CN101544954B - Recombinant pichia pastoris expressing non-starch polysaccharides complex enzymes and application thereof - Google Patents
Recombinant pichia pastoris expressing non-starch polysaccharides complex enzymes and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant pichia pastoris expressing non-starch polysaccharides complex enzymes and application thereof. The recombinant bacterium is formed by leading xylanase gene, cellulase gene and beta-mannanase gene into pichia pastoris. The recombinant bacterium can be used for producing the non-starch polysaccharides complex enzymes; and the yield of the xylanase gene, the cellulase gene and the beta-mannanase gene expressed by the recombinant bacterium can reach 5,012 U/ml, 4,109 U/ml and 2,017 U/ml respectively. The recombinant bacterium can be used for producing feed. The recombinant pichia pastoris has low nutrition requirement on a reactor, quick growth, low price of culture medium and very little protein secreted by self, is very favorable for purifying the complex enzymes, contributes to industrial production, and provides a novel method for producing the non-starch polysaccharides complex enzymes.
Description
Technical field
The present invention relates to a kind of reorganization bacterium and application thereof of expressing the non-starch polysaccharide prozyme, particularly a kind of reorganization pichia pastoris bacterium and application thereof of expressing the non-starch polysaccharide prozyme.
Background technology
Non-starch polysaccharide (NSP) is to be formed by connecting through glycosidic link by multiple monose and uronic acid in the plant tissue, the ramose chain-like structure is arranged mostly, normal and mineral ion is in the same place with protein bound, is the main component of cell walls, generally is difficult to be decomposed by simple stomach animal self excretory digestive ferment.
Many achievements in research show, add the NSP zymin in the feed, by the hydrolysis to antinutritional factor-NSP in the feed, can reduce animal gastrointestinal tract chyme viscosity, eliminate its anti-oxidant action, improve feed nutritive value, improve breeding performonce fo animals.Therefore for efficiently, eliminate the anti-oxidant action of NSP in the daily ration to greatest extent, improve the feed digestive utilization ratio, in feed, add application NSP prozyme and just seem very important, meanwhile can also give full play to different N SP enzyme and tie up to synergistic effect on the degradation of substrates, improve the biological value of fodder enzyme preparation.
The mode of production of current feeding non-starch polysaccharide compound enzymic preparation mainly contains following three kinds:
1. complete method of double crossing: refer to produce or buy various single enzyme preparations, carry out rational proportion, add certain dispersion agent, thinner again and through mixing according to the zymogram formula rate.This is the early stage method that generally adopts of producing of complex enzyme for feed.Method of double crossing requires the quality of each single enzyme preparation all reliable fully, should carry out in strict accordance with the zymogram prescription when each single enzyme is composite simultaneously.This method shortcoming is more, makes the appropriate design difficulty comparatively of complex enzyme for feed zymogram prescription as the shortage of basic data, and the expensive of the pure zymin of extensive stock makes that the cost of composite back complex enzyme for feed is higher; In addition, fully the complex enzyme for feed produced of method of double crossing is difficult to obtain to dezymotize some UGFs etc. of the promoted growth of animal beyond the function.
2. single strain fermentation method: at the occurring in nature screening bacterial strain of the multiple non-starch polysaccharide enzyme of secreting, expressing simultaneously, and utilize the method for various mutagenesis to obtain to meet the good superior strain that prescription requires, the thick enzyme of producing gained through fermentation is compound enzymic preparation.Yet most of bacterial strains can be when Secretases according to substratum in the composition of substrate carry out Secretases selectively, so the compound enzymic preparation that utilizes this method acquisition is in fact only based on one or both zymins, and the secretory volume of other corresponding zymin is less and active very low.The source of present industrial prozyme mainly is aerobic filamentous fungus Trichoderma and Aspergillus, has multiple mycotoxins in the Trichoderma tunning, toxic suspicion, and the difficult control of filamentous fungus fermentation, and bacterial classification is easily degenerated.
3. many bacterial strains mixed fermentation: with each bacterial strain combined inoculation, one time fermentation forms and meets the compound enzymic preparation product that prescription requires.Feasible on this law theory, but condition and the demand because of the different strain enzymatic production is different in the actual production operating process, and mutual to each other antagonism is so this technology application difficulty is very big.
Summary of the invention
The object of the present invention is to provide reorganization pichia pastoris bacterium and the application thereof of expressing the non-starch polysaccharide prozyme.
Reorganization bacterium of the present invention is the reorganization pichia pastoris bacterium that contains xylanase gene, cellulose enzyme gene and beta-mannase gene.
