CN102978223A - Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end - Google Patents
Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end Download PDFInfo
- Publication number
- CN102978223A CN102978223A CN 201210562539 CN201210562539A CN102978223A CN 102978223 A CN102978223 A CN 102978223A CN 201210562539 CN201210562539 CN 201210562539 CN 201210562539 A CN201210562539 A CN 201210562539A CN 102978223 A CN102978223 A CN 102978223A
- Authority
- CN
- China
- Prior art keywords
- aoxyn11a
- enzyme
- xyn11
- heterozygosis
- xylanase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for improving the thermal stability of Aspergillus oryzae xylanase through adding a disulfide bond on an N end. The invention provides a novel method for directionally transforming normal-temperature xylanase through utilizing a computer technology and a bioinformatics method, and a genetic engineering method is utilized to construct heterozygosis xylanse engineering bacteria with high-efficiency expression capability. A normal-temperature xylanase gene from an Aspergillusoryzae (China center of industrial culture collection) CICC 40186 strain is subjected to thermal stability directional transformation, so that the heterozygosis xylanse is obtained, the thermal stability of the heterozygosis xylanse is certainly improved, the activity of the heterozygosis xylanse is not reduced compared with the activity of an original enzyme, and the xylanse has large industrial production potential and economic value as a heat-resistant enzymic preparation.
Description
Technical field
Normal temperature zytase (AoXyn11A) gene that the present invention relates to be derived from aspergillus oryzae (Aspergillus oryzae) CICC 40186 bacterial strains carries out the thermostability directional transformation, the method that efficiently expresses of the structure of heterozygosis zytase engineering bacteria and restructuring heterozygosis zytase belongs to technical field of bioengineering.
Background technology
Zytase (EC 3.2.1.8) is the important industrial enzymes of a class.It mainly is to act on xylan backbone, cuts randomly the wood sugar glycosidic bond of xylan inside, and hydrolysate is mainly the wood oligose of different polymerization degree and a small amount of wood sugar, is the enzyme of most critical in the xylanolytic enzyme.In recent years, because zytase is in fodder industry, food-processing and the industry such as brewage and have potential industrial application and economic worth, the especially application in industrial papermaking reduced the environmental pollution that produces because of bleaching, had received increasing concern.Therefore yet in many industry, zytase all need to operate under hot conditions, and stable on heating research has also caused the great attention of domestic and international research to zytase.The research of exploitation heat resistant type zytase mainly comprises screens super heat-resisting xylan enzyme-producing bacteria and utilizes genetically engineered that the enzyme molecule is carried out directional transformation, and with respect to the blindness of sieve bacterium technology, the latter has stronger specific aim, and the parent who is subject to Chinese scholars looks at.
According to structure and the property analysis of zytase catalysis region, it can be divided into different families, wherein in the majority with glycoside hydrolase the 10th family and the 11st family.Because the zytase molecular weight of G/11 family, and relatively simple for structure, be more suitable for the molecular model as theoretical investigation, can be used for the research of zytase catalysis modular system and protein molecule folding mechanism etc. under the extreme condition.The fast development of genetic engineering technique, information biology and computer science is for new prospect has been opened up in molecular biological research.We can use extensive high performance computer and related software thereof, in conjunction with genetic engineering means, utilize simulation test that the enzyme molecule is carried out directional transformation to improve thermostability, accelerate the application process of zytase in various fields.
Summary of the invention
The purpose of this invention is to provide a kind of novel computer technology and bioinformatics method of utilizing the zytase of normal temperature is carried out design and rational, use engineered method to make up the method that efficiently expresses of heterozygosis zytase engineering bacteria and restructuring heterozygosis zytase.To be derived from the zytase called after AoXyn11A of the 11st family of A.oryzae CICC 40186, its corresponding unnamed gene is Aoxyn11A; The template that adopts is the super heat resistant xylanase with family, with its called after EvXyn11
TS, gene order is this laboratory synthetic gene Syxyn11 (GenBank accession NO.JX459567); The heterozygosis zytase called after AoXyn11A that is obtained by directional transformation
1, its corresponding gene is AoXyn11A
1, its thermostability improves, and the activity of enzyme does not descend than protoenzyme, and larger industrial application potentiality and economic worth are arranged.
