CN103525793A - Alkaline xylanase with improved heat stability, and coding gene and application thereof - Google Patents
Alkaline xylanase with improved heat stability, and coding gene and application thereof Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
- C12N9/2485—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1,3-beta-xylanase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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- Y02W30/64—Paper recycling
Abstract
The invention relates to the field of genetic engineering, specifically to alkaline xylanase with improved heat stability and a coding gene and application thereof. According to the invention, the crystal structure of Bacillus subtillis B230 xylanase xyn-B230 is used as a starting structure, and selected mutation sites are S23H and I129Y. Compared with the xylanase xyn-B230, mutated xylanase has substantially improved heat stability and can retain 90% of enzymatic activity when maintained at a temperature of 70 DEG C for 120 min and more than 65% of enzymatic activity when treated at a temperature of 75 DEG C for 30 min. The xylanase xyn-B230-m has good properties and is applicable to the industry of papermaking.
Description
Technical field
The present invention relates to genetically engineered field, particularly, relate to a kind of alkalescent xylanase and encoding gene and application of thermostability improvement.
Background technology
Xylan is the important composition of plant hemicellulose, is prevailing hemicellulose in terrestrial plant cell walls, and except Mierocrystalline cellulose, xylan is the abundantest polysaccharide of occurring in nature.In pourous wood and annual gramineae plant, xylan accounts for the 20%-30% of plant material dry weight, and in softwood, xylan content is less, is generally the 7%-10% of raw material dry weight.
Zytase is the general name of one group of enzyme of degradation of hemicellulose xylan, and it mainly comprises the inscribe-l that acts on main chain, 4-beta-xylanase and xylobiase and other coenzyme.After finding that zytase can the bleaching for paper pulp, to zytase, research and development is paid special attention to both at home and abroad, it can be applied to bio-pulping, association with pulp bleaching, the recovery of waste paper secondary stock, deinking processing and paper surface processing etc., and particularly its huge applications potentiality in pulp bio-bleaching cause showing great attention to of the colleague of all circles already.No matter be pourous wood or softwood sulfate pulp and other chemical pulp; No matter float with traditional chlorine that order matches or without chlorine, float order and match with advanced, adopt xylanase treatment all can impel the degraded of residual lignin in paper pulp and extracting of solvability xylogen, not only can improve whiteness and the retention of whiteness of paper pulp, improve drainability and the Papermaking Performance of fiber, and can greatly reduce muriatic consumption in following bleaching process, thereby greatly reduce slurrying and paper industry pollution on the environment.
Different with other zymin, for the zymin in papermaking, need to experience the treating processes of a high temperature highly basic, because pulping process is under alkaline condition, by the method for high temperature steaming, remove the xylogen in wood chip.Therefore, the zytase for association with pulp bleaching should be heat-resisting and alkaline-resisting.And up to the present, the industrial zytase for association with pulp bleaching is neutral meta-acid mostly, optimal reactive temperature is mostly below 45 ℃, and this just requires first the pH of paper pulp and temperature to be transferred to the scope that is applicable to zytase effect before enzyme-added, and this is time-consuming and a require great effort process.
Overcome the above problems, just need acquisition to be adapted at the heat-resisting and alkaline-resisting zytase using under paper industry condition.Obtain the zytase of special property, can consider to organize work from the following aspects: first, can from extreme environment, separation there is extremophile microbial strains, and therefrom clone the zytase of alkali-resistant.Meanwhile, also to actively strengthen comprising selection by mutation and the molecular breeding of traditional Microbial Breeding, constantly promote the performance of expression amount and the product of bacterial classification xylanase gene.Secondly, by modern biotechnology means such as genetically engineered and protein engineerings, xylanase gene is carried out to molecular modification, improve stability and the resistance to bad ambient conditions of zytase, filter out and meet the xylanase gene that different application field requires.Therefore improve the thermostability of this zytase, can effectively on each field, obtain better application.
The current means by protein engineering are there being a lot of successfully examples aspect enzyme molecular modification, particularly in the thermostability that improves enzyme.Rite-directed mutagenesis is that design and rational is a certain active group or the module that has object on the structure of known enzyme and the basis of function, change targetedly enzyme, the final particular amino acid residue that obtains must have the protein that character obtains expection change, and this technology has been widely used in the transformation of enzyme molecule.
