CN111424048A - Gene for expressing acidic β -mannase, vector and application thereof - Google Patents

Abstract

The application discloses a gene for expressing acid β -mannase, wherein the nucleotide sequence of the coding gene is SEQ ID NO. 3. the invention also discloses an expression cassette, a recombinant vector or a recombinant microorganism containing the gene and application thereof, and a method for preparing acid β -mannase by utilizing fermentation of a recombinant strainManTAfter being transferred into host cells, the gene can be stably and efficiently expressed and inherited, the expressed acidic β -mannase has good temperature resistance and acid resistance, the can-releasing enzyme activity can reach 32000U/m L,is 2 times of the enzyme activity before optimization.

Description

Gene for expressing acidic β -mannase, vector and application thereof

Technical Field

The application relates to the field of genetic engineering, in particular to a gene for coding acidic β -mannase, and a vector and application thereof.

Background

β -mannan is a main hemicellulose existing in cell wall and plant seed, β -mannase is an endoglycoside hydrolase, which hydrolyzes β -1, 4-D-mannoside bond of mannan to generate mannan oligosaccharide, β -mannase is rich in source, and is divided into plant source (such as beans, locust, straw and the like), animal source (such as snail, marine mollusk and the like) and microbial source, because the microbial resource is wide, the cultivation is easy, the operation and extraction process is simple, and the activity of the produced β -mannase is relatively higher, β -mannase with good tolerance to temperature, acidity and the like can be screened in extreme environment, so the β -mannase from the microbial source has wider application range, and is also popularized and applied in production and life.

For example, the enzyme activity of mannase bred by mutation breeding of Bacillus licheniformis AM-001 is improved from 36U/ml to 430U/ml, the mannase produced by Bacillus lentus bred by the company of U.S. chamGen has the enzyme activity of 400U/ml, along with the development of molecular biology technology, the method for producing mannase by utilizing engineering bacteria heterologous high-efficiency expression is a current main means, the mannase produced by utilizing Pichia pastoris has the enzyme activity of about 343U/ml (Li Song yoge, 2009, biotechnology communication), but the requirements of practical application cannot be completely met in the aspects of expression quantity and enzymatic properties, in the feed industry, the addition of β -mannase is mainly used for reducing the chyme viscosity, improving the utilization rate of animals on feed and the likeBecause the feed granulation needs to be carried out in a high-temperature environment, the activity of the mannanase which is not high in temperature resistance is greatly lost. In addition, although the main process of carbohydrate digestion occurs in the small intestine and other digestive tracts, which are slightly acidic, food or feed is subjected to the acidic environment of the stomach, and the loss of enzyme activity is reduced by the good acid resistance. At present, the strains capable of producing the mannanase include trichoderma reesei, aspergillus thiochrous, bacillus subtilis, aspergillus niger and the like. Wherein isolated Aspergillus nigerAspergillus niger) The produced mannase can better meet the production requirement, but the produced β -mannase has low yield and is difficult to meet the industrial production requirement, the optimum action temperature of β -mannase of a strain before optimization is 50 ℃, the optimum action pH is 5.50, the enzyme activity retention rate of β -mannase of the strain before optimization is only 32% after treatment for 30min at the pH value of 3.0, therefore, the optimization of enzyme genes by using a genetic engineering technology is still the key point of research on improving the enzymology property and the yield or the enzyme activity expression quantity of the acid mannase.

Disclosure of Invention

The invention optimizes the original β -mannase gene shown in SEQ ID NO.1 according to the pichia pastoris codon preference to synthesize an optimized β -mannase geneManTAfter the acidic β -mannase is transferred into a host cell, the stable and efficient expression and inheritance can be realized, the homology of the nucleotide sequence of the acidic β -mannase produced by fermentation and the nucleotide sequence before optimization is 75.34%, the amino acid sequence of the enzyme is not changed, and the pot-releasing enzyme activity and the enzymological property of the acidic β -mannase expressed by the acidic β -mannase are greatly improved.

The invention provides a gene for expressing acid β -mannase, wherein the nucleotide sequence of the coding gene is SEQ ID NO. 3.

The invention also provides an expression cassette, a recombinant vector or a recombinant microorganism containing the gene.

The invention further provides an expression vector, which comprises the gene.

In one embodiment according to the invention, the expression vector is a pPICZ α plasmid, preferably a pPICZ α A plasmid, comprising the genes as described above.

In one embodiment according to the invention, the gene is ligated into the plasmid pPICZ α A after restriction with EcoRI and NotI restriction enzymes.

