CN102433267A - Method for preparing beta-mannase and special strain - Google Patents
Method for preparing beta-mannase and special strain Download PDFInfo
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Abstract
The invention discloses a method for preparing beta-mannase and a special strain. The strain is Pichia pastoris GS4SMAN CGMCC NO.4095. The recombinant strain disclosed by the invention contains a plurality of copied mannase genes expressed under methanol induction. Experiments show that by means of a process, disclosed by the invention, for producing alkaline mannase through high-density fermentation by using the recombinant strain, the enzyme activity of the alkaline mannase can reach 6336u/ml finally in an induction period of 84 hours. The method disclosed by the invention has the advantages of low cost, simplicity for operation and high yield. Therefore, the method disclosed by the invention has a wide application prospect in the field of mannase preparation.
Description
Technical field
The present invention relates to a kind of method and special strain therefore for preparing 'beta '-mannase.
Background technology
'beta '-mannase (β-1,4-D-mannan mannohydrolase; EC31211178) be the main chain β-1 of a type of degraded mannosans, glucomannan, polygalactomannan and gala glucomannan; The enzyme of 4-D-mannopyranose, its various beta-mannase in the stem tuber of the nut, guar-bean, acacia, coffee tree and the konjaku that utilize the existing endosperm that is some plants such as copra, ivory palm has potential importance.The bleaching, the broken glue of oil well, the degrading plant glue that are mainly used in both at home and abroad paper industry paper pulp are produced oligose as the bifidus bacillus somatomedin at present, in protective foods that anti-disease, anti-aging is old and the fodder industry as ANFs.
'beta '-mannase has the branch of acidity, neutrality, alkalescence by the difference of its optimum pH; Wherein alkaline ' beta '-mannase is meant the highest 'beta '-mannase of catalysis activity under alkaline condition; Its research is later relatively, finds mainly to be produced by Alkaliphilic bacillus, and some Bacillus licheniformis also produces alkaline ' beta '-mannase; Reported that like Yang Wenbo etc. a bacillus licheniformis NK-27, its optimum pH are 9.0.Subtilis generally speaking, actinomycetes etc. produce neutral 'beta '-mannase, and mould produces acidic beta-mannase.First alkali mannanase is by discoveries such as Akino.Ma Yan with equal 1991 reported the outer alkali mannanase of three born of the same parents of Alkaliphilic bacillus N16-5 purifying and zymologic property, and reported wherein a kind of gene order of having a liking for the alkali mannase in 2004.Takeda equals to find in 2004 that the bacterial strain JAMB-602 of bacillus produces alkali mannanase, and this enzyme belongs to GH family 5, and ph optimum is 9.The optimum pH of being found at present of having a liking for the alkali mannase is up to 10, equals 2004 years reports by Takeda.
Alkaline ' beta '-mannase not only can produce as the bifidus bacillus somatomedin oligose, and be with a wide range of applications in the industrial aspect such as association with pulp bleaching of need alkaline environment.But up to the present, the preparation of alkaline ' beta '-mannase still is the bottleneck that limits its industrial application, no matter is to use the original bacterial strain of alkaline ' beta '-mannase also to be to use recombinant bacterial strain production in forefathers' the report, and its output is all lower.
Ma Yan with equal 1991 reported the outer alkali mannanase of three born of the same parents of Alkaliphilic bacillus N16-5 purifying and zymologic property; And reported wherein a kind of gene order of having a liking for the alkali mannase in 2004, the coding region sequence of its gene is shown in 7-1407 position among the SEQ ID NO:1.
PTM
1Solution: with Cu SO
45H
2O 6g, KI 0.08g, MnSO
42.68g, H
3BO
30.02g, Na
2MoO
42H
2O 0.2g, ZnSO
47H
2O 20g, FeSO
47H
2O 65g, CoCl
26H
2O 0.916, H
2SO
45mL, vitamin H 0.2g mixes, and water is settled to 1 liter, obtains PTM
1Solution.
Per 1 liter of BMGY liquid nutrient medium prepares according to following method: with 8 grams-12 grams or 10 gram yeast extracts, 18 grams-22 grams or 20 gram peptones, 12 grams-15 grams or 13.4 gram YNB, 3 * 10
-4Gram-5 * 10
-4Gram or 4 * 10
-4Gram vitamin H and 8 grams-12 grams or 10 gram glycerine are soluble in water, and water is settled to 1 liter, obtains the BMGY liquid nutrient medium.
Summary of the invention
An object of the present invention is to provide a kind of reorganization bacterium.
Reorganization bacterium provided by the present invention is the encoding sox importing starting strain with 'beta '-mannase, the reorganization bacterium that obtains; The coding gene sequence of said 'beta '-mannase is shown in 7-1407 position Nucleotide among the SEQ ID NO:1.
In the above-mentioned reorganization bacterium, the encoding sox of said 'beta '-mannase imports starting strain through recombinant vectors; Said recombinant vectors is as follows shown in (1) or (2) or (3):
(1) dna fragmentation shown in the SEQ ID NO:1 is inserted between the XhoI and EcoRI restriction enzyme site of pAO815alpha, the recombinant vectors that obtains, note is made pAO α-1sman; Wherein, the XhoI restriction enzyme site is positioned at 5 of dna fragmentation shown in the SEQ ID NO:1 ' end upper reaches, and the EcoRI restriction enzyme site is positioned at 3 of dna fragmentation shown in the SEQ ID NO:1 ' end downstream;
(2), reclaim the 2932bp fragment with BamHI/BglII double digestion pAO α-1sman; With BamHI single endonuclease digestion pAO α-1sman, reclaim the big fragment of carrier; Said 2932bp fragment is connected with the big fragment of said carrier, the recombinant vectors that obtains, note is made pAO α-2sman;
(3), reclaim the 5864bp fragment with BamHI/BglII double digestion pAO α-2sman; With BamHI single endonuclease digestion pAO α-2sman, reclaim the big fragment of carrier; Said 5864bp fragment is connected with the big fragment of said carrier, the recombinant vectors that obtains, note is made pAO α-4sman;
Said pAO815alpha obtains according to following method: carrier pPICZ α is used the SacI/EcoRI double digestion, collect the 1000bp fragment; With SacI/EcoRI double digestion carrier pAO815, reclaim the big fragment of carrier; Said 1000bp fragment is connected with the big fragment of said carrier, and the recombinant vectors that obtains is pAO815alpha;
Said starting strain is a yeast, is preferably Pichia yeast, is preferably pichia pastoris phaff bacterial strain GSl15 again.