The present invention for the material that sets out, by serial rite-directed mutagenesis, has finally obtained a zytase xyn-B230-m that thermostability improves with the current zytase xyn-B230 that derives from genus bacillus having used in paper pulp and papermaking.Xyn-B230 compares with zytase, and the thermostability of the enzyme after sudden change is significantly improved, and at 70 ℃ of insulation 120min, still can retain 90% enzymic activity.At 75 ℃, process 30min enzymic activity and still can keep more than 65% activity.Zytase xyn-B230-m has reasonable character, can meet the use at paper industry.
Summary of the invention
The object of the invention is by deriving from the transformation of the zytase xyn-B230 of genus bacillus, improved zytase xyn-B230-m stability when high temperature is improved, can better in the alkaline pulp bio-bleaching of high temperature, bring into play the effect of xylan in degraded raw material, impel the better degraded of residual lignin and extracting of solvability xylogen in paper pulp.
The object of this invention is to provide a kind of zytase xyn-B230-m that optimizes improvement
A further object of the present invention is to provide the xylanase gene of optimizing improvement.
A further object of the present invention is to provide the recombinant vectors that comprises above-mentioned xylanase gene.
A further object of the present invention is to provide the recombinant bacterial strain that comprises above-mentioned xylanase gene.
A further object of the present invention is to provide the application of above-mentioned zytase xyn-B230-m.
Take the crystalline structure (PDB:1IGO) of the Bacillus subtillis B230 zytase xyn-B230 structure as setting out, the mutational site of selecting, in order to improve the thermostability of the zytase xyn-B230 that derives from genus bacillus, utilize biological computation and protein engineering means to carry out mutation research to affecting the key amino acid of its thermostability in xyn-B230, these amino acid are respectively that the S of the 23rd sports H, the N of the 45th sports I, the D of the 55th sports P, the H of the 60th sports Y, the S of the 61st sports D, the N of the 65th sports D, the N of the 75th sports T, the I of the 129th sports Y, the T of the 149th sports D, the Y of the 185th sports F, and the I that the S of the 23rd further thermostability being increased sports H and the 129th sports Y, carry out combinatorial mutagenesis.The zytase xyn-B230-m improveing finally by mistake optimization is significantly improved the thermostability of 75 ℃, can in papermaking application, demonstrate huge application potential.
By each mutant being carried out to the analysis of zymologic property, finally obtained the zytase xyn-B230-m that thermostability obviously improves.Zytase xyn-B230-m of the present invention compares with original genus bacillus zytase xyn-B230, has two amino acid that sudden change has occurred, and mutational site is S23H and I129Y (mutational site indicates with italic and underscore).Aminoacid sequence after sudden change (not comprising signal peptide sequence) is as shown in SEQ ID NO.1:
ATTITSNQTGTHDGYDYELWKD
HGNTSMTLNSGGAFSAQWSNIGNALFRKGKKF?DSTKTHSQLGNISINYNATFNPGGNSYLCVYGWTKDPLTEYYIVDNWGTYRPTGT?PKGTFTVDGGTYDIYETTR
YNQPSIIGIATFKQYWSVRQTKRTSGTVSVSEHFKKW?ESLGMPMGKMYETALTVEGYQSNGSANVTANVLTIGGKPLAA
The present invention also provides the gene order of the fire resistant xylanase xyn-B230-m of above-mentioned improvement, and its base sequence is as shown in SEQ ID NO.2:
gctaccaccatcacatctaatcagacaggaacacacgacggttatgactatgaactttggaaggac
catggaaacacctctatgac?tttgaactctggtggagctttttcagcccaatggagtaacattggtaacgcattgtttagaaagggaaagaaattcgattctaccaaaa?ctcattcccagcttggtaacatctctatcaactacaacgctactttcaacccaggtggaaattcctacttgtgtgtttatggttggacaa?aggatcctcttaccgaatactatattgtcgacaactggggaacttatagaccaacaggtacccctaaaggaacttttacagttgatgg?tggaacatacgacatctacgagactacaagatataatcaaccatcaattatcggtatcgccacttttaagcaatactggtcagtcaga?cagaccaaaagaactagtggaacagtttctgtctccgaacacttcaagaaatgggagagtttgggtatgcctatgggaaagatgta?cgaaaccgctcttactgttgagggttatcagtcaaacggaagtgctaatgttactgctaatgtccttacaatcggtggaaaacctcttt?aa
The present invention also provides the recombinant vectors of the zytase xyn-B230-m that comprises above-mentioned optimization improvement, the zytase xyn-B230-m of optimization improvement of the present invention is inserted between the restriction enzyme site that expression vector is suitable, the exercisable expression regulation sequence of its nucleotide sequence is connected.As the most preferred embodiment of the present invention, be preferably zytase xyn-B230-m of the present invention is inserted between the EcoR I and Not I restriction enzyme site on plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-xyn-B230-m.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned zytase xyn-B230-m, and preferably recombinant bacterial strain is Pichi strain GS115.