In still another aspect of the present invention, there is provided a recombinant strain for expressing β -mannanase gene, the recombinant strain comprising the above-described expression vector.

In one embodiment according to the present invention, the host bacterium of the recombinant strain is pichia pastoris.

The invention also provides application of the expression vector or the recombinant strain in fermentation preparation of β -mannase.

In another aspect, the invention provides a fermentation method of acidic β -mannase, comprising:

inoculating the recombinant strain into a fermentation medium, wherein the pH value is maintained at 4.5-5.0 by ammonia water in the fermentation process, the culture temperature is 28-30 ℃, after the glycerol is exhausted, glycerol with the mass fraction of 50% is fed in at the feeding rate of 10-20 m L/L/h, the wet weight of the thalli is maintained at 200-250 g/L, then, methanol induction is started, the flow rate of the methanol is controlled at 3-7m L/L/h, the aeration ratio is maintained at 1:1.5 in the whole fermentation process, and the stirring speed is 140 rpm;

wherein the fermentation medium contains 20-50 g/L of glycerol, 30-60 g/L of ammonium dihydrogen phosphate, 5-15 g/L of monopotassium phosphate, 5-15 g/L of potassium sulfate, 10-20 g/L of magnesium sulfate, 0.5-2 g/L of calcium sulfate and 1-2 g/L14-7 g/L of potassium hydroxide.

The invention has the following beneficial effects:

the β -mannase complete gene sequence is obtained by a complete gene synthesis methodManTThe expressed acidic β -mannase can be stably and efficiently expressed and inherited after being transferred into host cells, the pot-placing enzyme activity of the expressed acidic β -mannase can reach 32000U/m L which is 2 times of the enzyme activity before optimization, the optimum pH value of the acidic β -mannase produced by recombinant strain fermentation is 3.5, the range of action pH is wide, the relative enzyme activity is more than 70 percent in the range of pH2.5-pH6.5, the enzyme activity retention rate is 94 percent after 30min of pH3.0 treatment, the temperature resistance is good, the optimum temperature is 60 ℃, and the enzyme is treated by water bath treatment at 75 ℃ for 90s, 75 ℃ for 3min and 80 ℃ for 5minThe survival rates of the post-enzyme activity are respectively 98%, 96% and 85%, and the acidic β -mannase provided by the invention has the most suitable pH value of acidity, realizes high-efficiency expression in pichia pastoris, does not need purification, and has very important industrial application value.

Drawings

FIG. 1 is an electrophoresis diagram of the acid β -mannanase gene of a recombinant strain verified by an a-factor primer/3' AOX primer, wherein M represents D L5000 DNA Marker, and sequence number 1 represents the amplification result by using the recombinant strain genome as a PCR template;

FIG. 2 is an SDS-PAGE pattern of recombinant acidic β -mannanase enzyme, wherein 1: protein Marker and 2: purified acidic β -mannanase enzyme;

FIG. 3 is a graph of pH activity of recombinant acidic β -mannanase;

FIG. 4 is a graph of temperature activity of recombinant acidic β -mannanase;

FIG. 5 is a temperature-resistant bar graph of recombinant acidic β -mannanase;

FIG. 6 is a bar graph of the acid resistance of recombinant acidic β -mannanase.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

Experimental materials and general experimental methods:

1. bacterial strain and carrier:

escherichia coli Strain (Escherichia coli) JM109, Pichia strain (Pichia pastoris) GS115 was purchased from Invitrogen plasmid pPICZ α A, a laboratory deposited plasmid.

2. Enzymes and other biochemical reagents:

the tool enzyme comprises restriction enzyme, DNA ligase and Taq enzyme, and DNA extraction, purification and gel recovery kits are all purchased from Takara company. Mannan, mannose were purchased from Sigma, and others were made-by-home reagents (all available from general biochemicals). 3. Culture medium:

the pichia pastoris strain produces an acidic β -mannase induction selective medium:

1) YPD culture medium comprising yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and agar powder 20 g/L;

2) induction expression culture medium BMGY yeast extract 10 g/L, peptone 20 g/L13.4.4 g/L, biotin 4 × 10^－4g/L, glycerol 10 m L/L, 100 mmol/L phosphate buffer (pH 6.0);

3) induction expression culture medium BMMY, yeast extract 10 g/L, peptone 20 g/L13.4.4 g/L, biotin 4 × 10^－4g/L, methanol 5m L/L, 100 mmol/L phosphate buffer (pH 6.0);

in the above culture medium, YNB, methanol and biotin are subjected to filtration sterilization, the glucose-containing medium is sterilized at 108 deg.C for 30min, and the rest is sterilized at 121 deg.C for 20 min.