In the above-mentioned reorganization bacterium, said reorganization bacterium is pichia pastoris phaff (Pichia pastoris) GS4SMAN, and its deposit number is CGMCC NO.4095.
Another object of the present invention provides a kind of method for preparing 'beta '-mannase.
The method for preparing 'beta '-mannase provided by the present invention comprises the steps: above-mentioned arbitrary said reorganization bacterium is inoculated in the fermention medium, and fermentation culture obtains 'beta '-mannase; All material notes in the fermenting container are made fermentation system.
In the aforesaid method, said fermention medium is the substratum that is used to cultivate Pichia yeast, and wherein contains glycerine;
Said fermentation culture is carried out according to following three phases successively:
Stage one: comprise the steps: to exhaust from the glycerine that inoculation begins to be cultured to the fermentation system;
Stage two: when comprising the steps: that glycerine in fermentation system exhausts, begin to flow glycerol adding or glycerine solution, be cultured to the OD of fermentation system
600Value reaches 200-350 or 200 or 250 or 320 or 350, stops to flow glycerol adding or glycerine solution;
Stage three: be following 1) or 2) shown in:
When 1) comprising the steps: that glycerine in fermentation system exhausts, in fermentation system, add mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, carry out abduction delivering;
When 2) comprising the steps: that glycerine in fermentation system exhausts; The temperature of fermentation system is transferred to 26 ℃～29 ℃ or 26 ℃ or 27 ℃ or 28 ℃ or 29 ℃; In fermentation system, add mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, carry out abduction delivering;
Said glycerine solution is by glycerine, PTM
1Solution and water are formed; Said methanol solution is by methyl alcohol, PTM
1Solution and water are formed.
In the aforesaid method, in the mixed aqueous solution of said sorbyl alcohol and methyl alcohol, the mass ratio of sorbyl alcohol and methyl alcohol is 1: 20～1: 5 or 1: 20 or 1: 10 or 1: 5.
In the aforesaid method, in the mixed aqueous solution of said sorbyl alcohol and methyl alcohol, the concentration of sorbyl alcohol is 36g/L-144g/L or 36g/L or 72g/L or 144g/L, and the concentration of methyl alcohol is 720g/L.
In the aforesaid method, the cultivation in said stage one and said stage two is 6.0 in the pH value, temperature is that 30 ℃, dissolved oxygen are to carry out under the condition more than 30%;
In the aforesaid method, 1 in said stage three) in, abduction delivering is 6.0 in the pH value, temperature is that 30 ℃, dissolved oxygen are to carry out under the condition more than 30%;
In the aforesaid method, 2 in said stage three) in, abduction delivering is 6.0 in the pH value, temperature is 26 ℃～29 ℃ or 26 ℃ or 27 ℃ or 28 ℃ or 29 ℃, dissolved oxygen are to carry out under the condition more than 30%.
In the aforesaid method, in the said stage three, the time of abduction delivering is 60h-160h, is specially 12h-84h or 36h-84h or 48h-84h or 60h-84h or 19h-134h or 43h-134h or 64h-134h or 86h-134h or 111h-134h; Be specially 12h, 36h, 48h, 60h, 72h, 84h or 19h, 43h, 64h, 86h, 111h or 134h again;
In the aforesaid method, in the said stage one, said inoculum size is 5%;
In the aforesaid method, in the said stage three, said glycerine in fermentation system exhausts when Shi Weicong stops to flow glycerol adding to be counted, behind 0.5～1h; (before adding methyl alcohol, cool the temperature to 26～29 ℃, thalline and hungry 0.5～1h to exhaust glycerine, reach the purpose that the AOX1 promotor derepresses).
In the aforesaid method, said dissolved oxygen is to be 30%-100% for dissolved oxygen more than 30%;
In the aforesaid method, said fermention medium is the BMGY liquid nutrient medium;
In the aforesaid method, said glycerine solution prepares according to following method: with 600g-650g or 625g glycerine, 10ml-14ml or 12mL PTM
1Solution mixes, and water is settled to 1 liter.
In the aforesaid method; In the said stage three; The said sorbyl alcohol and the mixed aqueous solution of methyl alcohol or the mode of methyl alcohol or methanol solution of in fermentation system, adding is: in fermentation system, add earlier methyl alcohol; Making the volumetric concentration of methyl alcohol in fermentation system is 3/1000-7/1000 or 5/1000,1.5 hour-2.5 hours or after 2 hours, and stream adds mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol in fermentation system again;
In the aforesaid method, in the said stage one, said dissolved oxygen be more than 30% be through bubbling air in fermentation system and the speed of regulating bubbling air with stir fermentation system and regulate the speed that stirs fermentation system and realize; Said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min;
In the aforesaid method; In the said stage two; Said dissolved oxygen be more than 30% be through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and the stream rate of acceleration of regulating glycerine or glycerine solution realizes that said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min; The speed of said stream glycerol adding or glycerine solution is 5ml glycerine or glycerine solution/L fermention medium/h-15ml glycerine or glycerine solution/L fermention medium/h;
In the aforesaid method; In the said stage three; Said dissolved oxygen be more than 30% be through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and regulate the stream rate of acceleration realization of mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min; Said stream adds the mixed aqueous solution of sorbyl alcohol and methyl alcohol or the speed of methyl alcohol or methanol solution is the mixed aqueous solution of 2ml sorbyl alcohol and methyl alcohol or mixed aqueous solution or the methyl alcohol or the methanol solution/L fermention medium/h of methyl alcohol or methanol solution/L fermention medium/h-6ml sorbyl alcohol and methyl alcohol.