The encoding sequence of above-mentioned xylanase gene is from the Genbank number nucleotide sequence for 5 of AJ292317 ' end 1-576 position; The nucleotide sequence of above-mentioned cellulose enzyme gene is from the Genbank number nucleotide sequence for 5 of EU022559 ' end 88-1500 position; The nucleotide sequence of above-mentioned beta-mannase gene is from the Genbank number nucleotide sequence for 5 of AY623903 ' end 122-1051 position.
Wherein, described xylanase gene imports in the described pichia pastoris bacterium by recombinant expression vector a, constitute reorganization pichia pastoris bacterium A, this recombinant expression vector a is the recombinant expression vector that described xylanase gene is inserted into the expressed xylanase that obtains among the expression vector pPIC9.
Described cellulose enzyme gene imports A in the described reorganization pichia pastoris bacterium by recombinant expression vector b, constitute reorganization pichia pastoris bacterium B, this recombinant expression vector b is the recombinant expression vector that described cellulose enzyme gene is inserted into the expression cellulase that obtains among the expression vector pPIC9K.
Described beta-mannase gene imports among the described reorganization pichia pastoris bacterium B by recombinant expression vector c and obtains reorganization bacterium of the present invention; Described recombinant expression vector c is the recombinant expression vector that described beta-mannase gene is connected to the expression 'beta '-mannase that obtains on the expression vector pPICZ α A.
Described pichia pastoris bacterium is pichia pastoris bacterium (Pichia pastoris) GS115.
Above-mentioned reorganization bacterium is pichia pastoris (Pichia pastoris) GS115-NSP, be preserved in Chinese microorganism strain preservation board of trustee reason person on 04 16th, 2009 and understood common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3025.
Another object of the present invention is to provide the application of described reorganization bacterium in producing the non-starch polysaccharide prozyme.
Described application is with reorganization bacterium of the present invention, at temperature 28-30 ℃, and pH5.0-6.0, dissolved oxygen is 30%-60%, induces fermentation, gives expression to the non-starch polysaccharide prozyme.
Described cultivation substratum is YPD substratum or BMGY substratum; Described BMGY substratum is 1% yeast extract, 2% peptone, 100mmol/L phosphoric acid buffer (pH6.0), 1.34%YNB, 0.00004% vitamin H (Biotin), 1% (volume percent) glycerine; Described YPD substratum is 1% yeast extract, 2% peptone, 2% glucose (except that glycerine, " % " of all the other materials all represents the quality percentage composition)
Described reorganization bacterium was cultivated 30-60 hour in the YPD substratum, preferred 48 hours.
Another purpose of the present invention is to provide the application of described reorganization bacterium in producing feed.
The present invention is by making up different pichia pastoris carrier for expression of eukaryon, the beta-mannase gene of the olive-green streptomycete xylanase gene that the clone is obtained, the cellulose enzyme gene of bacillus amyloliquefaciens and Bacillus circulans the turnover of recombinating is respectively sent out in the bacterial strain, obtain can be simultaneously the engineering strain GS115-NSP of secreting, expressing plurality of enzymes efficiently, wherein the output of zytase can reach 5012U/ml, the output of cellulase can reach 4109U/ml, and the output of 'beta '-mannase can reach 2017U/ml.Reorganization pichia pastoris bioreactor culture nutritional requirement of the present invention is low, growth is fast, substratum is cheap, himself excretory albumen is considerably less, very help the purifying of prozyme, be convenient to suitability for industrialized production, this provides a kind of novel method for producing the non-starch polysaccharide prozyme.
Embodiment
Among the following embodiment, if no special instructions, be ordinary method.
Among the following embodiment, described percentage composition is the quality percentage composition if no special instructions.
The equal water preparation of agents useful for same of the present invention, its solute is made up of the material of following final concentration respectively:
RDB solid medium: 1mol/L sorbyl alcohol, 1% glucose, 1.34%YNB (yeast nitrogen does not contain amino acid (U.S. company BD)), 0.00004% vitamin H, 0.005% L-glutamic acid, 0.005% methionine(Met), 0.005% Methionin, 0.005% leucine, 0.005% Isoleucine, 1.5% agar (above-mentioned " % " all represents the quality percentage composition).
The LB substratum: 0.5% yeast extract, 1.0% peptone, 1.0%NaCl transfers to pH7.0 with NaOH, (solid medium contains 1.5% agar) 121 ℃ of sterilizations 15min (above-mentioned " % " all represents the quality percentage composition).
YPD substratum: 1% yeast extract, 2% peptone, 2% glucose (above-mentioned " % " all represents the quality percentage composition).