4. The experimental method comprises the following steps:

standard Molecular manipulation techniques such as DNA extraction, gel electrophoresis, E.coli transformation, and yeast transformation were performed using standard techniques, such as those described in Molecular Cloning, A L laboratory Manual, 2002.

Example 1 β optimization of the mannanase Gene and construction of recombinant expression vectors

1) β optimization and synthesis of mannanase gene

Original β -mannanase geneManIs derived from Aspergillus niger which is obtained by screening and separating from natural soil. Core thereofThe nucleotide sequence is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. Under the premise of not changing the coded amino acid sequence, according to the codon bias of pichia pastorisManThe gene is subjected to codon optimization, and the whole gene synthesis method synthesizes the whole gene sequence of the acid β -mannaseManTAn artificially synthesized acidic β -mannanase geneManTThe nucleotide sequence of (A) is shown as SEQ ID NO. 3. SynthesizedManTThe two ends of the gene are also provided withEcoRI andNoti enzyme cutting site to facilitate connection with the expression vector. By usingManTβ -mannase amplified by using gene sequence as templateManTGenes, the primers used were:

ManT-up：5′- CCGGAATTCTCATTCGCTTCTACTTCAGGACTTCAGT TTACT-3′（SEQ ID NO.4），

ManT-dn：5′- TAAAGCGGCCGCTTATGCTGAACCAATAGCGGCAACGT GATC -3′（SEQ ID NO.5）。

underlined areEcoRI andNoti enzyme cutting site.

2) Construction of recombinant expression vectors

Will be provided withManTFor geneEcoRI andNoti after double cleavage withEcoRI andNotthe expression vector pPICZ α A after the enzyme digestion is connected to obtain the recombinant plasmid pPICZ α A-ManTRecombinant plasmid pPICZ α A-ManTTransforming Escherichia coli competent cell JM109, culturing on L B plate, screening positive clone, extracting plasmid of positive clone, transferring to Huada gene for sequencing, and sequencing to show that the β -mannase geneManTThe total length is 1038bp, and 345 amino acids are coded.

Example 2 acquisition and characterization of acid β -mannanase engineering bacteria

Transformation of Pichia pastorisP.pastorisGS115, transformation and screening the main procedures refer to the Invitrogen Pichia expression Manual, recombinant plasmid pPICZ α A-ManTBy usingSacI linearization, transformation of Pichia pastoris by electric shockP.pastorisGS115, transformed cells plated in YPD medium containing 100. mu.g/ml Zeocin antibiotic, was cultured at 30 ℃ for two days. Screening to obtainThe transformant of (1) was subjected to PCR amplification using an a-factor/3' AOX primer, and the PCR amplification product was identified by 1% agarose gel electrophoresis after the reaction was completed, by extracting the genome according to a yeast genome DNA extraction kit (see FIG. 1). And cutting the gel, recovering and purifying PCR products, sending the PCR products to Huada gene for sequencing, and determining whether the sequence is correct or not. The recombinant strain was stored at-70 ℃ for future use.

a-factor primer: CTACTATTGCCAGCATTGCTGCT (SEQ ID NO. 6)

3' AOX primer: GAGGATGTCAGAATGCCATTTGCC (SEQ ID NO. 7).

Example 3 high expression of recombinant acidic β -mannanase strains

1) Shake flask seed

The formula of the culture medium comprises 10 g/L of yeast powder, 20 g/L of peptone and 20 g/L of glucose, and the natural pH value is high.

The culture conditions are as follows: the culture temperature is 30 ℃, the rotating speed of a shaking table is 220rpm, the liquid loading amount of a shaking bottle is 15 percent, the pH value is natural, and the culture period is 20 hours. After the seeds are mature, flame inoculation is carried out on the first-stage seed tank.

2) First-level seed tank

The formula of the culture medium comprises 40 g/L g of glycerol, 30 g/L g of ammonium dihydrogen phosphate, 5 g/L g of potassium dihydrogen phosphate, 10 g/L g of potassium sulfate, 5 g/L g of magnesium sulfate, 0.5 g/L g of calcium sulfate and 1.5 g/L/L g of potassium hydroxide.

The culture conditions are as follows: the inoculation amount is 0.5 percent, and the ventilation volume is 40 m³The stirring speed is 300 rpm, the pH value is maintained at 5.0 by ammonia water during the culture process, and the culture temperature is 30 ℃. The culture period is 20 h. After the seeds are mature, all the seeds are transplanted to a secondary seed tank.

