CN102321646B - Beta-endoglucanase PEGase-2 gene, its expressed products and application - Google Patents

Abstract

The protein encoded by the gene comprises 305 amino acids, the amino acid sequence has an identity of 44 % with Paecilomyces thermophila. The gene is recombined on the expression vector, and then is respectively transformed into yeast and Trichoderma reesei, and the expressed protein can be secreted out of the cell. The protein can be prepared by carrying out fractional precipitation for crude separation by using ammonium sulfate with the concentration of 40-70 %, and purifying the crude product with HiTrapTMCaptoTM Q ion exchange chromatography glue. The expressed products can be used for specifically hydrolyzing beta-1,3(4)-glucan, have high activity for hydrolyzing laminarin, and the activity for hydrolyzing beta-1,3(4)-glucan can be improved in the presence of metal ions. The expressed products can be applied in brewing and can be used as additives of medicine, food and feed.

Description

Beta-glucan restriction endonuclease PEGase-2 gene and expression product and application

[technical field]

The present invention relates to molecular biology, relate in particular to a kind of beta-glucan restriction endonuclease PEGase-2 gene and expression product and application.

[background technology]

β-1,3 (4)-endoglucanases (Endo-β-1,3 (4)-glucanase, EGase, hereinafter to be referred as " beta-glucan restriction endonuclease ", the zymetology classification number is EC3.2.1.73) be occasionally specific hydrolysis β-1 of a class, glycoside hydrolase (the glycosylhydrolases of the β adjoined with β-1,3 glycosidic link in 3 (4)-dextran-Isosorbide-5-Nitrae glycosidic link, GH), hydrolysate is generally 3～5 oligosaccharides ^[1].

Fructus Hordei Germinatus is the main raw material of beer production, and barley endosperm cell walls approximately 70% component is β-1,3 (4)-dextran ^[2].In Process of Beer Brewing, the protein of saccharifying sex change can cause yeast to deposit in early days with excessive β-1,3 (4)-glucan binding domian, thus the impact fermentation, and cause the di-acetyl reduction period in Process of Beer Brewing to extend; Excessive β-1,3 (4)-dextran also can cause that beer is muddy and precipitate, affect the quality of beer.Therefore, beer, after the raw material saccharification, add appropriate beta-glucan restriction endonuclease with the β in hydrolysis material-1,3 (4)-dextran.Oatmeal is because fibre content is high, being of high nutritive value becomes the food that the elderly likes, but the oatmeal of excessive amount easily causes the elderly's maldigestion, therefore, add after appropriate beta-glucan restriction endonuclease or edible oatmeal the edible several polyzyme tabletses containing the beta-glucan restriction endonuclease again in oatmeal and can contribute to digestion.Also containing a large amount of β-1,3 (4)-dextran, easily cause maldigestion during feed that the son pig feed is main raw material in order to corn in the corn embryosperm cell walls.Therefore, add appropriate beta-glucan restriction endonuclease in feed, allow son pig edible digestible feed early, can allow son pig and early-weaning, thereby improve the production efficiency of live pig.

Plant, fungus and bacterium all contain the beta-glucan restriction endonuclease, and plant beta-glucan restriction endonuclease belongs to the GH17 family of glycoside hydrolase, and microorganism beta-glucan restriction endonuclease belongs to the GH16 family of glycoside hydrolase.Although the beta-glucan restriction endonuclease of GH16 and GH17 family has similar function, they structurally do not have similarity, and GH17 family member's tertiary structure is (beta/alpha) ₈tubbiness ^[3], GH16 family member's tertiary structure is the folding web-like of β-sandwich ^[4].Barley beta-glucan restriction endonuclease is the highest in seed germination early expression amount, its optimum pH (pH _opt) be 4.6, optimum temperuture (T _opt) be 45 ℃ ^[5,6].Genus bacillus beta-glucan restriction endonuclease also can special hydrolysis barley β-1,3 (4)-dextran, its pH _optbetween 6.0～7.5, T _optbetween 58 ℃～60 ℃ ^[7-13], but the large multipair acid environment sensitivity of such beta-glucan restriction endonuclease ^[1].Bacillus macerans beta-glucan restriction endonuclease is one of the most heat-resisting beta-glucan restriction endonuclease, T _optbe 65 ℃, also have 80% activity in the time of 75 ℃, pH _optfor neutrality.The pH of the beta-glucan restriction endonuclease of bacillus amyloliquefaciens _optbe 6.0, be subject to sour environment to affect minimum ^[14].The heterozyme H (A16-M) that 16 amino acid of 16 amino acid substitution Bacillus macerans beta-glucan restriction endonuclease N ends holding with bacillus amyloliquefaciens beta-glucan restriction endonuclease N are combined into also has 80% activity in the time of 80 ℃, the sour environment tolerance is increased, specificity to barley β-1,3 (4)-dextran is also also high by 40% than wild-type Bacillus macerans beta-glucan restriction endonuclease ^[15], but the intellecture property of domestic not this fermentoid, the at present main dependence on import of the beta-glucan restriction endonuclease that beer industry is used.The substrate of fungi beta-glucan restriction endonuclease hydrolysis is more extensive than barley and genus bacillus beta-glucan restriction endonuclease, can not only be hydrolyzed barley β-1,3 (4)-dextran, can also hydrolyzed carboxymethylcellulo, e and laminarin ^[16-19].On genus bacillus beta-glucan restriction endonuclease and fungi beta-glucan restriction endonuclease, certain research is also arranged although domestic ^{[11-13, 19,20]}, but the beta-glucan restriction endonuclease of energy import substitutes does not also occur.

[summary of the invention]

Based on this, be necessary to provide a kind of beta-glucan restriction endonuclease PEGase-2 gene that can express the albumen with beta-glucan endonuclease activity, and its expression product and application are provided.

A kind of beta-glucan restriction endonuclease PEGase-2 gene comprises following sequence:

(a), the polynucleotide of the polypeptide that forms of the aminoacid sequence shown in coding SEQ ID No.1, or the complementary strand of described polynucleotide;

And the polynucleotide of the polynucleotide of the polypeptide that forms of the aminoacid sequence shown in coding SEQ ID No.1 with at least 98% homology, or the complementary strand of described polynucleotide (b);

(c), the complementary strand of the polynucleotide of coded polypeptide or described polynucleotide, described polypeptide is comprised of the aminoacid sequence shown in SEQ ID No.1, wherein one or more amino acid are lacked, are substituted or increased, and described polypeptide has the beta-glucan endonuclease activity; Or

(d), the polynucleotide of coded polypeptide or the complementary strand of described polynucleotide, described polypeptide has at least 98% homology with the polypeptide be comprised of the aminoacid sequence shown in SEQ ID No.1.

