CN102311965A - Beta-endoglucanase PEGase-1 gene and expression product and application thereof - Google Patents

Abstract

The invention discloses a beta-endoglucanase PEGase-1 gene separated from physarum polycephalum; protein coded by the gene comprises 309 amino acids; the amino acid sequence is 44% homologous to paecilomyces thermophila; a simulated tertiary structure is as a beta-sandwich folded coil; the protein belongs to a GH16 family member of glucoside hydrolase. The gene is recombined to an expression vector and is transformed into yeast and trichoderma reesei respectively; the expressed protein is secreted outside the cells; the protein can be separated crudely by fractional precipitation with 40%-70% ammonium sulfate, can be purified by HiTrapTMCapto TM Q ion exchange chromatographic gel, and can hydrolyze barley beta-1,3(4)-glucan specifically; the hydrolytic activity can be increased in the presence of metal ions; therefore the protein is applicable to beer brewage, and can be used as an additive for medicine, food and feed.

Description

Beta-glucan restriction endonuclease PEGase-1 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-1 gene and expression product and application.

[background technology]

β-1,3 (4)-endoglucanase (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 one type of specific hydrolysis β-1 of ability idol; Glycoside hydrolase (the glycosylhydrolases of β-1,4 glycosidic link that adjoins with β-1,3 glycosidic link in 3 (4)-VISOSEs; GH), hydrolysate is generally 3～5 oligosaccharides ^[1]

Fructus Hordei Germinatus is the main raw material of beer prodn, and the component of barley endosperm cell walls about 70% is β-1,3 (4)-VISOSE ^[2]In the brewage process, the protein of saccharifying sex change can cause yeast to deposit in early days with excessive β-1,3 (4)-VISOSE combination, thus the influence fermentation, and cause the di-acetyl reduction period in the brewage process to prolong; Excessive β-1,3 (4)-VISOSE also can cause the muddy and deposition of beer, influences the quality of beer.Therefore, beer will add an amount of beta-glucan restriction endonuclease with the β in the hydrolysis material-1,3 (4)-VISOSE after the raw material saccharification.Oatmeal is because of fibre content is high, being of high nutritive value becomes the food that the elderly likes; But the oatmeal of excessive amount causes the elderly's maldigestion easily; Therefore, in oatmeal, add an amount of beta-glucan restriction endonuclease or edible oatmeal after the edible more several polyzyme tabletses that contain the beta-glucan restriction endonuclease can help digestion.Also contain a large amount of β-1,3 (4)-VISOSE in the corn embryosperm cell walls, the son pig feed causes maldigestion easily when being the feed of main raw material in order to corn.Therefore, in feed, add an amount of beta-glucan restriction endonuclease, let the edible early digestible feed of son pig, can let son pig and early-weaning, thereby improve the production efficiency of live pig.

Plant, fungi and bacterium all contain the beta-glucan restriction endonuclease, and plant beta-glucan restriction endonuclease belongs to the GH17 family of glycoside hydrolase, and mikrobe beta-glucan restriction endonuclease then 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 then 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)-VISOSE, its pH _OptBetween 6.0～7.5, T _OptBetween 58 ℃～60 ℃ ^[7-13], but such beta-glucan restriction endonuclease is responsive to acid environment mostly ^[1]Soaking numb genus bacillus beta-glucan restriction endonuclease is one of the most heat-stable beta-glucan restriction endonuclease, T _OptBe 65 ℃, in the time of 75 ℃, also have 80% activity, pH _OptBe neutrality.The pH of the beta-glucan restriction endonuclease of bacillus amyloliquefaciens _OptBe 6.0, receive sour environment to influence minimum ^[14]The heterozyme H (A16-M) that 16 amino acid that soak numb genus bacillus beta-glucan restriction endonuclease N end with 16 amino acid replacements of bacillus amyloliquefaciens beta-glucan restriction endonuclease N end 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)-VISOSE is also soaked numb genus bacillus beta-glucan restriction endonuclease also high 40% than wild-type ^[15], but domestic not this zymoid intellecture property, the at present main dependence on import of the beta-glucan restriction endonuclease that beer industry uses.The substrate of fungi beta-glucan restriction endonuclease hydrolysis is more extensive than barley and genus bacillus beta-glucan restriction endonuclease, can not only hydrolysis barley β-1,3 (4)-VISOSE, and can also hydrolyzed carboxymethylcellulo, e and laminarin ^[16-19]Although domestic also have certain research on genus bacillus beta-glucan restriction endonuclease and fungi beta-glucan restriction endonuclease ^{[11-13, 19,20]}, but the beta-glucan restriction endonuclease of ability import substitutes does not also occur.

[summary of the invention]

Based on this, be necessary to provide a kind of and can express proteic beta-glucan restriction endonuclease PEGase-1 gene, and its expression product and application are provided with beta-glucan endonuclease activity.

A kind of beta-glucan restriction endonuclease PEGase-1 gene comprises following sequence:

(a), the polynucleotide of the polypeptide formed by the aminoacid sequence shown in the SEQ ID No.1 of coding, the perhaps complementary strand of said polynucleotide;

(b) and the polynucleotide of the polynucleotide of the polypeptide formed by the aminoacid sequence shown in the SEQ ID No.1 of coding with at least 98% homology, the perhaps complementary strand of said polynucleotide;

(c), the complementary strand of the polynucleotide of coded polypeptide or said polynucleotide; Said polypeptide is made up of the aminoacid sequence shown in the SEQ IDNo.1; Wherein one or more amino acid are lacked, are substituted or increased, and said polypeptide has the beta-glucan endonuclease activity; Or

(d), the polynucleotide of coded polypeptide or the complementary strand of said polynucleotide, said polypeptide has at least 98% homology with the polypeptide of being made up of the aminoacid sequence shown in the SEQ IDNo.1.