3) Two-stage seed tank

The formula of the culture medium comprises 40 g/L of glycerol, 40 g/L of ammonium dihydrogen phosphate, 6 g/L of potassium dihydrogen phosphate, 10 g/L of potassium sulfate, 10 g/L of magnesium sulfate, 1 g/L of calcium sulfate and 1.5 g/L/L of potassium hydroxide.

The culture conditions are as follows: inoculation amount of 10 percent and ventilation amount of 400 m³The stirring speed is 300 rpm, the pH value is maintained at 5.0 by ammonia water during the culture process, and the culture temperature is 30 ℃. The culture period is 10 h, and the seed is completely transplanted to a fermentation tank after the seed is mature.

4) Fermentation tank

The formula of the culture medium comprises 30 g/L of glycerol, 50 g/L of ammonium dihydrogen phosphate, 10 g/L of potassium dihydrogen phosphate, 10 g/L of potassium sulfate, 15 g/L of magnesium sulfate, 1 g/L of calcium sulfate and 1.5 g/L16 g/L of potassium hydroxide.

The culture conditions are that ammonia water is used to maintain the pH value at 4.5 and the culture temperature at 28 ℃, after the glycerol is exhausted, glycerol with the mass fraction of 50% is fed in, the feeding rate is 16 m L/L/h, the wet weight of the bacteria is maintained at 220 g/L, then methanol induction is started, the methanol flow rate is controlled at 4 m L/L/h, the aeration ratio is maintained at 1:1.5 in the whole fermentation process, the stirring speed is 140rpm, induction is carried out for 120 h, the tank-placing enzyme activity is 32000U/m L, under the same fermentation conditions, the tank-placing enzyme activity of the pichia pastoris strain containing the original β -mannanase gene is 16500U/m L, and therefore, the enzyme production of the acid β -mannanase of the optimized strain is increased by 1 time.

Example 4 acidic β -mannanase SDS-PAGE analysis of recombinant strains

After 120 hours of inducible expression, fermentation supernatants were collected for purification of recombinant β -mannanase and subjected to SDS-PAGE, as shown in FIG. 2.

EXAMPLE 5 determination of β -mannanase Activity and evaluation of enzymatic Properties of recombinant strains

(1) β -mannase activity detection method

According to GB/T36861-2018 feed additive β -mannase activity spectrophotometry, a DNS method is adopted to determine reducing sugar generated by hydrolysis, at 37 ℃ and pH5.5, the enzyme amount required by releasing 1 mu mol of reducing sugar from a mannan solution with the concentration of 3 mg/m L per minute is β -mannase activity unit (U), three parallel tests are set for each sample to determine, and the relative error is controlled within 8%.

(2) β -mannanase pH Activity Curve

The 0.1M citric acid solution and the 0.2M disodium hydrogen phosphate solution are mixed according to a certain volume ratio, so that the pH values of the citric acid-disodium hydrogen phosphate solution are respectively 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 and 7, and the activity of β -mannanase is measured under different pH values, the result shows that (figure 3) the recombinant strain β -mannanase has the optimum action pH value of 3.5, and the β -mannanase of the recombinant strain has the relative enzyme activity of more than 70 percent in the pH range of 2.5-6.5, which shows that the β -mannanase of the recombinant strain has a broad-spectrum pH action range.

(3) β -mannanase temperature activity curve

The reaction temperature in the enzyme activity determination process is respectively set to 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃ and 80 ℃, and β -mannase activity is determined under different reaction temperature conditions, and the result shows that the optimal action temperature of the recombinant strain β -mannase (shown in figure 4) is 60 ℃.

(4) β -mannanase thermotolerance evaluation

Diluting an enzyme solution to be detected to about 1U/m L by using a pH5.5 acetic acid-sodium acetate buffer solution, respectively treating the enzyme solution at 75 ℃ for 90s, treating the enzyme solution at 75 ℃ for 3min and treating the enzyme solution at 80 ℃ for 5min, determining residual enzyme activity, and calculating relative enzyme activity by taking the enzyme activity of an untreated sample as 100 percent.