A kind of beta-glucan restriction endonuclease PEGase-2 gene comprises following sequence:

(a), the polynucleotide that formed by the nucleotide sequence shown in SEQ ID No.2, or the complementary strand of described polynucleotide;

And the polynucleotide of the polynucleotide that form of the nucleotide sequence shown in coding SEQ ID No.2 with at least 98% homology, or the complementary strand of described polynucleotide (b); Or

(c), the complementary strand of the polynucleotide of coded polypeptide or described polynucleotide, described polynucleotide sequence is comprised of the nucleotide sequence shown in SEQ ID No.2, wherein one or more codons are lacked, are substituted or increased, and described polypeptide has the beta-glucan endonuclease activity.

Preferably, described beta-glucan restriction endonuclease PEGase-2 gene comprises the polynucleotide that are comprised of the nucleotide sequence shown in SEQ ID No.2.

A kind of expression vector, comprise above-mentioned beta-glucan restriction endonuclease PEGase-2 gene.

A kind of host cell, comprise expression vector described above, and described host cell is eukaryotic cells.

Preferably, described eukaryotic cells is yeast cell or Trichodermareesei cell.

A kind of preparation method of protein, comprise the steps:

Step 1, provide above-mentioned host cell, abduction delivering obtains expressing liquid;

Step 2, utilize 40% and 70% saturated ammonium sulphate by described expression liquid fractionation precipitation, separate crude product;

Step 3, utilize HiTrap ^tMcapto ^tMq ion exchange chromatography glue purification crude product, obtain described protein.

A kind of protein comprises following sequence:

(a) polypeptide, formed by the aminoacid sequence shown in SEQ ID No.1;

And the polypeptide of the polypeptide formed by the aminoacid sequence shown in SEQ ID No.1 with at least 98% homology (b); Or

(c), the polypeptide that formed by the aminoacid sequence shown in SEQ ID No.1, wherein one or more amino acid are lacked, are substituted or increased, and described polypeptide has the beta-glucan endonuclease activity.

A kind of zymin, comprise above-mentioned protein; Described zymin is for being hydrolyzed β-1,3 (4)-dextran.

Preferably, described zymin also comprises for improving the metallic cation of enzymic activity.

Screening P14-3-3 related protein gene from the Physarum Polycephalum cDNA library, isolate one section coding have the beta-glucan endonuclease activity beta-glucan restriction endonuclease albuminoid 3 '-the cDNA fragment.This 3 '-the beta-glucan restriction endonuclease albuminoid of cDNA fragment expression has the aminoacid sequence as shown in SEQ ID No.1, this 3 '-the cDNA fragment has the nucleotide sequence as shown in SEQ ID No.2.Above-mentioned beta-glucan restriction endonuclease PEGase-2 gene can be expressed the protein with beta-glucan endonuclease activity, and expression product can be applied to brewage, and as medicine, food and feed additive.

[accompanying drawing explanation]

The aminoacid sequence comparison chart of the beta-glucan restriction endonuclease that Fig. 1 is PEGase-2 and thermophilic Paecilomyces varioti.

Fig. 2 is the beta-glucan restriction endonuclease of the thermophilic Paecilomyces varioti of simulation and the tertiary structure schematic diagram of PEGase-2; Wherein, A is the tertiary structure of the thermophilic Paecilomyces varioti beta-glucan restriction endonuclease of simulation, and B is the tertiary structure of the PEGase-2 of simulation.

Fig. 3 is the schematic diagram that the Trx-PEGase-2 that expresses in Bacillus coli cells of embodiment 2 and Trx label protein carry out Coomassie blue stain after SDS-PAGE.

Fig. 4 is after PEGase-2 that embodiment 3 expresses in yeast cell carries out SDS-PAGE, the schematic diagram of Coomassie blue stain; Wherein, the PEGase-2 that arrow points is expressed.

The schematic diagram of Fig. 5 Coomassie blue stain that is embodiment 4 after the PEGase-2 of Trichodermareesei cell inner expression and negative control group are carried out SDS-PAGE simultaneously; Wherein, the PEGase-2 that arrow points is expressed.

The T.reesei QM9414 that Fig. 6 is the expression PEGase-2 that obtains from embodiment 4 goes down to posterity 7 times, select 2nd generation and the 8th generation thalline extract genome, the electrophoresis schematic diagram obtained of pcr amplification PEGase-2 gene fragment rear electrophoresis.

The T.reesei QM9414 that Fig. 7 is the expression PEGase-2 that obtains from embodiment 4 goes down to posterity 7 times, select 2nd generation and the 8th generation the thalline expression-secretion the vigor of PEGase-2 hydrolysis barley β-glucan detect schematic diagram.

Fig. 8 is the supernatant liquor that in embodiment 4, experimental group and negative control group are collected, the schematic diagram of coomassie brilliant blue staining after crude enzyme liquid that in embodiment 6, roughing out obtains carries out SDS-PAGE together with the albumen obtained after purifying; Wherein, the PEGase-2 of arrow points purifying.

The two reciprocal equation graphic representations of Lineweaver-Burk that Fig. 9 is purifying PEGase-2 hydrolysis barley β-glucan of obtaining of embodiment 6.

Figure 10 is that the purifying PEGase-2 that embodiment 6 obtains is hydrolyzed the variation schematic diagram of the vigor of barley β-glucan with reaction system pH.

Figure 11 is that the purifying PEGase-2 that embodiment 6 obtains is hydrolyzed the variation diagram of the vigor of barley β-glucan with temperature of reaction system.

Figure 12 is that purifying PEGase-2 that embodiment 6 obtains is hydrolyzed barley β-glucan vigor and schemes over time under differing temps.

Figure 13 is hydrolysis barley β after purifying PEGase-2 that embodiment 6 obtains deposits 24 hours in different pH damping fluids-glucan vigor variation diagram.

Figure 14 is that the purifying PEGase-2 that embodiment 6 obtains is hydrolyzed the schematic diagram of barley β-glucan vigor under the different types of metal ion of different concns exists.

Figure 15 is the technology schematic diagram of PEGase-2 gene in Trichodermareesei QM9414 recombinant conversion.

[embodiment]

Below in conjunction with drawings and Examples, beta-glucan restriction endonuclease PEGase-2 gene and expression product thereof and application are further explained to explanation.

The Physarum Polycephalum 14-3-3 albumen P14-3-3 (GenBank sequence number EF140724) of take is bait albumen ^[21], by yeast two-hybrid, screening P14-3-3 related protein gene from the Physarum Polycephalum cDNA library ^[22], isolate one section coding have the beta-glucan endonuclease activity beta-glucan restriction endonuclease albuminoid 3 '-the cDNA fragment.This 3 '-the beta-glucan restriction endonuclease albuminoid of cDNA fragment expression has the aminoacid sequence as shown in SEQ ID No.1, this 3 '-the cDNA fragment has the nucleotide sequence as shown in SEQ ID No.2.

The beta-glucan restriction endonuclease PEGase-2 gene of one embodiment is provided, and this genes encoding has the protein of beta-glucan endonuclease activity.

