CN112813051B - Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application - Google Patents
Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application Download PDFInfo
- Publication number
- CN112813051B CN112813051B CN202110041031.1A CN202110041031A CN112813051B CN 112813051 B CN112813051 B CN 112813051B CN 202110041031 A CN202110041031 A CN 202110041031A CN 112813051 B CN112813051 B CN 112813051B
- Authority
- CN
- China
- Prior art keywords
- mutp124g
- mutant
- ala
- gly
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- 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
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
- C12N15/1031—Mutagenizing nucleic acids mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
-
- 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
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01007—Inulinase (3.2.1.7)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention relates to the technical field of genetic engineering and protein modification, in particular to a low Wen Waiqie inulinase mutant MutP124G with improved thermal adaptability and application thereof, wherein the amino acid sequence of the mutant MutP124G is obtained by mutating 124 th amino acid (proline) of wild exoinulinase InuAMN8 into glycine, and the sequence is shown as SEQ ID NO. 1. Compared with the wild enzyme InuAMN8, the mutant enzyme MutP124G has higher catalytic activity at low temperature and is more suitable for the biotechnology field under the low-temperature environment requirement. The low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability can be applied to industries such as food, 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 Wen Waiqie inulinase mutant MutP124G with improved thermal adaptability and application thereof.
Background
Jerusalem artichoke is an important crop for replacing grain starchy raw materials, and is easy to plant, high in yield, cold-resistant, barren-resistant and drought-resistant. The main component of Jerusalem artichoke tuber is inulin, and the inulin content is 19% of its wet weight or 70% of dry weight.
Inulin is a levan with one molecule of glucose residues. The exoinulase hydrolyzes beta-2, 1 glycosidic bonds from the non-reducing end of inulin one by one, fructose and a small part of glucose are finally generated, and the fructose content in the fructose syrup can reach 95% of the total sugar content. The hydrolysate of inulin, namely fructose, is widely used in industries of food, medicine, bioenergy and the like, can be used as a natural sweetener to replace sucrose, can be eaten by diabetics, and can be used for producing bioethanol and the like. Thus, exoinulase can be used in the industries of food, wine and bioenergy (Singh RS et al International Journal of Biological Macromolecules,2017, 96:312-322.).
Food treatment at low temperature can prevent microbial contamination, nutrient loss and food quality degradation, fermentation of sake and wine, aquaculture environment, washing, and sewage treatment are generally performed at low temperature; in addition, cryogenic treatment can reduce energy consumption (Cavicchioli et al microbial Biotechnology,2011,4 (4): 449-460.). Therefore, the low-temperature enzyme has important development value.
The low Wen Waiqie inulase can be used for fermenting wine, pickling food, decontaminating, hydrolyzing inulin at low temperature to prepare high concentration fructose syrup, etc. (Zhou et al journal ofBioscience and Bioengineering,2015,119 (3): 267-274). The low Wen Waiqie inulase has unique advantages in practical application, such as high catalytic activity in a low-temperature environment, energy conservation, cost saving, enzyme inactivation by simple heat treatment, and simple and effective operation. Thus, obtaining a mutant enzyme with higher activity at low temperatures would be more advantageous for the application of the enzyme in the field of low temperature biotechnology.
Disclosure of Invention
The invention aims to provide a low Wen Waiqie inulase mutant MutP124G with improved heat adaptability, which can be applied to industries such as food, brewing, washing and the like.
In order to achieve the technical aim, the invention is specifically realized by the following technical scheme:
the low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability, and the amino acid sequence of the mutant MutP124G is shown in SEQ ID NO. 1. In contrast to the exonuclease sequence AGC01505 (SEQ ID No. 3) recorded by GenBank, amino acid 124 of MutP124G is glycine and amino acid 124 of AGC01505 is proline.
The mutant MutP124G has an optimal temperature of 30 ℃, and has 48%, 67%, 89% and 69% of enzyme activities at 10 ℃,20 ℃, 25 ℃ and 40 ℃, respectively; after being treated for 10-60 min at 50 ℃, the MutP124G keeps more than 73% of enzyme activity; after the treatment at 55 ℃ for 10 to 60 minutes, the enzyme activity of MutP124G is reduced from 64 percent to 22 percent; the enzyme hydrolyzes inulin to produce fructose.
