CN104531732A - Optimized extremely-thermostable xylanase XYNH coding gene and application thereof - Google Patents
Optimized extremely-thermostable xylanase XYNH coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to an optimized extremely-thermostable xylanase XYNH coding gene and application thereof. The nucleotide sequence of the nucleotide sequence is shown as SEQ ID NO:1. By means of the optimized extremely-thermostable xylanase XYNH coding gene and application, the problem of expression efficiency reduction caused by the codon preference of extremely-thermostable xylanase genes coming from bacteria in an eukaryotic expression system is solved; and xylanase XYNH which is excellent in character and has not been used for pichia pastoris expressions in the global yet is provided.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of extremely thermostable xylanase XYNH encoding gene and application thereof of optimization.
Background technology
Xylan is topmost hemicellulose in plant cell wall, accounts for about 35% of vegetable cell dry weight, is the polysaccharide that occurring in nature goes out that outside Mierocrystalline cellulose, content is the abundantest.Xylan is by β-1 by wood sugar, the main chain that 4-glycosidic link is polymerized and the class heterozygosis saccharan that some side-chain radicals are formed jointly, as a kind of abundant biomass resource, the xylo-oligosaccharide that world market is badly in need of and wood sugar can be degraded under the effect of zytase.But occurring in nature a big chunk zytase is not yet used effectively, and causes the very large waste of this resource.
Microbial xylanase (EC 3.2.1.8) is important industrial enzymes, and the β-Isosorbide-5-Nitrae-D-wood sugar glycosidic bond of random catalytic hydrolysis xylan inside generates wood oligose.Wood oligose can be degraded by other xylan enzymes further as substrate, as (Khandeparker R such as β-D-xylosidase, α-l-arabfuranglycosidase and D-Glucose aldehydic acid enzymes, Numan MT.Bifunctionalxylanase and their potential use in biotechnology.J Ind Microbiol biotechnol 2008,35:635-644.).Based on the significant difference of functionally elementary and tertiary structure and model, zytase classification is glycoside hydrolases the 10th and 11 family, but in the enzyme of glycoside hydrolases the 5th, 7,8,16,26,30,43,52 and 62 family, have also discovered activity (http://www.cazy.org/fam/acc_GH.html) (the Collins T with hydrolyzed xylan, Gerday C, Feller G.Xylanase, xylanase families and extremophilic xylanases.FEMS Microbiol Rev 2005,29:3-23.).Wherein GH 10 family zytase relative molecular weight comparatively large (>30000), complex structure, usually be made up of multiple structural domain, comprise a catalyst structure domain (Catalysis Domain, CD) be zytase chief component, carry the hydrolysis properties of zytase.Although they are widely different in amino acid quantity and composition, but its catalytic structure is all very close in size, the main structure repeated with alpha-helix and α-pleated sheet, because close with TIM structure, belong to a family, be called (α/β) 8 pleated sheet structure, wherein the L-glutamic acid of specific position and aspartic acid very large on catalysis characteristics impact.The structural domain of this enzyme also containing non-catalytic, as polysaccharide Binding Capacity territory, thermally-stabilised structural domain, and multiple catalyst structure domains etc., give this enzyme decomposing soluble xylan, the functions such as insolubility zytase and other substrates.
The application of zytase originates from the use in animal-feed processing in 1980, is applied to papermaking, food, pharmacy thereupon gradually, brewages, weaves and the field such as biofuel.At present this enzyme is mainly used in pulping and paper-making, the industry such as feed and food, and application status in modern industry is more and more obvious.Zytase is one of key enzyme utilized in non-starchy raw material production alcohol process.Along with the needs of bioenergy career development, zytase certainly will be applied to field (Fawzi EM.Highly thermostable purified xylanase from Rhizomucor miehei NRRL 3169.Ann Microbiol 2010,60:363-368.) widely.In Industrial processes, often there is the extreme environments such as high temperature, hot environment can accelerate enzymatic reaction speed, improves the flowing property of liquid material, prevents harmful microorganism growth and breeding during the course etc.Common middle temperature zytase at high temperature can recurring structure change, thus loss of activity significantly.For preventing this hot inactivation process, just needing to add the method protective enzymes such as some chemical substances, so not only increasing production cost and having disadvantageous effect to quality product.
