CN111621486B

CN111621486B - Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method

Info

Publication number: CN111621486B
Application number: CN202010447957.6A
Authority: CN
Inventors: 熊海容; 王晶
Original assignee: South Central University for Nationalities
Current assignee: South Central Minzu University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2021-10-19
Anticipated expiration: 2040-05-25
Also published as: CN111621486A

Abstract

The invention belongs to the field of protein engineering and genetic engineering, and particularly relates to a high-enzyme-activity heat-resistant xylanase XYNB at a low temperature, a mutant gene thereof, an application thereof and a gene sequence preparation method.

Description

Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method

Technical Field

The invention belongs to the field of protein engineering and genetic engineering, and particularly relates to heat-resistant xylanase XYNB with high enzyme activity at low temperature, a mutant gene, application and a gene sequence preparation method thereof.

Background

Xylan is the most important hemicellulose in plant cell walls, accounts for about 35% of the dry weight of plant cells, and is the polysaccharide with the most abundant content except cellulose in nature. Xylan is a hybrid polysaccharide formed by a main chain formed by polymerization of xylose through beta-1, 4-glycosidic bonds and a plurality of side chain groups, is an abundant biomass resource, and can be degraded into xylooligosaccharide and xylose which are urgently needed in the international market under the action of xylanase. However, a large part of xylanase in nature is not effectively utilized, and a great waste of the resource is caused.

Microbial xylanases (EC 3.2.1.8) are important industrial enzymes that randomly catalyze the hydrolysis of beta-1, 4-D-xylosidic bonds within xylan to xylooligosaccharides. Xylo-oligosaccharides as substrates can be further degraded by other xylanases such as beta-D-xylosidase, alpha-L-arabinofuranosidase, D-glucuronidase, etc. (Khandepaker R, Numan MT. bifunctional and the said gene functional use in biotechnology J Ind microbial biotechnology 2008,35: 635-644.). Xylanases are mainly classified into families 10 and 11 of glycoside hydrolases based on significant differences in functional primary and tertiary structure and model, however xylanases with xylanolytic activity have also been found in enzymes of families 5, 7, 8, 16, 26, 30, 43, 52 and 62 of glycoside hydrolases (http:// www.cazy.org/fam/ac _ gh. html) (Collins T, Gerday C, Feller G. Xylanase, xylanase family and exophagic xylanases. FEMS Microbiol Rev 2005,29: 3-23.). The GH 10 family xylanase has a large relative molecular weight (>30000), a complex structure, and usually consists of multiple structural domains, and a Catalytic Domain (CD) is a main component of the xylanase and bears the hydrolysis characteristic of the xylanase. Although they vary greatly in the number and composition of amino acids, their catalytic structures are very close in size, mainly structures that occur repeatedly in α -helices and α -sheets, and belong to a family, called the (α/β) 8-sheet structure, because of structural similarity to TIM, where glutamate and aspartate at specific positions have a large influence on the catalytic properties. The enzyme also contains domains with non-catalytic activity, such as polysaccharide substrate binding domain, thermal stability domain, and a plurality of catalytic domains, etc., which endow the enzyme with the functions of decomposing soluble xylan, insoluble xylanase and other substrates, etc.

The application of xylanase originated from its use in animal feed processing in 1980 and was subsequently gradually applied in the fields of paper making, food, pharmacy, brewing, textile and biofuel, etc. At present, the enzyme is mainly applied to industries such as pulping and papermaking, feed, food and the like, and the application position in modern industry is more and more obvious. Xylanase is one of the key enzymes in the process of producing alcohol by using non-starch raw materials. With the development of bioenergy industry, xylanases have been applied in a wider field (Fawzi EM. high viscous purified xylanase from Rhizomucor miehei NRRL 3169, Ann Microbiol 2010,60: 363-368.). In the industrial production process, extreme environments such as high temperature and the like often exist, the high temperature environment can accelerate the enzymatic reaction, improve the flow property of liquid materials, prevent harmful microorganisms from growing and propagating in the process and the like. The common intermediate-temperature xylanase can generate structural change at high temperature, thereby greatly losing activity. To prevent this heat inactivation, some chemicals are added to protect the enzyme, which not only increases the production cost but also adversely affects the product quality. The heat-resistant xylanase produced by Thermotoga maritima MSB8 is used for hydrolysis, so that the problem that the added enzyme is quickly inactivated in a high-temperature environment can be fundamentally solved.

