CN100354424C - Expression method for high temperature resistant xylanase and specific expression carrier for same - Google Patents
Expression method for high temperature resistant xylanase and specific expression carrier for same Download PDFInfo
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- CN100354424C CN100354424C CNB2003101135629A CN200310113562A CN100354424C CN 100354424 C CN100354424 C CN 100354424C CN B2003101135629 A CNB2003101135629 A CN B2003101135629A CN 200310113562 A CN200310113562 A CN 200310113562A CN 100354424 C CN100354424 C CN 100354424C
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- xylanase
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Abstract
本发明公开了一种耐高温木聚糖酶的表达方法及其专用表达载体。本发明所提供的耐高温木聚糖酶表达载体，是含有耐高温木聚糖酶基因mxynB(64)的重组毕赤酵母表达载体pPIC9K-mxynB(64)。本发明所提供的表达耐高温木聚糖酶的方法，是将重组毕赤酵母表达载体pPIC9K-mxynB(64)转化到毕赤酵母中，得到阳性克隆，诱导培养所述阳性克隆，表达耐高温木聚糖酶。本发明的木聚糖酶分泌量达207mg/L培养液，酶活达522U/mg(蛋白)。该木聚糖酶的最适作用温度为90℃，在沸水浴中处理30分钟后，酶活力仍能保持最高酶活力的70%，该酶活性在90℃检测时，其pH值稳定性在6.2～10.0范围内。本发明的方法在耐高温木聚糖酶的生产中具有广阔的工业前景。The invention discloses a method for expressing a high-temperature resistant xylanase and a special expression carrier thereof. The high temperature resistant xylanase expression vector provided by the present invention is a recombinant Pichia pastoris expression vector pPIC9K-mxynB (64) containing the high temperature resistant xylanase gene mxynB ( 64) . The method for expressing high-temperature-resistant xylanase provided by the present invention is to transform recombinant Pichia pastoris expression vector pPIC9K-mxynB (64) into Pichia pastoris to obtain positive clones, induce and cultivate the positive clones, and express high-temperature-resistant Xylanase. The secretion amount of the xylanase of the present invention reaches 207 mg/L culture fluid, and the enzyme activity reaches 522 U/mg (protein). The optimum action temperature of this xylanase is 90°C. After being treated in a boiling water bath for 30 minutes, the enzyme activity can still maintain 70% of the highest enzyme activity. When the enzyme activity is tested at 90°C, its pH value is stable at In the range of 6.2 to 10.0. The method of the invention has broad industrial prospects in the production of high temperature resistant xylanase.
Description
Technical field
The present invention relates to a kind of expression method and dedicated expression vector therefor thereof of fire resistant xylanase.
Background technology
Zytase is mainly produced by saprophytic fungus and bacterium, its xylan composition in can degrading plant cell walls, and be proved in agricultural and industrial everyway and have important use value.Xylan is to be only second to the biological polymer that content of cellulose second enriches; and be hemicellulose polysaccharide main in the plant cell wall; this allos polysaccharide is by β-1 by D-xylopyranose residue; 4 glycosidic links are formed by connecting, and this residue can be substituted by ethanoyl, arabinose aldehydic acid base or glucuronyl in different plants.This heterology of xylan makes complete degradation of xylan need the acting in conjunction of plurality of enzymes, β-1 wherein, the 4-D-zytase is wherein a kind of key enzyme, it can open the β between the D-xylose residues-1 on the main chain, 4 glycosidic links, thus the skeleton of polysaccharide is degraded into the wood oligose of short chain.So β-1, the 4-D-zytase has a very wide range of applications in feed and the paper industry at food.The thermostability of zytase is restraining factors in production application.But heat-stable microorganism is often cultivated difficulty, and what have also need cultivate under the high temperature anaerobic condition, and yield of enzyme is low and enzyme system is complicated, is difficult to obtain pure enzyme.
The xylanase gene of having found at present has xynA, xynB, xynC, xynZ etc.Studying more is xynA, but mainly is confined to clone and the expression of the xynA of bacterium, and xynA is at expression in escherichia coli but be not secreted into the extracellular.Donald etc. express with the xynA of thermophilic bacterium clone and S. cervisiae first, and the active zytase of secreting to the nutrient solution that has reaches 10mg/l.Research and utilization Kluyveromyces lactis expression systems such as Bergquist are expressed the zytase XynA of Thermotoga strain FjSS3B.1, and expression amount is 120mg/l on the shake-flask culture level.Research and utilization pichia yeast expression systems such as Berrin are expressed aspergillus niger xylanase gene XylA, and enzymic activity is 350U/mg (albumen).
