CN112725305B - Thermohaline-sensitive inulase mutant MutY119D and preparation method thereof - Google Patents
Thermohaline-sensitive inulase mutant MutY119D and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering and protein modification, and discloses a thermohaline sensitive inulase mutant MutY119D and a preparation method thereof, wherein the amino acid sequence of the mutant MutY119D is obtained by mutating 119 th amino acid (tyrosine) of wild exoinulase InuAMN8 into aspartic acid, and the sequence of the MutY119D is shown as SEQ ID NO. 1. Compared with a wild enzyme InuAMN8, the mutant enzyme MutY119D has improved low-temperature activity, reduced thermal stability and reduced activity and stability in sodium chloride, and is beneficial to safe use of the enzyme and application in the field of biotechnology under the requirement of low-temperature environment. The heat-salinity-sensitive inulase mutant MutY119D can be applied to industries such as 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 thermohaline sensitive inulase mutant MutY119D and a preparation method 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 inulinase can be applied to industries such as food, wine and bioenergy (Singh RS et al. International Journal of Biological Macromolecules,2017, 96.
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 temperatures (if the juice is clear) can prevent microbial contamination, nutrient loss and food quality degradation, and conversion of the medium-temperature or high-temperature treatment mode to the low-temperature treatment mode can also serve to reduce energy consumption (cavcc hiei et al. Microbiological Biotechnology,2011,4 (4): 449-460.). 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 and salt is easier to denature, and the enzyme is easy to degrade due to denaturation, so that the catalytic reaction of the enzyme can be easily controlled, the enzyme can be inactivated by simple heat treatment or salt treatment, the operation is simple, convenient and effective, and the use of the enzyme is safer; the heat treatment and the salt treatment are also effective in preventing contamination by microorganisms. Therefore, the mutant enzyme sensitive to heat and salt is obtained, and the application of the enzyme in the industries of food, wine brewing, washing and the like is facilitated.
Disclosure of Invention
The invention aims to provide a heat-salinity-sensitive inulase mutant MutY119D 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 heat-salt sensitive inulase mutant MutY119D, wherein the amino acid sequence of the mutant MutY119D is shown as SEQ ID NO. 1. In contrast to the exoinulinase sequence AGC01505 (SEQ ID NO. 3) recorded in GenBank, amino acid 119 of MutY119D is aspartic acid, while amino acid 119 of AGC01505 is tyrosine.
The mutant MutY119D has an optimum temperature of 25 ℃ and has 15%, 43%, 71% and 48% of activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; after the treatment for 60min at 50 ℃, 9% of enzyme activity of MutY119D remains; adding 5.0-25.0% (w/v) NaCl into the enzymatic reaction system, and reducing the activity of MutY119D from 64% to 19%; after the MutY119D is treated by 5.0-25.0% (w/v) NaCl for 60min, the enzyme activity of the MutY119D is reduced from 83% to 44%; mutY119D can hydrolyze inulin to produce fructose.
The invention provides a coding gene mutY119D of the inulase mutant mutY119D, and the nucleotide sequence of the coding gene mutY119D is shown in SEQ ID NO. 2.
Another object of the present invention is to provide a recombinant vector comprising the inulase mutant encoding gene mutY119D.
Another purpose of the invention is to provide a recombinant bacterium containing the inulase mutant encoding gene mutY119D.
In addition, the application of the inulase mutant MutY119D in the preparation of food, wine and washing products is also within the protection scope of the invention.
The preparation method of the inulase mutant MutY119D 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 of 5 'TTACACCAGTGCCGACAGTGACGCCGCCGCTT 3' and 5 'TGTCGGCACTGTGTAAATGGCCACCAGCGGGG 3' by taking the plasmid pEasy-E1-inuAMN8 as a template, and obtaining a recombinant expression plasmid pEasy-E1-mutY119D containing mutY119D through PCR amplification;
3) Transforming the recombinant expression plasmid pEasy-E1-mutY119D into escherichia coli BL21 (DE 3) to obtain a recombinant strain containing mutY119D;
4) Culturing the recombinant strain, and inducing the expression of the recombinant inulase mutant MutY119D;
5) Recovering and purifying the expressed recombinant inulase mutant MutY119D;
6) And (4) measuring the activity.