Reorganization disclosed by the invention bacterium contains mannase gene a plurality of copies, that expressed by methanol induction.Experiment showed, and of the present inventionly carry out the technology that high density fermentation is produced alkali mannanase that can make final alkali mannanase in the induction duration of 84h, enzyme work reaches 6336u/ml with this reorganization bacterium.The inventive method cost is low, simple to operate, productive rate is high.Therefore, the inventive method has broad application prospects in the preparation field of mannase.
Description of drawings
Fig. 1 is the structure synoptic diagram of secreted expression carrier pAO815alpha.
Fig. 2 is recombinant expression vector pAO α-1sman, pAO α-2sman, the structure synoptic diagram of pAO α-4sman.
Fig. 3 is that reorganization bacterium GS4SMAN is in methanol induction high density fermentation result.
Fig. 4 for reorganization bacterium GS4SMAN in sorbyl alcohol mixing feed supplement, 30 ℃ of results of high density fermentation when inducing
Fig. 5 for reorganization bacterium GS4SMAN in sorbyl alcohol mixing feed supplement, 26 ℃ of results of high density fermentation when inducing
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The structure of embodiment 1, alkali mannanase secretion expression carrier
One, the clone of alkali mannanase gene
Design and synthetic primer
Primer 1 (Forward): 5 ' ATAT
CTCGAGAAAAGATCCCCATCAGAAGC 3 '
Primer 2 (Reverse): 5 ' CGGGC
GAATTCTATCTTAAAGTAACATTAT 3 '
In α-mating factor signal peptide-coding sequence downstream an XhoI restriction enzyme site (CTCGAG is arranged; Corresponding amino acid is Leu-Glu); Thereafter immediately following a Kex2 proteolytic enzyme recognition site (AAAAGA; Corresponding aminoacid sequence Lys-Arg), therefore, the design of primer 1 is before mannase gene, to introduce XhoI site and Kex2 proteolytic enzyme recognition site sequence.Through this design; Can mannase gene directly be connected after the signal peptide sequence; Like this behind the protein translation; Under the effect of pichia spp self Kex2 proteolytic enzyme, can cut and give expression to the constant target protein of amino acid (and can not introduce extra amino-acid residue) because of the reason of restriction enzyme site.Primer 2 comprises terminator codon TAG, and introduces an EcoRI restriction enzyme site.
With the total DNA of Alkaliphilic bacillus N16-5 is template (or be template with gene shown in the SEQ ID NO:1 of synthetic); Add primer 1, primer 2, HiFi Taq archaeal dna polymerase and dNTP mixture; Carry out touchdown PCR (touchdown PCR), reaction conditions is following: 94 ℃ of 5min; 94 ℃ of 30s, 60-50 ℃ of 30s, 72 ℃ of 90s, 1 ℃ of each cycle down, totally 10 circulations; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, totally 25 circulations.
Pcr amplification obtains the fragment of 1400bp; Called after sman; Behind Omega purification kit purifying, connect into pMD18-T carrier (available from TaKaRa company), obtain recombinant vectors pMD18-sman; Send Beijing AudioCodes to check order pMD18-sman, sequencing result shows in the fragment that pcr amplification obtains and contains sequence shown in the SEQ ID NO:1.
Two, the structure of secretion expression carrier
The structure flow process is as depicted in figs. 1 and 2.
The preparation of pAO815alpha: pPICZ α (available from Invitrogen company) is used the SacI/EcoRI double digestion, collect 1000bp fragment (fragment that promptly contains α-mating factor signal dna encoding peptide); With SacI/EcoRI double digestion pAO815 carrier (available from Invitrogen company), reclaim the big fragment of carrier; Said 1000bp fragment is connected with the big fragment of said carrier, and the recombinant vectors that obtains is pAO815alpha;
1, the structure of single copy alkali mannanase secretion expression carrier
The pMD18-sman that step 1 is obtained is connected with same pAO815alpha plasmid through XhoI and EcoRI double digestion with XhoI and EcoRI double digestion, forms singly copy alkali mannanase secretion expression carrier pAO α-1sman.
2, the acquisition of the bacterial strain of 2 copy sman
The recombinant expression vector pAO α-1sman that obtains in the step 1 is used the BamHI/BglII double digestion, reclaim 2932bp fragment (being the BamHI-sman-BglII fragment); With BamHI single endonuclease digestion pAO α-1sman, reclaim the big fragment of carrier; Said 2932bp fragment is connected with the big fragment of said carrier, get final product 2 the copy sman expression vector pAO α-2sman;
3, the acquisition of the bacterial strain of 4 copy sman
With BamHI/BglII double digestion pAO α-2sman, reclaim 5864bp fragment (being the BamHI-sman-sman-BglII fragment); With BamHI single endonuclease digestion pAO α-2sman, reclaim the big fragment of carrier; Said 5864bp fragment is connected with the big fragment of said carrier, get final product 4 the copy sman expression vector pAO α-4sman.
