CN104651383A - Recombinant pichia pastoris engineering bacteria and production method thereof - Google Patents
Recombinant pichia pastoris engineering bacteria and production method thereof Download PDFInfo
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Abstract
The invention discloses recombinant pichia pastoris engineering bacteria. The recombinant pichia pastoris engineering bacteria comprises a codon-optimized Richter trichoderma xylanase gene, and the nucleotide sequence of the gene is shown in SEQ:1 in a sequence table. The invention also discloses a production method of the recombinant pichia pastoris engineering bacteria. The production method comprises the following steps: (1) performing synonymous mutation on the Richter trichoderma xylanase gene according to pichia pastoris flavored codons so that the Richter trichoderma xylanase gene is completely mutated into the pichia pastoris favored codons; (2) synthesizing an optimized xylanase gene by utilizing a total-gene synthesis technology, and connecting the optimized xylanase gene with a pMD20-T vector to build a cloning vector pMD20T-Xynopti; (3) building a recombinant expression vector pPICZalpha-Xynopti; (4) transforming a pichia pastoris host; (5) performing induction culture of a positive converter; (6) producing xylanase by small-scale fermentation by adopting the recombinant pichia pastoris engineering bacteria. The recombinant pichia pastoris engineering bacteria disclosed by the invention can be used for secreting a great amount of xylanase.
Description
Technical field
The present invention relates to genetically engineered field, particularly relate to a kind of recombinant yeast pichia pastoris engineering bacteria and production method thereof.
Background technology
Hemicellulose is the second the utilized natural resources enriched being only second to content of cellulose.Xylan is the important composition composition of hemicellulose in vegetable matter cell walls, be present in xylogen and cellulosic below, and closely to combine with xylogen and Mierocrystalline cellulose.Xylan is a class poly five-carbon sugar, and the five-carbon sugar primarily of multiple identical or different kind is coupled together by Isosorbide-5-Nitrae-β-D-wood sugar glycosidic bond, is one of Main Antinutritional Factors in feed.Zytase (xylanase) is a class glycoside hydrolase, can in catalysis xylan molecule 1, the endo hydrolysis of 4-β-D-wood sugar glycosidic bond, its main hydrolysate is xylo-oligosaccharide more than xylo-bioses and xylo-bioses, also have a small amount of wood sugar and pectinose, this effect had both reduced the anti-oxidant action of xylan, was translated into nutritional substance again.
Large quantity research shows, zytase can improve the utilization ratio of all kinds feed, reduces the viscosity of gi tract chyme, promotes livestock birds health, and it is developed at feed resource and improves in the utilization ratio of cheap byproduct, has broad prospects.In addition zytase is in Pulp industry, is used as pulp bio-bleaching; The hydrolysate (wood sugar and xylo-oligosaccharide) of xylan can be applicable to food service industry, such as thickening material, fat substitute or anti-freezing foodstuff additive; In pharmaceutical industry, xylan is combined with other materials, can the release of slow pharmaceutical cpd; Also can be applicable to wine industry and energy conversion aspect.Visible zytase has huge using value and Research Prospects.
Abroad the research work of zytase is carried out comparatively early, and enter the suitability for industrialized production stage already, and China's research is more late, also not can be used in the bacterial strain of large-scale production zytase at present.Zytase produces mainly through fermentable, and comprise bacterium, actinomycetes, fungi and some yeast etc., filamentous fungus causes paying special attention to of people.This is mainly because filamentous fungus can by the xylanase secretion that produces in substratum, and the generation of zytase is higher compared with other fungus and bacteriums.
Although the Application Areas of zytase is very extensive, but because the enzyme that in Natural strains, zytase output is lower, production cost is expensive, be separated is not high, product enzyme efficiency is bad, zymologic property does not reach industrial application requirement, and these all govern the application and development of zytase.Along with the development of modern molecular biology technique, by genetic engineering technique, building the high-effective xylanase engineering strain with potential using value, carry out codon optimized, improving the expression level of zytase, to applying in the industrial production.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of recombinant yeast pichia pastoris engineering bacteria that is stable, that can secrete zytase in a large number and production method thereof.
A gene for codon optimized Li's Trichoderma zytase, the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
A kind of recombinant yeast pichia pastoris engineering bacteria, it comprises gene order of the present invention.