In embodiment preferably, this beta-glucan restriction endonuclease PEGase-2 gene comprises the polynucleotide of the polypeptide that the aminoacid sequence shown in coding SEQ ID No.1 forms, or the complementary strand of described polynucleotide.Especially, this beta-glucan restriction endonuclease PEGase-2 gene comprises the polynucleotide that are comprised of the nucleotide sequence shown in SEQ ID No.2, or the complementary strand of described polynucleotide.

The polynucleotide that this beta-glucan restriction endonuclease PEGase-2 gene can also have at least 98% homology for the polynucleotide that comprise the polypeptide formed with the aminoacid sequence shown in coding SEQ ID No.1, or the complementary strand of described polynucleotide; Be preferably and at least there is at least 99% homology.Especially, the polynucleotide that the polynucleotide that the nucleotide sequence shown in this beta-glucan restriction endonuclease PEGase-2 gene and coding SEQ ID No.2 forms have at least 98% homology, or the complementary strand of described polynucleotide; Be preferably and at least there is at least 99% homology.

This beta-glucan restriction endonuclease PEGase-2 gene can also be the polynucleotide that comprise coded polypeptide or the complementary strand of described polynucleotide, described polypeptide is comprised of the aminoacid sequence shown in SEQ ID No.1, wherein one or more amino acid are lacked, are substituted or increased, and described polypeptide has the beta-glucan endonuclease activity.Especially, this beta-glucan restriction endonuclease PEGase-2 gene comprises the polynucleotide of coded polypeptide or the complementary strand of described polynucleotide, described polynucleotide sequence is comprised of the nucleotide sequence shown in SEQ ID No.2, wherein one or more codons are lacked, are substituted or increased, and described polypeptide has the beta-glucan endonuclease activity.

This beta-glucan restriction endonuclease PEGase-2 gene can also be the polynucleotide that comprise coded polypeptide or the complementary strand of described polynucleotide, and described polypeptide has at least 98% homology with the polypeptide be comprised of the aminoacid sequence shown in SEQ ID No.1; Be preferably and at least there is at least 99% homology.

This beta-glucan restriction endonuclease PEGase-2 gene can be expressed the protein with beta-glucan endonuclease activity, and expression product can be applied to brewage, and as medicine, food and feed additive.

Due to polymorphism and the variation of protein coding gene, naturally occurring protein there will be amino acid whose disappearance, insertion, replacement or other variation, thereby causes the aminoacid sequence of protein to occur that one or more amino acid is lacked, substitutes or increases.In fact, exist on some physiology and biological activity and substantially be equal to the albumen without variant protein matter.These structures are different from corresponding protein but do not have the polypeptide of obvious function difference or albumen to be called the functional equivalent varient with this protein.

The varient of functional equivalent is equally applicable to change one or more codons by the artificial means such as disappearance, insertion and sudden change, thus the polypeptide of making to importing this class variation in a kind of aminoacid sequence of protein.Even now can obtain how multi-form varient, but the varient of gained is that its physiologically active is equal to the original activity without variant protein matter substantially as the prerequisite of functional equivalent varient.

For example, the heterozyme H (A16-M) that 16 amino acid of 16 amino acid substitution Bacillus macerans beta-glucan restriction endonuclease N ends holding with bacillus amyloliquefaciens beta-glucan restriction endonuclease N are combined into also has 80% activity in the time of 80 ℃, the sour environment tolerance is increased, specificity to barley β-1,3 (4)-dextran is also also high by 40% than wild-type Bacillus macerans beta-glucan restriction endonuclease ^[15].

In addition, when by gene engineering method, producing protein, often desired protein is expressed as to fused protein, for example, the N-terminal peptide chain that comes from other protein is added to the N-terminal of desired protein to improve the expression of desired protein, perhaps a suitable peptide chain is added to N or the C-terminal of desirable proteins, expresses this albumen and use a kind of carrier that added peptide chain is had to avidity that the purifying of desirable proteins is become being more prone to.

With regard to the codon (combination of triplet base) of specific amino acids on determining gene, there is 1 to 6 codon in every seed amino acid.Although therefore depend on aminoacid sequence, still have a kind of amino acid whose gene of many codings.At occurring in nature, gene is also unstable, and the nucleic acid variation often occurs.The variation of gene may not affect coded aminoacid sequence (silent variant), in this case, can produce the different genes of coding same acid sequence.Therefore, even isolated the gene of coding specific amino acid sequence, along with containing going down to posterity of this gene biological body, still inevitably can produce the different genes of coding same acid sequence.

The range gene that manually produces the coding same acid sequence by the range gene engineering is not difficult.For example, when the codon of the natural gene of coding desired protein when the protein content lower, that express of the host's utilizability for produce protein with genetic engineering technique is inadequate, can manually codon be transformed into to another codon that utilizability is high in this host and not change coded aminoacid sequence, can strengthen the expression of desired protein.Therefore, the artificial different polynucleotide of producing of this class are also included within scope of the present invention, the aminoacid sequence disclosed by the invention as long as it can be encoded out.

In addition, the activity that there is on function protein as described in being equal to by least one changes the polypeptide of (as disappearance, insertion or the replacement of one or more amino-acid residues are arranged in the aminoacid sequence of protein) gained or albumen, the gene of this class polypeptide or albumen of encoding is also included within scope of the present invention, no matter it separates or artificial production from natural origin.

General, the gene that encoding function is equal to varient is homology.The nucleic acid molecule that therefore, can have the protein of PEGase-2 activity with gene recombination of the present invention and coding is also included within scope of the present invention.

The protein of one embodiment is provided, is obtained by above-mentioned beta-glucan restriction endonuclease PEGase-2 genetic expression.

This protein is beta-glucan restriction endonuclease albuminoid and has the beta-glucan endonuclease activity.

In embodiment preferably, this protein comprises the polypeptide be comprised of the aminoacid sequence shown in SEQ ID No.1.

The polypeptide that this protein can also comprise and the polypeptide that is comprised of the aminoacid sequence shown in SEQ ID No.1 has at least 98% homology.

This protein can also comprise the polypeptide be comprised of the aminoacid sequence shown in SEQ ID No.1, and wherein one or more amino acid are lacked, substitute or increase, and described polypeptide has the beta-glucan endonuclease activity.

The expression vector of one embodiment is provided, comprises above-mentioned beta-glucan restriction endonuclease PEGase-2 gene.

General, can select to adopt standard program to be sufficient to various conventional criteria carriers above-mentioned beta-glucan restriction endonuclease PEGase-2 gene, such as: pPET-32a (+), pPICZ α A, pPIC-GST etc. obtain the said gene expression vector.

This expression vector is transformed in host cell, obtains expressing the above-mentioned beta-glucan restriction endonuclease albuminoid with beta-glucan endonuclease activity.