Three, express the acquisition of the yeast strain of mannase
1, electricity transforms the preparation of pichia pastoris phaff cell
Inoculation pichia pastoris phaff bacterial strain GSl15 (Invitrogen; Ca.18100) in 500ml YPD substratum (10 grams per liter yeast extract pastes, 20 grams per liter peptones, 20 grams per liter glucose, 15 grams per liter agaroses); 30 ℃, 200r/min is cultured to OD600=1.3-1.5.4 ℃, 1, the centrifugal 5min of 500g collects thalline, respectively washes once with the aqua sterilisa of 500ml, 250ml precooling and the 1mol/L sorbyl alcohol of 20ml precooling respectively.After washing at every turn all at 4 ℃, 1, centrifugal 5min collects thalline under the 500g, the 1mol/L sorbyl alcohol with the 1ml precooling suspends at last, promptly obtains the competent cell that shocks by electricity.
2, recombinant expression plasmid electricity transformed yeast cell
Use the SalI linearization for enzyme restriction respectively with making up correct each about 10ug of recombinant expression plasmid pAO α-1sman, pAO α-2sman and pAO α-4sman in the experiment two, ethanol sedimentation reclaims linear DNA and is dissolved in the 10ul sterilized water.Above-mentioned linearizing DNA is mixed with 80ul GS115 competent cell, change in the 0.2cm electricity revolving cup of precooling.Adopt U.S. BIO-RAD company electricity conversion instrument, the pichia spp parameter preset according to making electricity consumption conversion instrument shocks by electricity.Add immediately after electric shock finishes in 1M sorbyl alcohol to the electric shock cup of 1ml precooling, the solution in the cup that will shock by electricity then all is transferred in the aseptic centrifuge tube, hatches 1-2h for 37 ℃, gets 500ul coating MD (2 grams per liter glucose, 13.4 grams per liter YNB, 4 * 10
-4Grams per liter vitamin H, 1.5 grams per liter agar) flat board, be inverted cultivation in 30 ℃ and occurred until bacterium colony in 2-4 days.So far, obtain containing the recombinant pichia yeast strain of 1,2,4 copy sman.
The molecular assay method that contains the recombinant pichia yeast strain (promptly changing the recombinant pichia yeast strain of pAO α-1sman over to) of 1 copy sman: with the GAP gene is internal control gene, and the copy number that uses fluorescence quantitative PCR method to measure this bacterial strain sman is 1 copy.Concrete measuring method is referring to Taicheng Zhu et al.; Efficient generation of multi-copystrains for optimizing secretory expression of porcine insulin precursor in yeast Pichiapastoris; Journal of Applied Microbiology; 2009,107:954-963;
The molecular assay method that contains the recombinant pichia yeast strain (promptly changing the recombinant pichia yeast strain of pAO α-2sman over to) of 2 copy sman: with the GAP gene is internal control gene, and the copy number that uses fluorescence quantitative PCR method to measure this bacterial strain sman is 2 copies.
The molecular assay method that contains the recombinant pichia yeast strain (promptly changing the recombinant pichia yeast strain of pAO α-4sman over to) of 4 copy sman: with the GAP gene is internal control gene, and the copy number that uses fluorescence quantitative PCR method to measure this bacterial strain sman is 4 copies.
Four, the shake flask fermentation of recombinant bacterial strain
1, the fermentation culture of reorganization bacterium
The single bacterial strain of sman, the bacterial strain of 2 copy sman and bacterial strain of 4 copy sman of copying of the expression alkali mannanase that obtains in the step 3 is inoculated in (10 grams per liter yeast extracts, 20 grams per liter peptones in the 25ml BMGY liquid nutrient medium respectively; Behind 121 ℃ of sterilization 20min, when reducing to room temperature, temperature adds 13.4 grams per liter YNB, 4 * 10
-4Grams per liter vitamin H, 10 grams per liter glycerine), in 30 ℃, the 200r/min shaking table is cultivated 48h, OD
600Reach 20, the centrifugal 5min of 3000r/min collects thalline, abandons supernatant, washs thalline 1-2 time with sterile purified water.(10 grams per liter yeast extracts, 20 grams per liter peptones behind 121 ℃ of sterilization 20min, add 13.4 grams per liter YNB, 4 * 10 when temperature is reduced to room temperature with the BMMY inducing culture with thalline
-4Grams per liter vitamin H, 0.5% methyl alcohol), be diluted to OD
600=10, replace cotton plug with 4 layers of gauze, in 30 ℃, the 200r/min shaking table is cultivated.Adding methyl alcohol to final concentration every day is 0.5% (V/V)., get fermented liquid and carry out enzyme biopsy survey after 3 days 30 ℃ of cultivations.All material notes in the fermenting container are made fermented liquid.
2, extracellular enzyme mensuration alive
Draw fermented liquid 1mL in centrifuge tube,, draw supernatant in test tube with the centrifugal 10min of 10000r/min.The sample solution and the 0.19mL reaction solution that add the 0.01ml dilution, 72 ℃ of insulation 10min; Add 0.2ml DNS, boil 5min; Get supernatant and measure light absorption value at the 540nm place.The enzyme liquid of making a living to go out is done blank, and the treatment process of blank is the same with the processing of sample.
The enzyme definition of living: be under 9.0 the condition at 37 ℃, pH value, PM is degraded, and to discharge the needed enzyme amount of 1 μ mol reducing sugar be an enzyme activity unit u to mannosans.
The go out preparation method of the enzyme liquid of making a living: the supernatant that step 1 is obtained boils 5min at 100 ℃, the enzyme liquid that obtains going out and make a living.
Locust bean gum is available from Sigma, and catalog number is G0753.The staple of locust bean gum is a mannosans.
Reaction solution compound method: the 0.5g locust bean gum is put into the 250ml beaker; Add pH9.0,0.05M glycocoll-sodium hydrate buffer solution (the 50ml0.2M glycocoll+8.8ml0.2N sodium hydroxide of 90ml 70 ℃ of preheatings; Thin up is to 200ml) the magnetic agitation mixing; The water constant volume obtains reaction solution to 500ml then.