The invention provides a coding gene mutP124G of a low Wen Waiqie inulase mutant mutP124G, and the nucleotide sequence of the coding gene mutP124G is shown as SEQ ID NO. 2.
Another object of the present invention is to provide a recombinant vector comprising a gene mutP124G encoding a low-temperature exoinulase mutant.
Another object of the present invention is to provide a recombinant bacterium comprising a gene mutP124G encoding a low-temperature exoinulase mutant.
In addition, the application of the exoinulase mutant MutP124G in the preparation of food, brewing and washing products is also within the protection scope of the invention.
The preparation method of the low Wen Waiqie inulase mutant MutP124G provided by the invention 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 inuAMN8;
2) Designing mutation primers 5'TGAC GCCGCGGGACTTCCGGGCCGGCAGGCGC3' and 5'GAAGTCCCG CGGCGTCACTGTAGGCACTGGTG3' by taking a plasmid pEasy-E1-inuAMN8 as a template, and obtaining a recombinant expression plasmid pEasy-E1-mutP124G containing mu tP124G through PCR amplification;
3) Transforming the recombinant expression plasmid pEasy-E1-mutP124G into escherichia coli BL21 (DE 3) to obtain a recombinant strain containing mutP124G;
4) Culturing the recombinant strain, and inducing the expression of a recombinant exoinulase mutant MutP124G;
5) Recovering and purifying the expressed recombinant exoinulase mutant MutP124G;
6) And (3) activity measurement.
The beneficial effects of the invention are as follows:
compared with the wild enzyme InuAMN8, the mutant enzyme MutP124G has higher catalytic activity at low temperature. The purified wild-type enzyme InuAMN8 had an optimal temperature of 35℃and enzyme activities of 32%, 58%, 74% and 94% at 10℃and 20℃and 25℃and 40℃respectively, while the mutant enzyme MutP124G had an optimal temperature of 30℃and enzyme activities of 48%, 67%, 89% and 69% at 10℃and 20℃and 25℃and 40℃respectively. The low Wen Waiqie inulase mutant MutP124G can be applied to industries such as food, brewing, washing and the like.
Drawings
FIG. 1 is a SDS-PAGE analysis of the wild-type enzyme InuAMN8 and the mutant enzyme MutP124G, wherein M: protein markers;
FIG. 2 shows the thermal activity of purified wild-type enzyme InuAMN8 and mutant enzyme MutP124G;
FIG. 3 shows the stability of purified wild-type enzyme InuAMN8 and mutant enzyme MutP124G at 50 ℃;
FIG. 4 shows the stability of purified wild-type enzyme InuAMN8 and mutant enzyme MutP124G at 55deg.C;
FIG. 5 is a product analysis of purified wild-type enzyme InuAMN8 and mutant enzyme MutP124G hydrolyzed inulin, wherein W: the wild enzyme InuAMN8 hydrolyzes the inulin product; CK: a control group containing inulin and inactivated wild-type enzyme InuAMN8 (boiled for 10 min); f: fructose; g: glucose; mut: the mutase MutP124G hydrolyzes the inulin product.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental materials and reagents in the following examples of the invention:
1. strains and vectors: coli Escherichia coli BL (DE 3) and expression vector pEasy-E1 are available from Beijing all gold biotechnology Co., ltd; arthrobacter sp.
2. Enzymes and other biochemical reagents: nickel-NTAAgarose is available from QIAGEN, DNA polymerase, dNTPs and MutII Fast Mutagenesis Kit the kit is purchased from Nanjing Norvigator, inulin from AlfaAesar, and the bacterial genomic DNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd, all other being domestic testAgents (all available from general Biochemical agents Co.).
3. Culture medium
LB medium: peptone 10g,Yeast extract 5g,NaCl 10g, distilled water was added to 1000mL and the pH was natural (about 7). The solid medium was supplemented with 2.0% (w/v) agar.
Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
EXAMPLE 1 construction and transformation of wild-type enzyme InuAMN8 expression vector
1) Extracting genome DNA of arthrobacter: the bacterial liquid after 2d culture is centrifuged to obtain thalli, 1mL of lysozyme is added, the treatment is carried out for 60min at 37 ℃, and then the Arthrobacter genomic DNA is extracted according to the specification of a bacterial genomic DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.) and is placed at-20 ℃ for standby.
2) According to the exonuclease nucleotide sequence JQ863111 (SEQ ID NO. 4) recorded in GenBank, primers 5'ATGAATTCATTGACGACGGC 3' and 5'TCAACGGCCGACGACGTCGA 3' are designed, PCR amplification is carried out by taking the arthrobacter genomic DNA as a template, and the PCR reaction parameters are as follows: denaturation at 95℃for 5min; then denaturation at 95℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 1min for 30sec, and incubation at 72℃for 5min after 30 cycles. And obtaining the encoding gene inuAMN8 of the wild exoinulase inuAMN8 by a PCR result. Based on the exonuclease nucleotide sequence JQ863111, inuAMN8 can also be obtained by gene synthesis.
3) And (3) connecting the exoinulase gene inuAMN8 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN8.
4) pEasy-E1-inuAMN8 was transformed into E.coli BL21 (DE 3) by heat shock to obtain recombinant E.coli strain BL21 (DE 3)/inuAMN 8 containing inuAMN8.
EXAMPLE 2 construction and transformation of mutant enzyme MutP124G expression vector
1) Primers 5'TGACGCCGCGGGACTTCCGGGCCGGCAGGCGC3' and 5'GAAGTCCCGCGGCGTCACTGTAGGCACTGGTG3' were designed and PCR amplification was performed using plasmid pEasy-E1-inuAMN8 as template, with the following parameters: denaturation at 95℃for 30sec; then denaturation at 95℃for 15sec, annealing at 70℃for 15sec, elongation at 72℃for 3min 30sec, and incubation at 72℃for 5min after 30 cycles. The PCR result gave a recombinant expression linearized plasmid pEasy-E1-mutP124G containing mutP124G. mutP124G and pEasy-E1-mutP124G can also be obtained by gene synthesis.
2) To 50. Mu.L of the PCR product of linearized plasmid pEasy-E1-mutP124G, 1. Mu.L of DpnI enzyme was added and digested at 37℃for 1h.
3) By MutII Fast Mutagenesis Kit kit, the digestion products of (2) are ligated for 30min at 37 ℃.
4) The ligation product of (3) was transformed into E.coli BL21 (DE 3) by heat shock to obtain a recombinant strain BL21 (DE 3)/mutP 124G containing mutP124G.
Example 3 preparation of recombinant wild-type enzyme InuAMN8 and mutant enzyme MutP124G
Recombinant strains BL21 (DE 3)/inuAMN 8 and BL21 (DE 3)/mutP 124G were inoculated to LB (containing 100. Mu.g mL) at an inoculum size of 0.1%, respectively -1 Amp) medium, the medium was rapidly shaken at 37 ℃ for 16h.
The activated bacterial solutions were then inoculated into fresh LB (containing 100. Mu.g mL) at 1% of the inoculum size -1 Amp) was cultured in a medium for about 2 to 3 hours (OD 600 was 0.6 to 1.0) with shaking, and then induction was performed by adding IPTG at a final concentration of 0.7mM, followed by shaking at 20℃for about 20 hours. The cells were collected by centrifugation at 12000rpm for 5min. After the cells were suspended at an appropriate ph=7.0. 7.0McIlv aine buffer, the cells were sonicated in a low-temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the target protein was affinity purified with Nickel-NTA Agarose and 0-500mM imidazole, respectively.
SDS-PAGE results (FIG. 1) showed that both recombinant InuAMN8 and MutP124G were purified and the product was a single band.
EXAMPLE 4 characterization of purified recombinant wild-type enzyme InuAMN8 and mutant enzyme MutP124G
1) Activity analysis of purified recombinant wild-type enzyme InuAMN8 and mutant enzyme MutP124G
The activity determination method adopts a 3, 5-dinitrosalicylic acid (DNS) method: dissolving substrate inulin in buffer solution to a final concentration of 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 of DNS to terminate the reaction, boiling with boiling water for 5min, cooling to room temperature, and measuring an OD 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 under the given conditions to produce 1. Mu. Mol of reducing sugar (in fructose) per minute.