MM solid medium: 1.34%YNB, 0.00004% vitamin H (Biotin), 0.5% methyl alcohol, 1.5% agar (above-mentioned " % " all represents the quality percentage composition).
MD solid medium: 1.34%YNB, 0.00004% vitamin H (Biotin), 2% glucose, 1.5% agar (above-mentioned " % " all represents the quality percentage composition).
BMGY substratum: 1% yeast extract, 2% (quality percentage composition) peptone, 100mmol/L phosphoric acid buffer (pH6.0), 1.34% (quality percentage composition) YNB yeast nitrogen, do not contain amino acid (U.S. company BD), 0.00004% (quality percentage composition) vitamin H (Biotin), 1% (volume percent) glycerine.
BMMY methanol induction substratum: replace glycerine divided by 0.5% (volume percent) methyl alcohol, all the other compositions are identical with BMGY.
PTM salt: copper sulfate 6.0g/L, sodium iodide 0.08g/L, manganous sulfate 3.0g/L, Sodium orthomolybdate 0.2g/L, boric acid 0.02g/L, cobalt chloride 0.5g/L, zinc chloride 20g/L, ferrous sulfate 65g/L, vitamin H 0.25g/L, sulfuric acid 5ml/L.
The strain fermentation substratum: phosphoric acid 26.7ml/L, calcium sulfate 0.93g/L, vitriolate of tartar 18.2g/L, bitter salt 14.9g/L, potassium hydroxide 4.13g/L, glucose 50g/L also adds 1.5% (volume percent) PTM salt.
The acquisition of the reorganization bacterium of embodiment 1, expression non-starch polysaccharide prozyme
One, the structure of secreting, expressing zytase pichia pastoris reorganization bacterium GS115-xynb
1, the structure that contains olive-green streptomycete xylanase gene pichia pastoris carrier for expression of eukaryon pPIC9K-xynb
According to the olive-green streptomycete xylanase gene of having delivered on the net, its encoding sequence is to be for Genebank number: the nucleotide sequence of 5 of AJ292317 ' end 1-576 position, this sequence is carried out synthetic (Beijing AudioCodes), synthetic altogether 576 bases, and at synthetic gene fragment 5 ' end interpolation EcoR I restriction enzyme site, its 3 ' end adds Not I restriction enzyme site, and insert between the EcoR I of pichia pastoris expression vector pPIC9 (invitrogen company) and the Not I restriction enzyme site with correct reading frame and to obtain recombinant vectors, with the recombinant vectors called after pPIC9-xynb that obtains.
But the structure of the pichia pastoris engineering bacteria GS115-xynb of 2 expressed xylanase
1) preparation of pichia pastoris yeast GS115 competent cell
(1) single colony inoculation of picking pichia pastoris yeast GS115 is in the 10mlYPD substratum, 30 ℃ of shaking tables 12 hours.
(2) be forwarded to the 100mlYPD liquid nutrient medium with 1% (volume percent) inoculum size, 30 ℃ of shaking tables spend the night to OD
600=1.3-1.5.4 ℃ of centrifugal 5000rpm, 5min abandons supernatant, collecting precipitation (thalline).
(3) thalline that step (2) is obtained is resuspended with thalline with 100ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, and 10min abandons supernatant, collects thalline.Then that thalline is resuspended with thalline with 50ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, 10min abandons supernatant, collecting precipitation (thalline).
(4) step (3) is washed 1 time with the sorbyl alcohol of 20ml 1mol/L again.Be dissolved in the sorbyl alcohol of the ice precooling of 200 μ L 1mol/L then, obtain pichia pastoris yeast GS115 competent cell.
2) goal gene inserts pichia pastoris yeast GS115 competent cell karyomit(e)
Get the recombinant plasmid pPIC9-xynb of 5 μ g, and carry out enzyme with Bgl II and cut 2 hours.The endonuclease reaction system is as follows:
The eukaryotic expression that contains goal gene carries 20 μ l (5 μ g);
Body pPIC9-xynb
10×buffer 5μl；
Bgl?II 5μl；
ddH
2O 20μL；
Cumulative volume 50 μ L.
Add the NaAC of 5ul 3M pH=5.2 and the dehydrated alcohol of 100ul in the system that enzyme is cut ,-20 ℃ left standstill after 15 minutes 10000g centrifugal 1 minute, wash with 75% ethanol, oven dry is with the dissolving of 10ul aqua sterilisa again, obtain linearizing pPIC9-xynb, in order to transforming.
