CN100348720C - Mannase and its coding gene and uses - Google Patents
Mannase and its coding gene and uses Download PDFInfo
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- CN100348720C CN100348720C CNB2005101031258A CN200510103125A CN100348720C CN 100348720 C CN100348720 C CN 100348720C CN B2005101031258 A CNB2005101031258 A CN B2005101031258A CN 200510103125 A CN200510103125 A CN 200510103125A CN 100348720 C CN100348720 C CN 100348720C
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Abstract
The present invention discloses mannase, an encoding gene and the application. The mannase is a protein with one of the following amino acid residue sequence: (1)SEQ ID No. 2 in a sequence table; (2) the amino acid residue sequence of the SEQ ID No. 2 which carries out one or a plurality of amino acid residue replacementand/or deletion and/or addition, and has the mannase activity in the sequence table. The mannase of the present invention has high activity, the expression amount of the mannase in the Pichia pastoris MAN22 CGMCC No. 1389 can achieve 200000 IU/mL of fermentation liquor. Compared with the existing mannase, the output is high and the production cost is low. The mannase of the present invention can be used for preparing manna oligosaccharide.
Description
Technical field
The present invention relates to a kind of method and dedicated engineering bacteria thereof of producing mannase.
Background technology
Mannase (β-mannanase, EC 3.2.1.78) is a kind of hemicellulase, can the hydrolysis mannosans, vegetable polysaccharides such as glucomannan, polygalactomannan and gala glucomannan.Have significant application value (Tipdon, et al., Advances in Carbohydrate Chemistry and Biochemistry, 32:299-316,1976 at aspects such as the broken glue of food, medicine, papermaking, washing and petroleum fracturing liquid; Khanongnuch, et al.Biotechnol Lett.21 (1): 61-63,1999; Philippe, etal.EP1059351,2000; Kelly, et al.USP6,197,730,2001).
At present, from difference source biology separate and obtained mannase, as (Braithwaite, et al., Biochem J, 305:1005-1010,1995 such as genus bacillus, gas sporangium, shuttle spore bacterium, fungi and streptomycetes; Duffaud, et al., Appl Environ Microbiol, 63 (1): 169-177,1997; Reese and Shibata, Can J Microbiol, 11:167-183,1965; Akino, etal., ApplMicrobial Biotech, 26:323-327,1987).
About existing patent of mannase and bibliographical information.As: Chinese invention patent 03118984.9 discloses a kind of mannase, and this mannase is expressed enzymic activity and is up to 1030IU/ml on the lab scale level.Enzymic activity in the unit volume fermented liquid is still on the low side, thereby production cost is higher.Chinese invention patent 01128868.X discloses a kind of neutral mannase, and to express enzymic activity be 307IU/ml to this mannase shaking on bottle level; The neutral mannase that produces of Institute of Microorganism, Academia Sinica subtilis (Bacillus subtilis) BMG602 in addition, enzyme activity is 96IU/ml under optimum conditions; The vigor of the alkali mannanase of the Alkalophilic bacillus sp.N16-5 of Institute of Microorganism, Academia Sinica fermentative production is 160IU/ml. in these researchs, perhaps exists enzyme activity not high, instability, perhaps complex process, the high deficiency of cost.
Summary of the invention
An object of the present invention is to provide a kind of active height, the mannase that expression level is high.
Mannase provided by the present invention is β-1, the 4-mannase, and name is called BSMA, derives from subtilis (Bacillus subtilis), is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have the active protein of mannase.
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
Wherein, the sequence in the sequence table 2 is made up of 360 amino-acid residues.From sequence 2 N-terminal the 1st to the 24th amino acids residue be signal peptide sequence, from sequence 2 N-terminal the 25th to the 360th amino acids residue be the maturation protein sequence of BSMA.
This mannosans enzyme molecular weight is 40kda; The pH value scope of enzymic activity is 3.0～10.0, and the optimum pH of enzymic activity is 5.5; The stability boundary of pH value is 2.0～10.0; The temperature range of enzymic activity is 20～80 ℃, and the optimum temperuture of enzymic activity is 30～60 ℃, and this enzyme optimal reactive temperature is 50 ℃; The stability boundary of temperature is 20～80 ℃.
Above-mentioned beta-mannase coding gene (BSMA) also belongs to protection scope of the present invention.
Above-mentioned beta-mannase coding gene can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits;
4) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.
The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
From 5 of sequence 1 ' the 1st to the 72nd deoxynucleotide of end is the encoding sequence of signal peptide, from the encoding sequence that 5 of sequence 1 ' the 73rd at end is maturation proteins of BSMA to 1083 deoxynucleotides.
Contain expression carrier of the present invention (as pQY22), clone and engineering bacteria and all belong to protection scope of the present invention.
Described engineering bacteria is preferably pichia pastoris (Pichia pastoris) MAN22.
Pichia pastoris (Pichiapastoris) MAN22 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 06 16th, 2005, deposit number is CGMCCNo.1389.
Pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 is single celled eukaryote, is oval.Monogony is budding, and pseudohypha is arranged sometimes.Produce ascus during syngenesis, each ascus contains 1-4 piece of smooth circle, carnival hat shape or Saturn shape thecaspore.Pichia pastoris (Pichiapastoris) MAN22 has the physiological and biochemical property of pichia spp, because of inserting beta-mannase gene, has the characteristic that efficiently expresses beta-mannase gene simultaneously.
Another object of the present invention provides a kind of method of expressing mannase BSMA.
The method of expression mannase provided by the present invention may further comprise the steps:
1) before the inoculation, adding mass percentage concentration and be 25% ammoniacal liquor in 10 * Basal Salts makes the pH of substratum reach 5.0, the amount adding PTM1 that adds 4.35mL again by every liter of substratum, insert pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 then, regulating dissolved oxygen electrode is 100%, and the dissolved oxygen that is cultured in the culture system returns to 80%; Described 10 * Basal Salts is grouped into by the one-tenth of following mass percentage concentration: 2.67%85%H
3PO
4, 0.093%CaSO
4, 1.49%MgSO
4, 1.82%K
2SO
4, 0.413%KOH, 0.5% glucose; Described PTM1 is grouped into by the one-tenth of following mass percentage concentration: 0.6%CuSO4,0.008%KI, 0.3%MnSO
4, 0.02%Na
2MoO
4, 0.002% boric acid, 0.05%CoCl
2, 2%ZnCl
2, 6.5%FeSO
4, 0.0025% vitamin H, 0.5%H
2SO
4
2) in the culture system that step 1) obtains stream to add mass percent concentration be 25% D/W, the stream dosage is the 18mL/h/L nutrient solution, every liter of described mass percent concentration is to contain 12mL PTM1 in 25% the D/W; And make dissolved oxygen amount in this system greater than 20%, cultivate 6h;
3) to step 2) in the culture system that obtains stream to add mass percent concentration be 25% glucose and methyl alcohol (volume ratio is 8: 1), the stream dosage is the 9mL/h/L nutrient solution; And make dissolved oxygen amount in this system greater than 20%, cultivate 4h;
(4) add methyl alcohol in the culture system that step 3) obtains, contain 12mL PTM1 in every liter of methyl alcohol, make the methyl alcohol final concentration maintain 0.3%, and make dissolved oxygen amount in this system greater than 20%, inducing culture 12-96h obtains mannase.
Mannosans enzymic activity height of the present invention, the expression amount of mannase can reach the 200000IU/mL fermented liquid among pichia pastoris (Pichia pastoris) the MAN22 CGMCCNo.1389, and than the output height of existing mannase, production cost is low.Mannase of the present invention can be used to prepare mannooligo saccharide.
Description of drawings
Fig. 1 is containing the transparent circle that forms with congo red staining after the enzymolysis on the flat board of Rhizoma amorphophalli powder for the outer supernatant liquor of the born of the same parents of Bacillus subtilis22
Fig. 2 is the physical map of recombinant yeast expression vector pQY22
Fig. 3 is that the PCR of pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 identifies
Fig. 4 is the SDS-PAGE of different induction time institute cumulative mannase in the 5L fermentor tank
Fig. 5 produces the enzyme curve for pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 in the 5L fermentor tank induces
Embodiment
Material
1) bacterial strain and plasmid intestinal bacteria (Escherichia coli) DH5 α bacterial strain and plasmid pGEM-TVector are available from Promega company; Pichia spp Pichia Pastoris GS115 and pPIC9K secreted expression carrier are available from Invitrogen company;
2) enzyme and test kit restriction enzyme, Taq enzyme, Pyrobest archaeal dna polymerase, T
4Toolenzymes such as dna ligase are purchased TaKaRa company; The DNA purification kit is available from Shen, Shanghai energy lottery industry Bioisystech Co., Ltd;
3) biochemical reagents G418 is available from Invitrogen company; Protein molecular weight standard is available from Shanghai biochemical research institute; IPTG, X-Gal, SDS and time locust bean gum are the Sigma product.TEMED, ammonium persulphate, acrylamide, methylene diacrylamide are the Promega product.