Technical scheme of the present invention: the xylanase gene of a kind of A.oryzae of deriving from CICC 40186 (Aoxyn11A) carries out design and rational under area of computer aided, obtains a kind of heterozygosis zytase: AoXyn11A
1, the DNA that it is complete and aminoacid sequence are called SEQID NO:1 and SEQ ID NO:2.
The activity determination method of described recombined xylanase:
In 25mL tool plug test tube A and B, each adds with pH 5.5, citric acid-Na
2HPO
4The mass concentration of damping fluid preparation is 0.5% beech xylan (Sigma, USA) solution 2.4mL, and 50 ℃ of preheating 10min add the suitably enzyme liquid of dilution of 0.1mL, 50 ℃ of accurate response 10min in the A pipe; Respectively add immediately 2.5mL 3 ', 5 '-dinitrosalicylic acid (DNS) reagent, in the B pipe, add again 0.1mL enzyme liquid, A and B pipe all boil 7min; Respectively add deionized water 5mL after the cooling, shake up; 540nm sentences the B pipe and measures A pipe absorbance for blank, and finds corresponding reducing sugar (in wood sugar) content and be converted to unit of enzyme activity from the wood sugar typical curve.Unit of enzyme activity's definition: under this condition determination, produce the required enzyme amount of 1 μ mol reducing sugar with per minute and be defined as 1 xylanase activity unit (IU).
The design of described heat-resisting heterozygosis xylanase gene, Cloning and Expression:
(1) space structure of homology modeling Simulation AoXyn11A: login protein structure database Protein data bank (http://rcsb.org), through the BLAST comparison, find with AoXyn11A to have the height homologous sequence.This experimental selection has very high heat-staple EvXyn11
TS(Protein Data Bank accession number is 2VUL) is template, and both homologys are 66.49%, uses that the SWISS-MODEL server is on-line Full to be AoXyn11A structure space structure.Use software Verify 3D that the structural models of homology modeling is evaluated and optimized, obtain the higher space-filling model of accuracy.
(2) heterozygous genes that utilizes the method design and rational thermostability of molecular dynamics simulation to improve: by sequence relatively, we are EvXyn11
TS5 amino acid in front (NAQTC) add the N end of AoXyn11A to, again 32 T of heterozyme are changed into C, and then we are with EvXyn11
TSBe template, the heterozygosis zytase is carried out the homology modeling, use again molecular dynamics simulation respectively heterozyme and protoenzyme to be carried out total energy and the RMSD value is calculated, by as a result we to draw total energy and the RMSD value of heterozyme less for protoenzyme, namely thermostability is better.
(3) structure of heterozygosis zytase engineering bacteria and expression method:
(1) heterozygous genes AoXyn11A
1The structure of expression plasmid: design 3 primers according to the upper AoXyn11A gene order (GenBank accessionNo.JQ326257) of GenBank:
Xyn11-F:5 '-CTCGAGAAAAGAAACGCTCAAACTTGTTCCACACCCAGTAGCACGG-3 ' contains the XhoI restriction enzyme site
Xyn11-Rm：5’-GTATGAACCGCCGTTGCCGTTACAGTAAGTCACATCAC-3’
Xyn11-R:5 '-GCGGCCGCTCAATAAACAGTGATAGCAG-3 ' contains Not I restriction enzyme site
Adopt the large primer PCR technology that protogene is carried out directed mutagenesis, mainly be divided into for three steps: the recombinant plasmid that the carries the Aoxyn11A gene (number of patent application: 201110410412.9) as template that makes up take this laboratory, adopt primer Xyn11-F and Xyn11-Rm, carry out PCR first run amplification (94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min); To this PCR product nucleic acid electrophoresis and the recovery of tapping rubber, reclaim product and hold primer as N, C end primer is Xyn11-R, carries out second and takes turns pcr amplification (94 ℃ of 5min; 94 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 60s; 72 ℃ are extended 10min); Taking turns PCR purpose band with second clones, checks order; Correct pUCm-T-Aoxyn11A checks order
1With pPIC9K
M(patent applied for, the patent No. 201110410391.0) plasmid all carries out double digestion with Xho I and Not I, and the enzyme of recovery is cut product and connected under the effect of T4DNA ligase enzyme, obtains recombinant plasmid pPIC9K
M-AoXyn11A
1, and recombinant expression plasmid carried out sequencing;
(2) GS115/Aoxyn11A
1The mensuration that the structure of recon, expression and enzyme are lived: use Sac I to pPIC9K
M-Aoxyn11A
1Carry out linearizing, carry out electricity according to the Pichia anomala expression handbook and transform, screen, obtain the pichia spp recon GS115/Aoxyn11A of high copy
1This project bacterium is the recombined xylanase crude enzyme liquid with 1.0% methanol induction 96h, centrifuged supernatant, and the zymologic property of this enzyme and protoenzyme is compared: beech xylan substrate, reaction times 0.5% are in the situation of 10min, AoXyn11A
1Optimum temperuture rise to 58 ℃ by 55 ℃ of protoenzyme; Under 55 ℃ of preheatings, AoXyn11A
1Transformation period by protoenzyme～4min is raised to～28min.