The xylanase derived from bacteria has higher thermal stability than xylanase derived from fungi, and pichia pastoris has the potential of efficient secretion, correct folding protein and extremely high cell concentration culture and is often used as an expression system for producing exogenous protein on a large scale, and xylanase genes derived from bacteria are applied to pichia pastoris capable of being cultured at high density after sequence optimization, so that the xylanase is more suitable for industrial production. Xylanase 1VBR derived from Thermotoga maritima MSB8 has excellent enzymatic properties (Winterhalter C, Liebl W. two extreme thermal stable xylanases of the hyperthermophilic bacteria Thermoyoga maritima MSB8. apple Environ Microbiol.1995,61(5):1810-1815.), and has extreme thermal stability.

The error-prone PCR technique is an evolution of the normal PCR technique, which is a PCR technique that makes DNA more susceptible to mismatching during the course of replication amplification, and is also called mismatch PCR or error-prone PCR. Error-prone PCR generally utilizes low-fidelity Taq DNA polymerase and some means such as changing PCR reaction systems to reduce the fidelity of DNA replication in the PCR process and increase the mismatch rate, thereby obtaining a DNA sequence or gene different from the original one.

After the enzyme gene sequence is mutated by error-prone PCR, and is subjected to expression and enzyme characteristic identification and screening, the obtained xylanase mutant XYNB with high enzyme activity at low temperature is remarkably higher than the original enzyme in the enzyme activity below 80 ℃, simultaneously retains the heat resistance of the original enzyme, is suitable for the industrial fields of feed, food, brewing, medicine and the like, and for example, the feed enzyme hopes to simultaneously meet the special requirements of less enzyme inactivation during high-temperature granulation and high enzyme activity at normal temperature of animal digestive tracts.

Disclosure of Invention

The invention aims to obtain xylanase XYNB with high enzyme activity at low temperature through mutating a xylanase 1VBR gene sequence by error-prone PCR, expressing and screening after enzyme characteristic identification, wherein the enzyme activity at the temperature of below 80 ℃ is obviously higher than that of an original enzyme, and the heat resistance of the original enzyme is kept.

The invention provides heat-resistant xylanase XYNB with high enzyme activity at low temperature, which is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

Moreover, the optimal reaction temperature of the heat-resistant xylanase XYNB with high enzyme activity at the low temperature is 90 ℃, and the enzyme activity of the heat-resistant xylanase XYNB with high enzyme activity at the low temperature can be maintained above 50% under the conditions of pH 5.5 and 60 ℃.

A mutant gene of heat-resistant xylanase XYNB with high enzyme activity at low temperature codes the heat-resistant xylanase XYNB with high enzyme activity at low temperature, and the gene sequence of the mutant gene is shown as SEQ ID NO. 2.

And an expression vector containing the coding sequence of the heat-resistant xylanase XYNB with high enzyme activity at low temperature.

Wherein the expression vector is pPIC 9K-XYNB.

And a recombinant strain containing the coding sequence of the heat-resistant xylanase XYNB with high enzyme activity at low temperature.

Wherein the strain is recombinant escherichia coli or recombinant yeast.

And the application of the heat-resistant xylanase XYNB with high enzyme activity at low temperature in feed additives, foods, brewing or medicines.

A method for preparing a gene sequence of heat-resistant xylanase XYNB with high enzyme activity at low temperature comprises the steps of taking pET-22b (+) -1VBR plasmid as a template, using Taq enzyme, setting the concentrations of Mg2+ and Mn2+ in a PCR system to be 2.5mM and 0.8mM respectively, and setting the primer sequences to be 5'-CGCCATGGATTCTCAGAATGTATC-3' and 5'-TCGCGACTCGAGTTTTCTTTCTTC-3', carrying out two rounds of error-prone PCR, and screening to obtain a mutant gene sequence of the xylanase XYNB, wherein the mutation positions of the mutant gene sequence are N562D, T568M, R575G, D592G, V632A, D643G, K789E, Q792L and I837M.