The heat resistant xylanase that application Thermotoga maritima Thermotoga maritima MSB8 produces is hydrolyzed, and fundamentally can solve the problem of the very fast inactivation of enzyme that hot environment makes to add.The zytase deriving from bacterium generally has higher thermostability than the zytase deriving from fungi, and pichia spp has the expression system of the potential Chang Zuowei scale operation exogenous protein that efficient secretion, correct folded protein and high cell concn are cultivated, being applied to after sequence optimisation by the xylanase gene deriving from bacterium can the pichia spp of high-density culture, is more suitable for suitability for industrialized production.
Derive from the zytase 1VBR of Thermotoga maritima Thermotoga maritima MSB8 by having more excellent zymologic property (Winterhalter C, Liebl W.Two extremely thermostable xylanases of the hyperthermophilic bacterium Thermoyoga maritima MSB8.Appl Environ Microbiol.1995, 61 (5): 1810-1815.), belong to glycoside hydrolases the 10th family, its gene size is 984bp, aminoacid sequence size is 328aa, predict that its molecular weight is 40kDa, xylan main decomposition is xylo-bioses and wood sugar by it, also the researchs that conduct in-depth analysis such as xln three-dimensional structure and function, and there is extreme heat stability, all high enzyme activity can be maintained under wide pH value condition.Up to the present, Thermotoga maritima Thermotoga maritima MSB8 is not also utilized to produce the relevant report of zytase 1VBR.Current correlative study is confined to genetic analysis to this enzyme and enzyme specificity analysis.Containing multiple EcoR I and Bgl II restriction enzyme site in original 1VBR gene, when utilizing genetic engineering technique to build Pichia yeast engineering, directly utilizing original gene cannot complete enzyme and cut and be connected.Pichia yeast engineering can be successfully constructed after this original gene must be optimized.
After this enzyme gene order is optimized, high expression in pichia yeast expression system, and based on extremely heat-resisting advantageous property, can in fodder additives, protective foods, papermaking, wash, brewage, to weave and medicine and other fields has broad application prospects.
Summary of the invention
What an object of the present invention was to provide a kind of optimization can the extremely thermostable xylanase XYNH encoding gene of a kind of optimization of efficient secretory expression in pichia spp.
Another object of the present invention is to provide a kind of recombinant expression plasmid of the xylanase gene containing this optimization.
Another object of the present invention is to provide a kind of pichia spp recombinant bacterial strain producing this optimization zytase.
Another object of the present invention is to provide the application of the extremely thermostable xylanase XYNH encoding gene of described optimization.
Above object of the present invention is achieved through the following technical solutions:
According to the zytase 1VBR aminoacid sequence (SEQ ID NO.2) deriving from Thermotoga maritima Thermotoga maritima MSB8 delivered in PDB and Thermotoga maritima MSB8 genome sequence (CP007013.1:1, 869, 644bp), the amino acid sequence similarity of prediction zytase (EHA58720.1) is 100%, in order to improve the expression efficiency of xylanase gene in pichia spp, do not changing in zytase 1VBR aminoacid sequence situation, sequence reorganization and codon optimized and improve mRNA secondary structural stability is carried out to this xylanase sequence, eliminate the EcoR I in gene order and Bgl II site simultaneously, obtain the zytase XYNH gene (SEQ ID NO.1) (optimize site and have underscore) optimized.
SEQ ID NO.1：
SEQ ID NO.2
After gene optimization, the aminoacid sequence of this enzyme 1VBR does not change.
The invention provides a kind of recombinant expression plasmid containing above-mentioned optimization zytase XYNH gene to comprise: the zytase XYNH gene of optimization, terminator sequence, and 5 ' end and 3 ' holds restriction endonuclease sites EcoR I and Not I.
The zytase XYNH gene of full genome synthesis optimizing, comprises its terminator sequence, and two ends restriction enzyme site sequence.By double digestion, connect and full genome is synthesized in xylanase gene insertion Expression vector pPIC9K, build recombinant expression plasmid pPIC9K-XYNH.By this recombinant expression plasmid transformation of E. coli DH5 α competent cell, filter out positive colony bacterial strain with PCR proof method.