The present invention has carried out directional transformation to aspergillus oryzae normal temperature zytase, on the basis that keeps original enzyme activity, has improved this Thermostability, makes it have better prospects for commercial application
Description of drawings
Fig. 1: recombinant plasmid pPIC9K
M-Aoxyn11A
1The structure synoptic diagram.
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only are used for describing the present invention in detail, limit the scope of the invention and be not used in.
The space structure of embodiment 1 homology modeling Simulation AoXyn11A and hybrid protein thereof
Login protein structure database Protein data bank (http://rcsb.org) through the BLAST comparison, finds with AoXyn11A to have the height homologous sequence.This experimental selection has very high heat-staple EvXyn11
TS(Protein Data Bank accession number is 2VUL) is template, and both homologys are 66.49%, uses that the SWISS-MODEL server is on-line Full to be AoXyn11A structure space structure.Use software Verify 3D that the structural models of homology modeling is evaluated and optimized, obtain the higher space-filling model of accuracy.
The heterozygous genes that embodiment 2 utilizes the method design and rational thermostability of molecular dynamics simulation to improve
Respectively to crystalline structure and the heterozygous AoXyn11A of the wild-type AoXyn11A that obtains by the homology modeling
1Carry out molecular dynamics simulation.Mainly comprise following a few step:
At first we carry out pre-treatment to space structure, use the pdb2gmx order among the GROMACS to add the hydrogen atom of all disappearances, and export a .gro file that comprises all atom information.The form of this order is:
pdb2gmx-f?1lbs.pdb-o?1lbs.gro-p?1lbs.top-ignh-ter
Second step utilizes editconf and the genbox order among the GROMACS to simulate the motion of AoXyn11A in the aqueous solution for the AoXyn11A space structure adds the suitable aqueous solution.Wherein solvent adopts the TIP3P water model, for the system of simulation, adds the water molecule layer of 1.5nm in the solute periphery.Its detailed process is at first to use the size of editconf command definition box, then reads in the structured file of GROMACS with the genbox order, and reads in the size of water box, and output file comprises minute subfile and water box.The original top file of simultaneously genbox change makes it contain water molecules.The form of this order is:
Editconf-f?1lbs.gro-o?1lbs_box.gro-bt?cubic-d?1.5
Genbox-cp?1lbs_box.gro-cs-p?1lbs.top-o?1lbs_water.gro
The 3rd step was used the electric charge of cation equilibrium system, and command format is:
grompp-f?em.mdp-c?1lbs_water_ion.gro-p?1lbs.top-o?minimize_water.tpr
genion-s?minimize_water.tpr-o?1lbs_water_ion.gro-p?1lbs.top-pname?Na+-np?10-random-gtrp_ion.log
Genion-s minimize_water.tpr-o 1lbs_water_ion.gro-p 1lbs.top-nname CL--nn 1-random-g trp_ion.log and need manually the 1lbs_water_ion.gro that obtains to be made amendment with the atom number in the 1lbs.top file is complementary with the metal ion of interpolation.