Because Physarum Polycephalum is eukaryotic cell, host cell is generally selected eukaryotic cells, for example: yeast cell and Trichodermareesei cell.

Yeast cell during as host cell, not only can be expressed under 0.5% methanol induction, it can also be secreted into to extracellular.

The Trichodermareesei cell is as host cell, and the beta-glucan restriction endonuclease albuminoid of expression can, through chemically modified preferably, have higher activity.

The preparation method of the above-mentioned albuminoid of one embodiment is provided, comprises the steps:

Step 1, provide the host cell of the expression vector that comprises above-mentioned beta-glucan restriction endonuclease PEGase-2 gene, abduction delivering obtains expressing liquid.

Step 2, utilize 40% and 70% saturated ammonium sulphate by described expression liquid fractionation precipitation, separate crude product.

Step 3, utilize HiTrap ^tMcapto ^tMq ion exchange chromatography glue purification crude product, obtain described protein.

Prepare beta-glucan restriction endonuclease albuminoid by aforesaid method, also need the protein to obtaining to carry out zymologic property mensuration, mainly comprise the genetic stability of the suitableeest substrate, enzyme kinetics reaction constant, optimal reactive temperature, optimal reaction potential of hydrogen, temperature stability, pH stability, storage stability and the host cell of this beta-glucan restriction endonuclease albuminoid.

Concrete measuring method and determination data are introduced in specific embodiment.

The zymin of one embodiment is provided, is mainly used in being hydrolyzed β-1,3 (4)-dextran, this kind of enzyme preparation comprises above-mentioned beta-glucan restriction endonuclease albuminoid.

In embodiment preferably, zymin also comprises for improving the metallic cation of enzymic activity.

Under the existence of metallic cation, generally can make the activity of above-mentioned beta-glucan restriction endonuclease albuminoid be strengthened, concrete metallic cation can be: Cu ²⁺, Ni ²⁺, Zn ²⁺, Fe ²⁺, Ba ²⁺, Mg ²⁺, Ca ²⁺, Mn ²⁺and Co ²⁺.

It is below the specific embodiment part.

In following examples, if no special instructions, all operations all adopts this area standard openating procedure, the reagent adopted or carrier etc. are conventional reagent or conventional carrier, beta-glucan restriction endonuclease albuminoid has the aminoacid sequence as shown in SEQ ID No.1, and beta-glucan restriction endonuclease PEGase-2 gene has the nucleotide sequence as shown in SEQ ID No.2.

Embodiment 1

The screening of PEGase-2 gene, amplification and structural analysis.

Get 100mg Physarum Polycephalum (weight in wet base), with RNeasy Plant Mini Kit (QIAGEN, Germany), extract total RNA, use GeneRacer ^tMkit (Invitrogen, the U.S.) is prepared into cDNAs by the complete mRNAs of Physarum Polycephalum; By 5 '-RLM RACE (RNA ligase-mediated rapid amplification of 5 '-cDNA ends) technology, from Physarum Polycephalum global cDNA s 5 of amplification beta-glucan restriction endonuclease albuminoid '-the cDNA fragment; Separate the PCR product by 1% agarose gel electrophoresis, will be through AxyPrep with the Solution I of DNA Ligation Kit (Takara, Dalian) ^tMdNA Gel Extraction Kit (Axygen, the U.S.) the target dna fragment reclaimed is connected to pMD18T carrier (Takara, Dalian) on, to connect after product transforms intestinal bacteria E.coli Top10 and check order, splicing 5 '-cDNA and 3 '-cDNA sequence, the global cDNA sequence of acquisition Physarum Polycephalum beta-glucan restriction endonuclease albuminoid.This patent is by the albumen called after PEGase-2 (Physarum Endo-β-1,3 (4)-glucanase 2) of this cDNA coding.PEGase-2 has the aminoacid sequence as shown in SEQ ID No.1, and the PEGase-2 gene has the nucleotide sequence as shown in SEQ ID No.2.

According to ORF (the Open Reading frame) sequence of PEGase-2 global cDNA, design containing the primer 5 of BamH I restriction enzyme site (with the sequence of underscore) '-CGC gGATCCaTGTCCAGCAAGGTGCT-3 ' and containing the primer 5 of Hind III restriction enzyme site '-GGG aAGCTTtTACTGGTACACTTGGATG-3 ', take the Physarum Polycephalum global cDNA as masterplate, by round pcr, from Physarum Polycephalum global cDNA s, amplifies the PEGase-2 gene.

The beta-glucan restriction endonuclease aminoacid sequence of the aminoacid sequence of PEGase-2 genes encoding and thermophilic Paecilomyces varioti (Paecilomyces thermophila) is compared.

As shown in Figure 1, the aminoacid sequence of the beta-glucan restriction endonuclease of the aminoacid sequence of PEGase-2 genes encoding and thermophilic Paecilomyces varioti has 44% similarity.

The tertiary structure of the beta-glucan restriction endonuclease of the PEGase-2 tertiary structure of simulation and thermophilic Paecilomyces varioti is contrasted.

As shown in Figure 2, the tertiary structure of the PEGase-2 tertiary structure of simulation and the beta-glucan restriction endonuclease of thermophilic Paecilomyces varioti is similar, secondary structure be take β-lamella as main, but β-lamella secondary structure number is fewer than thermophilic Paecilomyces varioti beta-glucan restriction endonuclease, tertiary structure is loose than thermophilic Paecilomyces varioti beta-glucan restriction endonuclease also.

Explanation thus, PEGase-2's is not only different from thermophilic Paecilomyces varioti beta-glucan restriction endonuclease on primary structure, on higher structure, also with thermophilic Paecilomyces varioti beta-glucan restriction endonuclease, exists larger difference.The PEGase-2 higher structure does not have thermophilic Paecilomyces varioti beta-glucan restriction endonuclease rigorous, and thermostability can be poorer than thermophilic Paecilomyces varioti beta-glucan restriction endonuclease, but the substrate of effect may be more extensive.

Embodiment 2

Expression and the evaluation of PEGase-2 gene in Bacillus coli cells.

The PCR fragment that to cut through BamH I and Hind III enzyme with the Solution I of DNA Ligation Kit is connected on the pET32a of same endonuclease digestion (+), to connect product and be transformed into E.coli origami (DE3) (Invitrogen, the U.S.), at the upper screening positive clone of LB solid culture plate (containing 50 μ g/ml Amp and 30 μ g/ml Kan), extract recombinant plasmid pET32 (a)-pegase2 and carry out PCR, double digestion and order-checking evaluation.

The label protein (Thioredoxin, Trx, Trx) that extracts expression product Trx-PEGase-2 and this fusion rotein carries out SDS-PAGE simultaneously, after coomassie brilliant blue staining as shown in Figure 3.