The present invention also provides the application of above-mentioned zytase xyn-B230-m in pulp processing, relates generally to zytase and is using enzyme pre-treatment to improve bleaching and the deinking of fibre property, paper pulp.In these processes, adopt xylanase treatment, all can impel the degraded of residual lignin in paper pulp and extracting of solvability xylogen, not only can improve whiteness and the retention of whiteness of paper pulp, improve drainability and the Papermaking Performance of fiber, and can greatly reduce the follow-up consumption that floats order chemistry, thereby reduce the pollution that association with pulp bleaching produces environment.
The present invention utilizes genetic engineering means to deriving from the improveing of zytase xyn-B230 of genus bacillus, to solve this zytase too much defect of high temperature loss of enzyme activity in pulp treatment process, the thermostability of the zytase xyn-B230-m improveing through optimization under 75 ℃ of conditions is greatly improved, further met the requirement of paper grade (stock) enzyme, therefore, the zytase xyn-B230-m of optimization improvement of the present invention can demonstrate huge application potential in paper industry.
Accompanying drawing explanation
The protein SDS-PAGE electrophorogram of Fig. 1, zytase xyn-B230 and xyn-B230-m purifying
The pH stability of Fig. 2, zytase xyn-B230 and xyn-B230-m.
The optimum temperuture of Fig. 3, zytase xyn-B230 and xyn-B230-m.
Fig. 4, zytase xyn-B230 and xyn-B230-m are the thermostability of 70 ℃.
Embodiment
Experiment condition:
1, bacterial strain and carrier
Coli strain Topl0, Escherichia coli BL21 (DE3), expression vector pET-22b (+) (purchased from Novagen company).Pichia pastoris GS115, carrier pPIC9(are purchased from Invitrogen company).
2, enzyme and other biochemical reagents
Fast pfu is purchased from Quan Shi King Company, and restriction endonuclease is purchased from TaKaRa company, and ligase enzyme is purchased from Invitrogen company.The substrates such as X-Gal, IPTG, birch xylan are purchased from Sigma company, and other is all domestic reagent.
3, substratum
Escherichia coli culture medium is LB(1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).Yeast culture base is YPD(1% yeast extract, 2% peptone, 2% glucose).Yeast screening assay substratum is MD(glucose 20g/L, agar powder 20g/L, vitamin H 4 * 10
-4g/L, YNB13.4g/L).
Yeast inducing culture BMGY(1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V)) and BMMY(divided by 1% methyl alcohol, replace glycerine, all the other composition phases are identical with BMGY).
In the present invention, the genetic recombination of using learns a skill and is the routine techniques in this area.The technology not describing in detail in following examples, all carries out according to the related Sections in following laboratory manual or document or part, comprising: the people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With James M.Cregg, Pichia Protocols (first version, 1998).
Embodiment 1, zytase xyn-B230 gene are synthetic, expression vector establishment and mutation construction
(1) composition optimizes
Take its aminoacid sequence of Bacillus subtillis B230 zytase xyn-B230(as shown in SEQ ID NO.3) the structure of crystalline structure (PDB:1IGO) as setting out, the mutational site of selecting is: S23H, N45I, D55P, H60Y, S61D, N65D, N75T, I129Y, T149D, Y185F.
