CN101624574A - Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof - Google Patents
Yeast engineering bacteria for expressing high-stability xylanase, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a yeast engineering bacteria for expressing high-stability xylanase with a preserving number of CCTC NO: M209148. The yeast engineering bacteria is formed by transfecting a recombinant mutant plasmid pPIC9KXYN17 in a secretion type pichia pastoris recombinant strain, and the mutant recombinant plasmid pPIC9KXYN17 takes a gene of glyceraldehydes-3-phosphate dehydrogenase as a promoter and comprises xylanase gene and ZeocinTM resistance gene. The invention also discloses a construction method and the application of the yeast engineering bacteria. The recombinant engineering bacteria is characterized in that: enzymatic specific activity is increased to 856IU/mg from 203IU/mg; optimum temperature is increased to 60 DEG C from 50 DEG C; half life period at 70 DEG C is enhanced to 14 minutes from 1 minute; and the stability range of pH is expanded to 4.0-10.0 from 5.0-7.0 under 50 DEG C for 60 min.
Description
Technical field
The present invention relates to a kind of engineering bacteria and construction process and application of expressed xylanase, specifically, relate to a kind of Yeast engineering bacterium strain and construction process and the application that can express high-stability xylanase.
Background technology
Zytase (EC3.2.1.8) is the enzyme system of a class with β-1,4 xylan glycosidic bond in the internal-cutting way degradation of xylan molecule.This enzyme is a kind of important industrial enzymes, can be widely used in food, feed, brewages, industry such as medicine and papermaking.Particularly this enzyme is used for association with pulp bleaching in pulp and paper industry, can increase the paper whiteness, improves paper performance, reduces the consumption of bleaching with chemical substance, thereby effectively alleviates the huge pollution that paper-making industry causes environment.
Present business-like zytase mostly is acidity or neutral xylanase, and carrying out pulp processing about 60 ℃, under the alkaline condition during bio-bleaching, bio-bleaching technology is considered from the Technological Economy aspect to require to use zytase to have greater activity under comparatively high temps and wide in range pH condition.Therefore, screen or make up heat-resisting, alkali proof zytase and become one of focus of current research.
People such as the Yao Bin of the Chinese Academy of Agricultural Sciences carry out rite-directed mutagenesis to the Xylanase XYNB of Streptomyces olivaceoviridis, find that mutant enzyme N13D and S40E handle 5min at 70 ℃, and thermostability has improved 24.76% and 14.46% respectively than protoenzyme; The specific activity of mutant enzyme N13D has improved 22% than XYNB, and optimal pH rises to 5.8 from 5.2.
(the Aspergillus usam ii) zytase Xyn II of E001 carries out rite-directed mutagenesis to people such as the Li Wu of Southern Yangtze University in 2008 to Aspergillus usamii, the result shows that the optimal pH of Xyn IID37N is elevated to 5.3 by 4.2, the pH stable range is reduced to 3.0～5.5 by 3.0～7.5, but optimum temperuture and thermostability remain unchanged substantially.The result shows that there be the 37th Asp of a conservative amino acid sites in the catalyst structure domain of the 11st family's zytase between βZhe Die thigh A3 and B3, this site is relevant with the pH characteristic of zytase.
People such as Fred in 2004, xylanase I I to Trichoderma reesei has carried out rite-directed mutagenesis, made up muton Y5 (T2C, T28C, K58R ,+191D), the result shows, mutant enzyme is elevated to 20min 65 ℃ transformation period by 40s, and the transformation period in the time of 70 ℃ is by being elevated to 6min less than 10s, and pH stability and wild-type enzyme are consistent.Subsequently, people such as Fred have made up several mutons: P9 (N97R+F93W+H144K) again on the basis of Y5, P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C).The pH stability of these several mutant enzymes all has raising in various degree, and the B6b of beta-pleated sheet and B9 zone are very important for the stability of enzyme, especially to thermotolerance.
People such as Jian-Yi Sun in 2005 with the terminal corresponding position that substitutes Aspergillus niger zytase A (AnxA) of the N-of Thermomonospora fusca zytase A (TfxA), make up the zytase of a heterozygosis, are called ATx.Test-results shows that the thermotolerance of heterozyme improves, child care 1h under ℃ condition of pH3.0～10.0,25, and the residual enzyme of ATx is lived all more than 80%, and therefore, thermotolerance and the catalytic activity of AnxA have all improved.