As shown in Figure 3, the relative molecular mass of Trx-PEGase-2 (50kD) is than the label protein (Thioredoxin of this fusion rotein, Trx, Trx) the many 33kD of relative molecular mass (17kD), approach with the theoretical molecular mass of PEGase-2 albumen, illustrate that PEGase-2 is not subject to obvious chemically modified in Bacillus coli cells.Trx-PEGase-2 is not hydrolyzed the enzyme activity of barley β-1,3 (4)-dextran (β-glucan), laminarin (laminarin) and Walocel MT 20.000PV (carboxymethyl cellulose, CMC) yet.

Explanation thus, the PEGase-2 that prokaryotic cell prokaryocyte obtains as host cell expression does not have the beta-glucan endonuclease activity.

Embodiment 3

Expression and the evaluation of PEGase-2 gene in yeast cell.

Plasmid pET32 (a)-pegase2 of take is template, with containing the primer 5 in EcoR I site '-CCG gAATTCtCCAGCAAGGTGCTCCT-3 ' and containing the primer 5 in Xba I site '-GC tCTAGAthe encoding gene fragment of AACTGGTACACTTGGATGTAATTG-3 ' amplification PEGase-2; It is upper that the PCR fragment of EcoR I and Xba I enzyme being cut with the Solution I in DNA Ligation Kit is connected to the expression plasmid of yeast pPICZ α A (purchased from Invitrogen, the U.S.) that same enzyme cuts, and be transformed into clone in E.coli Top10; Extract recombinant plasmid pPICZ alpha A-pegase2 and carry out PCR, double digestion and order-checking evaluation; With restriction enzyme Sac I, by after its linearizing, electricity is transformed in competence P.pastoris GS115 (Invitrogen, the U.S.) cell, is containing screening positive clone bacterium colony on the YPDS culture plate of 50 μ g/ml Zeocin.Picking mono-clonal bacterium colony, in containing the liquid YPDS substratum of 50 μ g/ml Zeocin, cultivate (30 ℃, 250rpm) spend the night, then use CTAB (Hexadecyl trimethyl ammonium bromide) method ^[23]extract the recombinant bacterium genomic dna, detect the PEGase-2 gene be binned on Yeast genome by PCR.Be cultured to OD with the BMGY nutrient solution by the negative control yeast with containing the recombination yeast of PEGase-2 gene ₆₀₀it is 2～6 (30 ℃, 250rpm), thalline being transferred in BMMY inducing culture liquid [every 24hr, supplementing 0.5% (v/v) methyl alcohol once] induces PEGase-2 to express, secrete again, 0,6,12,24,36,48,60,72 and 84h after cultivation respectively get 2ml bacterium liquid, centrifugal collection supernatant, detect the PEGase-2 of expression of recombinant yeast secretion by SDS-PAGE, obtain Fig. 4, detect the enzyme activity of the PEGase-2 hydrolysis barley β-glucan in nutrient solution.

As shown in Figure 4, swimming lane M is standard molecular weight albumen, swimming lane 1～9 corresponds to respectively yeast cell induce after 0,6,12,24,36,48,60,72 and the bacterium liquid supernatant that obtains of 84h.

As seen from Figure 4, yeast cell to express obtains the PEGase-2 albumen that relative molecular mass is 44kD, than the many 11kD of relative molecular mass (33kD) of the PEGase-2 albumen of escherichia coli expression.The PEGase-2 albumen that the yeast expression secretion is described is chemically modified protein.This albumen has the vigor of hydrolysis barley β-1,3 (4)-dextran (β-glucan), but is not hydrolyzed the enzyme activity of laminarin and Walocel MT 20.000PV.

Explanation thus, the beta-glucan endonuclease activity that the PEGase-2 that yeast cell obtains as host cell expression has.

Embodiment 4

The PEGase-2 gene is in the intracellular expression of Trichodermareesei and evaluation.

Plasmid pET32 (a)-pegase2 of take is template, use primer 5 '-CGCAGCTAGTGTGCCTCTAGAGGAGCGGATGTCCAGCAAGGTGCTCCTG-3 ' and 5 '-TTACTGGTACACTTGGAT GTAATTGAAAATCCA-3 ' amplification PEGase-2 encoding gene fragment; With the Solution I in DNA Ligation Kit, this PCR fragment is connected on the plasmid pPIC-PST that the Eam1105I enzyme cuts, then will connect product Transformed E .coli Top10 clone recombinant plasmid pPIC-PST-pegase2, and extract plasmid pPIC-PST-pegase2 and carry out PCR and order-checking evaluation.Take plasmid pPIC-PST-pegase2 more afterwards as template, use primer 5 '-GAT aGATCTaAAAAAATATGAGCGCAGGGACAAGCA-3 ' and 5 '-AGTGTCGACTGGTACTG GGATACACGAAGAGCG-3 ', amplification PEGase-2 expression cassette P _gpd-pegase2-T _cbhI.The method of setting up with reference to Penttil and Liu Gang etc. ^[26,27], get 10 μ g expression cassette P _gpd-pegase2-T _cbhIwith 10 μ g plasmid PAN7-1 (cumulative volume is no more than 20 μ l), with 200 μ l concentration be 10 ⁸the T.reesei QM9414 of individual/ml (ATCC, the U.S.) protoplastis solution mixes, and after 48 ℃ of heat shock 2min, adds the PEG4000 of 50 μ l 60% (containing 50mmol/L CaCl ₂the damping fluid of 10mmol/L TrisCl, pH 7.5) mix, after 20min (room temperature), add again the PEG4000 of 2ml 60% to mix, adding 8ml STC after 5min mixes, 8, collect protoplastis after the centrifugal 15min of 000rpm, with the resuspended protoplastis of 1ml STC solution and with 10ml protoplast regeneration nutrient solution (containing the Mandels basic culture solution of 1.2mol/L sorbyl alcohol), mix, cultivate 24h under 28 ℃, centrifugal 20min collects thalline, with the resuspended thalline of 1ml STC solution and be coated on 5 containing on the PDA culture plate of 100 μ g/ml hygromycin B, cultivate 2 days under 28 ℃; Take mycelium inoculation on culture plate to new PDA solid culture plate, continue to cultivate 7 days under 28 ℃.Collect the mycelium on culture plate with sterilized water, ratio in 1～5% is inoculated in 20ml and (is seeded on the PDA culture plate containing the Mandels nutrient solution of 100 μ g/ml hygromycin B simultaneously, after it grows spore, collect spore for conservation), under 28 ℃, cultivate 2 days, again in 1～5% ratio transfer in 25ml Mandels produce the enzyme nutrient solution cultivate 5 days (28 ℃, 250rpm), collect supernatant liquor, detect the PEGase-2 of expression of recombinant yeast secretion by SDS-PAGE, and measure the enzyme activity of the PEGase-2 hydrolysis barley β-glucan in nutrient solution.