A kind of beta-glucan restriction endonuclease PEGase-1 gene comprises following sequence:

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

(b) and the polynucleotide of the polynucleotide formed by the nucleotide sequence shown in the SEQ ID No.2 of coding with at least 98% homology, the perhaps complementary strand of said polynucleotide; Or

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

Preferably, said beta-glucan restriction endonuclease PEGase-1 gene comprises the polynucleotide of being made up of the nucleotide sequence shown in the SEQ ID No.2.

A kind of expression vector comprises above-mentioned beta-glucan restriction endonuclease PEGase-1 gene.

A kind of host cell comprises that like above-mentioned expression vector said host cell is an eukaryotic cells.

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

A kind of proteinic preparation method comprises the steps:

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

Step 2, utilize 40% and 70% saturated ammonium sulphate with said expression liquid fractionation precipitation, crude product;

Step 3, utilize HiTrap ^TMCapto ^TMQ ion exchange chromatography glue purification crude product obtains said protein.

A kind of protein comprises following sequence:

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

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

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

A kind of zymin comprises above-mentioned protein; Said zymin is used for hydrolysis β-1,3 (4)-VISOSE.

Preferably, said zymin also comprises the metallic cation that is used to improve enzymic activity.

Screening PSRPK related protein gene from bull suede bubble bacterium 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 shown in SEQ ID No.1, this 3 '-the cDNA fragment has the nucleotide sequence shown in SEQ ID No.2.Above-mentioned beta-glucan restriction endonuclease PEGase-1 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.

[description of drawings]

Fig. 1 is the aminoacid sequence comparison figure of the beta-glucan restriction endonuclease of PEGase-1 and thermophilic Paecilomyces varioti.

Fig. 2 is the beta-glucan restriction endonuclease of the thermophilic Paecilomyces varioti of mimic and the tertiary structure synoptic diagram of PEGase-1; Wherein, A is the tertiary structure of the thermophilic Paecilomyces varioti beta-glucan of mimic restriction endonuclease, and B is the tertiary structure of mimic PEGase-1.

Trx-PEGase-1 that Fig. 3 expresses in Bacillus coli cells for embodiment 2 and Trx label protein carry out the synoptic diagram of Coomassie blue stain behind the SDS-PAGE.

The PEGase-1 that Fig. 4 expresses in yeast cell for embodiment 3 carries out the synoptic diagram of Coomassie blue stain behind the SDS-PAGE; Wherein, the PEGase-1 of arrow points expression.

Fig. 5 is the synoptic diagram of embodiment 4 Coomassie blue stain after the PEGase-1 of Trichodermareesei cell inner expression and negative control group are carried out SDS-PAGE simultaneously; Wherein, the PEGase-1 of arrow points expression.

Fig. 6 is for the T.reesei QM9414 of the expression PEGase-1 that obtains from embodiment 4 goes down to posterity after 7 times, select the 2nd generation and the 8th generation thalline extract genome, the electrophoresis synoptic diagram that obtains of pcr amplification PEGase-1 gene fragment rear electrophoresis.

Fig. 7 is for the T.reesei QM9414 of the expression PEGase-1 that obtains from embodiment 4 goes down to posterity after 7 times, select the 2nd generation and the 8th generation the thalline expression-secretion the vigor of PEGase-1 hydrolysis barley β-glucan detect synoptic diagram.

The supernatant that Fig. 8 collects for experimental group among the embodiment 4 and negative control group, the albumen that obtains behind crude enzyme liquid that roughing out obtains in embodiment 6 and the purifying carries out the synoptic diagram of coomassie brilliant blue staining behind the SDS-PAGESDS-PAGE; Wherein, the PEGase-1 of arrow points purifying.

The two reciprocal equation graphic representations of the Lineweaver-Burk of purifying PEGase-1 hydrolysis barley β-glucan that Fig. 9 obtains for embodiment 6.

The vigor of purifying PEGase-1 hydrolysis barley β-glucan that Figure 10 obtains for embodiment 6 is with the variation synoptic diagram of reaction system pH.

The vigor of purifying PEGase-1 hydrolysis barley β-glucan that Figure 11 obtains for embodiment 6 is with the variation diagram of temperature of reaction system.

The purifying PEGase-1 that Figure 12 obtains for embodiment 6 hydrolysis barley β-glucan vigor under differing temps is schemed over time.

The hydrolysis barley β-glucan vigor variation diagram purifying PEGase-1 that Figure 13 obtains for embodiment 6 deposits 24 hours in different pH damping fluids after.

Figure 14 is the synoptic diagram of purifying PEGase-1 hydrolysis barley β-glucan vigor in the presence of different concns different kinds of metals ion of obtaining for embodiment 6.

Figure 15 is the technological synoptic diagram of PEGase-1 gene in Trichodermareesei QM9414 recombinant conversion.

[embodiment]

Below in conjunction with accompanying drawing and embodiment beta-glucan restriction endonuclease PEGase-1 gene and expression product thereof are explained with being used as further.

With bull suede bubble bacterium SR protein kinase PSRPK (GenBank sequence number DQ140379) is bait albumen ^[21], through yeast two-hybrid, screening PSRPK related protein gene from bull suede bubble bacterium 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 shown in SEQ IDNo.1, this 3 '-the cDNA fragment has the nucleotide sequence shown in SEQ ID No.2.

The beta-glucan restriction endonuclease PEGase-1 gene of one embodiment is provided, and this genes encoding has the protein of beta-glucan endonuclease activity.

In more excellent embodiment, this beta-glucan restriction endonuclease PEGase-1 gene comprises the polynucleotide of the polypeptide that coding is made up of the aminoacid sequence shown in the SEQ IDNo.1, the perhaps complementary strand of said polynucleotide.Special, this beta-glucan restriction endonuclease PEGase-1 gene comprises the polynucleotide of being made up of the nucleotide sequence shown in the SEQ ID No.2, the perhaps complementary strand of said polynucleotide.

This beta-glucan restriction endonuclease PEGase-1 gene can also be the polynucleotide that comprise and the polynucleotide of the polypeptide be made up of the aminoacid sequence shown in the SEQ ID No.1 of encoding have at least 98% homology, the perhaps complementary strand of said polynucleotide; Be preferably and have at least 99% homology at least.Special, the polynucleotide that the polynucleotide that this beta-glucan restriction endonuclease PEGase-1 gene and coding are made up of the nucleotide sequence shown in the SEQ ID No.2 have at least 98% homology, the perhaps complementary strand of said polynucleotide; Be preferably and have at least 99% homology at least.