The application of recombinant yeast pichia pastoris engineering bacteria of the present invention in animal nutrition and feed industrial circle, described recombinant yeast pichia pastoris engineering bacteria can secrete zytase in a large number, and range of reaction temperature is 40-60 DEG C, and pH value range is 4-8.
A production method for recombinant yeast pichia pastoris engineering bacteria, comprises the steps:
(1) according to pichia spp preference codon, Xylanase from Trichoderma reesei gene is carried out same sense mutation, all sport pichia spp preference codon, the nucleotide sequence of the gene after sudden change is as shown in SEQ ID NO:1 in sequence table;
(2) utilize the xylanase gene after full genome synthetic technology synthesis optimizing, be connected with pMD20-T carrier, build cloning vector pMD20T-Xynopti;
(3) recombinant expression vector builds: M-Zyme gene fragment recovery obtained, after EcoRI and Kpn I double digestion, is connected with the plasmid pPICZ α A cut through same restriction enzyme, construction of expression vector pPICZ α A-Xynopti;
(4) conversion of pichia spp host: recombinant expression plasmid pPICZ α A-Xynopti after electric transformed wild type pichia pastoris X-33, and filters out positive transformant and carries out abduction delivering;
(5) positive transformant inducing culture: picking list yeast proceeds in BMMY substratum after transferring and cultivate 16-18h in BMGY substratum and carries out abduction delivering from flat board, culture obtains, containing the supernatant of heavy zytase, utilizing SDS-PAGE to detect it through centrifugal post analysis;
(6) recombinant yeast pichia pastoris engineering bacteria fermentative production zytase is on a small scale adopted.
The production method of recombinant yeast pichia pastoris engineering bacteria of the present invention, wherein adopt the method for recombinant yeast pichia pastoris engineering bacteria small-scale fermentative production zytase to carry out in 30L liquid fermenting system in step (6), process was divided into for three stages carried out:
First stage: the positive transformant list bacterium colony in inoculation step (5) in the 250mL shaking flask that 25mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as first order seed fermented liquid;
Subordinate phase: get described first order seed fermented liquid 20ml and be inoculated in the 2L shaking flask that 400mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as secondary seed fermented liquid;
Phase III:
Inoculum size with 2% is inoculated in the 30L fermentor tank be equipped with in 17L glycerine-BSM substratum, and 320r/min, cultivates under the condition of 29 DEG C, and the omnidistance adjustment carrying out pH value with 28% ammoniacal liquor, remains on 5.3; Described glycerine-BSM substratum comprises the composition of following mass percent: glycerine: 5%; Ammonium sulfate: 4%; K
2pO
4: 0.5%; CaSO
42H
2o:0.1%; K
2sO
4: 1.6%; MgSO
47H
2o:1.5%; KOH:0.15%; PTM1:0.43%; 0.02% vitamin H: 0.6%; Defoamer: 0.1%; Synergistic agent: 0.1%; The described glycerine of mentioned component formation all soluble in water-BSM substratum;
After cultivating 24h, carry out feed supplement, feeding volume is 3.4L, until feed supplement terminates, feed supplement formula is 50% glycerine; 1.5%PTM1; Glycerine and the described feed supplement of PTM1 formation soluble in water;
Treat that carbon source exhausts, when namely DO value rises rapidly, start induction after hungry 1h, the inductor formula used in Induction Process is 100% methyl alcohol, and wherein mass percent is 0.02% vitamin H of the PTM1 and 1% of 1.5% in addition.
The production method of recombinant yeast pichia pastoris engineering bacteria of the present invention, described in step (4), electric transformed wild type pichia pastoris X-33 specifically comprises the steps:
A) mixed with the above-mentioned cell suspension of 80 μ L by the linearizing recombinant plasmid of 10 ~ 20 μ g, the 2mm diameter electricity going to precooling transforms in cup, ice bath 5min;
B) parameters 25 μ F, voltage are that 1500V is electroporated;
C) after electric shock terminates, add rapidly the 1mol/L sorbyl alcohol of 1ml precooling, go in the EP pipe of 1.5mL after thalline is mixed, place 1 ~ 2h in 30 DEG C of incubators;
D) the bacterium liquid got after 200 ~ 300 μ L conversions is coated on YPD+Zeocin flat board, cultivates 2 ~ 3 days for 30 DEG C, until single bacterium colony occurs.