People such as Seok in 2008 think that the pKa value of catalytic residue directly affects the optimal pH of enzyme, therefore can determine mutational site and the prediction pH stability that mutant enzyme had by the calculating of pKa.They are with Bacillus circulans zytase (BCX) be fundamental construction two muton: A115X and S84X.Wherein the activity of A115X mutant enzyme descends, and may be to have destroyed the appropriate change of enzymatic structure in the enzyme-to-substrate cohesive process.Therefore illustrated that also the amino-acid residue far away from catalytic site also has a very important role to the pKa value of the amino-acid residue of catalytic site and the optimal pH of BCX.
Summary of the invention
Technical problem to be solved by this invention provides a kind of Yeast engineering bacteria of expressing high-stability xylanase, with realize effectively saving enzyme use in the soda acid that brings of pH and temperature control and energy consumption cost, the realization zytase industrial applications of producing at association with pulp bleaching and xylo-oligosaccharide be worth.
The technical problem that the present invention also will solve provides the construction process of above-mentioned Yeast engineering bacteria.
The technical problem that the present invention will solve at last provides the application of above-mentioned Yeast engineering bacteria.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of Yeast engineering bacteria of expressing high-stability xylanase, its called after pichia pastoris phaff (Pichia.pastoris) of classifying.Be preserved in Wuhan University China typical culture collection center (being called for short CCTCC), address: Wuhan Wuhan University; Postcode: 430072; The numbering of registering on the books: CCTCCNO:M209148; Preservation date: on July 14th, 2009.
The Yeast engineering bacteria of above-mentioned expression high-stability xylanase is secretor type pichia pastoris recombinant bacterial strain.In secretor type pichia pastoris recombinant bacterial strain, transform recombination mutation plasmid pPIC9KXYN17 is arranged, this recombination mutation plasmid pPIC9KXYN17 is a promotor with glyceraldehyde 3-phosphate dehydrogenase gene (DQ465985), and contains xylanase gene (XYN II) (S67387) and ZeocinTM resistant gene (Z46234).
A kind of method that makes up the Yeast engineering bacteria of above-mentioned expression high-stability xylanase may further comprise the steps:
(1) uses conventional construction of recombinant plasmid method and construct recombinant plasmid pPIC9K-XYN II;
(2) using primer-design software, is template with recombinant plasmid pPIC9K-XYN II, designs three mutant primers, is respectively:
T2C-gtggagaagcgccag
tgcattcagcccggcacg；
T28C-cggcgtgacgta?c
tgcaatggtcccggc；
S156F-cggcgaaccac
ttcaacgcgtgggc；
(3) the multiple rite-directed mutagenesis test kit of employing Stratagene company, with recombinant plasmid pPIC9K-XYN II is template, the primer of design in the applying step (2), the the 2nd and 28 Threonine of template is sported halfcystine, and then between these two amino acid, form disulfide linkage the N-end sequence of this enzyme is linked to each other with adjacent beta chain, and be phenylalanine with the 156th mutant serine; Prepare recombination mutation plasmid pPIC9KXYN17;
(4) with behind the recombination mutation plasmid pPIC9KXYN17 usefulness Sal I linearization for enzyme restriction, electric shock is transformed in the pichia spp host bacterium, promptly is built into the pichia spp recombinant bacterial strain of expressed xylanase.
(5) after the recombinant bacterial strain that step (4) is made up, process methyl alcohol utilize phenotypic screen, the screening of foreign gene multi-copy integration and abduction delivering screening, promptly select the recombinant bacterial strain of highly effective expression of xylanase.
Wherein, in the step (1), described recombinant plasmid pPIC9K-XYNII is that goal gene XYNII is inserted into the recombinant plasmid that forms among the plasmid pPIC9K.Concrete grammar is referring to the Multi-Copy PichiaExpression Kit operational manual of Invitrogen company.
In the step (2), primer-design software adopts the online primer-design software of Stratagene company
Primer Design Program.
In the step (4), after recombination mutation plasmid pPIC9KXYN17 is used Sal I linearization for enzyme restriction, before electric shock is transformed in the pichia spp host bacterium, mutant plasmid pPIC9KXYN17 can be imported intestinal bacteria and increase.