The 4th step was utilized respectively grompp and two orders of mdrun to carry out energy minimization and processes.At first retrain solute, optimized for 800 steps with method of steepest descent, optimized for 1200 steps with method of conjugate gradient again.Then go to carry out again the method for steepest descent optimization of 800 steps after the constraint, the method for conjugate gradient optimization of 1200 steps.Command format is:
grompp-f?emst1.mdp-c?1lbs_water_ion.gro-p?1lbs.top-o?minimize_water.tpr
mdrun-s?minimize_water.tpr-o?minimize_water.trr-c?minimize_water.gro-e?minimize_water.edr-gminimize_water.log
grompp-f?emst.mdp-c?minimize_water.gro-p?1lbs.top-o?minimize_water.tpr
mdrun-s?minimize_water.tpr-o?minimize_water.trr-c?minimize_water2.gro-e?minimize_water.edr-gminimize_water.log
The 5th step molecular dynamics simulation was divided into for two steps, at first carried out the MD simulation of the constraint solute of 20ps, and this moment, analog temperature progressively was elevated to 300K from 0K; Then carry out simulating without constraint constant temperature MD of 500ps.Command format is:
grompp-fpr.mdp-c?minimize_water2.gro-p?1lbs.top-o?minimize_water1.tpr
mdrun-s?minimize_water1.tpr-o?minimize_water1.trr-c?minimize_water1.gro-e?minimize_water1.edr-g?minimize_water1.log
grompp-f?full.mdp-c?minimize_water1.gro-p?1lbs.top-o?minimize_water2.tpr
mdrun-s?minimize_water2.tpr-o?minimize_water2.trr-c?minimize_water3.gro-e?minimize_water2.edr-g?minimize_water2.log
Obtain at last its total energy, each amino acid whose B-factor value of RMSD value and AoXyn11A.Command format is:
g_rms-s?minimize_water1.tpr-f?minimize_water1.trr-o?rms.xvg
xmgrace-nxy?rms.xvg
g_rmsf-s?minimize_water2.tpr-f?minimize_water2.trr-b?400-e?600-o?rmsf.xvg-oq?1.pdb
xmgrace-nxy?rmsf.xvg
g_energy-f?minimize_water2-o?energy.xvg
xmgrace-nxy?energy.xvg
The AoXyn11A that we obtain MD respectively and AoXyn11A
1Each amino acid whose B-factor value imports in the PDB file, uses B-FITTER software to derive each amino acid whose B-factor value, as seen from the figure AoXyn11A
1Each amino acid whose flexibility is less in the nitrogen terminal sequence, and fluctuating range is comparatively average, and AoXyn11A nitrogen terminal sequence is comparatively unstable, and total energy and the RMSD value less of heterozyme is described, namely thermostability is better.
Embodiment 3 heterozygous genes AoXyn11A
1And the structure of expression plasmid
Adopt the large primer PCR technology that protogene is carried out directed mutagenesis, mainly be divided into for three steps: take the protogene plasmid as template, adopt primer Xyn11-F and Xyn11-Rm, carry out PCR first run amplification (94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min); To this PCR product nucleic acid electrophoresis and the recovery of tapping rubber, reclaim product and hold primer as N, C end primer is Xyn11-R, carries out second and takes turns pcr amplification (94 ℃ of 5min; 94 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 60s; 72 ℃ are extended 10min); Taking turns PCR purpose band with second clones, checks order; Correct pUCm-T-Aoxyn11A checks order
1With pPIC9K
MPlasmid all carries out double digestion with Xho I and Not I, and the enzyme of recovery is cut product and connected under the effect of T4DNA ligase enzyme, obtains recombinant plasmid pPIC9K
M-AoXyn11A
1, and recombinant expression plasmid carried out sequencing;
Embodiment 4GS115/Aoxyn11A
1The mensuration that the structure of recon, expression and enzyme are lived
With Sac I to pPIC9K
M-Aoxyn11A
1Carry out linearizing, carry out electricity according to the Pichia anomala expression handbook and transform, screen, obtain the pichia spp recon GS115/Aoxyn11A of high copy
1This project bacterium is the recombined xylanase crude enzyme liquid with 1.0% methanol induction 96h, centrifuged supernatant, and the zymologic property of this enzyme and protoenzyme is compared: beech xylan substrate, reaction times 0.5% are in the situation of 10min, AoXyn11A
1Optimum temperuture rise to 58 ℃ by 55 ℃ of protoenzyme; Behind 55 ℃ of preheating 30min, AoXyn11A
1Transformation period by protoenzyme～4min is raised to～28min.
