu. Mol reducing sugars (calculated as fructose) per minute under the given conditions.
2) Determination of the thermal Activity of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5
The enzymatic reaction was carried out at 0 to 60 ℃ in a buffer of pH = 7.0. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5.
The results show that: the optimum temperature of the wild enzyme InuAMN8 is 35 ℃, the enzyme activity is 94% and 40% at 40 ℃ and 50 ℃, the temperature is increased from 0 ℃ to 20 ℃, and the activity of the InuAMN8 is increased from 15% to 58%; the mutant enzyme MutAP122EK5 has an optimum temperature of 30 ℃ and an enzyme activity of 67% and 18% at 40 ℃ and 50 ℃ respectively, and the activity of MutAP122EK5 increases from 22% to 76% when the temperature is increased from 0 ℃ to 20 ℃ (FIG. 2).
3) Thermostability assay of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5
The enzyme solutions with the same enzyme amount were treated at 50 ℃ and 55 ℃ for 10 to 60min, and then enzymatic reactions were carried out at pH =7.0 and 37 ℃ with the untreated enzyme solution as a control. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutAP122EK5.
The results show that: after the treatment at 50 ℃ for 10 to 60min, over 81 percent of enzyme activity of a wild enzyme InuAMN8 is remained, and over 73 percent of enzyme activity of a mutant enzyme MutAP122EK5 is remained (figure 3); after treatment for 10-60min at 55 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the enzyme activity of the mutant enzyme MutAP122EK5 is reduced from 54% to 7% (figure 4).
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Finally, it should be noted that: although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention and it is intended to cover in the claims the invention as defined in the appended claims.
Sequence listing
<110> university of Yunnan Master
<120> low-temperature inulase mutant MutAP122EK5 and application thereof
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<213> wild enzyme (InuAMN 8)
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Pro Thr Gly Gly Trp Arg Ser Ala Met Ser Leu Pro Arg Glu Val Ser
325 330 335
Leu Thr Arg Val Asp Gly Lys Val Met Leu Arg Gln Gln Ala Ile Asp
340 345 350
Pro Leu Pro Glu Arg Glu Thr Gly His Val Arg Leu Gly Pro Gln Pro
355 360 365
Leu Ala Ser Gly Val Leu Asp Val Pro Ala Ala Ala Ser Val Ala Arg
370 375 380
Ile Asp Val Glu Leu Glu Pro Gly Ala Ala Ala Gly Val Gly Leu Val
385 390 395 400
Leu Arg Ala Gly Asp Asp Glu Arg Thr Val Leu Arg Tyr Asp Thr Ser
405 410 415
Asp Gly Met Leu Arg Leu Asp Arg Arg Glu Ser Gly Gln Val Ala Phe
420 425 430
His Glu Thr Phe Pro Ser Ile Glu Ala Met Ala Val Pro Leu Gln Gly
435 440 445
Gly Arg Leu Arg Leu Arg Val Tyr Leu Asp Arg Cys Ser Val Glu Val
450 455 460
Phe Ala Gln Asp Gly Leu Ala Thr Leu Thr Asp Leu Val Phe Pro Gly
465 470 475 480
Glu Ala Ser Thr Gly Leu Ala Ile Phe Ala Glu Gly Glu Gly Ala His
485 490 495
Leu Val Val Leu Asp Val Val Gly Arg
500 505
<210> 4
<211> 1518
<212> DNA
<213> wild enzyme Gene (inuAMN 8)
<400> 4
atgaattcat tgacgacggc ggcgggcgcc acgttggctg ccaccgacca gtaccggccc 60
gcgttccact acaccgccga acggaactgg ttgaacgatc cgaacgggct ggtgtacctc 120
aacggcacct accacctctt ctaccagcac aacccgttcg gcgctgactg gggcaacatg 180
tcctgggggc acgccacctc gcgggacctg ctgcactggg acgagcagcc cgtggccatt 240
ccgtgcgacg aacacgaggc catcttctcc ggctcggcgg tattcgatca gcacaacacc 300
agcggcctcg gcacagcggc caatcccccg ctggtggcca tttacaccag tgcctacagt 360
gacgccgcgc cgcttccggg ccggcaggcg cagtcgctcg cctacagcct cgacgaaggc 420
cggacctgga ccaagtacca cggcaatccc gtgctggacc gcgcgtccgc tgacttccgc 480
gatccaaagg ttttttggta cgacggcggc gccggaagtt actgggtgat ggtcgccgtc 540
gaggcggtgc agcgccaggt agtgctgtac aagtcggccg acctgaaggc gtgggaacac 600
ctgagcacct ttggccctgc caacgccacc ggcggcgtct gggaatgccc ggacctgttt 660
gagctgcccg tggacgggaa tccggaggac aaccggtggg tcctcattgt gaacatcaac 720
ccgggcggca ttgccggcgg ctccgcggga cagtacttcg tgggagagtt cgacggcgtg 780