According to the specific embodiment of the present invention, construct the pichia spp gene work bacterial strain of efficient secretory expression zytase XYNH: with reference to Invitrogen Pichia anomala expression handbook, recombinant expression plasmid pPIC9K-ManB in the positives clone strain of extraction step two, after restriction enzyme Bgl II linearizing, electroporated Pichia pastoris GS115 competent cell, Geneticin G418 E-test is adopted to increase the method such as selective pressure and enzyme activity detection, filter out the pichia spp gene work strain X YNH-GS115 of multiple copied highly effective expression of xylanase XYNH.
According to the specific embodiment of the present invention, liquid fermenting also induces product enzyme: the above-mentioned gene work strain X YNH-GS115 built is inoculated in YPD substratum, and 8h is cultivated in concussion, obtains seed liquor.With 2% inoculum size, seed liquor is accessed BSM inorganic salt glycerin medium, use ammoniacal liquor regulation and control pH to maintain 5.5, when thalli growth is to logarithmic phase, stream adds 40% glycerine (containing 12mL/L PTM1), maintaining dissolved oxygen is about 60%, reaches 130g/L to thalline weight in wet base, stops glycerine feed supplement.Stream adds methyl alcohol (containing 12mL/L PTM1) induction and produces enzyme, regulates methanol feed rate that dissolved oxygen is maintained about 30% simultaneously, detects when Xylanase activity no longer raises and can stop induction.
First the present invention solves the expression efficiency that the Xylanase B Gene of Thermotoga maritima that derives from bacterium causes because of codon-bias in eukaryotic expression system and reduces, provide a kind of good properties and be not yet used to the zytase XYNH of Pichia anomala expression in the whole world at present, have stronger degradation capability to xylan, its Rate activity is 3054.725U/mg.This enzyme has stronger stability within the scope of the pH of broadness, optimal pH is 5.0, still more than 80% enzyme activity can be maintained process 1h under pH 4.0 ~ 9.0 condition after, optimum temperuture is 100 DEG C, under optimal pH condition, the half death time of 100 DEG C of process reaches 3h, has splendid adaptive faculty for the thermal extremes environment occurred normal in industrial application.
Host Strains used herein is pichia pastoris phaff GS115, the exogenous protein of appropriate glycosylation modified expression, and impels the correct folding of protein, effectively can improve the stability of foreign protein.A glycosylation site is only had through this enzyme of N-glycosylation analyses and prediction, meanwhile, the molecular size range of SDS-PAGE result display zytase XYNH is about 40kDa, consistent with this enzyme molecular weight of prediction, show that this enzyme does not exist excessive grooming, do not affect this enzyme-to-substrate avidity.
The engineering strain XYNH-GS115 of product zytase XYNH provided by the invention, the secreted zytase XYNH protein content in fermented liquid can reach the pure rank of electrophoresis, needs purifying hardly, reduces production cost.
Accompanying drawing explanation
The SDS-PAGE of Fig. 1 zytase XYNH analyzes;
The optimal pH (90 DEG C, 10 minutes) of Fig. 2 zytase XYNH;
The pH stability (90 DEG C, 1 hour) of Fig. 3 zytase XYNH;
The optimum temperuture (pH 5,10 minutes) of Fig. 4 zytase XYNH;
The thermal destruction curve (pH 5,100 DEG C) of Fig. 5 zytase XYNH.
Embodiment
Experiment material:
1) bacterial strain and plasmid: intestinal bacteria (Escherichia coli) DH5 α and Pichia pastoris GS115; PPIC9K secreted expression carrier is purchased from Invitrogen company.
2) enzyme and test kit: the toolenzymes such as restriction enzyme, Taq enzyme, Pyrobest archaeal dna polymerase, T4DNA ligase enzyme purchase TaKaRa company; DNA purification kit is purchased from liking to pursue progress Bioisystech Co., Ltd.
3) biochemical reagents: G418 is purchased from Invitrogen company; Protein molecular weight standard is purchased from Shanghai biochemical research institute; IPTG, X-Gal, SDS and carob bean gum available from Sigma; TEMED, ammonium persulphate, acrylamide, methylene diacrylamide are traditional Chinese medicines reagent.