The invention has the beneficial effects that:
compared with the wild enzyme InuAMN8, the mutant enzyme MutY119D has improved low-temperature activity, reduced thermal stability and reduced activity and stability in sodium chloride. The optimum temperature of the wild enzyme InuAMN8 is 35 ℃, and the wild enzyme InuAMN8 has the activity of 15%, 32%, 58% and 94% at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; the mutant enzyme MutY119D has an optimum temperature of 25 ℃ and has 15%, 43%, 71% and 48% of activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; after the treatment for 60min at 50 ℃, 82% of enzyme activity of wild enzyme InuAMN8 is remained, and 9% of enzyme activity of mutant enzyme MutY119D is remained; adding 5.0-25.0% (w/v) NaCl into the enzymatic reaction system, reducing the activity of a wild enzyme InuAMN8 from 79% to 15%, and reducing the activity of a mutant enzyme MutY119D from 64% to 19%; after the wild enzyme InuAMN8 is treated by NaCl of 5.0-25.0% (w/v) for 60min, the enzyme activity of the wild enzyme InuAMN8 is hardly lost, and the enzyme activity of the mutant enzyme MutY119D is reduced from 83% to 44%. The low-temperature exoinulase mutant MutY119D 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 MutY119D, wherein M: a protein Marker;
FIG. 2 shows the thermal activity of the purified wild-type enzyme InuAMN8 and the mutant enzyme MutY119D;
FIG. 3 shows the stability of the purified wild-type enzyme InuAMN8 and the mutant enzyme MutY119D at 50 ℃;
FIG. 4 shows the activity of the purified wild enzyme InuAMN8 and the mutant enzyme MutY119D in NaCl;
FIG. 5 shows the stability in NaCl of the purified wild-type enzyme InuAMN8 and the mutant enzyme MutY119D;
FIG. 6 is the product analysis of the hydrolysis of inulin by the purified wild enzyme InuAMN8 and the mutant enzyme MutY119D, wherein W: the product of the hydrolysis of inulin by the wild enzyme InuAMN8; CK: control group containing inulin and inactivated wild enzyme InuAMN8 (boiling for 10 min); f: fructose; g: glucose; mut: the mutant enzyme MutY119D hydrolyzes the product of inulin.
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 coli BL21 (DE 3) and expression vector pEasy-E1 are commercially available from Beijing Quanji gold Biotechnology, inc.; arthrobacter (Arthrobacter sp.) is supplied by university of faculty in Yunnan.
2. Enzymes and other biochemical reagents: nickel-NTAAgarose was purchased from QIAGEN, DNA polymerase, dNTP and Mut
II Fast Mutagenesis Kit was purchased from Nanjing Novosa, inulin was purchased from Alfaaesar, bacterial genomic DNA extraction Kit was purchased from Tiangen Biochemical technology (Beijing) Ltd, and the others were all domestic reagents (all of them are available from general Biochemical reagent Co.).
3. Culture medium
LB culture medium: peptone 10g, yeast extract 5g, naCl 10g, distilled water was added to 1000mL, and the pH was 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) Based on the nucleotide sequence JQ863111 (SEQ ID NO. 4) of exoinulase recorded in GenBank, primers 5'ATGAATTCATTGACGACGGC 3' and 5'TCAACGGCCGACGACGTCGA 3' were designed, PCR amplification was carried out using Arthrobacter genome DNA as a template, and the PCR reaction parameters were: denaturation at 95 deg.C for 5min; 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 according to the inulase exonuclease 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 mutant enzyme MutY119D
1) Designing primers of 5'TTACACCAGTGCCGACAGTGACGCCGCGCCGCTT 3' and 5 'TGTCGGCACTGTGTAAATGGCCACCAGCGGGG 3', and carrying out PCR amplification by using a plasmid pEasy-E1-inuAMN8 as a template, wherein the PCR reaction parameters are as follows: denaturation at 95 ℃ for 30sec; then denaturation at 95 ℃ for 15sec, annealing at 70 ℃ for 15sec, extension at 72 ℃ for 3min for 30sec, and heat preservation at 72 ℃ for 5min after 30 cycles. As a result of PCR, a recombinant expression linearized plasmid pEasy-E1-mutY119D containing mutY119D was obtained. mutY119D and pEasy-E1-mutY119D can also be obtained by gene synthesis.
2) mu.L of DpnI enzyme was added to 50. Mu.L of the PCR product of linearized plasmid pEasy-E1-mutY119D and digested at 37 ℃ for 1h.
3) Using Mut
II Fast Mutagenesis Kit, the digestion product in (2) was ligated for 30min at 37 ℃.
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)/mutY 119D comprising mutY119D.
Example 3 preparation of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119D
Recombinant strains BL21 (DE 3)/inuAMN 8 and BL21 (DE 3)/mutY 119D were inoculated to LB (containing 100. Mu.g mL) at an inoculum size of 0.1% respectively -1 Amp) culture solution, and rapidly shaking at 37 ℃ for 16h.