People such as Karen E.Nelson in 1999 and Claire M.Fraser have finished the gene complete sequence determination of Thermotoga maritima MSB8 (Thermotoga maritima MSB8), and open reading frame TM0061 (xynA) and TM0070 (xynB) control synthetic two independently zytase XynA and XynB respectively.The xynA gene of Thermotoga maritima has been cloned and at expression in escherichia coli.This enzyme has thermostability, and optimum temperuture is 90 ℃, and optimal reaction pH value is 6.2.
Pichia spp allogeneic gene expression system is the yeast expression system of using morely in recent years, and this system is considered to express the idealized system of foreign protein, has very high commercial application value.The advantage of this system has the following aspects: it has the AOX1 strong promoter, can effectively regulate expression of exogenous gene; Characteristic of division and molecular biological research thereof to this system are relatively more thorough, help engineered operation; This expression system can also carry out the thalline high-density culture, helps fermentative production.These advantages all help pichia yeast expression system becomes the heterologous gene expression system that is widely used, and is used to express various foreign proteins.
The innovation and creation content
The expression vector that the purpose of this invention is to provide a kind of fire resistant xylanase.Its expression product can be secreted into outside the born of the same parents, is beneficial to the extraction of zytase, is fit to suitability for industrialized production.
The expression vector of fire resistant xylanase provided by the present invention is to contain high temperature resistant xylanase gene mxynB
(64)Restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)
Wherein, mxynB
(64)In the GenBank registration, retrieval is numbered: AY340789.
Restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)Obtain by following step:
(1) be template with Thermotoga maritima MSB8 genomic dna, pcr amplification goes out xylanase gene mxynB
(64)
(2) recovery of the amplified production in the step (1) is connected on the carrier pGEM-T Easy, picks out positive colony pGEM-T-mxynB
(64)
(3) with positive colony pGEM-T-mxynB
(64)With yeast expression vector pPIC9K reorganization, obtain restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)
Wherein, the pcr amplification primer in the step (1) can be the SEQ ID № in the sequence table: 1 and SEQ ID №: 2, wherein, SEQ ID №: 1 and SEQ ID №: 2 be the restriction enzyme site of NotI from the 4th at 5 ' end to the 11st bit base.
Second purpose of the present invention provides a kind of method of expressing fire resistant xylanase.
A kind of method of expressing fire resistant xylanase is with restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)Be transformed in the pichia spp, obtain positive colony, the described positive colony of inducing culture is expressed fire resistant xylanase.
In order to obtain better effect, it is the methanol induction cultivation positive colony of 0.25-1% with final concentration also that pichia spp is preferably the GS115 bacterial strain, and wherein the final concentration of methyl alcohol is preferably 0.5%.
Described positive colony is for containing foreign gene mxynB
(64), can contain the transformant of growing on the YPD flat board of 6.0mg/ml G418: GS115-9k-mxynB
(64)I, GS115-9k-mxynB
(64)II, GS115-9k-mxynB
(64)III and GS115-9k-mxynB
(64)IV wherein is preferably GS115-9k-mxynB
(64)II.
The present invention adopts gene clone technology, has successfully made up Yeast expression carrier pPIC9K-mxynB
(64)Utilize the electroporation method for transformation, obtain having copy number of foreign gene, in 5 liters of fermentor tanks, cultivated 260 hours cell density OD through methanol induction approximately greater than 20 high copy number yeast transformant
600Reach 250, the zytase secretory volume reaches the 207mg/L nutrient solution, and xylanase activity is 522U/mg (albumen).The optimal reactive temperature of this zytase is 90 ℃, and (100 ℃) were handled after 30 minutes in boiling water bath, enzyme activity can keep still that maximum enzyme lives 70%, measure down at 90 ℃ and to decompose xylan when active, its pH value stabilization is in the 6.2-10.0 scope.Method of the present invention has wide industrial prospect in the production of fire resistant xylanase.