2) Thermal Activity assay of purified recombinant wild-type enzyme InuAMN8 and mutant enzyme MutP124G
The enzymatic reaction was carried out at 0-60 ℃ in a buffer at ph=7.0. Inulin is used as a substrate and reacts for 10min, and the enzymatic properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutP124G are measured.
The results show that: the optimal temperature for the wild-type enzyme InuAMN8 was 35℃and at 10℃20℃25℃and 40℃32%, 58%, 74% and 94% respectively, while the optimal temperature for the mutant enzyme MutP124G was 30℃and at 10℃20℃25℃and 40℃48%, 67%, 89% and 69% respectively (FIG. 2).
3) Thermal stability assay of purified recombinant wild-type enzyme InuAMN8 and mutant enzyme MutP124G
The enzyme solutions with the same enzyme amount are respectively treated at 50 ℃ and 55 ℃ for 10-60 min, and then the enzymatic reaction is carried out at the pH=7.0 and 37 ℃, and untreated enzyme solution is used as a control. Inulin is used as a substrate and reacts for 10min, and the enzymatic properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutP124G are measured.
The results show that: after 10-60 min of treatment at 50 ℃, the wild enzyme InuAMN8 maintains more than 81% of enzyme activity, and MutP124G maintains more than 73% of enzyme activity (figure 3); after treatment at 55℃for 10-60 min, the enzyme activity of the wild-type enzyme InuAMN8 was reduced from 70% to 17% and the enzyme activity of MutP124G was reduced from 64% to 22% (FIG. 4).
4) Analysis of the products of the hydrolysis of inulin by the purified recombinant wild-type enzyme InuAMN8 and the mutant enzyme MutP124G
The product analysis reaction system contained 450. Mu.L of 0.5% (w/v) inulin, and 50. Mu.L of an appropriate dilution enzyme solution (total 0.1U enzyme solution). The reaction was terminated after 4h of enzymatic reaction at pH7.0 and 37 ℃. The product was analyzed by thin layer chromatography (using high performance thin layer chromatography silica gel plate type G from Qingdao ocean chemical Co., ltd.).
The thin layer chromatography steps are as follows:
(1) preparing a developing agent (20 mL of glacial acetic acid, 20mL of double distilled water and 40mL of n-butanol, uniformly mixing), pouring a proper amount of the developing agent into a developing groove, and standing for about 30 min;
(2) activating silica gel plate in 110 deg.C oven for 30min, cooling, scribing, and spotting (0.5 μl each time, blow drying, and spotting for 3 times);
(3) placing the silica gel plate at one end of the sample application downwards into the unfolding groove, wherein the sample application point does not sink into the unfolding agent;
(4) when the spreading agent reaches 1.5cm from the upper edge of the silica gel plate, taking out the silica gel plate, drying, and spreading again;
(5) after the second expansion is finished, the silica gel plate is directly immersed into a proper amount of color reagent (1 g of diphenylamine is dissolved in 50mL of acetone, 1mL of aniline and 5mL of 85% phosphoric acid are added after the dissolution, and the mixture is uniformly mixed and prepared at present);
(6) after a few seconds, the silica gel plate was immediately removed and placed in an oven at 90℃for 10-15min to develop the spots.
The results show that: the products of the hydrolysis of inulin by the wild-type enzyme InuAMN8 and the mutant enzyme MutP124G were almost all fructose (fig. 5).
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, what should be said is: the above embodiments are only for illustrating the technical aspects of the present invention, and 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, which is intended to be encompassed by the claims.
