CN112646794B

CN112646794B - Exoinulase mutant MutY119V with improved low-temperature activity

Info

Publication number: CN112646794B
Application number: CN202110041022.2A
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: 2022-06-24
Anticipated expiration: 2041-01-13
Also published as: CN112646794A

Abstract

The invention discloses an exoinulase mutant MutY119V with improved low-temperature activity, wherein the mutant MutY119V has an amino acid sequence shown as SEQ ID NO. 1. Compared with a wild enzyme InuAMN8, the mutant enzyme MutY119V has changed thermal activity and thermal stability, has higher catalytic activity and is easier to thermally denature in a low-temperature environment, and is favorable for controlling the catalytic reaction of the enzyme through temperature change. After treatment at 50 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutY119V is reduced from 75% to 41%; after treatment at 55 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the mutant enzyme MutY119V is completely inactivated after treatment at 55 ℃ for 10 min. The mutant MutY119V can be applied to industries such as food, wine brewing, washing and the like.

Description

Exoinulase mutant MutY119V with improved low-temperature activity

Technical Field

The invention relates to an exoinulase mutant, in particular to an exoinulase mutant MutY119V with improved low-temperature activity.

Background

Inulin is the major ingredient constituting the tuber of jerusalem artichoke and may constitute 19% by wet weight or 70% by dry weight thereof. The jerusalem artichoke is easy to plant, high in yield, cold-resistant, barren-resistant and drought-resistant, and is an important non-grain crop.

Inulin is polymerized from fructose and is terminally attached to a molecule of glucose residues. The exoinulase hydrolyzes beta-2, 1 glycosidic bonds from the non-reducing end of the inulin one by one, and finally degrades the inulin into fructose and a small part of glucose, wherein the fructose content in the fructose syrup can reach 95 percent of the total sugar content. The fructose can be widely used in the industries of food, medicine, biological energy and the like, can be used as a natural sweetener to replace sucrose, can be eaten by diabetics, and can be used as a raw material for producing bioethanol and the like. Therefore, the inulase exonuclease can be applied to industries such as food, wine brewing and bioenergy (Singh RS et al. International Journal of Biological Macromolecules,2017,96: 312-.

Low temperature enzymes have important development value. Food treatment at low temperature can prevent microbial contamination, nutrient loss and food quality degradation, and fermentation, aquaculture environment, washing and sewage treatment of sake and wine are usually carried out at low temperature; in addition, low temperature treatment can also reduce energy consumption (Cavicchiali et al. Microbiological Biotechnology,2011,4(4): 449-.

Low temperature exoinulinase can be used for fermentation of wine, pickling of food, decontamination, hydrolysis of inulin at low temperature to produce high concentration fructose syrup, etc. (Zhou et al, journal of bioscience and Bioengineering,2015,119(3): 267-274.). The low-temperature inulase exonuclease 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, convenient and effective operation. Therefore, obtaining the exoinulase mutant which has higher activity and is easier to thermally inactivate in a low-temperature environment is more beneficial to the practical application of the enzyme.

Disclosure of Invention

The invention aims to provide an exoinulase mutant MutY119V with improved low-temperature activity, wherein the mutant MutY119V has changed thermal activity and thermal stability, has higher catalytic activity and easier thermal denaturation in a low-temperature environment, and can be applied to industries such as food, wine brewing, washing and the like.

In order to achieve the above object, the present invention provides an exoinulase mutant MutY119V with improved low temperature activity, wherein the mutant MutY119V has an amino acid sequence as 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 MutY119V is valine, while amino acid 119 of AGC01505 is tyrosine.

The mutant MutY119V has the optimum temperature of 25 ℃, and has enzyme activities of 29%, 62%, 91% and 62% respectively at 0 ℃, 10 ℃,20 ℃ and 35 ℃; after the treatment at 50 ℃ for 10-60 min, the enzyme activity of MutY119V is reduced from 75% to 41%; MutY119V was completely inactivated after 10min of treatment at 55 ℃.

Another objective of the invention is to provide a gene mutY119V encoding the mutant mutY 119V.

Preferably, the coding gene mutY119V has the nucleotide sequence shown in SEQ ID NO. 2.

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

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

Another objective of the invention is to provide a preparation method of the mutant MutY119V, wherein the method comprises the following steps: 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 inuAMN 8; the plasmid pEasy-E1-inuAMN8 is taken as a template, and mutation primers of nucleotide sequences shown in SEQ ID NO.7 and SEQ ID NO.8 are used for carrying out PCR amplification to obtain a recombinant expression plasmid pEasy-E1-mutY119V containing mutY 119V; transforming the recombinant expression plasmid pEasy-E1-mutY119V into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutY 119V; the recombinant strain is cultured and the expression of the recombinant exoinulase mutant MutY119V is induced to obtain the recombinant exoinulase mutant MutY 119V.