DNS: take by weighing Seignette salt 182.0g, be dissolved in the 500mL zero(ppm) water, heating (being no more than 50 ℃); In hot soln, add 3 successively, 5-dinitrosalicylic acid 6.3g, NaOH 21.0g; Phenol 5.0g, sodium sulphite anhydrous 99.3 5.0g is stirred to dissolving fully; The cooling back is settled to 1000mL with zero(ppm) water, is stored in the brown bottle room temperature preservation.
Enzyme is lived and OD
540Reduction formula be:
Fermentation broth enzyme work=[(OD
540+ 0.187)/0.025/18] * the fermented liquid extension rate.
3 repetitions are established in experiment, and the result takes the mean.
After inducing fermentation 72h, table 1 is seen in the work of 1-4 copy bacterial strain shake flask fermentation liquid enzyme.Be contrast with the Pichi strain GSl15 that changes pAO815alpha over to simultaneously, result's contrast comes to the same thing with blank, detects less than enzyme and lives.
The enzyme of the different copy of table 1. bacterial strain is lived
With an enzyme the highest strain bacterium called after GS4SMAN alive; This bacterial strain is the recombinant pichia yeast strain that changes pAO α-4sman over to, and this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 18th, 2010 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica; Postcode 100101), preserving number is CGMCC No.4095, classification called after pichia pastoris phaff (Pichia pastoris).
Embodiment 2, fermentation reorganization bacterium prepare mannase
In the following experiment, used reorganization bacterium is pichia pastoris phaff (Pichia pastoris) GS4SMANCGMCC NO.4095.
Yeast extract is available from OXOID company, and catalog number is LP0021.Peptone is available from BD company, and catalog number is 211677.YNB is available from BD company, and catalog number is 291940.Vitamin H is available from Beijing chemical reagents corporation, and catalog number is 67000294.
Fermention medium (BMGY liquid nutrient medium): 10 gram yeast extracts and 20 gram peptones are mixed, behind 121 ℃ of sterilization 20min, add 13.4 gram YNB, 4 * 10 when temperature is reduced to room temperature
-4Gram vitamin H and 10 gram glycerine, water is settled to 1 liter.
PTM
1(1L): with Cu SO
45H
2O 6g, KI 0.08g, MnSO
42.68g, H
3BO
30.02g, Na
2MoO
42H
2O 0.2g, ZnSO
47H
2O 20g, FeSO
47H
2O 65g, CoCl
26H
2O 0.916, H
2SO
45mL, vitamin H 0.2g mixes, and water is settled to 1 liter.
One, fermentation I:
(1) seed shake-flask culture: from the glycerine pipe, connect 200 microlitre bacterium liquid in 5 milliliters of YPD, cultivate that the inoculum size by 1% is inoculated among the 50mlBMGY behind the 12h, cultivate 24h in 30 ℃, 220r/min, to OD
600=20, obtain seed liquor.
(2) high density fermentation is cultivated:
Prepare: fermentation tank culture medium is pressed 50% liquid amount, and the demarcation of pH, dissolved oxygen electrode is carried out in 121 ℃ of sterilizations earlier before the sterilization, sterilized then 30 minutes, is cooled to 30 ℃, regulates the dissolved oxygen to 100% in the substratum.The method of regulating dissolved oxygen is: bubbling air and regulate Ventilation Rate and stir fermentation system and regulate stir speed (S.S.) in fermentation system; Stir speed (S.S.) is 200rpm-900rpm; The speed of bubbling air is 0.5-1.5VVM (L air/L fermention medium/min); The method of regulating the pH value is: with the pH value of 50% ammonia aqueous solution regulation system so that the pH value of system remains on 6.0.
Stage one: seed liquor is inserted 1L automatic fermenter (the 0.7L fermention medium wherein is housed) by 5% inoculum size, be 6.0 in the pH value, temperature is that 30 ℃, dissolved oxygen are to cultivate under the condition of (being 30%-100%) more than 30%, the glycerine to substratum exhausts.
In this process, when thalline began to grow, oxygen-consumption increased, and caused dissolved oxygen to begin to reduce by 100%, still regulated dissolved oxygen through regulating stirring velocity with the mode of regulating the blowing air amount, to keep dissolved oxygen more than 30%.The method of regulating dissolved oxygen is: bubbling air and regulate the speed of bubbling air and stir fermentation system and regulate the speed that stirs fermentation system in fermentation system; Stir speed (S.S.) is 200rpm-900rpm; The speed of bubbling air is 0.5-1.5VVM (L air/L fermention medium/min); The method of regulating the pH value is: with the pH value of 25% ammonia aqueous solution regulation system so that the pH value of system remains on 6.0.
Stage two: when the glycerine in fermentation system exhausts, begin to flow glycerol adding solution, the OD of fermentation culture to fermentation system
600Value reaches 250, stops to flow glycerol adding solution; In this phase process, the pH value of fermentation system is 6.0, temperature is that 30 ℃, dissolved oxygen are (being 30%-100%) more than 30%; Said glycerine solution prepares according to following method: with 625g glycerine, 12mL PTM
1Solution mixes, and water is settled to 1 liter.
The method of regulating dissolved oxygen is: through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and the stream rate of acceleration of regulating glycerine solution realize; When stir speed (S.S.) transfers to maximum and air flow when transferring to maximum, dissolved oxygen still is lower than at 30% o'clock, just begins to regulate the stream rate of acceleration of glycerine solution.Stir speed (S.S.) is 200rpm-900rpm; The speed of bubbling air is 0.5-1.5VVM; The speed of stream glycerol adding solution is 5ml glycerine solution/L fermention medium/h-15ml glycerine solution/L fermention medium/h;
The method of regulating the pH value is: identical with the stage one.
