CN112852781A

CN112852781A - Heat-sensitive inulase mutant MutY119N and application thereof

Info

Publication number: CN112852781A
Application number: CN202110041559.9A
Authority: CN
Inventors: 周峻沛; 张蕊; 黄遵锡; 岑潇龙; 唐湘华; 许波; 李俊俊; 韩楠玉; 吴倩; 高艳秀
Original assignee: Yunnan Normal University
Current assignee: Yunnan Normal University
Priority date: 2021-01-13
Filing date: 2021-01-13
Publication date: 2021-05-28
Anticipated expiration: 2041-01-13
Also published as: CN112852781B

Abstract

The invention discloses a heat-sensitive inulase mutant MutY119N and application thereof, wherein the amino acid sequence of the heat-sensitive inulase mutant MutY119N is shown as SEQ ID NO.1, and the amino acid sequence of the mutant MutY119N is obtained by mutating 119 th tyrosine of wild exoinulase InuAMN8 into asparagine. Compared with a wild enzyme InuAMN8, the mutant enzyme MutY119N has improved low-temperature activity and reduced thermal stability, and is beneficial to safe use of the enzyme and application in the field of biotechnology under the requirement of low-temperature environment. The low-temperature exoinulase mutant MutY119N can be applied to industries of food, wine brewing, washing and the like.

Description

Heat-sensitive inulase mutant MutY119N and application thereof

Technical Field

The invention relates to an inulase mutant, in particular to a heat-sensitive inulase mutant MutY119N 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, etc. after being fermented by yeast, etc. Therefore, the inulase exonuclease can be applied to industries such as food, wine brewing and Biological energy (Singh RS et al. International Journal of Biological Macromolecules,2017,96: 312-322).

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, the treatment at low temperature (if juice is clear) can prevent the pollution of microorganisms, the nutrient loss and the reduction of food quality, and the function of reducing energy consumption can be achieved by changing the medium-temperature or high-temperature treatment mode into the low-temperature treatment mode (Cavicchiali 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 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, the operation is simple, convenient and effective, and the use of the enzyme is safer; the heat treatment can also effectively prevent contamination by microorganisms. 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 heat-sensitive inulase mutant MutY119N and application thereof, wherein the mutant MutY119N has improved low-temperature activity and reduced thermal stability, and is beneficial to the safe use of enzyme and the application in the biotechnology field under the requirement of low-temperature environment.

In order to achieve the above object, the present invention provides a heat-sensitive inulinase mutant MutY119N with altered thermohality, the amino acid sequence of the heat-sensitive inulinase mutant MutY119N is shown in SEQ ID NO. 1. In contrast to the sequence of exoinulinase AGC01505(SEQ ID NO.3) recorded in GenBank, amino acid 119 of MutY119N is asparagine, while amino acid 119 of AGC01505 is tyrosine.

The mutant MutY119N of the present invention had an optimum temperature of 25 ℃ and 20%, 45%, 76% and 44% activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃, respectively; after the treatment for 60min at 50 ℃, 8 percent of enzyme activity of MutY119N is remained; adding 5.0-25.0% (w/v) NaCl into an enzymatic reaction system, and reducing the activity of MutY119N from 75% to 18%; after the MutY119N is treated by 5.0-25.0% (w/v) NaCl for 60min, the enzyme activity of the MutY119N is almost not lost; MutY119N can hydrolyze inulin to produce fructose.

Another object of the present invention is to provide the gene mutY119N encoding the heat-sensitive inulase mutant mutY 119N.

Preferably, the nucleotide sequence of the coding gene mutY119N is shown in SEQ ID NO. 2.

Another object of the present invention is to provide a recombinant vector comprising said gene mutY 119N.

Another purpose of the invention is to provide a recombinant bacterium containing the encoding gene mutY 119N.

Another object of the present invention is to provide a method for producing the heat-sensitive inulase mutant MutY119N, which comprises the following steps:

(1) connecting a wild exoinulase gene inuAMN8 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN 8;

(2) the plasmid pEasy-E1-inuAMN8 is used as a template, the nucleotide sequence of the adopted primer is shown in SEQ ID NO.7 and SEQ ID NO.8, and the recombinant expression plasmid pEasy-E1-mutY119N containing mutY119N is obtained through PCR amplification;

(3) transforming the recombinant expression plasmid pEasy-E1-mutY119N into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutY 119N;

(4) culturing the recombinant strain, and inducing the expression of the recombinant exo-thermo-sensitive inulase mutant MutY 119N;

(5) the expressed recombinant exothermo-sensitive inulase mutant MutY119N was recovered and purified.

Preferably, the recombinant strain comprising mutY119N is prepared by digesting the recombinant expression plasmid pEasy-E1-mutY119N with DpnI enzyme using Mut

II Fast Mutagenesis Kit digested products were ligated and transformed into E.coli BL21(DE3) by heat shock.

Preferably, the induction is performed using IPTG.

Preferably, the expression product of the recombinant strain containing mutY119N is subjected to affinity and purification respectively by Nickel-NTA Agarose and 0-500 mM imidazole to obtain the recombinant exo-thermo-sensitive inulase mutant mutY 119N.