Figure 15 is the technology schematic diagram of PEGase-2 gene in Trichodermareesei QM9414 recombinant conversion, recombinant plasmid pPIC-pegase2, by the signal peptide gene of the cellobiohydrolase II of the glyceraldehyde 3-phosphate dehydrogenase promotor of Puc ori, Zeocin, PEM7, PTEF1 and AOX1TT member and the Trichodermareesei of pPICZ α A carrier, Trichodermareesei and the cellobiohydrolase I terminator of Trichodermareesei.Wherein, the PEGase-2 gene is inserted between the signal peptide gene and cellobiohydrolase I terminator of cellobiohydrolase II.

Carry out negative control, control group adopts the single plasmid pAN7-1 that carries hygromycin gene to be transformed into Trichoderma reesei QM9414 (Trichodermareesei) simultaneously, and other conditions are identical with experimental group, collect supernatant liquor.

Adopt experimental group and negative control group to collect supernatant liquor and carry out SDS-PAGE, obtain Fig. 5, swimming lane M is standard molecular weight albumen, the fermented liquid that swimming lane 1 is collected for experimental group, the fermented liquid that the negative control group of swimming lane 2 is collected.

As seen from Figure 5, the relative molecular mass of the PEGase-2 of trichoderma reesei expression secretion is 65kD, than the many 30kD of PEGase-2 relative molecular mass of escherichia coli expression; Than the many 20kD of PEGase-2 relative molecular mass of yeast expression secretion, illustrate that the PEGase-2 of trichoderma reesei expression secretion is also higher than the chemically modified degree of the PEGase-2 of yeast expression secretion.The PEGase-2 of trichoderma reesei expression secretion has the vigor of hydrolysis barley β-1,3 (4)-dextran.

Embodiment 5

The genetic stability experiment of the Trichodermareesei containing the PEGase-2 encoding gene obtained in embodiment 4.

The inoculation of the T.reesei QM9414 recombinant bacterium of the expression PEGase-2 that obtains in embodiment 4, containing on antibiotic PDA culture plate, is inoculated after the spore grown on new PDA culture plate, so repeatedly, go down to posterity 7 times.

2nd generation and the 8th generation thalline carry genome by the CTAB method, pcr amplification PEGase-2 gene fragment identified, result as shown in Figure 6.

By 2nd generation and the 8th generation thalline receive liquid nutrient medium, the centrifuging and taking supernatant, survey enzyme activity according to a conventional method, result as shown in Figure 7.

As seen from Figure 6, go down to posterity after 7 times and still can from the genomic dna of recombinant bacterium, amplify the PEGase-2 gene.As seen from Figure 7, the PEGase-2 gone down to posterity after 7 times expresses the vigor that bacterium still has hydrolysis barley β-1,3 (4)-dextran.

Explanation thus, it is stable that PEGase-2 expresses bacterium in heredity, and is keeping the function of expression, secretion PEGase-2.

Embodiment 6

Roughing out and the purifying of the PEGase-2 of trichoderma reesei expression secretion in embodiment 4.

The roughing out of PEGase-2.

T.reesei QM 9414 recombinant bacteriums of expressing PEGase-2 are inoculated in containing in the Mandels basic culture solution of 100 μ g/ml hygromycin B, cultivating (28 ℃, 250rpm) after 2 days, the ratio in 1～5% connects bacterium in the Mandels culture medium, under 28 ℃, cultivate again 4～5 days, glucose to nutrient solution exhausts, afterwards under 4 ℃ centrifugal (10, 000rpm) 20min, collect fermented liquid supernatant and add ammonium sulfate to 40% saturation ratio, centrifugal collection supernatant after 2hr, add ammonium sulfate to 70% saturation ratio, centrifugal collecting precipitation, with 0.05mol/LHACNaAC damping fluid (pH5.0) dissolution precipitation albumen and dialyse 3 times, with 3, filter membrane (the Millipore in 000MW aperture, the U.S.) the dialyzate ultrafiltration and concentration is become to the 4ml crude enzyme liquid.By BCA (Bicinchonininc Acid) method ^[24]measure the protein content (test kit is produced by Beijing CWBIO company) of concentrated solution, and measure thick enzymic hydrolysis barley β-glucan vigor.

The purifying of PEGase-2.

The HiTrap be pre-installed by HACNaAC damping fluid (pH4.5) balance of 0.05mol/L ^tMcapto ^tMq ion exchange column (GE Healthcare, the U.S.), get 0.45 μ m membrane filtration upper prop for the 1ml crude enzyme liquid, with 0.05mol/L HACNaAC damping fluid (pH4.5) wash-out foreign protein, with containing 0～1mol/L NaCl damping fluid (0.05mol/L HACNaAC, pH4.5) gradient elution albumen, measure the protein content of each elution peak afterwards by the BCA method, and the specific activity of enzyme of hydrolysis barley β-glucan.

By the supernatant liquor that in embodiment 4, experimental group and negative control group are collected, the crude enzyme liquid obtained with roughing out in the present embodiment, together with the albumen obtained after purifying, carries out SDS-PAGE, and result as shown in Figure 8.Wherein, swimming lane M is standard molecular weight albumen, the supernatant liquor that swimming lane 1 is collected for negative control group in embodiment 4, and swimming lane 2 is the supernatant liquor that in embodiment 4, experimental group is collected, swimming lane 3 is HiTrap ^tMcapto ^tMq ion-exchange glue-line is analysed the PEGase-2 obtained after separation, purifying, the crude enzyme liquid that the sulphur ammonium fractionation precipitation that swimming lane 4 is 40%～70% obtains.

The BCA method is measured protein content, and shown in the specific activity of enzyme data plot following table of hydrolysis barley β-glucan:

Table 1

By Fig. 8 and upper table, can be drawn, by 40%～70% sulphur ammonium fractionation precipitation and HiTrap ^tMcapto ^tMq ion-exchange glue-line is analysed the PEGase-2 of separation, purifying trichoderma reesei expression, can reclaim 78.8% and 46.0% PEGase-2, and the ratio vigor of hydrolysis barley β-1,3 (4)-dextran can improve respectively 15.985 times and 25.966 times.

Concrete formula and the preparation method of part substratum, nutrient solution and the protoplastiss etc. of using in embodiment 1～6 are as follows.

YPDS solid medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, 1mol/L sorbyl alcohol, 20g/L agar powder.

The PDA solid medium: 20% potato leach liquor (potato that 200g smashs to pieces adds water 800ml, filters after boiling 30min, is settled to 1,000ml), and 10g/L glucose, 20g/L agar powder.

BMMY nutrient solution: 10g/L yeast extract, 20g/L peptone, 13.4g/L YNB, 0.5% methyl alcohol, 0.4mg/L vitamin H, 0.1mol/L potassium phosphate buffer.