50mM Sodium phosphate dibasic-citrate buffer solution: get 7.10g Sodium phosphate dibasic and be dissolved in 800mL distilled water, is in 4.0 ~ 7.5 scopes after arbitrary value with lemon acid for adjusting pH, is settled to 1L.
50mM Tris-HCl damping fluid: get 6.06g Tris and be dissolved in 800mL distilled water, after arbitrary value in the scope of 1M HCl joint pH to 7.5 ~ 9.0, is settled to 1L.
50mM Glycine-NaOH damping fluid: get 7.50g glycine and be dissolved in 800mL distilled water, after arbitrary value in the scope of 1M sodium hydroxide solution tune pH to 9.0 ~ 12, is settled to 1L.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is mass percentage.
Embodiment 1: the acquisition of the zytase XYNH gene order of optimization
According to sequence alignment, the zytase 1VBR aminoacid sequence (SEQ ID NO.2) deriving from Thermotoga maritima Thermotoga maritima MSB8 delivered in PDB and Thermotoga maritima MSB8 genome sequence (CP007013.1:1,869,644bp) in prediction zytase (EHA58720.1) amino acid sequence similarity be 100%.This prediction xylanase sequence is a fragment gene sequence (873,589 → 874,572bp) in Thermotoga maritima MSB8 genome sequence.
Based on pichia pastoris phaff codon usage bias, wherein will be converted to high frequency expression codon by rare codon, contrast the aminoacid sequence of zytase 1VBR simultaneously, carry out codon optimized to prediction zytase (EHA58720.1) gene order, obtain zytase XYNH gene order after optimizing.
Embodiment 2: the structure of the recombinant expression plasmid pPIC9K-XYNH containing the zytase XYNH gene optimized
The terminator sequence of pichia spp preference is added at the zytase XYNH gene 3 ' end optimized, and introduce restriction enzyme EcoR I and Not I site respectively at 5 ' end and 3 ' end, this gene order given Wuhan to hold up Kechuang true tumor Science and Technology Ltd. and complete full genome synthesis.
Adopt restriction enzyme EcoR I and Not I to complete zytase XYNH gene and the secreted expression carrier pPIC9K double digestion of optimization, re-use ligase enzyme and both are connected, build recombinant expression plasmid pPIC9K-XYNH.By this recombinant expression plasmid transformation of E. coli DH5 α competent cell, filter out positive colony bacterial strain pPIC9K-XYNH-DH5 α with PCR proof method.
Embodiment 3: the structure of the pichia spp gene work bacterial strain of efficient secretory expression zytase XYNH
Adopt LB liquid nutrient medium activation culture bacterial strain pPIC9K-XYNH-DH5 α, extract recombinant plasmid pPIC9K-XYNH.Adopt this recombinant plasmid of restriction enzyme Bgl II linearizing, and reclaim digestion products.With reference to EasySelect
tMpichia Expression Kit prepares Pichia pastoris GS115 competent cell.The competent cell getting about 10 μ g linearization plasmids and 80 μ L softly mixes and is placed on 15min on ice, be transferred in the 0.2cm electricity revolving cup of precooling, the 1mol/L sorbyl alcohol of 1mL precooling is added immediately after 1500V has shocked by electricity, 1h is left standstill in 30 DEG C of incubators, coat on MD flat board, cultivate about 48h in 30 DEG C of inversions and occur to transformant.
Picking list bacterium colony is inoculated on the MD flat board containing 0.25,0.5,1.0,2.0,3.0mg/mL G418 by corresponding numbering successively respectively, cultivates about 48h occur to single bacterium colony in 30 DEG C of inversions.The recombinant bacterial strain of screening strong resistance, be inoculated in 50mL BMGY liquid nutrient medium, 28 DEG C, 200r/min shaking table is cultured to OD600=20-30.Collect thalline, remove supernatant, cell precipitation is all transferred in 50mL BMMY liquid nutrient medium, and 200r/min cultivates on the constant-temperature table of 28 DEG C.Add methyl alcohol every 24h to induce to final concentration 1%, after induction 84h, collect supernatant.DNS method is adopted to detect enzyme activity to screen the higher positive recombinant bacterial strain XYNH-GS115 of yield of enzyme.