Preferably, the recombinant strain comprising mutY119V is prepared by digesting the recombinant expression plasmid pEasy-E1-mutY119V 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 product expressed by the recombinant exoinulase mutant MutY119V is respectively subjected to affinity and purification by Nickel-NTAAgarose and 0-500 mM imidazole to obtain the recombinant exoinulase mutant MutY 119V.

Another objective of the invention is to provide the application of the mutant MutY119V in food, wine brewing and washing.

The exoinulase mutant MutY119V with improved low-temperature activity has the following advantages:

compared with a wild enzyme InuAMN8, the mutant enzyme MutY119V has changed thermal activity and thermal stability, and the mutant enzyme MutY119V has higher catalytic activity and is easier to thermally denature in a low-temperature environment, so that the catalytic reaction of the enzyme can be controlled through temperature change. The optimal temperature of the purified wild enzyme InuAMN8 is 35 ℃, the enzyme activities of 15%, 32%, 58% and 74% are respectively at 0 ℃, 10 ℃,20 ℃ and 25 ℃, and the optimal temperature of the mutant enzyme MutY119V is 25 ℃, and the enzyme activities of 29%, 62%, 91% and 62% are respectively at 0 ℃, 10 ℃,20 ℃ and 35 ℃; after being treated at 50 ℃ for 10-60 min, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutY119V is reduced from 75% to 41%; after being treated at 55 ℃ for 10-60 min, the enzyme activity of the wild enzyme InuAMN8 is reduced from 70% to 17%, and the mutant enzyme MutY119V is completely inactivated after being treated at 55 ℃ for 10 min. The exoinulase mutant MutY119V with improved low-temperature activity can be applied to 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 MutY 119V.

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

FIG. 3 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutY119V at 50 ℃.

FIG. 4 shows the stability of the purified wild enzyme InuAMN8 and the mutant enzyme MutY119V at 55 ℃.

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:

1. bacterial strain and carrier

Escherichia coli BL21(DE3) and expression vector pEasy-E1 are commercially available from Beijing Quanjin Biotechnology, Inc.; arthrobacter sp (Arthrobacter sp.) is provided by university of Master in Yunnan, and is deposited in the culture Collection of institute of microbiological research in Yunnan province under the number YMF 4.00006.

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 Alfa Aesar, bacterial genomic DNA extraction Kit was purchased from Tiangen Biochemical technology (Beijing) Ltd, and 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 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 biology experiments, which are not specifically described in the following examples, were carried out according to the specific methods listed in molecular cloning, A laboratory Manual (third edition) J. Sambuchok, supra, 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, and carrying out PCR amplification by using Arthrobacter genome DNA 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 MutY119V

(1) Primers 5'-ACCAGTGCCGTAAGTGACGCCGCGCCGCTTCC-3' (SEQ ID NO.7) and 5'-TCACTTACGGCACTGGTGTAAATGGCCACCAG-3' (SEQ ID NO.8) were designed, and PCR amplification was performed using plasmid pEasy-E1-inuAMN8 as a template, with the PCR parameters: denaturation at 95 ℃ for 30 sec; then denaturation at 95 ℃ for 15sec, annealing at 68 ℃ 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-mutY119V comprising mut Y119V (SEQ ID NO. 2). mut Y119V and pEasy-E1-mutY119V can also be obtained by gene synthesis.

(2) mu.L of DpnI enzyme was added to 50. mu.L of the PCR product of the linearized plasmid pEasy-E1-mutY119V and digested 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)/mutY119V comprising mutY 119V.

Example 3 preparation of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119V

(1) Recombinant strains BL21(DE3)/inuAMN8 and BL21(DE3)/mutY119V 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) was rapidly cultured with shaking for about 2 to 3 hours (OD600 reached 0.6 to 1.0), then IPTG was added to a final concentration of 0.7mM for induction, and shaking culture was continued at 20 ℃ for about 20 hours. Centrifugation was carried out at 12000rpm for 5min to collect the cells. After the cells were suspended in an appropriate amount of McIlvaine buffer at pH 7.0, the cells were disrupted by ultrasonic waves in a low-temperature water bath. After the crude enzyme solution concentrated in the above cells 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 0-500 mM imidazole.