Sequence listing
<110> university of Yunnan teachers and students
<120> mutant MutP124G with improved thermal adaptability and low Wen Waiqie inulase and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 505
<212> PRT
<213> mutant enzyme (MutP 124G)
<400> 1
Met Asn Ser Leu Thr Thr Ala Ala Gly Ala Thr Leu Ala Ala Thr Asp
1 5 10 15
Gln Tyr Arg Pro Ala Phe His Tyr Thr Ala Glu Arg Asn Trp Leu Asn
20 25 30
Asp Pro Asn Gly Leu Val Tyr Leu Asn Gly Thr Tyr His Leu Phe Tyr
35 40 45
Gln His Asn Pro Phe Gly Ala Asp Trp Gly Asn Met Ser Trp Gly His
50 55 60
Ala Thr Ser Arg Asp Leu Leu His Trp Asp Glu Gln Pro Val Ala Ile
65 70 75 80
Pro Cys Asp Glu His Glu Ala Ile Phe Ser Gly Ser Ala Val Phe Asp
85 90 95
Gln His Asn Thr Ser Gly Leu Gly Thr Ala Ala Asn Pro Pro Leu Val
100 105 110
Ala Ile Tyr Thr Ser Ala Tyr Ser Asp Ala Ala Gly Leu Pro Gly Arg
115 120 125
Gln Ala Gln Ser Leu Ala Tyr Ser Leu Asp Glu Gly Arg Thr Trp Thr
130 135 140
Lys Tyr His Gly Asn Pro Val Leu Asp Arg Ala Ser Ala Asp Phe Arg
145 150 155 160
Asp Pro Lys Val Phe Trp Tyr Asp Gly Gly Ala Gly Ser Tyr Trp Val
165 170 175
Met Val Ala Val Glu Ala Val Gln Arg Gln Val Val Leu Tyr Lys Ser
180 185 190
Ala Asp Leu Lys Ala Trp Glu His Leu Ser Thr Phe Gly Pro Ala Asn
195 200 205
Ala Thr Gly Gly Val Trp Glu Cys Pro Asp Leu Phe Glu Leu Pro Val
210 215 220
Asp Gly Asn Pro Glu Asp Asn Arg Trp Val Leu Ile Val Asn Ile Asn
225 230 235 240
Pro Gly Gly Ile Ala Gly Gly Ser Ala Gly Gln Tyr Phe Val Gly Glu
245 250 255
Phe Asp Gly Val Ala Phe His Ser Gly Ser Thr Val Thr Glu Gly Leu
260 265 270
Gln Lys Asp Ser Ser Arg Met Arg Glu Tyr Gly Trp Leu Asp Trp Gly
275 280 285
Arg Asp Tyr Tyr Ala Ala Val Ser Phe Ser Asn Val Pro Asp Gly Arg
290 295 300
Arg Ile Met Ile Gly Trp Met Asn Asn Trp Asp Tyr Ala Arg Glu Thr
305 310 315 320
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> 2
<211> 1518
<212> DNA
<213> mutant enzyme Gene (mutP 124G)
<400> 2
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
gacgccgcgg gacttccggg 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> 3
<211> 505
<212> PRT
<213> wild-type enzyme (InuAMN 8)
<400> 3
Met Asn Ser Leu Thr Thr Ala Ala Gly Ala Thr Leu Ala Ala Thr Asp
1 5 10 15
Gln Tyr Arg Pro Ala Phe His Tyr Thr Ala Glu Arg Asn Trp Leu Asn
20 25 30
Asp Pro Asn Gly Leu Val Tyr Leu Asn Gly Thr Tyr His Leu Phe Tyr
35 40 45
Gln His Asn Pro Phe Gly Ala Asp Trp Gly Asn Met Ser Trp Gly His
50 55 60
Ala Thr Ser Arg Asp Leu Leu His Trp Asp Glu Gln Pro Val Ala Ile
65 70 75 80
Pro Cys Asp Glu His Glu Ala Ile Phe Ser Gly Ser Ala Val Phe Asp
85 90 95
Gln His Asn Thr Ser Gly Leu Gly Thr Ala Ala Asn Pro Pro Leu Val
100 105 110
Ala Ile Tyr Thr Ser Ala Tyr Ser Asp Ala Ala Pro Leu Pro Gly Arg
115 120 125