The application of the Yeast engineering bacteria of above-mentioned expression high-stability xylanase in producing high-stability xylanase.
In the BMG substratum, 28～30 ℃ are cultured to cell density OD with the recombinant bacterial strain of highly effective expression of xylanase of preparation
600Reach 2～6, use the BMM re-suspended cell to OD
600=1.0, carry out abduction delivering.Through 72 hours inducing culture (adding 100% methyl alcohol to final concentration every 24 hours to substratum is 1.0%), to finish the yeast gene engineering bacteria of cultivation through the centrifugal thalline of removing, collect supernatant liquor, with the molecular weight is the ultrafiltration pipe of 10KD, elimination small molecular weight protein and salt, through molecular sieve Superdex 75prep grade purifying, use deionized water, flow velocity 0.5ml/min wash-out, the sample in the elution peak is collected in distribution, through enzyme activity determination, determine the collection tube at purpose sample place, obtain electrophoretically pure target protein.
The present invention uses the method for transgenation to improve the industrial application stability of xylanase gene XYN II coded product; Pass through site-directed mutagenesis, obtain mutator gene, the xylanase gene of this sudden change thermostability and pH stability behind Pichia anomala expression increase substantially, make up the yeast gene engineering bacteria that efficiently expresses recombined xylanase by engineered means, and therefrom cultivation separation and purification is produced the recombined xylanase of alkali-resistant.
Beneficial effect: the resulting sudden change zytase of the present invention (below be designated as HA17) and the zytase that do not suddenly change (below be designated as H9K+-3) carry out zymologic property relatively, be increased to 856IU/mg than enzyme work by original 203IU/mg, optimal reaction pH is constant, is 5.0 (pH5.0-6.0 basically identicals); Optimal reactive temperature is brought up to 60 ℃ (55-65 ℃ of basically identicals) by 50 ℃, and the work of 60 ℃ of 2min enzymes of protoenzyme reduces to 59%, and 60 ℃ of 20min enzymes of mutant enzyme are lived and do not descended; 70 ℃ of transformation period were brought up to 14 minutes by 1 minute, and the pH stable range under 50 ℃ of 60min expands to 4.0～10.0 by 5.0～7.0.
Description of drawings
Fig. 1 is the structural representation of recombinant plasmid pPIC9K-XYN II.
Fig. 2 is the schema of multiple rite-directed mutagenesis method.
Fig. 3 is the SDS-PAGE collection of illustrative plates of purified enzyme liquid.
Fig. 4 is the optimal reactive temperature of the sudden change zytase and the zytase that do not suddenly change.
Fig. 5 is the influence of pH to xylanase activity.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1: construction of recombinant plasmid.
Construct recombinant plasmid pPIC9K-XYN II (the Multi-Copy Pichia Expression Kit operational manual of Invitrogen company), its structural representation as shown in Figure 1, this recombinant plasmid pPIC9K-XYN II is that goal gene XYN II is inserted into the recombinant plasmid that forms among the plasmid pPIC9K, and later mutation operation is finished on this recombinant plasmid.
Embodiment 2: the rite-directed mutagenesis of recombinant plasmid.
Use the online primer-design software of Stratagene company
Primer Design Program is a template with recombinant plasmid pPIC9K-XYN II, designs three mutant primers, is respectively:
T2C-gtggagaagcgccag
tgcattcagcccggcacg；
T28C-cggcgtgacgta?c
tgcaatggtcccggc；
S156F-cggcgaaccac
ttcaacgcgtgggc；
The 2nd, 28 of recombinant plasmid pPIC9K-XYN II and 156 amino acids are carried out rite-directed mutagenesis, prepare recombination mutation plasmid pPIC9KXYN17.Wherein, the 2nd and 28 s' Threonine sports halfcystine, and then forms disulfide linkage between these two amino acid the N-end sequence of this enzyme is linked to each other with adjacent beta chain; The 156th mutant serine is phenylalanine; The site-directed point mutation reaction system is as shown in table 1, and the PCR reaction parameter is as shown in table 2.
After PCR finishes, through Dpn I digestion, mutant plasmid is transformed in the intestinal bacteria body, the plasmid DNA that adopts TaKaRa company purification kit is in a small amount extracted plasmid, delivers to the order-checking of Jin Site company, selects and wants the plasmid that obtains.