A preparation method of the heat-resistant xylanase XYNB with high enzyme activity at low temperature comprises the steps of transforming host cells by using the expression vector to obtain a recombinant strain, and culturing the recombinant strain to express the heat-resistant xylanase XYNB with high enzyme activity at low temperature.

The xylanase XYNB with high enzyme activity at low temperature has the advantages that the enzyme activity at the temperature of below 80 ℃ is obviously higher than that of the original enzyme, the heat resistance of the original enzyme is kept, and the xylanase XYNB is suitable for the industrial fields of feed additives, foods, brewing, medicines and the like, for example, the enzyme for feed is expected to simultaneously meet the special requirements of less enzyme inactivation during high-temperature granulation and high enzyme activity at normal temperature of animal digestive tracts.

The invention firstly synthesizes xylanase 1VBR gene KR078269 by whole gene, which comprises terminator sequence and enzyme cutting site sequence at two ends thereof. Error-prone PCR mutation is carried out on the gene sequence to obtain the gene sequence of the xylanase XYNB. The whole gene synthetic xylanase gene is inserted into an expression vector pPIC9K through double enzyme digestion and connection to construct a recombinant expression plasmid pPIC 9K-XYNB. The recombinant expression plasmid is transformed into escherichia coli DH5 alpha competent cells, and a positive clone strain is screened out by a PCR verification method.

The protein content of the xylanase XYNB secreted by the gene engineering strain GS115-XYNB for producing the xylanase XYNB in the fermentation liquor can reach the electrophoresis purity level, and almost no purification is needed.

Drawings

FIG. 1 SDS-PAGE analysis of xylanase mutant XYNB;

FIG. 2 temperature optimum (pH 5.5, 10 min) for xylanase mutant XYNB;

FIG. 3 thermal death curve (pH 5.5, 100 ℃) of xylanase mutant XYNB.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Experimental materials:

1) strains and plasmids: escherichia coli (Escherichia coli) DH5 alpha and Pichia pastoris GS115 were gifted to the institute for feed, Chinese academy of agricultural sciences; the pPIC9K secretory expression vector was purchased from Invitrogen.

2) Enzymes and kits: restriction enzyme, Taq enzyme, Pyrobest DNA polymerase, T4 DNA ligase and other tool enzymes purchased from TaKaRa company; DNA purification kits were purchased from Seiki Biotechnology Ltd.

3) Biochemical reagents: g418 was purchased from Invitrogen; protein molecular weight standards were purchased from shanghai institute of biochemistry; IPTG, X-Gal, SDS and carob were purchased from Sigma; TEMED, ammonium persulfate, acrylamide and methylene bisacrylamide are used as traditional Chinese medicine reagents.

50mM disodium phosphate-citric acid buffer: dissolving 7.10g of disodium hydrogen phosphate in 800mL of double distilled water, adjusting the pH value to any value within the range of 4.0-7.5 by using citric acid, and then fixing the volume to 1L.

50mM Tris-HCl buffer: 6.06g of Tris is dissolved in 800mL of double distilled water, the pH value is adjusted to any value within the range of 7.5-9.0 by 1M HCl, and then the volume is adjusted to 1L.

50mM glycine-sodium hydroxide buffer: dissolving 7.50g of glycine in 800mL of double distilled water, adjusting the pH value to any value within the range of 9.0-12 by using 1M of sodium hydroxide solution, and then fixing the volume to 1L.

The experimental procedures in the following examples are conventional unless otherwise specified.

The percentages in the following examples are by mass unless otherwise specified.

Example 1: acquisition of xylanase mutant strain XYNB Gene sequence

According to the sequence alignment, the amino acid sequence of xylanase 1VBR from Thermotoga maritima MSB8 published in PDB has 100% similarity with the predicted xylanase (EHA58720.1) in Thermotoga maritima MSB8 genome sequence (CP 007013.1: 1,869,644 bp). The predicted xylanase gene sequence is a gene sequence (873,589 → 874,572bp) in the Thermotoga maritima MSB8 genome sequence.