Claims (3)
1. normal temperature xylanase gene Aoxyn11A who is derived from aspergillus oryzae (Aspergillus oryzae) CICC 40186, belongs to glycoside hydrolase the 11st family, with another super heat resistant xylanase gene Syxyn11 (GenBank accession NO.JX459567) template with family, utilize computer technology and related software thereof, the method of binding molecule dynamics simulation, the AoXyn11A molecule is carried out design and rational, and directional transformation is to improve thermostability.
2. utilize the heterozygous genes of the method design and rational thermostability raising of molecular dynamics simulation:
(1) space structure of homology modeling Simulation AoXyn11A: login protein structure database Protein data bank (http://rcsb.org), through the BLAST comparison, find the sequence that has the height homology with AoXyn11A; The thermally-stabilised very high EvXyn11 of this experimental selection
TS(Protein Data Bank accession number is 2VUL) is template, and both homologys are 66.49%, and using the SWISS-MODEL server on-line Full be A.oryzae normal temperature zytase mature peptide structure space structure; Use software Verify_3D that the structural models of homology modeling is evaluated and optimized, obtain the higher space-filling model of accuracy;
(2) heterozygous genes that utilizes the method design and rational thermostability of molecular dynamics simulation to improve: by sequence relatively, we are EvXyn11
TS5 amino acid in front (NAQTC) add the N end of AoXyn11A to, again 32 T of heterozyme are changed into C, and then we are with EvXyn11
TSBe template, the heterozygosis zytase is carried out the homology modeling, use again molecular dynamics simulation respectively heterozyme and protoenzyme to be carried out total energy and the RMSD value is calculated, by as a result we to draw total energy and the RMSD value of heterozyme less for protoenzyme, namely thermostability is better.
3. the structure of heterozygosis zytase engineering bacteria and expression method:
(1) heterozygous genes Aoxyn11A
1The structure of expression plasmid: design 3 primers according to the upper Aoxyn11A gene order (GenBank accessionNo.JQ326257) of GenBank:
Xyn11-F:5 '-
CTCGAGAAAAGAAACGCTCAAACTTGTTCCACACCCAGTAGCACGG-3 ' contains the XhoI restriction enzyme site
Xyn11-Rm：5’-GTATGAACCGCCGTTGCCGTTACAGTAAGTCACATCAC-3’
Xyn11-R:5 '-
GCGGCCGCTCAATAAACAGTGATAGCAG-3 ' contains Not I restriction enzyme site
Adopt the large primer PCR technology that protogene is carried out directed mutagenesis, mainly be divided into for three steps: take the protogene plasmid as template, adopt primer Xyn11-F and Xyn11-Rm, carry out PCR first run amplification (94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min); To this PCR product nucleic acid electrophoresis and the recovery of tapping rubber, reclaim product and hold primer as N, C end primer is Xyn11-R, carries out second and takes turns pcr amplification (94 ℃ of 5min; 94 ℃ of 30s, 45 ℃ of 30s, 72 ℃ of 60s; 72 ℃ are extended 10min); Taking turns PCR purpose band with second clones, checks order; Correct pUCm-T-Aoxyn11A checks order
1With carrier pPIC9K
M(patent applied for, the patent No. 201110410391.0) plasmid all carries out double digestion with Xho I and Not I, and the enzyme of recovery is cut product and connected under the effect of T4DNA ligase enzyme, obtains recombinant plasmid pPIC9K
M-Aoxyn11A
1, and recombinant expression plasmid carried out sequencing;
(2) GS115/Aoxyn11A
1The mensuration that the structure of recon, expression and enzyme are lived: use Sac I to pPIC9K
M-Aoxyn11A1 carries out linearizing, carries out electricity according to the Pichia anomala expression handbook and transforms, screens, and obtains the pichia spp recon GS115/Aoxyn11A of high copy
1This project bacterium is the recombined xylanase crude enzyme liquid with 1.0% methanol induction 96h, centrifuged supernatant, and the zymologic property of this enzyme and protoenzyme is compared: beech xylan substrate, reaction times 0.5% are in the situation of 10min, AoXyn11A
1Optimum temperuture rise to 58 ℃ by 55 ℃ of protoenzyme; Under 55 ℃ of preheatings, AoXyn11A