In the yeast competent cell GS115 that 80 μ l step 1) obtain, add the linearizing pPIC9-xynb of 1-5 μ g, placed 15 minutes on ice, add in the ice precooling 0.2cm electric shock cup (MicroPulserBio-Rad 165-2100) rapidly, electric shock transformed yeast competent cell (2.5KV, 5ms), in the electric shock cup, add the ice-cold 1mol/L sorbyl alcohol of 1ml immediately, behind the mixing, be applied on the RDB flat board with every plate 200 μ l bacterium liquid, 30 ℃ are cultured to and grow transformant.
Transformant is inoculated in 3ml BMGY substratum, 30 ℃ of shaking tables of 250rpm are cultivated 48h, the centrifugal 8min of 5000rpm, supernatant discarded is collected thalline, thalline is resuspended with 1ml BMMY methanol induction substratum, continuation centrifugal 8min of 5000rpm behind 28 ℃ of inducing culture 48h gets supernatant, and the enzyme work that supernatant is surveyed zytase as follows identifies that it whether can expressed xylanase, and the height of enzymatic productivity.The experiment triplicate, the measurement result of zytase output is as shown in table 1:
The measurement result (mean value) of table 1. zytase output
| No. 1 bacterial strain | No. 2 bacterial strains | No. 3 bacterial strains | |
| Zytase output | 600U/ml | 750U/ml | 1006U/ml |
The zytase measuring method: the enzyme liquid of drawing the suitable dilution of 0.2ml adds in the test tube, the xylan solution (1g oat xylan is dissolved in 100ml pH5.5 citric acid-disodium hydrogen phosphate buffer solution) that adds 1.8ml 1% again, electromagnetic oscillation 3s, 37 ℃ accurately are incubated 10min.Add 3ml DNS reagent, electromagnetic oscillation 3s boiling water bath heating 5min is cooled to room temperature, measures the reducing sugar amount of its generation.
The xylanase activity unit of force: under 37 ℃ and pH=5.5 condition, the per minute needed enzyme amount of release 1 μ mol reducing sugar of degrade from xylan solution is an enzyme activity unit U.
With enzyme the highest bacterial strain (No. 3) alive, called after GS115-xynb.
But the structure of the pichia pastoris engineering bacteria GS115-xynb-cen of two expressed xylanase and cellulase
1, the clone of bacillus amyloliquefaciens cellulose enzyme gene
According to the bacillus amyloliquefaciens cellulase gene sequence of having delivered on the net, the base of its coded signal peptide is removed, this sequence is the Genbank number nucleotide sequence for 5 of EU022559 ' end 88-1500 position, and designs primer with this, and primer sequence is as follows:
5 ' primer: 5 '-
GAATTCGCAGGGACAAAAACGCCA-3 ' (underscore mark be EcoR I restriction enzyme site);
3 ' primer: 5 '-
GCGGCCGCCTAATTTGGTTCTGTTCCCC-3 ' (underscore mark be Not I restriction enzyme site, italics is a terminator codon).
With above-mentioned 5 ' primer and 3 ' primer is primer, and ((numbering: CGMCC1.1226)) genomic dna is a template, carries out pcr amplification to understand the common micro-organisms center available from Chinese microorganism strain preservation board of trustee reason person with bacillus amyloliquefaciens.
PCR reaction system: the genomic dna 1 μ L (0.5 μ g) of bacillus amyloliquefaciens, 5 ' primer, 1 μ L (0.1 μ g), 3 ' primer, 1 μ L (0.1 μ g), 10 * PCR buffer, 2.5 μ L, Taq Plus (sky, Beijing root company) 1 μ L (2.5U), dNTP mix (2.5mmol/L) 4 μ L, ddH
2O 14.5 μ L, cumulative volume 25 μ L.
The pcr amplification condition is: 94 ℃ of 3min of elder generation; 94 ℃ of 30s then, 53.5 ℃ of 30s, 72 ℃ of 90s, totally 28 circulations; Last 72 ℃ of 10min.
Pcr amplification obtains the fragment of 1413bp, the PCR product that obtains is reclaimed, connect T carrier (pEASY-T3 of Beijing Quanshijin Biotechnology Co., Ltd), and sending Beijing AudioCodes to check order, sequencing result shows that the fragment that pcr amplification obtains has from the Genbank number nucleotide sequence for 5 of EU022559 ' end 88-1500 position.
Certainly, the plain enzyme gene of the above-mentioned bacillus amyloliquefaciens number of writing to peptide fiber also can prepare by the method for synthetic.