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
The acquisition of embodiment 1, mannase BSMA and encoding gene thereof
1, the screening of the bacterial strain of mannase
Strains tested comprises: subtilis (Bacillus subtilis) AS1.1849, Bacillusstearother-mophilus (thermophilic fat genus bacillus), Pseudomonas fluorescens (Pseudomonas fluorescens), Azotobacter chroococum (blown-ball Azotobacter), Micrococcus luteus (micrococcus luteus), and all available from Chinese common micro-organisms culture presevation administrative center.Picking strains tested list colony inoculation contains liquid inducing culture (0.5% Rhizoma amorphophalli powder, the 0.5%NH of Rhizoma amorphophalli powder in 5mL
4NO
3, 0.05%NaCl, 0.05%MgSO
4.0.03%MnSO
4, 0.03%FeSO
4, pH7.0) in, cultivated 2 days for 30 ℃, centrifuging and taking supernatant liquor 100 μ L add agar plate (0.5% Rhizoma amorphophalli powder, the 0.5%NH that contains 0.5% Rhizoma amorphophalli powder
4NO
3, 0.05%NaCl, 0.05%MgSO
4, 0.03%MnSO
4, 0.03%FeSO
4, 2% agar, in hole pH7.0), cultivate 1h for 50 ℃, the congo red staining 1h with 0.1% is again with the 1mol/L NaCl 40min that decolours.Observe the transparent circle (Fig. 1) that produces.Picking produces the supernatant of the bacterial strain of transparent circle and carries out enzyme activity determination: according to Akino (Agr.Biol.Chem.1988,52,773-779) method is accurately diluted enzyme liquid with 0.05mol/L Sodium phosphate dibasic-0.025mol/L citrate buffer solution (pH5.0); Add 0.5% locust bean gum solution of 1.8ml to test tube, put 50 ℃ of water bath heat preservation 5min, add 0.2ml dilution enzyme sample respectively, mixing is put in 50 ℃ of water-baths; Accurately be incubated 5min; Add 1.5ml DNS (3, the 5-dinitrosalicylic acid) reagent respectively, heating is 5 minutes in boiling water bath, and cool to room temperature immediately after the taking-up adds 21.5 ml distilled waters to every pipe again, shakes up; Survey the OD value in 520nm wavelength place; Blank enzyme liquid uses the inactivator sample, and original enzyme liquid after 10 minutes, is done same dilution operation with enzyme liquid to be measured 100 ℃ of heating.With reference to Akino (Agr.Biol.Chem.1988,52,773-779) method, enzyme unit (IU) that lives is defined as: the per minute hydrolysis substrate produces the enzyme amount that 1 μ g is equivalent to the reducing sugar of seminose and is defined as a mannosans unit of enzyme.The highest enzyme that measures is with this understanding lived and is 32IU/ml, and the bacterial strain of its generation is through being accredited as subtilis (Bacillus subtilis) AS1.1849 (available from Chinese common micro-organisms culture presevation administrative center).
2, the acquisition of mannase BSMA and encoding gene thereof
The CTAB method is taked in the extraction of subtilis (Bacillus subtilis) AS1.1849 genomic dna: 10 hours nutrient solutions of 100ml subtilis (Bacillus subtilis) AS1.1849, add 9.5mlTE suspension precipitation, and add 0.5ml 10%SDS, 50 μ l 20mg/ml (or 1mg dry powder) Proteinase Ks, mixing, 37 ℃ are incubated 1 hour; Add 1.5ml 5mol/L NaCl, mixing; Add 1.5ml 10%CTAB solution (with the preparation of 0.7mol/LNaCl solution) again, mixing, 65 ℃ are incubated 20 minutes; Use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, centrifugal 10 minutes of 5000rpm moves to clean centrifuge tube with supernatant liquor; Use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, supernatant liquor adds 1 times of volume Virahol, deposit D NA.
According to the different mannase gene sequence of having reported that derives from subtilis, design synthetic pcr primer thing MnF and MnR:
MnF：5’
GAATTCATGCTTAAAAAGTTAGCAGTCTGC?3’
MnR：5’TT
GCGGCCGCAAACCGATTTTCAAAAGAA?3’
These two sections design of primers all design outside mannase structure gene, and upstream primer has designed the EcoRI restriction enzyme site, and downstream primer has designed the NotI restriction enzyme site.The PCR reaction system is 50 μ L, contains 50ng subtilis (Bacillus subtilis) AS1.1849 genomic dna, 0.2m mol/L dNTP, every kind of primer of 25pmol, 1 * PCR reaction buffer, 2.5 U Deep Vent archaeal dna polymerases.Reaction conditions is: behind 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 1min, carry out 35 circulations; Extend 7min at 72 ℃ then.The dna fragmentation of the about 1.0Kb that amplifies reclaims test kit through DNA and reclaims, is connected on the pGEM-T Vector, and transformed into escherichia coli DH5 α, screening obtains positive colony, obtains recombinant plasmid pQY-1.By the DNA complete sequence analysis, obtained the global DNA sequence (sequence 1) of mannase gene (BSMA), 1083 Nucleotide of total length, coding has the mannase BSMA of the amino acid residue sequence of sequence 2 in the sequence table.Structure prediction according to signal peptide sequence, 24 amino acid of N end (from the 1st of the aminoterminal of sequence 2 to the 24th amino acids residue) are signal peptide, having typical signal peptide structure, is a hydrophobic core area behind N two hydrophilic amino acid of end (lys), and the C end is Ala.The cleavage site of signal peptide is between 25 His of the 24th Ala to the of aminoterminal.The molecular weight of mannase BSMA maturation protein is about 36960 Dal.
The expression of embodiment 2, mannase BSMA
1, the structure of Yeast expression carrier pQY22
For make BSMA can be in yeast heterogenous expression smoothly, removed signal coding sequence among the BSMA, concrete grammar is synthetic one 29 the oligonucleotide fragment MnM (5 ' CG of the nucleotide sequence after the contrast signal peptide-coding sequence
GAATTCCACACCGTTTATCCCGTCAAC 3 '),, increases the EcoRI restriction enzyme site, the downstream primer MnR of embodiment 1 as upstream primer.The method of PCR reaction is with example 1.The dna fragmentation of the about 1.0Kb that amplifies reclaims test kit through DNA and reclaims, is connected on the pGEM-T Vector, and transformed into escherichia coli DH5 α, screening obtains positive colony, obtains recombinant plasmid pQY-2.