Embodiment
Embodiment 1, restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)Structure
(1) xylanase gene mxynB
(64)The clone
According to the complete sequence of Thermotoga maritima MSB8 xylanase gene xynB and the feature of the multiple clone site on the yeast expression vector pPIC9K, the design synthetic primer:
FW：5’-ATA
gcggccgcTATGAAAATATTACCTTCTGTG-3’
REV：5’-ATA
gcggccgcTCATTTTCTTTCTTCTATCTTTT-3’
The restriction enzyme site of the synthetic NotI of primer two ends design is dwelt hot spore bacterium MSB8 genomic dna as template with the sea, amplifies xylanase gene mxynB by PCR method
(64), amplification condition is: 94 ℃ of pre-sex change 5 minutes, carry out 25 circulations again: 94 ℃ of sex change, 1 minute, extend 72 ℃, 1 minute 30 seconds, 72 ℃, extended 10 minutes 56 ℃ of 30 seconds, annealing.Amplified production (about about 1kb) reclaims and is connected on the carrier pGEM-T Easy, picks out positive transformant pGEM-T-mxynB through bacterium colony PCR
(64), and positive transformant is carried out the NotI enzyme cut detection.
By this gene being carried out sequential analysis and carrying out sequence homology relatively with xylanase gene (xynB) that people such as Karen E.Nelson and Claire M.Fraser announce, learn that this gene at the 64th bit base change has taken place, change into guanine (G) by original VITAMIN B4 (A), so the 22nd amino acids of this gene product zytase also changes asparagus fern amino acid (Asp) into by l-asparagine (Asn) accordingly.With this gene product called after mxynB
(64)This gene mxynB
(64)In the GenBank registration, retrieval is numbered: AY340789.
(2) positive colony pGEM-T-mxynB
(64)After cutting, the NotI enzyme, makes up restructured Pichia pastoris in expression carrier pPIC9K-mxynB with Yeast expression carrier pPIC9k (available from American I nvitrogen company) reorganization
(64), positive colony is cut detection through the NotI enzyme, and determines that by the method for sequencing the gene forward is connected on the expression plasmid.
Be cloned into the mxynB on the expression vector
(64)Gene passes through the determined dna sequence analysis once more, and the result shows that sequence is correct.
The expression of embodiment 2, fire resistant xylanase
(1) restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)Conversion and the screening of high copy number transformant
Restructured Pichia pastoris in expression carrier pPIC9K-mxynB
(64)After the linearizing of SalI complete degestion, electric shocking method transforms pichia spp GS115 bacterial strain, transforms the His that filters out the generation homologous recombination on the minimum medium MD flat board of Histidine (His) not containing
+The clone puts positive colony respectively then and is connected on MD and the MM flat board, carries out Mut
+/ Mut
sPhenotypic evaluation.
Utilization contains the YPD flat board of different concns G418, can filter out positive transformant fast, and can estimate the copy number that to be integrated into genomic foreign gene.Sterilized water with 2ml also suspends the sub-wash-out of single colony transformation on the MD flat board, and after the slight vibration, the dilution suitable multiple is with 10
5Cell concn be 0.25 containing G418 concentration respectively, 0.5,1.0,1.5,2.0,3.0,4.0mg/ml the YPD flat board on be coated with, random choose can contain 40 transformant list bacterium colonies of growing on the YPD flat board of 4.0mg/ml G418, the point be connected on the YPD flat board of the G418 that contains 6.0mg/ml, these 40 transformants can both be grown.Find that in screening process subsequently 4 transformant GS115-9k-mxynB are arranged
(64)I, II, III, IV can grow in the YPD of the G418 that contains 10mg/ml liquid nutrient medium, show that these 4 transformants contain the foreign gene of 40 copies at least.GS115-9k-mxynB wherein
(64)The growing way of II is best, the cell concn maximum.Therefore, select GS115-9k-mxynB
(64)II carries out inducing culture, express recombinant protein.
(2) abduction delivering of yeast positive transformant
Select His with high copy number
+/ Mut
+Positive transformant GS115-9k-mxynB
(64)II is inoculated in 25mlBMGY substratum [1% yeast extract, 2% peptone, 100mmol/l, potassiumphosphate pH6.0,1.34%YNB (YeastNitrogen Base), 1% glycerine, (4 * 10
-5) the % vitamin H] be cultured to OD in 30 ℃ of shaking tables
600Be about 2.0, centrifugal collection thalline is transferred to 100ml BMMY substratum [1% glycerine in the BMGY substratum changes 0.5% methyl alcohol into], continue to cultivate, per 24 hours sampling 1ml, and to add methyl alcohol be 0.5% in substratum to final concentration, abduction delivering 7 days, centrifugal collection supernatant.