The activated cell suspension was inoculated into fresh LB (100. Mu.g mL) at an inoculum size of 1% -1 Amp) culture solution, rapidly shaking and culturing for about 2-3 h (OD 600 reaches 0.6-1.0), adding IPTG with final concentration of 0.7mM for induction, and continuing shaking and culturing for about 20h at 20 ℃. Centrifugation was carried out at 12000rpm for 5min to collect the cells. After the cells were suspended in an appropriate amount of pH =7.0McIlvaine buffer, the cells were disrupted by ultrasonic waves in a low-temperature water bath. After the crude enzyme solution concentrated intracellularly above was centrifuged at 13,000rpm for 10min, the supernatant was aspirated and the objective protein was respectively subjected to affinity purification using Nickel-NTA Agarose and 0 to 500mM imidazole.
SDS-PAGE results (FIG. 1) showed that both recombinant InuAMN8 and MutY119D were purified and the product was a single band.
Example 4 determination of the Properties of the purified recombinant wild-type enzyme InuAMN8 and the mutant enzyme MutY119D
1) Activity analysis of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119D
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 to terminate the reaction, boiling in 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 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 MutY119D
The enzymatic reaction was performed 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 MutY119D.
The results show that: the wild enzyme InuAMN8 has the optimum temperature of 35 ℃ and has the activity of 15%, 32%, 58% and 94% at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; the mutant enzyme MutY119D has an optimum temperature of 25 ℃ and 15%, 43%, 71% and 48% activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively (FIG. 2).
3) Thermostability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119D
After treating the enzyme solution with the same amount of enzyme at 50 ℃ for 10 to 60min, the enzyme reaction was carried out at pH =7.0 and 37 ℃ with 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 MutY119D.
The results show that: after 60min of treatment at 50 ℃, 82% of the enzyme activity of the wild enzyme InuAMN8 is remained, and 9% of the enzyme activity of the mutant enzyme MutY119D is remained (figure 3).
4) Activity assay of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119D in NaCl
5.0-25.0% (w/v) NaCl is added to the enzymatic reaction system, and the enzymatic reaction is carried out at pH7.0 and 37 ℃. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119D.
The results show that: when NaCl was added to the enzymatic reaction system at 5.0-25.0% (w/v), the activity of the wild enzyme InuAMN8 was reduced from 79% to 15%, and the activity of the mutant enzyme MutY119D was reduced from 64% to 19% (FIG. 4).
5) Stability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119D in NaCl
The purified enzyme solution was placed in an aqueous NaCl solution of 5.0 to 25.0% (w/v), treated at 37 ℃ for 60min, and then subjected to enzymatic reaction at pH7.0 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 MutY119D.
The results show that: after the wild enzyme InuAMN8 is treated by NaCl of 5.0-25.0% (w/v) for 60min, the enzyme activity of the wild enzyme InuAMN8 is hardly lost, and the enzyme activity of the mutant enzyme MutY119D is reduced from 83% to 44% (figure 5).
6) Analysis of the products of hydrolysis of inulin by the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119D
Product analysis the reaction system contained 450. Mu.L of 0.5% (w/v) inulin, 50. Mu.L of an appropriate dilution of the enzyme solution (total 0.1U enzyme solution). The reaction was terminated after 4h of enzymatic reaction at pH7.0 and 37 ℃. The product analysis was carried out by thin layer chromatography (using high performance thin layer chromatography silica gel plate type G available from Qingdao ocean chemical Co., ltd.).
The thin layer chromatography procedure is as follows:
(1) preparing a developing solvent (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 solvent into a developing tank, and standing for about 30 min;
(2) activating the silica gel plate in a 110 deg.C oven for 30min, cooling, scribing, and spotting (0.5 μ L each time, blow drying, and spotting for 3 times);
(3) placing the silica gel plate at one end of the sample application downwards into an expansion tank, wherein the sample application point does not immerse a developing agent;
(4) when the developing agent is 1.5cm away from the upper edge of the silica gel plate, taking out the silica gel plate, drying and developing again;
(5) after the second unfolding is finished, directly immersing the silica gel plate into a proper amount of color developing agent (1 g of diphenylamine is dissolved in 50mL of acetone, 1mL of aniline and 5mL of 85% phosphoric acid are added after the solution is dissolved, and the mixture is uniformly mixed and prepared on site;
(6) after a few seconds, the silica gel plate was immediately removed and placed in an oven at 90 ℃ for 10-15min to allow the spots to develop color.
The results show that: the products of hydrolysis of inulin by the wild enzyme InuAMN8 and the mutant enzyme MutY119D were almost fructose (FIG. 6).