2, the structure that contains the plain enzyme gene pichia pastoris carrier for expression of eukaryon of the bacillus amyloliquefaciens number of writing to peptide fiber (pPIC9K-cen)
The plain enzyme gene recombination of the bacillus amyloliquefaciens number of the writing to peptide fiber that the sequencing result of step 1 is correct T carrier carries out double digestion with EcoR I and Not I, reclaim the plain enzyme gene fragment of the bacillus amyloliquefaciens number of writing to peptide fiber, be inserted between the EcoR I of pichia pastoris expression system carrier pPIC9K (invitrogen company) and the Not I restriction enzyme site and obtain recombinant vectors, with the recombinant vectors called after pPIC9K-cen that obtains.
But the structure of the pichia pastoris engineering bacteria GS115-xynb-cen of 3 expressed xylanase and cellulase
1) preparation of pichia pastoris yeast GS115-xynb competent cell
(1) single colony inoculation of picking pichia pastoris yeast GS115-xynb is in the 10mlYPD substratum, 30 ℃ of shaking tables 12 hours.
(2) be forwarded to the 100mlYPD liquid nutrient medium with 1% (volume percent) inoculum size, 30 ℃ of shaking tables spend the night to OD
600=1.3-1.5.4 ℃ of centrifugal 5000rpm, 5min abandons supernatant, collecting precipitation (thalline).
(3) thalline that step (2) is obtained is resuspended with thalline with 100ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, and 10min abandons supernatant, collects thalline.Then that thalline is resuspended with thalline with 50ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, 10min abandons supernatant, collecting precipitation (thalline).
(4) step (3) is washed 1 time with the sorbyl alcohol of 20ml 1mol/L again.Be dissolved in the sorbyl alcohol of the ice precooling of 200 μ L 1mol/L then, obtain pichia pastoris yeast GS115-xynb competent cell.
2) goal gene inserts pichia pastoris GS115-xynb competent cell karyomit(e)
Get the recombinant plasmid pPIC9K-cen of 5 μ g, and carry out enzyme with Bgl II and cut 2 hours.The endonuclease reaction system is as follows:
The carrier for expression of eukaryon 20 μ L that contain goal gene
pPIC9K-cen (5μg)
10×buffer 5μL
Bgl?II 5μL
ddH
2O 20μL
Cumulative volume 50 μ L.
Add the NaAC of 5 μ L 3M pH=5.2 and the dehydrated alcohol of 100 μ L in the system that enzyme is cut ,-20 ℃ left standstill after 15 minutes 10000g centrifugal 1 minute, wash with 75% ethanol, oven dry is dissolved with 10 μ L aqua sterilisas again, obtain linearizing pPIC9K-cen, in order to transforming.
In the pichia pastoris GS115-xynb competent cell suspension that 80 μ l step 1) obtain, the linearizing pPIC9K-cen that adds the above-mentioned acquisition of 1-5 μ g, placed on ice 15 minutes, add in the ice precooling 0.2cm electric shock cup (MicroPuLser Bio-Rad 165-2100) rapidly, electric shock transformed yeast competent cell (2.5KV, 5ms), in the electric shock cup, add the ice-cold 1mol/L sorbyl alcohol of 1ml immediately, behind the mixing, be applied on the RDB flat board with every plate 200 μ l bacterium liquid, 30 ℃ are cultured to and grow transformant.
Transformant obtains multi-copy strains by G418 (invitrogen company) screening of 8mg/ml, the positive strain that screening is obtained is inoculated in 3ml BMGY substratum respectively, 30 ℃ of shaking tables of 250rpm are cultivated 48h, to shake training then and obtain bacterium liquid in the centrifugal 8min of 5000rpm, supernatant discarded is collected thalline, thalline is resuspended with 1ml BMMY methanol induction substratum, continuation centrifugal 8min of 5000rpm behind 28 ℃ of inducing culture 48h gets supernatant, supernatant is surveyed the enzyme of cellulase and zytase and lived, wherein the enzyme work of zytase is measured according to the method that the step 2 in the step 1 provides; The enzyme of cellulase is lived and is measured as follows, and experiment repeats 3 times, and wherein the measurement result of zytase and yield of cellulase is as shown in table 2:
The measurement result of table 2. zytase and yield of cellulase (mean value)
| No. 1 | No. 2 | No. 3 | |
| Zytase | 100U/ml | 420U/ml | 1012U/ml |
| Cellulase | 15U/ml | 260U/ml | 710U/ml |
Cellulase activity measuring method: adopt the CMCNa-DNS method to measure, the enzyme liquid of drawing the suitable dilution of 2ml adds in the test tube, the carboxymethylcellulose sodium solution (0.8g CMC-Na is dissolved in 100ml pH5.2 acetate-sodium acetate buffer solution) that adds 2ml 0.8% again, electromagnetic oscillation 3s, 37 ℃ accurately are incubated 30min.Add 5ml DNS reagent, boiling water bath heating 5min is cooled to room temperature, adds water and is settled to 25ml, measures the reducing sugar amount of its generation.