Embodiment 4: the fermentor tank high density fermentation of pichia yeast genetic engineering bacteria XYNH-GS115
Adopt pichia yeast genetic engineering bacteria XYNH-GS115 at 10L fermentor tank middle-high density fermentative production zytase.Fermenting process is specific as follows:
1) seed liquor preparatory phase.The pichia yeast genetic engineering bacteria XYNH-GS115 of preservation is inoculated in YPD substratum, 28 DEG C, about 12h cultivated by 200rpm shaking table, obtains seed liquor.
2) earlier fermentation cultivation stage.With 2% inoculum size, seed liquor is accessed BSM inorganic salt glycerin medium, use ammoniacal liquor to regulate pH to 5.5 before inoculation, aeration-agitation is cultivated, and along with the growth of bacterial strain in culturing process, the dissolved oxygen amount in substratum reduces gradually by 100%.After carbon source runs out of, dissolved oxygen amount can raise once again, when dissolved oxygen is increased to more than 80%, starts the carbon source fed batch fermentation stage.
Wherein, BSM substratum consists of the following composition: 85%H
3pO
426.7mL/L, CaSO
42H
2o 0.93g/L, K
2sO
418.2g/L, MgSO
42H
2o 14.9g/L, KOH 4.13g/L, glycerine 40g/L, PMT1 4.0mL/L.Described PMT1 formula: CuSO
45H
2o 6.0g/L, KI 0.088g/L, MnSO
4h
2o 3.0g/L, Na
2moO
42H
2o 0.2g/L, H
3bO
30.02g/L, CoCl
26H
2o 0.5g/L, ZnCl
220.0g/L, FeSO
47H
2o 65.0g/L, Biotin 0.2g/L, dense H
2sO
45.0mL/L (filtration sterilization).
3) the fed batch fermentation stage.Stream adds 40% glycerine solution (containing 12mL/L PTM1), controls dissolved oxygen about 60% simultaneously, when rotating speed and air quantity reach maximum, when comparatively large decline of floating appears in dissolved oxygen, stops glycerine feed supplement.
4) the abduction delivering stage.Stream adds methyl alcohol (containing 12mL/L PTM1) induction and produces enzyme, regulate methanol feed rate that dissolved oxygen is maintained more than 15% simultaneously, pH maintains about 5.5, and every 24h sampling detection OD value, weight in wet base, enzyme are lived, and can stop induction when Xylanase activity no longer raises.
Detected result shows, and the protein concn of fermented liquid is 3.14mg/mL.Zytase XYNH vigor is 9540.47U/mL, and Rate activity is 3031.725U/mg.In addition, fermentation liquor SDS-PAGE detects, and result shows, and in fermented liquid, the protein content of zytase XYNH reaches the pure rank of electrophoresis, and molecular size range is about 40kDa, as shown in Figure 1.
Embodiment 5: the property analysis of the zytase XYNH of optimization
1) mensuration of zytase XYNH optimal pH: adopt DNS method to detect this zytase optimal pH, when 100 DEG C, zytase XYNH under different pH buffer conditions with 0.5% xylan solution accurate response 15min, add 2.5mL DNS reagent, boiling water bath 5min, be cooled to room temperature, add water and be settled to 12.5mL, in spectrophotometer, detect OD
540.Result shows, and zytase XYNH optimal pH is 5.0, all can keep the enzyme activity of more than 70%, as shown in Figure 2 in pH 5.0 ~ 7.5 scope.
2) mensuration of zytase XYNH pH stability: at 90 DEG C, accurately processes 1h by enzyme liquid, is placed in cooled on ice rapidly in different pH damping fluid.In 100 DEG C, pH 5.0 times, this enzyme and 0.5% xylan solution react 15min respectively at different temperatures, and add 2.5mL DNS reagent, boiling water bath 5min, is cooled to room temperature, adds water and is settled to 12.5mL.OD is detected in spectrophotometer
540.Result shows, and zytase XYNH still can maintain more than 80% enzyme activity process 1h under the condition of pH4.0 ~ 9.0 after, as shown in Figure 3.