This beta-glucan restriction endonuclease PEGase-1 gene can also be the polynucleotide that comprise coded polypeptide or the complementary strand of said polynucleotide; Said polypeptide is made up of the aminoacid sequence shown in the SEQ ID No.1; Wherein one or more amino acid are lacked, are substituted or increased, and said polypeptide has the beta-glucan endonuclease activity.Special; This beta-glucan restriction endonuclease PEGase-1 gene comprises the polynucleotide of coded polypeptide or the complementary strand of said polynucleotide; Said polynucleotide sequence is made up of the nucleotide sequence shown in the SEQ ID No.2; Wherein one or more codons are lacked, are substituted or increased, and said polypeptide has the beta-glucan endonuclease activity.

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

This beta-glucan restriction endonuclease PEGase-1 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.

Because the polymorphum and the variation of protein coding gene, amino acid whose disappearance, insertion, replacement or other variation can appear in naturally occurring protein, lacked, substitute or increase thereby cause proteinic aminoacid sequence one or more amino acid to occur.In fact, exist the albumen that is equal to no variant protein matter on some physiology and the biological activity basically.These structures are different from corresponding proteins matter but do not have the polypeptide of tangible 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 through artificial means such as disappearance, insertion and sudden changes, thereby in a kind of proteinic aminoacid sequence, imports this type variation and the polypeptide processed.Even now can obtain more how multi-form varient, but the varient of gained is the activity that its physiologically active is equal to original no variant protein matter basically as the prerequisite of functional equivalent varient.

For example; The heterozyme H (A16-M) that 16 amino acid that soak numb genus bacillus beta-glucan restriction endonuclease N end with 16 amino acid replacements of bacillus amyloliquefaciens beta-glucan restriction endonuclease N end 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)-VISOSE is also soaked numb genus bacillus beta-glucan restriction endonuclease also high 40% than wild-type ^[15]

In addition; When producing protein with gene engineering method; Often be expressed as fused protein to desired protein, for example, be added to of the expression of the N-terminal of desired protein with the raising desired protein coming from other proteinic N-terminal peptide chains; Perhaps be added to a suitable peptide chain N or the C-terminal of desirable proteins, express this albumen and use a kind of to adding the carrier of peptide chain with avidity the purifying of desirable proteins is become be 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 takes place.The variation of gene possibly not influence 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 acids sequence,, still can produce the different genes of coding same acid sequence inevitably along with containing going down to posterity of this gene biological body.

Range gene with the artificial generation coding of range gene engineering same acid sequence is not difficult.For example; When the coding desired protein natural gene codon be used for genetic engineering technique produce proteinic host's utilizability lower, when the expressed proteins amount is not enough; Can artificial codon be transformed into 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 type are also included within the scope of the present invention the aminoacid sequence disclosed by the invention as long as it can be encoded out.

In addition; By the polypeptide of at least a change (as disappearance, insertion or the replacement of one or more amino-acid residues are arranged in the proteinic aminoacid sequence) gained or albumen, have and be equal to said activity of proteins on the function; Encode this type polypeptide or proteic gene is also included within the scope of the present invention, still is artificial production no matter it separates from natural origin.

General, the gene that encoding function is equal to varient is a homologous.Therefore, can have the active proteinic nucleic acid molecule of PEGase-1 with gene recombination of the present invention and coding is also included within the scope of the present invention.

The protein of one embodiment is provided, obtains by above-mentioned beta-glucan restriction endonuclease PEGase-1 genetic expression.

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

In more excellent embodiment, this protein comprises the polypeptide of being made up of the aminoacid sequence shown in the SEQ ID No.1.

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

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

The expression vector of one embodiment is provided, comprises above-mentioned beta-glucan restriction endonuclease PEGase-1 gene.

General, can select to adopt standard program to be sufficient to various conventional criteria carriers above-mentioned beta-glucan restriction endonuclease PEGase-1 gene, for example: pPET-32a (+), pPICZ α A, pPIC-GST etc. obtain the said gene expression vector.

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

Because bull suede bubble bacterium is an eukaryotic cell, host cell is generally selected eukaryotic cells, for example: yeast cell and Trichodermareesei cell.

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

The Trichodermareesei cell is as host cell, and the beta-glucan restriction endonuclease albuminoid of expression can pass through better chemical and modify, and has higher activity.

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

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

Step 2, utilize 40% and 70% saturated ammonium sulphate with said expression liquid fractionation precipitation, crude product.

Step 3, utilize HiTrap ^TMCapto ^TMQ ion exchange chromatography glue purification crude product obtains said protein.

Obtain beta-glucan restriction endonuclease albuminoid through method for preparing; Also need carry out zymologic property to the protein that obtains and measure, mainly comprise the genetic stability of the righttest 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 hydrolysis β-1,3 (4)-VISOSE, this kind of enzyme preparation comprises above-mentioned beta-glucan restriction endonuclease albuminoid.

In more excellent embodiment, zymin also comprises the metallic cation that is used to improve enzymic activity.

Can make generally under the existence of metallic cation that the activity of above-mentioned beta-glucan restriction endonuclease albuminoid is 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⁺

Be the specific embodiment part below.

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

Embodiment 1

The screening of PEGase-1 gene, amplification and structural analysis.

Get 100mg bull suede bubble bacterium (weight in wet base), extract total RNA, use GeneRacer with RNeasy Plant Mini Kit (QIAGEN, Germany) ^TMKit (Invitrogen, the U.S.) is prepared into cDNAs with the complete mRNAs of bull suede bubble bacterium; Through 5 '-RLM RACE (RNA ligase-mediated rapid amplification of5 '-cDNA ends) technology, from bull suede bubble bacterium global cDNA s 5 of amplification beta-glucan restriction endonuclease albuminoid '-the cDNA fragment; Separate the PCR product through 1% agarose gel electrophoresis, will be with the Solution I of DNA Ligation Kit (Takara, Dalian) through AxyPrep ^TMDNA Gel Extraction Kit (Axygen; The U.S.) the target dna fragment that reclaims is connected to pMD18T carrier (Takara; Dalian) on; Check order after will connecting product transformed into escherichia coli E.coli Top10, splicing 5 '-cDNA and 3 '-cDNA sequence, obtain the global cDNA sequence that bull suede steeps bacterium beta-glucan restriction endonuclease albuminoid.This patent is with this cDNA encoded protein called after PEGase-1 (Physarum Endo-β-1,3 (4)-glucanase 1).PEGase-1 has the aminoacid sequence shown in SEQ ID No.1, and the PEGase-1 gene has the nucleotide sequence shown in SEQ ID No.2.

According to ORF (the Open Reading frame) sequence of PEGase-1 global cDNA, design contain the primer 5 of the BamHI restriction enzyme site sequence of underscore (band) '-CGC GGATCCATGAACCCTAAAGTGTTCATTTTTG-3 ' with contain the primer 5 of Hind III restriction enzyme site '-GGG AAGCTTTTACTGTTGGTACACTTTGATGG-3 ' is a masterplate with bull suede bubble bacterium global cDNA, from bull suede bubble bacterium global cDNA s, amplifies the PEGase-1 gene through round pcr.

The aminoacid sequence of PEGase-1 genes encoding and the beta-glucan restriction endonuclease aminoacid sequence of thermophilic Paecilomyces varioti (Paecilomyces thermophila) are compared.

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

The tertiary structure of the beta-glucan restriction endonuclease of mimic PEGase-1 tertiary structure and thermophilic Paecilomyces varioti is compared.

As shown in Figure 2, the tertiary structure of the beta-glucan restriction endonuclease of mimic PEGase-1 tertiary structure and thermophilic Paecilomyces varioti is similar, and secondary structure is main with β-lamella, all belongs to heat-staple protein structure.

Explanation thus, the thermostability of PEGase-1 is similar with the thermostability of thermophilic Paecilomyces varioti EGase.

Embodiment 2

Expression and the evaluation of PEGase-1 gene in Bacillus coli cells.

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

The label protein (Thioredoxin, Trx, Trx) that extracts expression product Trx-PEGase-1 and this fusion rotein carries out SDS-PAGE simultaneously, and is as shown in Figure 3 behind the coomassie brilliant blue staining.

As shown in Figure 3; The relative molecular mass of Trx-PEGase-1 (51kD) is than the label protein (Thioredoxin of this fusion rotein; Trx; Trx) the many 34kD of relative molecular mass (17kD), approaching with the proteic theoretical molecular mass of PEGase-1, explain that PEGase-1 does not receive tangible chemically modified in Bacillus coli cells.Trx-PEGase-1 does not have hydrolysis barley β-1,3 (4)-VISOSE (β-glucan), laminarin (laminarin) and Walocel MT 20.000PV (carboxymethyl cellulose, enzyme activity CMC) yet.

Explanation thus, prokaryotic cell prokaryocyte does not have the beta-glucan endonuclease activity as the PEGase-1 that host cell expression obtains.

Embodiment 3

Expression and the evaluation of PEGase-1 gene in yeast cell.

With plasmid pET32 (a)-pegasel is template, with contain the primer 5 in EcoR I site '-CCGGAATTCAA CCCTAAAGTGTTCATTTTTG-3 ' and the primer 5 that contains Xba I site '-the encoding sox fragment of GCTCTAGAAACTGT TGGTACACTTTGATGGA-3 ' amplification PEGase-1; The PCR fragment of EcoR I and Xba I enzyme being cut with the Solution I among the DNA Ligation Kit is connected on the expression plasmid of yeast pPICZ α A (available from Invitrogen, the U.S.) that same enzyme cuts, and is transformed into clone in the E.coli Top10; Extract recombinant plasmid pPICZ alpha A-pegasel and carry out PCR, double digestion and order-checking evaluation; With restriction enzyme Sac I with its linearizing after, electricity is transformed in competence P.pastoris GS115 (Invitrogen, the U.S.) cell, screening positive clone bacterium colony on the YPDS culture plate that contains 50 μ g/ml Zeocin.Picking mono-clonal bacterium colony, in the liquid YPDS substratum that contains 50 μ g/ml Zeocin, cultivate (30 ℃, 250rpm) spend the night, use CTAB (Hexadecyl trimethyl ammonium bromide) method then ^[23]Extract reorganization bacterium genomic dna, detect through PCR and be binned in the PEGase-1 gene on the yeast genes group.With the BMGY nutrient solution negative control yeast and the recombination yeast that contains the PEGase-1 gene are cultured to OD ₆₀₀It is 2～6 (30 ℃; 250rpm), thalline is transferred in the BMMY inducing culture liquid [every separated 24hr replenishes 0.5% (v/v) methyl alcohol once] induced PEGase-1 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 expression of recombinant yeast excretory PEGase-1 through SDS-PAGE, obtain Fig. 4, detect the enzyme activity of the PEGase-1 hydrolysis barley β-glucan in the nutrient solution.

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

Can find out that by Fig. 4 yeast cell to express obtains the PEGase-1 albumen that relative molecular mass is 45kD, many 11kD than the proteic relative molecular mass of the PEGase-1 of escherichia coli expression (34kD).Explain that yeast expression excretory PEGase-1 albumen is chemically modified protein.This albumen has hydrolysis barley β-1,3 (4)-VISOSE (vigor of β-glucan), but do not have the enzyme activity of hydrolysis laminarin and Walocel MT 20.000PV.

Explanation thus, the beta-glucan endonuclease activity that the PEGase-1 that yeast cell obtains as host cell expression has.

Embodiment 4

The PEGase-1 gene is in intracellular expression of Trichodermareesei and evaluation.

With plasmid pET32 (a)-pegasel is template, with primer 5 '-CGCAGCTAGTGTGCCTCTAGAGGAGCGGATGAACCCTAAAGTGTTCATTT-3 ' and 5 '-TTACTGTTGGTACACTTTGATGGAGTTGACC-3 ' amplification PEGase-1 encoding sox fragment; With the Solution I among the DNA Ligation Kit this PCR fragment is connected on the plasmid pPIC-PST that the Eam1105I enzyme cuts; To connect product Transformed E .coli Top10 clone recombinant plasmid pPIC-PST-pegasel then, and extract plasmid pPIC-PST-pegasel and carry out PCR and order-checking evaluation.Be template with plasmid pPIC-PST-pegasel more afterwards, with primer 5 '-GAT AGATCTAAAAAAATATGAGCGCAGGGACAAGCA-3 ' and 5 '-AGTGTCGACTGGTACTG GGATACACGAAGAGCG-3 ', amplification PEGase-1 expression cassette P _Gpd-pegasel-T _BhlMethod with reference to foundation such as Penttil and Liu Gang ^[26,27], get 10 μ g expression cassette P _Gpd-pegasel-T _CbhlWith 10 μ g plasmid PAN7-1 (TV 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 behind 48 ℃ of heat shock 2min, the PEG4000 that adds 50 μ l 60% (contains 50mmol/L CaCl ₂The damping fluid of 10mmol/L TrisCl, pH 7.5) mixing, add the PEG4000 mixing of 2ml 60% behind the 20min (room temperature) again; Add 8ml STC mixing behind the 5min; 8, collect protoplastis behind the centrifugal 15min of 000rpm, mix with the resuspended protoplastis of 1ml STC solution and with 10ml protoplast regeneration nutrient solution (the Mandels basic culture solution that contains the 1.2mol/L sorbyl alcohol); Cultivate 24h down in 28 ℃; Centrifugal 20min collects thalline, with the resuspended thalline of 1ml STC solution and be coated on 5 PDA culture plates that contain 100 μ g/ml HYGs, and 28 ℃ of cultivations 2 days down; Take mycelium inoculation on the culture plate to new PDA solid culture plate, 28 ℃ are continued down to cultivate 7 days.Collect the mycelium on the culture plate with sterilized water; Ratio in 1～5% be inoculated in 20ml contain 100 μ g/ml HYGs the Mandels nutrient solution (be seeded in simultaneously on the PDA culture plate, treat that it grows spore after, collect spore and be used for protecting kind); Cultivated 2 days down for 28 ℃; Transfer in 1～5% ratio again and produce enzyme nutrient solution cultivation 5 days (28 ℃ 250rpm), are collected supernatant in 25ml Mandels; Detect expression of recombinant yeast excretory PEGase-1 through SDS-PAGE, and measure the enzyme activity of the PEGase-1 hydrolysis barley β-glucan in the nutrient solution.

Figure 15 is the technological synoptic diagram of PEGase-1 gene in Trichodermareesei QM9414 recombinant conversion; Recombinant plasmid pPIC-pegasel is by the signal peptide gene of the cellobiohydrolase II of the glyceraldehyde 3-phosphate dehydrogenase promotor of Puc ori, Zeocin, PEM7, PTEF1 and the AOX1TT member and the Trichodermareesei of pPICZ α A carrier, Trichodermareesei and the cellobiohydrolase I terminator of Trichodermareesei.Wherein, the PEGase-1 gene is inserted between the signal peptide gene and cellobiohydrolase I terminator of cellobiohydrolase II.

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

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

Can find out that by Fig. 5 the relative molecular mass of trichoderma reesei expression excretory PEGase-1 is 66kD, many 32kD than the PEGase-1 relative molecular mass of escherichia coli expression; Many 21kD than yeast expression excretory PEGase-1 relative molecular mass, explain that trichoderma reesei expression excretory PEGase-1 is also higher than the chemically modified degree of yeast expression excretory PEGase-1.Trichoderma reesei expression excretory PEGase-1 has the vigor of hydrolysis barley β-1,3 (4)-VISOSE.

Embodiment 5

The genetic stability experiment of the Trichodermareesei that contains the PEGase-1 encoding sox that obtains among the embodiment 4.

The T.reesei QM9414 reorganization bacterium inoculation of the expression PEGase-1 that obtains among the embodiment 4 is not contained on the antibiotic PDA culture plate, inoculate behind the spore that grows on the new PDA culture plate, so repeatedly, go down to posterity 7 times.

The 2nd generation and the 8th generation thalline carry genome with the CTAB method, pcr amplification PEGase-1 gene fragment identifies that the result is as shown in Figure 6.

With the 2nd generation and the 8th generation thalline receive liquid nutrient medium, the centrifuging and taking supernatant is surveyed enzyme activity by ordinary method, the result is as shown in Figure 7.

Can find out that by Fig. 6 going down to posterity still can amplify the PEGase-1 gene after 7 times from the genomic dna of reorganization bacterium.Can find out that by Fig. 7 the PEGase-1 that goes down to posterity after 7 times expresses the vigor that bacterium still has hydrolysis barley β-1,3 (4)-VISOSE.

Explanation thus, it is stable in heredity that PEGase-1 expresses bacterium, and is keeping the function of expression, secretion PEGase-1.

Embodiment 6

Roughing out and the purifying of trichoderma reesei expression excretory PEGase-1 among the embodiment 4.

The roughing out of PEGase-1.

The T.reesei QM that expresses PEGase-1 9414 reorganization bacterium are inoculated in the Mandels basic culture solution that contains 100 μ g/ml HYGs cultivate (28 ℃, 250rpm) connect bacterium in 1～5% ratio after 2 days and produce the enzyme substratum in Mandels, cultivated again 4～5 days under 28 ℃; Glucose to nutrient solution exhausts, centrifugal under 4 ℃ afterwards (10,000rpm) 20min; Collect fermented liquid supernatant and add ammonium sulfate to 40% saturation ratio; Centrifugal collection supernatant adds ammonium sulfate to 70% saturation ratio, centrifugal collecting precipitation behind the 2hr; With 0.05mol/LHACNaAC damping fluid (pH5.5) dissolution precipitation albumen and dialyse 3 times; With 3, the filter membrane in 000MW aperture (Millipore, the U.S.) becomes the 4ml crude enzyme liquid with the dialyzate ultrafiltration and concentration.With BCA (BicinchoninincAcid) method ^[24]Measure the protein content (test kit is produced by Beijing CWBIO company) of liquid concentrator, and measure thick enzymic hydrolysis barley β-glucan vigor.

The purifying of PEGase-1.

With the good HiTrap of HACNaAC damping fluid (pH4.5) the balance prepackage of 0.05mol/L ^TMCapto ^TMQ ion exchange column (GE Healthcare; The U.S.), get the 1ml crude enzyme liquid, with 0.05mol/L HACNaAC damping fluid (pH4.5) wash-out foreign protein with 0.45 μ m membrane filtration and upper prop; With containing 0～1mol/L NaCl damping fluid (0.05mol/L HACNaAC; PH4.5) gradient elution albumen passes through the protein content that the BCA method is measured each elution peak afterwards, and the specific activity of enzyme of hydrolysis barley β-glucan.

With the supernatant that experimental group and negative control group among the embodiment 4 are collected, the albumen that obtains behind crude enzyme liquid that roughing out obtains in present embodiment and the purifying carries out SDS-PAGE, and the result is as shown in Figure 8.Wherein, swimming lane M is a standard molecular weight albumen, the supernatant that swimming lane 1 is collected for negative control group among the embodiment 4, and the supernatant that swimming lane 2 is collected for experimental group among the embodiment 4, swimming lane 3 is HiTrap ^TMCapto ^TMQ IX glue-line is analysed the PEGase-1 that obtains behind separation, the purifying, and swimming lane 4 is the crude enzyme liquid that 40%～70% sulphur ammonium fractionation precipitation 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

Can draw sulphur ammonium fractionation precipitation and HiTrap through 40%～70% by Fig. 8 and last table ^TMCapto ^TMQ IX glue-line is analysed the PEGase-1 of separation, purifying trichoderma reesei expression, can reclaim 62.0% and 48.0% PEGase-1, and the ratio vigor of hydrolysis barley β-1,3 (4)-VISOSE can improve 19.05 times and 27.125 times respectively.

The concrete prescription and the preparation method of part substratum, nutrient solution and the protoplastiss of using among the embodiment 1～6 etc. are following.

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, boils the 30min after-filtration, 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.

T, the preparation of reesei QM9414 protoplastis: the Trichodermareesei spore inoculating in the PDA culture plate, was cultivated 7 days for 28 ℃.Prepare spore suspension with sterilized water, get 0.8～1.0 * 10 ⁸Individual spore inoculating is cultivated 10～12hr down for 28 ℃ in the Mandels basic medium of 40ml; Treat that spore germination rate reaches at 99% o'clock, get the centrifugal collection thalline of 25ml bacterium liquid, use 1mol/L MgSO ₄Washing mycelia 2 times, centrifugal collection thalline (is dissolved in 1mol/L MgSO with the lywallzyme liquid of 10ml10mg/mL ₄In) resuspended mycelia; Behind 28 ℃ of following enzymolysis fungal cell wall 2～2.5hr, add the STC liquid of 20ml, centrifugal collection protoplastis; Behind twice of STC solution washing protoplastis; With 1ml STC liquid suspension protoplastis, calculate the concentration of protoplastis with blood counting chamber, with STC liquid with protoplastis concentration dilution to 10 ³Individual/ml, the protoplastis of getting after 100 μ l dilute is coated 3 repetitions of (2% agar powder, Mandels basic medium) last calculating protoplast regeneration rates on the regenerating and culturing plate.The average regeneration rate of protoplastis can be used for gene transformation greater than 10% protoplastis.

Embodiment 7

The detection of the PEGase-1 enzyme activity of the purifying that embodiment 6 obtains.

1, the making of glucose typical curve.

With reference to the method that Miller sets up, get 80 μ l concentration and be 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 after 5 days and is used) mixing, behind the boiling water bath 10min; Detect the absorbance (A of each reaction solution under 540nm with spectramax 190 (Molecular Devices, the U.S.) ELIASA ₅₄₀), be X-coordinate with the glucose concn, with A ₅₄₀Be ordinate zou, draw glucose concn and A ₅₄₀Relation curve, obtain reflection glucose concn and A ₅₄₀The regression equation of relation is in order to calculate the PEGase-1 hydrolysis barley reducing sugar amount that β-glucan produces.

2, the detection of PEGase-1 enzyme activity.

1. enzyme reaction: get 20 μ l enzyme liquid and 40 μ l enzyme reaction buffer solution (0.05mol/L HACNaAC, pH5.5) mixing is behind 55 ℃ of water-bath 5min; Substrate solution 20 μ l and the mixings that add 55 ℃ of water-bath 5min add 240 μ l 3,5-dinitrosalicylic acid (3 behind the reaction 10min again; 5-dinitro-salicyli acid, DNS) liquid, boiling water bath 10min termination reaction; Cooling back water is settled to 2ml, and on spectrophotometer, measures A ₅₄₀Value.

2. blank: get 20 μ l enzyme liquid and 40 μ l enzyme reaction buffer solution mixings; The substrate that adds 55 ℃ of water-bath 5min of 20 μ l behind 55 ℃ of water-bath 5min adds the DNS liquid of 240 μ l, boiling water bath 10min termination reaction afterwards immediately; Cooling back water is settled to 2ml, and on spectrophotometer, measures A 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, calculates the concentration of reduced sugar that PEGase-1 hydrolysis barley β-glucan generates, and presses 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-1 definition.

55 ℃ with the pH5.5 condition under, the required PEGase-1 enzyme amount of reducing sugar (with glucose meter) that PM hydrolysis barley β-glucan generates 1 μ mol is 1 unit of activity, representes with U.The enzyme activity of unit fermented liquid and unit zymoprotein preparation is represented with U/ml and U/mg.