The cellulase activity unit of force: under 37 ℃ and pH=5.5 condition, per minute from carboxymethylcellulose sodium solution degraded to discharge the needed enzyme amount of 1 μ g reducing sugar be an enzyme activity unit U.
Choose the highest reorganization pichia pastoris bacterium (No. 3) of above-mentioned expressed xylanase and cellulose enzyme activity, called after GS115-xynb-cen.
But the structure of the pichia pastoris engineering bacteria of three expressed xylanase, cellulase and 'beta '-mannase
1, Bacillus circulans goes the clone of signal peptide beta-mannase gene
According to the Bacillus circulans beta-mannase gene sequence of having delivered on the net, the base of its coded signal peptide is removed, this sequence is the Genbank number nucleotide sequence for 5 of AY623903 ' end 122-1051 position, and designs primer with this, and primer sequence is as follows:
5 ' primer: 5 '-
GAATTCCAAAACGGATTTCACGTATC-3 ' (underscore is EcoR I point of contact)
3 ' primer: 5 '-
GCGGCCGCTTAATCACTCTTAAGCCCAT-3 ' (underscore is a Not I restriction enzyme site, and italics is a terminator codon)
With above-mentioned 5 ' primer and 3 ' primer is primer, and (numbering: CGMCC1.2411)) genomic dna is a template, carries out pcr amplification to understand the common micro-organisms center with Bacillus circulans available from Chinese microorganism strain preservation board of trustee reason person.
PCR reaction system: the genomic dna 1 μ L (0.5 μ g) of Bacillus circulans, 5 ' primer, 1 μ L (0.1 μ g), 3 ' primer, 1 μ L (0.1 μ g), 10 * PCR buffer, 2.5 μ L, Taq Plus (sky, Beijing root company) 1 μ L (2.5U), dNTP mix (2.5mmol/L) 4 μ L, ddH
2O 14.5 μ L, cumulative volume 25 μ L.
Pcr amplification condition: 94 ℃ of 3min of elder generation; 94 ℃ of 30s then, 54 ℃ of 30s, 72 ℃ of 1min; Totally 28 circulations; Last 72 ℃ of 10min.
Pcr amplification obtains the fragment of 930bp, the PCR product that obtains is reclaimed, connect T carrier (pEASY-T3 of Beijing Quanshijin Biotechnology Co., Ltd), and sending Beijing AudioCodes to check order, sequencing result shows that the fragment that pcr amplification obtains has from the Genbank number nucleotide sequence for 5 of AY623903 ' end 122-1051 position.
Certainly, containing Bacillus circulans goes the signal peptide beta-mannase gene also can to prepare by the method for synthetic.
2, contain the structure that Bacillus circulans removes the beta-mannase gene pichia pastoris carrier for expression of eukaryon (pPICZ-man) of signal peptide
The Bacillus circulans that the sequencing result of step 1 is correct goes the beta-mannase gene reorganization T carrier of signal peptide to carry out double digestion with EcoR I and Not I, reclaim the beta-mannase gene that Bacillus circulans removes signal peptide, be inserted between the EcoRI of pichia pastoris expression system carrier pPICZ α A (invitrogen company) and the Not I restriction enzyme site and obtain recombinant vectors, the recombinant vectors that obtains is carried out enzyme with EcoR I and Not I cut and identify and order-checking is identified, enzyme is cut and checked order and identify and show the correct segmental recombinant vectors called after of the beta-mannase gene pPICZ-man that above-mentioned Bacillus circulans removes signal peptide that contains.
But the structure of the pichia pastoris engineering bacteria GS115-NSP of 3 expressed xylanase, cellulase and 'beta '-mannase
1) preparation of pichia pastoris yeast GS115-xynb-cen competent cell
(1) single colony inoculation of picking pichia pastoris yeast GS115-xynb-cen is in the 10mlYPD substratum, 30 ℃ of shaking tables spend the night (12 hours).
(2) be forwarded to the 100mlYPD liquid nutrient medium with 1% (volume percent) inoculum size, 30 ℃ of shaking tables spend the night to OD
600=1.3-1.5.4 ℃ of centrifugal 5000rpm, 5min abandons supernatant, collecting precipitation (thalline).
