CN101067117A - Method for producing thermostable xylanase by gene recombinant Pichia pastoris - Google Patents
Method for producing thermostable xylanase by gene recombinant Pichia pastoris Download PDFInfo
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Abstract
The present invention is process of producing heat resistant xylanase with genetically recombinant Pichia yeast. Xylanase gene is separated from Aspergillus niger var niger strain N402 and expressed effectively in a eukaryon expression system Pichia yeast. The genetic engineering yeast constructed with Aspergillus niger var niger strain N402 xylanase gene, Pichia yeast expressing vector PGAP Z alpha A and host strain Pichia pastoris SMD1168 and GS115 has capacity of producing heat resistant xylanase. The heat resistant xylanase produced with the genetically recombinant Pichia yeast has excellent heat resistance optimal acting temperature of 37-50 deg.c and excellent catalytic effect in the stomach of animal, and is especially suitable for use in feed industry.
Description
Technical field
The present invention relates to the method for producing heat resistant xylanase with genetically recombinant Pichia yeast.
Background technology
Xylan is the important component of plant hemicellulose, mainly is present in the cell walls of plant, accounts for 35% of dry cell weight, is occurring in nature the abundantest polysaccharide except that Mierocrystalline cellulose, also is a kind of huge renewable resources.But the xylan in wheat class (as wheat, rye, paddy, wheat bran, the rice bran etc.) feed as the non-starch polysaccharide antinutritional factor to the digestion of animal be absorbed with very strong anti-oxidant action.Mainly show as: in animal gastrointestinal tract, combine, reduce the absorption of nutritive ingredient with nutritive substance viscosity; In conjunction with digestive ferment and bile acide, influence digestion increases the defecation amount, and sticking excrement increases, contaminate environment; Cause that microbial reproduction increases in the animal gastrointestinal tract, and the animal products quality is caused disadvantageous effect.
Therefore, the most critical issue of affluent resources such as development and utilization wheat class cereal and bran is the antinutritional factor of eliminating in the feed.In these feeds, add effectively degradation of xylan of zytase (mainly being inscribe-β-1, the 4-zytase), weaken even eliminate its anti-oxidant action, improve the utilization ratio of feed, the concentration of ammonia and sulfide in the reduction air, pollution abatement, the health of promotion animal.Therefore, zytase has very considerable economic value, social effect and ecological benefits for resources such as exploitation cereal, brans.
The xylan degrading enzyme is that hydrolyzed xylan becomes xylo-bioses, wood oligose, and the prozyme system of a kind of complexity of a small amount of pectinose and wood sugar, extensively is present in natural bacterium and the fungi, has obtained the utmost point and use widely in fodder industry.Now become the focus of domestic and international research, and obtained a lot of impressive progresses, comprise that mainly the structure of zytase and the mechanism of action, zymetology and enzyme application characteristic, tool good characteristic zytase produce the screening of bacterial strain, produce bacterium and the aspects such as application of xylan in fodder industry by tradition and genetic engineering means improvement zytase.
Particularly Protocols in Molecular Biology develop rapidly in recent years, the research of xylan genetically engineered has obtained huge progress.For highly effective expression of xylanase with change its characteristic to adapt to commercial applications, separate at first from Bacillus subtilis PAPII5 from nineteen eighty-three Bernier etc. and to obtain xylanase gene so far, external scientific worker has cloned over one hundred kind of xylanase gene, and obtains activity expression in various host bacterium such as prokaryotic organism, filamentous fungus and yeast.In the prokaryotic expression system, the expression amount of recombined xylanase gene in intestinal bacteria is generally than low in the parent, even if increase, its expressed proteins also is difficult to be secreted in the substratum, but is limited in tenuigenin or the pericentral siphon chamber.From helping producing and further improving the angle of expression amount level, many researchers are attempted utilizing genus bacillus, streptomycete, aspergillus and the yeast etc. of secretor type to express as recipient bacterium.The expression system of having reported at present has Streptomyces parullus, Bacillus subtilis, Saccharomycescerevisiae, Pichia yeast, Kluyveromyces lactis, T.reesei, A.niger, A.oryzae etc., and the high expression level amount of the zytase of report is 20000 units per ml fermented liquids.