Contain the yeast expression justacrine in the supernatant nutrient solution to the outer zytase of born of the same parents, nutrient solution was handled 10 minutes in 70 ℃ of water-baths, remove foreigh protein removing.In order to detect the efficient of yeast secreted expression, with the cell of centrifugal collection, through enzymolysis (Zymolyase) broken wall, and in 70 ℃ of water-baths after 10 minutes, centrifugal collection supernatant is the thick zyme extract of expressing in the yeast born of the same parents.The crude enzyme liquid that born of the same parents are interior and born of the same parents are outer is all through SDS-PAGE electrophoresis detection expressing quantity, the crude enzyme liquid that electrophoresis result shows outside the born of the same parents and born of the same parents are interior can both obtain electrophoretically pure zytase XynB after handling in 10 minutes through 70 ℃ of water-baths, its relative molecular weight is 42kDa, matches with the molecular weight (42.067KDa) of theoretical calculate.
Embodiment 3, fermentation culture GS115-9k-mxynB
(64)II produces fire resistant xylanase
(1) xylanase activity is measured
Oat xylan with 0.25% (oat spelts xylan, Sigma Chemical St.Louis, MO.USA) be substrate, adopt P-hydroxybenzoic acid hydrazides method (p-hydroxybenzoic acid hydrazide, PHBAH method) to measure the fire resistant xylanase vigor.Concrete operations are as follows:
1). get storage liquid 0.5M Trisodium Citrate in order respectively, the 1M S-WAT, 0.2M calcium chloride, each 10ml of 5M sodium hydroxide adds while stirring, to 100ml, adds 1.52 gram PHBAH with the distilled water constant volume again, the stirring that does not stop, the necessary matching while using of this solution;
2). weighing 1 gram xylan substrate is dissolved in the 100ml 50mM MES damping fluid (pH6.2), after fully stirring, the centrifugal precipitation of removing, supernatant liquor is about 0.25% zytase reaction substrate;
3). get xylan substrate insulation 10min under 50 ℃ of 190 μ l 0.25%, add the 10 μ l enzyme solution of dilution rationally,, add 400 μ l PHBAH solution again, in boiling water bath, keep 6min behind the mixing in 50 ℃ of following accurate response 10min;
4). after the sample cooling, with spectrophotometric determination absorbance (OD
405).The barren operation replaces the enzyme solution of dilution rationally with above-mentioned the same with 10 μ l water.1 enzyme activity unit (unit) is defined as: under above-mentioned experiment condition, it is 1 enzyme activity unit that per minute produces the required enzyme amount of 1 μ mol wood sugar, and OD
405Be equivalent to 0.067 μ mol wood sugar at=1 o'clock.
(2) fermentation culture (5L fermentor tank)
Substratum:
Seed culture medium: BMGY (1% yeast extract; 2% peptone; The 10mM potassiumphosphate, pH6.0; 1.34%YNB (Yeast Nitrogen Base); 4 * 10
(5)The % vitamin H; 1% glycerine);
Fermention medium: FBS basis salt [glycerine 4% (w/v)+3.5ml/L PTM
1];
Supplemented medium: (W/V contains 12mL/L PTM to 50% glycerine
1) feed supplement liquid;
Inducing culture: 100% methyl alcohol (contains 12mL/L PTM
1) induced liquid.
FBS basis salt: 85%H
3PO
4, 26.7mL; CaSO
4, 0.93g; K
2SO
4, 18.2g; MgSO
4.7H
2O, 14.9g; KOH, 4.13g; Glycerine, 40.0g; Water, 1000ml.
PTM
1Trace salts (filtration sterilization): CuSO
4.5H
2O, 6.0g; NaI, 0.08g; MnSO
4.H
2O, 3.0g; NaMoO
4.2H
2O, 0.2g; Boric acid, 0.02g; CoCl
2, 0.5g; ZnCl
2, 20.0g; FeSO
4.7H
2O, 65.0g; Vitamin H, 0.2g; H
2SO
4, 5.0mL; Water, 1000ml.
Seed culture: choose single bacterium colony from flat board, be linked in the seed culture medium (100ml/500ml triangular flask), 30 ℃, 220r/min cultivates 18h, OD
600About about 8.0.