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 any modifications and equivalents.
Sequence listing
<110> university of Yunnan Master
<120> thermohaline sensitive inulase mutant MutY119D and preparation method thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 505
<212> PRT
<213> mutant enzyme (MutY 119D)
<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
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Asp Pro Asn Gly Leu Val Tyr Leu Asn Gly Thr Tyr His Leu Phe Tyr
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Gln His Asn Pro Phe Gly Ala Asp Trp Gly Asn Met Ser Trp Gly His
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Ala Thr Ser Arg Asp Leu Leu His Trp Asp Glu Gln Pro Val Ala Ile
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Pro Cys Asp Glu His Glu Ala Ile Phe Ser Gly Ser Ala Val Phe Asp
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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 Asp 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
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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
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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
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Pro Thr Gly Gly Trp Arg Ser Ala Met Ser Leu Pro Arg Glu Val Ser
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Leu Thr Arg Val Asp Gly Lys Val Met Leu Arg Gln Gln Ala Ile Asp
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Pro Leu Pro Glu Arg Glu Thr Gly His Val Arg Leu Gly Pro Gln Pro
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Leu Ala Ser Gly Val Leu Asp Val Pro Ala Ala Ala Ser Val Ala Arg
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Ile Asp Val Glu Leu Glu Pro Gly Ala Ala Ala Gly Val Gly Leu Val
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Leu Arg Ala Gly Asp Asp Glu Arg Thr Val Leu Arg Tyr Asp Thr Ser
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Asp Gly Met Leu Arg Leu Asp Arg Arg Glu Ser Gly Gln Val Ala Phe
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His Glu Thr Phe Pro Ser Ile Glu Ala Met Ala Val Pro Leu Gln Gly
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Phe Ala Gln Asp Gly Leu Ala Thr Leu Thr Asp Leu Val Phe Pro Gly
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Glu Ala Ser Thr Gly Leu Ala Ile Phe Ala Glu Gly Glu Gly Ala His
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Leu Val Val Leu Asp Val Val Gly Arg
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<211> 1518
<212> DNA
<213> mutant enzyme Gene (mutY 119D)
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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 tgccgacagt 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> 3
<211> 505
<212> PRT
<213> wild 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 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
ttacaccagt gccgacagtg acgccgcgcc gctt 34
<210> 8
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tgtcggcact ggtgtaaatg gccaccagcg gg 32
Claims (6)
1. A thermohaline sensitive inulase mutant MutY119D is characterized in that the amino acid sequence of the mutant MutY119D is shown as SEQ ID NO. 1.
2. The mutant MutY119D according to claim 1, wherein the nucleotide sequence of the gene mutY119D is as shown in SEQ ID NO. 2.
3. A recombinant vector comprising the gene mutY119D according to claim 2.
4. A recombinant bacterium comprising the encoded gene mutY119D according to claim 2.
5. A process for the preparation of inulase mutant MutY119D as claimed in claim 1, comprising the steps of:
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 'TTACACCAGTGCCGACAGTGACGCCGCGCCGCTT' and 5 'TGTCGGCACTGGTGTAAATGGCCACCAGCGGG' by taking the plasmid pEasy-E1-inuAMN8 as a template, and obtaining a recombinant expression plasmid pEasy-E1-mutY119D containing mutY119D through PCR amplification;
3) Converting the recombinant expression plasmid pEasy-E1-mutY119D into escherichia coli to obtain a recombinant strain containing mutY119D;
4) Culturing the recombinant strain, and inducing the expression of the recombinant inulase mutant MutY119D;
5) Recovering and purifying the expressed recombinant inulase mutant MutY119D;
6) And (4) measuring the activity.
6. Use of the mutant MutY119D according to claim 1 for brewing wine.
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| CN112813050B (en) * | 2021-01-13 | 2022-08-30 | 云南师范大学 | Exo-inulinase mutant MutP126Q with reduced thermostability |
| 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 |
| 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 |
| 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 |
| CN112725304B (en) * | 2021-01-13 | 2022-10-18 | 云南师范大学 | Low-temperature inulase exonuclease mutant MutAP122EK5 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 |
| CN112813052B (en) * | 2021-01-13 | 2022-08-26 | 云南师范大学 | Exo-inulase mutant MutDP121ET6 with improved low-temperature activity |
| CN112980814B (en) * | 2021-01-13 | 2023-06-27 | 云南师范大学 | Exoinulase mutant MutV268 delta 13 with improved low-temperature adaptability |
| CN112813053B (en) * | 2021-01-13 | 2022-06-24 | 云南师范大学 | Inulase mutant MutY119H 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 |
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