The aminoacid sequence of Bacillus subtillis B230 zytase xyn-B230 is as shown in SEQ ID NO.3:
ATTITSNQTGTHDGYDYELWKDSGNTSMTLNSGGAFSAQWSNIGNALFRKGKKFDSTKTHSQ?LGNISINYNATFNPGGNSYLCVYGWTKDPLTEYYIVDNWGTYRPTGTPKGTFTVDGGTYDIYE?TTRINQPSIIGIATFKQYWSVRQTKRTSGTVSVSEHFKKWESLGMPMGKMYETALTVEGYQSN?GSANVTANVLTIGGKPLAA
(2) gene optimization is synthetic
By coming from the xylanase gene xyn-B230 of subtilis Bacillus subtillis B230, according to the preferences of pichia spp, under the prerequisite that does not change its aminoacid sequence, carry out sequence transformation.In transformation, avoid GT ... the site of this form of AG, attention can not affect the stability of mRNA.Sending Nanjing Jin Ruisi company to carry out full gene the good gene order of improvement and design synthesizes.
(3) structure of expression vector
According to the sequences Design PCR primer 5 ' end of synthetic gene, contain Nco I restriction enzyme site, 3 ' end is containing EcoR I restriction enzyme site, and primer sequence is as follows:
5 ' end primer pET-xyn-F:GCAC
cCATGGgCTACCACCATCACATCTAATCA; 3 ' end primer pET-xyn-R:GCAC
gAATTCtTAAAGAGGTTTTCCACCGATTG; Take synthetic gene as template, with above-mentioned primer, carry out pcr amplification, it is upper that the fragment that amplification is obtained is cloned into carrier pET-22b (+), obtains recombinant vectors pET-22b (+)-xyn-B230.
(4) structure of mutant
Adopt the Fast Mutagenesis System of TransGen company to carry out rite-directed mutagenesis to the xylanase gene xyn-B230 gene of Bacillus subtillis B230, the primer (table 1) that partly overlaps that comprises mutational site, requirement according to this product description designs, and the relating operations such as PCR are undertaken by the specification sheets of this product.The mutant that screening is obtained, through the exactness of definite mutator gene sequence that checks order.
Table 1 mutant primer
Embodiment 2 zytase xyn-B230 and the expression of mutant in intestinal bacteria thereof
Two kinds of expression vector pET-22b (+)-xyn-B230 described in embodiment 1 and mutant S23H, N45I, D55P, H60Y, S61D, N65D, N75T, I129Y, T149D and Y185F, totally 11 expression plasmids transform bacillus E.coli BL21 (DE3) by thermal shock, obtain respectively the recombinant bacterial strain that contains protogene and each mutant.
Get BL21 (DE3) bacterial strain that contains recombinant plasmid and BL21 (DE3) bacterial strain (in contrast) that contains pET-22b (+) empty plasmid, be inoculated in respectively 3mL LB(containing 100 μ g/mL Amp) in nutrient solution, 37 ℃ of quick oscillation overnight incubation.Get respectively 100 μ L incubated overnight liquid and add in the 10mL LB nutrient solution (1% inoculum size) containing Amp, quick oscillation is cultivated about 2-3h(OD
600reach 0.6-0.8), after add the inductor IPTG of final concentration 1mmol/L, 37 ℃ are continued the about 4h of shaking culture, or 30 ℃ of about 6h of shaking culture.The centrifugal 5min of 12,000rpm, collects respectively cleer and peaceful precipitation on substratum, citric acid-Na of pH7.0 for cell precipitation
2hPO
4damping fluid is resuspended, after ultrasonic disruption 12, and the centrifugal 10min of 000rpm.Detect respectively on substratum the activity of zytase in cleer and peaceful cytoclasis liquid.Xylanase activity power is measured the DNS method that adopts.Concrete grammar is as follows: in test tube, add 900 μ L1% oat xylan substrates, be placed in 37 ℃ of water-baths and be incubated in advance 5min, add the suitably enzyme liquid of dilution of 100 μ L, in 37 ℃ of water-baths, be accurately incubated 30min, add 1.5mL DNS termination reaction, boiling water boiling 5min.After cool to room temperature, 540nm measures OD value.Through calculating zytase xyn-B230 and mutant thereof in intestinal bacteria, the supernatant liquor xylanase activity of expression is between 12.3 to 18.2U/mL.Simultaneously the various 20 μ L that get add 5 * electrophoresis sample-loading buffer, and boiling water boiling 5min is usingd the thalline BL21 that contains empty plasmid pET-22b (+) that disposes equally as negative control, carries out SDS-PAGE electrophoresis detection.Enzyme activity determination result and SDS-PAGE result all show, zytase xyn-B230 and mutant thereof equal successful expression in intestinal bacteria.