BSMA is inserted into the signal peptide sequence downstream of expression vector pPIC9k (Invitrogen), form correct reading frame with signal peptide, make the goal gene stable integration to yeast chromosomal by the homologous recombination between carrier and the yeast chromosomal genome then.Concrete process is: with the BSMA gene from plasmid pQY-2 through EcoRI, insert between the multiple clone site EcoRI and NotI recognition site of yeast expression vector pPIC9K behind the NotI double digestion, and be positioned at the downstream of α-factor signal peptide sequence, obtain recombinant plasmid pQY22, pQY22 is checked order, sequencing result shows that pQY22 contains the mannase BSMA maturation protein coding gene sequence from 5 of sequence 1 ' 1083 deoxynucleotides of the 73rd deoxynucleotide to the of end, and the signal peptide of this mannase BSMA maturation protein coding gene sequence and pPIC9k (Invitrogen) forms correct open reading frame (Fig. 2).The goal gene that so just will have secretion signal has been cloned into AOX1 promotor downstream.
2, the acquisition of yeast conversion and engineering bacteria pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389
The DNA of plasmid pQY22 is behind the PEG transformed yeast cell, and by recombinating in the body, goal gene can be incorporated in the acceptor yeast genes group.Under the condition that exogenous induction material methyl alcohol exists, the AOX1 promotor can start the expression of its downstream gene, and signal peptide can instruct expression product to enter the zymic Secretory Pathway, through cutting, the foreign protein product is finally secreted to born of the same parents, and mannosans enzyme amino acid sequence that is produced and naturally occurring ripe mannase are identical.Foreign protein can carry out posttranslational modification through such pathways metabolism, for example glycosylation, forms disulfide linkage, thereby obtains the protein product of biologically active.
At first cut the DNA of pQY22 with 2～3 times of excessive restriction endonuclease BglII enzymes, whether the electrophoresis detection enzyme is cut complete, makes it linearizing.Use phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, ethanol sedimentation, the 70% ethanol desalinization of soil by flooding or leaching, dissolve with sterilized water dry back, and-20 ℃ of preservations are standby.
Pichia pastoris bacterial strain GS115 is inoculated in 10mL YPD (1% yeast extract, 2% peptone, 2% glucose, regulating pH is 7.0) in 30 ℃ cultivated 8 hours, get culture and be transferred to fresh 10mL YPD (1% yeast extract, 2% peptone, 2% glucose, regulating pH is 7.0) in, make its OD value between 0.1-0.2, continue 30 ℃ of shaking culture up to the OD value between 0.6-1.0.(Sorbitol Solution USP also contains polyoxyethylene glycol and DMSO to centrifugal thalline with the 10mL solution I, provide by the Pichia Easy CompTMTransformation kit of Invitrogen company) resuspended, again thalline is resuspended in the above-mentioned 1mL solution I after centrifugal, be distributed into 50 μ L-200 μ L/ pipe, yeast competent cell preparation finishes, be stored in-80 ℃ standby.
Room temperature is melted 1 pipe competent cell and is added the linearizing pQY22 plasmid of 3 μ g, and (PEG solution is by the Pichia Easy Comp of Invitrogen company to add the 1mL solution II
TMTransformation kit provides) mixing, to hatch 1 hour in 30 ℃, per 15 minutes vibration mixings are once.
42 ℃ of heat shocks are centrifugal after 10 minutes, and (salts solution is by the Pichia Easy Comp of Invitrogen company with the 10mL solution III for thalline
TMTransformation kit provides) mixing) resuspended, use the resuspended thalline of 150 these solution of μ L after centrifugal again, coated plate is in RDB solid medium (18.6% sorbyl alcohol, 1.34%YNB, 2% glucose, 0.00004% vitamin H, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, agar powder 2%, regulating the pH value is 7.0) on, cultivated 2-4 days, and occurred for 30 ℃ until transformant.Transformant can owing to do not have the yeast replicon in the carrier, could be expressed so the His4 gene must be integrated in the yeast genes group in minimum medium (not containing Histidine) growth.In addition, because the AOX1 gene is damaged in the transformed yeast cells, so it just can not utilize methyl alcohol as carbon source again.Like this with methyl alcohol as the substratum of sole carbon source on transformant just can not grow (perhaps growth is extremely slow), show as methyl alcohol and utilize defective type (Mut
-).In addition, be incorporated in the yeast genes group, given the ability of the anti-G418 of yeast, in containing the substratum of G418 so transformant can be grown owing to have the gene of coding resistance in the carrier.