Since nineteen ninety-eight, there are Finland international corporation, Denmark NOVO company to produce and the large-scale popularization zytase respectively, obtained effect preferably in China.The domestic during this period research work that also has many research units and company to begin to carry out this respect, and obtained a large amount of achievements in research, some product comes into the market, no matter but present product be home and overseas do not possess heat-resistant quality.All to experience an of short duration high temperature granulating technology (75-93 ℃) in the course of processing of granulated feed, general zytase is understood loss of activity significantly under this high temperature, therefore can must have good thermostability by the zytase of real utilization and extention in feed; The final effect place of zytase in the feed is in the stomach of animal normal body temperature (37 ℃) on the other hand, and zytase also must keep greater activity simultaneously at normal temperatures.Therefore, how to solve and have active this contradiction of high enzyme at granulation high temperature with under the animal normal body temperature simultaneously, become present fodder enzyme preparation key in application sport technique segment.
At present, by genetic engineering technique xylanase gene being transformed on molecular level will be the effective means that addresses this problem, and also be the trend and the key of feeding from now on zytase development.
Summary of the invention
The method that the purpose of this invention is to provide a kind of producing heat resistant xylanase with genetically recombinant Pichia yeast.It is to isolate xylanase gene from aspergillus niger (Aspergillus niger var niger strain N402), successfully realizes xylanase gene efficiently expressing in the eukaryotic expression system pichia spp.
The present invention isolates xylanase gene from aspergillus niger (Aspergillus niger var niger strain N402), efficiently express in the eukaryotic expression system pichia spp.
Used aspergillus niger Aspergillus niger var niger strain N402 xylanase gene, its amino acid sequence coded such as accompanying drawing 6, the yeast expression vector PGAP Z alpha A and host strain Pichia pastoris (Pichia pastoris) SMD1168 that buy from Invitrogen Co. have been adopted, GS115, constructed gene engineering yeast has the ability of producing fire resistant xylanase, constructed gene engineering yeast removes has the form of host strain SMD1168 and GS115, outside heredity and the physiological and biochemical property, also comprise aspergillus niger Aspergillus niger var niger strain N402 xylanase gene, can the encode aminoacid sequence of accompanying drawing 6 of this gene has the ability of producing fire resistant xylanase.
Concrete grammar of the present invention and step:
One, the structure of engineering bacteria
(1) extraction of aspergillus niger genomic dna;
(2) adopt PCR reaction cloned DNA gene segment from the aspergillus niger genomic dna, primer and reaction conditions are as follows:
Primer 1 (F): 5 '-cggaattcgctcctgtgccggaacctg-3 '
Primer 2 (R): 5 '-cggaattcagaggagatcgtgacactggcgcg-3 '
Reaction conditions is: 94 ℃ of sex change 1min, and 50 ℃ of renaturation 1min30s, 72 ℃ are extended 2min30s, after 30 circulations, 72 ℃ of insulation 20min.
Also can be by the synthetic full gene of TaKaRa Dalian company, aspergillus niger xylanase gene (as shown in Figure 5), this gene can be optimized by gene site-directed technology and DNA total synthesis technology, must the encode aminoacid sequence of accompanying drawing 6 of gene after the optimization (is seen Van, D.B., De, G.L., Visser.J.and Van O.A..Patent:WO 9783153-A 5,1997).
(3) glue reclaims dna fragmentation: get an amount of pcr amplification product and carry out the agarose gel electrophoresis detection, reclaim test kit with the centrifugal glue of a small amount of post and carry out the glue recovery;
(4) structure of recombinant yeast expression vector: the xylanase gene fragment that the clone is obtained is connected construction of expression vector PGAP Z alpha-Xyl with expression vector PGAP Zalpha A by EcoR I site;
(5) pichia spp host's conversion: recombinant expression vector transforms pichia spp SMD1168 and GS115 respectively by electroporation, adopts pichia spp electricity Transformation Program, filters out respectively then and efficiently expresses transformant;
(6) shake flask fermentation of yeast transformant is cultivated: be inoculated in the BMGY substratum and cultivated 2 days from the flat board bacterium that takes a morsel, changed in the BMMY substratum inducing culture again over to two days, centrifugal removal cell obtains containing the supernatant liquor of zytase, then fermented liquid is carried out the alive and zymologic property detection of enzyme.
Two, adopt the engineering bacterium fermentation of above-mentioned structure to cultivate the method (seeing David R.Higgins, PichiaProtocols, Humana Press, new Jersey, USA, 1998) of producing zytase.