(3) SDS-PAGE results (FIG. 1, M: protein Marker) showed that both recombinant InuAMN8(SEQ ID NO.3) and MutY119V (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 MutY119V

1. Analysis of the Activity of the purified recombinant wild enzyme InuAMN8 and the mutant enzyme MutY119V

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 MutY119V

The enzymatic reaction was carried out at 0-60 ℃ in a buffer at 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 MutY 119V.

The results show that: the wild enzyme InuAMN8 has an optimum temperature of 35 ℃ and 15%, 32%, 58% and 74% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 25 ℃ respectively, while the mutant enzyme MutY119V has an optimum temperature of 25 ℃ and 29%, 62%, 91% and 62% of enzyme activity at 0 ℃, 10 ℃,20 ℃ and 35 ℃ respectively (FIG. 2).

3. Thermostability assay of purified recombinant wild enzyme InuAMN8 and mutant enzyme MutY119V

The enzyme solutions were treated at 50 ℃ and 55 ℃ for 10-60 min, respectively, and then enzymatically reacted at pH 7.0 and 37 ℃ with the untreated enzyme solution as a control. Reacting for 10min by taking inulin as a substrate, and determining the enzymological properties of the recombinant wild enzyme InuAMN8 and the mutant enzyme MutY 119V.

The results show that: after treatment for 10-60 min at 50 ℃, the enzyme activity of the wild enzyme InuAMN8 is reduced from 88% to 81%, and the enzyme activity of the mutant enzyme MutY119V is reduced from 75% to 41% (figure 3); after treatment at 55 ℃ for 10-60 min, the enzyme activity of the wild enzyme InuAMN8 was reduced from 70% to 17%, while the mutant enzyme MutY119V was completely inactivated after treatment at 55 ℃ for 10min (FIG. 4).

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 limited only by the attached claims.

Sequence listing

<110> university of Yunnan Master

<120> exoinulase mutant MutY119V with improved low temperature activity

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 505

<212> PRT

<213> mutant enzyme (MutY119V)

<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 Val 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> 2

<211> 1518

<212> DNA

<213> mutant enzyme Gene (mutY119V)

<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 tgccgtaagt 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 InuAMN8(AGC01505)

<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 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

accagtgccg taagtgacgc cgcgccgctt cc 32

<210> 8

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcacttacgg cactggtgta aatggccacc ag 32

Claims

1. The exoinulase mutant MutY119V with improved low-temperature activity is characterized in that the amino acid sequence of the mutant MutY119V is shown as SEQ ID number 1.

2. The mutant MutY119V coding gene MutY119V as claimed in claim 1, wherein the nucleotide sequence of the coding gene MutY119V is shown in SEQ ID number 2.

3. A recombinant vector comprising the gene mutY119V according to claim 2.

4. Recombinant bacterium comprising the gene mutY119V according to claim 2.

5. A method of making the mutant MutY119V of claim 1, comprising:

connecting a wild exoinulase gene inuAMN8 with a nucleotide sequence shown as SEQ ID number 4 with an expression vector pEasy-E1 to obtain a recombinant expression plasmid pEasy-E1-inuAMN8 containing inuAMN 8;

the plasmid pEasy-E1-inuAMN8 is used as a template, and mutation primers of nucleotide sequences shown as SEQ ID number 7 and SEQ ID number 8 are used for carrying out PCR amplification to obtain a recombinant expression plasmid pEasy-E1-mutY119V containing mutY 119V;

transforming the recombinant expression plasmid pEasy-E1-mutY119V into Escherichia coli BL21(DE3) to obtain a recombinant strain containing mutY 119V;

culturing the recombinant strain, and inducing the expression of the recombinant exoinulase mutant MutY119V to obtain the recombinant exoinulase mutant MutY 119V.

6. The method according to claim 5, wherein the recombinant strain containing mutY119V is prepared by digesting the recombinant expression plasmid pEasy-E1-mutY119V with DpnI enzyme, ligating the digested products with Mut Express II Fast mutagenetics Kit, and transforming into Escherichia coli BL21(DE3) by heat shock.

7. The method of claim 5, wherein the induction is carried out using IPTG.

8. The process of claim 5, wherein the product of the expression of the mutant MutY119V is subjected to affinity by Nickel-NTA Agarose and then to elution and purification by 0-500 mM and not 0mM of imidazole to obtain the mutant MutY 119V.

9. Use of the mutant MutY119V according to claim 1 in the food industry.

10. The use according to claim 9, wherein the food industry comprises: the wine industry.