The production method of recombinant yeast pichia pastoris engineering bacteria of the present invention, in step (4), the method for screening positive transformant specifically comprises the steps: that the screening of recombinant yeast pichia pastoris positive transformant adopts the single bacterium colony on picking YPD+Zeocin flat board to carry out bacterium colony PCR method; The screening of speed spot is carried out on MD/MM flat board; For determining that selected bacterium colony is positive transformant further, by its abduction delivering in shaking flask, by SDS-PAGE and enzyme analyzing and testing expression product alive.
The production method of recombinant yeast pichia pastoris engineering bacteria of the present invention, described in step (5), positive transformant inducing culture specifically comprises the steps:
A) picking positive transformant is inoculated in the 250mL triangular flask containing 50mL liquid B MGY substratum, 30 DEG C, 220r/min, cultivates 24h;
B) the centrifugal 10min of 4000rpm, collects thalline, and with containing the resuspended thalline of 50mL liquid B MMY substratum in 250mL triangular flask, 28 DEG C, 220r/min, in the process of cultivating, adding a methyl alcohol every 24h, is 1% (V/V) to final concentration;
C) while adding methanol induction, get 1mL substratum in centrifuge tube every 24h, 4 DEG C of centrifugal 10min of 4000rpm, draw supernatant in clean centrifuge tube, for analyzing the Best Times of expression level and the rear collecting cell of induction.All supernatants or cell precipitation are stored in-80 DEG C.
The production method difference from prior art of recombinant yeast pichia pastoris engineering bacteria of the present invention is:
Pichia spp heterologous expression system is a kind of novel expression system being developed the beginning of the eighties and developing.This system expresses with prokaryotic expression, yeast saccharomyces cerevisiae and mammalian cell expression system compares, and has many advantages: 1. toxigenic capacity is low.Can grow in the substratum of cheapness, same with E.coli and yeast saccharomyces cerevisiae simple during operation; 2. heterogenous expression level is high.Exogenous gene expression product not only in born of the same parents' inner accumulated but also can be secreted in substratum, and the secretory volume of the foreign protein of secretion is large, is beneficial to industrial production and separation and purification; 3. containing strong promotor.Expression vector contains distinctive alcohol oxidase (AOX) gene promoter.4. genetic stability is good.Build bacterial strain Stability Analysis of Structures, expression vector can directly by exogenous origin gene integrator to yeast chromosomal, not easily degenerate; 5. secreting, expressing.Have employed in the present invention at present powerful secreted expression carrier system it, pPICZ α A, this carrier introduces the α-mating factor (α-factor) of yeast saccharomyces cerevisiae, recombinant protein secretion capacity is strengthened greatly, can reach 10g/L.6. the purity of protein of pichia spp secretion is high.Pichia spp self only secretes small amount of albumen, and separation and purification foreign protein is easier to.7. carry out protein modified, play protected protein effect.Compared with yeast saccharomyces cerevisiae, the albumen of pichia spp to secretion carries out that N-, 0-are glycosylation modified and degree is moderate, and this contributes to the activity increasing secretory protein.
Host Strains pichia spp of the present invention itself can not secrete zytase, utilizes genetic engineering technique, proceeds in pichia spp genome, can secrete zytase in a large number, for suitability for industrialized production by the Xylanase from Trichoderma reesei gene of optimization.Genetically engineered recombinant yeast pichia pastoris only in shaking flask the secretion of zytase just reach 13014U/mL; And recombined xylanase has high purity (more than 90%), therefore can omit purification procedures in developmental process, larger reduces production cost.
The zytase stability that in the present invention, recombinant yeast pichia pastoris is produced is better, and its suitable range of reaction temperature is 40-60 DEG C, pH scope is 4-8, well corresponding function can occur, be more suitable in animal nutrition and feed industrial circle in animal digestive tract.
Be described further below in conjunction with the production method of accompanying drawing to recombinant yeast pichia pastoris engineering bacteria of the present invention.
Accompanying drawing explanation
Fig. 1 is restructured Pichia pastoris in expression product electrophoretic analysis result in the present invention;
Fig. 2 is recombinant yeast pichia pastoris xylanase secretion discharge curve figure in the present invention;
Fig. 3 is the analysis chart of recombined xylanase optimal reaction pH value in the present invention;
Fig. 4 is the analysis chart of recombined xylanase optimal reactive temperature in the present invention;
Fig. 5 is the analysis chart of recombined xylanase temperature stability in the present invention;
Fig. 6 is the analysis chart of recombined xylanase pH stability in the present invention.