Another objective of the invention is to provide the application of the mutant MutY119N in food, wine brewing and washing.

The heat-sensitive inulase mutant MutY119N and the application thereof have the following advantages:

compared with the wild enzyme InuAMN8, the mutant enzyme MutY119N of the invention has improved low-temperature activity and reduced thermal stability. The wild enzyme InuAMN8 has an optimum temperature of 35 ℃ and has 15%, 32%, 58% and 94% of activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; the mutant enzyme MutY119N of the present invention has an optimum temperature of 25 ℃ and has 20%, 45%, 76% and 44% of activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃, respectively; after the treatment for 60min at 50 ℃, 82% of the enzyme activity of the wild enzyme InuAMN8 is remained, and 8% of the enzyme activity of the mutant enzyme MutY119N is remained. Therefore, the low-temperature exoinulase mutant MutY119N can be applied to industries such as food, wine brewing and washing.

Drawings

FIG. 1 is an SDS-PAGE analysis of the wild-type enzyme InuAMN8 and the mutant enzyme MutY 119N.

FIG. 2 shows the thermal activity of the purified wild enzyme InuAMN8 and the mutant enzyme MutY 119N.

FIG. 3 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutY119N at 50 ℃.

FIG. 4 shows the activity of the purified wild enzyme InuAMN8 and the mutant enzyme MutY119N in NaCl.

FIG. 5 shows the stability in NaCl of the purified wild-type enzyme InuAMN8 and the mutant enzyme MutY 119N.

FIG. 6 is an analysis of the products of hydrolysis of inulin by the purified wild enzyme InuAMN8 and the mutant enzyme MutY 119N.

Reference numbers: in fig. 1, M: a protein Marker; in fig. 6, W: the wild enzyme InuAMN8 hydrolyzes the product of inulin; CK: control group, containing inulin and inactivated wild enzyme InuAMN8 (boiled for 10 min); f: fructose; g: glucose; mut: the mutant enzyme MutY119N hydrolyzes the product of inulin.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Experimental materials and reagents in the following examples of the invention:

1. bacterial strain and carrier:

escherichia coli BL21(DE3) and expression vector pEasy-E1 are commercially available from Beijing Quanyujin Biotechnology, Inc.; arthrobacter (Arthrobacter sp.) is supplied by university of teachers and universities 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 Novovin, inulin was purchased from Alfa Aesar, bacterial genomic DNA extraction Kit was purchased from Tiangen Biochemical technology (Beijing) Ltd, and the others were made of domestic reagents (all of which can be purchased from general Biochemical reagent Co.).

3. Culture medium:

LB culture medium: peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, natural pH (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) Designing primers 5'-ATGAATTCATTGACGACGGC-3' (SEQ ID NO.5) and 5'-TCAACGGCCGACGACGTCGA-3' (SEQ ID NO.6) according to an inulinase exonuclease nucleotide sequence JQ863111(SEQ ID NO.4) recorded by GenBank, carrying out PCR amplification by using Arthrobacter genome D NA as a template, wherein 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 obtained the coding gene inuAMN8 of the wild exoinulase inuAMN 8. 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 inuAMN 8.

(4) Coli BL21(DE3) was transformed with pEasy-E1-inuAMN8 by heat shock to obtain recombinant E.coli strain BL21(DE3)/inuAMN8 comprising inuAMN 8.

Example 2 construction and transformation of the expression vector for the mutant enzyme MutY119N

(1) Designing primers 5'-ACCAGTGCCAACAGTGACGCCGCGCCGCTTCC-3' (SEQ ID NO.7) and 5'-TCACTGTTGGCACTGGTGTAAATGGCCACCAG-3' (SEQ ID NO.8), 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 30 sec; 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. The PCR result gave a recombinant expression linearized plasmid pEasy-E1-mutY119N containing mutY119N (SEQ ID NO. 2). mutY119N and pEasy-E1-mutY119N can also be obtained by gene synthesis.

(2) mu.L of the PCR product of linearized plasmid pEasy-E1-mutY119N was digested with 1. mu.L of DpnI enzyme at 37 ℃ for 1 h.

(3) Using Mut

II Fast Mutagenesis Kit, the digestion product in step (2) was ligated for 30min at 37 ℃.

(4) The ligation product in step (3) was transformed into E.coli BL21(DE3) by heat shock to obtain recombinant strain BL21(DE3)/mutY119N containing mutY 119N.

Example 3 preparation of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119N

(1) Recombinant strains BL21(DE3)/inuAMN8 and BL21(DE3)/mutY119N were inoculated to LB (containing 100. mu.g mL) at an inoculum size of 0.1% respectively^-1Amp) in the culture medium, the mixture was rapidly shaken at 37 ℃ for 16 hours.