1Transformation period by protoenzyme～4min is raised to～28min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210562539 CN102978223A (en) | 2012-12-24 | 2012-12-24 | Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210562539 CN102978223A (en) | 2012-12-24 | 2012-12-24 | Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102978223A true CN102978223A (en) | 2013-03-20 |
Family
ID=47852561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210562539 Pending CN102978223A (en) | 2012-12-24 | 2012-12-24 | Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102978223A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388412A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Method for improving heat stability of AoXyn11A through N-terminal replacement |
| CN104862290A (en) * | 2015-06-12 | 2015-08-26 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN111640466A (en) * | 2020-06-04 | 2020-09-08 | 山东大学 | Method for obtaining stable DNA tetrahedron synthesis parameters based on modeling |
-
2012
- 2012-12-24 CN CN 201210562539 patent/CN102978223A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388412A (en) * | 2014-12-10 | 2015-03-04 | 江南大学 | Method for improving heat stability of AoXyn11A through N-terminal replacement |
| CN104862290A (en) * | 2015-06-12 | 2015-08-26 | 江南大学 | 1,3-1,4-beta-glucanase mutant |
| CN104862290B (en) * | 2015-06-12 | 2017-11-17 | 江南大学 | A kind of 1,3 1,4 beta glucan enzyme mutants |
| CN111640466A (en) * | 2020-06-04 | 2020-09-08 | 山东大学 | Method for obtaining stable DNA tetrahedron synthesis parameters based on modeling |
| CN111640466B (en) * | 2020-06-04 | 2022-03-15 | 山东大学 | Method for obtaining stable DNA tetrahedron synthesis parameters based on modeling |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102719457A (en) | Rational design of heat resistance improvement of beta-1,4-endoxylanase (Aor Xyn11A) gene and preparation method of hybrid enzyme | |
| CN103642777A (en) | Method of improving thermal stability of aspergillus oryzae xylanase | |
| CN103003423B (en) | Improved xylose utilization in recombinant zymomonas having additional xylose isomerase activity | |
| CN102787130B (en) | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method | |
| CN104164375B (en) | The structure of ethanol high temperature high-yielding engineering bacterial strain and application | |
| CN103397064A (en) | Method for preparing rebaudioside M through enzyme method | |
| CN101531988B (en) | Alkaline pectinase genetic engineering bacteria and construction method thereof | |
| CN105143447B (en) | Protein and application thereof with xylose isomerase activity | |
| MX2014000665A (en) | Method for the production of cellulases by a filamentous fungus adapted to a fermenter having a low volumetric oxygen transfer coefficient kla. | |
| CN105441415A (en) | Preparation method and application of pullulanase mutant PulB-d99-D436H | |
| CN103642778A (en) | Beta-endoglucanase mutant and application thereof | |
| CN102703481A (en) | Method for heat resistance modification of aspergillus oryzae GH11 xylanase gene and preparation of mutant enzyme | |
| CN102978223A (en) | Method for improving thermal stability of Aspergillus oryzae xylanase through adding disulfide bond on N end | |
| CN104630183A (en) | Heat-resisting xylanase as well as coding gene and application thereof | |
| CN102260694A (en) | Acidproof medium-temperature alpha-amylase and preparation method thereof | |
| CN105255951B (en) | A method of Alcohol Production efficiency is improved by overexpression HAC1 genes | |
| CN105368731A (en) | Saccharomyces cerevisiae strain for expressing xylose isomerase and construction method | |
| CN105112433A (en) | Novel coding gene of Type-I pullulanase, and recombinant expression and application thereof | |
| CN102994476B (en) | Saccharifying enzyme | |
| CN103642776A (en) | Directed evolution method for improving heat resistance of aspergillus oryzae GH11 xylanase | |
| CN103045575B (en) | Recombinant L-arabinose isomerase as well as gene and application thereof | |
| CN103614354A (en) | Diastatic enzyme and recombined expression bacterial strain thereof | |
| CN103421761A (en) | Heatproof trehalose synthase fusion enzyme mutant gene and application thereof | |
| CN101886064B (en) | Acid amylase AMYA4 and gene and application thereof | |
| CN102492676A (en) | Directional transformation of computer-assisted beta-1,4-endo-xylanase (Aus Xyn11A) heat stability |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130320 |