Gln Ala Gln Ser Leu Ala Tyr Ser Leu Asp Glu Gly Arg Thr Trp Thr
130 135 140
Lys Tyr His Gly Asn Pro Val Leu Asp Arg Ala Ser Ala Asp Phe Arg
145 150 155 160
Asp Pro Lys Val Phe Trp Tyr Asp Gly Gly Ala Gly Ser Tyr Trp Val
165 170 175
Met Val Ala Val Glu Ala Val Gln Arg Gln Val Val Leu Tyr Lys Ser
180 185 190
Ala Asp Leu Lys Ala Trp Glu His Leu Ser Thr Phe Gly Pro Ala Asn
195 200 205
Ala Thr Gly Gly Val Trp Glu Cys Pro Asp Leu Phe Glu Leu Pro Val
210 215 220
Asp Gly Asn Pro Glu Asp Asn Arg Trp Val Leu Ile Val Asn Ile Asn
225 230 235 240
Pro Gly Gly Ile Ala Gly Gly Ser Ala Gly Gln Tyr Phe Val Gly Glu
245 250 255
Phe Asp Gly Val Ala Phe His Ser Gly Ser Thr Val Thr Glu Gly Leu
260 265 270
Gln Lys Asp Ser Ser Arg Met Arg Glu Tyr Gly Trp Leu Asp Trp Gly
275 280 285
Arg Asp Tyr Tyr Ala Ala Val Ser Phe Ser Asn Val Pro Asp Gly Arg
290 295 300
Arg Ile Met Ile Gly Trp Met Asn Asn Trp Asp Tyr Ala Arg Glu Thr
305 310 315 320
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-type 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
atgaattcat tgacgacggc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tcaacggccg acgacgtcga 20
<210> 7
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tgacgccgcg ggacttccgg gccggcaggc gc 32
<210> 8
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gaagtcccgc ggcgtcactg taggcactgg tg 32
Claims (6)
1. The mutant MutP124G with improved thermal adaptability and low Wen Waiqie inulase is characterized in that the amino acid sequence of the mutant MutP124G is shown as SEQ ID NO. 1.
2. The mutant mutP124G encoding gene mutP124G of claim 1, wherein the nucleotide sequence of said encoding gene mutP124G is shown in SEQ ID NO. 2.
3. A recombinant vector comprising the coding gene mutP124G of claim 2.
4. A recombinant bacterium comprising the coding gene mutP124G of claim 2.
5. The method for preparing the low Wen Waiqie inulase mutant MutP124G as claimed in claim 1, comprising the following steps:
1) Connecting a wild exoinulase gene inuAMN8 with a nucleotide sequence shown as SEQ ID NO.4 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN8;
2) Designing mutation primers 5 'TGACGCCGCGGGACTTCCGGGCCGGCAGGC3' and 5 'GAAGTCCCCGCGGCGTCACCTGTAGGCACTGGTG3' by taking plasmid pEasy-E1-inuAMN8 as a template, and obtaining recombinant expression plasmid pEasy-E1-mutP124G containing mutP124G through PCR amplification;
3) Transforming the recombinant expression plasmid pEasy-E1-mutP124G into escherichia coli BL21 (DE 3) to obtain a recombinant strain containing mutP124G;
4) Culturing the recombinant strain, and inducing the expression of a recombinant exoinulase mutant MutP124G;
5) Recovering and purifying the expressed recombinant exoinulase mutant MutP124G;
6) And (3) activity measurement.