The purifying of embodiment 3, zytase xyn-B230 and mutant thereof
The detailed process of zytase xyn-B230 and mutant purifying thereof is substantially the same, flow process is as follows substantially: the supernatant liquor that shaking flask is expressed concentrates with tubular fibre, then in the concentrated solution of every 100mL, slowly add ammonium sulfate, and constantly stir 3h, carry out fractionation precipitation, follow the centrifugal 10min of 12,000rpm, collecting precipitation.Precipitation is dissolved with the 20mM Tris-HCl damping fluid of a small amount of pH10.0, and in this damping fluids in a large number dialysed overnight.Solution after dialysis is crossed anion column (HiTrap Q Sepharose XL5mL) and is carried out purifying.Anion column is used the 20mM Tris-HCl damping fluid balance of pH10.0 in advance, after loading, with the NaCl eluant solution of 0～1.0mol/L linear gradient, collects the wash-out pipe that has xylanase activity, through SDS-PAGE, analyzes its purity, after for the analysis of zymologic property.
The some properties analysis of embodiment 4, zytase xyn-B230 and mutant thereof
(1) optimum temperuture of zytase xyn-B230 and mutant thereof and thermostability
Zytase xyn-B230 and mutant thereof that purifying is good are diluted to desired concn, get 50 μ L and at 40,50,55,60,65,70,80,90 ℃ of temperature, measure respectively enzyme work, calculate relative enzyme and live, to determine the optimum temperuture of this enzyme.Its result shows, the optimum temperuture of the mutant of part zytase xyn-B230 is significantly improved, and brought up to 65 ℃ from 60 ℃, and the relative enzyme in the time of 70 ℃ is lived and still can be maintained more than 95%.As can be seen here, the mutant optimum temperuture of the zytase xyn-B230 by design and rational is significantly improved.
Zytase xyn-B230 and mutant thereof that purifying is good are diluted to desired concn, get 2mL 70 ℃ of insulations.Then respectively 5,10,20,30,60 and 120min take out 100 μ L enzyme liquid be diluted to shown in concentration.At 37 ℃, under the condition of pH7.0, measure enzyme and live, using untreated original enzyme liquid as 100% contrast.As shown in Figure 4, the mutant of zytase xyn-B230 (S23H and I129Y) is significantly improved the thermostabilitys of 70 ℃ its measurement result, at 70 ℃, processes the enzymic activity that 120min can also retain 50-60%.And zytase xyn-B230 has just lost 75% 70 ℃ of processing 10min enzymic activitys, 10min enzymic activity is lost completely.Stability experiment result at 70 ℃ shows, the mutant of this zytase xyn-B230 can be resisted the high temperature in pulp processing for a long time.
(2) optimal pH of zytase xyn-B230 and mutant thereof and pH stability
Purified zytase xyn-B230 and mutant thereof, at 37 ℃, carry out enzymatic reaction to measure its optimal pH under different pH.Damping fluid used is citric acid-Na of pH3.0-8.0
2hPO
4damping fluid, the Tris-HCl damping fluid of the 0.1mol/L of pH8.0-9.0, and the Gly-NaOH damping fluid of pH9.0-10.0.Result shows, the optimal pH curvilinear motion of zytase xyn-B230 and mutant thereof is little, and optimum pH is still 6.5, at alkaline condition, has higher activity, when pH9.0, can keep the relative reactivity of 40% left and right; When pH10.0, can keep the relative reactivity of 20% left and right.
By purifying good zytase xyn-B230 and mutant thereof, be diluted to finite concentration, in the damping fluid of pH4-10, place 1h for 37 ℃.And then at 37 ℃, under the condition of pH6.5, measure enzyme and live, by calculating relative enzyme work, analyze the stability of zytase under condition of different pH, result shows, the pH stability of zytase xyn-B230 and mutant thereof very, after treatment, Enzyme activity, basic not loss, and improve, even under the condition of pH10.0, process after 1 hour, still can keep more than 97.5% relative reactivity.