(2) Then, the activated bacterial suspension was inoculated into fresh LB (containing 100. mu.g mL) in an amount of 1% of the inoculum size^-1Amp) culture solution, rapidly shaking and culturing for about 2-3 h (OD)₆₀₀0.6-1.0), adding IPTG with the final concentration of 0.7mM for induction, and continuing shaking culture at 20 ℃ for about 20 hours. Centrifuging at 12000 rpm for 5min, and collecting thallus. After the cells were suspended in an appropriate amount of pH7.0 McIlvaine buffer, the cells were disrupted by ultrasonic waves in a low-temperature water bath. Centrifuging the crude enzyme solution concentrated in the cells at 13,000rpm for 10min, sucking the supernatant, and respectively carrying out affinity and purification on the target protein by using Nickel-NTA Agarose and 0-500 mM imidazole.

(3) SDS-PAGE results (see FIG. 1) showed that both recombinant InuAMN8(SEQ ID NO.3) and MutY119N (SEQ ID NO.1) were purified and the product was a single band.

EXAMPLE 4 determination of the Properties of the purified recombinant wild-type enzymes InuAMN8 and the mutant enzyme MutY119N

1. Analysis of the Activity of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119N

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 MutY119N

Carrying out an enzymatic reaction at 0-60 ℃ in a buffer solution with a pH of 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 MutY 119N.

The results show that: the wild enzyme InuAMN8 has an optimum temperature of 35 ℃ and has 15%, 32%, 58% and 94% of activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively; the mutant enzyme MutY119N had an optimum temperature of 25 ℃ and 20%, 45%, 76% and 44% activity at 0 ℃, 10 ℃,20 ℃ and 40 ℃ respectively (see FIG. 2).

3. Thermostability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119N

Treating the enzyme solution with the same amount of enzyme at 50 deg.C for 10-60 min, and performing enzymatic reaction at pH7.0 and 37 deg.C, wherein untreated enzyme solution is used as 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 MutY 119N.

The results show that: after 60min of treatment at 50 ℃, 82% of the enzyme activity of the wild enzyme InuAMN8 remains, and 8% of the enzyme activity of the mutant enzyme MutY119N remains (see FIG. 3).

4. Activity assay of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119N in NaCl

Adding 5.0-25.0% (w/v) NaCl into the enzymatic reaction system, and performing enzymatic reaction 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 MutY 119N.

The results show that: and (3) adding 5.0-25.0% (w/v) NaCl into an enzymatic reaction system, wherein the activity of a wild enzyme InuAMN8 is reduced from 79% to 15%, and the activity of a mutant enzyme MutY119N is reduced from 75% to 18% (see figure 4).

5. Stability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119N in NaCl

The purified enzyme solution was placed in 5.0-25.0% (w/v) NaCl aqueous solution, treated at 37 ℃ for 60min, and then subjected to enzymatic reaction at pH7.0 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 MutY 119N.

The results show that: after the wild enzyme InuAMN8 and the mutant enzyme MutY119N are treated by NaCl of 5.0-25.0% (w/v) for 60min, the enzyme activities of the wild enzyme InuAMN8 and the mutant enzyme MutY119N are almost not lost (see figure 5).

6. Analysis of the products of hydrolysis of inulin by the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119N

The product analysis 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 of the 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, directly immersing the silica gel plate into a proper amount of color developing agent (1g 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 on site;

(6) after a few seconds, the silica gel plate was immediately removed and placed in an oven at 90 ℃ for 10-15 min to develop the spots.

The results show that: the products of hydrolysis of inulin by the wild enzyme InuAMN8 and the mutant enzyme MutY119N were almost all fructose (see FIG. 6).

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

<110> university of Yunnan Master

<120> heat-sensitive inulase mutant MutY119N and application thereof

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<213> wild enzyme InuAMN8(AGC01505)

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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 inuAMN8(JQ863111)

<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

accagtgcca acagtgacgc cgcgccgctt cc 32

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcactgttgg cactggtgta aatggccacc ag 32

Claims

1. A heat-sensitive inulase mutant MutY119N with altered thermohality, wherein the amino acid sequence of the heat-sensitive inulase mutant MutY119N is shown in SEQ ID NO. 1.

2. The heat-sensitive inulase mutant MutY119N of claim 1 encoding gene mutY 119N.

3. The encoding gene mutY119N of claim 2, wherein the nucleotide sequence of the encoding gene mutY119N is shown in SEQ ID No. 2.

4. A recombinant vector comprising the gene mutY119N according to claim 2 or 3.

5. Recombinant bacterium comprising the gene mutY119N according to claim 2 or 3.

6. The method of producing the heat-sensitive inulinase mutant MutY119N according to claim 1, comprising:

7. The method according to claim 6, wherein the recombinant strain containing mutY119N is prepared by digesting the recombinant expression plasmid pEasy-E1-mutY119N with DpnI enzyme using Mut

8. The method of claim 6, wherein the induction is performed using IPTG.

9. The preparation method of claim 6, wherein the expression product of the recombinant strain containing mutY119N is subjected to affinity and purification by Nickel-NTAAgarose and 0-500 mM imidazole respectively to obtain the recombinant exo-thermo-sensitive inulase mutant mutY 119N.

10. The use of the mutant MutY119N according to claim 1 in food, brewing and washing.