Embodiment
The invention discloses a kind of codon optimized Li's Trichoderma xylanase gene, the nucleotide sequence of described gene as shown in SEQ ID NO:1 in sequence table, its coding aminoacid sequence (i.e. aminoacid sequence of Li's Trichoderma zytase) as shown in SEQ ID NO:2 in sequence table.The nucleotide sequence of the xylanase gene before optimization is as shown in SEQ ID NO:3 in sequence table.
Application claims protects a kind of recombinant yeast pichia pastoris engineering bacteria, and it comprises gene order of the present invention.
The application of recombinant yeast pichia pastoris engineering bacteria of the present invention in animal nutrition and feed industrial circle, described recombinant yeast pichia pastoris engineering bacteria can secrete zytase in a large number, and range of reaction temperature is 40-60 DEG C, and pH value range is 4-8.
Trichodermareesei (Trichoderma reesei) xylanase gene codon by full genome synthetic technology, is all optimized for pichia spp preference codon by the present invention, and in eukaryotic expression system pichia spp high expression.
Present invention uses the xylanase gene of optimization and the aminoacid sequence of coding thereof, utilize Invitrogen company expressive host bacterium P.pastoris X-33 and efficient expression vector pPICZ α A, according to the Preference of pichia spp codon, zytase codon is optimized, constructed gene engineering microzyme is except having the ability of efficient secretory expression recombined xylanase, also there is the form of Host Strains X-33, heredity and physiological and biochemical property, the xylanase gene of Trichoderma.reesei is contained in its genome, the aminoacid sequence of this gene encodes as shown in SEQ ID NO:2 in sequence table, there is the ability of High-efficient Production zytase.
The production method of recombinant yeast pichia pastoris engineering bacteria of the present invention comprises the steps:
(1) according to pichia spp preference codon, Xylanase from Trichoderma reesei gene is carried out same sense mutation, all sport pichia spp preference codon, the nucleotide sequence of the gene after sudden change is as shown in SEQ ID NO:1 in sequence table;
(2) utilize the xylanase gene after full genome synthetic technology synthesis optimizing, be connected with pMD20-T carrier, build cloning vector pMD20T-Xynopti;
(3) recombinant expression vector builds: M-Zyme gene fragment recovery obtained, after EcoR I and Kpn I double digestion, is connected with the plasmid pPICZ α A cut through same restriction enzyme, construction of expression vector pPICZ α A-Xynopti;
(4) conversion of pichia spp host: recombinant expression plasmid pPICZ α A-Xynopti after electric transformed wild type pichia pastoris X-33, and filters out positive transformant and carries out abduction delivering;
(5) positive transformant inducing culture: a small amount of yeast of picking proceeds in BMMY substratum after transferring and cultivate 16-18h in BMGY substratum and carries out abduction delivering from flat board, culture obtains the supernatant containing heavy zytase through centrifugal post analysis, SDS-PAGE is utilized to detect it, as shown in Figure 1;
(6) recombinant yeast pichia pastoris engineering bacteria fermentative production zytase is on a small scale adopted.
More specifically, the detailed step of the construction process of recombinant yeast pichia pastoris engineering bacteria of the present invention is as follows:
Plasmid double digestion and segment reclaim:
By the T cloned plasmids containing xylanase gene with containing after carrier pPICZ α A plasmid extraction, carry out double digestion with restriction enzyme EcoRI and Kpn I, digestion products reclaims for subsequent use after agarose gel electrophoresis purifying.
Double digestion system is as follows:
Be placed in 37 DEG C of enzymes and cut through night.
DNA ligation:
The gene fragment reclaimed through gel and expression vector pPICZ α A carrier carry out ligation by T4 ligase enzyme.