Go up the His of picking growth with aseptic toothpick from transforming flat board (RDB solid medium)
+Recon, at first be inoculated into MM solid medium (1.34%YNB, 0.5% methyl alcohol, 0.00004% vitamin H, 2% agar powder, regulating the pH value is 7.0) on, inoculate MD solid medium (1.34%YNB (yeast nitrogen), 2% glucose, 0.00004% vitamin H, 2% agar powder, regulating the pH value is 7.0) on, picking His like this
+Recon was cultivated 2 days for 30 ℃.Searching is at clone's normal but that some growth is arranged on the MM flat board or do not grow fully of growing on the MD flat board.
In order to screen the restructuring yeast strains that obtains high expression level, again with His
+Mut
-Transformant is put plate respectively on the YPD solid plate that contains 500 μ g/mL, 1000 μ g/mL, 2000 μ g/mL G418 (1% yeast extract, 2% peptone, 2% glucose, 2% agar powder, regulating the pH value is 7.0).Select the 6 strain recombinant bacterial strains of on the YPD solid plate of 2000 μ g/mL G418, growing and carry out abduction delivering.At first at BMGY substratum (1% glycerine, 1% yeast extract, 2% peptone, 1.34% yeast nitrogen (YNB), 0.00004% vitamin H, 1% glycerine, regulating pH is 6.0) (is carbon source with glycerine) middle cultivation, treat that it grows into state of saturation, remove BMGY, change to inducing culture BMMY (1% yeast extract, 2% peptone, 1.34% yeast nitrogen (YNB), 0.00004% vitamin H, 0.5% methyl alcohol, pH6.0) (with methyl alcohol as inductor) reached supernatant in 36 hours and carries out the mannosans enzyme activity assay inducing.Enzyme assay by the expression mannase screens the highest recombinant bacterial strain of a strain enzyme activity (200000IU/ml nutrient solution), names to be MAN22.Pichia pastoris (Pichiapastoris) MAN22 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 06 16th, 2005, deposit number is CGMCC No.1389.
3, confirming reorganization and the translation of mannase gene in yeast on the molecular level
The methyl alcohol of restructuring yeast strains utilizes defective type explanation foreign gene accurately to be incorporated into AOX1 gene locus in the yeast genes group, thereby has destroyed the function of this gene.Also confirmed this point by Molecular Detection.Extract pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 genomic dna, concrete grammar is to get the centrifugal collection thalline of cultivating 8 hours of 5mL yeast cell, the washing of 1mL sterilized water is centrifugal, and precipitation adds 200 μ L broken bacterium damping fluid (SDS1%, Triton X-100 2%, NaCl 100mM, Tris-Cl (pH8.0) 10mM, EDTA 1mM), thermal agitation adds 200 μ L phenol/chloroforms/primary isoamyl alcohol (25: 24: 1) again, the 1-2min that vibrates at a high speed on vortex gets supernatant.The dehydrated alcohol precipitation, the centrifugal drying genomic dna.With pichia pastoris (Pichiapastoris) MAN22 CGMCC No.1389 genomic dna is template, utilize primer MnM and MnR pcr amplification to remove signal coding sequence among the BSMA, wherein, the PCR reaction system is 50 μ L, contain the 50ng genomic dna, 0.2m mol/LdNTP, 25 pmolMnM and MnR primer, 1 * PCR reaction buffer, the 2.5UTaq archaeal dna polymerase.Reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min again, 57 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 35 circulations; Extend 7min at 72 ℃ then.PCR result as shown in Figure 3, PCR obtains the dna fragmentation of 1.0Kb, shows to have removed that signal coding sequence has been incorporated in the recombination yeast genome among the BSMA.Among Fig. 3, swimming lane 1 is a dna molecular amount standard, and swimming lane 2,3,4 is respectively pichia pastoris GS115, pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 and plasmid pQY22 is the PCR product of template.
The inducing culture liquid of getting 10 μ L and do not have thalline carries out SDS-PAGE and analyzes (acrylamide: methylene diacrylamide is 30: 1).Used separation gel is 123.5%, and concentrated glue is 4%.Electrophoresis finishes the back with Kao Masi light blue R250 dyeing 30 minutes, then decolours with 10% glacial acetic acid.The result shows that the molecular weight of the mannase of expression is about 40kD, conform to theoretical value, and the mannase of expressing does not carry out glycosylation modified.
The property analysis of the mannase that embodiment 3, pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 express
The mannase that pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 is expressed is incubated 30min for 50 ℃ at the damping fluid (pH2.0-7.0 Sodium phosphate dibasic-citrate buffer solution, pH8.0-9.0 Tris-hydrochloride buffer, pH9.0-11.0 glycine-sodium hydrate buffer solution) of different pH values, detect remaining enzyme activity, the result shows that the pH value scope that this enzyme has an enzymic activity is 3.0-10.0, still have the enzyme activity more than 70% behind insulation 30min under the condition of pH5.0-10.0, the enzymic activity optimal pH is 5.5; The stability boundary of enzyme pH value is 2.0-10.0.
Under condition of different temperatures, measure the enzyme activity of the mannase of pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 expression, the result shows that the temperature range of enzymic activity is 20-80 ℃, the optimum temperature range of enzymic activity is 30-60 ℃, this enzyme optimal reactive temperature is 50 ℃, and this mannase still has about enzyme activity more than 40% behind the insulation 30min under the condition of 60 ℃ and 70 ℃; The stability boundary of temperature is 20-80 ℃.