This multiple rite-directed mutagenesis method flow as shown in Figure 2.The first step serves as to instruct and is that template is carried out pcr amplification with plasmid pPIC9K-XYN II with the mutant primer that designs; In second step, the plasmid that amplification is good digests with the DpnI enzyme, removes the DNA chain identical with fundamental chain, is only contained the DNA chain in mutational site; In the 3rd step, the plasmid DNA that digestion is good transforms into and increases in the XL10-Gold competent cell.
Table 1 site-directed point mutation reaction system
Table 2PCR reaction parameter
Embodiment 3: the preparation of target DNA and the electricity of pichia spp transform.
Resulting intestinal bacteria transformant is cultivated, extracted plasmid, adopt Sal I to carry out enzyme and cut, obtain linear target DNA by phenol extracting and ethanol sedimentation; Produce the electroreception attitude cell of pichia spp; The electroreception attitude cell of 80 μ l is mixed with the linearizing DNA of 10 μ l, change the 0.2cm electricity over to and transform in the cup, under voltage 2.0KV condition, shock by electricity.
Embodiment 4: the screening of transformant.
(1) methyl alcohol of yeast transformant utilizes the screening of phenotype.
With sterilization toothpick picking mono-clonal, on MM and MD flat board, rule in some way or some His
+Transformant is guaranteed point on the MM flat board earlier; For separating Mut
+And Mut
sPhenotype, contrast (GS115/His on each point on MD and the MM flat board
+Mut
sAblumin) reach (GS115/His
+Mut
+β-gal); 30 ℃, hatched 2 days; Two days later, the big or small also counting of observation bacterium colony, (phenotype is His to the yeast transformant of screening equal energy normal growth on MD flat board and MM flat board
+Mut
+).
(2) foreign gene multi-copy integration transformant screening.
Prepare an amount of YPD substratum, high-temperature sterilization postcooling to 55 ℃～60 ℃ is pressed the 100mg/mlG418 stock solution that table 3 adds proper volume, mixing shop system is dull and stereotyped immediately, contain different concns G418 (0mg/ml, 0.25mg/ml, 0.75mg/ml with preparation, 1.00mg/ml, 1.50mg/ml, 2.00mg/ml, 3.00mg/ml, 4.00mg/ml) the YPD flat board, note to reduce bubble and produce; Prepare 96 porocyte culture plates, in each hole, add 200 μ l YPD; In first group of flat board, every hole inoculate single HIS with the sterilization toothpick
+Transformant stirs gently with suspension cell; Covering 96 orifice plates hatched 2 days at 30 ℃; After 2 days, get 96 new orifice plates, every hole adds 190 μ l YPD; From first group of plate, draw 10 μ l nutrient solutions in second group of plate of correspondence with pipettor; Cover second group of plate, 30 ℃ of overnight incubation; Second day, in the 3rd group of plate, repeat the operation of second group of plate; After hatching, get the 3rd group of plate, blow and beat suspension cell again up and down with the nutrient solution that pipettor is drawn in the hole; Get that liquid dot is 0,0.25,0.5,0.75,1.00,2.00,3.00 containing Geneticin concentration in the every hole of 10 μ l, on the YPD flat board of 4.00mg/ml; Treat liquid-absorbent, cultivated 2～5 days, and checked the colony growth situation every day for 30 ℃; Picking has the bacterium colony of G418 resistance, and purification process is carried out in line on the YPD flat board.
Table 3G418 stock solution
(3) recombination microzyme is expressed.
The picking mono-clonal is seeded to 25ml MGY, and among BMG or the BMGY, 28-30 ℃, 250-300rpm shakes to OD
600=2-6; The centrifugal 5min of room temperature 1500-3000g, collecting cell is removed supernatant, uses MM, and BMM or BMMY re-suspended cell are to OD
600=1.0, carry out abduction delivering; Shake the above-mentioned culture of adding in the bottle at 1L, add a cover two-layer sterile gauze, put into the shaking table continued growth; Per 24 hours, add methyl alcohol to final concentration and be 1.0% and induce continuing; Finished in 72 hours to express, collect fermented liquid, measure enzymic activity.