(5) β -mannanase acid resistance evaluation

Diluting the enzyme solution to be detected by 20 times with citric acid-disodium hydrogen phosphate buffer solution with the pH value of 3.0, the pH value of 4.0 and the pH value of 5.0 respectively, placing the diluted solution in an electric heating constant-temperature water tank at 40 ℃ for 30min, taking the enzyme solution out of the constant-temperature water tank, immediately diluting the enzyme solution by acetic acid-sodium acetate buffer solution with the pH value of 5.5 appropriately, measuring the enzyme activity after acid-resistant treatment, and calculating the relative enzyme activity by taking the enzyme activity of a sample which is not treated by the citric acid-disodium hydrogen phosphate as 100 percent.

Example 6 evaluation of Bionical digestion enzymolysis efficiency of acid β -mannanase expressed by recombinant Strain

A bionic digestion method is used for measuring repeatability and additive research on reducing sugar release amount of pig feed raw materials [ J ] animal nutrition report, 029(1): 168-.

TABLE 1 Effect of different β -mannanase on reducing sugar release from soybean meal

The β -mannan content in the non-peeled soybean meal is 1.33% -2.12%, the β -mannan content in the peeled soybean meal is 1.02% -1.50% (Xun Yue et al, 2011, Jiangxi feed.) under the action of β -mannase, β -mannan in the soybean meal is degraded to release reducing sugar, the bionic digestion method is used for determining the reducing sugar release amount of the feed raw material, and the evaluation on the nutritional value of the feed and the effect of the feed enzyme preparation are of great significance.

Although the present application has been described in detail with respect to the general description and the specific examples, it will be apparent to those skilled in the art that certain changes and modifications may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.

Sequence listing

β -mannanase gene nucleotide sequence SEQ ID NO. 1:

1 TCCTTCGCTA GCACCTCCGG CCTCCAATTC ACCATTGATG GCGAAACTGG

51 CTACTTCGCC GGAACGAACA GCTACTGGAT CGGTTTCCTC ACTGACAACG

101 CGGACGTCGA CCTCGTCATG GGCCACCTGA AGTCGTCCGG CCTCAAGATC

151 CTCCGCGTGT GGGGCTTCAA CGATGTCACC TCGCAGCCCT CCTCCGGCAC

201 AGTTTGGTAC CAACTGCACC AGGACGGCAA ATCGACAATC AACACGGGTG

251 CCGACGGTCT CCAGCGCCTC GACTACGTCG TCTCGTCTGC CGAACAGCAC

301 GACATCAAAC TCATCATCAA CTTCGTCAAC TACTGGACCG ATTACGGTGG

351 TATGTCTGCG TACGTGAGCG CGTATGGCGG ATCCGGCGAG ACGGATTTCT

401 ATACCAGTGA TACCATGCAG AGTGCCTATC AGACATATAT CAAGACGGTC

451 GTGGAGCGGT ACAGTAACTC CTCGGCGGTG TTTGCGTGGG AGTTGGCGAA

501 TGAGCCGAGA TGTCCGAGTT GCGATACTTC TGTGTTGTAT AACTGGATTG

551 AGAAGACGAG TAAGTTTATT AAGGGGTTGG ATGCGGATCG TATGGTTTGT

601 ATTGGTGATG AGGGCTTCGG TCTCAACATC GACTCGGACG GCAGCTACCC

651 TTATCAATTC TCCGAGGGCT TGAACTTTAC GATGAACCTC GGTATCGATA

701 CTATTGACTT TGGTACCCTC CACTTGTACC CTGATAGCTG GGGCACCTCC

751 GACGACTGGG GCAACGGCTG GATCACCGCC CACGGCGCAG CCTGCAAAGC

801 GGCCGGCAAG CCATGTCTCC TGGAGGAATA CGGAGTCACC TCGAACCACT

851 GCAGTGTGGA GGGCTCGTGG CAGAAGACAG CGCTCAGCAC AACGGGCGTC

901 GGCGCGGATC TGTTCTGGCA GTATGGTGAT GATTTGAGTA CCGGGAAGTC

951 GCCGGATGAT GGGTTTACTA TCTACTATGG GACTAGTGAT TATCAGTGTC

1001 TGGTGACGGA TCATGTTGCT GCTATTGATA GCGCCTAA

amino acid sequence of β -mannase before optimization SEQ ID NO. 2:

1 SFASTSGLQF TIDGETGYFA GTNSYWIGFL TDNADVDLVM GHLKSSGLKI

51 LRVWGFNDVT SQPSSGTVWY QLHQDGKSTI NTGADGLQRL DYVVSSAEQH

101 DIKLIINFVN YWTDYGGMSA YVSAYGGSGE TDFYTSDTMQ SAYQTYIKTV

151 VERYSNSSAV FAWELANEPR CPSCDTSVLY NWIEKTSKFI KGLDADRMVC

201 IGDEGFGLNI DSDGSYPYQF SEGLNFTMNL GIDTIDFGTL HLYPDSWGTS

251 DDWGNGWITA HGAACKAAGK PCLLEEYGVT SNHCSVEGSW QKTALSTTGV

301 GADLFWQYGD DLSTGKSPDD GFTIYYGTSD YQCLVTDHVA AIGSA

the optimized β -mannase nucleotide sequence SEQ ID NO. 3:

1 TCATTCGCTT CTACTTCAGG ACTTCAGTTT ACTATTGATG GAGAGACAGG

51 ATACTTCGCT GGAACCAACT CTTACTGGAT TGGATTTTTG ACTGATAACG

101 CTGATGTTGA CTTGGTCATG GGTCATCTTA AATCTTCCGG ATTGAAGATC

151 CTTAGAGTTT GGGGTTTCAA TGATGTCACA AGTCAACCTT CAAGTGGAAC

201 CGTTTGGTAC CAATTGCATC AGGACGGTAA ATCTACCATT AACACTGGTG

251 CCGATGGATT GCAAAGACTT GACTATGTTG TCTCTTCCGC AGAACAGCAC

301 GATATCAAGT TGATTATCAA CTTCGTTAAC TACTGGACTG ACTATGGTGG

351 AATGTCAGCT TACGTCAGTG CCTATGGTGG TTCTGGTGAA ACTGATTTCT

401 ACACATCCGA CACCATGCAA TCAGCTTACC AGACTTACAT CAAGACAGTT

451 GTCGAGAGAT ACTCAAACTC AAGTGCCGTT TTTGCATGGG AATTGGCCAA

501 TGAGCCAAGA TGTCCTTCTT GCGATACCTC CGTTCTTTAC AACTGGATCG

551 AGAAGACTTC TAAGTTTATT AAGGGATTGG ATGCTGACAG AATGGTTTGT

601 ATTGGTGACG AAGGTTTCGG ATTGAACATC GATTCTGACG GTTCTTACCC

651 ATATCAATTT TCCGAGGGTT TGAACTTCAC TATGAATCTT GGAATTGATA

701 CCATCGACTT TGGTACTTTG CATCTTTATC CTGATTCATG GGGTACAAGT

751 GATGACTGGG GTAATGGATG GATTACCGCT CACGGAGCTG CCTGTAAAGC

801 AGCTGGTAAA CCATGCTTGC TTGAAGAGTA CGGAGTTACT TCTAACCACT

851 GCTCCGTCGA AGGTTCATGG CAAAAGACTG CATTGTCTAC TACAGGTGTT

901 GGAGCTGATT TGTTCTGGCA GTATGGAGAT GACCTTAGTA CTGGAAAGTC

951 TCCAGATGAC GGTTTTACAA TCTACTATGG TACTTCAGAC TATCAGTGTT

1001 TGGTTACAGA TCACGTTGCC GCTATTGGTT CAGCATAA

SEQ ID NO.4：

ManT-up：5′- CCGGAATTCTCATTCGCTTCTACTTCAGGACTTCAGTTTACT-3′

SEQ ID NO.5：

ManT-dn：5′- TAAAGCGGCCGCTTATGCTGAACCAATAGCGGCAACGTGATC -3′。

SEQ ID NO.6：

a-factor primer: CTACTATTGCCAGCATTGCTGCT

SEQ ID NO.7

3' AOX primer: GAGGATGTCAGAATGCCATTTGCC

Sequence listing

<110> Beijing-challenged agriculture technology Co., Ltd

<120> gene for expressing acid β -mannase, vector and application thereof

<130>200528

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>1038

<212>DNA

<213>Aspergillus niger

<400>1

tccttcgcta gcacctccgg cctccaattc accattgatg gcgaaactgg ctacttcgcc 60

ggaacgaaca gctactggat cggtttcctc actgacaacg cggacgtcga cctcgtcatg 120

ggccacctga agtcgtccgg cctcaagatc ctccgcgtgt ggggcttcaa cgatgtcacc 180

tcgcagccct cctccggcac agtttggtac caactgcacc aggacggcaa atcgacaatc 240

aacacgggtg ccgacggtct ccagcgcctc gactacgtcg tctcgtctgc cgaacagcac 300

gacatcaaac tcatcatcaa cttcgtcaac tactggaccg attacggtgg tatgtctgcg 360

tacgtgagcg cgtatggcgg atccggcgag acggatttct ataccagtga taccatgcag 420

agtgcctatc agacatatat caagacggtc gtggagcggt acagtaactc ctcggcggtg 480

tttgcgtggg agttggcgaa tgagccgaga tgtccgagtt gcgatacttc tgtgttgtat 540

aactggattg agaagacgag taagtttatt aaggggttgg atgcggatcg tatggtttgt 600

attggtgatg agggcttcgg tctcaacatc gactcggacg gcagctaccc ttatcaattc 660