6. Use of the mutant MutP124G of claim 1 in the preparation of wine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110041031.1A CN112813051B (en) | 2021-01-13 | 2021-01-13 | Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110041031.1A CN112813051B (en) | 2021-01-13 | 2021-01-13 | Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112813051A CN112813051A (en) | 2021-05-18 |
| CN112813051B true CN112813051B (en) | 2023-07-28 |
Family
ID=75869056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110041031.1A Active CN112813051B (en) | 2021-01-13 | 2021-01-13 | Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112813051B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN112852782B (en) * | 2021-01-13 | 2023-07-28 | 云南师范大学 | Low-temperature adaptive improved low Wen Waiqie inulase mutant MutDL121EK5 and application thereof |
| CN112725304B (en) * | 2021-01-13 | 2022-10-18 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof |
| CN112813050B (en) * | 2021-01-13 | 2022-08-30 | 云南师范大学 | Exo-inulinase mutant MutP126Q with reduced thermostability |
| CN112813052B (en) * | 2021-01-13 | 2022-08-26 | 云南师范大学 | Exo-inulase mutant MutDP121ET6 with improved low-temperature activity |
| 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 |
| CN112725305B (en) * | 2021-01-13 | 2022-11-04 | 云南师范大学 | Thermohaline-sensitive inulase mutant MutY119D and preparation method thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170000890A (en) * | 2015-06-24 | 2017-01-04 | 경상북도(관련부서:경상북도산림자원개발원) | Fermented alcoholic beverage using Jerusalem artichoke and method of manufacturing the same |
| MX2016016731A (en) * | 2016-12-15 | 2018-06-14 | Centro De Investig Y Asistencia En Tecnologia Y Diseno Del Estado De Jalisco A C | Fructans enzymatic hydrolysis process through the use of a b-fructofuranosidase or inulinase of the aspergillus kawachii fungi. |
| CN112646792A (en) * | 2021-01-13 | 2021-04-13 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutA122 delta 5 with reduced thermal stability and application |
| CN112646793A (en) * | 2021-01-13 | 2021-04-13 | 云南师范大学 | Inulase mutant MutS120D with improved low-temperature adaptability and salt adaptability and application thereof |
| CN112708607A (en) * | 2021-01-13 | 2021-04-27 | 云南师范大学 | Inulase mutant MutS120R with changed thermal adaptability and application thereof |
| CN112725307A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutG169 delta 4 with reduced heat resistance and application thereof |
| CN112725304A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | High-activity low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof |
| CN112725308A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutA118H and application thereof |
| CN112725306A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Inulase mutant MutY119T with changed thermal salinity and application thereof |
| CN112813054A (en) * | 2021-01-13 | 2021-05-18 | 云南师范大学 | Inulase mutant MutS117N with changed low-temperature salt tolerance and application thereof |
| CN112831485A (en) * | 2021-01-13 | 2021-05-25 | 云南师范大学 | Exo-inulase mutant MutDR121EH9 with improved low-temperature activity |
| CN112852782A (en) * | 2021-01-13 | 2021-05-28 | 云南师范大学 | Low-temperature-adaptively-improved low-temperature inulinase mutant MutDL121EK5 and application thereof |
-
2021
- 2021-01-13 CN CN202110041031.1A patent/CN112813051B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20170000890A (en) * | 2015-06-24 | 2017-01-04 | 경상북도(관련부서:경상북도산림자원개발원) | Fermented alcoholic beverage using Jerusalem artichoke and method of manufacturing the same |
| MX2016016731A (en) * | 2016-12-15 | 2018-06-14 | Centro De Investig Y Asistencia En Tecnologia Y Diseno Del Estado De Jalisco A C | Fructans enzymatic hydrolysis process through the use of a b-fructofuranosidase or inulinase of the aspergillus kawachii fungi. |
| CN112646792A (en) * | 2021-01-13 | 2021-04-13 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutA122 delta 5 with reduced thermal stability and application |
| CN112646793A (en) * | 2021-01-13 | 2021-04-13 | 云南师范大学 | Inulase mutant MutS120D with improved low-temperature adaptability and salt adaptability and application thereof |
| CN112708607A (en) * | 2021-01-13 | 2021-04-27 | 云南师范大学 | Inulase mutant MutS120R with changed thermal adaptability and application thereof |
| CN112725307A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutG169 delta 4 with reduced heat resistance and application thereof |