Stage three: when the glycerine in fermentation system exhausts (behind the hungry 0.5～1h of thalline), in fermentation system, add methyl alcohol, carry out abduction delivering; The pH value of the fermentation system in this stage is 6.0, temperature is that 30 ℃, dissolved oxygen are (being 30%-100%) more than 30%;
The mode that in fermentation system, adds methyl alcohol is: in fermentation system, add methyl alcohol earlier, making the volumetric concentration of methyl alcohol in fermentation system is (purpose is to make bacterium adapt to methyl alcohol) after 5/1000,2 hour, and stream adds methyl alcohol in fermentation system again.
The method of regulating dissolved oxygen is: be through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and the stream rate of acceleration of regulating methyl alcohol realized; When stir speed (S.S.) transfers to maximum and air flow when transferring to maximum, dissolved oxygen still is lower than at 30% o'clock, just begins to regulate the stream rate of acceleration of methyl alcohol.Said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5-1.5VVM; The speed that said stream adds methyl alcohol is 2ml methyl alcohol/L fermention medium/h-6ml methyl alcohol/L fermention medium/h.
The method of regulating the pH value is: identical with the stage one.
At the inductive different time, get 10mL fermented liquid detection enzyme and live.All material notes in the fermentor tank are made fermented liquid.
Enzyme biopsy survey method: with identical described in the embodiment 1.
3 repetitions are established in experiment, and the result takes the mean.
It is as shown in Figure 3 that the result is surveyed in the enzyme biopsy, and during abduction delivering 19h, enzyme is lived and is the 1355u/ml fermented liquid in the fermented liquid, during abduction delivering 43h; Enzyme is lived and is the 1286u/ml fermented liquid in the fermented liquid, and during abduction delivering 64h, enzyme is lived and is the 1066u/ml fermented liquid in the fermented liquid; During abduction delivering 86h, enzyme is lived and is the 2063u/ml fermented liquid in the fermented liquid, during abduction delivering 111h; Enzyme is lived and is the 2921u/ml fermented liquid in the fermented liquid, and during abduction delivering 134h, enzyme is lived and is the 3918u/ml fermented liquid in the fermented liquid.
Ferment with following method: the OD that removes fermentation system when stopping to flow glycerol adding solution in the stage two
600Value is outside 200, and all the other conditions are all identical among the I with fermentation.And living with the enzyme that the identical induction time of fermentation I is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation I.Work as OD
600Value is 320 or 350 o'clock, and the result does not all have significant difference yet.
Two, fermentation II:
Remove in the stage three, the inducing solution of adding is outside the mixed aqueous solution (mass ratio of sorbyl alcohol and methyl alcohol is 1: 10, and the concentration of sorbyl alcohol is 72g/L, and the concentration of methyl alcohol is 720g/L) of sorbyl alcohol and methyl alcohol, and all the other are all identical with method described in the experiment one.
3 repetitions are established in experiment, and the result takes the mean.
The enzyme biopsy is surveyed the result shown in Fig. 4 A, and during abduction delivering 19h, enzyme is lived and is the 1076u/ml fermented liquid in the fermented liquid, during abduction delivering 43h; Enzyme is lived and is the 1081u/ml fermented liquid in the fermented liquid, and during abduction delivering 64h, enzyme is lived and is the 1145u/ml fermented liquid in the fermented liquid; During abduction delivering 86h, enzyme is lived and is the 3258u/ml fermented liquid in the fermented liquid, during abduction delivering 111h; Enzyme is lived and is the 3485u/ml fermented liquid in the fermented liquid, and during abduction delivering 134h, enzyme is lived and is the 4316u/ml fermented liquid in the fermented liquid.
Ferment with following method: the OD that removes fermentation system when stopping to flow glycerol adding solution in the stage two
600Value is outside 200, and all the other conditions are all identical among the II with fermentation.And living with the enzyme that the identical induction time of fermentation II is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation II.Work as OD
600Value is 320 or 350 o'clock, and the result does not all have significant difference yet.
Ferment with following method: the sorbyl alcohol that in the stage three, adds was different with the mixed aqueous solution of methyl alcohol, all the other were all identical with method described in the real fermentation II.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, the mass ratio of sorbyl alcohol and methyl alcohol is 1: 5 (concentration of sorbyl alcohol is 144g/L, and the concentration of methyl alcohol is 720g/L).And living with the enzyme that the identical induction time of fermentation II is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation II.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, when the mass ratio of sorbyl alcohol and methyl alcohol was 1: 20 (concentration of sorbyl alcohol is 36g/L, and the concentration of methyl alcohol is 720g/L), the result did not all have significant difference yet.
Three, fermentation III:
Remove the OD of fermentation system when in the stage two, stopping to flow glycerol adding solution
600Value is outside 320, and all the other conditions are all identical in two with experiment.
3 repetitions are established in experiment, and the result takes the mean.
The enzyme biopsy is surveyed the result shown in Fig. 4 B, and during abduction delivering 12h, enzyme is lived and is the 1196u/ml fermented liquid in the fermented liquid, during abduction delivering 36h; Enzyme is lived and is the 1742u/ml fermented liquid in the fermented liquid, and during abduction delivering 48h, enzyme is lived and is the 2382u/ml fermented liquid in the fermented liquid; During abduction delivering 60h, enzyme is lived and is the 2122u/ml fermented liquid in the fermented liquid, during abduction delivering 72h; Enzyme is lived and is the 2822u/ml fermented liquid in the fermented liquid, and during abduction delivering 84h, enzyme is lived and is the 4364u/ml fermented liquid in the fermented liquid.
Ferment with following method: the OD that removes fermentation system when stopping to flow glycerol adding solution in the stage two
600Value is outside 200, and all the other conditions are all identical among the III with fermentation.And living with the enzyme that the identical induction time of fermentation III is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation III.Work as OD
600Value is 250 or 350 o'clock, and the result does not all have significant difference yet.