Enzyme unit definition alive: 1 xylanase activity unit (IU) is 1% soluble xylan (4-O-Me-D-glucurono-D-xylan, Sigma From Birchwood) be substrate, per minute decomposes xylan and generates the required enzyme amount of 1 μ mol wood sugar under pH4.8,50 ℃ of conditions.
Embodiment 5: the purifying of recombined xylanase.
Yeast fermentation broth centrifugal 10 minutes through 4000rpm/min, is supernatant liquor that the ultrafiltration pipe of 10KD carries out ultrafiltration by molecular weight, some small molecular proteins of elimination and salt are got the liquid 2ml of ultra filtration, change through molecular sieve Superdex 75prep grade pure 0, use deionized water, flow velocity 0.5ml/min wash-out, the sample in the collection elution peak that distributes is through enzyme activity determination, determine the collection tube at purpose sample place, obtain electrophoretically pure target protein.As shown in Figure 3, be the SDS-PAGE collection of illustrative plates of purified enzyme liquid.Wherein M is a standard molecular weight albumen, 1 is HA17,2 is H9K+-3,3 is that HA17 handles (because untreated HA17 has two bands with deglycosylating enzyme, an only surplus band after deglycosylating enzyme is handled, illustrate an other band by glycosylation enzyme), 4 is deglycosylating enzyme (Deglycosylase).
Embodiment 6: the analysis of zymologic property and comparison.
Mutant enzyme behind the purifying and wild-type enzyme are carried out the comparative studies of zymologic property.The optimum temperuture and thermostability, optimal pH and pH stability, the specific activity etc. that comprise enzyme.Be increased to 856IU/mg than enzyme work by original 203IU/mg, optimal reaction pH is constant, is 5.0 (pH5.0-6.0 basically identicals); Optimal reactive temperature is brought up to 60 ℃ (55-65 ℃ of basically identicals) by 50 ℃, and the work of 60 ℃ of 2min enzymes of protoenzyme reduces to 59%, and 60 ℃ of 20min enzymes of mutant enzyme are lived and do not descended; 70 ℃ of transformation period were brought up to 14 minutes by 1 minute, and the pH stable range under 50 ℃ of 60min expands to 4.0～10.0 by 5.0～7.0.
Fig. 4 can learn that for the optimal reactive temperature of sudden change zytase with the zytase that do not suddenly change optimal reactive temperature is brought up to 60 ℃ by 50 ℃ from figure.Fig. 5 is the influence of pH to xylanase activity, and the pH stable range under 50 ℃ of 60min expands to 4.0～10.0 by 5.0～7.0.Wherein, do not adding under the substrate condition, placing 50 ℃ of water-bath incubations to measure enzyme after 30 minutes enzyme and live.
SEQUENCE?LISTING
＜110〉Nanjing Forestry University
＜120〉a kind of Yeast engineering bacteria and construction process and application of expressing high-stability xylanase
<130>njfu090724
<160>3
<170>PatentIn?version?3.3
<210>1
<211>33
<212>DNA
<213>Artificial
<220>
<223>T2C
<400>1
gtggagaagc?gccagtgcat?tcagcccggc?acg 33
<210>2
<211>28
<212>DNA
<213>Artificial
<220>
<223>T28C
<400>2
cggcgtgacg?tactgcaatg?gtcccggc 28
<210>3
<211>25
<212>DNA
<213>Artificial
<220>
<223>S156F
<400>3
cggcgaacca?cttcaacgcg?tgggc 25
Claims (7)
1, a kind of Yeast engineering bacteria of expressing high-stability xylanase has been preserved in Wuhan University China typical culture collection center, and its deposit number is CCTCC NO:M 209148.
2, the Yeast engineering bacteria of expression high-stability xylanase according to claim 1 is characterized in that described Yeast engineering bacteria is a secretor type pichia pastoris recombinant bacterial strain.
3, the Yeast engineering bacteria of expression high-stability xylanase according to claim 2, it is characterized in that described secretor type pichia pastoris recombinant bacterial strain conversion has recombination mutation plasmid pPIC9KXYN17, described recombination mutation plasmid pPIC9KXYN17 is a promotor with the glyceraldehyde 3-phosphate dehydrogenase gene, and contains xylanase gene and ZeocinTM resistant gene.