| CN112725304A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | High-activity low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof |
| CN112725308A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutA118H and application thereof |
| CN112725306A (en) * | 2021-01-13 | 2021-04-30 | 云南师范大学 | Inulase mutant MutY119T with changed thermal salinity and application thereof |
| CN112813054A (en) * | 2021-01-13 | 2021-05-18 | 云南师范大学 | Inulase mutant MutS117N with changed low-temperature salt tolerance and application thereof |
| CN112831485A (en) * | 2021-01-13 | 2021-05-25 | 云南师范大学 | Exo-inulase mutant MutDR121EH9 with improved low-temperature activity |
| CN112852782A (en) * | 2021-01-13 | 2021-05-28 | 云南师范大学 | Low-temperature-adaptively-improved low-temperature inulinase mutant MutDL121EK5 and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| Junpei Zhou等.Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp. MN8 and its potential for use in the production of fructose at low temperatures.《Journal of Bioscience and Bioengineering》.2014,第119卷(第3期),第267-274页. * |
| Zhou,J.等.Arthrobacter sp. MN8 glycosyl hydrolase family 32 exo-inulinase (inuA) gene, complete cds.《Genbank Database》.2015,Accession No：JQ863111.1. * |
| Zhou,J.等.glycosyl hydrolase family 32 exo-inulinase [Arthrobacter sp. MN8].《Genbank Database》.2015,Accession No：AGC01505.1. * |
| 唐艳斌等.产菊粉酶曲霉菌株的筛选和发酵条件的优化.《饲料研究》.2008,(第07期),第1-4页. * |
| 岑潇龙.外切菊粉酶InuAMN8的热盐耐受性研究.《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑》.2021,A006-138. * |
| 李建颖.《食品添加剂速查手册》.南开大学出版社,2017,(2017年11月第1版),第103-104页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112813051A (en) | 2021-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112813051B (en) | Low Wen Waiqie inulase mutant MutP124G with improved thermal adaptability and application | |
| CN112725308B (en) | Low-temperature inulase exonuclease mutant MutA118H and application thereof | |
| CN112813054B (en) | Inulase mutant MutS117N with low-temperature salt tolerance changed and application thereof | |
| CN112708607B (en) | Inulase mutant MutS120R with changed thermal adaptability and application thereof | |
| CN112646793B (en) | Inulase mutant MutS120D with improved low-temperature adaptability and salt adaptability and application thereof | |
| CN112646792B (en) | Low-temperature inulase exonuclease mutant MutA122 delta 5 with reduced thermal stability and application | |
| CN112725305B (en) | Thermohaline-sensitive inulase mutant MutY119D and preparation method thereof | |
| CN112725306B (en) | Inulase mutant MutY119T with changed thermal salinity and application thereof | |
| CN112813053B (en) | Inulase mutant MutY119H and preparation method thereof | |
| CN112980813B (en) | Low-temperature modified exoinulase mutant MutS117G | |
| CN112852781B (en) | Heat-sensitive inulase mutant MutY119N and application thereof | |
| CN112646794B (en) | Exoinulase mutant MutY119V with improved low-temperature activity | |
| CN112725307B (en) | Low-temperature inulase exonuclease mutant MutG169 delta 4 with reduced heat resistance and application thereof | |
| CN112852782B (en) | Low-temperature adaptive improved low Wen Waiqie inulase mutant MutDL121EK5 and application thereof | |
| CN112725304B (en) | Low-temperature inulase exonuclease mutant MutAP122EK5 and application thereof | |
| CN112831485B (en) | Low-temperature activity improved exoinulase mutant MutDR121EH9 | |
| CN112813052B (en) | Exo-inulase mutant MutDP121ET6 with improved low-temperature activity | |
| CN111647579B (en) | Thermolabile exoinulase mutant MutQ23 delta 9 and preparation and application thereof | |
| CN112725310B (en) | Thermolabile low-temperature exoinulase mutant MutG360 delta 9 | |
| CN112725309B (en) | Low-temperature inulase exo-mutant MutP126R stable at medium temperature | |
| CN112980814B (en) | Exoinulase mutant MutV268 delta 13 with improved low-temperature adaptability | |
| CN108707593B (en) | Low-temperature inulase exonuclease mutant MutE137 delta 5 and application thereof | |
| CN112813050B (en) | Exo-inulinase mutant MutP126Q with reduced thermostability | |
| CN111621489B (en) | Thermostable inulase exonuclease mutant MutQ23 delta 6 and preparation and application thereof | |
| CN111690628B (en) | Exo-inulinase mutant MutQ23 delta 2 unstable at medium temperature |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |