CN100420744C

CN100420744C - Beta-mannase, its expression and special engineering bacterium

Info

Publication number: CN100420744C
Application number: CNB2006100731604A
Authority: CN
Inventors: 李德发; 陈小玲; 曹云鹤; 陆文清; 赖长华; 朴香淑; 贺平丽; 乔家运
Original assignee: China Agricultural University
Current assignee: Beijing Yang long view Biotechnology Co.,Ltd.
Priority date: 2006-04-10
Filing date: 2006-04-10
Publication date: 2008-09-24
Anticipated expiration: 2026-04-10
Also published as: CN1834237A

Abstract

The present invention discloses a beta-mannose, an expression method thereof and a special engineering bacterium thereof. The beta-mannase is a protein with one of the following amino acid residue sequence: (1)SEQ ID No. 1 in a sequence table; (2) the amino acid residue sequence which has the replacement, deletion or addition of one to ten amino acid residue, and has the degradation function on the mannan in the SEQ ID No. 1. The special engineering bacterium is prepared by leading a pichia pastoris expression vector containing the maturation protein coding sequence of the beta-mannase into a pichia pastoris. The special engineering bacterium realizes the high efficiency expression of the beta-mannase, has simple fermentation technology and low extraction cost, and is suitable for the large-scale industrial production. The beta-mannase and the engineering bacterium of the present invention perform important function in the industrial production of beta-mannase and the preparation of animal feed additives, and have wide application prospect.

Description

'beta '-mannase and expression method thereof and dedicated engineering bacteria

Technical field

The present invention relates to enzyme and expression method thereof and dedicated engineering bacteria thereof, particularly relate to a kind of 'beta '-mannase and expression method thereof and dedicated engineering bacteria.

Background technology

Plant cell wall mainly is made of materials such as hemicellulose, Mierocrystalline cellulose and xylogen.Mannosans is the important component of plant hemicellulose, is by β-1, and the wire polymer that the 4-D-seminose is formed by connecting mainly contains substituted radicals such as ethanoyl and galactosyl on the side chain of polysaccharide.Mannosans has the wetting ability of height, thereby suction in a large number in the digestive tube of monogastric animal, the viscosity of alimentary canal content is increased, thereby the wriggling of stomach and intestine is produced resistant function, has directly influenced digestion and the absorption of animal to nutritive substance.In recent years, along with the widespread use of bean products (dregs of beans etc.) in diet, the anti-nutrition problem of mannosans also more and more comes into one's own, and adding zymin in feed is the main path that addresses this problem.The complete enzymolysis of mannosans needs the synergy of 'beta '-mannase (EC3.2.1.78), beta-Mannosidase (EC3.2.1.25), beta-glucosidase enzyme (EC3.2.1.21), alpha-galactosidase (EC3.2.1.22) and deacetylase (EC3.1.1.6), and wherein the application of 'beta '-mannase in feed is more extensive.

'beta '-mannase is widespread in nature, and in lower animal, higher plant seeds germinated, and bacterium and fungi are medium that discovery all arranged.Microbe-derived 'beta '-mannase is because active high, advantage such as extracts, purifying is convenient, and temperature that the performance enzyme is imitated and pH scope are wide, enjoys researchist's attention.Particularly more to the 'beta '-mannase research of producing such as mould of genus bacillus, aspergillus and wood, the 'beta '-mannase and the characteristic thereof of part production by biological are as shown in table 1.All there is very big-difference in the 'beta '-mannase that different microorganisms is produced at aspects such as molecular weight, the suitableeest catalytic temperature, optimum pH, substrate specificities.Fungi produces 'beta '-mannase effect slant acidity, and bacterium and actinomycetes produce the nearly neutrality of pH or the meta-alkalescence of 'beta '-mannase effect, but stability better.

The 'beta '-mannase and the character thereof of table 1 part microorganisms

In recent years, along with to the exploitation of nature hemicellulose resource with to the going deep into of mannosans antinutritional factor research in the feed, the researchist has also launched the molecular biology research to mannase gradually.But present research also mainly concentrate on zymoprotein separation and purification, zymologic property analysis and encoding gene the clone and express, about the research report of enzymatic molecular mechanism and enzymatic property transformation aspect also seldom.(Wu's flap such as Wu's flap, He Bingwang, 2000, the chemically modified of nocardia actinomycete 'beta '-mannase and the research in active centre, Chinese biological chemistry and molecular biosciences journal, 16 (2): 227-230.) utilize the method for chemically modified that the structure and the function of the seminase of Nocardia bacteria shape actinomycetes product have been carried out preliminary study, sulfydryl, tyrosine residues and the tryptophan residue that has proved zymoprotein is to keep the essential group of enzymic activity, and the disulfide linkage that further studies confirm that active site of protein is the important factor that influences this enzyme heat stability.(Cann IKO such as Cann, Kocherginskaya S, King MR et al, 1999, Molecularcloning, sequencing, and expression of a novel multidomain mannanase gene fromThermoanaerobacterium polysaccharolyticum, Journal of Bacteriology, 181 (5): 1643-1651.) be cloned into a kind of high molecular (119.6kDa) mannase from Thermoanaerobacterium polysaccharolyticum, deletion mutantion proves that this enzyme contains two cellulose bindings and a catalysis district.

At present, the solution fermentation of bacterium or genus bacillus is mainly adopted in the production of 'beta '-mannase, and main drawback is to need substrate for induction, and the zymotechnique complexity, the cost height, and productive rate is low, and enzymic activity is low etc.

Summary of the invention

The purpose of this invention is to provide the higher 'beta '-mannase of a kind of activity.

'beta '-mannase provided by the present invention, derive from sulfuraspergillus (Aspergillus Sulphureus, preserving number: CGMCC No.0608), be protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 1;

2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein that mannosans is had Degradation.

SEQ ID № in the sequence table: 1 is made up of 383 amino-acid residues, from aminoterminal (N end) 1-21 amino acids residue is signal peptide, from aminoterminal 22-383 amino acids residue is maturation protein, is typical mannase conserved sequence from aminoterminal 205-207 amino acids residue.

The encode gene of above-mentioned 'beta '-mannase is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

2) SEQ ID № in the sequence table: 3 dna sequence dna;

3) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;

4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of 2 and SEQ ID №: the 3 dna sequence dnas hybridization that limit.

The rigorous condition of described height is at 6 * SSC or (6 * SSPE), 0.1%SDS in 2 * Denhardt solution, is hybridized under 65 ℃ of conditions; At 0.1 * SSC, in the 0.1%SDS solution, wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 2 and SEQ ID №: 3 by 1327 based compositions, encoding sequence is from 5 ' end 25-1176 bit base, all coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, from 5 ' end 1-24 bit base is 5 ' end non-translational region, from 5 ' end 25-87 bit base is signal coding sequence, from 5 ' end 88-1176 bit base is mature protein coding sequence, from 5 ' the end 613-621 bit base typical mannase conserved sequence of encoding, be 3 ' end non-translational region from 5 ' end 1177-1327 bit base.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention in the amplification beta-mannase gene.

The 3rd purpose of the present invention provides a kind of dedicated engineering bacteria of expressing 'beta '-mannase.

The engineering bacteria of expression 'beta '-mannase provided by the present invention, be will contain the yeast expression vector of above-mentioned 'beta '-mannase mature protein coding sequence import in the pichia spp and obtain; Described 'beta '-mannase mature protein coding sequence is positioned at the downstream of alpha factor sequence in yeast expression vector; Described 'beta '-mannase mature protein coding sequence is the SEQ ID № in the sequence table: 4 or SEQ ID №: 5, be preferably SEQ ID №: 5.

SEQ ID № in the sequence table: 4 and SEQ ID №: 5 by 1089 based compositions, the maturation protein of the above-mentioned 'beta '-mannase of encoding.

The carrier that sets out that is used to make up the yeast expression vector that contains the 'beta '-mannase mature protein coding sequence can be the yeast expression vector that any one is suitable for exogenous gene expression, as pPICz α A, pPICZA or pPIC9K etc.

Be the carrier that sets out with pPICz α A, the yeast expression vector that contains above-mentioned 'beta '-mannase mature protein coding sequence of structure is pPIC-Mann.

Described pichia spp can be the Pichi strain that is suitable for protein expression arbitrarily, as pichia spp X-33 or GS115 bacterial strain etc.

With pichia spp X-33 is starting strain, and it is X-33/pPIC-Mann that the conversion of structure has the recombinant pichia yeast strain of pPIC-Mann.

Above-mentioned restructured Pichia pastoris in expression carrier can make up according to the ordinary method in the bioengineering field.

The method that the restructured Pichia pastoris in expression carrier is transformed pichia spp is preferably the electric shock conversion method, but also can adopt method commonly used in other bioengineering field, for example, and lithium chloride conversion method, protoplast transformation method etc.

Another object of the present invention provides a kind of expression method of 'beta '-mannase.

The expression method of 'beta '-mannase provided by the present invention is the dedicated expression engineered bacteria of the above-mentioned 'beta '-mannase of fermentation, obtains 'beta '-mannase.

Need add the methanol induction agent when fermenting above-mentioned recombinant yeast pichia pastoris, add methyl alcohol concentration be 0.8-1.2%, be preferably 1%.

For the volatilization loss of compensation methyl alcohol, also to add methyl alcohol in per 24 hours, make the methanol concentration in the bacterium liquid remain on 0.8-1.2%, inducing temperature is 28-30 ℃.

Above-mentioned percentage concentration is concentration expressed in percentage by volume.

The invention provides a kind of 'beta '-mannase and encoding gene thereof that derives from sulfuraspergillus, and codon preference according to pichia spp, synthesized encoding sequence, and then constructed the pichia pastoris engineered strain of sulfuraspergillus 'beta '-mannase through this codon optimized enzyme maturation protein.After the 10L fermentation cylinder for fermentation, the enzyme activity of fermented liquid can reach more than the 1100IU/mL with this bacterial strain, has realized efficiently expressing of 'beta '-mannase, and zymotechnique is simple, and extraction cost is cheap, is suitable for large-scale industrial production.In addition, this enzyme has the acid acceptance height, and catalytic pH range is wide, specific activity height, the characteristics good to metal ion tolerance.'beta '-mannase of the present invention and engineering bacteria thereof will play a significant role in the preparation of the suitability for industrialized production of 'beta '-mannase and animal feedstuff additive, have a extensive future.

Below in conjunction with specific embodiment the present invention is described in further detail.

Description of drawings

Fig. 1 is the PCR qualification result of pichia spp recon

Fig. 2 is the PCR qualification result of beta-mannase gene genetic stability among the recombinant yeast pichia pastoris X-33/pPIC-Mann

Fig. 3 is the SDS-PAGE detected result of reorganization strain X-33/pPIC-Mann expression product

Fig. 4 is the optimum pH and the pH Detection of Stability result of the sulfuraspergillus 'beta '-mannase of expression

Fig. 5 is the optimum temperuture and the temperature stability detected result of the sulfuraspergillus 'beta '-mannase of expression

Fig. 6 for the sulfuraspergillus 'beta '-mannase of expressing kinetic parameter

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the solvent in the described substratum is water, and described primer, sequence are synthesized and examining order is finished by the living worker's biotechnology in Shanghai company limited.

Embodiment 1, the present invention are from the clone of the beta-mannase gene of sulfuraspergillus

Acquisition from the full length cDNA sequence of the beta-mannase gene of sulfuraspergillus may further comprise the steps:

1, the medicine of sulfuraspergillus is joined and the extraction of RNA always

With sulfuraspergillus (Aspergillus Sulphureus, preserving number: CGMCC No.0608) be inoculated into its growth medium (with 400mL tap water dissolving 85g wheat bran, 10g soybean cake powder, 0.2g KH ₂PO ₄, 0.3g NaCl and 10g sucrose, 121 ℃ of boiling 30min cross the leaching supernatant, autoclaving again after the packing) in, 30 ℃ of shaking tables are cultivated 48h.After cultivating end, the centrifugal 1min of 5000r/min collects thalline, places liquid N ₂The middle grinding.Get the thalline after 100mg grinds, place the 1.5mL centrifuge tube, add 500 μ L RNA extraction buffer (20mmol/LTris-HCl, pH8.0,1%SDS, 200mmol/L NaCl, 5mmol/L EDTA), add 500 μ L phenol/imitative (1: 1) again, 4 ℃, the centrifugal 5min of 12000r/min, supernatant adds equal-volume 4mol/L LiCl, 4 ℃ of precipitations 4h, the centrifugal 15min of 12000r/min then, abandon supernatant, precipitation is washed twice with 70% ethanol, and vacuum is drained, and is dissolved in the ddH that 20 μ l DEPC handle ₂Among the O, obtain total RNA of purpose bacterial strain.

2, the segmental clone of beta-mannase gene cDNA

To the aspergillus echinulatus announced among the GenBank (the GenBank accession number: L35487) and Rui Shi wood mould (the GenBank accession number: L25310) the mRNA sequence of beta-mannase gene is analyzed, analytical results shows at interlude one section height homologous nucleotide sequence, according to the nucleotide sequence design pair of degenerate primers of this high conservative, primer sequence is as follows:

P1 (upstream primer): 5 '-TTCAACGACGTCAACAC-3 ';

P2 (downstream primer): 5 '-A (T/C) CCAGCC (G/A) TTGCCCCA-3 '

The total RNA of sulfuraspergillus that extracts with step 1 is a template, with synthetic its first chain cDNA of M-MLV ThermoScript II (Promega).Be template with the reverse transcription synthetic first chain cDNA again, carry out pcr amplification under the guiding of primer 1 and primer 2,50 μ L PCR reaction systems are: 10 * PCR damping fluid, 5 μ L, primer P1 and P2 each 1 μ L (20 μ M), Taq enzyme 1 μ L (5 μ/μ L), dNTPs 1 μ L (2.5mM), template cDNA 1 μ L, H ₂O 40 μ L; The PCR reaction conditions is: 94 ℃ of pre-sex change 3min at first; 94 ℃ of pre-sex change 45s then, 49 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are extended 10min.After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, reclaim the purpose fragment of the about 600bp of length and it is carried out purifying, to reclaim fragment cloning in carrier pUCm-T (worker's biotechnology company limited is given birth in Shanghai), obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that amplified fragments contains the nucleotide sequence of above-mentioned high conservative, and length is 608bp.

3, the clone of the 3 ' UTR (non-translational region) of beta-mannase gene cDNA

The Partial cDNA Sequence of the beta-mannase gene that obtains according to step 2 is designed 3 '-RACE special primer MAN-3-2:5 '-TACCCGTACCAATTCGC-3 ' that increases.Be template with total RNA then, with 3 ' terminal fragment, reaction system and the reaction conditions reference reagent box specification sheets of 3 '-Full RACE test kit (TaKaRa company) amplification beta-mannase gene cDNA.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 600bp of purifying length, it is cloned into carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that 3 ' the UTR fragment length of the beta-mannase gene cDNA of amplification is 151bp.

4, the clone of the 5 ' UTR of beta-mannase gene cDNA

The Partial cDNA Sequence of the beta-mannase gene that obtains according to step 2 is designed 5 '-RACE increase special primer Race-1, Race-2, Race-3, Race-4 and RT-P, and primer sequence is as follows:

Race-1：5’-GTATTTGCGTGGGAGTTGGC-3’；

Race-2：5’-TACCCGTACCAATTCGCCGA-3’；

Race-3：5’-TGCAATCTGCGTATCAGACG-3’；

Race-4：5’-ACGAGACGGATTTCTATACC-3’；

RT-P：5’-CCAACTCCCACGCAA-3’。

Be template with the total RNA of sulfuraspergillus then, with 5 ' terminal fragment, reaction system and the reaction conditions reference reagent box specification sheets of 5 '-Full RACE test kit (TaKaRa company) amplification beta-mannase gene cDNA.After should finishing, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 500bp of purifying length, it is cloned on the carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, it is checked order, and sequencing result shows that 5 ' the UTR fragment length of the beta-mannase gene cDNA of amplification is 24bp.

5, the acquisition of beta-mannase gene full length cDNA sequence

5 ' and 3 ' end sequence according to

step

3 and 4 beta-mannase genes that obtain designs a pair of primer, and primer sequence is as follows:

F1 (upstream primer): 5-cggaattcatgaagctctccagctc-3;

F2 (downstream primer): 5-cccaagcttttaggcgctatcaatagc-3

With the total RNA of sulfuraspergillus is template, under the guiding of primers F 1 and F2, the full-length cDNA of pcr amplification beta-mannase gene, 10 * PCR buffer, 5 μ L, primer (F1/F2) each 1 μ L (20 μ M), Taq enzyme 1 μ L (5 μ/μ L), dNTPs 1 μ L (2.5mM), template cDNA 1 μ L, H ₂O 40 μ L, total reaction system 50 μ L, response procedures are 94 ℃ of pre-sex change 3min, carry out amplified reaction by following loop parameter then: 94 ℃ of pre-sex change 45s, 49 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations, last 72 ℃ are extended 10min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also amplified fragments of the about 1152bp of purifying length, it is cloned on the carrier pUCm-T, obtain containing the segmental recombinant plasmid of recovery, with M13+/-primer checks order to it, sequencing result shows that sulfuraspergillus beta-mannase gene full-length cDNA has SEQ ID № in the sequence table: 2 nucleotide sequence, SEQ ID № in the sequence table: 2 by 1327 based compositions, encoding sequence is from 5 ' end 25-1176 bit base, coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence, from 5 ' end 1-24 bit base is 5 ' end non-translational region, from 5 ' end 25-87 bit base is signal coding sequence, from 5 ' end 88-1176 bit base is mature protein coding sequence, from 5 ' the end 613-621 bit base typical mannase conserved sequence of encoding, be 3 ' end non-translational region from 5 ' end 1177-1327 bit base.SEQ ID № in the sequence table: 1 is made up of 383 amino-acid residues, from aminoterminal (N end) 1-21 amino acids residue is signal peptide, from aminoterminal 22-383 amino acids residue is maturation protein, is typical mannase conserved sequence from aminoterminal 205-207 amino acids residue.The molecular weight of sulfuraspergillus 'beta '-mannase maturation protein is 41kDa.The aminoacid sequence of clone's the nucleotide sequence of sulfuraspergillus beta-mannase gene and proteins encoded thereof is carried out homology with the sequence among the GenBank to be compared, comparison result shows that the sibship of this enzyme and aspergillus echinulatus 'beta '-mannase is nearest, the nucleotide sequence similarity reaches 71%, and amino acid sequence similarity reaches 72%.

The structure of the expression engineering bacteria of embodiment 2, sulfuraspergillus 'beta '-mannase

One, the structure of sulfuraspergillus beta-mannase gene yeast expression vector

1, sulfuraspergillus 'beta '-mannase mature protein gene is synthetic

Under the prerequisite that does not change aminoacid sequence, only the proteic codon of encoding mature in embodiment 1 clone's the sulfuraspergillus beta-mannase gene is replaced with the preference codon of pichia spp, thereby obtain the nucleotide sequence of an other sulfuraspergillus beta-mannase gene, be the SEQ ID № in the sequence table: 3, SEQ ID № in the sequence table: 3 by 1327 based compositions, encoding sequence is from 5 ' end 25-1176 bit base, coding has SEQ ID №: the protein of 1 amino acid residue sequence, from 5 ' end 1-24 bit base is 5 ' end non-translational region, from 5 ' end 25-87 bit base is signal coding sequence, from 5 ' end 88-1176 bit base is mature protein coding sequence, from 5 ' the end 613-621 bit base typical mannase conserved sequence of encoding, be 3 ' end non-translational region from 5 ' end 1177-1327 bit base.Synthetic SEQ ID № then: the gene fragment of coding sulfuraspergillus 'beta '-mannase maturation protein in 3 (SEQ ID №: in 3 from 5 ' end 88-1176 bit base), and add restriction enzyme EcoR I and Xba I restriction enzyme site respectively at 5 ' and 3 ' end.

2, the acquisition of sulfuraspergillus beta-mannase gene yeast expression vector

After with restriction enzyme EcoR I and Da I step 1 synthetic gene being carried out double digestion, it is connected into the downstream of alpha factor sequence in the carrier pPICz of same enzyme double digestion α A (Invitrogen), obtain the yeast expression vector of sulfuraspergillus beta-mannase gene, called after pPIC-Mann, to connect product transformed into escherichia coli DH 5 α competent cells then, filter out positive colony, the upgrading grain with the LB resistant panel that contains 100 μ g/mL microbiotic Zeocin.

Two, the acquisition of the expression engineering bacteria of sulfuraspergillus 'beta '-mannase

1, the acquisition of the Pichia anomala expression plasmid of linearizing sulfuraspergillus beta-mannase gene

Get the Pichia anomala expression plasmid pPIC-Mann of the sulfuraspergillus beta-mannase gene of 10 μ g step 1 acquisition, it was cut 12-24 hour at 37 ℃ of following enzymes with restriction enzyme BspHI, then enzyme is cut product and precipitate, be dissolved in the 10 μ L aqua sterilisas with dehydrated alcohol.

2, the preparation of pichia spp X-33 competent cell

Pichia spp X-33 is inoculated in the YPD substratum (2% glucose, all the other are water for 1% yeast extract, 2% peptone), at 28 ℃ of following shake-flask culture to OD ₆₀₀Behind 1.3-1.5, the centrifugal 5min of 5000rpm collects somatic cells, with aqua sterilisa washing 2 times, thalline is suspended in the sorbitol solution of 0.5mL 1mol/L.

3, transform

Get the pichia spp X-33 competent cell of 80 μ L steps 2 preparation, add the linearizing plasmid pPIC-Mann that 10 μ g steps 1 obtain, mixing, place 0.2cm electric shock cup, ice bath 5min, under 2000 volts of voltages (25 μ F) condition, shock by electricity, add 1mL 1mol/L sorbitol rapidly, place 28 ℃ of static cultivation 2h.Then culture is coated on the YPDS that contains microbiotic Zeocin (100 μ g/mL) (2% glucose, 2% agar, all the other are water for 1% yeast extract, the 2% peptone) flat board, cultivated 3-4 days down at 28 ℃, until growing bacterium colony clearly.

4, evaluation, the screening of reorganization bacterium

Single bacterium colony that picking grows is inoculated in it in YPD liquid nutrient medium, at 28 ℃ of following shake-flask culture 2-3 days, extracts total DNA of recombination microzyme and as template, at primer

QJ-F-1 (5-ccggaattcttgccaaaggctttc-3) and

Under the guiding of QJ-R-1 (5-gctctagattaagcagaatcaatagcag-3),, PCR identifies positive recombinant, qualification result is (swimming lane 1: the pichia spp X-33 (contrast) that transforms pPICz α A empty carrier as shown in Figure 1; Swimming lane 2:DNA Marker: λ DNA/EcoRI+HindIII double digestion; Swimming lane 3 and swimming lane 4: recombination yeast), amplify the positive recon of about 1000bp dna fragmentation.The picking positive recombinant is inoculated in it and contains 30mL BMGY (1% yeast extract, 2% peptone, 1.34%YNB, 4 * 10 ^-5%biotin, 1% glycerine, 100mM pH6.0 phosphate buffered saline buffer, all the other are water) 250mL of substratum shakes in the bottle, is cultured to OD in 28 ℃, 250-300rpm ₆₀₀Be 2-6, centrifugal collection thalline under the room temperature with the resuspended thalline of BMMY (1% yeast extract, 2% peptone, 0.1mol/L phosphoric acid buffer pH6.0,1.34%YNB, 4 * 10-5% vitamin H), makes OD then ₆₀₀Being 1.0, is that the methyl alcohol of 0.5% (V/V) is that sole carbon source carries out inducing culture with final concentration again, efficiently expresses recombinant bacterial strain by SDS-PAGE and determination of activity screening, and the result is sieved to the 1 strain enzyme higher reorganization bacterium that lives, called after X-33/pPIC-Mann.To the cultivation of going down to posterity of this strain bacterium, passed for 20 generations altogether, respectively at reach the 1st, 5,10,15,20 generation collected specimens, extract total DNA, under the guiding of primer QJ-F-1 (5-ccggaattcttgccaaaggctttc-3) and QJ-R-1 (5-gctctagattaagcagaatcaatagcag-3), PCR identifies the stability of sulfuraspergillus beta-mannase gene, and the result is (swimming lane 1: the pichia spp X-33 (contrast) that transforms pPICz α A empty carrier as shown in Figure 2; Swimming lane 2-6: the X-33/pPIC-Mann that reached for the 1st, 5,10,15,20 generations; Swimming lane 7:DNAMarker: λ DNA/EcoRI+HindIII double digestion), show that foreign gene does not take place loses phenomenon, this bacterial strain has higher genetic stability.

The abduction delivering of embodiment 3, sulfuraspergillus 'beta '-mannase

The recombinant yeast pichia pastoris yeast strain X-33/pPIC-Mann that picking embodiment 2 obtains is inoculated in the 3L that 200mL BMGY substratum is housed with it and shakes in the bottle, and jolting obtained OD more than 24 hours under 28-30 ℃, 280rpm ₆₀₀Be about 3.0 seed liquor.Prepare 4L BMGY substratum then, the 10L that packs into controls in the fermentor tank automatically, after sterilization under 121 ℃, is cooled to 28.5 ℃, adds above-mentioned seed liquor, regulates pH to 5.5 with ammoniacal liquor and phosphoric acid, by regulating rotating speed and air flow control dissolved oxygen greater than 30%.After measured, behind the access seed liquor 23h, glycerine consumption is (dissolved oxygen is labeled as 100%) fully, enters stream and adds 50% (W/V) the glycerine stage, and flow velocity is 60mL/h.After stream added 4h, glycerine consumption is (dissolved oxygen was labeled as 100%) fully, enters methyl alcohol stream and adds the stage, and flow velocity is 12mL/h, control dissolved oxygen simultaneously and be 20% or more (as dissolved oxygen is remained on more than 20%, stop to add methyl alcohol, until the dissolved oxygen rise).Behind the 3h, the methyl alcohol flow velocity is transferred to 24mL/h, after 2h, flow velocity is transferred to 30mL/h, and keeps to the final, simultaneously, gather fermented liquid after 1,2,3,4 days, wait until detection respectively at abduction delivering.After cultivating end, detect expressed proteins content in the fermented liquid by SDS-PAGE, detected result is (swimming lane 1:Marker as shown in Figure 3; Swimming lane 2: the pichia spp X-33 (contrast) that transforms pPICz α A empty carrier; Swimming lane 3: induce the sample of gathering after 4 days; Swimming lane 4: induce the sample of gathering after 3 days; Swimming lane 5: induce the sample of gathering after 2 days; Swimming lane 6: induce after 1 day the sample of gathering), the result has obtained the recombinant protein of about 45KD through abduction delivering, conform to expected results, and the expressing quantity of abduction delivering after 4 days is the highest.

The zymologic property analysis of embodiment 4, sulfuraspergillus 'beta '-mannase

Now the sulfuraspergillus 'beta '-mannase that embodiment 3 is expressed carries out following zymologic property analysis:

1, optimum pH and pH stability analysis

The mensuration of optimum pH: be 2.4,3.0,4.0,5.0,6.0,6.0,7.0,8.0 Sodium phosphate dibasic-citrate buffer solution preparation mannosans substrate with the pH value respectively, concentration is 0.8g/mL, measure relative enzyme activity down in 40 ℃, with high enzymatic activity is 100%, determines the optimum pH of this enzyme.Detected result as shown in Figure 4, after measured, the optimum pH of sulfuraspergillus 'beta '-mannase of the present invention is 2.4.

The detection of pH value stabilization: after the 'beta '-mannase of expressing is Sodium phosphate dibasic-citrate buffer solution processing 30min of 2.4,3.0,4.0,5.0,6.0,7.0,8.0 with pH value respectively, under 40 ℃ of temperature of reaction, measure residual relative enzyme activity, with high enzymatic activity is 100%, detected result as shown in Figure 4, after measured, sulfuraspergillus 'beta '-mannase of the present invention has higher pH stability.

2, optimum temperuture and temperature stability analysis

The mensuration of optimum temperuture: with 100mM sodium-acetate buffer preparation mannosans substrate, concentration is 0.8g/mL, measuring down relative enzyme activities at 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ respectively, is 100% with high enzymatic activity, determines the optimal reactive temperature of this enzyme.Detected result as shown in Figure 5, after measured, the optimal reactive temperature of sulfuraspergillus 'beta '-mannase of the present invention is 50 ℃.

The detection of temperature stability: with the 'beta '-mannase of expressing (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ of differing tempss, 90 ℃) following insulation 30min, putting into frozen water then cools off, under optimum temperuture (50 ℃) and optimum pH (2.4) condition, measure relative enzyme activity, with high enzymatic activity is 100%, detected result as shown in Figure 5, after measured, sulfuraspergillus 'beta '-mannase of the present invention has higher temperature stability.

3, the mensuration of enzyme kinetics parameter

Kinetic parameter (K _m: Michaelis-Menton constant, V _Max: mensuration maximum speed of reaction): be mixed with the mannan solution of different concns (1-20mg/mL) and as substrate with Sodium phosphate dibasic-citrate buffer solution (optimum pH 2.4), react the enzyme activity that 10min measures the 'beta '-mannase of expressing down in optimum temperuture (50 ℃), utilize Michaelis-Menton equation double-reciprocal plot method to determine K _m, V _MaxValue.According to Michaelis-Menton equation: V=-K _mXV/[S]+V _MaxThe rectilinear of drawing as shown in Figure 6, K as seen from the figure _mValue is 0.91mg/mL, V _MaxValue shows that for 909.09U/mg sulfuraspergillus 'beta '-mannase of the present invention has higher enzyme activity.

4, metal ion and sequestrant (EDTA) are to the influence of enzyme activity

Different compound (the MgCl that in Sodium phosphate dibasic-citrate buffer (optimum pH 2.4), add 1mM ₂, FeCl ₂, MnSO ₄, CuSO ₄, ZnCl ₂, CaCl ₂, NaCl, EDTA), the mannase of expressing handled 30min in above-mentioned solution after, under optimum temperuture, measure the relative enzyme activity of the 'beta '-mannase of expressing, not add metal ion and sequestrant etc. is blank (100%), the result is as shown in table 2, show that metal ion and sequestrant (EDTA) are little to the influence alive of the enzyme of sulfuraspergillus 'beta '-mannase of the present invention, this enzyme has higher metal ion stability.

The metal ion stability detected result of the sulfuraspergillus 'beta '-mannase that table 2 is expressed

Sequence table

<160>5

<210>1

<211>383

<212>PRT

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>1

Met?Lys?Leu?Ser?Ser?Ser?Leu?Leu?Thr?Leu?Ala?Ser?Leu?Ala?Leu?Ala

1 5 10 15

Asn?Leu?Ser?Thr?Ala?Leu?Pro?Lys?Ala?Ser?Pro?Ala?Pro?Ser?Thr?Ser

20 25 30

Ser?Ser?Ser?Ala?Ser?Thr?Ser?Phe?Ala?Ser?Thr?Ser?Gly?Leu?Gln?Phe

35 40 45

Thr?Ile?Asp?Gly?Glu?Thr?Gly?Tyr?Phe?Ala?Gly?Thr?Asn?Ser?Tyr?Trp

50 55 60

Ile?Gly?Phe?Leu?Thr?Asp?Asp?Ser?Asp?Val?Asp?Leu?Val?Met?Ser?His

65 70 75 80

Leu?Lys?Ser?Ser?Gly?Leu?Lys?Ile?Leu?Arg?Val?Trp?Gly?Phe?Asn?Asp

85 90 95

Val?Thr?Thr?Gln?Pro?Ser?Ser?Gly?Thr?Val?Trp?Tyr?Gln?Leu?His?Gln

100 105 110

Asp?Gly?Lys?Ser?Thr?Ile?Asn?Thr?Gly?Ala?Asp?Gly?Leu?Gln?Arg?Leu

115 120 125

Asp?Tyr?Val?Val?Ser?Ser?Ala?Glu?Gln?His?Gly?Ile?Lys?Leu?Ile?Ile

130 135 140

Asn?Phe?Val?Asn?Tyr?Trp?Thr?Asp?Tyr?Gly?Gly?Met?Ser?Ala?Tyr?Val

145 150 155 160

Ser?Ala?Tyr?Gly?Gly?Ser?Asp?Glu?Thr?Asp?Phe?Tyr?Thr?Ser?Asp?Thr

165 170 175

Met?Gln?Ser?Ala?Tyr?Gln?Thr?Tyr?Ile?Lys?Thr?Val?Val?Glu?Arg?Tyr

180 185 190

Ser?Asn?Ser?Ser?Ala?Val?Phe?Ala?Trp?Glu?Leu?Ala?Asn?Glu?Pro?Arg

195 200 205

Cys?Pro?Ser?Cys?Asp?Thr?Thr?Val?Leu?Tyr?Asp?Trp?Ile?Glu?Lys?Thr

210 215 220

Ser?Lys?Phe?Ile?Lys?Gly?Leu?Asp?Ala?Asp?His?Met?Val?Cys?Ile?Gly

225 230 235 240

Asp?Glu?Gly?Phe?Gly?Leu?Asn?Thr?Asp?Ser?Asp?Gly?Ser?Tyr?Pro?Tyr

245 250 255

Gln?Phe?Ala?Glu?Gly?Leu?Asn?Phe?Thr?Met?Asn?Leu?Gly?Ile?Asp?Thr

260 265 270

Ile?Asp?Phe?Ala?Thr?Leu?His?Leu?Tyr?Pro?Asp?Ser?Trp?Gly?Thr?Ser

275 280 285

Asp?Asp?Trp?Gly?Asn?Gly?Trp?Ile?Ser?Ala?His?Gly?Ala?Ala?Cys?Lys

290 295 300

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

305 310 315 320

His?Cys?Ser?Val?Glu?Ser?Pro?Trp?Gln?Gln?Thr?Ala?Leu?Asn?Thr?Thr

325 330 335

Gly?Val?Ser?Ala?Asp?Leu?Phe?Trp?Gln?Tyr?Gly?Asp?Asp?Leu?Ser?Thr

340 345 350

Gly?Glu?Ser?Pro?Asp?Asp?Gly?Asn?Thr?Ile?Tyr?Tyr?Gly?Thr?Ser?Asp

355 360 365

Tyr?Glu?Cys?Leu?Val?Thr?Asp?His?Val?Ala?Ala?Ile?Asp?Ser?Ala

370 375 380

<210>2

<211>1327

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>2

aatacagcca?agcaaaccac?cacaatgaag?ctctccagct?ccctcctcac?cctggccagc 60

ctggcgctgg?ccaacctctc?cacggccctg?cccaaagcct?ctcctgcacc?aagcaccagc 120

agcagcagcg?cctccacctc?cttcgccagc?acctcgggcc?tccaattcac?catcgacggc 180

gaaaccggct?acttcgccgg?aacgaacagt?tactggatcg?gtttcctgac?cgacgactcc 240

gatgtcgacc?tggtgatgag?ccacctgaag?tcatccggcc?tcaaaatcct?ccgcgtatgg 300

ggcttcaacg?acgtcaccac?gcagccctct?tccggcacag?tctggtacca?actgcaccag 360

gacggcaaat?caaccatcaa?cactggcgcc?gacggtctcc?agcgcctcga?ctacgtcgtc 420

tcctccgccg?aacagcacgg?catcaaactc?atcatcaact?tcgtcaacta?ctggaccgac 480

tacggcggta?tgtccgcgta?cgtgagcgca?tatggcggat?ccgacgagac?ggatttctat 540

accagtgata?cgatgcaatc?tgcgtatcag?acgtatatta?agacggtcgt?ggagcggtat 600

agtaactcct?ctgcggtatt?tgcgtgggag?ttggcgaatg?agccgagatg?tcctagttgt 660

gatactaccg?tgttgtatga?ttggattgag?aagacgagta?aatttattaa?ggggttggat 720

gccgatcata?tggtttgtat?tggtgatgag?ggcttcggcc?tcaacaccga?ctcggacggc 780

agctacccgt?accaattcgc?cgagggtctc?aacttcacca?tgaacctcgg?tatcgatact 840

attgactttg?ctaccctcca?cttgtatcct?gatagctggg?gcacctccga?cgactggggc 900

aacggatgga?tcagcgcaca?cggcgcggca?tgcaaagcgg?ccggcaagcc?ctgtctactg 960

gaggaatacg?gagtcacctc?gaaccactgt?agtgtggaga?gcccgtggca?gcagacggcc 1020

ctcaacacga?ctggcgtcag?cgcagatctc?ttctggcagt?atggtgatga?tttgagcacg 1080

ggcgagtcgc?cggatgatgg?aaataccatt?tactacggga?cgagcgatta?tgagtgtttg 1140

gtcacggatc?atgtggctgc?tattgatagc?gcctaaggga?tagggagatg?tggatatatc 1200

tagttgatac?attatgaggg?gtgctgtaca?taagaatgcg?ccggagggag?ggtgtgaagg 1260

atgataactg?atctttgagt?ttggatagga?tacaattcga?gaggcaaatc?atcccttcga 1320

ttaccat 1327

<210>3

<211>1327

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>3

aatacagcca?agcaaaccac?cacaatgaag?ctctccagct?ccctcctcac?cctggccagc 60

ctggcgctgg?ccaacctctc?cacggccttg?ccaaaggctt?ctccagctcc?atctacttct 120

tcttcttctg?cttctacttc?ttttgcttct?acttctggtt?tgcaatttac?tattgatggt 180

gaaactggtt?actttgctgg?tactaactct?tactggattg?gttttttgac?tgatgattct 240

gatgttgatt?tggttatgtc?tcatttgaag?tcttctggtt?tgaagatttt?gagagtttgg 300

ggttttaacg?atgttactac?tcaaccatct?tctggtactg?tttggtacca?attgcatcaa 360

gatggtaagt?ctactattaa?cactggtgct?gatggtttgc?aaagattgga?ttacgttgtt 20

tcttctgctg?aacaacatgg?tattaagttg?attattaact?ttgttaacta?ctggactgat 480

tacggtggta?tgtctgctta?cgtttctgct?tacggtggtt?ctgatgaaac?tgatttttac 540

acttctgata?ctatgcaatc?tgcttaccaa?acttacatta?agactgttgt?tgaaagatac 600

tctaactctt?ctgctgtttt?tgcttgggaa?ttggctaacg?aaccaagatg?tccatcttgt 660

gatactactg?ttttgtacga?ttggattgaa?aagacttcta?agtttattaa?gggtttggat 20

gctgatcata?tggtttgtat?tggtgatgaa?ggttttggtt?tgaacactga?ttctgatggt 780

tcttacccat?accaatttgc?tgaaggtttg?aactttacta?tgaacttggg?tattgatact 840

attgattttg?ctactttgca?tttgtaccca?gattcttggg?gtacttctga?tgattggggt 900

aacggttgga?tttctgctca?tggtgctgct?tgtaaggctg?ctggtaagcc?atgtttgttg 960

gaagaatacg?gtgttacttc?taaccattgt?tctgttgaat?ctccatggca?acaaactgct 1020

ttgaacacta?ctggtgtttc?tgctgatttg?ttttggcaat?acggtgatga?tttgtctact 1080

ggtgaatctc?cagatgatgg?taacactatt?tactacggta?cttctgatta?cgaatgtttg 1140

gttactgatc?atgttgctgc?tattgattct?gcttaaggga?tagggagatg?tggatatatc 1200

tagttgatac?attatgaggg?gtgctgtaca?taagaatgcg?ccggagggag?ggtgtgaagg 1260

atgataactg?atctttgagt?ttggatagga?tacaattcga?gaggcaaatc?atcccttcga 1320

ttaccat 1327

<210>4

<211>1089

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>4

ctgcccaaag?cctctcctgc?accaagcacc?agcagcagca?gcgcctccac?ctccttcgcc 60

agcacctcgg?gcctccaatt?caccatcgac?ggcgaaaccg?gctacttcgc?cggaacgaac 120

agttactgga?tcggtttcct?gaccgacgac?tccgatgtcg?acctggtgat?gagccacctg 180

aagtcatccg?gcctcaaaat?cctccgcgta?tggggcttca?acgacgtcac?cacgcagccc 240

tcttccggca?cagtctggta?ccaactgcac?caggacggca?aatcaaccat?caacactggc 300

gccgacggtc?tccagcgcct?cgactacgtc?gtctcctccg?ccgaacagca?cggcatcaaa 60

ctcatcatca?acttcgtcaa?ctactggacc?gactacggcg?gtatgtccgc?gtacgtgagc 420

gcatatggcg?gatccgacga?gacggatttc?tataccagtg?atacgatgca?atctgcgtat 480

cagacgtata?ttaagacggt?cgtggagcgg?tatagtaact?cctctgcggt?atttgcgtgg 540

gagttggcga?atgagccgag?atgtcctagt?tgtgatacta?ccgtgttgta?tgattggatt 600

gagaagacga?gtaaatttat?taaggggttg?gatgccgatc?atatggtttg?tattggtgat 660

gagggcttcg?gcctcaacac?cgactcggac?ggcagctacc?cgtaccaatt?cgccgagggt 720

ctcaacttca?ccatgaacct?cggtatcgat?actattgact?ttgctaccct?ccacttgtat 780

cctgatagct?ggggcacctc?cgacgactgg?ggcaacggat?ggatcagcgc?acacggcgcg 840

gcatgcaaag?cggccggcaa?gccctgtcta?ctggaggaat?acggagtcac?ctcgaaccac 900

tgtagtgtgg?agagcccgtg?gcagcagacg?gccctcaaca?cgactggcgt?cagcgcagat 960

ctcttctggc?agtatggtga?tgatttgagc?acgggcgagt?cgccggatga?tggaaatacc 1020

atttactacg?ggacgagcga?ttatgagtgt?ttggtcacgg?atcatgtggc?tgctattgat 1080

agcgcctaa 1089

<210>5

<211>1089

<212>DNA

＜213〉sulfuraspergillus (Aspergillus Sulphureus)

<400>5

ttgccaaagg?cttctccagc?tccatctact?tcttcttctt?ctgcttctac?ttcttttgct 60

tctacttctg?gtttgcaatt?tactattgat?ggtgaaactg?gttactttgc?tggtactaac 120

tcttactgga?ttggtttttt?gactgatgat?tctgatgttg?atttggttat?gtctcatttg 180

aagtcttctg?gtttgaagat?tttgagagtt?tggggtttta?acgatgttac?tactcaacca 240

tcttctggta?ctgtttggta?ccaattgcat?caagatggta?agtctactat?taacactggt 300

gctgatggtt?tgcaaagatt?ggattacgtt?gtttcttctg?ctgaacaaca?tggtattaag 360

ttgattatta?actttgttaa?ctactggact?gattacggtg?gtatgtctgc?ttacgtttct 420

gcttacggtg?gttctgatga?aactgatttt?tacacttctg?atactatgca?atctgcttac 480

caaacttaca?ttaagactgt?tgttgaaaga?tactctaact?cttctgctgt?ttttgcttgg 540

gaattggcta?acgaaccaag?atgtccatct?tgtgatacta?ctgttttgta?cgattggatt 600

gaaaagactt?ctaagtttat?taagggtttg?gatgctgatc?atatggtttg?tattggtgat 660

gaaggttttg?gtttgaacac?tgattctgat?ggttcttacc?cataccaatt?tgctgaaggt 720

ttgaacttta?ctatgaactt?gggtattgat?actattgatt?ttgctacttt?gcatttgtac 780

ccagattctt?ggggtacttc?tgatgattgg?ggtaacggtt?ggatttctgc?tcatggtgct 840

gcttgtaagg?ctgctggtaa?gccatgtttg?ttggaagaat?acggtgttac?ttctaaccat 900

tgttctgttg?aatctccatg?gcaacaaact?gctttgaaca?ctactggtgt?ttctgctgat 960

ttgttttggc?aatacggtga?tgatttgtct?actggtgaat?ctccagatga?tggtaacact 1020

atttactacg?gtacttctga?ttacgaatgt?ttggttactg?atcatgttgc?tgctattgat 1080

tctgcttaa 1089

Claims

1. 'beta '-mannase, its amino acid residue sequence is shown in SEQ ID NO:1.

2. the gene of coding claim 1 described 'beta '-mannase.

3. gene according to claim 2 is characterized in that: the base sequence of described gene is shown in SEQ ID NO:2 or SEQ ID NO:3.

4. contain the described expression carrier of claim 2.

5. the transgenic cell line that contains the described gene of claim 2.

6. the engineering bacteria that contains the described gene of claim 2.

7. engineering bacteria according to claim 6, it is characterized in that: expressing the engineering bacteria of the described 'beta '-mannase of claim 1, is that the yeast expression vector that will contain described 'beta '-mannase mature protein coding sequence imports in the pichia spp and obtains; Described 'beta '-mannase mature protein coding sequence is positioned at the downstream of alpha factor sequence in yeast expression vector; Described 'beta '-mannase mature protein coding sequence is SEQID NO:4 or the SEQ ID NO:5 in the sequence table.

8. engineering bacteria according to claim 7 is characterized in that: being used to make up the described carrier that sets out that contains the yeast expression vector of 'beta '-mannase mature protein coding sequence is pPICz α A, pPICZA or pPIC9K; With pPICz α A is that the set out yeast expression vector of vector construction is pPIC-Mann.

9. engineering bacteria according to claim 7 is characterized in that: the starting strain that is used for the engineering bacteria of the described 'beta '-mannase of construction expression is pichia spp X-33 or GS115; With pichia spp X-33 is that the engineering bacteria that starting strain makes up is X-33/pPIC-Mann.

10. a method of expressing the described 'beta '-mannase of claim 1 is the engineering bacteria of the described expression 'beta '-mannase of fermentation claim 6, obtains 'beta '-mannase.

11. the application of the described 'beta '-mannase of claim 1 in the preparation animal feedstuff additive.