tccgagggct tgaactttac gatgaacctc ggtatcgata ctattgactt tggtaccctc 720

cacttgtacc ctgatagctg gggcacctcc gacgactggg gcaacggctg gatcaccgcc 780

cacggcgcag cctgcaaagc ggccggcaag ccatgtctcc tggaggaata cggagtcacc 840

tcgaaccact gcagtgtgga gggctcgtgg cagaagacag cgctcagcac aacgggcgtc 900

ggcgcggatc tgttctggca gtatggtgat gatttgagta ccgggaagtc gccggatgat 960

gggtttacta tctactatgg gactagtgat tatcagtgtc tggtgacgga tcatgttgct 1020

gctattgata gcgcctaa 1038

<210>2

<211>345

<212>PRT

<213>Aspergillus niger

<400>2

Ser Phe Ala Ser Thr Ser Gly Leu Gln Phe Thr Ile Asp Gly Glu Thr

1 5 10 15

Gly Tyr Phe Ala Gly Thr Asn Ser Tyr Trp Ile Gly Phe Leu Thr Asp

20 25 30

Asn Ala Asp Val Asp Leu Val Met Gly His Leu Lys Ser Ser Gly Leu

35 40 45

Lys Ile Leu Arg Val Trp Gly Phe Asn Asp Val Thr Ser Gln Pro Ser

50 55 60

Ser Gly Thr Val Trp Tyr Gln Leu His Gln Asp Gly Lys Ser Thr Ile

65 70 75 80

Asn Thr Gly Ala Asp Gly Leu Gln Arg Leu Asp Tyr Val Val Ser Ser

85 90 95

Ala Glu Gln His Asp Ile Lys Leu Ile Ile Asn Phe Val Asn Tyr Trp

100 105 110

Thr Asp Tyr Gly Gly Met Ser Ala Tyr Val Ser Ala Tyr Gly Gly Ser

115 120 125

Gly Glu Thr Asp Phe Tyr Thr Ser Asp Thr Met Gln Ser Ala Tyr Gln

130 135 140

Thr Tyr Ile Lys Thr Val Val Glu Arg Tyr Ser Asn Ser Ser Ala Val

145 150 155 160

Phe Ala Trp Glu Leu Ala Asn Glu Pro Arg Cys Pro Ser Cys Asp Thr

165 170 175

Ser Val Leu Tyr Asn Trp Ile Glu Lys Thr Ser Lys Phe Ile Lys Gly

180 185 190

Leu Asp Ala Asp Arg Met Val Cys Ile Gly Asp Glu Gly Phe Gly Leu

195 200 205

Asn Ile Asp Ser Asp Gly Ser Tyr Pro Tyr Gln Phe Ser Glu Gly Leu

210 215 220

Asn Phe Thr Met Asn Leu Gly Ile Asp Thr Ile Asp Phe Gly Thr Leu

225 230 235 240

His Leu Tyr Pro Asp Ser Trp Gly Thr Ser Asp Asp Trp Gly Asn Gly

245 250 255

Trp Ile Thr Ala His Gly Ala Ala Cys Lys Ala Ala Gly Lys Pro Cys

260 265 270

Leu Leu Glu Glu Tyr Gly Val Thr Ser Asn His Cys Ser Val Glu Gly

275 280 285

Ser Trp Gln Lys Thr Ala Leu Ser Thr Thr Gly Val Gly Ala Asp Leu

290 295 300

Phe Trp Gln Tyr Gly Asp Asp Leu Ser Thr Gly Lys Ser Pro Asp Asp

305 310 315 320

Gly Phe Thr Ile Tyr Tyr Gly Thr Ser Asp Tyr Gln Cys Leu Val Thr

325 330 335

Asp His Val Ala Ala Ile Gly Ser Ala

340 345

<210>3

<211>1038

<212>DNA

<213>SFASTSGLQF TIDGETGYFA GTNSYWIGFL TDNADVDLVM GHLKSSGLKI

<400>3

tcattcgctt ctacttcagg acttcagttt actattgatg gagagacagg atacttcgct 60

ggaaccaact cttactggat tggatttttg actgataacg ctgatgttga cttggtcatg 120

ggtcatctta aatcttccgg attgaagatc cttagagttt ggggtttcaa tgatgtcaca 180

agtcaacctt caagtggaac cgtttggtac caattgcatc aggacggtaa atctaccatt 240

aacactggtg ccgatggatt gcaaagactt gactatgttg tctcttccgc agaacagcac 300

gatatcaagt tgattatcaa cttcgttaac tactggactg actatggtgg aatgtcagct 360

tacgtcagtg cctatggtgg ttctggtgaa actgatttct acacatccga caccatgcaa 420

tcagcttacc agacttacat caagacagtt gtcgagagat actcaaactc aagtgccgtt 480

tttgcatggg aattggccaa tgagccaaga tgtccttctt gcgatacctc cgttctttac 540

aactggatcg agaagacttc taagtttatt aagggattgg atgctgacag aatggtttgt 600

