CN101775385B - Heat-resisting beta-1, 3-1, 4-dextranase and encoding gene thereof - Google Patents

Abstract

The invention discloses a beta-1, 3-1, 4-dextranase and an encoding gene thereof. The beta-1, 3-1, 4-dextranase provided by the invention is protein in 1 or 2 or 3 as follows: 1. the protein which is formed from an amino acid sequence shown in 19th-314th bit of a sequence 2 in a sequence table; 2. the protein which is formed from the amino acid sequence shown in the sequence 2 in the sequence table; and 3. the protein which is formed from an amino acid residue sequence of the sequence 2 in the sequence table through the substitution and/or deletion and/or addition of one or more amino acid residues, has the activity of the beta-1, 3-1, 4-dextranase and is derived from 1. A recombined pichia formed by leading the encoding gene of the protein into pichia also belongs to the protecting range of the invention. Experiments prove that the highest enzyme activity of a recombined pichia fermented liquid supernatant can be 55,300U/mL, and crude protein is 9.1g/L. The optimum reaction temperature of the beta-1, 3-1, 4-dextranase of the invention is 70 DEG C, and the optimum pH value is 7.0. The beta-1, 3-1, 4-dextranase has great generalization and application potential in production.

Description

A kind of heat-resisting β-1,3-1,4-dextranase and encoding gene thereof

Technical field

The present invention relates to β-1,3-1,4-dextranase and encoding gene thereof and efficiently express.

Background technology

Dextran be by glucose by the base polymer that the different sugar glycosidic bond couples together, be the abundantest glycan of occurring in nature content.β-1,3-1, the 4-D-dextran is a nature synthetic saccharan, with the structural non-starch polysaccharide of solubility in the equal platymiscium cell walls such as araboxylan, piperylene, Mierocrystalline cellulose, pectin.Most of cereal all contains β-1,3-1, and the 4-D-dextran, wherein the content in the barley is up to 5-8%, mainly is present in the barley laticiferous cell wall.β-1,3-1, the 4-D-dextran is to pass through β-1 by glucose with beta comfiguration, 3-and β-1, the linear chain structure that 4-mixing key forms approximately contains 70% β-1,4-key and 30% β-1, the 3-key, their molecular weight and the different to some extent differences of configuration because of factors such as barley variety, physical environment, growth phases.

Specificity according to the hydrolysis beta-glucan can mainly be divided into specific β-1 with dextranase, 3-1,4-D-dextranase or lichenstarch lytic enzyme (EC 3.2.1.73), inscribe β-1,4-D-dextranase (EC 3.2.1.4), β-1,3 (4)-dextranases (EC 3.2.1.6) and β-1,3-D-dextranase four classes such as (EC 3.2.1.39).So far, the microorganism β-1 of report, 3-1,4-dextranase belong to glycoside hydrolase 16 families of Production by Bacteria mostly, comprise bacterium, fungi and rumen microorganism.Existing multiple genus bacillus β-1,3-1, the 4-glucanase gene is cloned and is expressed, as bacillus B.amyloliquefaciens, B.circulans (Kim JY et al.Biotechnol Lett, 2003,25:1445-1449), B.licheniformis EGW039 (Teng et al.Applied Microbiology andBiotechnology, 2006,72 (4): 705-712); Class bacillus Paenibacillus polymyxa (Gosalbes MJ et al.J Bacteriol, 1991,173 (23): 7705-7710).These dextranases all have similar Nucleotide and aminoacid sequence, and a conservative aminoacid sequence EIDIEF is all arranged, and the wherein hydrolysis of two L-glutamic acid participation enzymes (Hahn M et al.J Biol Chem, 1995,270:3081-3088).Also the clone obtains β-1 from the bacterium of some non-bacilluss, 3-1, the 4-glucanase gene, as streptococcus bovis Streptococcus bovis (Ekinci MS et al.Appl Environ Microbiol, 1997,63:3752-3756), produce the thread bacillus Fibrobacter of succsinic acid succinogenes (Chen JL et al.J BiolChem, 2001,276:17895-17901).In recent years, the outer β-1 of some report research fungi born of the same parents is arranged also, 3-1, the purifying of 4-dextranase and character, as Orpinomyces sp.PC-2, Cochliobolus carbonum (

Et al.Appl Environ Microbiol, 1998,64 (2): 385-391), Talaromyces emersonii (Murray PG et al.Enzyme and Microbial Technology, 2001,29 (1) 90-98) and Rhizopus microspores var.microsporus (Celestino et al.BMC biochemistry, 2006,7:23).But have only several fungi β-1,3-1,4-glucanase gene clone and the report of expressing, these fungies comprise Orpinomyces sp.PC-2 (Chen H et al.Journal of Bacteriology, 1997,179:6028-6034), Aspergillus japonicas (Grishutin SG et al.Carbohydr Res, 2006,341:218-229) with Bispora sp.MEY-1 (Luo et al.J Agric Food Chem, 2009,57 (12): 5535-5341).

In recent years, some patents have been introduced the β-1 of different sources, 3-1, and the 4-glucanase gene obtains expressing at different carriers and host, and as intestinal bacteria, pichia spp etc., but expression level is low.The β-1 of series bacillus Paenibacillus spF-40,3-1,4-dextranase in pichia spp, express (patent: 200610112092.8), the thick enzyme of the fermented liquid 4553.7U/mL that lives.The β-1 of starch liquefacation bacillus B.amyloliquefaciens, 3-1, the 4-dextranase expression in escherichia coli (patent: 200910031553.2), the total enzyme of the LB substratum 322.0U/mL that lives, the extracellular enzyme 127.5U/mL that lives.From the patent analyses of domestic and international announcement, about fungi β-1,3-1, the patent of 4-glucanase gene is less, and is also bigger in the dextranase of different expression systems difference alive.About the β-1 of thermophilic fungus, 3-1, the report of 4-glucanase gene, clonal expression and patent.Therefore clone and expression have height ratio work, Heat stability is good and the strong thermophilic fungus β-1 of substrate specificity, 3-1, and the 4-dextranase has important effect for industries such as feed, food.

Summary of the invention

The object of the present invention is to provide a kind of β-1,3-1, the 4-dextranase derives from thermophilic Paecilomyces varioti (Paecilomyces thermophila).

β provided by the invention-1,3-1, the 4-dextranase is following 1) or 2) or 3) protein:

1) protein of forming by the aminoacid sequence shown in the 19-314 position of sequence in the sequence table 2;

2) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

3) with the amino acid residue sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have β-1,3-1, the 4-dextranase activity by 1) deutero-protein.

Wherein, the sequence 2 in the sequence table is made up of 314 amino acid, and molecular weight is about 34.54 * 10 ³KDa.

In order to make 1) or 2) in albumen be convenient to purifying, can be in 1 by sequence table) or 2) proteinic N-terminal or C-terminal connect and go up label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag?II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

Above-mentioned 3) but in the albumen synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 3) the proteic encoding gene in can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

Above-mentioned proteic encoding gene also belongs within protection scope of the present invention.

Above-mentioned proteic encoding gene is following 1)-4) in arbitrary described gene:

1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 55-945 position;

2) its encoding sequence is a sequence 1 in the sequence table;

3) under stringent condition with 1) or 2) gene recombination and the gene of encoding said proteins;

4) with 1) or 2) gene have the homology more than 90% and the gene of encoding said proteins.

Above-mentioned stringent condition can be that (or 0.1 * SSC), the solution of 0.1%SDS is hybridized under 65 ℃ and washed film with 0.1 * SSPE in DNA or RNA hybrid experiment.

Sequence 1 has 945 bases in the sequence table, the albumen shown in the sequence 2 in the codified sequence table.The signal peptide of the 1-18 position shown in the sequence 2 in 5 ' end 1-54 position code sequence tabulation of sequence 1 in the sequence table wherein; The protein that aminoacid sequence shown in the 19-314 position of sequence 2 in the dna fragmentation codified sequence table shown in 5 ' the end 55-945 position of sequence 1 is formed.

Amplification said gene total length or its arbitrary segmental primer are to also belonging to protection scope of the present invention.

The recombinant vectors that contains the above gene also belongs within protection scope of the present invention.

Above-mentioned recombinant vectors can be that above-mentioned gene is inserted the recombinant expression vector that the multiple clone site of pPIC9K obtains.Particularly will, above-mentioned gene is to insert between the SnaBI of pPIC9K and the AvrII restriction enzyme site, makes nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it.

The expression cassette, transgenic cell line or the reorganization bacterium that contain said gene also belong within protection scope of the present invention.

Above-mentioned reorganization bacterium specifically is that above-mentioned gene is imported in the pichia spp (Pichiapastoris) by described recombinant expression vector, the transgenosis reorganization bacterium that obtains.

Above-mentioned pichia spp is GS115.

Another object of the present invention is to provide above-mentioned proteic preparation method.

Method provided by the invention is described reorganization bacterium to be fermented obtain, and described fermentation condition is to get the GS115 bacterial strain that contains recombinant plasmid, is inoculated in the 200mL BMGY substratum, and 30 ℃, the 250rpm shaken overnight is cultured to OD ₆₀₀10.0 about, be inoculated in the fermentation minimum medium then in the 5L fermentation cylinder for fermentation.Cultivate and the methanol induction three phases through basis cultivation, glycerine batch feeding, make recombinant beta-1,3-1, the 4-dextranase is efficiently expressed.The whole fermentation process temperature is controlled at 30 ℃, and by adjusting rotating speed, air flow and feed rate control dissolved oxygen＞20%, the three phases pH of fermentation is controlled at 4.0,5.0 and 6.0 respectively.

Experimental results show that: but method efficient production recombinant beta-1 provided by the invention, 3-1, the 4-dextranase, the work of recombinant yeast pichia pastoris fermented liquid supernatant enzyme is up to 55,300U/mL, crude protein 9.1g/L.β of the present invention-1,3-1,4-dextranase optimal reactive temperature is 70 ℃, optimum pH is 7.0.β of the present invention-1,3-1, the 4-dextranase has expression level height and heat-resistant quality, can be widely used in feed, food and brewing industry etc., has the very big potentiality of applying aborning.

Description of drawings

Fig. 1 is heat-resisting β-1 of the present invention, 3-1,4-glucanase gene conserved regions pcr amplification product agarose gel electrophoresis figure (row M:marker, row 1:PCR amplified production).

Fig. 2 is heat-resisting β-1 of the present invention, 3-1,4-glucanase gene coding region pcr amplification product agarose gel electrophoresis figure (row M:marker, row 1:PCR amplified production).

Fig. 3 is the present invention heat-resisting β-1,3-1,4-dextranase fermentor tank process of high-density fermentation figure (◆ OD ₆₀₀■ protein content ● enzyme is lived).

Fig. 4 is the present invention heat-resisting β-1,3-1,4-dextranase fermentor tank process of high-density fermentation SDS-PAGE figure (row M: lower molecular weight standard; Row 1,2,3,4,5 are respectively fermentation 24h, 48h, 60h, 72h, 84h sample, applied sample amount 1 μ L fermented liquid).

Fig. 5 is heat-resisting β-1 of the present invention, 3-1,4-dextranase purge process figure (row M: lower molecular weight standard; Row 1: fermented liquid supernatant; Row 2: cross QSFF post sample; Row 3: cross the S-100 purifying protein).

Fig. 6 is heat-resisting β-1,3-1,4-dextranase optimal pH (■: Citric-Na wherein ₂HPO ₄Damping fluid; ●: the MES damping fluid; ▲: phosphoric acid buffer; ▼: the Glycine-NaOH damping fluid).

Fig. 7 is heat-resisting β-1 of the present invention, 3-1, the optimum temperuture of 4-dextranase.

Embodiment

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

Among the following embodiment, if no special instructions, be ordinary method.

Thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18: China Agricultural University, non-patent literature is: Jiang Zhengqiang etc. the character of thermophilic Paecilomyces varioti xylosidase and with the synergy of zytase, food and fermentation industries, 2008 (07): 12-16.

Embodiment 1, β-1,3-1, the discovery of 4-glucanase gene

One, glucanase gene genome conserved regions segment pcr amplification

With thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18 (DSMZ of institute of microbiology of the Chinese Academy of Sciences, preserving number is CGMCC 6885) genomic dna be template, according to the aminoacid sequence of the filamentous fungus beta-glucanase of GeneBank report, utilize online software Block Maker ( Http:// blocks.fhcrc.org/blocks/blockmkr/make blocks.html) the search conserved regions, utilize online primer-design software CODEHOP (http://blocks.fhcrc.org/codehop.html) design degenerate primer PtLic16CP1 (forward) again: 5 '-CGGCGAGATCGACATCATHGARGGNGT-3 ', Pt Lic16CP2 (oppositely): 5 '-GCCCAGTCGC CGCARAANGTNRTRTC-3 '.PCR reaction mixture (50 μ L): water 37.25 μ L; 10 * Ex Taq buffer, 2.5 μ L; 2.5mmol/L dNTPs, 4 μ L; Primer (10 μ mol/L), each 2.5 μ L; Dna profiling (50-100ng), 1 μ L; Ex Taq (5U/ μ L), 0.25 μ L.PCR reaction conditions: 94 ℃ of 5min; 30s is landed 0.5 ℃ in 61 ℃ of-55 ℃ each circulations; 72 ℃ of 1min, 10 circulations; 94 ℃ of 5min; 55 ℃ of 30s; 72 ℃ of 1min, 20 circulations, 72 ℃ of 10min.Get 5 μ L after the PCR reaction finishes and carry out the agarose gel electrophoresis detection, the result shows a specific band (Fig. 1) about 500bp, be connected into the pMD-18T carrier after sepharose reclaims the test kit recovery, through heat shock method transformed into escherichia coli JM109, bacterium colony PCR checks order after identifying recon.Sequencing result is through NCBI Blast search comparison, with β-1,3 (4)-glucanase gene of other originated from fungus higher homology arranged.

Two, β-1,3-1, the amplification of 4-glucanase gene full-length cDNA

The dna fragmentation that obtains according to the clone and read the frame analysis designs 3 ' RACE and 5 ' RACE primer respectively, utilize the SMARTRACE method (SMART RACE cDNA Amplification Kit, Takara) 3 of amplification cDNA ' and 5 ' end.3 ' RACE special primer: PtLic16AGSP2:5 '-CGATAACGACGGCTTCACGGGCAAT-3 '; 5 ' RACE special primer: PtLic16AGSP1:5 '-CTTGGCAGCGGGCGTGTCCCAGTTC-3 ', PtLic16ANGSP1:5 '-CGTAGATGCCGCCGCCAATGCTGTT-3 '.Sepharose reclaims test kit recovery target fragment and is connected into the pMD-18T carrier, heat shock method transformed into escherichia coli JM109, and after bacterium colony PCR identifies recon, order-checking respectively.In order to obtain 5 ' terminal maximum length, wherein, last for 10 clones of 5 ' RACE product order-checking, according to sequencing result splicing 5 ' terminal and 3 ' end, obtain full length cDNA sequence.Gene order is through NCBI Blast search compare of analysis, and its full-length cDNA comprises the complete open reading frame of a 945bp (ORF) (sequence 1), 314 amino acid of encoding.And Aspergillus fumigatus (Aspergillusfumigatus) inscribe β-1,3 (4)-glucanase gene (EAL88240.1) homology is the highest, and similarity reaches 69%.Aminoacid sequence and Fei Xierxinsatuo bacterium (Neosartorya fischeri) inscribe β-1,3 (4)-dextranase (EAW23242) homology is the highest, and similarity reaches 61%.Through the albumen database compare of analysis, belong to glycoside hydrolase 16 families.

Embodiment 2, recombinant beta-1,3-1, the expression of 4-glucanase gene

One, makes up the reorganization bacterium

Complete open reading frame (ORF) coding protein sequence is analyzed through SignalP 3.0, and a signal peptide is arranged, and possible cleavage site is between 18 and 19 amino acids (AAA-YH).Design primer in view of the above, remove the protogene terminator codon, two pairs of primers are respectively: PtLic16ASnaBIF:5 '-TACGTATATCATCTTGTTGACGACTACG-3 ' (containing the SnaBI restriction enzyme site); PtLic16AAvr II R:5 '- CCTAGGTTAGGGTGCGTACACGCGGAG-3 ' (containing Avr II restriction enzyme site) uses above-mentioned primer, is that template is carried out pcr amplification with the cDNA of thermophilic Paecilomyces varioti (Paecilomyces thermophila) J18.Pcr amplification product agarose gel electrophoresis such as Fig. 2 reclaim after the TA clone connects into the pMD-18T carrier through the gel reagents box, and heat shock method transformed into escherichia coli JM109 identifies recon, positive recombinant plasmid called after pMD-18T-Glu through bacterium colony PCR.Behind the extraction of pMD-18T-Glu plasmid, SnaBI and Avr II double digestion, reclaim object tape, connection is by the expression vector pPIC9K (Invitrogen company) of SnaBI and AvrII double digestion, after PCR and sequence verification, and positive recombinant plasmid called after pPIC9K-Lic16A.Sequencing result shows, pPIC9K-Lic16A is that the dna fragmentation from shown in 5 ' end 55-945 position of sequence 1 inserts the recombinant expression vector that constitutes between the SnaBI of pPIC9K and the Avr II restriction enzyme site in the sequence table.Sequence 1 from 5 ' the dna fragmentation codified sequence table shown in the end 55-945 position in the protein formed of the aminoacid sequence shown in the 19-314 position of sequence 2.

Pichia methanolica GS115 was had a liking in the electricity conversion after recombinant plasmid pPIC9K-Lic16A adopted the SacI linearizing, constituted the reorganization bacterium.The reorganization bacterium that obtains is coated with the MD flat board, and (1.34% yeast does not have amino nitrogen source YNB, 4 * 10 ^-5% vitamin H, 2% glucose), the His that obtains from the MD flat board ⁺Transformant is scraped YPD-G418 flat board (1% yeast powder, 2% peptone, 2% glucose of getting 100 μ L coating different concns after getting through aqua sterilisa, 2% agarose, the G418 of different concns), picking transformant behind the 3-5d, (1.34% yeast does not have amino nitrogen source YNB, 4 * 10 to dibbling MM respectively ^-5The % vitamin H, 0.5% methyl alcohol), MD plate, induce target protein to express by shake-flask culture respectively then, every sampling in 24 hours, the centrifugal 10min of 1000rpm, get supernatant and live, through screening the GS115 bacterial strain that obtains containing recombinant plasmid pPIC9K-Lic16A from YPD-G418 (8mg/mL) flat board, bacterial strain phenotype Mut by following DNS method mensuration enzyme ⁺His ⁺

Two, shake-flask culture

Above-mentioned screening enzyme is lived the highest bacterial strain in BMGY substratum (1% yeast powder, 2% peptone, the phosphate buffered saline buffer of 100mM, pH7.0,1.34%YNB, 4 * 10 ^-5The % vitamin H, 1% glycerine) shake-flask culture 16-18h, the centrifugal 5min of 3000g collects thalline, with BMMY substratum (1% yeast powder, 2% peptone, the phosphate buffered saline buffer of 100mM, pH7.0,1.34%YNB, 4 * 10 ^-5The % vitamin H, 0.5% methyl alcohol) resuspended thalline OD ₆₀₀To about 1.0, induce target protein to express.Above culture condition is a 500mL triangular flask loading amount 100mL substratum, 30 ℃ of temperature, rotating speed 250rpm.Induce 96h, enzyme work can reach the 1698U/mL supernatant liquor.

Enzyme activity is by DNS method (Miller, G.L.1959.Use of dinitrosalicylic acid reagentfor determinat ion of reduc ing sugars.Anal.Chem.31,426-428) measure: the barley substrate is mixed with 1g/100mL concentration, in small test tube, add 50 μ L substrates during mensuration, the MES damping fluid that adds 100 μ L pH 7.0,75mM then, it is 50mM that mixing makes buffer concentration, and concentration of substrate is 0.25g/100ml.70 ℃ of preheating 3min add the suitably enzyme liquid of dilution of 50 μ L, add 200 μ LDNS reagent behind the reaction 10min, add the saturated potassium sodium tartrate solution of 200 μ L after boiling 15min, and the 540nm absorbance is measured in the cooling back, with glucose as standard.

The unit definition of 1 enzyme activity (U) is: (pH7.0,70 ℃ of temperature) under these conditions, per minute generates the needed enzyme amount of 1 μ mol glucose.

Three, high density fermentation

This step is provided with the empty carrier contrast: according to the method for embodiment 2 step 1 acquisition reorganization bacterium, pichia methanolica GS115 is had a liking in electricity conversion behind the pPIC9K plasmid linearization, obtain empty carrier contrast pPIC9K/GS115.

1, the fermentor tank high density fermentation is expressed

Adopt the fermentor tank (BIOTECH, Shanghai Baoxing Biology Equipment Engineering Co., Ltd) of 5L.

Seed culture medium BMGY;

Fermentation minimum medium BSM (85%H ₃PO ₄, 26.7mL; CaSO ₄, 0.93g; K ₂SO ₄, 18.2g; MgSO ₄7H ₂O, 14.9g; KOH, 4.13g; Glycerine, 40.0g, adding distil water water is to 1L).

Glycerine batch feeding substratum: behind the 50g/100ml glycerine autoclaving, add PTM1 (CuSO47H2O, 6.0g; NaI, 0.08g; MnSO4H2O, 3.0g; Na2MoO42H2O, 0.2g; Boric acid, 0.02g; CoCl2,0.5g; ZnCl2,20.0g; FeSO47H2O, 65.0g; Vitamin H, 0.2g; The vitriol oil, 5.0mL; Add water to 1L) 12mL/L glycerine.

100% methanol induction substratum: 100% methyl alcohol adds PTM ₁12mL/L methyl alcohol.

Fermenting process:

(1) seed culture: draw 0.2mL bacterium liquid inoculation 200mL BMGY substratum from the glycerine pipe of preservation, 30 ℃, the 250rpm incubated overnight is to OD ₆₀₀10.0 about.

(2) batch culture: loading amount 2.0L (fermentation minimum medium BSM), the fermentor tank sterilization, 28% strong aqua is transferred pH 4.0, adds PTM ₁4.35mL/L starting fermentation liquid (2L fermentation minimum medium BSM), inoculum size 10%, rotating speed 700rpm, air flow 1.0vvm, fermentation 18-24h.

(3) the glycerine batch feeding is cultivated: treat that batch culture to glycerine exhausts and (operate according to DO spikes, dissolved oxygen rises to rapidly near 100% and descends rapidly again in the 30s), stream glycerol adding batch feeding substratum, flow velocity 18.4mL/h/L starting fermentation liquid, all the time monitor DO, add, adjust maintenance DO＞20% such as rotating speed and air flow by stopping stream.Stream adds 4 hours time, treats OD ₆₀₀About 220, stop stream and add.

(4) 100% methanol inductions are cultivated: after stopping to flow glycerol adding, according to DO spikes, about hungry 30min, stream adds 100% methanol induction substratum, flow velocity is increased to about 10.9mL/h/L starting fermentation liquid, monitoring dissolved oxygen (DO)＞20% from 3.6mL/h/L starting fermentation liquid.Sampling analysis cell concn and enzyme are lived (the same step 2 of measuring method) in the fermenting process, and the result shows, reaches the highest enzyme at 72h and lives, and the fermented liquid supernatant enzyme lives 55,300U/mL, crude protein 9.1g/L.Protein concentration is with reference to (Lowry such as Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J.Protein measurement with the folin phenolreagent.J Biol Chem, 1951, method 193:265-275), with bovine serum albumin as standard protein).And the no enzyme of empty carrier contrast is lived, so not shown in the diagram.

Albumen is through Gelpro 4.5 gel analysis software analysis, and target protein accounts for outer more than 80% of total protein of born of the same parents, and Fermentation Process of Parameter is seen Fig. 3, Fig. 4.

Four, recombinant beta-1,3-1,4-dextranase purifying

Get fermentation (72h is put jar) the supernatant liquor 10mL in the step 3, the centrifugal 10min of 10000rpm/min, get supernatant 10mL, with pH 9.0Tris-Cl level pad exchange three times, finally be concentrated into 1mL, last sample is through pH 9.0, the Tris-Cl level pad equilibrated Q-Sepharose Fast Flow reinforcing yin essence ion exchange column (1 * 10cm) of 20mM, 200mM NaCl washs 5-10 column volume, 300mM NaCl wash-out, flow velocity 1mL/min, collect activated part, ultrafiltration and concentration is gone up sample through pH 7.2 at twice to 1mL, 20mM phosphoric acid buffer (containing 100mMNaCl) equilibrated Sephacryl S-100HR (1 * 100cm), with same phosphoric acid buffer wash-out, flow velocity 0.3mL/min, electrophoresis detection is collected target protein.Purifying protein is through SDS-PAGE (Laemmli UK.Cleavageof structural proteins during the assembly of the head of bacteriophage T4.Nature, 1970,277:680-685) detection reaches electrophoresis pure (Fig. 5).Purification result sees Table 2.Wherein the measuring method of enzyme work and protein content is respectively with step 2 and step 3.

Table 2 recombinant protein purification table

^aThe 10mL fermented supernatant fluid

Embodiment 3, β-1,3-1, the character of 4-dextranase

One, β-1,3-1,4-dextranase optimal pH

Albumen behind the purifying among the embodiment 2 is dissolved in (Citric-Na in the different buffer solution systems of 4 kinds of 50mM of different pH values ₂HPO ₄, pH 2.5-5.5; MES, pH 5.0-6.5; Phosphoric acid buffer, pH 6.5-8.5; Glycine-NaOH, pH 8.5-11.0), under 70 ℃ of conditions, measure enzyme activity (the same step 2 of enzyme activity determination method) then, map as 100% with the enzyme activity vertex.The result shows recombinant beta-1, and 3-1, the optimal pH of 4-dextranase are 7.0 (Fig. 6).

Two, β-1,3-1,4-dextranase optimal reactive temperature

Albumen behind the purifying among the embodiment 2 suitably is diluted in the MES damping fluid of 50mM pH 7.0, under 40-100 ℃ of differing temps, measures β-1 according to the method described above respectively then, 3-1, the enzyme activity of 4-dextranase (the same step 2 of enzyme activity determination method).Map as 100% with the enzyme activity vertex.The result shows that β-1,3-1, the optimal reactive temperature of 4-dextranase are 70 ℃ (Fig. 7).

Sequence table

＜110〉China Agricultural University

＜120〉a kind of heat-resisting β-1,3-1,4-dextranase and encoding gene thereof

 

<130>CGGNARL102007

 

<160>2

 

<210>1

<211>945

<212>DNA

＜213〉thermophilic Paecilomyces varioti (Paecilomyces thermophila)

 

<400>1

atgcgttccc?ttcccatcct?tttcgccggt?ttgacctctc?aactggccgc?ggcgtatcat 60

cttgttgacg?actacggccg?gggcaatggc?ttcttcgaca?agttcaactt?cttcaccggc 120

gacgatccca?cccatgggta?cgtcgactat?gtgagccggg?atgtggctgc?aggcgccggc 180

ctcatcggtg?agcgcgacgg?tcgcacatac?atgggtgtcg?acttcaccaa?tcccgcttcg 240

ggccgtggcc?ggcggagcgt?gcgattggag?agcaagaaca?cgtatgagca?cggcctgatt 300

gtgatcgatc?ttgctcatat?gccaggctcg?gtctgcggca?cctggccggc?cttctggacc 360

ctgggcaccg?gtgactggcc?gtacggcggg?gagattgaca?tcattgaggg?tgtcaacgac 420

aataccttca?accacatggt?gctccacacc?agcgatggtt?gcaccatcga?taacgacggc 480

ttcacgggca?atctgaagac?gtccaactgc?tacgtgtacg?cccccggcca?ggacgccaac 540

gccggctgtg?gcattgaggc?taccgacccg?aattcctacg?gcaaaggttt?caacagcatt 600

ggcggcggca?tctacgccac?ggagatcacc?cccaacggga?tcagcatctg?gttcttccct 660

cgtggctccg?agcccggtga?cgtcctcggc?gacaacccga?acccggcgaa?ctgggacacg 720

cccgctgcca?agttcgcggg?aggtggctgc?gactgggagg?gcaagttcaa?cgcccagaga 780

ctgatctttg?acgtcacctt?ctgcggcgat?tgggccggca?atgtttgggg?cattggtggc 840

tgcgccagcc?gtgcggccaa?ctgcgtggac?ttcgttcgcg?ataacccgtc?cgccttcgcc 900

gagtcttact?ggctggtgaa?ctcgctccgc?gtgtacgcac?cctaa 945

<210>2

<211>314

<212>PRT

＜213〉thermophilic Paecilomyces varioti (Paecilomyces thermophila)

 

<400>2

Met?Arg?Ser?Leu?Pro?Ile?Leu?Phe?Ala?Gly?Leu?Thr?Ser?Gln?Leu?Ala

1 5 10 15

Ala?Ala?Tyr?His?Leu?Val?Asp?Asp?Tyr?Gly?Arg?Gly?Asn?Gly?Phe?Phe

20 25 30

Asp?Lys?Phe?Asn?Phe?Phe?Thr?Gly?Asp?Asp?Pro?Thr?His?Gly?Tyr?Val

35 40 45

Asp?Tyr?Val?Ser?Arg?Asp?Val?Ala?Ala?Gly?Ala?Gly?Leu?Ile?Gly?Glu

50 55 60

Arg?Asp?Gly?Arg?Thr?Tyr?Met?Gly?Val?Asp?Phe?Thr?Asn?Pro?Ala?Ser

65 70 75 80

Gly?Arg?Gly?Arg?Arg?Ser?Val?Arg?Leu?Glu?Ser?Lys?Asn?Thr?Tyr?Glu

85 90 95

His?Gly?Leu?Ile?Val?Ile?Asp?Leu?Ala?His?Met?Pro?Gly?Ser?Val?Cys

100 105 110

Gly?Thr?Trp?Pro?Ala?Phe?Trp?Thr?Leu?Gly?Thr?Gly?Asp?Trp?Pro?Tyr

115 120 125

Gly?Gly?Glu?Ile?Asp?Ile?Ile?Glu?Gly?Val?Asn?Asp?Asn?Thr?Phe?Asn

130 135 140

His?Met?Val?Leu?His?Thr?Ser?Asp?Gly?Cys?Thr?Ile?Asp?Asn?Asp?Gly

145 150 155 160

Phe?Thr?Gly?Asn?Leu?Lys?Thr?Ser?Asn?Cys?Tyr?Val?Tyr?Ala?Pro?Gly

165 170 175

Gln?Asp?Ala?Asn?Ala?Gly?Cys?Gly?Ile?Glu?Ala?Thr?Asp?Pro?Asn?Ser

180 185 190

Tyr?Gly?Lys?Gly?Phe?Asn?Ser?Ile?Gly?Gly?Gly?Ile?Tyr?Ala?Thr?Glu

195 200 205

Ile?Thr?Pro?Asn?Gly?Ile?Ser?Ile?Trp?Phe?Phe?Pro?Arg?Gly?Ser?Glu

210 215 220

Pro?Gly?Asp?Val?Leu?Gly?Asp?Asn?Pro?Asn?Pro?Ala?Asn?Trp?Asp?Thr

225 230 235 240

Pro?Ala?Ala?Lys?Phe?Ala?Gly?Gly?Gly?Cys?Asp?Trp?Glu?Gly?Lys?Phe

245 250 255

Asn?Ala?Gln?Arg?Leu?Ile?Phe?Asp?Val?Thr?Phe?Cys?Gly?Asp?Trp?Ala

260 265 270

Gly?Asn?Val?Trp?Gly?Ile?Gly?Gly?Cys?Ala?Ser?Arg?Ala?Ala?Asn?Cys

275 280 285

Val?Asp?Phe?Val?Arg?Asp?Asn?Pro?Ser?Ala?Phe?Ala?Glu?Ser?Tyr?Trp

290 295 300

Leu?Val?Asn?Ser?Leu?Arg?Val?Tyr?Ala?Pro

305 310

Claims (9)

1. albumen, the protein of forming by the aminoacid sequence shown in the 19-314 of sequence in the sequence table 2.

2. the described proteic encoding gene of claim 1.

3. encoding gene as claimed in claim 2 is characterized in that: the encoding sequence of described proteic encoding gene be in the sequence table sequence 1 from 5 ' terminal 55-945 position.

4. the recombinant vectors that contains claim 2 or 3 described genes.

5. recombinant vectors as claimed in claim 4 is characterized in that, described recombinant vectors is that claim 2 or 3 described genes are inserted the recombinant expression vector that the multiple clone site of pPIC9K obtains.

6. contain claim 2 or 3 described expression of gene box or transgenic cell lines.

7. the reorganization bacterium that contains claim 2 or 3 described genes.

8. reorganization bacterium as claimed in claim 7 is characterized in that: described reorganization bacterium is that claim 2 or 3 described genes are imported in the pichia spp by claim 4 or 5 described recombinant vectorss, the transgenosis reorganization bacterium that obtains.

9. the described proteic preparation method of claim 1 is claim 7 or 8 described reorganization bacterium to be fermented obtain, and described fermentation condition is that temperature is 30 ℃, dissolved oxygen＞20%.

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