Reaction system is as follows:
16 DEG C are reacted 5 ~ 6 hours.
Bacillus coli DH 5 position competence makes and transforms:
Adopt Calcium Chloride Method to prepare bacillus coli DH 5 alpha competent cell, concrete grammar is as follows:
1) the mono-colony inoculation of DH5 α that grows fine on the LB flat board of antibiotic-free of picking is in 10mL LB liquid nutrient medium, and 37 DEG C, 190r/min shaking culture is spent the night;
2) draw 2mL and activate the bacterium liquid that spends the night in the fresh LB liquid nutrient medium of 50mL, 37 DEG C, 190r/min continues to be cultured to OD
600value about 0.3 ~ 0.4;
3), after the bacterium liquid after cultivation being transferred to aseptic 50mL centrifuge tube mid-ice bath on ice 10min, 4 DEG C, the centrifugal 7min of 5000r/min, abandons supernatant liquor;
4) the 0.1mol/L CaCl of the aseptic precooling of 25mL is added
2solution in centrifuge tube, Eddy diffusion thalline, ice bath 20min, 4 DEG C, the centrifugal 7min of 5000r/min;
5) abandon supernatant liquor, Xiang Guanzhong adds the 0.1mol/L CaCl of the aseptic precooling of 2mL
2solution is suspended bacteria body again;
6) competent cell just done can be directly used in conversion test, also remaining competent cell can be added final concentration be 15 ~ 20% glycerine be sub-packed in 1.5mL centrifuge tube (100 μ L/ manage) and put-70 DEG C of preservations.
7) 10 μ L are joined in 100 μ L DH5 α competence containing the connection product of goal gene segment, mix gently with rifle, ice bath 30min;
8) pipe is placed in 42 DEG C of water-baths, thermal shock 100s, is then placed in ice bath 5min on ice;
9) add the LB liquid nutrient medium of 850 μ L, mix gently, 37 DEG C, 170r/min, cultivate about 1h;
10) 5000r/mim, centrifugal 4min, remove part supernatant, and the bacterium liquid drawing 200 μ L with rifle coats LB+Amp flat board, is inverted cultivation 13 ~ 17h for 37 DEG C.Observe bacterium colony formational situation on substratum, preliminary screening positive colony.
The screening of positive conversion:
Select single bacterium colony of normal growth on 25 μ g/mL Zeocin flat boards, carry out PCR, qualification is cut and checked order to enzyme.
Recombinant expression plasmid pPICZ α A-Xynopti is prepared and linearizing in a large number:
Extract plasmid by after the positive list bacterium colony enlarged culturing picked out, with Sac I, linearization process carried out to it for subsequent use.
Reaction system is as follows:
37 DEG C are reacted 3 ~ 4 hours.
Pichia spp competent cell makes:
1) the mono-bacterium colony of picking yeast X33, is seeded in the 50ml triangular flask containing 5ml YPD substratum, 30 DEG C, 250rpm overnight incubation;
2) inoculation culture cell is in 50mL fresh YPD medium, 30 DEG C, and 250rpm overnight incubation, to OD
600reach 1.3 ~ 1.5;
3) in 4 DEG C, the centrifugal 5min of 4000rpm abandons supernatant and collects thalline, and adds the sterilized water 50mL of precooling, abundant suspension cell;
4) in 4 DEG C, the centrifugal 5min of 4000rpm abandons supernatant and collects thalline, and adds the sterilized water 20mL of precooling, abundant suspension cell;
5) in 4 DEG C, the centrifugal 5min of 4000rpm collects thalline, adds the resuspended thalline of 1mol/L sorbyl alcohol of 5mL precooling;
6) in 4 DEG C, the centrifugal 5min of 4000rpm collects thalline, and the 1mol/L sorbyl alcohol adding 200 μ L precoolings is blown and beaten gently, abundant suspension cell, packing, and the same day uses.
Pichia pastoris X-33 is electroporated:
A) mixed with the above-mentioned cell suspension of 80 μ L by the linearizing recombinant plasmid of 10 ~ 20 μ g, the 2mm diameter electricity going to precooling transforms in cup, ice bath 5min;
B) parameters 25 μ F, voltage are that 1500V is electroporated;
C) after electric shock terminates, add rapidly the 1mol/L sorbyl alcohol of 1ml precooling, go in the EP pipe of 1.5mL after thalline is mixed, place 1 ~ 2h in 30 DEG C of incubators;
D) the bacterium liquid got after 200 ~ 300 μ L conversions is coated on YPD+Zeocin flat board, cultivates 2 ~ 3 days for 30 DEG C, until single bacterium colony occurs.