3) mensuration of zytase XYNH optimum temperuture: in pH 5.0 times, this enzyme and 0.5% xylan solution react 15min respectively at different temperatures, and add 2.5mL DNS reagent, boiling water bath 5min, is cooled to room temperature, adds water and is settled to 12.5mL.OD is detected in spectrophotometer
540.With most high enzymatic activity for 100%, calculate the relative residual enzyme activity under other conditions.Result shows, and zytase XYNH optimal reactive temperature is 100 DEG C, can maintain the enzyme activity of more than 50%, as shown in Figure 5 as extremely high temperature resistant enzyme zytase XYNH at 80 DEG C ~ 100 DEG C.
4) mensuration of zytase XYNH thermal destruction curve: by xylanase solution after the dilution of pH 5 damping fluid, be placed in 100 DEG C of water-baths, process 0 respectively, 30,60,120 and 180min after, under optimum reaction conditions, detect Xylanase activity, calculate the transformation period of zytase accordingly.Result shows, and under optimal pH condition, after 100 DEG C of process 2h, this enzyme still can keep about 50% enzyme activity, shows that this enzyme half death time at 100 DEG C reaches 2h, as shown in Figure 5.
The zytase 1VBR of the zymologic property of the zytase 1VBR that Pichia yeast engineering gives expression to and the Thermotoga maritima Thermotoga maritima MSB8 of original report is built without noticeable change after gene optimization, its optimal reactive temperature is not less than 100 DEG C, and thermotolerance is fabulous.Illustrate that the Pichia yeast engineering constructed can produce zytase 1VBR by normal expression, make this zytase of commercial scale production become possibility.
Claims (7)
1. the extremely thermostable xylanase XYNH encoding gene optimized, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO.1.
2. comprise the expression vector of the extremely thermostable xylanase XYNH encoding gene optimized described in claim 1.
3. comprise the Expression vector pPIC9K-XYNH of the extremely thermostable xylanase XYNH encoding gene optimized described in claim 1.
4. comprise described in claim 1 optimize extremely thermostable xylanase XYNH encoding gene recombinant bacterial strain.
5. recombinant bacterial strain according to claim 4, is characterized in that, described bacterial strain is recombination bacillus coli, or recombination yeast.
6. a method of high expression extremely thermostable xylanase XYNH, is characterized in that, said method comprising the steps of:
(1) transformed host strain is carried with expressing described in claim 2;
(2) host strain after transforming is cultivated;
(3) Purification of Xylanases XYNH.
7. the extremely thermostable xylanase XYNH encoding gene optimized described in claim 1 is for expressing the application of hot zytase XYNH.
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| CN107267487A (en) * | 2017-07-18 | 2017-10-20 | 广东溢多利生物科技股份有限公司 | A kind of preparation method of recombinant heat-proof zytase |
| CN108018274A (en) * | 2018-02-01 | 2018-05-11 | 中南民族大学 | A kind of mutant XYNH of extremely thermostable xylanase 1VBR and application thereof |
| CN111549016A (en) * | 2020-05-25 | 2020-08-18 | 中南民族大学 | Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof |
| CN111621486A (en) * | 2020-05-25 | 2020-09-04 | 中南民族大学 | Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267487A (en) * | 2017-07-18 | 2017-10-20 | 广东溢多利生物科技股份有限公司 | A kind of preparation method of recombinant heat-proof zytase |
| CN108018274A (en) * | 2018-02-01 | 2018-05-11 | 中南民族大学 | A kind of mutant XYNH of extremely thermostable xylanase 1VBR and application thereof |
| CN111549016A (en) * | 2020-05-25 | 2020-08-18 | 中南民族大学 | Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof |
| CN111621486A (en) * | 2020-05-25 | 2020-09-04 | 中南民族大学 | Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method |
| CN111621486B (en) * | 2020-05-25 | 2021-10-19 | 中南民族大学 | Heat-resistant xylanase XYNB with high enzyme activity at low temperature, mutant gene, application and gene sequence preparation method |
| CN111549016B (en) * | 2020-05-25 | 2021-11-26 | 中南民族大学 | Extreme heat-resistant xylanase XYNA and mutant gene, application and preparation method thereof |
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