5, the preparation of substrate solution.

1. 1% barley β-glucan solution: take by weighing 0.1g barley β-glucan (Megazyme; Ireland), it is wetting to add the 1ml absolute ethyl alcohol, adds 8ml enzyme reaction buffer solution (HACNaAC of 0.05mol/L) then; In boiling water bath, dissolve; After the 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 by weighing 0.1g palmate sea-tangle laminarin (Sigma; The U.S.), it is wetting to add the 1ml absolute ethyl alcohol, adds 8ml enzyme reaction buffer solution (HACNaAC of 0.05mol/L) then; In boiling water bath, dissolve; After the cooling room temperature, be settled to 10ml, be mixed with the substrate solution that concentration is 10mg/ml.

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

Through aforesaid method, (result is as shown in table 2 for carboxymethyl cellulose, the CMC) reaction that is hydrolyzed with barley β-glucan, laminarin (laminarin) and Walocel MT 20.000PV respectively for the PEGase-1 of purifying.

Table 2

Can find out by table 2; The ratio vigor of PEGase-1 hydrolysis barley β-glucan, laminarin and the CMC of purifying is respectively 2.335,0.441 and 0.234U/mg; The specific substrate that the PEGase-1 of trichoderma reesei expression is described is barley β-1; 3 (4)-VISOSEs, but laminarin is also had certain hydrolysis vigor.

Select barley β-glucan as substrate, the vigor of test PEGase-1 hydrolysis barley β-glucan under different test conditions, detected result such as Fig. 9～shown in Figure 14.

Can find out the K of PEGase-1 hydrolysis barley β-glucan by Fig. 9 _mValue is 0.882mg/ml, V _MaxValue is 0.691mg/min.

Can find out that by Figure 10 the optimum pH of PEGase-1 hydrolysis barley β-glucan is 5.5, and PEGase-1 is lower than under 5.5 environment at pH more stable than being higher than at pH under 7.0 environment.

Can find out that by Figure 11 the optimum temperuture of PEGase-1 hydrolysis barley β-glucan is 55 ℃, and PEGase-1 is more stable than being higher than in 55 ℃ the environment in being lower than 55 ℃ environment.

Figure 12 is PEGase-1 hydrolysis barley β-time dependent synoptic diagram of glucan vigor under differing temps, by finding out that PEGase-1 stability is best in the time of 40 ℃ among the figure.

Figure 13 deposits 24h posthydrolysis barley β-glucan vigor synoptic diagram for PEGase-1 under different pH damping fluids, by finding out that pH is at 5.5 o'clock among the figure, the PEGase-1 vigor keeps better.

Figure 14 be PEGase-1 in the presence of different concns, different sorts positively charged ion, the variation synoptic diagram of the vigor of hydrolysis barley β-glucan.

Can find out that by Figure 14 metals ion is to PEGase-1 hydrolysis barley β-the glucan vigor is influential, the Cu of 1mmol/L ²⁺, Ni ²⁺, Zn ²⁺, Fe ²⁺, Ba ²⁺, Mg ²⁺And Ca ²⁺Can improve 49.1%, 43.3%, 29.6%, 27.7%, 26.8%, 23.8% and 19.0% PEGase-1 hydrolysis vigor, the Mn of 1mmol/L ²⁺And Co ²⁺Can suppress 64.6% and 74.5% PEGase-1 hydrolysis vigor, the Ni of 5mmol/L ²⁺And Ca ²⁺Can improve 20.9% and 12.9% PEGase-1 hydrolysis vigor, but the Zn of 5mmol/L ²⁺, Ba ²⁺And Mg ²⁺Little to PEGase-1 hydrolysis effect of vigor, the Cu of 5mmol/L ²⁺, Fe ²⁺, Mn ²⁺And Co ²⁺Suppressed 58.4%, 42.6%, 73.4% and 75.4% PEGase-1 hydrolysis vigor on the contrary.

Explain that thus above-mentioned metallic cation is relevant with ionic concn to the influence of PEGase-1 hydrolysis vigor, the Cu of suitable concentration ²⁺, Ni ²⁺, Zn ²⁺, Fe ²⁺, Ba ²⁺, Mg ²⁺And Ca ²⁺Can improve the hydrolysis vigor of PEGase-1.

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 claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, 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 accompanying claims.

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SEQUENCE?LISTING

< 110>Shenzhen University

< 120 >-endoglucanase PEGase-1 gene and expression product and application

<160>2

<170>PatentIn?version?3.3

<210>1

<211>309

<212>PRT

< 213>the beta-glucan restriction endonuclease PEGase-1 of bull suede bubble bacterium