(3) thalline that step (2) is obtained is resuspended with thalline with 100ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, and 10min abandons supernatant, collects thalline.Then that thalline is resuspended with thalline with 50ml ice precooling sterilized water, 4 ℃ of centrifugal 5000rpm, 10min abandons supernatant, collecting precipitation (thalline).
(4) step (3) is washed 1 time with the sorbyl alcohol of 20ml 1mol/L again.Be dissolved in the sorbyl alcohol of the ice precooling of 200 μ L 1mol/L then, obtain pichia pastoris yeast GS115-xynb-cen competent cell.
2) goal gene inserts pichia pastoris GS115-xynb-cen competent cell karyomit(e)
Get recombinant plasmid pPICZ-man of 5 μ g, and carry out enzyme with Bgl II and cut 2 hours.The endonuclease reaction system is as follows:
The carrier for expression of eukaryon 20 μ L that contain goal gene
pPICZ-man (5μg)
10×buffer 5μL
Bgl?II 5μL
ddH
2O 20μL
Cumulative volume 50 μ L.
Add the NaAC of 5 μ L 3M pH=5.2 and the dehydrated alcohol of 100 μ L in the system that enzyme is cut ,-20 ℃ left standstill after 15 minutes 10000g centrifugal 1 minute, wash with 75% ethanol, oven dry is dissolved with 10 μ L aqua sterilisas again, obtain linearizing pPICZ-man, in order to transforming.
In the pichia pastoris GS115-xynb-cen competent cell suspension that 80 μ l step 1) obtain, the linearizing pPICZ-man that adds the above-mentioned acquisition of 1-5 μ g, placed on ice 15 minutes, add in the ice precooling 0.2cm electric shock cup (MicroPuLser Bio-Rad 165-2100) rapidly, electric shock transformed yeast competent cell (2.5KV, 5ms), in the electric shock cup, add the ice-cold 1mol/L sorbyl alcohol of 1ml immediately, behind the mixing, be applied on the RDB flat board with every plate 200 μ l bacterium liquid, 30 ℃ are cultured to and grow transformant.
Transformant obtains positive recombinant bacterial strain by Zeocin (invitrogen company) screening of 300 μ g/ml, the positive strain that screening is obtained is inoculated in 3ml BMGY substratum respectively, 30 ℃ of shaking tables of 250rpm are cultivated 48h, the centrifugal 8min of 5000rpm, supernatant discarded, resuspended with 1ml BMMY methanol induction substratum, continuation centrifugal 8min of 5000rpm behind 28 ℃ of inducing culture 48h gets supernatant.
Supernatant is surveyed enzyme work identify whether it can express 'beta '-mannase, cellulase and zytase, and the height of enzymatic productivity, wherein, the enzyme activity determination of cellulase and zytase detects according to the described method of the step 3 in the step 1, 'beta '-mannase measuring method alive: the enzyme liquid of drawing the suitable dilution of 0.2ml adds in the test tube, the 1 acacia bean powder solution 1.8ml (0.5g acacia bean powder is dissolved in 100ml pH5.5 acetate-sodium acetate buffer solution) that adds 5mg/m again, electromagnetic oscillation 3s, 37 ℃ accurately are incubated 10min.Add 3ml DNS reagent, boiling water bath heating 5min is cooled to room temperature, adds water and is settled to 15ml, measures the reducing sugar amount of its generation.The beta-mannase enzyme activity unit: under 37 ℃ and pH=5.5 condition, the per minute needed enzyme amount of release 1 μ mol reducing sugar of degrade from acacia bean powder solution is an enzyme activity unit U.Result's screening obtains the strain bacterial strain of expressed xylanase, cellulase and 'beta '-mannase simultaneously, with its called after pichia pastoris (Pichiapastoris) GS115-NSP, its expressed xylanase, cellulase and 'beta '-mannase enzyme activity determination repeat 3 times, and the result of mean value is as shown in table 3:
The measurement result that zytase, cellulase and the 'beta '-mannase enzyme of table 3.GS115-NSP lived
| Zytase | 1124U/ml |
| Cellulase | 723U/ml |
| 'beta '-mannase | 312U/ml |
Pichia pastoris (Pichia pastoris) GS115-NSP, be preserved in Chinese microorganism strain preservation board of trustee reason person on 04 16th, 2009 and understood common micro-organisms center (abbreviation CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preserving number is CGMCC № .3025.