Combinatorial mutagenesis and the thermal stability analysis in the site that embodiment 5, zytase xyn-B230 thermostability improve
Through thermostability initial analysis, the mutant of zytase xyn-B230 (S23H and I129Y) thermostability, is all significantly improved.In order further to improve the thermostability of zytase xyn-B230, the mutational site that thermostability is improved, S23H and I129Y are incorporated on same molecule, on the basis of S23H mutant, further introduce I129Y catastrophe point, finally obtain the zytase xyn-B230-m of two point mutation.And the technological method that adopts above embodiment to provide, zytase xyn-B230-m to be expressed and purifying (Fig. 1), pH characteristic is determined in the pacing of going forward side by side, optimum temperature and thermostability.
PH characteristic measurement result shows, zytase xyn-B230-m optimal pH curve does not change substantially, and optimum pH is still 6.5.The pH stability of zytase xyn-B230-m very, although be slightly worse than zytase xyn-B230 at neutrallty condition, with high, under the condition of pH10.0, is processed after 1 hour when alkaline condition, still can keep more than 108% relative reactivity (Fig. 2).
Temperature characteristic measurement result shows, the optimum temperuture of xylanase mutant xyn-B230-m has obtained further raising, from 65 ℃, 70 ℃ have further been brought up to, than the original the most applicable temperature of zytase xyn-B230, improved 10 ℃, and the relative enzyme work in the time of 75 ℃ still can maintain more than 60% (Fig. 3).Thermal stability analysis shows, zytase xyn-B230-m, at the non-normal temperature of 70 ℃, still can retain 90% enzymic activity (Fig. 4) at 70 ℃ of insulation 120min.At 75 ℃, process 30min enzymic activity and still can keep more than 65% activity.Stability experiment result at 70 ℃ shows, this xylanase mutant xyn-B230-m can resist the alkaline-resisting and high temperature capabilities in pulp processing for a long time, and can narrow spectrum xylan effectively be degraded.Therefore, the zytase B230 variant of optimization improvement of the present invention can demonstrate huge application potential in paper industry.
Embodiment 6, the zytase xyn-B230-m high efficient expression in pichia spp
(1) structure of expression vector and in the expression of yeast
Do not changing under the condition of aminoacid sequence, the preferences of using according to pichia spp codon, design synthetic the full gene of zytase xyn-B230-m, and at the two ends of xyn-B230-m gene, added respectively EcoR I and Not I restriction enzyme site.And by EcoR I, cut to be connected with Not I enzyme and enter expression vector pPIC9 (Invitrogen, San Diego), make zytase xyn-B230-m with correct reading frame, be inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, be built into Yeast expression carrier pPIC9-xyn-B230-m, transform competent escherichia coli cell JM109.A large amount of recombinant plasmids that extract in positive transformant, carry out linearization process with restriction enzyme BglII, and transformed yeast GS115 competent cell, and the MD that coats histidine defect is dull and stereotyped, cultivate 2-3 days for 30 ℃.
(2) screening of high xylanase activity transformant
With sterilized toothpick picking list bacterium colony from the long MD plate that has a transformant, according to numbering, first to put MM upper, then put on the MD flat board of corresponding numbering, 100 single bacterium colonies of each dull and stereotyped upper point, amount to 500 transformants; There are MM, the MD flat board of transformant to be placed in 30 ℃ of incubators point and cultivate 1-2 days, grow to bacterium colony.From MD flat board, picking transformant is inoculated in the centrifuge tube that 3mL BMGY substratum is housed by number, 30 ℃, 220rpm shaking table cultivation 48h; The centrifugal 5min of bacterium liquid 3,000 * g that shaking table is cultivated to 48h, removes supernatant, and the BMMY substratum that adds again 1mL to contain 0.5% methyl alcohol in centrifuge tube, at 30 ℃, 200rpm inducing culture; After inducing culture 48h, the centrifugal 5min of 3,000 * g, gets supernatant and detects for enzymic activity, therefrom filters out and has the transformant of high xylanase activity, and it is numbered xyl-B230-m-74.