STC solution: 1.2mol/L Sorbitol, 50mmol/L CaCl ₂, 10mmol/L TrisCl, pH 7.5

The Mandels basic culture solution ^[28]: 100ml Mandels concentrated nutrient solution [14g/L (NH4) ₂sO ₄, 3g/L urea, 20g/L KH ₂pO ₄, 4g/L CaCl ₂2H ₂o (or 3g/L CaCl ₂), 3g/L MgSO ₄7H ₂o], 1.0ml Mandels concentrates liquid microelement [5g/L FeSO ₄7H ₂o, 1.7g/L ZnSO ₄7H ₂o (or 0.7g/L ZnCl ₂), 3.7g/L CoCl ₂6H ₂o (or 2g/L CoCl ₂), 1.6g/L MnSO ₄h ₂o (or 1.67g/LMnCl ₂, 2.6g/L MnSO ₄7H ₂o], the 10g dextrose anhydrous, the 1.0g peptone, the citrate buffer solution of 50ml 1mol/L (pH 4.5), 1.0～2.0g tween 80, water is settled to 1,000ml.

Mandels produces the enzyme nutrient solution: 200ml Mandels concentrated nutrient solution, and 2.0ml Mandels concentrates liquid microelement, 30g Microcrystalline Cellulose, 3g glucose, 1.0g peptone, 50ml 1mol/L citrate buffer solution (pH 4.5), 1.0～2.0g tween 80, water is settled to 1,000ml.

The preparation of T.reesei QM9414 protoplastis: the Trichodermareesei spore inoculating, in the PDA culture plate, is cultivated 7 days for 28 ℃.Prepare spore suspension with sterilized water, get 0.8～1.0 * 10 ⁸individual spore inoculating, in the Mandels basic medium of 40ml, is cultivated 10～12hr under 28 ℃; When spore germination rate reaches 99%, get the centrifugal collection thalline of 25ml bacterium liquid, use 1mol/L MgSO ₄washing mycelia 2 times, centrifugal collection thalline, (be dissolved in 1mol/L MgSO with the lywallzyme liquid of 10ml10mg/mL ₄in) resuspended mycelia, after 28 ℃ of lower enzymolysis fungal cell wall 2～2.5hr, the STC liquid that adds 20ml, centrifugal collection protoplastis, with after twice of STC solution washing protoplastis, with 1ml STC liquid suspension protoplastis, calculate the concentration of protoplastis with blood counting chamber, use STC liquid by protoplastis concentration dilution to 10 ³individual/ml, the protoplastis of getting after 100 μ l dilute is coated 3 repetition protoplast regeneration rates of (2% agar powder, Mandels basic medium) upper calculating on the regeneration culture plate.The average regeneration rate of protoplastis is greater than 10% protoplastis and can be used for gene transformation.

Embodiment 7

The detection of the PEGase-2 enzyme activity of the purifying that embodiment 6 obtains.

1, the making of glucose typical curve.

The method of setting up with reference to Miller, getting 80 μ l concentration is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 and glucose solution and the 240 μ l 3 of 2.1mg/ml, 5-dinitrosalicylic acid (3, 5-dinitro-salicyli acid, DNS) liquid (7.5g DNS, 14.0g NaOH, 216.0g Seignette salt, 5.6ml liquid phenol and 6.0g Sodium Metabisulfite, water is settled to 1, 000ml, brown bottle is preserved and is used afterwards in 5 days) mix, after boiling water bath 10min, with spectramax 190 (Molecular Devices, the U.S.) microplate reader detects the absorbance (A of each reaction solution under 540nm ₅₄₀), take glucose concn as X-coordinate, with A ₅₄₀for ordinate zou, draw glucose concn and A ₅₄₀relation curve, obtain reflection glucose concn and A ₅₄₀the regression equation of relation, in order to calculate the PEGase-2 hydrolysis barley reducing sugar amount that β-glucan produces.

2, the detection of PEGase-2 enzyme activity.

1. enzyme reaction: get 20 μ l enzyme liquid and 40 μ l enzyme reaction buffer solution (0.05mol/L HACNaAC, pH5.0) mix, after 55 ℃ of water-bath 5min, add the substrate solution 20 μ l of 55 ℃ of water-bath 5min and mix, after reaction 10min, adding again 240 μ l 3,5-dinitrosalicylic acid (3,5-dinitro-salicyli acid, DNS) liquid, boiling water bath 10min termination reaction, cooling rear water is settled to 2ml, and measures A on spectrophotometer ₅₄₀value.

2. blank: get 20 μ l enzyme liquid and 40 μ l enzyme reaction buffer solution mix, the substrate that adds 55 ℃ of water-bath 5min of 20 μ l after 55 ℃ of water-bath 5min, add the DNS liquid of 240 μ l, boiling water bath 10min termination reaction afterwards immediately, cooling rear water is settled to 2ml, and measures A on spectrophotometer ₅₄₀value.

3, enzyme activity method of calculation.

A with enzyme reaction solution ₅₄₀value deducts the A of blank ₅₄₀value, the equation of linear regression of glucose concn is calculated in substitution afterwards, calculate the PEGase-2 hydrolysis barley concentration of reduced sugar that β-glucan generates, press the enzyme activity calculation formula: enzyme activity=(50*m)/(180*t) [wherein, m is the reducing sugar amount (μ g) that enzyme reaction discharges, 50 is extension rate, and 180 is the glucose molecule amount, and t is enzyme reaction time (min)] the calculating enzyme activity.

4, the enzyme activity of PEGase-2 definition.

Under 55 ℃ and pH5.0 condition, per minute hydrolysis barley β-glucan generates the required PEGase-2 enzyme amount of the reducing sugar (take glucose meter) of 1 μ mol as 1 unit of activity, with U, means.The enzyme activity of unit fermented liquid and unit zymoprotein preparation means with U/ml and U/mg.

5, the preparation of substrate solution.

1. 1% barley β-glucan solution: take 0.1g barley β-glucan (Megazyme, Ireland), add the 1ml dehydrated alcohol wetting, then add 8ml enzyme reaction buffer solution (HACNaAC of 0.05mol/L), in boiling water bath, dissolve, after cooling room temperature, be settled to 10ml, be mixed with the substrate solution that concentration is 10mg/ml.

2. 1% palmate sea-tangle laminarin solution: take 0.1g palmate sea-tangle laminarin (Sigma, the U.S.), add the 1ml dehydrated alcohol wetting, then add 8ml enzyme reaction buffer solution (HACNaAC of 0.05mol/L), in boiling water bath, dissolve, after cooling room temperature, be settled to 10ml, be mixed with the substrate solution that concentration is 10mg/ml.

3. 1%CMC solution: take 0.1g CMC, add 8ml enzyme reaction buffer solution (HAC-NaAC of 0.05mol/L), after fully dissolving, be settled to 10ml, be mixed with the substrate solution that concentration is 10mg/ml.

By aforesaid method, the PEGase-2 of purifying respectively with the reaction that is hydrolyzed of barley β-glucan, laminarin (laminarin) and Walocel MT 20.000PV (carboxymethyl cellulose, CMC), result is as shown in table 2.