(1) shake flask fermentation of engineering bacteria is cultivated the method for production zytase with above-mentioned (six) step.
(2) the engineering bacteria method that enlarged culturing is produced zytase in the different scales fermentor tank: substratum is every liter of phosphoric acid 30ml, calcium sulfate 1g, vitriolate of tartar 18g, sal epsom 15g, potassium hydroxide 4g, glycerine 40g, 0.0004g vitamin H; Zymotechnique is 30 ℃ of temperature, pH5.0, and ventilation is 0.5vvm, stirs 100～500 rev/mins, and dissolved oxygen is controlled at more than 20%.Fermentation is divided into two stages, and the fs is the thalli growth stage, and behind 5% access seed, seed begins to grow in above-mentioned substratum, incubation time 48 hours, and when glycerine exhausted, dissolved oxygen can rise rapidly; Change subordinate phase this moment over to, and stream adds inducing culture (90% methyl alcohol, 0.004% vitamin H), and dissolved oxygen is maintained more than 20% all the time, and incubation time is 100 to 200 hours, and micro-filtration is removed the supernatant liquor that yeast cell obtains containing zytase.
Three, enzyme activity determination method and thick enzyme zymologic property
(1) enzyme activity determination method
1, principle:
Under certain conditions, the xylanase hydrolysis xylan produces reducing sugar, and reducing sugar can be with 3, nitroreduction in the 5-dinitrosalicylic acid becomes orange-yellow aminocompound, so can under the 550nm wavelength, carry out colorimetric, measure absorbancy, absorbancy is directly proportional with enzyme work.
2, enzyme reaction system:
Get the suitably fermented liquid adding test tube of dilution of 1ml,, add the substrate solution 1ml of similarity condition preheating then, behind 50 ℃ of accurate response 30min, add DNS reagent 3ml, in boiling water, boil 7min at 50 ℃ of preheating 2min.Take out the back and add 10mlddH
2O is 15ml to final volume, and OD is measured in the cooling back rapidly
550Control group is 100 ℃ of dilution enzyme liquid that boil 5min, and other steps are identical.
3, enzyme is lived and is defined:
To generate the amount of 1 μ mol wood sugar be an enzyme activity unit (U) to the every min catalytic substrate of (pH4.8,50 ℃) every ml enzyme liquid to be measured under certain condition.
(2) thick enzyme zymologic property
1, the optimal pH of enzyme effect: see Table 1 and Fig. 1
The optimal pH of table 1. enzyme effect
| pH | Relative enzyme (%) alive |
| 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 | 94.8 100 97.8 91.6 85.7 77.3 57.9 41 27.5 7.1 |
2, pH is to the influence of enzyme stability: see Table 2 and Fig. 2
Table 2.pH is to the influence of enzyme stability
| pH | Relative enzyme (%) alive |
| 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 | 91.1 93.8 90.3 85.8 81.2 77.4 72.3 66.4 62.1 57.8 |
3, the optimum temperuture of enzyme effect: see Table 3 and Fig. 3
The optimum temperuture of table 3. enzyme effect
| Temperature () | Relative enzyme (%) alive |
| 28 37 42 50 55 65 | 54.4 82.8 97.5 100 25.8 2.5 |
4, temperature is to the influence of enzyme stability: enzyme liquid is lived measuring enzyme again behind the heat treated different time respectively under 100 ℃ of conditions, sees Table 4 and Fig. 4
Table 4. temperature is to the influence of enzyme stability
| Time (min) | Relative enzyme (%) alive |
| 0 1 2 3 4 5 6 7 8 9 10 | 100 98 95 93 88 65 53 45 30 21 12 |
The beneficial effect that the present invention is compared with prior art had:
Pichia pastoris is the eukaryotic gene novel expression system.There is unique advantage in this system, and potentiality are very big, utilizes it to express foreign protein and has following advantage: 1. high expression level.Because expression vector utilizes the alcohol oxidase gene promoter very strong, the cell speed of growth is very fast, so the foreign protein output that this expression system is expressed is very high.For example the proteic output of tetanus toxin reaches 12g/L, and expression system commonly used at present is generally in the milligram level.So its expression amount is higher 10 times even 100 times than other system, this is a quantum jump aspect expression amount.2. high stable.Do not exist because the expression vector of this system is not the plasmid form with self-replicating, but be incorporated on the karyomit(e), so the bacterial strain that makes up is very stable.3. high secretion.Some signals in yeast saccharomyces cerevisiae series and leader are used for this expression system, it self biological characteristics in addition, its secreting, expressing can reach 10g/L, and this is very rare in known secreting, expressing system.In view of the big potentiality of tool of this expression system, so this expression system is significant for the exploitation of novel enzyme.