Positive transformant selection systems:
The screening of recombinant yeast pichia pastoris positive transformant adopts the single bacterium colony on picking YPD+Zeocin flat board to carry out bacterium colony PCR method; The screening of speed spot is carried out on MD/MM flat board; For determining that selected bacterium colony is positive transformant further, by its abduction delivering in shaking flask, by SDS-PAGE and enzyme analyzing and testing expression product alive.
The inducing culture of yeast-positive transformant:
A) picking positive transformant is inoculated in the 250mL triangular flask containing 50mL liquid B MGY substratum, 30 DEG C, 220r/min, cultivates 24h;
B) the centrifugal 10min of 4000rpm, collects thalline, and with containing the resuspended thalline of 50mL liquid B MMY substratum in 250mL triangular flask, 28 DEG C, 220r/min, in the process of cultivating, adding a methyl alcohol every 24h, is 1% (V/V) to final concentration;
C) while adding methanol induction, get 1mL substratum in centrifuge tube every 24h, 4 DEG C of centrifugal 10min of 4000rpm, draw supernatant in clean centrifuge tube, for analyzing the Best Times of expression level and the rear collecting cell of induction.All supernatants or cell precipitation are stored in-80 DEG C.
The method adopting recombinant yeast pichia pastoris engineering bacteria to produce zytase in step (6) is carried out in 30L liquid fermenting system, and process was divided into for three stages carried out:
First stage: the positive transformant list bacterium colony in inoculation step (5) in the 250mL shaking flask that 25mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as first order seed fermented liquid;
Subordinate phase: get described first order seed fermented liquid 20ml and be inoculated in the 2L shaking flask that 400mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as secondary seed fermented liquid;
Phase III:
Inoculum size with 2% is inoculated in the 30L fermentor tank be equipped with in 17L glycerine-BSM substratum, and 320r/min, cultivates under the condition of 29 DEG C, and the omnidistance adjustment carrying out pH value with 28% ammoniacal liquor, remains on 5.3; Described glycerine-BSM substratum comprises the composition of following mass percent: glycerine: 5%; Ammonium sulfate: 4%; K
2pO
4: 0.5%; CaSO
42H
2o:0.1%; K
2sO
4: 1.6%; MgSO
47H
2o:1.5%; KOH:0.15%; PTM1:0.43%; 0.02% vitamin H: 0.6%; Defoamer: 0.1%; Synergistic agent: 0.1%; The described glycerine of mentioned component formation all soluble in water-BSM substratum;
After cultivating 24h, carry out feed supplement, feeding volume is 3.4L, until feed supplement terminates, feed supplement formula is 50% glycerine; 1.5%PTM1; Glycerine and the described feed supplement of PTM1 formation soluble in water;
Treat that carbon source exhausts, when namely DO value rises rapidly, start induction after hungry 1h, the inductor formula used in Induction Process is 100% methyl alcohol, and wherein mass percent is 0.02% vitamin H of the PTM1 and 1% of 1.5% in addition.
Induction flow acceleration formula: additional amount per hour (mL/h)=(aperture/cycle) × flow × 60min, (aperture: s; Cycle: s; Flow: mL/min.)
Methanol feed rate: carry out in three stages, 24mL/h, 36mL/h, 48mL/h.And speed regulates aperture and cycle accordingly.(produce enzyme curve and see Fig. 3)
Host Strains pichia spp of the present invention itself can not secrete zytase, utilizes genetic engineering technique, proceeds in pichia spp genome, can secrete zytase in a large number, for suitability for industrialized production by the Xylanase from Trichoderma reesei gene of optimization.Genetically engineered recombinant yeast pichia pastoris only in shaking flask the secretion of zytase just reach 13014U/mL; And recombined xylanase has high purity (more than 90%), therefore can omit purification procedures (see Fig. 1 and Fig. 2) in developmental process, larger reduces production cost.
Enzyme activity determination method and characterization analysis:
(1) enzyme activity determination method
1, measuring principle: under certain condition, substrate xylan under the effect of zytase catalytic hydrolysis under discharge reducing sugar, reducing sugar is by 3, the nitroreduction of 5-dinitrosalicylic acid becomes orange-yellow aminocompound, the absorbance of reducing sugar can be measured under 550nm wavelength, compared with known typical curve, thus calculate enzymic activity.