High density fermentation: by 10% inoculum size the seeding tank bacterial classification is linked in the 5L fermentor tank of dress 2.5L batch fermentation substratum, carries out batch culture (transferring about pH to 5.0) with 25% ammoniacal liquor.After about 20 hours, beginning flow feeding growth medium (the stream rate of acceleration is kept dissolved oxygen greater than 20%).Stop feed supplement after 4 hours, glycerine exhausts the back and mends the fermentation inducement substratum, carries out abduction delivering and cultivates (pH 25%NH
3H
2O is controlled to be 6.0), add methyl alcohol speed by adjusting rotating speed, tank pressure, air flow quantity and stream and make dissolved oxygen, about inducing culture 260h greater than 20%.It is alive that timing sampling is measured cell growth, secretory protein amount and enzyme thereof in the culturing process.Final zytase secretory volume can reach the 207mg/L nutrient solution, and enzyme is lived and is 522U/mg (albumen).
Utilize the standard method of known protein materialization to measure the fundamental characteristics of this zytase, the optimum temperature that shows this zytase after measured is 90 ℃, decomposes the xylan activity and stablizes in the pH value is the 6.2-8.0 scope.
Sequence table
<160>2
<210>1
<211>33
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
atagcggccg?ctatgaaaat?attaccttct?gtg 33
<210>2
<211>34
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
atagcggccg?ctcattttct?ttcttctatc?tttt 34
Claims (7)
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| CN1322110C (en) * | 2005-04-19 | 2007-06-20 | 湖北大学 | Yeast gene engineering bacteria and heat resistant alkali resistant xylanase preparation and application method |
| CN100532560C (en) * | 2005-05-18 | 2009-08-26 | 中国农业科学院饲料研究所 | Expression vector of high specific activity xylanase |
| CN100400665C (en) * | 2005-07-08 | 2008-07-09 | 中国农业大学 | A kind of constitutive expression vector and its application |
| CN101372694B (en) * | 2007-08-22 | 2013-03-27 | 中国农业大学 | Expression of high temperature resistant xylanase gene in Kluyveromyces lactis |
| CN101418308B (en) * | 2008-12-10 | 2011-08-03 | 北京德宝群兴科技有限公司 | Xylanase coding gene and use thereof |
| CN101838636B (en) * | 2009-06-11 | 2011-09-07 | 江苏奕农生物工程有限公司 | High-specific-activity xylanase XYN11F63 and genes and application thereof |
| CN102757977B (en) * | 2012-07-19 | 2014-04-09 | 中国科学技术大学 | Expression and preparation method of granulysin |
| CN102994480A (en) * | 2012-12-03 | 2013-03-27 | 青岛蔚蓝生物集团有限公司 | Xylanase and pichia pastoris engineering bacteria for recombining and expressing xylanase |
| CN105154463B (en) * | 2015-09-30 | 2018-10-02 | 西北大学 | A kind of structure of bacterial strain and application thereof being overexpressed Thermotoga maritima acetolactate synthase catalytic subunit |
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| US5489526A (en) * | 1990-06-08 | 1996-02-06 | Novo Nordisk A/S | Thermostable xylosidase produced by Bacillus stearothermophilus NRRL B-18659, Bacillus stearothermophilus NRRL B-18660 and Bacillus stearothermophilus NRRL B-18661 |
| CN1177639A (en) * | 1996-09-09 | 1998-04-01 | 加拿大国立研究院 | Modification of xylanase to improve thermophilicity, alkophilicity and thermostability |
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| US5489526A (en) * | 1990-06-08 | 1996-02-06 | Novo Nordisk A/S | Thermostable xylosidase produced by Bacillus stearothermophilus NRRL B-18659, Bacillus stearothermophilus NRRL B-18660 and Bacillus stearothermophilus NRRL B-18661 |
| CN1177639A (en) * | 1996-09-09 | 1998-04-01 | 加拿大国立研究院 | Modification of xylanase to improve thermophilicity, alkophilicity and thermostability |
Non-Patent Citations (3)
| Title |
|---|
| COEXPRESSION OF THE BACILLUS PUMILUSB-XYLOSIDASE (XYNB) GENE WITH THE TRICHODERMAREESEI B-XYLANASE(XYN2)GENE IN THE YEASTSACCHAROMYCES CEREVISAE. LA GRANGE DC ETAL.APPL MICROBIOL BIOTECHNOL,Vol.54. 2000 * |
| 毕氏酵母KM71和GS115发酵工艺的比较 张维延，江阳，许志祥，时宏珍，李新燕，强亦忠，张学光.苏州医学院学报，第3期 2001 * |
| 江正兵，宋慧婷，马立新.生物工程学报，第19卷第1期 2003 * |
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