attggtgacg aaggtttcgg attgaacatc gattctgacg gttcttaccc atatcaattt 660

tccgagggtt tgaacttcac tatgaatctt ggaattgata ccatcgactt tggtactttg 720

catctttatc ctgattcatg gggtacaagt gatgactggg gtaatggatg gattaccgct 780

cacggagctg cctgtaaagc agctggtaaa ccatgcttgc ttgaagagta cggagttact 840

tctaaccact gctccgtcga aggttcatgg caaaagactg cattgtctac tacaggtgtt 900

ggagctgatt tgttctggca gtatggagat gaccttagta ctggaaagtc tccagatgac 960

ggttttacaa tctactatgg tacttcagac tatcagtgtt tggttacaga tcacgttgcc 1020

gctattggtt cagcataa 1038

<210>4

<211>42

<212>PRT

<213>primer

<400>4

Cys Cys Gly Gly Ala Ala Thr Thr Cys Thr Cys Ala Thr Thr Cys Gly

1 5 10 15

Cys Thr Thr Cys Thr Ala Cys Thr Thr Cys Ala Gly Gly Ala Cys Thr

20 25 30

Thr Cys Ala Gly Thr Thr Thr Ala Cys Thr

35 40

<210>5

<211>42

<212>DNA

<213>PRIMER

<400>5

taaagcggcc gcttatgctg aaccaatagc ggcaacgtga tc 42

<210>6

<211>23

<212>DNA

<213>PRIMER

<400>6

ctactattgc cagcattgct gct 23

<210>7

<211>24

<212>DNA

<213>PRIMER

<400>7

gaggatgtca gaatgccatt tgcc 24

Claims (9)

1. A gene for expressing acid β -mannase is characterized in that the nucleotide sequence of the gene is SEQID NO. 3.

2. An expression cassette, recombinant vector or recombinant microorganism comprising the gene of claim 1.

3. An expression vector comprising the gene of claim 1.

4. The expression vector of claim 3, wherein the expression vector is the pPICZ α A plasmid comprising the gene of claim 1.

5. The expression vector of claim 4, wherein the gene of claim 1 is ligated into pPICZ α A plasmid after restriction with EcoRI and NotI restriction enzymes.

6. A recombinant strain for expressing β -mannanase gene, comprising the expression vector of any one of claims 3-5.

7. The recombinant strain of claim 6, wherein the host bacterium of the recombinant strain is Pichia pastoris.

8. Use of the expression vector of any one of claims 3-5 or the recombinant strain of any one of claims 6-7 for the fermentative preparation of β -mannanase.

9. A fermentation method of acidic β -mannase, which comprises the following steps:

inoculating the recombinant strain as claimed in claim 6 or 7 into a fermentation medium, wherein the pH value is maintained at 4.5-5.0 by ammonia water during fermentation at 28-30 ℃, glycerol with the mass fraction of 50% is fed in after the glycerol is exhausted, the feeding rate is 10-20 m L/L/h, the wet weight of the cells is maintained at 200-250 g/L, then methanol induction is started, the flow rate of methanol is controlled at 3-7m L/L/h, the aeration ratio is maintained at 1:1.5 during the whole fermentation process, and the stirring speed is 140 rpm;

wherein the fermentation medium contains 20-50 g/L of glycerol, 30-60 g/L of ammonium dihydrogen phosphate, 5-15 g/L of monopotassium phosphate, 5-15 g/L of potassium sulfate, 10-20 g/L of magnesium sulfate, 0.5-2 g/L of calcium sulfate and 1-2 g/L14-7 g/L of potassium hydroxide.

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