Table 2

As can be seen from Table 2, the ratio vigor of the PEGase-2 of purifying hydrolysis barley β-glucan, laminarin and CMC is respectively 1.677,0.516 and 0.214U/mg, the specific substrate that the PEGase-2 of trichoderma reesei expression is described is barley β-1,3 (4)-dextran, but laminarin is also had to certain hydrolysis vigor.

Select barley β-glucan as substrate, the vigor of test PEGase-2 hydrolysis barley β-glucan under different test conditions, detected result is as shown in Fig. 9～Figure 14.

As seen from Figure 9, the K of PEGase-2 hydrolysis barley β-glucan _mvalue is 0.973mg/ml, V _maxvalue is 0.513mg/min.

As seen from Figure 10, the optimum pH of PEGase-2 hydrolysis barley β-glucan is 5.0, and PEGase-2 pH lower than 5.0 environment under than stable under higher than 7.0 environment at pH.

As seen from Figure 11, the optimum temperuture of PEGase-2 hydrolysis barley β-glucan is 55 ℃, and PEGase-2 stablizes in the environment lower than 55 ℃ than higher than the environment of 55 ℃.

Figure 12 is that PEGase-2 is hydrolyzed barley β-time dependent schematic diagram of glucan vigor under differing temps, and as can be seen from Figure, in the time of 40 ℃, PEGase-2 stability is best.

Figure 13 is hydrolysis barley β-glucan vigor schematic diagram after PEGase-2 deposits 24h under different pH damping fluids, and as can be seen from Figure, pH is 5.0 o'clock, and the PEGase-2 vigor keeps better.

Figure 14 is PEGase-2 under different concns, different sorts positively charged ion exist, the variation schematic diagram of the vigor of hydrolysis barley β-glucan.

As seen from Figure 14, β-the glucan vigor is influential to PEGase-2 hydrolysis barley for metal ion, the Cu of 1mmol/L ²⁺, Ni ²⁺, Zn ²⁺, Fe ²⁺, Ba ²⁺, Mg ²⁺, Ca ²⁺, Mn ²⁺and Co ²⁺can suppress 18.8%, 28.3%, 16.8%, 33.2%, 75.0%, 30.8%, 13.4%, 70.2% and 26.0% PEGase-2 hydrolysis vigor, the Cu of 5mmol/L ²⁺, Ni ²⁺, Zn ²⁺, Fe ²⁺, Ba ²⁺, Mn ²⁺and Co ²⁺can suppress 44.5%, 10.8%, 20.1%, 40.0%, 16.6%, 79.9% and 85.1% PEGase-2 hydrolysis vigor, but the Mg of 5mmol/L ²⁺and Ca ²⁺can improve 7.2% and 12.4% PEGase-2 hydrolysis vigor.

Illustrate that thus above-mentioned metallic cation is relevant with ionic concn on the impact of PEGase-2 hydrolysis vigor, the Mg of suitable concentration ²⁺and Ca ²⁺could improve the hydrolysis vigor of PEGase-2.

The above embodiment has only expressed one or more embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

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SEQUENCE LISTING

<110 > Shenzhen University

<120 >-endoglucanase PEGase-2 gene and expression product and application

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<170>PatentIn version 3.3

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<213 > the beta-glucan restriction endonuclease PEGase-2 of Physarum Polycephalum

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Met Ser Ser Lys Val Leu Leu Val Leu Leu Leu Gly Val Ala Ala Cys

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

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Asn Phe Asp Tyr Phe Thr Gly Asn Asp Pro Thr His Gly Cys Val Tyr

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atgtccagca aggtgctcct ggttttgctt ttgggggttg ctgcctgttc tgcctacact 60

cttcaggata actggcaagg agggcaaatt ttcgataatt tcgattactt cacgggcaat 120

gaccccacgc atggatgcgt gtactatgct tccaaggcag aagccaccca gtggtcgtac 180

acctacgagg ccaacggtca ggcgtggatc agatccgatg actctactat tgcccaaggt 240

tctggaagag gatctgtgag attgcaaagc cagaaggcct acacacacgg tttgttcatt 300

gctgacatcg aacacatgcc atttggggct ggtacatggc ccgctttttg gactaccaac 360

ggagctgttt ggccaaatgg tggagagatc gatattattg agggtgtgaa taccaacacc 420

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atttatgatg ctgactcatg gtcctacgga gatggcttca acaacaatgg aggtggtgtg 600

tgggccatgc aatgggagga aagtggtgta tacatctggc tctgggcacg caactacatc 660

cccgcggata tcaagagcaa caaccctgat ccgtctactt ggggagctcc tcgtggcagg 720

ttcacatttg accaaggatg cacagattcc caacacttct acaatcacaa catcattatt 780

gatctgacct tctgtggtga ctgggcagga ggtgtgtacc ctggtggaaa tggtgcttgc 840

tgtaactttg tctacaataa cccaaccgcc ttcgccgatg cctactggat tttcaattac 900

atccaagtgt accagtaa 918

Claims (9)

1. a beta-glucan restriction endonuclease PEGase-2 gene, is characterized in that, is following sequence:

The polynucleotide of the polypeptide that the aminoacid sequence shown in coding SEQ ID No.1 forms, or the complementary strand of described polynucleotide.

2. a beta-glucan restriction endonuclease PEGase-2 gene, is characterized in that, is following sequence:

The polynucleotide that formed by the nucleotide sequence shown in SEQ ID No.2, or the complementary strand of described polynucleotide.

3. beta-glucan restriction endonuclease PEGase-2 gene as claimed in claim 2, is characterized in that, described beta-glucan restriction endonuclease PEGase-2 gene is the polynucleotide that are comprised of the nucleotide sequence shown in SEQ ID No.2.

4. an expression vector, is characterized in that, comprises beta-glucan restriction endonuclease PEGase-2 gene as claimed in claim 1 or 2; Expression vector is eukaryotic gene expression vector.

5. a host cell, is characterized in that, comprises expression vector as claimed in claim 4, and described host cell is eukaryotic cells.

6. host cell as claimed in claim 5, is characterized in that, described eukaryotic cells is yeast cell or Trichodermareesei cell.

7. the preparation method of a protein, is characterized in that, comprises the steps:

Step 1, provide host cell as claimed in claim 5, abduction delivering obtains expressing liquid;

Step 2, utilize 40% and 70% saturated ammonium sulphate by described expression liquid fractionation precipitation, separate crude product;

Step 3, utilize HiTrap ^tMcapto ^tMq ion exchange chromatography glue purification crude product, obtain described protein.

8. a protein, is characterized in that, is following sequence:

The polypeptide formed by the aminoacid sequence shown in SEQ ID No.1; Protein is that eukaryotic expression obtains.

9. a zymin, is characterized in that, comprises protein as claimed in claim 8; Described zymin is for being hydrolyzed β-1,3 (4)-dextran.

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