Utilize pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389 at 5 liters of fermentor tank middle-high density fermentative production mannases.Fermenting process is divided into four-stage.Specific as follows: (1) strain culturing stage.Before the inoculation, at 2.5 liters of 10 * Basal Salts (2.67%85%H
3PO
4, 0.093%CaSO
4, 1.49% MgSO
4, 1.82%K
2SO
4, 0.413%KOH, 0.5% glucose) in to add mass percentage concentration be that 25% ammoniacal liquor makes the pH of substratum reach 5.0 (ammoniacal liquor is simultaneously also as the nitrogenous source of strain growth), the amount that adds 4.35mL by every liter of substratum adds PTM1 (0.6%CuSO4,0.008%KI, 0.3%MnSO again
4, 0.02%Na
2MoO
4, 0.002% boric acid, 0.05%CoCl
2, 2%ZnCl
2, 6.5%FeSO
4, 0.0025% vitamin H, 0.5%H
2SO
4).By 8% (volume ratio) inoculation seed liquor, regulate dissolved oxygen electrode, dissolved oxygen is set to 100%, and 16h is cultivated in aeration-agitation, and along with the growth of bacterial strain, the dissolved oxygen amount in the substratum reduces gradually by 100% in culturing process.Dissolved oxygen amount can raise once again after carbon source runs out of, and when dissolved oxygen is increased to 80% when above, the beginning carbon source is fed the stage.(2) carbon source is fed the stage.Stream adds 25% D/W (containing 12mLPTM1 in every liter), and the stream dosage is the 18mL/h/L nutrient solution, cultivates 6h.Adjusting air flow makes dissolved oxygen amount all the time greater than 20%.(3) the carbon source-methanol mixing is fed the stage.Stream adds 25% D/W: methyl alcohol (volume ratio is 8: 1) is cultivated 4h, and the stream dosage is 9mL/h/L, and the control dissolved oxygen amount is all the time greater than 20%.(4) the abduction delivering stage.Add inductor methyl alcohol (containing 12mL PTM1 in every liter), make the methyl alcohol final concentration maintain 0.3%, dissolved oxygen amount is all the time greater than 20%.The SDS-PAGE that every 12h takes a sample and once measures the accumulation volume of the seminase of expressing and carry out expressing protein in inducing process.SDS-PAGE result as shown in Figure 4, the enzyme activity determination curve of mannase as shown in Figure 5, the mannase that shows expression accumulates with the increase of induction time, peaks in 96 hours inducing, at this moment biomass reaches the 220g/L fermented liquid, and enzymic activity reaches 2.0 * 10
5The IU/ml fermented liquid, the zymoprotein molecular weight size of expression is about 40kD (arrow shows among Fig. 4).Above result proves that mannase gene has not only obtained expression, effectively secretion, and the mannase of expressing has normal biologic activity.Among Fig. 4, swimming lane 1-8 is respectively the electrophoresis result of inducing 12,24,36,48,60,72,96,0 hours, and 9 is the standard protein molecule.Fermented liquid after the fermentation ends with the absorption of 2kg corn cob, is made mannase BSMA solid phase prod behind the Air drying.
Adding in the konjaku powder is the water of 4 times of quality of konjaku powder, heated and boiled is fully dissolved powder, be cooled to 40 ℃, adding respectively is the mannase BSMA that obtains among 2%, 5% and 8% the embodiment 4 of konjaku powder quality, stir, reacted respectively 48,27,10 hours, sampling detects sugar and forms.The result shows that adding is 2% BSMA of konjaku powder quality, reacts 48 hours, and 2-20 glycosyl oligosaccharides accounts for total reducing sugar 80%; Adding is 5% BSMA of konjaku powder quality, reacts 27 hours, and 2-20 glycosyl oligosaccharides accounts for total reducing sugar 82%; Adding is 8% BSMA of konjaku powder quality, reacts 10 hours, and 2-20 glycosyl oligosaccharides accounts for total reducing sugar 85%.The reaction solution drying is made mannooligo saccharide.Made oligosaccharides can be used for animal-feed, biological pesticide etc.
Adding in the sesbania gum is the water of sesbania gum 4-8 times quality, and heated and boiled is fully dissolved glue, is cooled to 60 ℃, adding respectively is the mannase BSMA that obtains among 2%, 4% the embodiment 4 of sesbania gum quality, stir, reacted 48,24 hours, sampling detects sugar and forms.The result shows that adding is 2% BSMA of sesbania gum quality, reacts 48 hours, and 2-20 glycosyl oligosaccharides accounts for total reducing sugar 82%; Adding is 4% BSMA of sesbania gum quality, reacts 24 hours, and 2-20 glycosyl oligosaccharides accounts for total reducing sugar 81%.The reaction solution drying is made mannooligo saccharide.Made oligosaccharides can be used for animal-feed, biological pesticide etc.