Pichia pastoris (Pichia pastoris) itself can not synthesize zytase, and the present invention imports the aspergillus niger xylanase gene by engineered means in Pichia pastoris, make Pichia pastoris produce zytase in a large number.By our experimental results show that, the xylanase gene that clones from Aspergillus niger var niger strain N402, its amino acids coding such as accompanying drawing 6, when being connected the recon that is constituted with yeast expression vector PGAP Z alphaA, when transforming the different host bacterium of pichia spp SMD1168 with GS115, can both obtain producing the transformant of zytase, and the enzyme that produces has above-mentioned identical characteristic.Because Pichia pastoris is very easy to realize high density fermentation to have high excretory characteristics, therefore be easy to realize a large amount of cheap aspergillus niger zytases of producing of industrialization.
The fire resistant xylanase of gene recombined Pichia pastoris production has fabulous heat-resistant quality among the present invention, heat treated is after 5 minutes under 100 ℃ of conditions, still has 65% remnant enzyme activity, and its optimum temperature scope is 37～50 ℃, can bring into play katalysis preferably in the stomach of animal, the fire resistant xylanase that the present invention produced is particularly suitable for being applied to fodder industry.
Description of drawings
Fig. 1 is the optimal pH of enzyme effect of the present invention.
Fig. 2 is the influence of pH of the present invention to enzyme stability.
Fig. 3 is the optimum temperuture of enzyme effect of the present invention.
Fig. 4 is the influence of temperature of the present invention to enzyme stability.
Fig. 5 is the coded zytase dna sequence dna of Aspergillus niger var niger strain N402.
Fig. 6 is Aspergillus niger var niger strain N402 and the coded zytase aminoacid sequence of recombinant yeast pichia pastoris.
Embodiment
Embodiment 1:
One, the structure of engineering bacteria
(1) extraction of aspergillus niger genomic dna: extracting method is seen Yao Bin etc., the Aspergillus niger strain screening of phytase generating and phytase gene clone thereof, Journal of Agricultural Biotechnology, 1998,6 (1): 1-6
(2) from the aspergillus niger genomic dna, adopt PCR reaction cloned DNA gene segment: the PCR concrete grammar see " CW Dieffenbach, round pcr experiment guide, Science Press " and＜TaKaRa Taq test kit specification sheets, primer and reaction conditions are as follows:
Primer 1 (F): 5 '-cggaattcgctcctgtgccggaacctg-3 '
Primer 2 (R): 5 '-cggaattcagaggagatcgtgacactggcgcg-3 '
Reaction conditions is: 94 ℃ of sex change 1min, and 50 ℃ of renaturation 1min30s, 72 ℃ are extended 2min30s, after 30 circulations, 72 ℃ of insulation 20min.
Also can be by the synthetic full gene of TaKaRa Dalian company, aspergillus niger xylanase gene (as shown in Figure 5), this gene also can be optimized by gene site-directed technology and DNA total synthesis technology, must the encode aminoacid sequence (Van of accompanying drawing 6 of gene after the optimization, D.B., De, G.L., Visser.J.and Van O.A..Patent:WO 9783153-A 5,1997).
(3) glue reclaims dna fragmentation: get an amount of pcr amplification product and carry out the agarose gel electrophoresis detection, reclaim test kit (reclaiming the test kit specification sheets by Shanghai China Shun glue operates) with the centrifugal glue of a small amount of post and carry out the glue recovery.
(4) structure of recombinant yeast expression vector: the xylanase gene fragment that the clone is obtained by EcoR I site and expression vector PGAP Zalpha A (available from Invitrogen Co., related base basic sequence and preparation method etc. see the Invitrogen specification sheets) be connected, with construction of expression vector PGAP Z alpha-Xyl; Concrete enzyme is cut and " molecular cloning experiment guide " [J.Sambrook, Science Press published in 1996] and carrier specification sheets are seen in method of attachment.