(3) high xylanase activity transformant is expressed at the lab scale of fermentor tank level
High cell density fermentation is tested with 3.7-L fermentor tank, according to the complete red fermenting process of Invirogen company, instructs (Pichia Fermentation Process Guidelines) operation.Xyl-B230-m-74 bacterial strain is carried out to the horizontal high density fermentation test of fermentor tank.Along with the prolongation of methanol induction time, in fermented supernatant fluid, xylanase activity power continues to increase, and induces and within 108 hours, reaches 5,864U/mL, and under pH9.0 condition determination, enzymic activity still has 2,300U/mL.In pH9.0 condition, there is very high expression level, solved its application cost problem, for the utilization of zytase xyn-B230-m in paper industry laid a good foundation.
Claims (10)
1. the xylanase mutant xyn-B230-m of thermostability improvement, it is characterized in that, zytase to aminoacid sequence as shown in SEQ ID No.3 carries out S23H and I129Y sudden change, obtains the xylanase mutant xyn-B230-m of described thermostability improvement.
2. the xylanase mutant xyn-B230-m of thermostability improvement according to claim 2, is characterized in that, its aminoacid sequence is as shown in SEQ ID No.1.
3. the xylanase mutant gene of thermostability improvement, is characterized in that, the xylanase mutant xyn-B230-m of the thermostability improvement claimed in claim 1 of encoding.
4. the xylanase mutant gene of thermostability improvement according to claim 3, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.2.
5. the recombinant vectors that comprises the xylanase mutant gene of thermostability improvement described in claim 3.
6. the recombinant vectors pPIC9-xyn-B230-m that comprises the xylanase mutant gene of thermostability improvement described in claim 3.
7. the recombinant bacterial strain that comprises the xylanase mutant gene of thermostability improvement described in claim 3.
8. the recombinant pichia yeast strain GS115 that comprises the xylanase mutant gene of thermostability improvement described in claim 3.
9. a method of preparing the zytase of thermostability improvement, is characterized in that, described method comprises step:
(1) with recombinant vectors transformed host cell claimed in claim 4;
(2) cultivate described host cell;
(3) zytase of separation and purification thermostability improvement.
10. the application of the xylanase mutant xyn-B230-m of thermostability improvement claimed in claim 1.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450650A (en) * | 2014-12-01 | 2015-03-25 | 青岛蔚蓝生物集团有限公司 | Alkaline xylanase mutant and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703481A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Method for heat resistance modification of aspergillus oryzae GH11 xylanase gene and preparation of mutant enzyme |
| CN103343113A (en) * | 2013-07-16 | 2013-10-09 | 中国农业科学院饲料研究所 | Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof |
-
2013
- 2013-10-29 CN CN201310519982.0A patent/CN103525793B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703481A (en) * | 2012-06-05 | 2012-10-03 | 江南大学 | Method for heat resistance modification of aspergillus oryzae GH11 xylanase gene and preparation of mutant enzyme |
| CN103343113A (en) * | 2013-07-16 | 2013-10-09 | 中国农业科学院饲料研究所 | Thermal stability improved xylanase XynAS9-m mutant V81P/G82E as well as gene and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 付正 等: ""嗜热真菌木聚糖酶1YNA的表达和定点突变"", 《湖北农业科学》, vol. 50, no. 7, 30 April 2011 (2011-04-30), pages 1488 - 1493 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104928270A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院天津工业生物技术研究所 | Recombinant basic xylanase resistant to chelating agent ethylenediamine tetraacetic acid and construction method thereof |
| CN104928270B (en) * | 2014-03-21 | 2018-04-17 | 中国科学院天津工业生物技术研究所 | The recombinant basic zytase and its construction method of one group of ethylenediamine tetra-acetic acid of resistance to chelating agent |
| CN107201352A (en) * | 2014-09-29 | 2017-09-26 | 中国农业科学院生物技术研究所 | A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application |
| CN107201352B (en) * | 2014-09-29 | 2020-05-12 | 中国农业科学院生物技术研究所 | β -galactosidase combined mutant with high transglycosidic activity and preparation method and application thereof |
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