Based on the codon usage preference of Pichia pastoris, rare codons in the Pichia pastoris are converted into high-frequency expression codons, and meanwhile, the amino acid sequence of xylanase 1VBR is contrasted to perform codon optimization on the predicted xylanase (EHA58720.1) gene sequence to obtain the optimized xylanase 1VBR gene sequence (NCBI gene number: KR 078269). Using pET-22b (+) -1VBR plasmid as a template, using Taq enzyme, setting the concentrations of Mg2+ and Mn2+ in a PCR system to be 2.5mM and 0.8mM respectively, setting the primer sequences to be 5'-CGCCATGGATTCTCAGAATGTATC-3' and 5'-TCGCGACTCGAGTTTTCTTTCTTC-3', carrying out two rounds of error-prone PCR, and screening to obtain the mutant gene sequence of the xylanase XYNB. FIG. 1 shows SDS-PAGE analysis of xylanase mutants XYNB.

Example 2: construction of recombinant expression plasmid pPIC9K-XYNB containing xylanase mutant strain XYNB gene

Adding a terminator sequence preferred by pichia pastoris at the 3 ' end of xylanase XYNB gene, respectively introducing restriction enzyme EcoR I and Not I sites at the 5 ' end and the 3 ' end, and handing the gene sequence with Wuhan engine scientific creative biotechnology limited company to complete the whole gene synthesis. The optimized xylanase XYNB gene and a secretory expression vector pPIC9K are subjected to double enzyme digestion by using restriction enzymes EcoR I and Not I, and then are connected by using ligase to construct a recombinant expression plasmid pPIC 9K-XYNB. The recombinant expression plasmid is transformed into escherichia coli DH5 alpha competent cells, and a positive clone strain pPIC9K-XYNB-DH5 alpha is screened out by a PCR verification method.

Example 3: construction of Pichia pastoris gene engineering strain for efficient secretory expression of xylanase XYNB

The strain pPIC9K-XYNB-DH5 alpha is subjected to activated culture by adopting an LB liquid culture medium, and the recombinant plasmid pPIC9K-XYNB is extracted. The recombinant plasmid was linearized with the restriction enzyme Bgl II and the digested product was recovered. Pichia pastoris GS115 competent cells were prepared with reference to the EasySelect Pichia Expression Kit. And (3) gently and uniformly mixing about 10 mu g of linearized plasmid and 80 mu L of competent cells, placing the mixture on ice for 15min, transferring the mixture into a precooled 0.2cm electric rotating cup, immediately adding 1mL of precooled 1mol/L sorbitol after 1500V electric shock is finished, standing the mixture in an incubator at 30 ℃ for 1h, coating the mixture on an MD (MD) plate, and performing inversion culture at 30 ℃ for about 48h until a transformant appears.

Single colonies were picked and inoculated in the order of the numbers on MD plates containing 0.25, 0.5, 1.0, 2.0, 3.0 and 4.0mg/mL G418, respectively, and cultured by inversion at 30 ℃ for about 48h until single colonies appeared. The recombinant strain with the strongest resistance is selected and inoculated in a BMGY medium containing 3mL of the corresponding number, and is subjected to shake cultivation at 30 ℃ and 200rpm for about 48 hours until the OD600 reaches 1.8-6.0. The cells were collected, resuspended in 1mL BMMY medium, and induced in 0.5% methanol for about 48 h.

Collecting supernatant, and detecting enzyme activity by using a DNS method to screen a positive recombinant strain GS115-XYNB with higher enzyme yield.

Example 4: property analysis of xylanase mutant strain XYNB

FIG. 1 is an SDS-PAGE analysis of xylanase mutants XYNB, where M is a molecular weight marker.

1) Determination of xylanase XYNB optimum temperature: reacting the enzyme with 0.5% xylan solution at different temperatures for 15min at pH 5.5, adding 2.5mL DNS reagent, boiling water bath for 5min, cooling to room temperature, and adding water to reach volume of 12.5 mL. OD540 was detected in a spectrophotometer. The relative residual enzyme activity under other conditions is calculated by taking the highest enzyme activity as 100 percent. The result shows that the optimum reaction temperature of the xylanase XYNB is 90 ℃, the enzyme activity of the xylanase XYNB can be maintained by more than 50% under the condition of 60 ℃, and is remarkably higher than the relative enzyme activity of the original enzyme, as shown in figure 2, and figure 2 shows the optimum temperature (pH 5.5 and 10 minutes) of the xylanase mutant XYNB, wherein 1VBR is represented by ●, and XYNB is represented by a tangle-solidup.