2, enzyme reaction system: get the enzyme liquid 0.5mL of suitably dilution in test tube, preheating 2min at 50 DEG C, then add 1% preheated xylan substrate of 1.5mL.Add 3mL DNS reagent react 10min in 50 DEG C of water-baths after, in boiling water, boil 5min, after add 10mL distilled water, in 540nm measure absorbancy.
3, Mei Huo unit: it is 1 unit of enzyme activity that unit enzymic activity is defined as the per minute hydrolyzed xylan enzyme amount formed required for 1 μm of oL wood sugar.
(2) zymologic property (the results are shown in accompanying drawing 3,4,5 and 6)
1, optimal pH is determined: respectively with the buffer xylan substrate solution of different pH (3.0 ~ 8.0), measures its enzyme and lives, determine its optimal reaction pH, see Fig. 3 at 50 DEG C;
2, optimal reactive temperature: with the buffer xylan substrate solution of optimum pH, measures relative activity respectively, measures its optimal reactive temperature, see Fig. 4 under differing temps (20 ~ 90 DEG C);
3, temperature stability: after zytase is incubated 30min respectively at 50 DEG C, 60 DEG C and 70 DEG C, measures its remnant enzyme activity (with uninsulated enzyme in contrast), sees Fig. 5;
4, pH stability: after zytase hatches 30min in different pH (3.0 ~ 8.0) damping fluid, measures its remnant enzyme activity (with the enzyme of not hatching in contrast), sees Fig. 6;
Adopt aforesaid method to zytase characterization analysis of the present invention:
Xylanase activity detects and adopts international reducing sugar method, and during mensuration, time of enzymatic reacting is 10min, pH is 5.0, and temperature is 50 DEG C.1 unit of xylanase activity (U) is defined as under certain condition, and per minute hydrolyzed xylan forms the enzyme amount required for 1 μm of oL wood sugar.Respectively with the buffer xylan substrate solution of pH 3.0 ~ 8.0, at 40 DEG C, measure its enzyme live; With the xylan substrate solution of the buffer 1% of optimum pH, at 30 ~ 90 DEG C of temperature, measure relative activity respectively, measure its optimal reactive temperature.
In sum, the present invention utilizes full genome to synthesize and codon-optimization techniques has prepared the Pichia yeast engineering of expressed xylanase, change the zytase that engineering bacterium expression obtains similar to native xylanases character, expression amount and purity high, suitability for industrialized is produced, and has a good application prospect.
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Claims (8)
1. a gene for codon optimized Li's Trichoderma zytase, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:1 in sequence table.
2. a recombinant yeast pichia pastoris engineering bacteria, is characterized in that, it comprises gene order according to claim 1.
3. the application of recombinant yeast pichia pastoris engineering bacteria in animal nutrition and feed industrial circle described in claim 2, described recombinant yeast pichia pastoris engineering bacteria can secrete zytase in a large number, and range of reaction temperature is 40-60 DEG C, and pH value range is 4-8.
4. a production method for recombinant yeast pichia pastoris engineering bacteria, is characterized in that: comprise the steps:
(1) according to pichia spp preference codon, Xylanase from Trichoderma reesei gene is carried out same sense mutation, all sport pichia spp preference codon, the nucleotide sequence of the gene after sudden change is as shown in SEQ ID NO:1 in sequence table;
(2) utilize the xylanase gene after full genome synthetic technology synthesis optimizing, be connected with pMD20-T carrier, build cloning vector pMD20T-Xynopti;
(3) recombinant expression vector builds: M-Zyme gene fragment recovery obtained, after EcoR I and Kpn I double digestion, is connected with the plasmid pPICZ α A cut through same restriction enzyme, construction of expression vector pPICZ α A-Xynopti;
(4) conversion of pichia spp host: recombinant expression plasmid pPICZ α A-Xynopti after electric transformed wild type pichia pastoris X-33, and filters out positive transformant and carries out abduction delivering;
(5) positive transformant inducing culture: picking list yeast colony proceeds in BMMY substratum after transferring and cultivate 16-18h in BMGY substratum and carries out abduction delivering from flat board, culture obtains, containing the supernatant of heavy zytase, utilizing SDS-PAGE to detect it through centrifugal post analysis;
(6) recombinant yeast pichia pastoris engineering bacteria fermentative production zytase is on a small scale adopted.