gcgttccatt ccggatcgac tgtcaccgag ggcctccaga aggacagcag ccggatgcgg 840
gagtacggct ggctggactg ggggcgggac tactacgccg ccgtttcgtt cagcaacgtg 900
ccggacgggc gccggatcat gatcggctgg atgaacaact gggactacgc ccgcgagacg 960
cccaccggcg gctggcgcag cgccatgtcc ctgccgcggg aggtgtcgct gacccgggta 1020
gacgggaaag tgatgcttcg gcagcaagcc attgatccgt tgccggagcg ggaaacaggg 1080
cacgtccggc tggggccgca gcccttggcg tccggcgttc tggacgttcc ggccgccgca 1140
tccgtggcgc ggatcgacgt tgagctggag ccgggcgctg ccgcgggagt gggactggtg 1200
cttcgggcgg gggacgatga gcggacggtc ctccgctacg acacttcgga cgggatgctg 1260
cggctggacc gccgcgaatc cgggcaggtt gccttccacg aaaccttccc gtcgatcgaa 1320
gccatggccg tgcccttgca gggaggccgg ctgcgcctgc gggtctacct ggaccgctgc 1380
tcggtggagg ttttcgccca ggacgggctc gccacgctca ctgacctggt gttccccggg 1440
gaggcgagca cgggcctggc catcttcgcc gaaggtgagg gggcgcacct cgtggtgctc 1500
gacgtcgtcg gccgttga 1518
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cgaagaagac cgaaagggcc ggcaggcgca gtcg 34
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cctttcggtc ttcttcgtca ctgtaggcac tggtgtaaat g 41
Claims (6)
1. The low-temperature inulase mutant MutAP122EK5 is characterized in that the amino acid sequence of the mutant MutAP122EK5 is shown as SEQ ID No. 1.
2. The mutant MutAP122EK5 encoding gene MutAP122EK5 as claimed in claim 1, wherein the nucleotide sequence of the encoding gene MutAP122EK5 is as shown in SEQ ID No. 2.
3. A recombinant vector comprising the gene mutAP122EK5 according to claim 2.
4. A recombinant bacterium comprising the encoded gene mutAP122EK5 according to claim 2.
5. The method for preparing the low temperature exoinulase mutant MutAP122EK5 as claimed in claim 1, characterized in that it comprises the following steps:
1) Connecting a wild exoinulase gene inuAMN8 shown as SEQ ID NO.4 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing the inuAMN8;
2) Designing mutation primers 5 'CGAAGAGACCGAAAGGCCGGCAGGCGCAGTCG 3' and 5 'CCTTTCGGCTTCTCGTCACTGTAGGCACTGGTGTAAATG 3' by taking the plasmid pEasy-E1-inuAMN8 as a template, and obtaining a recombinant expression plasmid pEasy-E1-mutAP122EK5 containing mutAP122EK5 through PCR amplification;
3) Transforming the recombinant expression plasmid pEasy-E1-mutAP122EK5 into escherichia coli BL21 (DE 3) to obtain a recombinant strain containing the mutAP122EK5;
4) Culturing the recombinant strain, and inducing the expression of a recombinant exoinulase mutant MutAP122EK5;
5) Recovering and purifying the expressed recombinant exoinulase mutant MutAP122EK5;
6) And (4) measuring the activity.
6. Use of the mutant MutAP122EK5 according to claim 1 for brewing wine.
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| CN112725305B (en) * | 2021-01-13 | 2022-11-04 | 云南师范大学 | Thermohaline-sensitive inulase mutant MutY119D and preparation method thereof |
| CN112725306B (en) * | 2021-01-13 | 2022-06-24 | 云南师范大学 | Inulase mutant MutY119T with changed thermal salinity and application thereof |
| CN112813053B (en) * | 2021-01-13 | 2022-06-24 | 云南师范大学 | Inulase mutant MutY119H and preparation method thereof |
| CN112813050B (en) * | 2021-01-13 | 2022-08-30 | 云南师范大学 | Exo-inulinase mutant MutP126Q with reduced thermostability |
| CN112813054B (en) * | 2021-01-13 | 2023-07-28 | 云南师范大学 | Inulase mutant MutS117N with low-temperature salt tolerance changed and application thereof |
| CN112813052B (en) * | 2021-01-13 | 2022-08-26 | 云南师范大学 | Exo-inulase mutant MutDP121ET6 with improved low-temperature activity |
| CN112813051B (en) * | 2021-01-13 | 2023-07-28 | 云南师范大学 | Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application |
| CN112725307B (en) * | 2021-01-13 | 2022-09-16 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutG169 delta 4 with reduced heat resistance and application thereof |
| CN112852781B (en) * | 2021-01-13 | 2023-06-27 | 云南师范大学 | Heat-sensitive inulase mutant MutY119N and application thereof |
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