Ferment with following method: the sorbyl alcohol that in the stage three, adds was different with the mixed aqueous solution of methyl alcohol, all the other were all identical with method described in the real fermentation III.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, the mass ratio of sorbyl alcohol and methyl alcohol is 1: 5 (concentration of sorbyl alcohol is 144g/L, and the concentration of methyl alcohol is 720g/L).And living with the enzyme that the identical induction time of fermentation III is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation III.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, when the mass ratio of sorbyl alcohol and methyl alcohol was 1: 20 (concentration of sorbyl alcohol is 36g/L, and the concentration of methyl alcohol is 720g/L), the result did not all have significant difference yet.
Four, fermentation IV:
Remove in the stage three before the mixing solutions that adds sorbyl alcohol and methyl alcohol, the temperature with fermentation system transfers to 26 ℃ earlier, and all remains on outside 26 ℃ in the whole temperature of fermentation system in the process of inducing, and all the other are all with to test method described in three identical.
3 repetitions are established in experiment, and the result takes the mean.
It is as shown in Figure 5 that the result is surveyed in the enzyme biopsy, and during abduction delivering 12h, enzyme is lived and is the 1172u/ml fermented liquid in the fermented liquid, during abduction delivering 36h; Enzyme is lived and is the 2078u/ml fermented liquid in the fermented liquid, and during abduction delivering 48h, enzyme is lived and is the 2402u/ml fermented liquid in the fermented liquid; During abduction delivering 60h, enzyme is lived and is the 3252u/ml fermented liquid in the fermented liquid, during abduction delivering 72h; Enzyme is lived and is the 6020u/ml fermented liquid in the fermented liquid, and during abduction delivering 84h, enzyme is lived and is the 6336u/ml fermented liquid in the fermented liquid.
Ferment with following method: the OD that removes fermentation system when stopping to flow glycerol adding in the stage two
600Value is outside 200, and all the other conditions are all identical among the IV with fermentation.The result does not have significant difference among result and the fermentation IV.Work as OD
600Value is 250 or 350 o'clock, and the result does not all have significant difference yet.
Ferment with following method: the sorbyl alcohol that in the stage three, adds was different with the mixed aqueous solution of methyl alcohol, all the other were all identical with method described in the real fermentation III.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, the mass ratio of sorbyl alcohol and methyl alcohol is 1: 5 (concentration of sorbyl alcohol is 144g/L, and the concentration of methyl alcohol is 720g/L).And living with the enzyme that the identical induction time of fermentation III is selected in the detection fermented liquid, the result does not have significant difference among result and the fermentation III.In the mixed aqueous solution of sorbyl alcohol and methyl alcohol, when the mass ratio of sorbyl alcohol and methyl alcohol was 1: 20 (concentration of sorbyl alcohol is 36g/L, and the concentration of methyl alcohol is 720g/L), the result did not all have significant difference yet.
Claims (10)
1. a reorganization bacterium is the encoding sox importing starting strain with 'beta '-mannase, the reorganization bacterium that obtains; The coding gene sequence of said 'beta '-mannase is shown in 7-1407 position Nucleotide among the SEQ ID NO:1.
2. reorganization bacterium according to claim 1 is characterized in that: the encoding sox of said 'beta '-mannase imports starting strain through recombinant vectors; Said recombinant vectors is as follows shown in (1) or (2) or (3):
(1) dna fragmentation shown in the SEQ ID NO:1 is inserted between the XhoI and EcoRI restriction enzyme site of pAO815alpha, the recombinant vectors that obtains, note is made pAO α-1sman; Wherein, the XhoI restriction enzyme site is positioned at 5 of dna fragmentation shown in the SEQ ID NO:1 ' end upper reaches, and the EcoRI restriction enzyme site is positioned at 3 of dna fragmentation shown in the SEQ ID NO:1 ' end downstream;
(2), reclaim the 2932bp fragment with BamHI/BglII double digestion pAO α-1sman; With BamHI single endonuclease digestion pAO α-1sman, reclaim the big fragment of carrier; Said 2932bp fragment is connected with the big fragment of said carrier, the recombinant vectors that obtains, note is made pAO α-2sman;
(3), reclaim the 5864bp fragment with BamHI/BglII double digestion pAO α-2sman; With BamHI single endonuclease digestion pAO α-2sman, reclaim the big fragment of carrier; Said 5864bp fragment is connected with the big fragment of said carrier, the recombinant vectors that obtains, note is made pAO α-4sman;
Said pAO815alpha obtains according to following method: carrier pPICZ α is used the SacI/EcoRI double digestion, collect the 1000bp fragment; With SacI/EcoRI double digestion carrier pAO815, reclaim the big fragment of carrier; Said 1000bp fragment is connected with the big fragment of said carrier, and the recombinant vectors that obtains is pAO815alpha;
Said starting strain is a yeast, is preferably Pichia yeast, is preferably pichia pastoris phaff bacterial strain GSl15 again.
3. reorganization bacterium according to claim 1 and 2 is characterized in that: said reorganization bacterium is pichia pastoris phaff (Pichia pastoris) GS4SMAN, and its deposit number is CGMCC NO.4095.
4. a method for preparing 'beta '-mannase comprises the steps: arbitrary said reorganization bacterium among the claim 1-3 is inoculated in the fermention medium, and fermentation culture obtains 'beta '-mannase; All material notes in the fermenting container are made fermentation system.