5. the production method of recombinant yeast pichia pastoris engineering bacteria according to claim 4, it is characterized in that: adopt the method for recombinant yeast pichia pastoris engineering bacteria small-scale fermentative production zytase to carry out in 30L liquid fermenting system in step (6), process was divided into for three stages carried out:
First stage: the positive transformant list bacterium colony in inoculation step (5) in the 250mL shaking flask that 25mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as first order seed fermented liquid;
Subordinate phase: get described first order seed fermented liquid 20ml and be inoculated in the 2L shaking flask that 400mL YPD substratum is housed, 250r/min, 29 DEG C of shaking culture 18h, as secondary seed fermented liquid;
Phase III:
Inoculum size with 2% is inoculated in the 30L fermentor tank be equipped with in 17L glycerine-BSM substratum, and 320r/min, cultivates under the condition of 29 DEG C, and the omnidistance adjustment carrying out pH value with 28% ammoniacal liquor, remains on 5.3; Described glycerine-BSM substratum comprises the composition of following mass percent: glycerine: 5%; Ammonium sulfate: 4%; K
2pO
4: 0.5%; CaSO
42H
2o:0.1%; K
2sO
4: 1.6%; MgSO
47H
2o:1.5%; KOH:0.15%; PTM1:0.43%; 0.02% vitamin H: 0.6%; Defoamer: 0.1%; Synergistic agent: 0.1%; The described glycerine of mentioned component formation all soluble in water-BSM substratum;
After cultivating 24h, carry out feed supplement, feeding volume is 3.4L, until feed supplement terminates, feed supplement formula is 50% glycerine; 1.5%PTM1; Glycerine and the described feed supplement of PTM1 formation soluble in water;
Treat that carbon source exhausts, when namely DO value rises rapidly, start induction after hungry 1h, the inductor formula used in Induction Process is 100% methyl alcohol, and wherein mass percent is 0.02% vitamin H of the PTM1 and 1% of 1.5% in addition.
6. the production method of recombinant yeast pichia pastoris engineering bacteria according to claim 4, is characterized in that: described in step (4), electric transformed wild type pichia pastoris X-33 specifically comprises the steps:
A) mixed with the above-mentioned cell suspension of 80 μ L by the linearizing recombinant plasmid of 10 ~ 20 μ g, the 2mm diameter electricity going to precooling transforms in cup, ice bath 5min;
B) parameters 25 μ F, voltage are that 1500V is electroporated;
C) after electric shock terminates, add rapidly the 1mol/L sorbyl alcohol of 1ml precooling, go in the EP pipe of 1.5mL after thalline is mixed, place 1 ~ 2h in 30 DEG C of incubators;
D) the bacterium liquid got after 200 ~ 300 μ L conversions is coated on YPD+Zeocin flat board, cultivates 2 ~ 3 days for 30 DEG C, until single bacterium colony occurs.
7. the production method of recombinant yeast pichia pastoris engineering bacteria according to claim 4, is characterized in that: in step (4), the method for screening positive transformant specifically comprises the steps: that the screening of recombinant yeast pichia pastoris positive transformant adopts the single bacterium colony on picking YPD+Zeocin flat board to carry out bacterium colony PCR method; The screening of speed spot is carried out on MD/MM flat board; For determining that selected bacterium colony is positive transformant further, by its abduction delivering in shaking flask, by SDS-PAGE and enzyme analyzing and testing expression product alive.
8. the production method of recombinant yeast pichia pastoris engineering bacteria according to claim 4, is characterized in that: described in step (5), positive transformant inducing culture specifically comprises the steps:
A) picking positive transformant is inoculated in the 250mL triangular flask containing 50mL liquid B MGY substratum, 30 DEG C, 220r/min, cultivates 24h;
B) the centrifugal 10min of 4000rpm, collects thalline, and with containing the resuspended thalline of 50mL liquid B MMY substratum in 250mL triangular flask, 28 DEG C, 220r/min, in the process of cultivating, adding a methyl alcohol every 24h, is 1% (V/V) to final concentration;
C) while adding methanol induction, get 1mL substratum in centrifuge tube every 24h, 4 DEG C of centrifugal 10min of 4000rpm, draw supernatant in clean centrifuge tube, for analyzing the Best Times of expression level and the rear collecting cell of induction, all supernatants or cell precipitation are stored in-80 DEG C.
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