<400>1

Met?Asn?Pro?Lys?Val?Phe?Ile?Phe?Val?Leu?Leu?Gly?Ala?Ser?Leu?Cys

1 5 10 15

Ser?Ala?Gly?Tyr?Asn?Leu?Lys?Asp?Ser?Trp?Ser?Gly?Gln?Gln?Ile?Phe

20 25 30

Asp?Gln?Phe?Asn?Phe?Trp?Ser?Gly?Pro?Asp?Pro?Thr?His?Gly?Tyr?Val

35 40 45

Tyr?Tyr?Ala?Ser?Lys?Ser?Glu?Ser?Gln?Gln?Trp?Gly?Tyr?Thr?Ser?Val

50 55 60

Gln?Asn?Gly?Val?Ala?Tyr?Ile?Arg?Ser?Asp?Ser?Thr?Ser?Val?Ser?Ser

65 70 75 80

Gly?Ala?Gly?Arg?Gly?Ser?Val?Arg?Leu?Thr?Ser?Gln?Lys?Thr?Tyr?Thr

85 90 95

His?Gly?Leu?Phe?Ile?Phe?Asp?Val?Ala?His?Met?Pro?Trp?Gly?Gln?Gly

100 105 110

Thr?Trp?Pro?Ala?Ile?Trp?Thr?Thr?Arg?Gly?Asp?Gly?Trp?Pro?Ala?Gly

115 120 125

Gly?Glu?Ile?Asp?Ile?Ile?Glu?Gly?Val?Asn?Ser?Asn?Arg?Asn?Asn?Gln

130 135 140

Met?Thr?Leu?His?Thr?Ser?Pro?Gly?Cys?Tyr?Val?Pro?Thr?Asn?Lys?Asp

145 150 155 160

Ala?Glu?Ser?Gly?Asn?Pro?Gly?Ser?Gly?Asp?Cys?Gly?Ala?Asn?Gly?Gly

165 170 175

Ser?Ala?Gly?Cys?Gly?Ile?Asn?Asp?Pro?Asp?Ser?Trp?Ser?Tyr?Gly?Pro

180 185 190

Asp?Phe?Asn?Asn?Asn?Gly?Gly?Gly?Thr?Trp?Ala?Met?Gln?Trp?Glu?Ser

195 200 205

Ser?Gly?Ile?Tyr?Ile?Trp?Leu?Trp?Ala?Lys?Gly?Tyr?Val?Pro?Gln?Asp

210 215 220

Val?Leu?Asn?Gly?Gln?Pro?Asn?Pro?Ala?Gly?Trp?Gly?Ile?Pro?Arg?Gly

225 230 235 240

Arg?Ser?Thr?Phe?Asn?Gln?Gly?Cys?Val?Asp?Ser?Gln?Tyr?Phe?Tyr?Asn

245 250 255

His?Gly?Ile?Ile?Ile?Asp?Asn?Thr?Phe?Cys?Gly?Asp?Trp?Ala?Gly?Asn

260 265 270

Val?Tyr?Pro?Gly?Gly?Gln?Asp?Ala?Cys?Lys?Ser?Phe?Val?Gln?Asn?Lys

275 280 285

Thr?Thr?Pro?Ser?Ala?Phe?Thr?Glu?Ala?Tyr?Trp?Ala?Val?Asn?Ser?Ile

290 295 300

Lys?Val?Tyr?Gln?Gln

305

<210>2

<211>930

<212>DNA

< 213>the beta-glucan restriction endonuclease PEGase-1 gene of bull suede bubble bacterium

<400>2

atgaacccta?aagtgttcat?ttttgtgctc?ttgggtgcct?ccttgtgctc?tgcagggtac 60

aacctgaagg?atagctggtc?aggacagcaa?atctttgacc?agttcaactt?ctggagtggc 120

cctgacccta?ctcacggata?cgtgtactat?gcttccaagt?ccgagagcca?acaatgggga 180

tacacctctg?tccaaaatgg?agttgcttac?atccgatcag?attctacttc?tgtctcttct 240

ggtgctggaa?gaggatcagt?taggttgacc?agccaaaaga?cctacaccca?tggtctgttc 300

atctttgatg?ttgcccatat?gccctggggt?cagggaactt?ggccagccat?ctggaccact 360

cgcggcgacg?gatggcccgc?gggaggagaa?atcgatatca?ttgagggagt?aaactcgaac 420

agaaacaacc?aaatgaccct?gcatacctct?cccgggtgct?acgtccccac?caacaaggat 480

gccgaatccg?gaaaccccgg?atctggcgat?tgcggtgcga?acggaggaag?tgctggatgt 540

ggaatcaatg?accctgactc?ctggtcatac?ggcccggact?tcaacaacaa?cggaggtggc 600

acatgggcca?tgcaatggga?gtccagtggc?atttacatct?ggctctgggc?taagggatac 660

gtcccccagg?acgtcttgaa?cggccaaccc?aaccccgccg?gatggggcat?tccccgcgga 720

aggtccacct?tcaaccaagg?ctgcgttgac?tcccaatact?tctacaacca?cggaatcatc 780

atcgacaaca?ccttctgcgg?tgactgggcc?ggaaacgtct?accctggtgg?ccaagatgct 840

tgcaagagct?tcgtccaaaa?caaaacaacc?cctagcgctt?tcactgaggc?gtactgggcg 900

gtcaactcca?tcaaagtgta?ccaacagtaa 930

Claims (10)

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

(a), the polynucleotide of the polypeptide formed by the aminoacid sequence shown in the SEQ ID No.1 of coding, the perhaps complementary strand of said polynucleotide;

(b) and the polynucleotide of the polynucleotide of the polypeptide formed by the aminoacid sequence shown in the SEQ ID No.1 of coding with at least 98% homology, the perhaps complementary strand of said polynucleotide;

(c), the complementary strand of the polynucleotide of coded polypeptide or said polynucleotide; Said polypeptide is made up of the aminoacid sequence shown in the SEQ IDNo.1; Wherein one or more amino acid are lacked, are substituted or increased, and said polypeptide has the beta-glucan endonuclease activity; Or

(d), the polynucleotide of coded polypeptide or the complementary strand of said polynucleotide, said polypeptide has at least 98% homology with the polypeptide of being made up of the aminoacid sequence shown in the SEQ IDNo.1.

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

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

(b) and the polynucleotide of the polynucleotide formed by the nucleotide sequence shown in the SEQ ID No.2 of coding with at least 98% homology, the perhaps complementary strand of said polynucleotide; Or

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

3. beta-glucan restriction endonuclease PEGase-1 gene as claimed in claim 2 is characterized in that, said beta-glucan restriction endonuclease PEGase-1 gene comprises the polynucleotide of being made up of the nucleotide sequence shown in the SEQ ID No.2.

4. an expression vector is characterized in that, comprises beta-glucan restriction endonuclease PEGase-1 gene according to claim 1 or claim 2.

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

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

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

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

Step 2, utilize 40% and 70% saturated ammonium sulphate with said expression liquid fractionation precipitation, crude product;

Step 3, utilize HiTrap ^TMCapto ^TMQ ion exchange chromatography glue purification crude product obtains said protein.

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

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

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

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

9. a zymin is characterized in that, comprises protein as claimed in claim 8; Said zymin is used for hydrolysis β-1,3 (4)-VISOSE.

10. zymin as claimed in claim 9 is characterized in that said zymin also comprises the metallic cation that is used to improve enzymic activity.

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