2) Determination of the xylanase XYNB thermal lethality curve: diluting the xylanase solution with a buffer solution with pH 5.5, placing the diluted xylanase solution in a water bath at 100 ℃, respectively treating for different times, detecting the activity of the xylanase under the optimal reaction condition, and calculating the half-life period of the xylanase according to the activity. The results showed that the mutant enzyme XYNB had slightly inferior heat resistance to the original enzyme 1VBR under the optimum pH conditions, but could retain a relative enzyme activity of 50% or more after 1 hour of treatment. As shown in FIG. 3, FIG. 3 is the thermal death curve (pH 5.5, 100 ℃) of xylanase mutant XYNB, wherein 1VBR is represented by ● and XYNB is represented by a-solidup.

SEQ ID NO.3 of the sequence Listing is the original amino acid sequence of the extreme heat-resistant xylanase 1VBR (PDB code: 1VBR, amino acid sequence 517-840); SEQ ID NO.1 is an error-prone PCR mutant XYNB (amino acid sequence 517-840, mutation sites N562D, T568M, R575G, D592G, V632A, D643G, K789E, Q792L, I837M) mutated at 9 amino acid positions.

The mutation sites are marked with underlining and bolding below, SEQ ID NO. 1:

SEQ ID NO.4 is the original gene sequence information of the extreme heat-resistant xylanase 1VBR (NCBI gene number: KR078269, base sequence 9-981); SEQ ID NO.2 is the sequencing information of the mutant XYNA gene generated by error-prone PCR: among them, the mutation sites are underlined, and among them, the effective mutation sites are marked by bold face (there are 9 effective mutation sites), and the ineffective mutation sites are marked by italic face and not bold face (there are 4 ineffective mutation sites).

SEQ ID NO.2：

Sequence listing

<110> university of the south China nationality

<120> heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Ser Leu Arg Glu Leu Ala Glu Lys Leu Asn Ile Tyr Ile Gly Phe

1 5 10 15

Ala Ala Ile Asn Asn Phe Trp Ser Leu Ser Asp Ala Glu Lys Tyr Met

20 25 30

Glu Val Ala Arg Arg Glu Phe Asn Ile Leu Thr Pro Glu Asp Gln Met

35 40 45

Lys Trp Asp Met Ile His Pro Glu Arg Asp Gly Tyr Asn Phe Thr Pro

50 55 60

Ala Glu Lys His Val Glu Phe Ala Glu Glu Asn Gly Met Ile Val His

65 70 75 80

Gly His Thr Leu Val Trp His Asn Gln Leu Pro Gly Trp Ile Thr Gly

85 90 95

Arg Glu Trp Thr Lys Glu Glu Leu Leu Asn Val Leu Glu Asp His Ile

100 105 110

Lys Thr Ala Val Ser His Phe Lys Gly Arg Val Lys Ile Trp Gly Val

115 120 125

Val Asn Glu Ala Val Ser Asp Ser Gly Thr Tyr Arg Glu Ser Val Trp

130 135 140

Tyr Lys Thr Ile Gly Pro Glu Tyr Ile Glu Lys Ala Phe Arg Trp Ala

145 150 155 160

Lys Glu Ala Asp Pro Asp Ala Ile Leu Ile Tyr Asn Asp Tyr Ser Ile

165 170 175

Glu Glu Ile Asn Ala Lys Ser Asn Phe Val Tyr Asn Met Ile Lys Glu

180 185 190

Leu Lys Glu Lys Gly Val Pro Val Asp Gly Ile Gly Phe Gln Met His

195 200 205

Ile Asp Tyr Arg Gly Leu Asn Tyr Asp Ser Phe Arg Arg Asn Leu Glu

210 215 220

Arg Phe Ala Lys Leu Gly Leu Gln Ile Tyr Ile Thr Glu Met Asp Val

225 230 235 240

Arg Ile Pro Leu Ser Gly Ser Glu Glu Tyr Tyr Leu Lys Lys Gln Ala

245 250 255

Glu Val Cys Ala Lys Ile Phe Asp Ile Cys Leu Asp Asn Pro Ala Val

260 265 270

Glu Ala Ile Leu Phe Trp Gly Phe Thr Asp Lys Tyr Ser Trp Val Pro

275 280 285

Gly Phe Phe Lys Gly Tyr Gly Lys Ala Leu Leu Phe Asp Glu Asn Tyr

290 295 300

Asn Pro Lys Pro Cys Tyr Tyr Ala Ile Lys Glu Val Leu Glu Lys Lys

305 310 315 320

Met Glu Glu Arg

<210> 2

<211> 972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtatctctga gagaactcgc agaaaagctg aacatctata ttggttttgc cgcaatcaac 60