4, a kind of method that makes up the Yeast engineering bacteria of the described expression high-stability xylanase of claim 1 is characterized in that this method may further comprise the steps:
(1) construction recombination plasmid pPIC9K-XYN II;
(2) using primer-design software, is template with recombinant plasmid pPIC9K-XYN II, designs three mutant primers, is respectively: T2C-gtggagaagcgccag
TgcAttcagcccggcacg; T28C-cggcgtgacgta c
TgcAatggtcccggc; S156F-cggcgaaccac
TtcAacgcgtgggc;
(3) the multiple rite-directed mutagenesis test kit of employing Stratagene company, with recombinant plasmid pPIC9K-XYN II is template, the primer of design in the applying step (2), template the 2nd and 28 s' Threonine is sported halfcystine, and then between these two amino acid, form disulfide linkage the N-end sequence of this enzyme is linked to each other with adjacent beta chain, and be phenylalanine with the 156th mutant serine; Prepare recombination mutation plasmid pPIC9KXYN17;
(4) with behind the recombination mutation plasmid pPIC9KXYN17 usefulness Sal I linearization for enzyme restriction, electric shock is transformed in the pichia spp host bacterium, promptly is built into the Yeast engineering bacteria of expressed xylanase;
(5) after the recombinant bacterial strain that step (4) is made up, process methyl alcohol utilize phenotypic screen, the screening of foreign gene multi-copy integration and abduction delivering screening, promptly select the Yeast engineering bacteria of highly effective expression of xylanase.
5, the method for the Yeast engineering bacteria of construction expression high-stability xylanase according to claim 4, it is characterized in that in the step (1) that described recombinant plasmid pPIC9K-XYN II is that goal gene XYNII is inserted into the recombinant plasmid that forms among the plasmid pPIC9K.
6, the method for the Yeast engineering bacteria of construction expression high-stability xylanase according to claim 4, it is characterized in that in the step (4), after recombination mutation plasmid pPIC9KXYN17 is used Sal I linearization for enzyme restriction, electric shock is transformed into before the pichia spp host bacterium, mutant plasmid pPIC9KXYN17 is imported intestinal bacteria increase.
7, the Yeast engineering bacteria of the described expression high-stability xylanase of claim 1, the application in producing high-stability xylanase.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899454B (en) * | 2010-02-05 | 2012-05-23 | 杭州宝晶生物化工有限公司 | 2-D-arbaitol dehydrogenase gene, recombinant protein thereof, escherichia coli containing gene and application thereof |
| CN105505806A (en) * | 2016-01-16 | 2016-04-20 | 新乡医学院 | Construction method of xylanase hybrid enzyme engineering strain |
| CN105349511B (en) * | 2015-12-16 | 2018-10-12 | 南京工业大学 | Zytase and the gene for encoding the enzyme and its application in deinking |
| CN105368804B (en) * | 2015-12-16 | 2018-10-12 | 南京工业大学 | A kind of high temperature resistant zytase of resistance to highly basic and its application in deinking |
| CN110607291A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103555749B (en) * | 2012-12-29 | 2015-06-24 | 湖北大学 | Method for in vitro efficient construction of multi-copy Pichia expression vector |
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2009
- 2009-08-06 CN CN2009101844533A patent/CN101624574B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899454B (en) * | 2010-02-05 | 2012-05-23 | 杭州宝晶生物化工有限公司 | 2-D-arbaitol dehydrogenase gene, recombinant protein thereof, escherichia coli containing gene and application thereof |
| CN105349511B (en) * | 2015-12-16 | 2018-10-12 | 南京工业大学 | Zytase and the gene for encoding the enzyme and its application in deinking |
| CN105368804B (en) * | 2015-12-16 | 2018-10-12 | 南京工业大学 | A kind of high temperature resistant zytase of resistance to highly basic and its application in deinking |
| CN105505806A (en) * | 2016-01-16 | 2016-04-20 | 新乡医学院 | Construction method of xylanase hybrid enzyme engineering strain |
| CN105505806B (en) * | 2016-01-16 | 2019-03-01 | 新乡医学院 | A kind of construction method of zytase heterozyme engineered strain |
| CN110607291A (en) * | 2018-06-14 | 2019-12-24 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
| CN110607291B (en) * | 2018-06-14 | 2022-05-31 | 青岛蔚蓝生物集团有限公司 | Heat-resistant xylanase mutant |
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