The pichia pastoris CGMCC № .3025 GS115-NSP that makes up or pichia pastoris GS115 be inoculated in the YPD substratum cultivated 48 hours, as fermentation seed liquid.Switching goes in the 5L fermentor tank to cultivate, inoculum size is 5-10%, temperature 28-30 ℃, pH5.0-6.0, dissolved oxygen (D0) is controlled at 30%-60%, and induce fermentation 132 hours, every sampling (fermented supernatant fluid) in 24 hours, measure zytase, cellulase and 'beta '-mannase enzyme according to the method described above and live.Aforesaid method repeats five times, and the result is as shown in table 4: the supernatant liquor of pichia pastoris GS115 fermentation in the time of 132 hours do not have enzyme to live.
Table 4. pichia pastoris CGMCC № .3025 GS115-NSP expresses enzyme qualification result (mean value) alive
| Fermentation time (hour) | Xylanase activity (U/ml) | Cellulose enzyme activity (U/ml) | 'beta '-mannase enzyme (U/ml) alive |
| 12 | 614 | 429 | 290 |
| 24 | 1190 | 788 | 591 |
| 36 | 1687 | 1143 | 917 |
| 48 | 2110 | 1620 | 1194 |
| 60 | 2625 | 2001 | 1442 |
| 72 | 3179 | 2396 | 1723 |
| 84 | 3910 | 2781 | 2012 |
| 96 | 4416 | 3115 | 2278 |
| 108 | 4726 | 3652 | 2145 |
| 120 | 4980 | 3988 | 2104 |
| 132 | 5012 | 4109 | 2017 |
Claims (9)
1. the reorganization bacterium is the reorganization pichia pastoris bacterium that contains xylanase gene, cellulose enzyme gene and beta-mannase gene;
The encoding sequence of described xylanase gene is the Genbank number nucleotide sequence for 5 of AJ292317 ' end 1-576 position; The nucleotide sequence of described cellulose enzyme gene is the Genbank number nucleotide sequence for 5 of EU022559 ' end 88-1500 position; The nucleotide sequence of described beta-mannase gene is the Genbank number nucleotide sequence for 5 of AY623903 ' end 122-1051 position;
Described pichia pastoris bacterium is pichia pastoris bacterium GS115.
2. reorganization pichia pastoris bacterium as claimed in claim 1 is characterized in that: described xylanase gene imports in the pichia pastoris bacterium by recombinant expression vector a, constitutes reorganization pichia pastoris bacterium A; Described recombinant expression vector a is the recombinant expression vector that described xylanase gene is inserted into the expressed xylanase that obtains on the expression vector pPIC9.
3. reorganization pichia pastoris bacterium as claimed in claim 2 is characterized in that: described cellulose enzyme gene imports among the reorganization pichia pastoris bacterium A by recombinant expression vector b, constitutes reorganization pichia pastoris bacterium B; Described recombinant expression vector b is the recombinant expression vector that described cellulose enzyme gene is inserted into the expression cellulase that obtains on the expression vector pPIC9K.
4. reorganization pichia pastoris bacterium as claimed in claim 3 is characterized in that: described beta-mannase gene imports among the described reorganization pichia pastoris bacterium B by recombinant expression vector c and obtains the described reorganization of claim 1 bacterium; Described recombinant expression vector c is the recombinant expression vector that described beta-mannase gene is connected to the expression 'beta '-mannase that obtains on the expression vector pPICZ α A.
5. as any described reorganization pichia pastoris bacterium among the claim 1-4, it is characterized in that: described reorganization bacterium is pichia pastoris (Pichia pastoris) CGMCC № .3025.
6. the application of any described reorganization pichia pastoris bacterium in producing the non-starch polysaccharide prozyme among the claim 1-5.
7. application as claimed in claim 6 is characterized in that: described application is with any described reorganization pichia pastoris bacterium among the claim 1-5, at temperature 28-30 ℃, pH5.0-6.0, dissolved oxygen is 30%-60%, induces fermentation, gives expression to the non-starch polysaccharide prozyme; Described cultivation substratum is YPD substratum or BMGY substratum; Described BMGY substratum contains the material of following final concentration: mass percentage concentration is 1% yeast extract, mass percentage concentration is 2% peptone, 100mmol/L pH6.0 phosphoric acid buffer, mass percentage concentration is 1.34% YNB, mass percentage concentration is the substratum of the glycerine of 0.00004% vitamin H and volume percent 1%; Described YPD substratum contains the material of following final concentration: mass percentage concentration is 1% yeast extract, and mass percentage concentration is 2% peptone, and mass percentage concentration is the substratum of 2% glucose; Described reorganization bacterium was cultivated 30-60 hour in the YPD substratum.
8. application according to claim 7 is characterized in that: described reorganization bacterium was cultivated 48 hours in the YPD substratum.
9. the application of any described reorganization pichia pastoris bacterium in producing feed among the claim 1-5.
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