aacttttggt ctctttccga cgcagaaaag tacatggaag ttgcaagaag agagttcaac 120

atcctgaccc ctgaggacca gatgaagtgg gatatgattc atccagaaag agacggatac 180

aatttcactc ccgctgaaaa acacgttgag tttgcggaag aaaacggcat gatcgtgcat 240

ggacacactc ttgtctggca caaccagctt cctggatgga tcactggtag agaatggaca 300

aaggaagaac ttttgaacgt tcttgaagac cacataaaaa cggtggcgtc tcatttcaaa 360

ggtagagtga agatttgggg tgtggtgaac gaagcggtga gcgattctgg aacctacagg 420

gaaagcgtgt ggtacaagac gatcggtcct gaatacattg aaaaagcgtt cagatgggca 480

aaagaagccg atccagatgc gattctcatc tacaacgact acagcataga agaaatcaac 540

gcaaagtcga acttcgtcta caacatgata aaagagctga aagaaaaggg agtacctgtt 600

gatggaatag gatttcagat gcacatagac tacagagggc tcaactatga cagtttcaga 660

aggaatttgg agagatttgc gaaactcggt cttcaaatat acatcactga gatggatgtg 720

agaatccctc tcagtggttc ggaggagtat tatttgaaaa aacaggctga agtttgtgcg 780

aagattttcg atatatgctt ggacaaccct gcagttgaag cgatcctatt ttggggattc 840

acagacaaat actcctgggt tcccggcttt ttcaaagggt acgggaaagc gttgctcttc 900

gatgagaatt acaaccccaa gccttgttat tacgcgataa aagaggtgct ggagaaaaag 960

atggaagaaa ga 972

<210> 3

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Val Ser Leu Arg Glu Leu Ala Glu Lys Leu Asn Ile Tyr Ile Gly Phe