Sequence table
<160>2
<210>1
<211>1083
<212>DNA
＜213〉subtilis (Bacillus subtilis)
<400>1
atgcttaaaa?agttagcagt?ctgcctgtct?atcgttttat?tactcttagg?agccgccagt 60
ccgatatcgg?ctcacaccgt?ttatcccgtc?aacccaaatg?cccagcagac?gacaaaagat 120
atcatgaact?ggctggccca?cctgcccaac?cgttcagaaa?acagggtcat?gtccggagcg 180
ttcggcgggt?acagcgatgt?cactttttca?atgacagagg?aaaaccgctt?gaaaaacgcg 240
acgggacagt?ctcccgccat?ctacggctgt?gactatggga?gagggtggct?ggaaacagcg 300
gatatcaccg?atactatcga?ttactgctgc?aacagcagct?taatctcata?ctggaaaagc 360
ggcggcctcc?ctcaggtcag?cctgcatctc?gcaaatccgg?cctttccatc?cggaaactat 420
aaaacggcca?tctcaaacag?ccagtacaaa?aacatccttg?acccttcaac?tgtggaagga 480
aaacggcttg?aggcgctgct?cagcaaaatt?gccgacggcc?ttactcagct?gaaaaatcaa 540
ggcgtcaccg?ttctgttcag?accgctgcat?gaaatgaacg?gcgagtggtt?ctggtggggg 600
ctgacaggct?acaaccaaaa?agacaatgag?agaatctcgc?tgtacaaaga?gctttacaag 660
aaaatatacc?gctatatgac?agaaacaaga?ggattggata?accttttgtg?ggtgtattcg 720
ccggatgcca?acagagactt?taaaacagac?ttctacccag?gctcatctta?tgtagatatt 780
accggtctgg?acgcttactt?cactgatccg?tatgcgatat?caggctatga?tgaaatgctg 840
tctctgaaaa?aaccgtttgc?ctttgccgaa?accggtccgt?ccggcaatat?cggaagcttt 900
gattatgctg?cttttattaa?tgcgatcagg?caaaaatacc?ctcagaccac?gtactttttg 960
acatgggatg?aacaattaag?tccggcggcc?aatcaaggcg?cgcaaagcct?ttatcaaaac 1020
agctggacgc?tgaacaaggg?cgaaatatgg?aacggcgggt?ccttgacgcc?gatcgcggaa 1080
taa 1083
<210>2
<211>360
<212>PRT
＜213〉subtilis (Bacillus subtilis)
<400>2
Met?Leu?Lys?Lys?Leu?Ala?Val?Cys?Leu?Ser?Ile?Val?Leu?Leu?Leu?Leu
1 5 10 15
Gly?Ala?Ala?Ser?Pro?Ile?Ser?Ala?His?Thr?Val?Tyr?Pro?Val?Asn?Pro
20 25 30
Asn?Ala?Gln?Gln?Thr?Thr?Lys?Asp?Ile?Met?Asn?Trp?Leu?Ala?His?Leu
35 40 45
Pro?Asn?Arg?Ser?Glu?Asn?Arg?Val?Met?Ser?Gly?Ala?Phe?Gly?Gly?Tyr
50 55 60
Ser?Asp?Val?Thr?Phe?Ser?Met?Thr?Glu?Glu?Asn?Arg?Leu?Lys?Asn?Ala
65 70 75 80
Thr?Gly?Gln?Ser?Pro?Ala?Ile?Tyr?Gly?Cys?Asp?Tyr?Gly?Arg?Gly?Trp
85 90 95
Leu?Glu?Thr?Ala?Asp?Ile?Thr?Asp?Thr?Ile?Asp?Tyr?Cys?Cys?Asn?Ser
100 105 110
Ser?Leu?Ile?Ser?Tyr?Trp?Lys?Ser?Gly?Gly?Leu?Pro?Gln?Val?Ser?Leu
115 120 125
His?Leu?Ala?Asn?Pro?Ala?Phe?Pro?Ser?Gly?Asn?Tyr?Lys?Thr?Ala?Ile
130 135 140
Ser?Asn?Ser?Gln?Tyr?Lys?Asn?Ile?Leu?Asp?Pro?Ser?Thr?Val?Glu?Gly
145 150 155 160
Lys?Arg?Leu?Glu?Ala?Leu?Leu?Ser?Lys?Ile?Ala?Asp?Gly?Leu?Thr?Gln
165 170 175
Leu?Lys?Asn?Gln?Gly?Val?Thr?Val?Leu?Phe?Arg?Pro?Leu?His?Glu?Met
180 185 190
Asn?Gly?Glu?Trp?Phe?Trp?Trp?Gly?Leu?Thr?Gly?Tyr?Asn?Gln?Lys?Asp
195 200 205
Asn?Glu?Arg?Ile?Ser?Leu?Tyr?Lys?Glu?Leu?Tyr?Lys?Lys?Ile?Tyr?Arg
210 215 220
Tyr?Met?Thr?Glu?Thr?Arg?Gly?Leu?Asp?Asn?Leu?Leu?Trp?Val?Tyr?Ser
225 230 235 240
Pro?Asp?Ala?Asn?Arg?Asp?Phe?Lys?Thr?Asp?Phe?Tyr?Pro?Gly?Ser?Ser
245 250 255
Tyr?Val?Asp?Ile?Thr?Gly?Leu?Asp?Ala?Tyr?Phe?Thr?Asp?Pro?Tyr?Ala
260 265 270
Ile?Ser?Gly?Tyr?Asp?Glu?Met?Leu?Ser?Leu?Lys?Lys?Pro?Phe?Ala?Phe
275 280 285
Ala?Glu?Thr?Gly?Pro?Ser?Gly?Asn?Ile?Gly?Ser?Phe?Asp?Tyr?Ala?Ala
290 295 300
Phe?Ile?Asn?Ala?Ile?Arg?Gln?Lys?Tyr?Pro?Gln?Thr?Thr?Tyr?Phe?Leu
305 310 315 320
Thr?Trp?Asp?Glu?Gln?Leu?Ser?Pro?Ala?Ala?Asn?Gln?Gly?Ala?Gln?Ser
325 330 335
Leu?Tyr?Gln?Asn?Ser?Trp?Thr?Leu?Asn?Lys?Gly?Glu?Ile?Trp?Asn?Gly
340 345 350
Gly?Ser?Leu?Thr?Pro?Ile?Ala?Glu
355 360
Claims (2)
1, pichia pastoris (Pichia pastoris) MAN22 CGMCC No.1389.