In addition, the preparation of the dephosphorylation of digestion with restriction enzyme, carrier, connection, competent escherichia coli cell, conversion and plasmid extraction etc. are all with reference to " molecular cloning experiment guide " [J.Sambrook, Science Press published in 1996].
(5) pichia spp host's conversion: recombinant expression vector transforms pichia spp SMD1168 and GS115 (available from Invitrogen Co.) respectively by electroporation, concrete grammar is seen Bio-Rad Gene-Pulser (electric shock instrument) working instructions, adopt pichia spp electricity Transformation Program, filter out respectively then and efficiently express transformant.
(6) shake flask fermentation of yeast transformant is cultivated: (see David R.Higgins, Pichia Protocols, Humana Press, new Jersey, USA, 1998) be inoculated in BMGY substratum [1% yeast extract from the flat board bacterium that takes a morsel, 2% peptone, 10ml 1Mol phosphate buffered saline buffer, 2ml 500 * vitamin H (20mg/100ml), 2% glycerine] the middle cultivation 2 days, change BMMY substratum [1% yeast extract again over to, 2% peptone, 10ml 1Mol phosphate buffered saline buffer, 2ml 500 * vitamin H (20mg/100ml), 2% methyl alcohol] in inducing culture two days, centrifugal removal cell obtains containing the supernatant liquor of zytase.Then fermented liquid is carried out the alive and zymologic property detection of enzyme.
Two, the engineering bacterium fermentation that adopts this patent to make up is cultivated the method (seeing David R.Higgins, PichiaProtocols, Humana Press, new Jersey, USA, 1998) of producing zytase.
(1) shake flask fermentation of engineering bacteria is cultivated the method for production zytase with above-mentioned (six) step.
(2) the engineering bacteria method that enlarged culturing is produced zytase in the different scales fermentor tank: substratum is every liter of phosphoric acid 30ml, calcium sulfate 1g, vitriolate of tartar 18g, sal epsom 15g, potassium hydroxide 4g, glycerine 40g, 0.0004g vitamin H.Zymotechnique is 30 ℃ of temperature, pH5.0, and ventilation is 0.5vvm, stirs 100～500 rev/mins (deciding according to the big small fermentor of difference), and dissolved oxygen is controlled at more than 20%.Fermentation is divided into two stages, and the fs is the thalli growth stage, and behind 5% access seed, seed begins to grow in above-mentioned substratum, incubation time 48 hours, and when glycerine exhausted, dissolved oxygen can rise rapidly; Change subordinate phase this moment over to, and stream adds inducing culture (90% methyl alcohol, 0.004% vitamin H), and dissolved oxygen is maintained more than 20% all the time, and incubation time is 100 to 200 hours, and micro-filtration is removed the supernatant liquor that yeast cell obtains containing zytase.
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| CN101880681A (en) * | 2010-04-30 | 2010-11-10 | 成都大学 | Maltoligosaccharide-based trehalose hydrolase gene sequence and preparation method of recombinant protein thereof |
| CN109652393A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院微生物研究所 | A kind of xylanase-m with high thermostability and its encoding gene and application |
| CN113430182A (en) * | 2021-08-09 | 2021-09-24 | 云南师范大学 | Bacterial laccase from Astrospiraceae of elephant intestinal tract and gene thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101880681A (en) * | 2010-04-30 | 2010-11-10 | 成都大学 | Maltoligosaccharide-based trehalose hydrolase gene sequence and preparation method of recombinant protein thereof |
| CN101880681B (en) * | 2010-04-30 | 2013-06-12 | 成都大学 | Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof |
| CN109652393A (en) * | 2017-10-12 | 2019-04-19 | 中国科学院微生物研究所 | A kind of xylanase-m with high thermostability and its encoding gene and application |
| CN113430182A (en) * | 2021-08-09 | 2021-09-24 | 云南师范大学 | Bacterial laccase from Astrospiraceae of elephant intestinal tract and gene thereof |
| CN114774461A (en) * | 2022-04-06 | 2022-07-22 | 暨南大学 | Application of Ash1p as negative regulatory factor in improving protein expression in host cell |
| CN114774461B (en) * | 2022-04-06 | 2023-05-26 | 暨南大学 | Application of Ash1p as a negative regulator in improving protein expression in host cells |
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