5. method according to claim 4 is characterized in that: said fermention medium is the substratum that is used to cultivate Pichia yeast, and wherein contains glycerine;
Said fermentation culture is carried out according to following three phases successively:
Stage one: comprise the steps: to exhaust from the glycerine that inoculation begins to be cultured to the fermentation system;
Stage two: when comprising the steps: that glycerine in fermentation system exhausts, begin to flow glycerol adding or glycerine solution, be cultured to the OD of fermentation system
600Value reaches 200-350 or 200 or 250 or 320 or 350, stops to flow glycerol adding or glycerine solution;
Stage three: be following 1) or 2) shown in:
When 1) comprising the steps: that glycerine in fermentation system exhausts, in fermentation system, add mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, carry out abduction delivering;
When 2) comprising the steps: that glycerine in fermentation system exhausts; The temperature of fermentation system is transferred to 26 ℃～29 ℃ or 26 ℃ or 27 ℃ or 28 ℃ or 29 ℃; In fermentation system, add mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, carry out abduction delivering;
Said glycerine solution is by glycerine, PTM
1Solution and water are formed; Said methanol solution is by methyl alcohol, PTM
1Solution and water are formed.
6. according to claim 4 or 5 described methods, it is characterized in that: in the mixed aqueous solution of said sorbyl alcohol and methyl alcohol, the mass ratio of sorbyl alcohol and methyl alcohol is 1: 20～1: 5 or 1: 20 or 1: 10 or 1: 5.
7. according to arbitrary described method among the claim 4-6, it is characterized in that: in the mixed aqueous solution of said sorbyl alcohol and methyl alcohol, the concentration of sorbyl alcohol is 36g/L-144g/L or 36g/L or 72g/L or 144g/L, and the concentration of methyl alcohol is 720g/L.
8. according to arbitrary described method among the claim 4-7, it is characterized in that: the cultivation in said stage one and said stage two is 6.0 in the pH value, temperature is that 30 ℃, dissolved oxygen are to carry out under the condition more than 30%;
In the said stage three 1) in, abduction delivering is 6.0 in the pH value, temperature is that 30 ℃, dissolved oxygen are to carry out under the condition more than 30%;
In the said stage three 2) in, abduction delivering is 6.0 in the pH value, temperature is 26 ℃～29 ℃ or 26 ℃ or 27 ℃ or 28 ℃ or 29 ℃, dissolved oxygen are to carry out under the condition more than 30%.
9. according to arbitrary described method among the claim 4-8; It is characterized in that: in the said stage three; The time of abduction delivering is 60h-160h, is specially 12h-84h or 36h-84h or 48h-84h or 60h-84h or 19h-134h or 43h-134h or 64h-134h or 86h-134h or 111h-134h; Be specially 12h, 36h, 48h, 60h, 72h, 84h or 19h, 43h, 64h, 86h, 111h or 134h again;
In the said stage one, said inoculum size is 5%;
In the said stage three, said glycerine in fermentation system exhausts when Shi Weicong stops to flow glycerol adding to be counted, behind 0.5～1h;
Said dissolved oxygen is to be 30%-100% for dissolved oxygen more than 30%;
Said fermention medium is the BMGY liquid nutrient medium;
Said glycerine solution prepares according to following method: with 600g-650g or 625g glycerine, 10ml-14ml or 12mLPTM
1Solution mixes, and water is settled to 1 liter.
10. according to arbitrary described method among the claim 4-9, it is characterized in that:
In the said stage three; The said sorbyl alcohol and the mixed aqueous solution of methyl alcohol or the mode of methyl alcohol or methanol solution of in fermentation system, adding is: in fermentation system, add earlier methyl alcohol; Making the volumetric concentration of methyl alcohol in fermentation system is 3/1000-7/1000 or 5/1000; 1.5 hours-2.5 hours or after 2 hours, stream added mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol in fermentation system again;
In the said stage one, said dissolved oxygen be more than 30% be through bubbling air in fermentation system and the speed of regulating bubbling air with stir fermentation system and regulate the speed that stirs fermentation system and realize; Said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min;
In the said stage two; Said dissolved oxygen be more than 30% be through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and the stream rate of acceleration of regulating glycerine or glycerine solution realizes that said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min; The speed of said stream glycerol adding or glycerine solution is 5ml glycerine or glycerine solution/L fermention medium/h-15ml glycerine or glycerine solution/L fermention medium/h;
In the said stage three; Said dissolved oxygen be more than 30% be through bubbling air in fermentation system and regulate bubbling air speed, stir fermentation system and regulate the speed that stirs fermentation system and regulate the stream rate of acceleration realization of mixed aqueous solution or the methyl alcohol or the methanol solution of sorbyl alcohol and methyl alcohol, said stir speed (S.S.) is 200rpm-900rpm; The speed of said bubbling air is 0.5L/L fermention medium/min-1.5L/L fermention medium/min; Said stream adds the mixed aqueous solution of sorbyl alcohol and methyl alcohol or the speed of methyl alcohol is 2ml/L fermention medium/h-6ml/L fermention medium/h.
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| CN104099262A (en) * | 2013-04-09 | 2014-10-15 | 中国科学院微生物研究所 | Recombinant bacterium for constitutive expression of alkaline beta-mannanase |
| CN104559994A (en) * | 2013-10-14 | 2015-04-29 | 中国石油集团川庆钻探工程有限公司长庆井下技术作业公司 | Biological gel breaker |
| CN104762243A (en) * | 2014-01-08 | 2015-07-08 | 中国科学院微生物研究所 | Recombinant strain and method for preparing alkaline beta-mannanase |
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| CN103146724A (en) * | 2012-12-29 | 2013-06-12 | 湖北大学 | Reorganized mannase, genetically-engineered bacteria of recombined mannose and hydrolyzing preparation mannan oligosaccharide method |
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| CN104099262B (en) * | 2013-04-09 | 2017-02-01 | 中国科学院微生物研究所 | Recombinant bacterium for constitutive expression of alkaline beta-mannanase |
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| CN105754970B (en) * | 2014-12-19 | 2019-07-12 | 中国科学院微生物研究所 | A kind of application of alkaline ' beta '-mannase and its encoding gene and they |
| CN105754970A (en) * | 2014-12-19 | 2016-07-13 | 中国科学院微生物研究所 | Alkaline beta-mannase, encoding genes thereof and application of encoding genes |
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