2, a kind of method of utilizing the described pichia pastoris of claim 1 (Pichia pastoris) MAN22 to produce mannase may further comprise the steps:
1) before the inoculation, adding mass percentage concentration and be 25% ammoniacal liquor in 10 * Basal Salts makes the pH of substratum reach 5.0, the amount adding PTM1 that adds 4.35mL again by every liter of substratum, insert pichia pastoris (Pichia pastoris) MAN22 then, the dissolved oxygen that is cultured in the culture system returns to 80%; Described 10 * Basal Salts is grouped into by the one-tenth of following mass percentage concentration: 2.67%85%H
3PO
4, 0.093% CaSO
4, 1.49% MgSO
4, 1.82% K
2SO
4, 0.413% KOH, 0.5% glucose; Described PTM1 is grouped into by the one-tenth of following mass percentage concentration: 0.6% CuSO
4, 0.008% KI, 0.3% MnSO
4, 0.02% Na
2MoO
4, 0.002% boric acid, 0.05% CoCl
2, 2% ZnCl
2, 6.5% FeSO
4, 0.0025% vitamin H, 0.5% H
2SO
4
2) in the culture system that step 1) obtains stream to add mass percent concentration be 25% D/W, the stream dosage is the 18mL/h/L nutrient solution, every liter of described mass percent concentration is to contain 12mL PTM1 in 25% the D/W; And make dissolved oxygen amount in this system greater than 20%, cultivate 6h;
3) to step 2) in the culture system that obtains stream to add volume ratio be that 8: 1 mass percent concentration is 25% D/W and methyl alcohol, the stream dosage is the 9mL/h/L nutrient solution; And make dissolved oxygen amount in this system greater than 20%, cultivate 4h;
4) add methyl alcohol in the culture system that step 3) obtains, contain 12mL PTM1 in every liter of methyl alcohol, make the methyl alcohol final concentration maintain 0.3%, and make dissolved oxygen amount in this system greater than 20%, inducing culture 12-96h obtains mannase.
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| CN102943068A (en) * | 2012-09-05 | 2013-02-27 | 周海燕 | Mannase Man23 and gene modification thereof |
| CN103725621B (en) * | 2013-11-27 | 2016-02-03 | 青岛蔚蓝生物集团有限公司 | A kind of engineering strain of recombinant expressed mannase |
| CN104726481A (en) * | 2015-02-27 | 2015-06-24 | 中国农业科学院油料作物研究所 | Super-high-temperature-resistant and alkali-resistant beta-mannase gene, amino acid sequence and application of beta-mannase gene |
| CN109536395A (en) * | 2018-02-07 | 2019-03-29 | 四川润格生物科技有限公司 | It is a kind of it is high expression 'beta '-mannase pichia pastoris engineered strain and application |
| CN112029751B (en) * | 2019-06-03 | 2022-05-03 | 中国农业大学 | Production method and application of thermophilic fungus mannase |
| CN115161336A (en) * | 2022-08-02 | 2022-10-11 | 青岛蔚蓝生物集团有限公司 | Pichia pastoris strain and application thereof in production of mannanase |
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Non-Patent Citations (3)
| Title |
|---|
| Cloning and sequencing of beta-mannanase gene fromBacillus subtilis NM-39. Mendoza,N.S. 等.NCBI:D37964. 2000 * |
| Ooi,T.NCBI:AB005755. 1997;CLONING AND SEQUENCING OF BETA-MANNANASEGENE FROM BACILLUS SUBTILISNM-39 MENDOZA,N.S等.BIOCHIM.BIOPHYS.ACTA,Vol.1243 No.3. 1995 * |
| 毕赤酵母表达***研究进展 齐连权等.中国生物工程杂志，第22卷第6期 2002 * |
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