1 5 10 15

Ala Ala Ile Asn Asn Phe Trp Ser Leu Ser Asp Ala Glu Lys Tyr Met

20 25 30

Glu Val Ala Arg Arg Glu Phe Asn Ile Leu Thr Pro Glu Asn Gln Met

35 40 45

Lys Trp Asp Thr Ile His Pro Glu Arg Asp Arg Tyr Asn Phe Thr Pro

50 55 60

Ala Glu Lys His Val Glu Phe Ala Glu Glu Asn Asp Met Ile Val His

65 70 75 80

Gly His Thr Leu Val Trp His Asn Gln Leu Pro Gly Trp Ile Thr Gly

85 90 95

Arg Glu Trp Thr Lys Glu Glu Leu Leu Asn Val Leu Glu Asp His Ile

100 105 110

Lys Thr Val Val Ser His Phe Lys Gly Arg Val Lys Ile Trp Asp Val

115 120 125

Val Asn Glu Ala Val Ser Asp Ser Gly Thr Tyr Arg Glu Ser Val Trp

130 135 140

Tyr Lys Thr Ile Gly Pro Glu Tyr Ile Glu Lys Ala Phe Arg Trp Ala

145 150 155 160

Lys Glu Ala Asp Pro Asp Ala Ile Leu Ile Tyr Asn Asp Tyr Ser Ile

165 170 175

Glu Glu Ile Asn Ala Lys Ser Asn Phe Val Tyr Asn Met Ile Lys Glu

180 185 190

Leu Lys Glu Lys Gly Val Pro Val Asp Gly Ile Gly Phe Gln Met His

195 200 205

Ile Asp Tyr Arg Gly Leu Asn Tyr Asp Ser Phe Arg Arg Asn Leu Glu

210 215 220

Arg Phe Ala Lys Leu Gly Leu Gln Ile Tyr Ile Thr Glu Met Asp Val

225 230 235 240

Arg Ile Pro Leu Ser Gly Ser Glu Glu Tyr Tyr Leu Lys Lys Gln Ala

245 250 255

Glu Val Cys Ala Lys Ile Phe Asp Ile Cys Leu Asp Asn Pro Ala Val

260 265 270

Lys Ala Ile Gln Phe Trp Gly Phe Thr Asp Lys Tyr Ser Trp Val Pro

275 280 285

Gly Phe Phe Lys Gly Tyr Gly Lys Ala Leu Leu Phe Asp Glu Asn Tyr

290 295 300

Asn Pro Lys Pro Cys Tyr Tyr Ala Ile Lys Glu Val Leu Glu Lys Lys

305 310 315 320

Ile Glu Glu Arg

<210> 4

<211> 972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtatctctga gagaactcgc agaaaagctg aacatctata ttggttttgc cgcaatcaac 60

aacttttggt ctctttccga cgcagaaaag tacatggaag ttgcaagaag agagttcaac 120

atcctgaccc ctgagaacca gatgaagtgg gatacgattc atccagaaag agacagatac 180

aatttcactc ccgctgaaaa acacgttgag tttgcagaag aaaacgacat gatcgtgcat 240

ggacacactc ttgtctggca caaccagctt cctggatgga tcactggtag agaatggaca 300

aaggaagaac ttttgaacgt tcttgaagac cacataaaaa cggtggtgtc tcatttcaaa 360

ggtagagtga agatttggga tgtggtgaac gaagcggtga gcgattctgg aacctacagg 420

gaaagcgtgt ggtacaagac gatcggtcct gaatacattg aaaaagcgtt cagatgggca 480

aaagaagccg atccagatgc gattctcatc tacaacgact acagcataga agaaatcaac 540

gcaaaatcga acttcgtcta caacatgata aaagagctga aagaaaaggg agtacctgtt 600

gatggaatag gatttcagat gcacatagac tacagagggc tcaattatga cagtttcaga 660

aggaatttgg agagatttgc gaaactcggt cttcaaatat acatcacaga gatggatgtg 720

agaatccctc tcagtggttc ggaggagtat tatttgaaaa aacaggctga agtttgtgcg 780

aagattttcg atatatgctt ggacaaccct gcagttaaag cgatccagtt ttggggattc 840

acagacaaat actcctgggt tcccggcttt ttcaaagggt acgggaaagc gttgctcttc 900

gatgagaatt acaaccccaa gccttgttat tacgcgataa aagaggtgct ggagaaaaag 960

atagaagaaa ga 972

Claims

1. A heat-resistant xylanase XYNB with high enzyme activity at low temperature is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. The heat-resistant xylanase XYNB with high enzyme activity at low temperature according to claim 1, characterized in that: the optimal reaction temperature of the heat-resistant xylanase XYNB with high enzyme activity at the low temperature is 90 ℃, and the enzyme activity of the heat-resistant xylanase XYNB with high enzyme activity at the low temperature can be maintained above 50% under the conditions of pH 5.5 and 60 ℃.

3. A mutant gene of heat-resistant xylanase XYNB with high enzyme activity at low temperature is characterized in that: the mutant gene codes the heat-resistant xylanase XYNB with high enzyme activity at low temperature as claimed in claim 1, and the gene sequence is shown in SEQ ID No. 2.

4. An expression vector comprising the coding sequence of the thermostable xylanase XYNB with high enzyme activity at low temperature according to claim 1.

5. The expression vector of claim 4, wherein: the expression vector is pPIC 9K-XYNB.

6. A recombinant strain comprising the coding sequence of the thermostable xylanase XYNB with high enzyme activity at low temperature according to claim 1.

7. The recombinant strain of claim 6, wherein: the recombinant strain is recombinant escherichia coli or recombinant yeast.

8. The use of the heat-resistant xylanase XYNB with high enzyme activity at low temperature according to claim 1 in feed additives, foods, brewing or medicines.

9.A preparation method of heat-resistant xylanase XYNB with high enzyme activity at low temperature is characterized by comprising the following steps: transforming a host cell by using the expression vector of claim 4 to obtain a recombinant strain, and culturing the recombinant strain to express the heat-resistant xylanase XYNB with high enzyme activity at low temperature.